研究成果の刊行に関する一覧表
雑誌
発表者氏名
Kasuga M.
Tokudome S.
Tsuboyama-Kasaoka N,
Tsubota-Utsugi M, Imai E,
Nakade M, Kasuga M.
Sasaki S.

Sasaki S.
Tabata I, Ebine N,
Kawashima Y,
Ishikawa-Takata K, Tanaka
S, Higuchi M, Yoshitake Y.
Kido Y, Shizuka F,
Shimomura Y, Sugiyama
T.
Ezaki O, Miyake Y, Sato S,
Iso H.
Yamada K,
Tsuboyama-Kasaoka N,
Goda T, Saito K,
Yamanouchi T, Yokoyama
T, Chonan O, Imai E,
Nakade M, Aoe S.
Tanaka K, Terao J, Shidoji
Y, Tamai H, Imai E, Okano
T.
Shibata K, Fukuwatari T,
Imai E, Hayakawa T,
Watanabe F, Takimoto H ,Watanabe T, Umegaki K.
Uenishi K, Ishimi Y,
Nakamura K, Kodama H,
Esashi T.
Yoshida M, Kikunaga S,
Yamauchi J,
Tsubota-Utsugi M,
Kodama H, Morita A,
Esashi T.
Morita A, Yoshiike N,
Takimoto H,
Tsubota-Utsugi M,
Kodama H, Shimizu T,
Sugiyama T, Tabata I,
Tamai H, Tsutsumi C,
Hirahara F, Kuzuya M,
Gomi I, Nakamura K,
Yoshida H, Nakamura M.

論文タイトル名

発表誌名

巻号

ページ

出版年

S1

2013

S2

2013

S6-S8

2013

Dietary Reference Intakes for Japanese.
Preface.
Dietary Reference Intakes for Japanese.
Foreword.
Historical overview of theestablishment of Dietary Reference
Intakes for Japanese.
Dietary Reference Intakes for Japanese
2010: basic theories for thedevelopment.
Dietary Reference Intakes for Japanese
2010: basic concepts for application.
Dietary Reference Intakes for Japanese
2010: energy.

J Nutr Sci
Vitaminol
J Nutr Sci
Vitaminol
J Nutr Sci
Vitaminol

59,
suppl
59,
suppl
59,
suppl

J Nutr Sci
Vitaminol

59,
suppl

S9-S172013

J Nutr Sci
Vitaminol
J Nutr Sci
Vitaminol

59,
suppl
59,
suppl

S18-S25

2013

S26-S35

2013

Dietary Reference Intakes for Japanese
2010: protein.

J Nutr Sci
Vitaminol

59,
suppl

S36-S43

2013

Dietary Reference Intakes for Japanese
2010: fat.
Dietary reference intakes for Japanese
2010: carbohydrates.

J Nutr Sci
Vitaminol
J Nutr Sci
Vitaminol

59,
suppl
59,
suppl

S44-S522013

S53-S56

2013

Dietary Reference Intakes for Japanese
2010: fat-soluble vitamins.

J Nutr Sci
Vitaminol

59,
suppl

S57-S66

2013

Dietary Reference Intakes for Japanese
2010: water-soluble vitamins.

J Nutr Sci
Vitaminol

59,
suppl

S67-S82

2013

Dietary Reference Intakes for Japanese
2010: macrominerals.

J Nutr Sci
Vitaminol

59,
suppl

S83-S902013

Dietary Reference Intakes for Japanese
2010: microminerals.

J Nutr Sci
Vitaminol

59,
suppl

S91-S102

2013

Dietary Reference Intakes for Japanese
2010: lifestage.

J Nutr Sci
Vitaminol

59,
suppl

S103-S109

2013

吉田宗弘.
Shibata K, Shimizu A,
Fukuwatari T.
Shibata K, Fukuwatari T,
Sasaki S, Sano M, Suzuki
K, Hiratsuka C, Aoki A,
Nagai C.
Fukumoto A, Asakura K,
Murakami K, Sasaki S,
Okubo H, Hirota N, Notsu
A, Todoriki H, Miura A,
Fukui M, Date C.
Nagae A, Kuwabara A,
Tozawa K, Kumode M,
Takeuchi Y, Tanaka K.
小林奈穂, 村山伸子, 稲村雪子, 久保田恵, 神田知子, 高橋孝子, 石田裕
美.
小林奈穂, 村山伸子, 稲村雪子, 久保田恵, 神田知子, 高橋孝子, 金光秀
子, 辻ひろみ, 石田裕美.
孫田みなみ, 笠岡(坪山)宜代, 瀧沢あす香, 坪田(宇津木)恵, 今井絵理, 岡純.
Tokudome S, Nishi N,
Tanaka H.
Shibata K, Imai E, Sano M,
Fukuwatari T.

Miyazaki A, Sano M,
Fukuwatari T, Shibata K.
Yoshida M, Fukuwatari T,
Sakai J, Tsuji T, Shibata
K.
Moriya A, Fukuwatari T,
Sano M, Shibata K.
Saito K, Yokoyama T,
Yoshida H, Kim H,
Shimada H, Yoshida Y,
Iwasa H, Shimizu Y,
Yoshitaka K, Handa S,
Maruyama N, Ishigami A,
Suzuki T.

微量ミネラルの過剰摂取 − 日本人のヨウ素摂取は過剰水準か.
Vitamin B1 deficiency does not affectthe liver concentrations of the otherseven kinds of B-group vitamins in rats.
Urinary excretion levels ofwater-soluble vitamins in pregnant andlactating women in Japan.

FFI ジャーナ
ル
Nutrition
Insights
J Nutr Sci
Vitaminol

in press

Within-and between-individual
variation in energy and nutrient intake
in Japanese adults: effect of age and sex
difference on the group size andnumber of records required foradequate dietary assessment.
Enteral nutrition and antibiotic use
increase the risk for vitamin K
deficiency in patients with severe motorand intellectual disabilities.
給食施設における「日本人の食事摂取基準」の活用の現状（第一報）―
病院および介護老人保健施設を対象とした質問紙調査―.
給食施設における「日本人の食事摂取基準」の活用の現状（第二報）－
高齢者施設を対象としたインタビュ
ー調査―.
政府が策定する食事指針・ガイドにおける食事摂取基準の活用状況.

J Epidemiolin press

e-SPEN
Journalin press

Towards a better National Health andNutrition Survey in Japan.
The urinary excretory ratio of
nicotinamide catabolites was associatedwith the conversion ratio of tryptophan
to nicotinamide in growing rats fed a
niacin-free 20% casein diet.
Effects of ethanol consumption on the
B-group vitamin contents of liver,blood and urine in rats.
Correlation between Mineral Intake andUrinary Excretion in Free-living
Japanese Young Women.
Different variations of tissue B-groupvitamin concentrations in short- andlong-term starved rats.
A significant relationship between
plasma vitamin C concentration andphysical performance among Japaneseelderly women.

Lancet

379

e44

2012

Biosci
Biotechnol
Biochem

76

186-1882012

Br J Nutr

1081034-1041

2012

Food andNutrition
Sciences
Br J Nutr

3

123-128

2012

107

52-60

2012

67

295-301

2012

218

12-19

2013
in Press

栄養学雑誌

70 記
念号

印刷中

栄養学雑誌

70 記
念号

印刷中

栄養学雑誌

J Gerontol A
Biol Sci Med
Sci

印刷中

Murakami K, Sasaki S,
Okubo H, the Freshmen in
Dietetic Courses Study II
Group.
Yoh K, Uzawa T, Orito T,
Tanaka K.

善方裕美, 渡辺優奈, 上西一弘他．

神田知子, 高橋孝子, 久
保田恵, 小林奈穂, 村山
伸子, 齊藤陽子, 増田利孝, 河野美穂, 石田裕美.
吉田宗弘, 増田卓也, 高
橋健哉, 福永健治.
吉田宗弘.
吉田宗弘.
吉田宗弘.
Ezaki O.

Ogawa A, Naruse Y,
Shigemura Y, Kobayashi
Y, Suzuki I, Wada S,
Hayamizu K, Kuwahata M,
Kido Y.
Imai E, Tsuji T, Sano M,
Fukuwatari T, Shibata K.

Endo M, Sano M,
Fukuwatari T, Shibata K.
Shibata K, Yasui M, Sano
M, Fukuwatari T.

Tsuji T, Fukuwatari T,
Sasaki S, Shibata K.

Characteristics of under- andover-reporters of energy intake among
young Japanese Women.

J Nutr Sci
Vitaminol

58

253-62

2012

Improvement of quality of life
（QOL） in osteoporotic patients byelcatonin treatment: a trial taking theparticipants’ preference into account.
妊娠初期の骨密度とライフスタイ
ル, 栄養摂取状態についての検討－
SKY Study（Saitama, Kobe, Yokohama
Pregnant Cohort Study）第 1 報－.
栄養管理報告書を用いた特定給食施設における食事摂取基準の活用に関する調査.

Japanese
ClinicalMedicine

2

9-14

2012

Osteoporosis
Japan

20

514-516

2012

栄養学雑誌

70

140-1512012

兵庫県の都市部在住の乳幼児に対する自家製離乳食のミネラル含有量の評価．
微量ミネラルの食事摂取基準：ヨウ素, セレン, クロム, モリブデン.
クロムはヒトの栄養にとって必須の微量元素だろうか？
セレンとモリブデンの生理機能と適切な摂取量の範囲.
The optimal dietary fat to carbohydrate
ratio to prevent obesity in Japanese
population: a review of theepidemiological, physiological andmolecular evidence.
An evaluation of protein intake formetabolic demands and the quality of
dietary protein in rats using an indicatoramino acid oxidation method.

微量栄養素
研究

29

67-71

2012

New Diet
Therapy
日本衛生学
雑誌
ビタミン

28

13-17

2012

67

485-4912012

86

548-557

2012

J Nutr Sci
Vitaminol

57383-3932011

J Nutr Sci
Vitaminol

57

418-425

2011

Association between 24 h urinaryalpha-tocopherol catabolite,
2,5,7,8-tetramethyl-2(2'-carboxyethyl) -6-hydroxychroman(alpha-CEHC) and alpha-tocopherol
intake in intervention andcross-sectional studies.
Vitamin contents in rat milk and effects
of dietary vitamin intakes of dams on
the vitamin contents in their milk.
Fluorometric determination of
2-oxoadipic acid, a common metaboliteof tryptophan and lysine, by
high-performance liquid
chromatography with pre-chemical
derivatization.
Twenty-four-hour urinary water-soluble
vitamin levels correlate with theirintakes in free-living Japanese school
children.

Asia Pac J
Clin Nutr

20507-5132011

J Nutr Sci Vi
taminol

57

203-208

2011

Biosci
Biotechnol
Biochem

75

185-1872011

Public Health
Nutr

14

327-333

2011Yoshida M, Ôgi N,
Iwashita Y.

Nakano T, Tsugawa N,
Kuwabara A, Kamao M,
Tanaka K, Okano T.
Kuwabara A, Fujii M,
Kawai N, Tozawa K, Kido
S, Tanaka K.
Kuwabara A,
Ogawa-Shimokawa Y,
Tanaka K.
Kuwabara A, Nakase H,
Tsuji H, Shide K, Chiba T,
Inagaki N, Tanaka K.
Yamada C, Fujimoto S,
Ikeda K, Nomura Y,
Matsubara A, Kanno M,
Shide K, Tanaka K, Imai E,
Fukuwatari T, Shibata K,
Inagaki N.
Nakano T, Kuwabara A,
Tanaka K.
木戸康博.
柴田克己, 福渡努, 吉田宗弘.
今井絵理，佐野光枝，福渡努，柴田克己.
柴田克己，坂崎愛，佐野光枝，福渡努.

Estimation of Mineral and Trace
Element Intake in Vegans Living inJapan by Chemical Analysis of
Duplicate Diets.
High prevalence of hypovitaminosis D
and K in patients with hip fracture.

Health3672-676

2011

Asia Pac J
Clin Nutr

20

56-61

2011

Bone is more susceptible to vitamin K
deficiency than liver in theinstitutionalized elderly.
Body weight divided by squared kneeheight as an alternative to body massindex.
Fat restriction is associated with
impaired quality of life (QOL) inpatients with ulcerative colitis andCrohn’s disease.
Relation of homocysteine andhomocysteine-related vitamins to bonemineral density in Japanese patientswith type 2 diabetes.

Asia Pac J
Clin Nutr

20

50-55

2011

Med
Hypotheses

76

336-3382011

ID5945322011

J Diabetes
Invest2

233-239

2011

Overestimated serum albumin levels
in patients with hip fracture.
たんぱく質・アミノ酸の必要量に関する研究．
生化学検査．（3）ビタミンと微量ミ
ネラル.
ストレプトゾトシン誘導糖尿病ラットのトリプトファン－ニコチンアミ
ド代謝－摂取ビタミン量との関係.
ビタミン B1 最小必要量飼料投与ラットあるいは十分量飼料投与ラット
を寒冷曝露させた時の肝臓，血液および尿中のビタミン B1 量.
ビタミン B12 欠乏ラットの種々の臓器，血清，尿中の B 群ビタミン含量.

Clin Nutr

30

261

2011

栄養学雑誌

69

285-2932011

栄養−評価と治療
日本栄養・食糧学会誌

28143-1472011

64

313-3212011日本栄養・食糧学会誌

64329-334

2011

ビタミン

85

18-22

2011

Osteoporosis
Japan

19

225-228

2011

循環器内科

70

607-614

2011

日本栄養士
会雑誌

54

556-565

2011

微量栄養素
研究

28

27-31

2011

微量栄養素
研究

28

79-83

2011

柴田克己，河田哲典，石田香織，清水篤史，守谷彩，寺方美希，佐野光枝，
福渡努.
鈴木隆雄, 島田裕之, 清
地域在住の女性後期高齢者における
水容子, 金憲経, 吉田英
血中ビタミン D 濃度と転倒発生に関世.
する縦断研究．
坪田（宇津木）恵.
欧米の循環器疾患予防のための食事ガイドラインの現状.
笠岡（坪山）宜代, 桑木 諸外国における栄養士養成のための泰子, 瀧沢あす香, 田
臨地･校外実習の現状に関する調査
中律子，藤生惠子, 斎藤
研究.
トシ子，恩田理恵，山岸博之，江田節子，木村祐子，小谷一子，小田光子，
田代晶子，池本真二.
吉田宗弘, 児島未希奈,
病院および介護施設の食事からの微三由亜耶, 森田明美.
量ミネラル摂取量の計算値と実測値との比較.
吉田宗弘, 野崎詩乃, 乾
市販離乳食からのヨウ素とクロムの由衣子.
摂取量の推定.

Ulcers Article

田中清, 桑原晶子.

Uenishi K, Fujita T, Ishida
H, et al.

Kuwabara A, Himeno M,
Tsugawa N, Kamao M,
Fujii M, Kawai N, Fukuda
M, Ogawa Y, Kido S,
Okano T, Tanaka K.
吉田宗弘.
田中清, 桑原晶子.

日本人の食事摂取基準における目安
量は健康人の摂取の中央値でよいのか？
Fractional absorption of active
absorbable algal calcium （AAACa）
and calcium carbonate measured by adual stable-isotope method.
Hypovitaminosis D and K are highly
prevalent and independent of overallmalnutrition in the institutionalized
elderly.

ビタミン85608-6092011

Nutrients

2752-764

2010

Asia Pac J
Clin Nutr

19

49-56

2010

食事摂取基準（2010）の策定 − ビタ
ミンとミネラル − 微量ミネラル.
ビタミン D による骨折予防効果の意義：医療経済の視点から.

ビタミン

84

240-245

2010

ビタミン

84

128-129

2010

書籍
書籍

論文タイトル名Yoshida M.

Molybdenum in
Biological Samples andClinical Significance ofSerum Molybdenum.

Kuwabara A,
Tsugawa N, Kamao
M, Nakase H, Chiba
T, Okano T, Tanaka
K.

Patients with Crohn’s
Disease Have
Hypovitaminosis D andK, which is Independentof General Malnutrition.

編集者
名
Caroli S,
Záray G

書籍名
Analytical
Techniques forClinicalChemistry:
Methods andApplications
Vitamin D:
Nutrition, Side
Effects andSymptoms.

出版社
名
John
Wiley &
Sons

Nova
Scientific
Publishersページ
393-404

出版
年
2012

2010

Correspondence

Towards a better
National Health andNutrition Survey in Japan
In his Comment (Oct 1, p 1205),1
Satoshi Sasaki doubts the value of the
National Health and Nutrition Survey
in Japan2 (hereafter, the Survey),
mentioning that “as long as the Survey
continues to be done and reported in
the current manner, it will not fulﬁlits potential as a valuable resource forhealth.” He raises three points. First, theuse of data from the Survey is limited;
second, there are problems withmethods and quality control; and third,
access to Survey information is limited.
We would like to address the ﬁrst point,
and oﬀer proposals as to the other two
points, in light of his comments.
Since 1948, the Survey has been
carried out annually by the Ministry of
Health, Labour and Welfare, together
with the National Institute of Healthand Nutrition and in collaboration
with local or registered dietitians andrandomly selected Japanese people(currently about 9000 individuals of4000 households). The Survey is, apriori, meant to obtain a set of national
statistics to get an overview of thepresent status of health and nutritionin Japan, and of long-term trends
for launching governmental policy
and initiatives. It is also concurrently
serving to provide a wide range of basic
information for setting dietary reference
intakes for Japanese people;3 an exercise
and physical activity reference forhealth promotion;4 regulatory measures
for food additives and contamination
with insecticides or pesticides, organic
mercury, and radioactive substances;and reference values for consumptionof energy and nutrients for the victimsof the Great Eastern Japan Earthquakeof March 11, 2011.
Sasaki’s ﬁrst comments do not seemreasonable because the Survey is a set ofcross-sectional observations that show
the status quo of health and nutritionin Japan as a whole, and has its own
limits in showing how the traditionale44Japanese diet has contributed towards
achieving the world’s highest longevity.
Furthermore, the Survey cannot becounted on to have a role in analyticalepidemiological approaches, including
case-control studies, cohort studies,or randomised controlled trials, toinvestigate the associations between
individual health and disease andphysical activity and nutrition, and theinteractions between environmental
factors and host genetic factors.
Second as Sasaki points out, theparticipation rate is rather low atabout 60%, suggesting that there
is non-response bias. Descriptions
remain somewhat unclear about
presently adopted semi-weighed
food records, assessment of individual
intake from household data, standardisation of consumption data,
validity, and reproducibility. Thus,
there might be issues of generalisability. We at the National Institute of
Health and Nutrition have committed
ourselves to managing data quality
control and standardisation of theSurvey methods, but we should keepon exerting every eﬀort to improve
the Survey. Since information onenergy and nutrients is scarcely given
for cooked dishes and prepared food,in particular, in the Standard Tables ofFood Composition in Japan, the quality
and quantity of table data should be
improved with all due speed.
A research group under the auspicesof the Ministry of Health, Labour andWelfare suggested transfer of theSurvey method from semi-weighed
food records to 1-day (or multiple-day)24-h dietary recall (with or without
photos),5 which is currently adopted
worldwide, making international
comparisons possible. This approachallows us to estimate individual
consumption of energy, food, andnutrients; clarify the causative factors
for health promotion and preventionof diseases; and elucidate the factors
associated with life expectancy.
The third point relates to governmental statistics: that is, secondary(post-tabulated) data are provided

to researchers. Thus, to obtain theSurvey primary data, researchers mustgo through formalities and secure
approval from the Ministry. Round tablediscussion on tabulation items to meetthe current needs, open access to theSurvey information, and provision of
the primary data (or setting-up a dataarchive) should be made. The National
Institute of Health and Nutrition
proposes to launch a cohort study
based on the Survey individual data toverify the associations between healthand disease, physical activity and sports,
and consumption of food and nutrientsalong with information on smoking,alcohol drinking, anthropometricmeasurements, and blood biomarkers.
Sasaki’s comments serve to alert
the Ministry and the Institute tomodify the framework of the Survey,
including replacement of the Survey
methods, and to guarantee quality
control, standardisation, and accessto the Survey data.
We declare that we have no conﬂicts of interest.

*Shinkan Tokudome, Nobuo Nishi,
Heizo Tanaka
tokudome@nih.go.jp
National Institute of Health and Nutrition, 1-23-1,
Toyama, Shinjuku-ku, Tokyo 162-8636, Japan (ST);Center for International Collaboration andPartnership, National Institute of Health andNutrition, Tokyo, Japan (NN); and Department of
Nutrition and Life Science, Faculty of Applied
Bioscience, Kanagawa Institute of Technology,
Atsugi City, Kanagawa, Japan (HT)
1

2

3

4

5

Sasaki S. The value of the National Health andNutrition Survey in Japan. Lancet 2011;
378: 1205–06.
Ministry of Health, Labour and Welfare, Japan.
The National Health and Nutrition Survey inJapan 2008. Tokyo: Oﬃce for Life-style RelatedDiseases Control, General Aﬀairs Division,
Health Service Bureau, Ministry of Health,Labour and Welfare, 2011 (in Japanese).
Ministry of Health, Labour and Welfare, Japan.
Dietary reference intakes for Japanese 2010.
Report from the Scientiﬁc Committee of Dietary
Reference Intakes for Japanese. Tokyo: Oﬃce forLife-style Related Diseases Control, General Aﬀairs
Division, Health Service Bureau, Ministry of
Health, Labour and Welfare, 2011 (in Japanese).
Ministry of Health, Labour and Welfare, Japan.
Exercise and physical activity reference for healthpromotion 2006: physical activity, exercise, andphysical ﬁtness. Tokyo: Oﬃce for Life-style
Related Diseases Control, General Aﬀairs
Division, Health Service Bureau, Ministry of
Health, Labour and Welfare, 2006 (in Japanese).
Willett W. Nutritional epidemiology, 2nd edn.
New York: Oxford University Press, 1998.

www.thelancet.com Vol 379 March 24, 2012

British Journal of Nutrition (2012), 108, 1034–1041
q The Authors 2011

doi:10.1017/S0007114511006192

Effects of ethanol consumption on the B-group vitamin contents of liver,blood and urine in rats
Aiko Miyazaki, Mitsue Sano, Tsutomu Fukuwatari and Katsumi Shibata*
Department of Food Science and Nutrition, School of Human Cultures, The University of Shiga Prefecture Hikone,
Shiga 522-8533, Japan

British Journal of Nutrition(Submitted 11 July 2011 – Final revision received 14 October 2011 – Accepted 17 October 2011 – First published online 15 December 2011)

Abstract
Several studies have shown that blood vitamin levels are lower in alcoholic patients than in control subjects. Acute ethanol exposure
enhances the release of vitamins from liver cells in vitro. The aim of the present study is to confirm the effects of ethanol consumption
on vitamin contents in vivo. We compared the contents of B-group vitamins in the liver, blood and urine between ethanol-fed and control
rats fed a diet containing a sufficient- and low-vitamin mixture. The experimental rats were fed a 15 % ethanol solution freely for 28 d, andthen 24 h urine samples were collected, after which the animals were killed. The B-group vitamin contents in the liver, blood andurine were measured. No differences in liver, blood and urine contents were observed between the control and ethanol-fed rats fed adiet containing a sufficient-vitamin mixture. On the contrary, in rats fed a diet containing a low-vitamin mixture, consumption of ethanolcaused a decrease in the contents of vitamins B1, B2 and pantothenic acid in the liver; however, the contents of the other vitamins did notdecrease. In the blood, the contents of vitamins B1, B2, B6 and pantothenic acid were lower in the ethanol-fed rats than in the controls.
Urinary excretion of the B-group vitamins, except for niacin, was lower in the ethanol-fed rats. These results show that ethanol consumption affects the absorption, distribution and excretion of each of the vitamins in rats fed a diet containing a low-vitamin mixture.
Key words: Vitamins: Urine: Blood: Liver: Ethanol

Numerous studies have shown that vitamin status of alcoholicpatients differs from non-drinking subjects(1 – 7), and the
majority have shown that blood vitamin levels are lower inalcoholic patients than in controls(8 – 10). In addition, several
reports have suggested that chronic alcohol feeding may leadto a significant inhibition of carrier-mediated thiamin(11,12) andfolate(13 – 19) uptake in the intestine and kidney. This phenomenon is observed only in alcoholic patients who drink ethanol
chronically. On the contrary, a reduction in circulating levelsof B-complex vitamins often occurred without clinical evidence of hypovitaminosis(20). Sorrell et al.(21) reported thatthe in vitro perfusion of rat liver with ethanol caused therelease of all B-vitamins except biotin from the liver stores.
Israel & Smith(22) reported that acute ethanol feeding to rats
inhibited the conversion of pantothenic acid to CoA. Thesestudies in animal models suggested that acute ethanol intake
results in an increased hepatic release of vitamins and an
impaired utilisation, which means increased levels of freeforms of vitamins in the liver which can in turn permeate
the cell membranes(21,22). This might lead to increases inblood vitamin contents and in urinary excretion. Although
there are many reports concerning the effects of ethanol on

the absorption and metabolism of vitamins, the conclusion
concerning the controversy remains elusive. The reason
might be that there is no study regarding the simultaneous
measurement of vitamin contents of liver (as a biomarker ofthe storage amount of vitamins), blood (as a biomarker ofthe circulation amount of vitamins) and urine (as a biomarkerof the reabsorption ability of kidney and an extra amount ofvitamins).
In the present study, we examined the effects of ethanol
consumption on the contents of B-group vitamins of theliver, blood and urine in rats fed two kinds of diets containing
either a sufficient- or a low-vitamin mixture.

Materials and methods
Chemicals
Vitamin-free milk casein, sucrose and L -methionine were purchased from Wako Pure Chemical Industries. Maize oil waspurchased from Ajinomoto. Gelatinised maize starch, a mineral mixture (AIN-93G mineral mixture)(23) and a vitamin mixture (nicotinic acid-free AIN-93 vitamin mixture containing

Abbreviations: 2-Py, N 1-methyl-2-pyridone-5-carboxamide; 4-Py, N 1-methyl-4-pyridone-3-carboxamide.
* Corresponding author: K. Shibata, fax þ 81 749 28 8499, email kshibata@shc.usp.ac.jp

B-group vitamin contents in ethanol-fed rats

British Journal of Nutrition

25 % choline bitartrate)(23) were obtained from Oriental Yeast
Company, Limited.
Thiamin hydrochloride (C12H17ClN4OS-HCl; molecularweight 337·27), riboflavin (C17H20N4O6; 376·37), pyridoxinehydrochloride (C8H11NO3-HCl; 205·63), cyanocobalamin(C63H88CoN14O14P; 1355·40), nicotinamide (C6H6N2O;
122·13), calcium pantothenate (C18H32N2O10-Ca; 476·54),
folic acid (C19H19N7O6; 441·40) and D (þ )-biotin (C10H16
N2O3S; 244·31) were purchased from Wako Pure Chemical
Industries. 4-Pyridoxic acid (C8H9NO4 ¼ 183·16) was madeby ICN Pharmaceuticals and obtained through Wako Pure
Chemical Industries.
N 1-Methylnicotinamide chloride (C7H9N2O-HCl; 159·61)
was purchased from Tokyo Kasei Kogyo. N 1-Methyl-2-pyridone-5-carboxamide (2-Py, C7H8N2O2 152·15) and N 1-methyl4-pyridone-3-carboxamide (4-Py, C7H8N2O2 152·15) were synthesised by the methods of Pullman & Colowick(24) and Shibata
et al.(25), respectively. All other chemicals used were of highest
purity available from commercial sources.

Animals and treatment
The care and treatment of the experimental animals conformed to the University of Shiga Prefecture guidelines forthe ethical treatment of laboratory animals. The animals
were maintained under controlled temperature (228C), 60 %humidity and light conditions (12 h light – 12 h dark cycle).

Effects of ethanol feeding on the B-group vitamin contentsof liver, blood and urine in rats fed a diet containing
a sufficient-vitamin mixture (Expt 1)
Male Wistar rats (3 weeks old) obtained from CLEA Japan were
fed freely with a conventional purified diet, consisting of 20 %vitamin-free milk casein, 0·2 % L -methionine, 46·9 % gelatinised
maize starch, 23·4 % sucrose, 5 % maize oil, 3·5 % AIN-93-G mineral mixture(14) and 1 % AIN-93 vitamin mixture(14) containing
chorine bitartrate, but without nicotinic acid, to acclimatisefor 7 d. Nicotinic acid had not been added to this diet because
it is supplied enough from tryptophan in casein(26), and a dietary fibre-free diet was used because it is a tradition not to use
dietary fibre in our laboratory which is not essential fornormal growth(27).
The rats were divided into two groups (n 5 each). Group 1
was fed with a diet containing the 1 % vitamin mixture (a sufficient-vitamin diet) and allowed to drink water for 28 d.
Group 2 was fed with a diet containing the 1 % vitaminmixture (a sufficient-vitamin diet) and forced to drink a 15 %ethanol solution instead of water for 28 d. The 24 h urine
samples were collected in amber bottles containing 1 ml of
1 M -HCl at 09.00– 09.00 hours of the last day and werestored at 2 258C until required. The rats were killed at about
09.00 hours; blood was collected and tissues were taken tomeasure the weights and the contents of B-group vitaminsin the liver, blood and urine. Liver samples were preserved
at 2258C until required.

1035

Effects of ethanol feeding on the B-group vitamin contentsof liver, blood and urine in rats fed a diet containing
a low-vitamin mixture (Expt 2)
A preliminary experiment revealed that the body-weight gainof young rats was the same when fed a diet containing the 1 %AIN-93 vitamin mixture and the 0·3 % AIN-93 vitamin mixture,
whereas the body-weight gain was lower in rats fed a dietcontaining the 0·2 % AIN-93 vitamin mixture than in those
fed a diet containing the 1 or 0·3 % diets. Thus, we determined
tentatively whether the diet containing the 0·3 % AIN-93
vitamin mixture could supply a minimum amount of vitamins
for the growing rats.
Male Wistar rats (3 weeks old) obtained from CLEA Japan
were fed freely with the conventional purified diet (mentioned
above) to acclimatise for 7 d. The rats were then divided into
two groups (n 5 each). Group 1 was fed a diet containing the
0·3 % vitamin mixture and allowed to drink water for 28 d.
Group 2 was fed a diet containing the 0·3 % vitamin mixture
and forced to drink a 15 % ethanol solution instead of waterfor 28 d. The 24 h urine samples and tissues were collected.
Levels of alanine aminotransferase, aspartate aminotransferase
and g-glutamyltranspeptidase were measured at Mitsubishi
Chemical Medience (Tokyo, Japan).

Measurement of B-group vitamins in urine and blood
Preparation and measurement of the extracts of the B-groupvitamins from the urine and blood are described as follows(28).

Vitamin B1
Frozen liver samples, about 0·5 g, were thawed, minced, andthen added to ten volumes of 5 % ice-cold TCA and homogenised with a Digital Homogenizer Hom (Iuchi). The acidified
homogenate was centrifuged at 10 000 g for 10 min at 48C,
and the supernatant was retained and used for the measurement of vitamin B1(29).

Vitamin B2
Frozen liver samples, about 0·5 g, were thawed, minced, andthen added to ten volumes of 50 mM -KH2PO4 – K2HPO4
buffer (pH 7·0) and homogenised with a Teflon/glass
homogeniser (Nikko Hansen). To 0·1 ml of the homogenate,
0·44 ml of water and 0·26 ml of 0·5 M -H2SO4 were added andthen kept at 808C for 15 min. After cooling, 0·2 ml of 10 %TCA were added and centrifuged at 10 000 g for 3 min at
48C. From the supernatant obtained, 0·2 ml was withdrawn
and added to 0·2 ml of 1 M -NaOH. The alkalised mixture wasirradiated with a fluorescent lamp for 30 min and then
0·02 ml of glacial acetic acid were added to the mixture. The
neutralised mixture was passed though a 0·45 mm microfilter
and the filtrate was directly injected into the HPLC system
for measuring lumiflavin(30).

1036

A. Miyazaki et al.

Vitamin B6

Frozen liver samples, about 0·5 g, were thawed, minced, andthen added to 2·5 ml of 0·57 M -acetic acid– sodium acetate
buffer (pH 4·5) plus 5 ml of water and 0·1 ml of 0·05 % potassium cyanide (KCN). The suspension was homogenised with a
Teflon/glass homogeniser. The homogenate was then put into
a boiling water-bath for 5 min. After cooling, 0·15 ml of 10 %metaphosphoric acid were added and made up to 10 mlwith water. The solution was filtered with qualitative filter
no. 2 (ADVANTEC MFS, Inc.). The filtrate was used formeasuring vitamin B12 as described previously(32).

(pH 6·1). The suspension was homogenised with a Teflon/glass homogeniser. The homogenate was autoclaved at
1218C for 5 min. After cooling, 2·5 ml of pronase (5 mg/ml;
Pronase MS; Kaken Pharmaceutical Company, Limited) wereadded and then incubated at 378C for 3 h. The reaction was
stopped by putting it into a boiling water-bath for 10 min.
After cooling, 0·5 ml of conjugase (extract from porcine kidneyacetone powder, Type II; Sigma-Aldrich) were added and incubated at 378C overnight. The reaction was stopped by putting it
into a boiling water-bath for 10 min. After cooling, the mixture
was centrifuged at 10 000 g for 10 min at 48C. The supernatant
was retained, and the precipitated materials were extracted
again with 3 ml of water, and the supernatant was retained.
Both the retained supernatants were combined, and the extract
was used for measuring folate as described previously(34). The
conjugase solution was made as follows: 60 ml of 50 mM KH2PO4 – K2HPO4 buffer (pH 7·0) were added to 20 g porcine
kidney acetone powder and stirred for 30 min at 48C. The suspension was centrifuged at 10 000 g for 10 min at 48C. The supernatant was dialysed against a large amount of 50 mM -KH2PO4 –K2HPO4 buffer (pH 7·0) to remove endogenous folate of thekidney acetone powder. The dialysed conjugase solution wasused.

Nicotinamide

BiotinFrozen liver samples, about 0·6 g, were thawed, minced, andthen added to five volumes of 0·1 g/ml isonicotinamide. The
suspension was homogenised with a Teflon/glass homogeniser. The homogenate (1 ml) was withdrawn and added to
4 ml of water, and then autoclaved at 1218C for 10 min. Aftercooling, the mixture was centrifuged at 10 000 g for 10 min
at 48C. The supernatant was retained and the precipitatedmaterials were extracted again with 5 ml of water, and thesupernatant was retained. Both the retained supernatants
were combined, and the extract was used for measuringnicotinamide as described previously(25).

Frozen liver samples, about 0·5 g, were thawed, minced,
and then added to two volumes of 2·25 M -H2SO4 and thenhomogenised with a Waring blender. The suspension washydrolysed by autoclaving for 1 h at 1218C. After cooling, thesuspension was centrifuged at 10 000 g for 10 min at 48C,
and the supernatant was used for measuring biotin(35).

Frozen liver samples, about 0·5 g, were thawed, minced, andthen added to 90 ml of 55 mM -HCl and homogenised with aWaring blender. The homogenate was autoclaved at 1218C
for 3 h. After cooling, the mixture was adjusted to pH 5·0
with 1 M -NaOH and then made up to 100 ml with water. The
solution was filtered with qualitative filter no. 2 (ADVANTEC
MFS, Inc.). The filtrate was used for measuring vitamin B6 as
described previously(31).

British Journal of Nutrition

Vitamin B12

Pantothenic acid
Frozen liver samples, about 0·2 g, were thawed, minced, andthen added to ten volumes of 50 mM -KH2PO4 –K2HPO4
buffer (pH 7·0). The suspension was homogenised with a
Teflon/glass homogeniser. The homogenate was incubatedat 378C overnight to convert free pantothenic acid from thebound type of pantothenate compounds. The reaction was
stopped by putting it into a boiling water-bath for 5 min.
After cooling, the mixture was centrifuged at 10 000 g for
10 min at 48C. The supernatant was retained and the precipitated materials were extracted again with 2 ml of water, andthe supernatant was retained. Both the retained supernatants
were combined, and the extract was used for measuringpantothenic acid as described previously(33).

FolateFrozen liver samples, about 0·5 g, were thawed, minced, andthen added to ten volumes of 0·1 M -KH2PO4 – K2HPO4 buffer

Analyses
The measurements of the B-group vitamins except for vitamin
B6 were described previously(19). The urinary excretion of
4-pyridoxic acid, a catabolite of vitamin B6, was measured
according to the method of Gregory & Kirk(36).

Statistical analysis
Mean values between the treatment groups were comparedusing the Mann– Whitney U two-tailed t test. P, 0·05 wasconsidered to be statistically significant. All statistical analyses
were performed using GraphPad Prism version 5.0 (GraphPad
Software).

Results
Effects of ethanol feeding on the B-group vitamin contentsof liver, blood and urine in rats fed a diet containing
a sufficient-vitamin mixture (Expt 1)
There were no differences in body-weight gain and liver
weights between the groups. No differences in the levels ofvitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide,
pantothenic acid, folate and biotin were observed in the liverB-group vitamin contents in ethanol-fed rats

and blood. Although the 24 h urinary excretion of some of thevitamins was slightly lower in the ethanol-treated group thanin the control, the differences were not significant (datanot shown). Thus, ethanol consumption did not affect the
B-group vitamin contents in the liver, blood and urine when
the rats were fed a diet containing sufficient amounts ofthe vitamins.

British Journal of Nutrition

Effects of ethanol feeding on the B-group vitamin contentsof liver, blood and urine in rats fed a diet containing
a low-vitamin mixture (Expt 2)
As shown in Table 1, body-weight gain, food intake and liver
weights were lower in the ethanol-fed group than in thecontrols. The overall food intake was lower in the ethanolfed group than in the controls, but energy intake was almost
the same because of ethanol intake.
The effects of ethanol consumption on the activities ofalanine aminotransferase, aspartate aminotransferase andg-glutamyltranspeptidase in plasma are shown in Table 2.
No significant effects of ethanol consumption were observed
for these indices of liver function.
The effects of ethanol consumption on the B-group vitamincontents of the liver are shown in Table 3. The contents of thevitamins in liver are measured as storage amounts of the vitamins, thus are expressed as mol/liver. The contents of vitamin
B1, vitamin B2 and pantothenic acid were lower in the ethanol-fed group than in the controls, whereas the contents ofvitamin B6, vitamin B12, nicotinamide, folate and biotin were
not significantly different.
The effects of ethanol consumption on the B-group vitamincontents of the blood are shown in Table 4. The contents ofvitamin B1, vitamin B2, vitamin B6 and pantothenic acid
were lower in the ethanol-fed group than in the controls,

Table 1. Effects of ethanol consumption on rat body-weight gain, foodintake, ethanol intake, water intake, energy intake, food efficiency ratio
and liver weight (Expt 2)(Mean values with their standard errors for five rats per group)
Control

Initial body weight (g)Final body weight (g)Body-weight gain (g/28 d)Food intake (g/28 d)
Ethanol intake† (g/28 d)
Water intake (ml/28 d)
Energy intake‡ (kcal/28 d)
Energy intake‡ (kJ/28 d)Food efficiency ratio§
Energy efficiency ratiok
Liver weight (g)15 % Ethanol

Mean

SEM

Mean

SEM

36
204
168
363

1
7
7
14

36
164*
128*
258*
45

1
8
3
6
3

–
396
1488
6230
0·46
0·113
9·70

26
58
242
0·01
0·020
0·55

Table 2. Effects of ethanol consumption on the activities of alanine
aminotransferase, aspartate aminotransferase and g-glutamyltranspeptidase in plasma(Mean values with their standard errors for five rats per group)
Control

Alanine aminotransferase (IU/l)
Aspartate aminotransferase (IU/l)
g-Glutamyltranspeptidase (IU/l)

56
234
0·00
0·006
0·36

* Mean values were significantly different from those of the control group (P, 0·05;
Mann– Whitney U two-tailed t test).
† The value is expressed in g of pure ethanol and not as the volume of 15 %ethanol.
‡ Energy of 1 g ethanol was calculated as 29·3 kJ (7 kcal)/g.
§ ( Body-weight gain/food intake) £ 100.
k (Body-weight gain/energy intake) £ 100.

15 % Ethanol

Mean

SEM

Mean

SEM

22·4
157
3·2

1·9
11
0·9

24·8
136
3·2

2·0
10
0·9

whereas the contents of vitamin B12, nicotinamide, folate
and biotin were not significantly different.
The effects of ethanol consumption on the 24 h urinary
excretion of the B-group vitamins are shown in Table 5. The
excretion of vitamin B1, vitamin B2, 4-pyridoxic acid (a catabolite of vitamin B6), vitamin B12, pantothenic acid, folate andbiotin was lower in the ethanol-fed group than in the controls,
whereas the contents of nicotinamide (sum of the contents of
nicotinamide and its catabolites such as N 1-methylnicotinamide, 2-Py and 4-Py) were not significantly different.
Food intake was different in the two groups, so that urinary
excretion ratios of the vitamins were calculated. As shown in
Table 5, the excretion ratios of all vitamins except for vitamin
B12 were lower in the ethanol-fed group.

Discussion
An ordinary diet for rats generally contains sufficient amountsof nutrients including vitamins(23). Under well-nourishedconditions, rats are generally little affected by factors such as
ethanol consumption. In fact, the present study proves thatethanol consumption did not affect the body-weight gain or
the vitamin contents in the liver and blood when rats were
fed a diet containing sufficient amounts of vitamins. On theother hand, when rats were fed a diet low in vitamins,
body-weight gain was lower in the ethanol-fed group thanin the control group and some vitamin contents of theliver and blood, and urinary excretion were decreased.
These results show that chronic ethanol consumption affects

Table 3. Effect of ethanol consumption on liver B-group vitamincontents (Expt 2)(Mean values with their standard errors for five rats per group)
Control

–
1396
5845
0·50
0·092
8·47

1037

Vitamin B1 (nmol/liver)
Vitamin B2 (nmol/liver)
Vitamin B6 (nmol/liver)
Vitamin B12 (nmol/liver)
Niacin (mmol/liver)
Pantothenic acid (mmol/liver)
Folate (nmol/liver)
Biotin (nmol/liver)

15 % Ethanol

Mean

SEM

Mean

SEM

127
686
229
0·39
18·2
3·16
70·0
9·31

6
62
16
0·03
1·8
0·19
9·7
1·10

100*
422*
281
0·38
16·6
2·42*
73·6
9·65

4
16
23
0·02
1·3
0·18
9·3
0·46

* Mean values were significantly different from those of the control group (P,0·05;
Mann –Whitney U two-tailed t test).

1038

A. Miyazaki et al.

Table 4. Effect of ethanol consumption on blood B-group vitamincontents (Expt 2)(Mean values with their standard errors for five rats per group)
Control

Vitamin B1 (pmol/ml)
Vitamin B2 (pmol/ml)
Vitamin B6 (nmol/ml)
Vitamin B12 (pmol/ml)
Niacin (nmol/ml)
Pantothenic acid (nmol/ml)
Folate (pmol/ml)
Biotin (pmol/ml)

15 % Ethanol

Mean

SEM

Mean

SEM

159
177
0·49
1·55
127
1·13
149
30·4

4
5
0·04
0·03
6
0·04
4
3·4

139*
142*
0·34*
1·41
117
0·89*
138
25·9

6
4
0·02
0·01
2
0·04
10
1·0

British Journal of Nutrition* Mean values were significantly different from those of the control group (P, 0·05;
Mann– Whitney U two-tailed t test).

absorption, distribution and excretion of vitamins, as reported
previously(1 – 19). The present findings are not consistent with
the in vitro perfusion of rat liver with ethanol, which caused
the release of all B-vitamins except biotin from the liver
stores(23). This phenomenon was not observed in the present
whole-body experiment, because the vitamin contents ofthe blood were not increased by ethanol consumption. In thepresent in vivo experiment, any vitamins released from theliver were quickly absorbed by non-hepatic tissues. In humans,
the typical dietary vitamin intakes are generally around theminimum requirements. Thus, the nutritional status of rats
fed a diet low in vitamins was similar to that of humans. Ethanol consumption was 45 g over 28 d, so that daily averageethanol consumption was about 1·6 g/d, which corresponds
to an energy intake of 46·9 kJ (11·2 kcal)/d. The energy
intake in the ethanol-fed group, including ethanol energy,
was 5845 kJ (1396 kcal) over 28 d (about 209 kJ (50 kcal)/d).
Thus, ethanol accounted for 20 % of dietary energy. Under
these conditions, liver functions in rats were not injured.
If humans were to consume 10 467 kJ (2500 kcal)/d, the equivalent ethanol consumption would be about 70 g/d, whichcorresponds to 1 litre of typical beer.
Vitamin depletion, common in malnourished alcoholicpatients(10), can occur despite vitamin supplementation.
Vitamin malabsorption(37), exacerbated by malnutrition, contributes to this depletion(38). Also, in alcoholic patients, theimpaired ability of the liver to bind and store vitamins might
contribute to this depletion. This may probably be due tothe hepatotoxicity of ethanol, which impairs not only the vitamin-binding capacity but also the vitamin storage of the liver.
In the present study, a diet containing 20 % casein supplemented with methionine was used, which is an excellent
protein source from a nutritional standpoint. This suggest
the reasons why ethanol consumption did not cause any
severe damage, such as an extremely low food intake andbody-weight gain and roughness of fur for the rats, evenwhen they were fed a low-vitamin diet.
Sorrell et al.(21) reported that the in vitro perfusion of rat
liver with ethanol caused the release of all vitamins from theliver stores, especially thiamin. It is generally consideredthat this phenomenon causes increased urinary excretionof vitamins, but in the present in vivo experiments, ethanol
consumption did not cause increased urinary excretion, butrather decreased it. This discrepancy between the expected
and the actual findings may be attributed to the difference
between the in vitro and in vivo experiments. Moreover,there are differences in short-term and long-term adjustment
mechanisms for ethanol toxicity. The protein nutritional
status was high in the present study because the diet used
20 % casein supplemented with methionine. Protein plays apivotal role in vitamin absorption and storage in hepatocytes.
Protein malnutrition causes malabsorption, reduced storageand impaired utilisation of vitamins. Thus, an adequate
intake of vitamins, and also protein, is essential for preventing
ethanol toxicity.
In the present study on the low-vitamin diet, vitamin B1,
vitamin B2 and pantothenic acid contents in the liver andblood were lower in the ethanol-fed group than in the controls, even when rats were fed a high-protein diet. Furthermore, the total urinary excretion and excretion ratios of all
three vitamins were also lower in the ethanol-fed group.
Thus, ethanol consumption reduced the intestinal absorptionof these vitamins, as reported by Subramanya et al.(12),Hamid et al.(13,14,16,17) and Wani & Kaur(19). Vitamins such as
Table 5. Effect of ethanol consumption on urinary B-group vitaminexcretion (upper row) and urinary excretion ratio (lower row) for each of
the vitamins (Expt 2)†(Mean values with their standard errors for five rats per group)
Control

Vitamin B1
nmol/d%Vitamin B2
nmol/d
%4-PIC‡
nmol/d%Vitamin B12
pmol/d%Niacin§mmol/d%Pantothenic acidnmol/d%Folatenmol/d%Biotinnmol/d
%15 % Ethanol

Mean

SEM

Mean

SEM

3·5
3·4

0·1
0·2

1·8*
2·7*

0·1
0·2

3·6
3·8

0·3
0·2

0·15*
0·24*

0·04
0·05

29·4
15·6

1·9
0·5

7·3*
4·5*

0·5
0·3

9·1
8·9

0·4
0·3

6·7*
9·1

0·2
0·2

0·16

1·82

2·00
–k

0·24
–k

24·3
6·5

2·4
0·5

6·3*
2·4*

0·3
0·2

1·85
7·3

0·19
0·7

0·77*
4·4*

0·11
0·6

0·21
5·0

0·02
0·4

0·09*
3·0*

0·01
0·25

4-PIC, 4-pyridoxic acid.
* Mean values were significantly different from those of the control group (P, 0·05;
Mann– Whitney U two-tailed t test).
† Percentage urinary excretion ratio was calculated using the following equation:(24 h urinary excretion (mol/d)/intake of the vitamin during urine collection(mol/d)) £ 100.
‡ A catabolite of vitamin B6.
§ Niacin content was calculated as the sum of the nicotinamide content and its
catabolites such as N 1-methylnicotinamide, N 1-methyl-2-pyridone-5-carboxamide
and N 1-methyl-4-pyridone-3-carboxamide.
k Urinary excretion ratio was not calculated as niacin was derived from tryptophan.

British Journal of Nutrition

B-group vitamin contents in ethanol-fed rats

vitamin B1, vitamin B2 and pantothenic acid might be directly
and/or indirectly involved in the metabolism of ethanol, indicating that the vitamin catabolites increased and were excreted
into the urine. Of these three vitamins, only the catabolic fateof vitamin B1 is relatively well known. It has been reported
that the excretion of vitamin B1 metabolites usually exceeds
by far the excretion of intact vitamin B1 using radioactive
tracer experiments(39). The major metabolites of vitamin B1 in
rat urine are 2-methyl-4-amino-5-pyridinecarboxylic acid(40),4-methylthiazole-5-acetic acid(41) and thiamine acetic acid(42).
Pearson(39) reported that the sum of the metabolites accounted
for about 50 % of the total urinary excretion of vitamin B1 andits catabolites from radioactive tracer experiments. Although
we cannot measure the catabolites of vitamin B1, these metabolites might increase in the urine of the ethanol-fed rats. It is
likely that a similar phenomenon would apply for the fatesof vitamin B2 and pantothenic acid.
The content of vitamin B6 in the blood was lower inthe ethanol-fed group, but the content of vitamin B6 in theliver was slightly higher in the ethanol-fed group than in thecontrol. The urinary excretion of vitamin B6, determinedfrom its catabolite 4-pyridoxic acid, was much lower inthe ethanol-fed group than in the control. Probably ethanol
consumption resulted in an increased storage of vitamin B6
in the liver.
Other B-group vitamin contents in the liver and blood, suchas vitamin B12, nicotinamide, folate and biotin, were notaffected by ethanol consumption. The lack of any effect of
ethanol consumption on the niacin content in this experiment
was probably because nicotinamide was synthesised fromtryptophan, which was present in the diet as casein and was
supplied adequately(43). For rats, NAD precursors such as nicotinic acid and nicotinamide are not essential. In fact, the urinary excretion of nicotinamide did not differ between the twogroups. Concerning the effect of ethanol consumption on
biotin, Sorrell et al.(21) reported that the in vitro perfusion of
rat liver with ethanol did not cause the release of biotin, butcaused the release of vitamin B12 first. In the present experiment, a similar phenomenon was observed for biotin, but
not for vitamin B12. Frank et al.(44) reported that the first vitamin released into the circulation during hepatic insult by ethanol is vitamin B12. This disparity between the reported and thepresent findings might also arise from the difference in protein
nutritional status.
There are many reports concerning how ethanol consumption affects folate absorption and metabolism(13 – 18,45 – 53).
Some studies have reported that ethanol consumption
increased the urinary excretion of folates(46,47,50 – 53) andcaused decreased serum folate levels. Romanoff et al.(53)reported that acute ethanol exposure inhibits the apical transport of 5-methyltetrahydrofolate in cultured human proximal
tubule cells, and in subchronic ethanol studies, increasing
concentrations of ethanol resulted in an up-regulation of
folate transporters. Furthermore, Romanoff et al.(53) reported
that both the folate receptor and reduced folate carrier transporter proteins were up-regulated in rats receiving an ethanol
diet. On the contrary, Hamid et al.(13,14,16,17) and Wani &Kaur(19) reported that ethanol reduced the intestinal uptake1039

of folate by altering the binding and transport kinetics of thefolate transport system and also the expression of folate transporters in the intestine. In addition, Hamid & Kaur(15) reported
that ethanol consumption reduces folate re-uptake in the renal
absorption system by the decreased expression of transporters. The present data for folate are not consistent withprevious reports(13 – 18,45 – 53); the contents of folate in theliver and blood were not affected by ethanol consumption,and the urinary excretion of folate and the excretion ratio
were decreased markedly. A study(52) reported that urinary
folate excretion increased in ethanol-fed rats consuming
folate-containing diets, but not in rats fed folate-deficient
diets. In the present study, the urinary excretion of folate
did not increase, but decreased. This was because the diet
was low in folate. In the present study, the urinary excretionof folate was lower in the ethanol-fed group than in the
non-ethanol group, suggesting that ethanol consumption
and the feeding of a low-folate diet up-regulated the folate
receptor and reduced folate carrier transporter proteins. This
up-regulation was probably a compensatory response tocounteract the effects of ethanol in inhibiting the reabsorptionof folate. Therefore, the effects of ethanol would depend on
the dose and duration of treatment.
In summary, these results show that ethanol consumption
affects the absorption, distribution and excretion of each of
the vitamins in rats fed a diet containing a low-vitaminmixture. On the other hand, when rats were fed a 20 %casein diet containing a sufficient amount of vitamins, ethanol
consumption did not affect any factors that we measured.

Acknowledgements
This study was part of the project ‘Studies on the Dietary
Reference Intakes for Japanese’ (principal investigator, K. S.),
which was supported by a Research Grant for Comprehensive
Research on Cardiovascular and Life-Style Related Diseases
from the Ministry of Health, Labour and Welfare of Japan.
The authors’ responsibilities are as follows: A. M. designed
the study, performed the experiments and prepared the manuscript; M. S. and T. F. helped in the study design, performed
the experiments and assisted with the data analysis; K. S. contributed to the study design and supervised the study. The
authors declare that they have no conflict of interest.

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Food and Nutrition Sciences, 2012, 3, 123-128
doi:10.4236/fns.2012.31018 Published Online January 2012 (http://www.SciRP.org/journal/fns)

123

Correlation between Mineral Intake and Urinary
Excretion in Free-Living Japanese Young Women
Munehiro Yoshida1*, Tsutomu Fukuwatari2, Junya Sakai2, Tomiko Tsuji2,3, Katsumi Shibata2
1

Laboratory of Food and Nutritional Sciences, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Japan;
Laboratory of Food Science and Nutrition, Department of Lifestyle Studies, School of Human Cultures, The University of Shiga
Prefecture, Hikone, Japan; 3Department of Health and Nutrition, School of Health and Human Life, Nagoya Bunri University, Inazawa, Japan.
Email: *hanmyou4@kansai-u.ac.jp

2

Received October 8th, 2011; revised November 8th, 2011; accepted November 25th, 2011

ABSTRACTTo clarify whether the urinary excretion of calcium, magnesium, phosphorus, iron, zinc, copper, manganese, selenium
and molybdenum can be used as an index of their intake, the association between urinary excretion and intake infree-living individuals was examined. A total of 102 healthy free-living female university dietetics students aged 18 - 33
years voluntarily participated in this study, of which 76 students were eligible for this assessment. All food consumed
for four consecutive days was recorded accurately by a weighed food record method. A 24-h urine sample was collectedon the fourth day, and the urinary levels of sodium, potassium, calcium, magnesium, phosphorus, iron, zinc, copper,
manganese, selenium and molybdenum were measured. Significant correlation between urinary excretion and intake
was observed in sodium (r = 0.596, p < 0.001), potassium (r = 0.583, p < 0.001), calcium (r = 0.402, p < 0.001), magnesium (r = 0.365, p < 0.01), phosphorus (r = 0.509, p < 0.001), selenium (r = 0.349, p < 0.01) and molybdenum (r =0.265, p < 0.01). On the other hand, urinary excretion was very low and completely independent of the intake in iron,zinc, copper and manganese. These results indicate that urinary calcium, magnesium, phosphorus, selenium and molybdenum can be used as an index of their intake, similarly to sodium and potassium.
Keywords: Mineral Intake; Trace Elements; Urinary Excretion; Assessment; Japanese Young Women

1. Introduction
To assess the nutritional status of healthy free-living humans, the weighed food record method has been usedwidely to record the dietary intake and to calculate nutrient intake [1]. Although this method can provide relatively precise information regarding dietary intake compared with other dietary assessment [2], substantial effort
is required for respondents to complete the dietary records and to weigh all food consumed. This often leads
to errors in the records, which reveals the limitation of aweighed food record method in terms of accuracy [3].
Alternatively, other methods using quantitative biological information, such as urinary excretion, or concentrations of nutrient or their metabolites in blood, as biomarkers to assess dietary intake or nutritional status havebeen well studied in recent years.
Many preceding studies have investigated urinary excretion as a biomarker for assessing dietary intake. For
example, 24-h urinary nitrogen is established as a marker
for protein intake [4], urinary sugars for sugar intake*

Corresponding author.

Copyright © 2012 SciRes.

[5,6], and urinary thiamine for thiamine intake [7]. As regards minerals, urinary potassium is established as a marker for potassium intake [8] and urinary iodine for iodine
intake [9] as well as urinary sodium for sodium intake[10,11].
In the present study, we measured sodium, potassium,
calcium, magnesium, phosphorus, iron, zinc, copper, manganese, selenium and molybdenum in 24-h urine and examined the association between urinary mineral excretionand their intake in free-living individuals. In addition, we
examined whether the urinary excretion of calcium, magnesium, phosphorus, iron, zinc, copper, manganese, selenium and molybdenum can be used as an index of theirintake, similarly to sodium and potassium.

2. Subjects and Methods
2.1. Subjects
This study was reviewed and approved by the Ethics
Committee of The University of Shiga Prefecture. A totalof 102 healthy free-living female university dietetics students aged 18 - 33 years voluntarily participated in this
FNS

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Correlation between Mineral Intake and Urinary Excretion in Free-Living Japanese Young Womenstudy. The purpose and protocol of this study was explained to all participants before joining the study, andwritten informed consent was obtained from each participant, and from parents of participants aged < 20 years.
We excluded participants diagnosed with cold or influenza, and those who had taken mineral supplements atleast once during the previous month. In addition, we
excluded participants whose 24-h urine collection or dietary records were considered as incomplete, with a collection time outside the 22 - 26 h range, urine volume <250 mL, creatinine excretion in relation to body weightoutside the 10.8 - 25.2 mg/kg range [12], or extremely
low or high energy intake (<500 or >4000 kcal/d). Afterscreening, 76 participants were found to be eligible. Anthropometric profiles of the 76 participants are shownand compared with those of general Japanese young women in Table 1. No difference was observed between
subjects and general women.

2.2. Dietary Records
This was a 4-day dietary assessment in which the participants were living freely at college and consuming
their normal diet. The first day (Monday) of the experimental period was defined as Day 1, etc. To measure
dietary intake during the 4-day period precisely, we used
a weighed food record method, which is the highest quality in Japan at this time [13,14]. A digital cooking scale(1 g unit; Tanita Inc., Tokyo, Japan), a set of dietary record forms, a dietary record manual, and a disposable
camera were distributed to the participants in advance.
Upon entry of the dietary record, the status of food atoral intake was identified as “raw”, “cooked”, “the presence of skin”, “cooking ingredient”, or “with or without
seasoning”, and coded according to the Fifth Revised andEnlarged Edition of the Standard Tables of Food Composition in Japan [15]. The participants took photographswith a disposable camera of the dish before and after
eating. Several experienced dietitians used the photographs to complete the data, and asked the participants toresolve any discrepancies or to obtain further information
when needed. The food that remained after eating wasmeasured by a digital scale and was deduced from the
dietary record. Food, nutrient and energy intake was calculated using the Standard Tables of Food Composition
Table 1. Comparison of anthropometric profiles between
subjects and general Japanese young women.
Subjects (n = 76) NHNSJ-20081 (n = 284)
Age
20.1 ± 2.3
20 - 29Height (cm)158.3 ± 5.0
158.3 ± 5.4
Weight (kg)50.8 ± 5.2
51.9 ± 9.5
Body mass index (kg/m2)
20.2 ± 1.7
20.7 ± 3.6
Values are the means ±SD. 1Values for general Japanese young women aged
20 to 29 years described in the National Health and Nutrition Survey ofJapan in 2008.

Copyright © 2012 SciRes.

in Japan. For mineral intake, sodium, potassium, calcium,
phosphorus, iron, zinc, copper and manganese were assessed. Because selenium and molybdenum are not designated in the Standard Table of Food Composition in
Japan, intake of these microminerals was calculated using averaged values of the contents for every food groups
described in the literature [16,17].

2.3. 24-h Urine Sampling
A single 24-h urine sample was collected on Day 4 tomeasure urinary mineral excretion. In the morning, participants were asked to discard the first specimen and torecord the time on the sheet. The next morning, participants were asked to collect the last specimen at the sametime as when the specimen had been discarded the previous morning, and to record the time on the sheet. Afterthe urine sample had been collected, the volume of thesample was measured. The urine samples were stored at–20˚C until analysis.

2.4. Measurement of Urinary Minerals
Urine samples were diluted with 9 or more volumes of
0.1 M HNO3 and filtrated through a 0.45-µm-membranefilter. Filtrate thus obtained was used for the measurement of minerals. Sodium, potassium, calcium and magnesium were determined by atomic absorption spectrometer (AA-6300; Shimadzu, Kyoto, Japan). Phosphorus,
iron, zinc and copper were determined by inductively
coupled plasma-atomic emission spectrometer (ULTIMA2;
Horiba Ltd., Kyoto, Japan). Manganese, selenium andmolybdenum were determined by inductively coupledplasma-mass spectrometer (ICPM-8500; Shimadzu) using rhodium (for manganese and molybdenum) and tellurium (for selenium) as internal standards. In these urinalyses, recovery of each mineral adding urine was 97%
to 101%.

2.5. Statistical Analysis
For each subject, means of daily nutrient and energy intake were calculated from the consecutive 4-day dietary
records. The mean values of the subjects were calculated
based on the resulting individual mean values. Pearson
correlation coefficients were calculated to determine theassociation between urinary and dietary measurements ofminerals. These statistical tests were performed using apersonal computer (eMac; Apple Computer, Cupertino,
CA, USA) with the operating system Mac OS 9.2 andstatistical program package StatView-J version 5.0 (Abacus Concept, Berkeley, CA).

3. Results and Discussion
In Table 2, the daily energy and nutrient intake of the 76
FNSCorrelation between Mineral Intake and Urinary Excretion in Free-Living Japanese Young Women
Table 2. Daily intake of energy, major nutrients and minerals of subjects at experimental period.

Energy (kcal)
Protein (g)Lipid (g)Carbohydrate (g)Minerals
Sodium (mg)
Potassium (mg)Calcium (mg)Magnesium (mg)
Phosphorus (mg)Iron (mg)
Zinc (mg)Copper (mg)Manganese (mg)Selenium (µg)
Molybdenum (µg)

Subjects1(n = 76)

NHNSJ-20082(n = 418)

1658 ± 302
57.3 ± 11.9
52.8 ± 15.5
232.8 ± 39.8

1669 ± 475
61.0 ± 21.4
53.7 ± 22.6
227.3 ± 66.6

2923 ± 834
1873 ± 472
503 ± 142
194 ± 53852 ± 193
6.7 ± 1.9
6.9 ± 1.5
0.90 ± 0.21
2.8 ± 0.8
189 ± 67272 ± 773617 ± 1415 31886 ± 710
406 ± 205 3
201 ± 70844 ± 292
6.7 ± 2.7
7.2 ± 2.6
0.98 ± 0.34-

Values are the means ±SD. 1Daily intake was assessed from the consecutive
4-day dietary records. 2Values for general Japanese young women aged 18
to 29 years described in the National Health and Nutrition Survey of Japan
in 2008. 3Significant difference was observed between subjects and general
Japanese young women at p < 0.001 by Student’s t-test.

eligible participants is presented and compared with thoseof general Japanese young women described in the National Health and Nutritional Survey of Japan (NHNSJ)[18]. Similarity was observed between the subjects andgeneral Japanese in the intake of energy and macronutrients. Among minerals, no difference was observed in potassium, magnesium, phosphorus, iron, zinc and copper
intake. In addition, manganese and molybdenum intake
in the participants was close to the reported values forgeneral Japanese [19,20]. On the other hand, lower sodium intake and higher calcium intake were observed in
the subjects than in general young women. In Japan, because excess intake of sodium and low intake of calcium
have been major nutritional problems, dietetics students
have received education so that sodium intake is reduced
and calcium intake is increased; therefore, it is thoughtthat the subjects made efforts to reduce their sodium intake and increase their calcium intake intentionally. Selenium intake in the participants was quite a bit higher
than the reported value for general Japanese [16,21]. This
indicates that overestimation arose in selenium intake
roughly calculated using averaged values of the contents
for every food group because no difference was observed
between the subjects and general Japanese adolescents in
the intake of energy and many nutrients.
Table 3 shows 24-h urinary excretion and the apparent
urinary excretion rate of minerals. As regards manganese,
since almost all samples showed less than the detection
limit (<10 µg/L), it is excluded from the table.
Copyright © 2012 SciRes.

125

Table 3. Daily urinary mineral excretion in subjects.

SodiumPotassium
Calcium
Magnesium
Phosphorus

Excretion amounts

Apparent excretion rate

(mg/d)(%)

2616 ± 1010
1456 ± 498
100.5 ± 36.4
39.9 ± 16.4
660 ± 223

90.7 ± 30.8
79.5 ± 23.0
20.9 ± 8.2
22.4 ± 15.4
79.1 ± 23.8(µg/d)
Iron
Zinc
CopperSelenium
Molybdenum

220 ± 138374 ± 12552.5 ± 37.1
84.8 ± 26.6
211 ± 93

3.6 ± 2.5
6.3 ± 2.8
6.3 ± 5.1
49.7 ± 21.3
82.2 ± 44.3

Values are the means ±SD. Apparent excretion rate was calculated as follows: (daily urinary excretion amounts)/(daily intake) × 100.

A high rate of urinary excretion (>70%) was observed
for sodium and potassium, which intake has been assessed using urine. In addition, phosphorus and molybdenum also showed a high excretion rate, parallel to sodium and potassium. Because most phosphorus and molybdenum ingested from food are absorbed in the intestine and their main excretion route is urine [20,22], thishigh excretion rate is valid. Although dietary selenium isalso mostly absorbed and its main excretion route is urine[23], the excretion rate was 50%, which was lower thanseveral reported values [24]. This was surely caused byan overestimation of selenium intake; if the excretion
rate were 70%, selenium intake would be estimated to beabout 120 µg/d, which is almost coincident with the reported value for general Japanese [16,21].
The apparent urinary excretion rate of calcium andmagnesium was about 20%, which was coincident withthe reported value [22,25]. On the other hand, urinary excretion of iron, zinc and copper was very low, which reflects that urine is not the main excretion route of theseminerals [26-28].
Figure 1 shows the correlation between daily intake
and 24-h urinary excretion of sodium, potassium, calcium, magnesium and phosphorus. Significant correlation was observed with all of these five minerals. In particular, a strong correlation (r > 0.5) was observed for
sodium, potassium and phosphorus; therefore, in these threeminerals, intake could be estimated from the amount ofurinary excretion for every individual with high accuracy.
Urinary sodium and potassium are already used as important indices of their intake for individuals [10,11]. In
addition, urinary phosphorus could also be used as anindex of its intake.
Also, in the case of calcium and magnesium, a significant correlation between urinary excretion and intake
was observed. The intestinal absorption rate of calcium
and magnesium is 30% to 50% and the main excretion
FNS

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Correlation between Mineral Intake and Urinary Excretion in Free-Living Japanese Young Women

Figure 1. Correlation between daily intake and urinary excretion of sodium, potassium, calcium, magnesium and phosphorus in subjects.

route is urine [22,25]; therefore, urinary excretion of theseminerals reflects absorption amounts. Since intestinal absorption of these minerals changes with various factors[29], it may be difficult to estimate the intake of theseminerals from the urinary excretion for every individual.
Nevertheless, it will be possible to estimate the intake
from urinary excretion at least in a group.
Figure 2 shows correlation between intake and urinary
excretion in iron, zinc, copper, selenium and molybdenum. In iron, zinc and copper, the scale is changed between the X- and Y-axis since their excretion rate tourine is very low. In these three minerals, urinary excretion was almost completely independent of the intake.
Accordingly, intake of these minerals cannot be estimated from urinary excretion. In addition, because urinary manganese excretion was very low, similarly to iron,zinc and copper, it may be difficult to use urinary manganese as an index of manganese intake. Probably, it is
the reason that their urinary excretion is constantly low
regardless of the intake, since they are bound to protein
in blood. In the case of selenium and molybdenum, asignificant correlation was observed; however, in spite of
having said that a large part of ingested selenium andmolybdenum was excreted into urine, similarly to potas
Copyright © 2012 SciRes.

Figure 2. Correlation between intake and urinary excretionof iron, zinc, copper, selenium and molybdenum of subjects.

sium, sodium and phosphorus [20,23], the correlation coefficients were smaller than those of calcium and magnesium. Probably, these weak correlations were due to roughintake estimation using averaged values of the contents
for every food group; therefore, it is considered that agreater correlation coefficient was obtained when intake
was estimated using the content of every food, as forother minerals.
In the present study, it was confirmed that excretion
amounts in 24-h urine were good indices of daily intakeof phosphorus, calcium, magnesium, selenium and molybdenum similarly to sodium and potassium. In minerals,
estimation of the intake using 24-h urine is possible whenthe main excretion route is urine. To estimate the intakeof these minerals from the urinary excretion, the precise
regression between intake and urinary excretion needs tobe established by a balance test in the future.

4. Acknowledgements
This study was supported by a grant for comprehensive
research on cardiovascular and lifestyle disease from the
Ministry of Health, Labour and Welfare of Japan.
FNSCorrelation between Mineral Intake and Urinary Excretion in Free-Living Japanese Young Women

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FNSBritish Journal of Nutrition (2012), 107, 52–60
q The Authors 2011

doi:10.1017/S0007114511002339

Different variations of tissue B-group vitamin concentrations in short- andlong-term starved rats
Aya Moriya1, Tsutomu Fukuwatari1,2, Mitsue Sano1,2 and Katsumi Shibata1,2*
1

Division of Life Style Studies, Section of Nutritional Sciences, Graduate School of Human Cultures, The University of Shiga
Prefecture, 2500 Hassaka, Hikone, Shiga 522-8533, Japan2
Department of Food Science and Nutrition, Graduate School of Human Cultures, The University of Shiga Prefecture,2500 Hassaka, Hikone, Shiga 522-8533, Japan

British Journal of Nutrition(Received 3 November 2010 – Revised 24 March 2011 – Accepted 25 March 2011 – First published online 27 June 2011)

Abstract
Prolonged starvation changes energy metabolism; therefore, the metabolic response to starvation is divided into three phases according tochanges in glucose, lipid and protein utilisation. B-group vitamins are involved in energy metabolism via metabolism of carbohydrates,
fatty acids and amino acids. To determine how changes in energy metabolism alter B-group vitamin concentrations during starvation,
we measured the concentration of eight kinds of B-group vitamins daily in rat blood, urine and in nine tissues including cerebrum,heart, lung, stomach, kidney, liver, spleen, testis and skeletal muscle during 8 d of starvation. Vitamin B1, vitamin B6, pantothenic acid,
folate and biotin concentrations in the blood reduced after 6 or 8 d of starvation, and other vitamins did not change. Urinary excretion
was decreased during starvation for all B-group vitamins except pantothenic acid and biotin. Less variation in B-group vitamin concentrations was found in the cerebrum and spleen. Concentrations of vitamin B1, vitamin B6, nicotinamide and pantothenic acid increasedin the liver. The skeletal muscle and stomach showed reduced concentrations of five vitamins including vitamin B1, vitamin B2, vitamin B6,
pantothenic acid and folate. Concentrations of two or three vitamins decreased in the kidney, testis and heart, and these changes showed
different patterns in each tissue and for each vitamin. The concentration of pantothenic acid rapidly decreased in the heart, stomach,kidney and testis, whereas concentrations of nicotinamide were stable in all tissues except the liver. Different variations in B-group vitaminconcentrations in the tissues of starved rats were found. The present findings will lead to a suitable supplementation of vitamins for theprevention of the re-feeding syndrome.
Key words: Starvation: Fasting: Energy metabolism

Starvation produces a series of metabolic changes that lead toa reduction in body weight, alterations in body composition
and metabolic gene expression(1,2). In mammals and birds,
three distinct levels of energy depletion have been established(3 – 10). The first phase (phase 1) is a rapid period ofadaptation marked by an increase in mobilisation of fat
stores and a lowering in protein utilisation. During thesecond phase (phase 2), which is a long period of thrift,
most of the energy expenditure is derived from fats, andthen fat stores are progressively exhausted, while bodyproteins are efficiently spared. The third phase (phase 3) ischaracterised by an increase in protein utilisation. In
humans, the negative energy balance resulting from starvation
can arise due to disease, eating or psychological disorders,or hunger strikes. Starvation and consequent re-feeding
syndrome can lead to electrolyte disorders, especially

Abbreviations: 2-Py, N 1-methyl-2-pyridone-5-carboxamide;
N 1-methylnicotinamide.

4-Py,hypophosphataemia, along with neurological, pulmonary,cardiac, neuromuscular and haematological complications(11).
To avoid the re-feeding syndrome, an additional load ofvitamins has been suggested to correct the vitamin deficiencies(11). However, little is known about B-group vitaminstatus during starvation.
Several B-group vitamins take part in energy metabolism.
For instance, vitamin B2 functions as FAD and FMN in redox
reactions including the electron transport chain and fatty acidoxidation. Nicotinamide is involved in more than 200 reactions,
including the metabolism of carbohydrates, amino acids andfatty acids, and also in the electron transport chain. Vitamin
B1 catalyses carbohydrate metabolism including decarboxylation of a-ketoacids and trans-ketolation as a cofactor thiamin
diphosphate; vitamin B6 functions as pyridoxal 50 -phosphatein amino acid metabolism including aminotransferases,

N 1-methyl-4-pyridone-5-carboxamide;* Corresponding author: K. Shibata, fax þ 81 749 28 8499, email kshibata@shc.usp.ac.jp

3-HBA,

3-hydroxybutyric

acid;

MNA,

B-group vitamin contents in starved rats

British Journal of Nutrition

decarboxylases, racemases and dehydratases as pyridoxal
50 -phosphate; and pantothenic acid is involved in fatty acidmetabolism such as oxidation and synthesis. For these reasons,in the ‘Dietary Reference Intakes for Japanese, 2010’, dietaryrequirements for vitamin B1, vitamin B2 and niacin are
expressed per 4186 kJ (1000 kcal), and the requirement for vitamin B6 is expressed in terms of protein intake(12).
As mentioned earlier, prolonged starvation sifts the energy
source from glucose to fats and then to protein, andB-group vitamins are involved in the metabolism of carbohydrates, fatty acids and amino acids. Thus, in the present
study, we investigated how changes in energy metabolism
altered B-group vitamin utilisation during starvation. We
comprehensively determined eight kinds of B-group vitaminconcentrations in rat blood, urine and tissues including thebrain, heart, lung, stomach, kidney, liver, spleen, testis andskeletal muscle during 8 d of starvation.

Materials and methods
Diets
The composition of the purified diet is shown in Table 1.
Vitamin-free milk casein, L -methionine and sucrose werepurchased from Wako Pure Chemical Industries Limited(Osaka, Japan). Maize oil was purchased from Nisshin OilliO
Group, Limited (Tokyo, Japan). Gelatinised maize starch, themineral mixture (AIN-93G) and the vitamin mixture(AIN-93VX) were obtained from Oriental Yeast Company,
Limited (Tokyo, Japan).

53

by decapitation as a control group (CONT, n 5). The otherrats were deprived from food for 1 d (S1, n 5), 2 d (S2, n 5),
6 d (S6, n 5) or until they had been in phase 3 for 2 d; thatis, they were starved for a total duration of 6– 9 d (P3, n 5).
The starving phase was determined by calculating the specificdaily rate of body mass loss dM/Mdt (g/kg per d) for eachanimal (dM represents the loss of body mass during dt ¼ t1-t0
and M is the body mass of the rat at t0 (10,14). Blood was
taken from the tail vein at 09.00 hours every day, and 3-hydroxybutyric acid (3-HBA) concentration in the blood wasmeasured with a 3-HBA Kit (Abbott Japan Company, Limited,Tokyo, Japan) to confirm the metabolic state of each animal
because blood 3-HBA reflects fatty acid oxidation.
After the animals were killed, blood samples were collected
into EDTA-2K tubes from the carotid artery and were centrifuged at 1700 g for 10 min at 48C. Plasma glucose, TAG, urea
N, aspartate aminotransferase and alanine aminotransferase
were measured with FUJI DRI-CHEM (FUJIFILM Company,
Tokyo, Japan).
The cerebrum, heart, lungs, stomach, kidneys, liver, spleen,
testes and leg muscles were dissected and weighed(^ 0·001 g). The stomach was cleared of its contents. All
tissue samples were immediately homogenised in ultra-purewater at 1:10 (w/v) using a Teflon glass homogeniser andstored at 2 208C until needed. The present study was
conducted according to the guidelines for the care and useof laboratory animals, and was approved by the Ethics
Committee of the University of Shiga Prefecture (Shiga, Japan).

Analytical methods
Animals
Male rats of the Wistar strain, weighing 225– 235 g, wereobtained from CLEA Japan, Inc. (Tokyo, Japan). The rats
were individually housed in a temperature-controlled room
(22 ^ 28C and 50 –60 % humidity) with a 12 h light – 12 hdark cycle and were allowed to acclimate to the environment
for 7 d before starting the experiment. Body mass, foodconsumption and water intake were recorded daily (^0·1 g).
We also collected 24 h urine samples every day.

Experimental procedures
A total of twenty-five rats were randomly divided into fivegroups. After 1 week of acclimatisation, five rats were killed
Table 1. Composition of the diet(g/100 g)
Vitamin-free milk casein
L -MetGelatinised maize starch
SucroseMaize oil
Mineral mixture (AIN-93-G)
Vitamin mixture (AIN-93VX)*

20·0
0·2
46·9
23·4
5·0
3·5
1·0

* The composition of the vitamin mixture is described byReeves et al.(13).

Vitamin B1. Thiamin in urine was measured directly. The vitamin B1 content in the blood and tissue was determined as thesum of thiamin, thiamin monophosphate and thiamin diphosphate and was expressed as total thiamin. TCA (5 %) was
added to whole blood and tissue homogenates, and theblood and homogenates were centrifuged for 5 min at
20 000 g, and the supernatant of the mixture was used formeasurement. Vitamin B1 levels in the urine, blood andtissue were determined by the HPLC post-labelled fluorescence method(15).
Vitamin B2. Riboflavin in urine was measured directly byHPLC(16). Riboflavin, FMN and FAD in blood and tissue wereconverted to lumiflavin by photolysis. Briefly, the supernatantfrom a TCA-treated blood or tissue sample was added to an
equal volume of 1 M -NaOH. The alkalised mixture was irradiated with a fluorescent lamp for 30 min, and acetic acidwas added to the mixture. The neutralised mixture was filteredwith a 0·45 mm microfilter and the filtrate was directly injected
into the HPLC system for the measurement of lumiflavin(17).
The measured lumiflavin was expressed as total vitamin B2.
Vitamin B6. 4-Pyridoxic acid, a catabolite of vitamin B6, in
urine was measured directly by HPLC(18). Serum pyridoxal
and pyridoxal 50 -phosphate were determined by the HPLC
method(19). Vitamin B6 vitamers, including phosphate esters
in the tissue, were converted to free vitamin B6 vitamerssuch as pyridoxal and pyridoxamine using an autoclave
under acidic conditions. Briefly, the homogenate was added

British Journal of Nutrition

54

A. Moriya et al.

to 0·06 M -HCl at 1:8 (v/v) and autoclaved at 1218C for 3 h, andthe mixture was adjusted to pH 5·0 using 1 M -NaOH. These
were measured as total vitamin B6 by the microbioassay
method using Saccharomyces carlsbergensis strain 4228
ATCC 9080(20).
Vitamin B12. Urine, plasma and tissue homogenates wereadded to a 0·2 mM -acetate buffer (pH 4·8) with 0·0006 % potassium cyanide. These were put into a boiling water bathfor 5 min to be converted to cyanocobalamin, and then 10 %metaphosphoric acid was added to be neutralised. Cyanocobalamin was determined by the microbioassay method using
Lactobacillus leichmannii ATCC 7830(21).
Niacin. Nicotinamide(22) and its catabolites, N 1-methylnicotinamide (MNA)(23), N 1-methyl-2-pyridone-5-carboxamide(2-Py) and N 1-methyl-4-pyridone-3-carboxamide (4-Py)(22),in urine were measured directly by HPLC. For measuring thetotal nicotinamide content in blood and tissues, the whole
blood and tissue homogenates were autoclaved at 1218C
for 20 min to convert the coenzymes to nicotinamide.
The resulting nicotinamide was then determined by the
HPLC method(22,24).
Pantothenic acid. Pantothenic acid in urine was determinedby HPLC(25). To digest the bound pantothenic acid including
coenzyme A and phosphopantetheine in tissue and plasma
to free form, the homogenate or blood was incubated at
378C for 24 h. Pantothenic acid in the plasma and tissue wasdetermined by the microbioassay method using Lactobacillus
plantarum ATCC 8014(26).
Folate. Folate in urine and plasma was directly determinedby the microbioassay method using Lactobacillus casei ATCC
27 773(27). Folate in tissues was digested to monoglutamate
forms by treatment with protease and conjugase. Briefly,
1 M -KH2PO4 –K2HPO4 buffer (pH 6·1) was added to thetissue homogenate at 1:9 (v/v), and the homogenate was autoclaved at 1218C for 5 min. Proteinase MS (Kaken Pharmaceutical Company, Limited, Tokyo, Japan) was added to thehomogenate at a final concentration of 2·5 mg/ml and thenincubated at 378C for 3 h. The reaction mixture was added

to the conjugase solution (extract from porcine kidneyacetone powder, Sigma, Porcine, Type II) at 30:1 (v/v) andincubated at 378C for 12 h. After centrifugation at 10 000 g for
10 min, the supernatant was used for determination by the
microbioassay.
Biotin. Bound biotin in tissues was converted to thefree form using autoclave under acidic conditions. Briefly,
1·5 M -H2SO4 was added to the homogenate at 1:1 (v/v), andthe homogenate was autoclaved for 1 h at 1218C. The suspension was centrifuged at 10 000 g for 10 min at 48C, and thesupernatant was used to measure biotin. Biotin in urine andplasma was measured directly. The biotin content in urine,
plasma and tissue was determined by the microbioassay
method using L. plantarum ATCC 8014(28).

Statistical analysis
Values are expressed as means with their standard errors. P3
rats (starved for 6– 9 d) were expressed at 8 d on the graphfor convenience. To test the significance of the differencesin mean values among all groups, one-way ANOVA with
Tukey’s post hoc test was employed. Repeated ANOVA with
Bonferroni’s post hoc test was used to analyse urinary
excretion of B-group vitamins in P3 rats, and individual data
points were compared with their data at day 0. All differences
at P , 0·05 were considered to be statistically significant. Prism
software (version 5; obtained from GraphPad Software, Inc.,San Diego, CA, USA) was used for all analyses.

Results
Changes in body mass during starvation
Changes in body mass during starvation are shown in Table 2.
Starvation for the first 24 h produced a weight loss of 7 %.
From the second day to the last day of starvation, the rats
lost 5 % weight for each 24 h (data not shown). The specificdaily rate of body mass loss (dM/Mdt) v. time in starved rats
is presented in Fig. 1. The pattern of dM/dMt showed a

Table 2. Body mass and organ mass in the control and starved rats
(Mean values with their standard errors, n 5)
CONT*Initial body mass (g)Final body mass (g)Organ mass (g, wet wt)
Cerebrum
Heart
Lungs
StomachKidneys
SpleenTestes
Liver

S1

S2

S6

P3†

Mean

SE

Mean

SE

Mean

SE

Mean

SE

Mean

SE

252·9
252·9a

3·3
3·3

253·1
235·1a,b

2·8
2·3

246·4
219·3b

6·2
5·6

252·2
182·7c

3·1
2·5

249·0
166·6c

3·2
5·4

1·29
0·84a
1·28
1·16
1·94a
0·75a
2·75a,b
11·18a

0·02
0·04
0·08
0·02
0·05
0·04
0·07
0·23

1·30
0·87a
1·19
1·15
1·89a,b0·67a2·66b,c
7·25b

0·01
0·03
0·10
0·04
0·6
0·03
0·05
0·22

0·02
0·02
0·13
0·05
0·02
0·01
0·03
0·12

1·23
0·58b
0·95
1·19
1·43d
0·25c
2·45c
3·83c

0·03
0·02
0·06
0·08
0·04
0·04
0·06
0·42

1·28
0·81a
1·09
1·14
1·69b,c
0·50a2·66b,c
6·07b

0·01
0·04
0·06
0·04
0·03
0·05
0·04
0·19

1·27
0·66b
1·09
1·20
1·53c,d0·30b2·47b,c4·69c

CONT, non-starved control rats; S1, 1-day starved rats; S2, 2-day starved rats; S6, 6-day starved rats; P3, starved to phase 3 rats.
a,b,c
Mean values within a row with unlike superscript letters were significantly different determined by one-way ANOVA with Tukey’s multiple comparison tests(P, 0·05).
* Since the control rats were killed at the beginning of the experiment, the initial body weight was same as the final body weight.
† Phase 3 is determined by the rapid increase in dM/dMt (refer to Fig. 1).

Phase 2

Phase 3Phase 2Phase 370

60

Blood/plasma parameters
50

40

30
1
2
First

3

4

5

6

7

8

9

–4

–3

–2
–1
Last

Days of starvation

Fig. 1. Rate of body mass loss (dM/Mdt) in starved rats. Values are means
with their standard errors (n 1–20 per d in the left; n 5 per d in the right).
dM/Mdt (dM represents the loss of body mass during dt ¼ t1 2 t0 and M isthe body mass of rat at t0) was calculated for each animal. Abscissa: left,days counted from the beginning of starvation in all rats; right, counted to theend of starvation in the P3 group.

British Journal of Nutrition

55

decreased. Heart and spleen mass decreased from 6 d. Testes
mass decreased in P3 rats. Prolonged starvation reduced thespleen and liver weight the most.

80

Phase 1

Rate of body mass loss (g/kg per d)

B-group vitamin contents in starved rats

sharp decrease during the first hours of starvation and a steadyrate at days 2 – 6 of starvation. Since obvious rapid increase
was not observed after day 7 of starvation, we showed thatthe last part of starvation was counted to the end of starvationin the P3 group. The dM/dMt in the P3 group clearly showed a
rapid increase from 3 d before the end of starvation. These
patterns were exactly consistent with previous reports(10,14).
Therefore, we defined the phase at first decrease as phase 1,
that of steady rate as phase 2 and the third part of the curveas phase 3 according to previous reports(10,14). The S6 group
showed the steady rate of body mass loss and the low
blood 3-HBA concentration, and these were characteristics
of both phases 2 and 3. These results showed that the S1
group was representative of phase 1, the S2 was of phase 2,
the S6 group was in the marginal range between phases 2
and 3, and the P3 group was in phase 3.

Changes in mass of individual organs during starvation
Table 2 shows the changes in the mass of individual organs
during starvation. The cerebrum, lung and stomach mass
was not affected by starvation. The liver weight was gradually
reduced by starvation, and that in the P3 group was 30 % of
the control group. From 2 d of starvation, kidney mass

Table 3 shows the blood parameters. Blood 3-HBA increased
more in S1 and S2 rats than in control rats. In contrast, the urea
concentration in plasma was significantly higher in S6 and P3
rats, whereas there was a non-significant increase in the S1and S2 rats. Plasma glucose level was 60 % significantly
lower in the S1, S2 and S6 rats than in the control rats. Interestingly, plasma glucose returned to the basal level in the P3
rats. Plasma TAG was dramatically decreased after 1 d ofstarvation and then continued to decrease gradually throughout the remainder of the starvation period. Plasma aspartate
aminotransferase was not affected by starvation. Plasma alanine aminotransferase began to increase after 6 d of starvation.

Effect of starvation on vitamin status
Table 4 shows B-group vitamin content in tissue, blood andurine in the control rats. We determined the B-group vitamincontents in nine tissues including the cerebrum, heart, lung,stomach, kidney, spleen, testis, skeletal muscle and liver,and five tissues were selected as representative variations in
Fig. 2.
Cerebrum (Fig. 2(A)) and spleen. With the exception of
biotin, all vitamin concentrations were unchanged by starvation. Biotin concentration was initially elevated to 150 % in
the S1 rats, and then returned to basal level. B-group vitaminconcentrations in the spleen showed a similar pattern that starvation did not affect their concentrations except for vitamin
B2. Vitamin B2 concentration in the testis was elevated to
130 % after 6 d of starvation.
Heart (Fig. 2(B)). Vitamin B1 and folate concentrations
significantly decreased to approximately 60 % after 6 d ofstarvation. Pantothenic acid concentration was significantly
lower in the S2 and S6 rats than in the control rats. Biotin
and vitamin B6 concentrations significantly increased to
160 and 250 % in the S1 and S6 rats, respectively. The otherB-group vitamin concentrations were unchanged.

Table 3. Blood parameters in the control and starved rats
(Mean values with their standard errors, n 5)
CONT
Mean
3-HBA (mmol/l)*
Glucose (mmol/l)
TAG (mmol/l)Urea (mmol/l)
AST (U/l)
ALT (U/l)

a

0·1
6·36a
3·01a
7·45a
263
39·8a

S1
SE

0·0
0·28
0·45
0·27
21
3·8

Mean
a

1·8
3·86b
0·82b
6·21a
245
29·4a

S2
SE

0·1
0·32
0·08
0·42
10
1·9

Mean
b

2·5
3·84b
0·81b
6·21a
242
31·8a

S6
SE

0·2
0·29
0·07
0·30
21
1·8

Mean
a

0·7
3·71b
0·54b
8·23b
240
65·5b

P3
SE

0·1
0·37
0·10
2·36
10
10·0

Mean

SE

a

0·1
0·60
0·05
0·68
8
5·9

0·3
6·44a
0·46b
13·08b252
73·3b

CONT, non-starved control rats; S1, 1-day starved rats; S2, 2-day starved rats; S6, 6-day starved rats; P3, starved to phase 3 rats; 3-HBA, 3-hydroxybutyrate;
AST aspartate aminotransferase; ALT, alanine aminotransferase.
a,b
Mean values within a row with unlike superscript letters were significantly different determined by one-way ANOVA with Tukey’s multiple comparison tests(P,0·05).
* 3-HBA was measured in whole blood, and the others in serum.

56

A. Moriya et al.
(A)

(B)

100

50

c

300

200
h
100

cg
f
f

f

ag

a
f

0
0

abcd
0

0

abg

2
4
6
8
Days of starvation

f

f
fg

f
g

2
4
6
8
Days of starvation

(F)
200

300
f
200
ce
100

0

0

2
4
6
8
Days of starvationceaRelative value of vitaminsin blood (%)

50

c
g
a
f

Relative value of vitamins
in liver (%)

100

b
50

0

400

150

100

2
4
6
8
Days of starvation

(E)
200

150

0
0

2
4
6
8
Days of starvation(D)
Relative value of vitaminsin skeletal muscle (%)

200
Relative value of vitaminsin kidney (%)

Relative value of vitaminsin heart (%)

Relative value of vitaminsin cerebrum (%)

h
150

0

British Journal of Nutrition

(C)
400

200

150

100
ac
50
f
0

0

a
c
h
g
f

2
4
6
8
Days of starvation

Fig. 2. Relative value of B-group vitamin concentrations in (A) cerebrum, (B) stomach, (C) kidney, (D) skeletal muscle, (E) liver and (F) blood of rats during starvation. * Sum of serum pyridoxal and pyridoxal-50 -phosphate is expressed as serum vitamin B6. Values are reported as means with their standard errors, n 5 per
d. Values of control rats are expressed as 100 %. P3 is expressed at 8 d of starvation. Means with unlike letters were significantly different from day 0 in avitamin
B1 (W), bvitamin B2 (X), cvitamin B6* (D), dvitamin B12 (O), enicotinamide (A), fpantothenic acid (B), gfolate ( £ ) and hbiotin (þ ; P, 0·05).

Kidney (Fig. 2(C)) and testis. Pantothenic acid concentration dramatically decreased to 50 % of control during starvation. Folate concentration significantly decreased to 40 %in the S6 and P3 rats. Vitamin B2 concentration significantly
decreased to 70 % in the P3 rats. The reduction in pantothenic
acid concentration from day 1 of starvation was also observedin the stomach and testis, and their maximal reduction was
60 %. In the testis, vitamin B2 concentration also significantly
reduced to 50 % during starvation, and other vitamin concentrations were not changed.
Skeletal muscle (Fig. 2(D)) and stomach. Concentrations ofvitamin B1, vitamin B2 and vitamin B6 significantly decreased
to 50 % from 2 d of starvation, but only vitamin B2 concentration returned to control levels in the P3 rats. Vitamin B12
concentration significantly decreased to 40 % only in the S2
rats. Pantothenic acid concentration significantly decreasedin the P3 rats. Folate concentration significantly decreased to
50 % in the S6 and P3 rats. Pantothenic acid concentration
was same as control until 6 d of starvation, and then dramatically decreased to 30 % in the P3 rats. Similar pattern wasobserved in the stomach that starvation reduced several
B-group vitamin concentrations. In brief, pantothenic acidand biotin concentrations reduced to 30 and 60 % from day
1 of starvation, respectively. Vitamin B6 concentration significantly decreased to 50 % from day 2, and vitamin B2 did to40 % from day 6 of starvation.
Liver (Fig. 2(E)). Vitamin B6 concentration was significantly
higher in the S1, S6 and P3 rats, and the relative value in the P3

rats was 160 % of the control animals. Nicotinamide concentration increased in the S2, S6 and P3 rats. Vitamin B1 andpantothenic acid concentrations were higher in the P3 rats,
and their values were 150 and 250 %, respectively. Other
B-group vitamins concentrations were unchanged.
Blood (Fig. 2(F)). Whole blood vitamin B1, serum vitamin
B6 and plasma pantothenic acid concentrations decreased in
the S6 and P3 rats. Plasma folate and biotin concentrations
decreased in the P3 rats. The relative values of vitamin B1
and vitamin B6 in the P3 rats were 60 % of control, thoseof folate and biotin were 50 %, and those of pantothenic
acid were 30 %.
Urinary contents of B-group vitamins (Fig. 3). Vitamin B1
excretion acutely decreased to 10 % after 1 d of starvation.
Urinary excretion of riboflavin, pyridoxal metabolite 4-pyridoxic acid and vitamin B12 gradually decreased during 4 d ofstarvation. Subsequently these values were stable, at approximately 20, 20 and 50 % of each control value. Urinary folate
was initially unchanged in the S1 rats and then decreased to
40 % of the baseline value. Urinary pantothenic acid wasincreased to 170 % in 3rd and 4th days of starvation, andthen returned to the control level. Although biotin excretion
increased to 460 % during the first 3 d of starvation, it subsequently returned to the basal level.
Urinary contents of nicotinamide and its catabolites (Fig. 4).
Nicotinamide excretion increased after 1 d of starvation andthen returned to the basal level. 2-Py and 4-Py decreased
after an initial increase on day 1. In contrast, MNA excretion

57

0·25

increased during the starvation period. Urinary excretion of
the sum of nicotinamide and its catabolites increased 1·4fold after 1 d of starvation and then decreased by less thanhalf of the food sufficient state.

2·96

0·01
0·04
0·02
0·01
0·12
0·01
0·01
0·00
0·11
0·19
0·23
0·13
0·27
1·40
0·05
0·08
0·03
1·84

(pmol/ml
plasma)54·6
3·5(pmol/d)SE

Mean

Biotin (nmol/g
tissue)B-group vitamin contents in starved rats

0·68
6·20
68
567
3·93
2
38
4
345
12
65
WB, whole blood.

96
Urine

4

285
Blood

15
(nmol/d)208

12(nmol/d)1·46
0·16(nmol/d)

4·97
0·13(pmol/d)

121(mmol/d)

5

0·17

7·02
0·39
(nmol/d)(pmol/ml
plasma)43·2
3·6(nmol/d)(nmol/ml plasma)(nmol/ml WB)(pmol/ml plasma)(pmol/ml serum)(pmol/ml WB)(pmol/ml WB)

0·02
0·03
0·03
0·02
0·35
0·04
0·00
0·01
0·69
0·52
0·75
0·78
1·14
6·47
1·17
0·07
0·30
14·14
3
9
8
5
18
4
8
2
10
61
166
59
51
250
33
101
22
242
12
46
11
22
27
26
7
24
34
292
578
375
221
920
645
186
400
862
2
20
4
12
19
8
7
5
10
35
202
49
77
269
74
61
38
144
0·4
1·6
0·3
0·6
1·1
0·2
0·4
1·8
0·6
18·3
14·5
3·6
3·4
18·8
1·4
13·9
35·5
17·1
0·1
1·6
0·6
0·3
2·7
0·4
0·8
0·5
3·2
6·6
34·4
8·4
11·5
57·2
6·4
9·6
7·5
68·0
0·4
1·5
0·5
1·7
3·1
0·6
6·4
0·2
1·8
9·1
18·9
5·7
11·7
16·8
7·0
25·2
4·3
32·2
CerebrumHeart
Lung
StomachKidneySpleenTestisSkeletal muscle
Liver

SE

Mean
SE

Mean
SE

Mean
SE

Mean
Mean
SE

SE

Mean
Mean

SE

Nicotinamide(nmol/g tissue)
Vitamin B12(pmol/g tissue)
Vitamin B6(nmol/g tissue)
Vitamin B2(nmol/g tissue)
Vitamin B1(nmol/g tissue)(Mean values with their standard errors, n 4 – 5)

Table 4. Contents of B-group vitamins in each tissue, blood and urine of control rats

British Journal of Nutrition

Pantothenic acid(nmol/g tissue)

Folate (nmol/g
tissue)Discussion
The effects of metabolic changes, which are designated as
the changes in the main energy sources such as glucose,lipids and protein, during starvation on the tissue and urine
vitamin concentrations are currently poorly understood(11).
Elucidation of the effects will lead to a suitable supplementation for preventing the refeeding syndrome. Therefore,
we investigated the effects of short- and long-term starvationon the vitamin concentrations in organs, muscle, blood andurine in rats.
Vitamin concentrations in organs and muscle showed different patterns for each vitamin. For noticeable characteristics,
biotin concentration, which means the value in terms of gtissue, was increased in most organs of the S1 rats. A part of
the reason is a reduced organ mass at S1. It was unclear
why the biotin concentrations in organs remained at thesame level regardless of organ mass during starvation. Vitamin
B1 is the vitamin that has the most rapid turnover(30), but thelevels in the kidney were maintained. This may point to thenecessity of vitamin B1 in kidneys of starving rats. In termsof the metabolic state, vitamin B1 was expected to decreasein the early days of starvation, because glucid is the mainenergy source in this period(3). However, vitamin B1 concentrations in tissues and blood were stable in the S1 rats. Thisis due to the sharp decrease in liver weight and in the urinary
excretion of vitamin B1. Along with the shifts in the mainenergy source from glucid to fat, vitamin demands appear tochange. Next to vitamin B1, pantothenic acid requirement
may be the highest because it is involved in the metabolism
of fatty acids(31), and also, biotin requirement may be higher
because the gluconeogenesis is more active at the deficientstate of glucose(32). However, the present results were contrary
to our expectations. Pantothenic acid concentrations in theheart, stomach, kidneys and testes were decreased in the S1
rats, and a similar phenomenon was observed in biotin concentrations. The urinary excretion of pantothenic acid andbiotin was significantly increased by starvation. A similar
phenomenon was already reported by Fukuwatari et al.(30).
Shibata and co-workers reported(33 – 38) that the urinary excretion of water-soluble vitamins reflects recent intake of the vitamins over the last few days, and in addition, the decreased
urinary excretion of vitamins means the elevated demand forvitamins, whereas the increased urinary excretion of vitamins
means the reduced demand for vitamins when their intake ofvitamins is almost the same(33). The increased urinary excretionsuggests that the requirement of pantothenic acid and biotin
was reduced by starvation, that is, lower concentrations of pantothenic acid and biotin in the body might prefer to live for along time during starvation. A possible inferable reason for
the increase in urinary pantothenic acid and biotin might be amechanism to prevent the stored fat in the body from overspending or to decrease the amount of acetyl-coenzyme A,

58

A. Moriya et al.
(A)

(B)
500

e

Relative value of vitamins in urine (%)

Relative value of vitamins in urine (%)

150

100

b

be

dg

bd

d

50
bceg

0

a

a

0

be beg
cg

ceg

a

a

2

c

c

c
a

a

4

d
e
b
g

a

6

h
h
400

300

h
h
h

200
f

0

8

0

2

4

Days of starvation

6

8

Days of starvation

Fig. 3. Relative value of urinary B-group vitamin contents in P3 rats during starvation. Those of vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide andfolate are shown in (A), and pantothenic acid and biotin in (B). Thiamin is expressed as vitamin B1, riboflavin as vitamin B2, 4-pyridoxic acid as vitamin B6, andsum of nicotinamide and its catabolites as nicotinamide. Values are reported as means with their standard errors, n 5 per d. Values of control rats are expressed
as 100 %. P3 is expressed at 8 d of starvation. Means with unlike letters were significantly different from day 0 in avitamin B1 (W), bvitamin B2 (X), cvitamin B6* (D),dvitamin B12 (O), enicotinamide (A), fpantothenic acid (B), gfolate ( £ ) and hbiotin (þ ; P, 0·05).

which modifies several functional proteins such as histone(39)and some enzymes(40), and in addition, to decrease holoenzymes of carboxylases(41,42). Acetylation generally activatedsome enzymes in fatty acid oxidation(40). The physiologically
active form of biotin is covalently attached at the active site ofa class of important metabolic enzymes in gluconeogenesis,

lipogenesis and amino acid metabolism(41,42). Accordingly,
decreased acetylation and biotin-dependent enzymes lead to
reduced fatty acid oxidation and to save fat in the body.
Vitamin B6 concentrations, expected to be the last vitamindecreased in tissues by starvation(3), decreased in the stomach,skeletal muscle and serum of the S2 rats. Vitamin B6 in the

(A)

(B)
Relative value of urinary MNA (%)

250
Relative value of
urinary nicotinamide (%)

*
200
150
100
50
0

0

2

4

6

8

250
200
150
100
*

*

*

50
0

0

2

Days of starvation

4

6

8

Days of starvation

(C)

(D)
250

Relative value of urinary 4-Py (%)

Relative value of urinary 2-Py (%)

British Journal of Nutrition

f

100

200
*
150
100

*

*

50
0

*

*

0

2

4

*

*

6

Days of starvation

8

250
200
*

150
100

*
*
50
0

*

0

2

4

*

*

6

*

8

Days of starvation

Fig. 4. Relative value of urinary nicotinamide (a) and its catabolites MNA (b), 2-Py (c) and 4-Py (d) contents in P3 rats during starvation. Values are reported asmeans with their standard errors, n 5 per d. * Mean values were significantly different from day 0 determined by one-way ANOVA with Tukey’s multiple comparison
tests (P, 0·05). MNA, N 1-methylnicotinamide; 2-Py, N 1-methyl-2-pyridone-5-carboxamide; 4-Py, N 1-methyl-4-pyridone-3-carboxamide.

British Journal of Nutrition

B-group vitamin contents in starved rats

lung and serum decreased in the S6 rats. The differences in thepattern of vitamin B6 decline may be due to the fat content ofeach tissue. Nicotinamide concentrations in tissues and blood
were unchanged by starvation, despite this vitamin being
involved in energy metabolism. Since nicotinamide is biosynthesised from tryptophan(43), nicotinamide concentrations
in organs and blood were maintained. Urinary excretion of
the sum of nicotinamide and its catabolites was high after
1 d of starvation and subsequently decreased. These results
are in agreement with those reported by a previous
study(30). The proportion of nicotinamide, MNA, 2-Py and
4-Py in urine is controlled by enzymes involved in the metabolism of tryptophan to niacin. Starvation or food restriction
induces a decline in MNA oxidase activity(44) and an elevationin nicotinamide methyltransferase(44). This may explain whylevels of 2-Py and 4-Py in urine decreased while those of
MNA increased. MNA is an inhibitor of nicotinamide methyltransferase(45). Therefore, an accumulation of MNA inhibits
the activity of nicotinamide methyltransferase and leads
to an increase in free form of nicotinamide, which inhibits
the activities of histone deacetylase(46) and poly(ADP-ribose)synthetase(47). This control might be suitable for living long
during starvation.
To our knowledge, the present study presents the firstdata on vitamin status during the three phases of starvation.
The changes in B-group vitamin concentrations in tissues
and blood did not always correspond to metabolic states.
The changes in vitamin content can be divided into threegroups. First, vitamin B1, vitamin B2, nicotinamide and biotin
levels declined gradually. Second, vitamin B6 and vitamin
B12 levels rapidly decreased after 1 d of starvation and thenremained at a steady level. Finally, pantothenic acid andfolate initially decreased in the S1 rats, then returned to nearbasal levels in the next day of starvation, then subsequently
decreased again. This might mean that pantothenic acidand folate were mobilised to other tissues. We are unsure
why such complicated changes occur. It is clear that further
investigation, such as separate measurement of the freeforms of the vitamins and of coenzymes, into the changes inthe vitamin requirements of starving rats would be useful forthe prevention of vitamin deficiency during starvation or forconsequent refeeding.

References

Acknowledgements

16.

The present study was supported by a Research Grant for
Comprehensive Research on Cardiovascular and Lifestyle
Related Diseases from the Ministry of Health, Labour and Welfare of Japan. The title of the project was ‘Studies on Construction of Evidence to revise the Dietary Reference Intakes forJapanese – Elucidation of balance between macronutrientand micronutrient’ (Principal Investigator, K. S.). A. M. and
T. F. were responsible for designing the study strategies,
retrieving the references and writing the manuscript. M. S. participated in retrieving references and critical reading. K. S.
directed and designed the study. The authors have no conflictof interests to report.

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Journal of Gerontology: MEDICAL SCIENCES
Cite journal as: J Gerontol A Biol Sci Med Sci
doi:10.1093/gerona/glr174© The Author 2011. Published by Oxford University Press on behalf of The Gerontological Society of America.
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Significant Relationship between Plasma Vitamin C
Concentration and Physical Performance among Japanese
Elderly Women
Kyoko Saito,1,2 Tetsuji Yokoyama,2 Hideyo Yoshida,1 Hunkyung Kim,1 Hiroyuki Shimada,3 Yuko Yoshida,1
Hajime Iwasa,4 Yoko Shimizu,1 Kondo Yoshitaka,5 Setsuko Handa,5 Naoki Maruyama,5 Akihito Ishigami,5and Takao Suzuki6
1Research Team for Promoting Independence of the Elderly, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
2Department of Human Resources Development, National Institute of Public Health, Saitama, Japan.
3Section for Health promotion, Department of Health and Medical Care, Center for Development of Advanced Medicine

for Dementia, National Center for Geriatrics and Gerontology, Aichi, Japan.
4Department of Social Gerontology, School of Public Health, Tokyo University, Tokyo, Japan.
5Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.
6Research Institute, National Center for Geriatrics and Gerontology, Aichi, Japan.
Downloaded from biomedgerontology.oxfordjournals.org by guest on October 11, 2011

Address correspondence to Kyoko Saito, PhD, Research Team for Promoting Independence of the Elderly, Tokyo Metropolitan
Institute of Gerontology, 35-2, Sakaecho, Itabashi, Tokyo, 173-0015, Japan. Email:kyon@tmig.or.jp

Background. Maintenance of physical performance could improve the quality of life in old age. Recent studies suggested a beneficial relationship between antioxidant vitamin (eg, vitamin C) intake and physical performance in elderly
people. The purpose of this study was to examine the relationship between plasma vitamin C concentration and physicalperformance among Japanese community-dwelling elderly women.
Methods. This is a cross-sectional study involving elderly females residing in an urban area in Tokyo, Japan, in
October 2006. We examined anthropometric measurements, physical performance, lifestyles, and plasma vitamin C
concentration of participants.
Results. A total of 655 subjects who did not take supplements were analyzed. The mean age (±standard deviation) of
participants was 75.7 ± 4.1 years in this study. The geometric mean (geometric standard deviation) of plasma vitamin C
concentration was 8.9 (1.5) mg/mL. The plasma vitamin C concentration was positively correlated with handgrip strength,
length of time standing on one leg with eyes open and walking speed, and inversely correlated with body mass index.
After adjusting for the confounding factors, the quartile plasma vitamin C level was significantly correlated with thesubject’s handgrip strength (p for trend = .0004) and ability to stand on one leg with eyes open (p for trend = .049).
Conclusions. In community-dwelling elderly women, the concentration of plasma vitamin C related well to theirmuscle strength and physical performance.
Key Words: Plasma vitamin C—Physical performance—Elderly women—Japanese.
Received January 26, 2011; Accepted August 30, 2011
Decision Editor: Luigi Ferrucci, MD, PhD

P

HYSICAL performance and physical ability are themost important indicators of health status in elderly
people and are also closely related to the quality of life.
Declines in physical performance and physical activity,
whether from specific disease, fall, fracture, poor nutrition,or aging itself, are associated with future disability, morbidity, and death (1,2).
In recent years, many studies have examined the roles of
diet, protein, and vitamins in physical performance andphysical activity(3–5). Several studies have associated low
serum albumin concentration with deteriorated muscle
strength and function (6,7). Some other studies have examined the relationship between serum vitamin D level andphysical performance such as muscle mass, muscle strength,
handgrip, walking speed, and functional capacity (8,9).
Cesari et al. (3) examined the relationship between antioxidant vitamin intake (vitamin C, vitamin E, b-carotene, andretinol) and physical performance in elderly people andshowed significant positive correlations between mostantioxidants, especially vitamin C, and higher skeletal muscular strength in this group of people.
There are a number of mechanistic hypotheses about thepotential beneficial effects of antioxidant vitamins(10–12).
Vitamin C, vitamin E, b-carotene, and retinol are importantantioxidants that are not synthesized by humans and, therefore, are mainly supplied via dietary intake. Vitamin C
1

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SAITO ET AL.

Subjects and Methods

Study Subjects
The present cross-sectional study was carried out as part ofa project involving mass health examination of communitydwelling people (“Otasha-kenshin” in Japanese) aged 70 years
and older living in Itabashi-ku, Tokyo. “Otasha-kenshin,”which literally means “health examination for successful
aging,” is a comprehensive health examination program forcommunity-dwelling older adults aimed at preventing geriatric syndromes including falls and fractures, incontinence, mild
cognitive impairment, depression, and undernutrition (16).
The eligible subjects were all female residents, aged between 70 and 84 years, living in the Itabashi area, an urban
part of Itabashi-ku, Tokyo, Japan in October 2006. The population of women belonging to this age range and residingin the Itabashi area was 5937, and they were recruitedby invitation through postal mail. Of them, 1,112 women
applied for admission and 957 women ultimately participatedin this study. The participants who were taking vitamin C

supplements (n = 238) were excluded from the primary
analyses for examination of the relationship between plasma
vitamin C and physical performance because intake of supplements could strongly influence the plasma vitamin C
level. Thus, data from 655 subjects were ultimately used forthe primary analysis. However, data from the 238 supplement users were also used for subanalysis to determine
whether any relationship exists between vitamin C supplementation and physical performance.
All participants were examined at the Tokyo Metropolitan
Institute of Gerontology’s hall. Physical performance, blood
examinations, lifestyle assessments, and anthropometricmeasurements were performed as described below (9).
The present study was approved by the ethics review
committee of the Tokyo Metropolitan Institute of Gerontology. All subjects gave written informed consent.
Anthropometric Measurements
Height and weight of each participant were measured, andbody mass index was defined as weight/height2 (kg/m2). Body
composition measurements (percent body fat) were obtainedby segmental bioelectrical impedance using eight tactile
electrodes according to the manufacturer’s instructions (InBody 3.0; Biospace, Seoul, Korea). Measurements for the triceps surae muscles were taken between the knee and the ankle,
at the level of maximum circumference of the medial and anterior calf of the left leg of each participant at sitting position.
Physical Performance
Physical performance was assessed by muscle strength(handgrip strength), balance capability, and usual and maximal walking speeds, without prior practice before the actual
measurements. These assessments are routinely conducted
for the elderly community as described previously (9). Handgrip strength (kg) was measured once for the dominant handwith the subjects in a standing position using a Smedley’s
Hand Dynamometer (Yagami, Tokyo, Japan). Grip devices
were calibrated with known weights. Subjects held the dynamometer at thigh level and were encouraged to exert thestrongest possible force. Balance capability was measured interms of the length of time standing on one leg, that is, we
asked the subjects to look straight ahead at a dot 1 m in frontof them and to stand on the preferred leg with their eyes open
and hands down alongside the trunk. The time until balance
was lost (or maximum 60 seconds) was recorded. We used
the better of two trials in the analysis. To determine the walking speed, participants were asked to walk on a flat surface attheir “usual and maximum walking speeds.” Two marks
were used to delineate the start and end of a 5-m path. The
start mark was preceded by a 3-m approach to ensure that theparticipants achieved their pace of usual or maximum before
entering the test path. The participants were also instructedto continue walking past the end of the 5-m path for a further
3 m to ensure that their walking pace was maintained

Downloaded from biomedgerontology.oxfordjournals.org by guest on October 11, 2011(ascorbic acid) is a water-soluble antioxidant present in thecytosol and extracellular fluid and can directly react withfree radicals such as superoxide (O2·−) and hydroxyl radicals (·OH) (13,14). Each one of these oxygen-derived intermediates is considered highly reactive because of their
unstable electron configurations, which could attract electrons from other molecules, resulting in another free radicalthat is capable of reacting with yet another molecule. This
chain reaction is thought to contribute to lipid peroxidation,
DNA damage, and protein degradation during oxidative
stress. Oxidative damage is thought to play an importantrole in the age-related decline of functional activity in human skeletal muscle (15). Concentration of plasma vitaminC, which has potent antioxidant activity, is known to increase after exercise (4).
An increase in the amount of blood vitamin C content has
been used as an indicator of increased oxidative reaction (11).
Previous studies have examined the effects of vitamin C supplementation on physical performance and exercise (4,11).
Although findings from some of the previous studies do notsupport any beneficial effect of increased antioxidant intake
on physical performance, other studies have shown improved
recovery from exercise with antioxidant intake and havealso shown a preventive role of antioxidant supplementation
against oxidative damage. These studies were carried out on
athletes after heavy exercise. So far, however, there has been
no study examining the relationship between physical performance and blood levels of vitamin C, which may be a more
direct marker of the antioxidative ability of the human body.
The present study, to the best of our knowledge, is thefirst report that examines the relationship between plasma
vitamin C concentration and physical performance in Japanese community-dwelling elderly women.

3

VITAMIN C AND PHYSICAL PERFORMANCE

throughout the test path. The time taken to complete the 5-m
walk was measured by an investigator and used for analysis.
Walking test at maximum speed was repeated twice, and thefaster speed was recorded for the test.
All physical performance tests were performed between
9 am and 4 pm during the day. We have no data on the reproducibility of the measurements. To reduce interexaminer
variation, each test was conducted by the same staff member specifically trained for this study.

Lifestyle Assessment
Information regarding the participants’ general health (suchas medical history, smoking habits, alcohol drinking habits,
regular exercise habits, vegetable intake, fruit intake and use ofvitamin C supplement) was collected by interview, and historyof medical conditions including hypertension, stroke, heart attack, diabetes mellitus, and hyperlipidemia was self-reported.
Alcohol drinking habits of the subjects were classified as
nondrinker, current drinker, or ex-drinker. Smoking habitsof the subjects were classified using three categories: neversmokers, current smokers, and ex-smokers. The frequency
of vegetable and fruit intake was asked using four categories: almost every day, once every two days, once or twice
per week, and almost never. Subsequently, for analysis, thecategories were summarized as almost every day and others.
Statistical Analysis
Data were summarized as mean and standard deviation or
percentage values. The data of plasma vitamin C concentration was logarithmically transformed to approximate a normal distribution and was summarized as the geometricmean and geometric standard deviation.

Characteristic

Mean (SD)Age (y)Height (cm)Weight (kg)Body mass index (kg/m2)
Triceps surae muscle (cm)
Plasma vitamin C (mg/ml)*
Serum albumin (mg/dL)
Body composition
Percent body fat (%)
Physical performance tests
Handgrip strength (kg)
One leg standing with eyes open (s)Usual walking speed (m/s)
Maximal walking speed (m/s)

75.7 (4.1)
149.1 (5.7)
51.0 (8.3)
22.9 (3.4)
33.1 (2.8)
8.9 (1.5)
4.3 (0.2)
32.2 (7.0)
18.7 (4.4)
35.2 (23.5)
1.2 (0.3)
1.8 (0.4)%

Medical history
Hypertension
StrokeHeart attack
Diabetes mellitus
Hyperlipidemia
Alcohol drinking habit
Current
FormerNever
Smoking habit
Current
FormerNeverRegular exercise habit
Yes
No
Vegetable intake
Everyday
Others†Fruit intake
Everyday
Others†

50.7
6.6
21.2
9.0
34.7
25.3
5.0
69.6
3.7
5.7
90.7
69.2
30.8
84.2
15.8
81.8
18.2

Notes: Data of vitamin C supplement users were excluded.
* The geometric mean and geometric SD.
† Including participants taking vegetables/fruits not everyday or almost never.

The age-adjusted Pearson’s correlation coefficient between
the plasma vitamin C concentration and other factors were
calculated. The least square means and SEs adjusted forpotential confounders were calculated and compared between
categories by analysis of covariance. To examine the relationship between plasma vitamin C concentration and physicalperformance, statistical adjustment was done by analysis ofcovariance for variables (except for other physical performance variables) that were correlated to plasma vitamin C
concentration with p < .20. The same analyses were repeated
for the 238 users of vitamin C supplement. All statistical analyses were performed using the SAS (version 9.0; SAS Institute Inc., NC).
Results
Table 1 summarizes the basic characteristics of the subjects. As shown, the mean age (±standard deviation) of the

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Blood Examinations
Blood samples (nonfasting) were collected from the subjects
between 9 am and 4 pm during the day. There was no difference in mean plasma vitamin C concentration with regard to
the time of collection (data not shown). Venous blood samples
were drawn into Ethylene diamine tetraacetic acid tubes.
Plasma was then obtained by centrifugation at 3,000 rpm
for 15 min at 4°C and subsequently used for biochemical
assays. Plasma was treated with Ethylene diamine tetraacetic
acid to prevent the spontaneous vitamin C degradation. Next,
100 ml of the plasma was dispensed into storage tubes, towhich 450 ml of 3% metaphosphoric acid solution was added,
and the mixture was stored at −80°C until further use. Vitamin
C concentration was determined by an High performance liquid chromatography-electrochemical detection–based method(17). The analysis was carried out centrally in our laboratory.
Serum albumin concentration was measured by the Bromocresol Green method (Special Reference Laboratories Inc.,Tokyo, Japan). The coefficient of variation for serum albuminfound using this method was less than 1% (9).

Table 1. Characteristics of Study Subjects (N = 655)

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SAITO ET AL.

Table 2. Correlation between Plasma Vitamin C Concentration and
Selected Factors (N = 655)
Correlation*
FactorAge
HeightWeightBody mass index
Triceps surae muscle
Serum albumin
Percent body fat
Handgrip strength
One leg standing with eyes open
Usual walking speed
Maximal walking speedrp−0.004
0.04
−0.05
−0.08
0.001
−0.04
−0.12
0.16
0.15
0.14
0.09

.91
.27
.19
.054
.98
.33
.002
<.001
<.001
<.001
.036

Notes: Number of subjects is slightly different for the selected factors becauseof missing values.
* Age-adjusted Pearson’s correlation coefficient between logarithm of vitamin C concentration and each factor.

Discussion
A previous study has shown an association between
higher daily dietary intake of vitamin C and skeletal muscle
strength in elderly people (3). Results described in the present study indicated that plasma vitamin C concentration was
positively related with muscle and physical performance incommunity-dwelling elderly women. To the best of ourknowledge, this is the first study showing a significant

Table 3. Relationship between Plasma Vitamin C Concentration and Physical Performance Adjusted for Potential Confounder
Quartile of plasma vitamin C level
Q1

Q2

Q3

Q4

Physical performance

Mean ± SE

Mean ± SE

Mean ± SE

Mean ± SE

Handgrip strength (kg), N
Age adjusted
Multivariate adjusted*
One leg standing with eyes open† (s), N
Age adjusted
Multivariate adjusted*
Usual walking speed (m/s), N
Age adjusted
Multivariate adjusted*
Maximal walking speed (m/s), N
Age adjusted
Multivariate adjusted*

154
17.70 ± 0.34
17.83 ± 0.34
162
31.44 ± 1.71
33.39 ± 1.74
146
1.13 ± 0.02
1.18 ± 0.02
146
1.70 ± 0.03
1.76 ± 0.03

159
18.75 ± 0.33
18.83 ± 0.32
163
33.98 ± 1.70
34.08 ± 1.67
154
1.19 ± 0.02
1.19 ± 0.02
154
1.76 ± 0.03
1.77 ± 0.03

154
18.75 ± 0.34
18.89 ± 0.33
16437.70 ± 1.70
37.63 ± 1.67
145
1.23 ± 0.02
1.22 ± 0.02
154
1.82 ± 0.03
1.80 ± 0.03

152
19.60 ± 0.34
19.60 ± 0.33
161
37.83 ± 1.71
37.50 ± 1.70
147
1.21 ± 0.02
1.21 ± 0.02
147
1.76 ± 0.03
1.75 ± 0.03

p for trend
.0001
.0004
.003
.049
.008
.23
.15
.94

Notes: Values are least squares mean and SE adjusted for the factors by analysis of covariance. Q1–Q4: first to fourth quartile groups of plasma vitamin C concentration, respectively.
* Adjusted for age, body mass index, percent body fat, hypertension, diabetes mellitus and fruit intake.
† Length of time standing on one leg with eyes open.

Downloaded from biomedgerontology.oxfordjournals.org by guest on October 11, 2011subjects was 75.7 ± 4.1 years. The geometric mean (geometric standard deviation) of plasma vitamin C concentration was 8.9 (1.5) mg/mL. The prevalence of women eating
vegetables everyday was 84.2% and those eating fruits
everyday was 81.8%.
The age-adjusted geometric mean of plasma vitamin C
concentration was significantly lower in subjects who had a
medical history of hypertension (8.53 vs 9.22, p = .0015)
and diabetes mellitus (7.59 vs 9.00, p = .002) as comparedwith those who did not. A history of stroke, heart attack, or
hyperlipidemia was not associated with plasma vitamin C
concentration. Subjects who took fruits every day had a significantly higher concentration of vitamin C than those who
did not (9.14 vs 7.78, p < .0001). Vegetable intake, alcohol
drinking habit and smoking habit were not related to plasma
vitamin C concentration (not shown in table).
Table 2 shows the age-adjusted correlations between theplasma vitamin C concentration and selected factors. As

shown, the plasma vitamin C concentration was positively
but modestly correlated with handgrip strength, length oftime standing on one leg with eyes open, as well as usual
walking speed and maximal walking speed, and modestly
inversely correlated with body mass index and percent bodyfat of the subjects.
Table 3 shows the relationship between plasma vitamin C
concentration and each physical performance after adjustingfor confounding factors. Results obtained after the adjustment for potential confounders confirmed that the plasma
vitamin C concentration was correlated with the handgrip
strength independently from the other factors (eg, p fortrend = .0004 after adjusting for age, body mass index,
percent body fat, hypertension, diabetes mellitus, and fruit
intake; Table 3). There was also a significant relationshipbetween the plasma vitamin C level and the subject’s lengthof time standing on one leg with eyes open after adjustments
for age, body mass index, percent body fat, hypertension,
diabetes mellitus, and fruit intake (Table 3; p for trend =.049). We did not observe any significant association between the plasma vitamin C level and the usual or the maximal walking speed of the subjects.
A subanalysis using data from the 238 vitamin C supplement users showed almost null relationship between handgrip strength and plasma vitamin C concentration (data notshown).

VITAMIN C AND PHYSICAL PERFORMANCE

habitual exercise reduces systemic inflammation and oxidative stress as the production of endogenous antioxidants areenhanced, acute exercise increases the generation of oxygen-free radicals and lipid peroxidation (4,25). Strenuous
physical performance can increase oxygen consumption by
10- to 15-folds over the resting state to meet the energy
demands and results in muscle injury (26). Prolonged submaximal exercise was shown to increase the amount ofboth whole-body and skeletal muscle lipid peroxidation
by-products; in the case of the former, the increase wasindicated by greater exhalation of pentane but not of ethane(4,27,28). Supplementation with vitamin C was shown todecrease the exercise-induced increase in the rate of lipid
peroxidation (27,28). Several studies suggested that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging(15). Consistent with this idea, several studies showed significantly lower plasma vitamin C level in the elderly population than in the younger adult population (29–31). Because
the plasma vitamin C levels in these apparently healthyelderly persons rose markedly after an oral dose of vitamin C,
their initially low plasma levels can be attributed to the low
intake rather than to an age-related physiological defect.
In fact, the relationship between handgrip strength andplasma vitamin C concentration was significantly different
between supplement users and nonusers, that is, an almost
null relationship in the former and a positive relationship in
the latter (data not shown). This finding suggested that vitamin C supplementation did not have any beneficial effect on
the physical performance and muscle strength despite the increased plasma level of vitamin C. A number of studies
reported that vitamin C supplement users had significantly
higher blood vitamin C concentration than non-users (29, 32,
33). Several studies have examined the effects of exercise onchanges in the serum vitamin C concentration (34–36).
Some other experimental studies have shown that vitamin C
supplementation can reduce symptoms or indicators of exercise-induced oxidative stress (37–40). However, the results
regarding vitamin C supplementation are equivocal, andmost well-controlled intervention studies report no beneficial
effect of vitamin C supplementation on either endurance orstrength performance (41,42). Likewise, vitamin C restriction studies showed that a marginal vitamin C deficiency did
not affect the physical performance (43). Although evidence
from a number of studies show that vitamin C is a powerfulantioxidant in biological systems in vitro, its antioxidant rolein humans has not been supported by currently available
clinical studies.
Vitamin C is especially plentiful in fresh fruits and vegetables. Plasma vitamin C concentration may be merely amarker for intake of other nutrients that are abundant infruits and vegetables. However, the statistical adjustment
for fruit intake did not attenuate the relationship between
plasma vitamin C and physical performance (Table 3),
suggesting that vitamin C did have some beneficial effects

Downloaded from biomedgerontology.oxfordjournals.org by guest on October 11, 2011

correlation between plasma vitamin C concentration andhandgrip strength and ability to stand on one leg with eyes
open. We, however, were unable to find any relationshipbetween skeletal muscle mass and plasma vitamin C concentration. Handgrip strength has been found to correlate wellwith the strength of other muscle groups and is thus a good
indicator of overall strength (18). Consistent with this idea,
handgrip strength was found to be a strong and consistentpredictor of all-cause mortality and morbidity of Activities
of Daily Living in middle-aged people (19). The handgrip
test is considered an easy and inexpensive screening tool
to identify elderly people at risk of disability. Handgrip
strength, an indicator of overall muscle strength, is thought
to predict mortality through mechanisms other than underlying disease that could cause muscle impairment (18,19). The
one leg standing test is one of the balance tests (20). The test
is a clinical tool to assess postural steadiness in a static position by quantitative measurement. Many studies have shownthat the decreased one leg standing time is associated withdeclines in Activities of Daily Living and increases in othermorbidities including osteoporosis and fall (20).
Our findings suggest that vitamin C may play an important role in maintaining physical performance and thereby
may help to improve healthy life expectancy in the elderly.
However, the usual and maximal walking speeds did not
relate to plasma vitamin C concentration. Walking speed
test may be an efficient tool in screening older persons withhigher risk of mortality and may easily identify highrisk groups in the community (21). Walking is a rhythmic,
dynamic, and aerobic activity of the large skeletal muscles
that confers multifarious benefits with minimal adverse
effects. Muscles of the legs, limbs, and lower trunk arestrengthened, and the flexibility of their joints are preserved(22). One of the reasons why walking speed was not related
to vitamin C concentration may be because walking
requires coordinated movements of arms, legs, and many
parts of the body rather than a simple muscle and balance
function. Previous reports showed that walking balance
function did not correlate with standing balance function(23). Although we did not find any clear association
between walking and plasma vitamin C concentration inthis study, vitamin C may still have effects on relatively
simple strength and balance functions.
One of the possible explanations for the observed relationship between vitamin C and physical performance, especially handgrip strength and the ability to stand on one
leg with eyes open, may be the potential protective effectsof the antioxidant vitamins against muscle damage (4,11).
Vitamin C is a six-carbon lactone that is synthesized fromglucose in the liver of most mammalian species, but not inhumans (12). Vitamin C is an antioxidant because, by donating its electrons, it prevents other compounds from being
oxidized (12). Thus, vitamin C readily scavenges reactive
oxygen and nitrogen species, thereby effectively protects
other substrates from oxidative damage (10,24). Although

5

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SAITO ET AL.

Funding
Ministry of Education, Culture, Sports, Science and Technology, Japan(grant-in-aid 16390187 for Scientific Research [B] and grant-in-aid[21790591] for Young Scientists [B]).
Acknowledgment
We offer special thanks to Dr. Satoshi Sasaki for his suggestions anduseful comments to this paper; we are indebted to Dr. Shoji Sinkai, Dr.
Yoshinori Fujiwara, Mr Toshiro Tango, Mrs Sachiko Imai, Mrs Turuko
Maekawa, Miss Erika Hosoi and the staff of the Otasha-kenshin who participated in this study.
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study. Am J Clin Nutr. 2004;79:289–294.
4. Evans WJ. Vitamin E, vitamin C, and exercise. Am J Clin Nutr.
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independently of other nutrients. A number of biochemical, clinical, and observational epidemiologic studies haveindicated that diets rich in fruits, vegetables, and vitamin C
may be of benefit for the prevention of chronic diseases
such as cardiovascular disease and cancer (44,45). Several
cohort studies have examined associations between plasma
vitamin C concentration and mortality from stroke or coronary heart disease (30,46,47). The effects of vitamin C
supplementation are, however, still unclear. A pooled study
suggested reduced incidences of coronary heart disease
events with higher intake of vitamin C supplement (48),while another study showed that a high intake of vitamin C
supplement is associated with an increased risk of mortality due to cardiovascular diseases in postmenopausal
women with diabetes (49). A randomized placebo controlled 5-year trial, however, did not show any significant
reduction in the mortality from, or incidence of, any typeof vascular disease or cancer (50). These studies, in fact,
have failed to demonstrate any benefit from such supplementation.
There are a number of potential weaknesses in our study
that should be mentioned here. The subjects used in thisstudy were not selected randomly from the study population, and they may be relatively healthy elderly women who
were able to come to the health examination hall from theirhomes. A previous study assessed the correlation of antioxidants with physical performance and muscular strength(3) and demonstrated that a higher daily intake of vitamin C
and carotene associated with skeletal muscle strength.
However, we have no data regarding the presence of otherdietary antioxidants in blood such as vitamin E, retinol, andcarotene. In our questionnaire, participants were asked torespond “Yes” or “No” to whether they took supplements,
and not about the frequency and quantity of intake of the
supplements. Thus, we were unable to examine the reason
why plasma vitamin C was not related to the handgrip
strength in the supplement users by considering the dose ofvitamin C they took.
This study was a cross-sectional study and, therefore,
does not provide cause/effect relationships, although wedemonstrated a significant correlation between physicalperformance and concentration of plasma vitamin C. Therefore, longitudinal follow-up studies and controlled clinical
trials are necessary to confirm the role of plasma vitamin C
and physical performance of the elderly women. These limitations should be considered in future studies.
In conclusion, we found a strong correlation of a higher
plasma vitamin C concentration with handgrip strength andone leg standing time in community-dwelling elderly
women. Although the elderly are prone to vitamin C deficiency, and they appear to have a higher dietary requirement for vitamin C, the beneficial effects of vitamin C
supplementation to maintain physical performance in elderly people are equivocal and thus, need further in-depth
studies.

VITAMIN C AND PHYSICAL PERFORMANCE

36. Duthie GG, Robertson JD, Maughan RJ, Morrice PC. Blood antioxidant status and erythrocyte lipid peroxidation following distance running. Arch Biochem Biophys. 1990;282:78–83.
37. Maxwell SR, Jakeman P, Thomason H, Leguen C, Thorpe GH.
Changes in plasma antioxidant status during eccentric exercise and theeffect of vitamin supplementation. Free Radic Res Commun. 1993;19:
191–202.
38. Jakeman P, Maxwell S. Effect of antioxidant vitamin supplementation
on muscle function after eccentric exercise. Eur J Appl Physiol Occup
Physiol. 1993;67:426–430.
39. Kaminski M, Boal R. An effect of ascorbic acid on delayed-onset
muscle soreness. Pain. 1992;50:317–321.
40. Bryer SC, Goldfarb AH. Effect of high dose vitamin C supplementation on muscle soreness, damage, function, and oxidative stressto eccentric exercise. Int J Sport Nutr Exerc Metab. 2006;16:
270–280.
41. Thompson D, Williams C, Kingsley M, et al. Muscle soreness anddamage parameters after prolonged intermittent shuttle-runningfollowing acute vitamin C supplementation. Int J Sports Med. 2001;
22:68–75.
42. Gey GO, Cooper KH, Bottenberg RA. Effect of ascorbic acid on
endurance performance and athletic injury. Jama. 1970;211:105.
43. van der Beek EJ, van Dokkum W, Schrijver J, Wesstra A, Kistemaker C,
Hermus RJ. Controlled vitamin C restriction and physical performance in volunteers. J Am Coll Nutr. 1990;9:332–339.
44. Ness AR, Powles JW. Fruit and vegetables, and cardiovascular disease:a review. Int J Epidemiol. 1997;26:1–13.
45. Genkinger JM, Platz EA, Hoffman SC, Comstock GW, Helzlsouer KJ.
Fruit, vegetable, and antioxidant intake and all-cause, cancer, and cardiovascular disease mortality in a community-dwelling populationin Washington County, Maryland. Am J Epidemiol. 2004;160:1223–
1233.
46. Yokoyama T, Date C, Kokubo Y, Yoshiike N, Matsumura Y, Tanaka H.
Serum vitamin C concentration was inversely associated with subsequent 20-year incidence of stroke in a Japanese rural community. The
Shibata study. Stroke. 2000;31:2287–2294.
47. Gale CR, Martyn CN, Winter PD, Cooper C. Vitamin C and risk ofdeath from stroke and coronary heart disease in cohort of elderly people. BMJ. 1995;310:1563–1566.
48. Knekt P, Ritz J, Pereira MA, et al. Antioxidant vitamins and coronary
heart disease risk: a pooled analysis of 9 cohorts. Am J Clin Nutr.
2004;80:1508–1520.
49. Lee DH, Folsom AR, Harnack L, Halliwell B, Jacobs DR Jr. Does
supplemental vitamin C increase cardiovascular disease risk in womenwith diabetes? Am J Clin Nutr. 2004;80:1194–1200.
50. Heart Protection Study Collaborative Group. MRC/BHF Heart Protection
Study of antioxidant vitamin supplementation in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet. 2002;360:23–33.

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19. Rantanen T, Volpato S, Ferrucci L, Heikkinen E, Fried LP,
Guralnik JM. Handgrip strength and cause-specific and total mortality
in older disabled women: exploring the mechanism. J Am Geriatr Soc.
2003;51:636–641.
20. Michikawa T, Nishiwaki Y, Takebayashi T, Toyama Y. One-leg standing
test for elderly populations. J Orthop Sci. 2009;14:675–685.
21. Rolland Y, Lauwers-Cances V, Cesari M, Vellas B, Pahor M,
Grandjean H. Physical performance measures as predictors of mortality in a cohort of community-dwelling older French women. Eur J
Epidemiol. 2006;21:113–122.
22. Morris JN, Hardman AE. Walking to health. Sports Med. 1997;23:
306–332.
23. Shimada H, Obuchi S, Kamide N, Shiba Y, Okamoto M, Kakurai S.
Relationship with dynamic balance function during standing andwalking. Am J Phys Med Rehabil. 2003;82:511–516.
24. Halliwell B. Vitamin C: antioxidant or pro-oxidant in vivo? Free Radic
Res. 1996;25:439–454.
25. Petersen AM, Pedersen BK. The anti-inflammatory effect of exercise.
J Appl Physiol. 2005;98:1154–1162.
26. Clarkson PM. Antioxidants and physical performance. Crit Rev FoodSci Nutr. 1995;35:131–141.
27. Gee DL, Tappel AL. The effect of exhaustive exercise on expired
pentane as a measure of in vivo lipid peroxidation in the rat. Life Sci.
1981;28:2425–2429.
28. Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals andtissue damage produced by exercise. Biochem Biophys Res Commun.
1982;107:1198–1205.
29. Blanchard J, Conrad KA, Mead RA, Garry PJ. Vitamin C disposition
in young and elderly men. Am J Clin Nutr. 1990;51:837–845.
30. Fletcher AE, Breeze E, Shetty PS. Antioxidant vitamins and mortality
in older persons: findings from the nutrition add-on study to the Medical Research Council Trial of Assessment and Management of Older
People in the Community. Am J Clin Nutr. 2003;78:999–1010.
31. Jacob RA, Otradovec CL, Russell RM, et al. Vitamin C status andnutrient interactions in a healthy elderly population. Am J Clin Nutr.
1988;48:1436–1442.
32. Sinha R, Block G, Taylor PR. Determinants of plasma ascorbic acid ina healthy male population. Cancer Epidemiol Biomarkers Prev.
1992;1:297–302.
33. Newton HM, Schorah CJ, Habibzadeh N, Morgan DB, Hullin RP. The
cause and correction of low blood vitamin C concentrations in the elderly. Am J Clin Nutr. 1985;42:656–659.
34. Gleeson M, Robertson JD, Maughan RJ. Influence of exercise on
ascorbic acid status in man. Clin Sci (Lond). 1987;73:501–505.
35. Camus G, Felekidis A, Pincemail J, et al. Blood levels of reduced/oxidized glutathione and plasma concentration of ascorbic acid during
eccentric and concentric exercises of similar energy cost. Arch Int
Physiol Biochim Biophys. 1994;102:67–70.

7Japanese Clinical Medicine

O r i g i n al R e s e a r c hOpen Access
Full open access to this andthousands of other papers at
http://www.la-press.com.

Improvement of Quality of Life (QOL) in Osteoporotic Patientsby Elcatonin Treatment: A Trial Taking the Participants’
Preference into AccountK. Yoh1, T. Uzawa2, T. Orito3 and K. Tanaka4
1

Orthopedic Department, Hyogo Medical College Sasayama Medical Center, Sasayama, Hyogo, Japan. 2Asahi Kasei
Pharma Co. Ltd. Pharmaceuticals Sales Division, 3Orito Orthopedic Surgery Clinic, 4Department of Food and Nutrition,
Kyoto Women’s University. Corresponding author email: tanakak@kyoto-wu.ac.jp

Abstract: Osteoporosis is associated with compromised quality of life (QOL), to which pain has the most important contribution.
Elcatonin, a derivative of calcitonin, is widely used in the treatment of osteoporosis in two ways. One is as the inhibitor of osteoclastic
bone resorption. The other is for osteoporosis-related pain based on the unique analgesic effects of elcatonin. Since pain is subjective
in nature, and QOL is the only clinical outcome representing the patients’ subjective perception of health status, pain associated withosteoporosis would be best evaluated based on QOL assessment. Evidence based medicine gives the highest remarks to the doubleblinded, randomized controlled trial, which, however, cannot be free from methodological problems on some occasions. For example,
it is practically impossible to remain blinded in the trial of a potent analgesia, which in turn causes biases. Thus, the significance of­taking the patients’ preference into account is increasingly acknowledged. In this study, 45 osteoporotic patients were given brochuresdescribing the pros and cons on the three treatment choices; calcium and alfacalcidol, additional use of elcatonin, and additional useof bisphosphonate. Those who favored elcatonin were older, had more vertebral fractures, and lower QOL scores. QOL was evaluatedbefore and three months after the treatment using SF-8; the most widely used generic questionnaire, and RDQ; a lumbago-specific­measure. Elcatonin treatment improved physical function, general health, and vitality of SF-8, and RDQ score. Although this is apreliminary study, our results suggest that patients with vertebral fracture(s) have impaired QOL and more likely to favor elcatonintreatment expecting analgesia.
Keywords: osteoporosis, elcatonin, patient preference trial, quality of life

Japanese Clinical Medicine 2012:2 9–14
doi: 10.4137/JCM.S8291
This article is available from http://www.la-press.com.
© the author(s), publisher and licensee Libertas Academica Ltd.
This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited.

Japanese Clinical Medicine 2012:2

9

Yoh et al

Introduction
Calcitonin is a hormone secreted from the para­follicular cells of the thyroid gland. It is involved incalcium homeostasis by inhibiting osteoclast-­mediatedbone resorption and decreasing serum calcium levels.
It is clinically used in various clinical conditions. For
example, it is prescribed to osteoporotic patients,1–3
especially those with enhanced bone resorption.
Additionally, it is a therapeutic drug in the treatmentof malignancy associated hypercalcemia.4
Elcatonin is a derivative of calcitonin5 with potent
analgesic action.6 Therefore, it is clinically administered to osteoporotic patients with pain.7,8 Japaneseguidelines for the prevention and treatment of
osteoporosis have given grade A, the highest possible
mark, to the analgesic action of elcatonin.9
Of the various measurement scales clinicallyavailable, QOL (Quality of Life) is the only ­clinicaloutcome representing the patients’ subjective perception of health.10 Pain is subjective in its nature,and could be evaluated only by subjective index:
QOL. It ­follows that the analgesic effect of elcatoninwould be best evaluated with QOL as the outcome.
A ­theoretical problem arises, however, if one attempts
to evaluate the analgesic effects of a certain drug
based on QOL scores.
Recently, the concept of evidence based ­medicine(EBM) has been advocated in which, a hierarchy of­evidence exists, with double-blinded randomized
controlled trial (hereafter in this paper, abbreviated as
DB-RCT) given the highest remark. DB-RCT is considered to be least affected by various biases. In some
types of trials, however, double-blindness or randomization cannot be strictly guaranteed with DB-RCT,and even DB-RCT cannot be free from such biases.
As will be detailed in the “Discussion, some formsof bias” to skew the results is known to take place instudies in which blindness or randomization is disrupted.11,12 Thus in this paper, we have attempted to
study the effects of elcatonin treatment on the QOL of­osteoporotic patients taking the patients’ ­preference
into account.

Subjects and Methods
Subjects

Forty-five (42 females, 3 males) osteoporotic patientswith back pain or lumbago were encouraged to participate
in the study. The diagnosis of osteoporosis was made10

based on the diagnostic criteria for primary osteoporosis
in Japan.13 The purpose of the study was explained, andwritten ­consent was obtained. Ethical approval was
obtained for the use of humans in this research
The exclusion criteria were as follows. Patients
receiving bone-active drugs were excluded unless they
were drug free for 48 weeks for bisphosphonates andfor 8 weeks for other drugs. Patients under ­sustained
analgesic treatment were also excluded unless theanalgesics were halted for at least 2 weeks. Patientswere also excluded when the attending ­physician
made a judgment that the patient was not eligible forentry based on the clinical condition.
Vertebral fractures were diagnosed based on aplain roentgenogram. The diagnosis of compression
fracture was made by one of the authors (KY) before
obtaining the information on QOL.

Intervention Protocol
The study design was an open-labeled one based on thepatients’ preference, further details of which will be
described below. There were three ­treatment groups;
group (1): calcium lactate 1 g or ­calcium aspartate
and 1 µg of alacalcidol daily, group (2): once-weekly
injection of elcatonin in addition to the regimen ingroup (1), and group (3): alendronate 5 mg daily or
risedronate 2.5 mg daily in addition to the regimen ingroup (1). Patients were given brochures describing
the pros and cons of each treatment, and asked to
select the treatment of their preference. Six, twentyseven, and twelve patients chose group (1), group (2),and group (3) treatment, respectively.

QOL (Quality of Life) Evaluation
QOL was evaluated with two questionnaires; SF-8
and RDQ (Roland Morris Disability Questionnaire).
SF-36 is a generic (non-disease specific) QOL questionnaire, and one of the most widely used worldwide.
SF-8 is an abridged edition of SF-36. According to
the authorized instruction, the data were transformed
to the deviation value adjusted by Japanese national
norms.14,15 Eight subscales were obtained: PF (physicalfunction), RP (role physical), BP (bodily pain), GH(general health), VT (vitality), SF (social function),
RE (role emotional), and MH (mental health). Thesescores are further summarized into two summaryscores: PCS (physical component summary) andMCS (mental component summary). These subscales
Japanese Clinical Medicine 2012:2

QOL improvement by elcatonin treatment in osteoporotic patientsand summary scores are interpreted as follows: 50
corresponding to the national norms, and 40 indicating one standard deviation lower than the norm.
RDQ is a questionnaire specifically targeted tolumbago. It is composed of 24 questions. The subjects
are asked to give “yes” or “no” to each question.
Total number of questions given “yes” is calculated.
Thus higher number is associated with more severe
lumbago.16,17
QOL evaluation was made before and three months
after initiating the intervention.

Statistical Analyses
Data were analyzed using SPSS 17.0J for ­Windows.
Comparison of three independent groups wasdone with one-way analysis of variance (ANOVA)­followed by Tukey’s test as the post-test. Contingent
tables were analyzed by the chi-square test. Statisticalsignificance was judged based on P , 0.05.

Results
Table 1 shows patients characteristics. Patients ingroup (2) were significantly older than those in othergroups, but there was no significant difference in theirheight or weight. All patients in group (1) and mostin group (3) had no ­vertebral fractures. In contrast,
most patients in group (2) had vertebral fracture(s).
These differences were statistically significant basedon chi-square test.
Baseline QOL scores are shown in Table 2. There
was a significant difference between the three groups
in four of the eight subscales of SF-8; RP, BP, GH,
and VT and total scores of RDQ. Patients in group (2)had worse QOL scores. Although not statisticallysignificant, those in group (2) had worse QOL scoresin practically all scales.
In Table 3 is shown the post-intervention changes
in the QOL scores. Elcatonin treatment markedly
improved in several subscales of QOL score.

Discussion

Osteoporosis is defined as a skeletal disordercharacterized by compromised bone strength­predisposing a person to an increased risk of fracture including vertebral, hip and wrist fractures.18
Hip fracture is associated with high mortality andmarkedly impaired activity of daily living (ADL). It
is not only a tragedy for the elderly individual, butalso a great burden to society.19 Vertebral fracture is
the most prevalent osteoporosis-related fracture.20
Recent ­studies have clarified that it is a fracture ofgreat clinical importance associated with increased
mortality, ­co-morbidity, and compromised ADL.21–23­Nevertheless, vertebral fracture has not received
much attention until recently. One of the reasons forthe ignorance of the importance of vertebral fractureis that approximately two-thirds of patients arewithout overt clinical signs. Its lack, however, does
not necessarily mean that the patients are symptom
free or subjectively well. Since QOL is the only index
representing the patients’ subjective status, QOL
would be quite suitable in the evaluation of patientswith vertebral fracture.
Recently, many questionnaires for QOL evaluation have become available. They are classified into
two major categories; generic and disease-targeted.
Generic ones, by their definition, only consist ofquestions related to the subjects’ general status, andTable 1. Baseline characteristics of participants in three treatment groups.
Group (1)Group (2)Group (3)

P

N
6
Age
72.3 ± 9.8
Height144.2 ± 6.5
Weight49.2 ± 6.0
Vertebral fracture number0
5 (+2.7)
1
0 (-1.4)≥2
0 (-1.6)

27
77.6 ± 7.0*
147.7 ± 7.9
50.7 ± 7.3

1269.4 ± 9.8*
149.2 ± 6.6
47.3 ± 6.8

0.017
0.536
0.458

5 (-4.2)
10 (+2.8)
11 (+1.9)

8 (+2.6)
0 (-2.1)
2 (-0.9)

(2–3)

0.001

Notes: The treatment groups were as follows; group (1): calcium and alfacalcidol, group (2) elcatonin, group (3): bisphosphonate. Age, height, and weight
were compared by ANOVA followed by Tukey’s test. Vertebral fracture number in each group was analyzed by chi-square test. Figures in the parentheses
represent the adjusted residual; those above +1.96 denotes that more subjects were distributed in the cell than expected, and those below -1.96 lesssubjects than expected.

Japanese Clinical Medicine 2012:2

11

Yoh et al
Table 2. QOL scores at entry.

PF
RP
BP
GH
VT
SF
RE
MH
PCS
MCS
RDQ

Group (1)Group (2)Group (3)

Total

P

41.4 ± 13.9
42.0 ± 12.4
47.2 ± 7.7
47.8 ± 7.7
50.7 ± 9.9
48.5 ± 12.8
43.1 ± 15.0
49.8 ± 8.5
41.9 ± 11.3
49.6 ± 10.1
6.3 ± 5.7

37.4 ± 8.2
36.4 ± 10.1
36.8 ± 7.5
40.4 ± 7.9
42.9 ± 7.3
41.1 ± 11.6
38.0 ± 14.0
43.9 ± 9.6
34.9 ± 7.6
44.0 ± 12.4
14.0 ± 5.8

43.6 ± 3.9
45.4 ± 6.1
42.3 ± 6.1
47.9 ± 5.9
48.1 ± 5.3
48.1 ± 5.6
48.7 ± 6.9
50.9 ± 7.5
41.2 ± 5.3
51.8 ± 7.0
9.5 ± 4.4

39.6 ± 8.8
39.6 ± 10.2
39.8 ± 8.1
43.5 ± 8.1
45.4 ± 7.9
44.0 ± 11.0
41.5 ± 13.3
46.6 ± 9.4
37.7 ± 8.3
46.9 ± 11.3
11.7 ± 6.1

0.123
0.037
0.003
0.009
0.023
0.107
0.077
0.063
0.032
0.127
0.003

(2–3)
(1–2)
(2–3)
(1–2)

(1–2)

Notes: The treatment groups were as follows; group (1): calcium and alfacalcidol, group (2) elcatonin, group (3): bisphosphonate. Age, height, andweight were compared by ANOVA. The results from post-test by Tukey’s test are shown in the parentheses. For example, (2–3) indicates the statisticallysignificant difference between groups (2) and (3).

do not include questions related to the features which
are specific to a certain disease. Therefore, they areapplicable to such studies as comparing the impact
of various ­diseases on QOL, or even to the evaluation of healthy subjects. In contrast, disease-targeted
ones include items specific to a certain disease. They
can be more sensitive than the generic ones in detecting the QOL impairment closely related to a certain
disease state, but are not applicable to the evaluationof patients with other diseases. The most widely usedgeneric QOL questionnaire is SF-36 and its abridged
form, SF-8.14
Roland-Morris Disability Questionnaire (RDQ)
is rather different from the above-mentioned

questionnaires, and unique in that it is specific tolumbago.16
We have recently shown that patients with ­vertebral
fracture(s) had compromised quality of life (QOL)
and elcatonin treatment remarkably improved it.7 In
the course of the study, we have noticed a ­challenging
problem. QOL evaluation could be done by giving
the questionnaire to the subjects and asking themto mail it back. In this study, however, the patients’
QOL was evaluated by the interviewers consideringthat most subjects are elderly osteoporotic patients.
The attending physician only obtained the consent toparticipate in the study without participating in theinterview. The interviewers were blinded about thetreatment regimen for each patient to minimize bias.

Table 3. Incremental QOL scores after the intervention.

PF
RP
BP
GH
VT
SF
RE
MH
PCS
MCS
RDQ

Group (1)Group (2)Group (3)P  -9.3 ± 17.4
3.4 ± 5.1  -1.6 ± 7.2  -3.8 ± 5.3  -6.6 ± 6.4  -6.9 ± 6.7-10.0 ± 14.8  -3.8 ± 5.5  -1.7 ± 5.1  -7.8 ± 10.4
0.4 ± 2.3

5.3 ± 10.7
4.8 ± 11.9
9.6 ± 11.2
7.4 ± 8.9
6.5 ± 8.4
3.1 ± 12.3
6.6 ± 15.8
5.7 ± 11.3
6.6 ± 10.7
4.4 ± 14.0
-5.6 ± 5.7

2.8 ± 6.9
3.0 ± 7.6
7.2 ± 7.5
1.4 ± 8.1
2.1 ± 3.7
-4.9 ± 10.4
3.0 ± 6.9
2.6 ± 10.9
4.0 ± 7.5
-0.4 ± 10.1
-2.7 ± 1.8

0.043
0.912
0.098
0.021
0.004
0.102
0.079
0.197
0.221
0.161
0.042

(1–2)

(1–2)
(1–2)

(1–2)

Notes: The treatment groups were as follows; group (1): calcium and alfacalcidol, group (2) elcatonin, group (3): bisphosphonate. Age, height, andweight were compared by ANOVA. The results from post-test by Tukey’s test are shown in the parentheses. For example, (1–2) indicates the statisticallysignificant difference between groups (1) and (2).

12Japanese Clinical Medicine 2012:2

QOL improvement by elcatonin treatment in osteoporotic patients­ evertheless, the blindness could not be ­maintained,Nsince elcatonin treatment markedly relieved pain insome patients, whereas such marked improvement
occurred in none of the subjects in the control groups.
Recently, such phenomena have been reported
to be a challenging problem to DB-RCT, and theimportance of patients’ preference has been recognized.11,24,25 Clinical studies are performed based on
the assumption that subjects both in ­intervention andcontrol arms are randomly sampled from a single
population. In RCT, patients are conceptualized
as relatively passive recipients of intervention. In
reality however patients are far from passive, andin fact are quite active participants in the research.
Therefore, patients often have preferences for acertain intervention, and will prefer one over the others where they are given the opportunity to choose.
In RCT, the possible interference is minimized byblinding. In some cases, however, participants know
which intervention they have received. Then it is possible that patients not allocated to the intervention of
their choice have lower compliance to the therapy,
which will impair the validity of the study. Several
theoretical frameworks have been developed to takethe patients’ preference into account.24
In this paper, we have attempted to study the analgesic effects of elcatonin considering the participants’
preference. When the patients were given brochuresdescribing the benefit and possible side effects ofeach treatment, and asked which treatment regimen
they would like to receive, those who preferred elcatonin were significantly older and more likely to havevertebral fracture(s). Those in the calcitonin group
had lower SF-8 scores and higher RDQ scores, indicating more compromised QOL. Thus subjects withvertebral fracture(s) have impaired QOL, and are
more likely to prefer elcatonin treatment expecting
its analgesic effects.
Elcatonin treatment resulted in more pronounced
improvement in the subjects’ QOL scores, althoughstatistically significant in only some of the scores­probably due to the limited number of patients
studied.
This is a preliminary study to identify the analgesic effects of elcatonin taking the patients’ preference
into account. Despite the limitations described above,
patients with vertebral fracture(s) have compromised

Japanese Clinical Medicine 2012:2

QOL, and are more likely to prefer ­elcatonin ­treatment,
and more likely to be benefited by its ­treatment due toits potent analgesic effects.

Disclosures

Author(s) have provided signed confirmations to
the publisher of their compliance with all applicablelegal and ethical obligations in respect to ­declarationof conflicts of interest, funding, ­authorship and­contributorship, and compliance with ethical­requirements in respect to treatment of human andanimal test subjects. If this article contains ­identifiable
human subject(s) author(s) were required to supply
signed patient ­consent prior to publication. Author(s)have confirmed that the published article is uniqueand not under consideration nor published by anyother ­publication and that they have consent to reproduce any copyrighted material. The peer reviewers
declared no conflicts of interest.

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18. NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis,
and Therapy. NIH consensus. Osteoporosis prevention, diagnosis, and­therapy. JAMA. 2001;285:785–95.
19. Burge R, Dawson-Hughes B, Solomon DH, Wong JB, King A, Tosteson A.
Incidence and economic burden of osteoporosis-related fractures in the
United States, 2005–2025. J Bone Miner Res. 2007;22:465–75.

20. Chen P, Krege JH, Adachi JD, et al; CaMOS Research Group. Vertebral­fracture status and the World Health Organization risk factors for predicting
osteoporotic fracture risk. J Bone Miner Res. 2009;24:495–502.
21. Cooper C, Atkinson EJ, Jacobsen SJ, O’Fallon WM, Melton LJ 3rd.
­Population-based study of survival after osteoporotic fractures. Am J
­Epidemiol. 1993;137:1001–5.
22. Ioannidis G, Papaioannou A, Hopman WM, et al. Relation between ­fracturesand mortality: results from the Canadian Multicentre Osteoporosis Study.
CMAJ. 2009;181:265–71.
23. Cauley JA, Thompson DE, Ensrud KC, Scott JC, Black D. Risk of mortality
following clinical fractures. Osteoporos Int. 2000;11:556–61.
24. King M, Nazareth I, Lampe F, et al. Conceptual framework and systematic review of the effects of participants’ and professionals’ preference in­randomized controlled trials. Health Technol Assess. 2005;9(35).
25. Howard L, Thornicroft G. Patient preference randomised controlled trials inmental health research. Br J Psychiatry. 2006;188:303–4.

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14

Japanese Clinical Medicine 2012:2

36

第22回婦人科骨粗霧症研究会●ワークショップ皿一1

伽艶。囎樋s

妊娠初期の骨密度とライフスタイル，
栄養摂取状態についての検討
一SKY

Study

（Saitarna，

ヨ目Pregnant

CohortStudy）

第1報一

渡辺優弧4）
竹島和美2）
野村可之5）
茶木修6）

岡野登志夫3）

平原史樹2）
1『Yokohaエna

子雄雄也弘

尚幸敦秀一

合田麻由2）
時長亜弥2）
菊地律子2）

川木崎原西
津鈴吉榊上

善方裕美1・2）

Kobe，

日照時間（UVケアの有無を確認）のほか，骨代的

妊娠・授乳期の骨代謝動態は，胎盤・母乳を介して，カルシウム，ビタミンD，Kなどの栄養

謝関連マーカーを測定した。研究プロトコール
を図1に示す。

なお，本研究は横浜市立大学倫理委員会の認

素を児へ大量に供給するために大きく変化する1，2）。近年，世界各国においてビタミンD不足可のもとに行われた。

者が高頻度に存在することが問題となっており3～6），小児くる病や，新生児頭蓋ろうの発生率増加は，妊婦・授乳婦のビタミンD，カルシウム不足と関連する可能性が高い7）。現在われわれは，

3

結

果

全対象の背景は，年齢32±3．7歳，身長159．2±4．9cm，

BMI（body

massindex）20．3±2．3で，ライフスタイル，栄養摂取状態と，減量，血清QUSによる骨密度の指標は，マーカーの推移について妊婦対象のコホート研

BUA

115±15．5，

究を行っている。今回は，妊娠初期の研究結果

SOS

1539±23．70m／sec，について報告する。

った。妊娠初期の踵骨骨密度は平均では良好なSOS値を示したが，T－score2

方

stiffness

92±14．2，1553±31．30m／sec，標準化
T－score100±15．5％であ80％未満の低骨密度妊婦

が6名（5％）存在した。

法

対象は2010年11，月から2011年2，月に当院をライフスタイル調査の結果は，運動回数7±受診した，妊娠5週から12週で，本研究内容に同意の得られた妊婦160名である。糖尿病，腎疾患など骨代謝に関連する慢性疾患を有する妊33．9回／月，運動時間9±82．9時間／E，運動力量婦，およびステロイド剤，ビタミン剤を服用す

妊娠初期のFFQ法による食物摂取頻度調査の結果では，2010年版栄養摂取基準に従い総エネる妊婦は除外した。

1±0．6／月，日照時間38±49．8時間／，月（UVケア
なし14±29．3時間／月）であった。

測定項目は，定量的超音波骨密度測定（QUS：

ルギーは1610±293．OkcaY日で必要量以下，タンGEヘルスケア・ジャパン社A－1000），食物摂取

パク質65±16．1g／日，脂質61±14．2疹日，糖質204頻度調査（FFQ法：上西らによる），運動量調査，±41．2g／日は目標範囲内，食塩10±1．69／日は摂取

1）よしかた産婦人科，2）横浜市立大学産婦人科，3）神戸薬科大学衛生化学研究室，4）女子栄養大学栄養生理学
研究室，5）横須賀共済病院産婦人科，6）横浜労災病院産婦人科

138（514）

OsteoPorosisJapan

vol．20

no．3

Presented by Medical*Online

2012

第22回婦人科骨粗霧症研究会

e
e

e
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e

e

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i52名）

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ρ過多，カルシウム418±141．4mg／日は目安量以す。それぞれ栄養摂取やライフスタイルに差は下，鉄8±4．8mgl日目安量以下，ビタミンA767

みられなかったが，踵骨骨密度は35歳以上妊婦±525．6μgRE／日，ビタミンD9±1．9μg／日，ビと経産婦で有意に低かった。

タミンK204±101．7μg／日で推奨量を充足していた。カルシウム摂取は94％の妊婦において不足していたが（図2），ビタミンD，ビタミンKの摂取量は推奨量に達していた。

対象を初産・経産で比較した結果を表1に，35歳以上と35歳未満で比較した結果を表2に示
OsteoPorosis／mρan

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考察および結語
カルシウム摂取不足は妊娠適齢期女性の多くにみられる傾向にある。2010年版国民栄養摂取基準では，妊娠・授乳期の付加量はゼロである
が，積極的なカルシウム摂取が推奨される。
vol．20

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Presented by Medical*Online

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139（515）38

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Asia Pac J Clin Nutr 2011;20 (4):507-513

Original Article

Association between 24 hour urinary α-tocopherolcatabolite, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6hydroxychroman (α-CEHC) and α-tocopherol intake inintervention and cross-sectional studies
Eri Imai MSc1, Tomiko Tsuji PhD1,2, Mitsue Sano PhD1, Tsutomu Fukuwatari PhD1,
Katsumi Shibata PhD1
1

Department of Food Science and Nutrition, School of Human Cultures, The University of Shiga Prefecture,
Shiga, Japan2
Department of Health and Nutrition, School of Health and Human Life, Nagoya Bunri University, Aichi,Japan
The objective is to determine the association between the 24 hour urinary α-tocopherol catabolite, 2,5,7,8tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (α-CEHC) and α-tocopherol intake in an intervention and across-sectional studies. In the 4-weeks intervention study, Japanese men (n = 10) consumed the test diet in week
1, and the test diet plus varying amounts of α-tocopherol in the three subsequent weeks: 21 μmol/d α-tocopherol
in week 2, 63 μmol/d in week 3, and 125 μmol/d in week 4. A significant association between α-tocopherol intake and urinary α-CEHC was observed in this strictly controlled experiment (r = 0.99, p<0.001). In the crosssectional study, all foods consumed over 4 consecutive days were recorded in 76 free-living young subjects (1833 years). The association was weak, but a significant relationship was observed (r = 0.29, p<0.05) even in the
cross-sectional study. In the cross-sectional study adults, mean estimated α-tocopherol intake calculated by urinary α-CEHC and the excretory ratio was 91% of their mean intake over the 4 days. The results show that urinary α-CEHC level reflected recent α-tocopherol intake in free-living young Japanese adults, and could be used
as a measure of intake during the previous few days, both for group means and for individual rankings within agroup.

Key Words: α-tocopherol, catabolism, CEHC, urine, biomarker

INTRODUCTION
Measurements of food intake are widely used for surveys
of nutritional assessment. However, there are limitationsin assessing only information from food surveys,1 andmethods that measure biological parameters can reveal
new information. Urine, which is a noninvasive bio-sample,
might overcome the limitations of nutritional assessment
by food survey. For example, 24-hour urinary nitrogenlevel is established as a marker for protein intake, 2 urinary potassium level as a marker for potassium intake,3and urinary sugar level as a marker for sugar intake. 4,5 In
previous studies, we investigated the relationship between
water-soluble vitamin intake and their urinary excretionof these nutrients. We clarified that urinary water-soluble
vitamin levels are strongly correlated with their intake.6-9
These studies 6-9 have indicated that 24-h urinary excretion of water-soluble vitamins is a potential biomarker forrecent vitamin intake in both intervention and crosssectional studies.
Generally, fat-soluble vitamins are not excreted inurine. However, In 1995, Schultz et al,10 reported that a
catabolite of α-tocopherol, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (α-CEHC), which is a metabo-lite with an intact chroman ring, is excreted in urine. Previously, α-CEHC was proposed as a potential excretion
product of α-tocopherol in 1965 by Schmandke et al,11but had not been described again until Schultz’s report.
Schultz et al,10 suggested that α-CEHC excretion indicates the saturated binding capacity of α-tocopherol in theplasma, and thus may be considered to be a marker of
optimum α-tocopherol intake. This proposal was strengthened by Shuelke et al,12 who found that α-CEHC was
excreted into the urine of patients with α-tocopherol binding protein defects regardless of the plasma α-tocopherol
concentration, whereas it was not excreted by healthy
subjects until the plasma α-tocopherol concentration

Corresponding Author: Dr Katsumi Shibata, Department ofFood Science and Nutrition, School of Human Cultures, The
University of Shiga Prefecture, 2500 Hassaka, Hikone, Shiga
522–8533, Japan.
Tel: +81–749–28–8449; Fax: +81–749–28–8499
Email: kshibata@shc.usp.ac.jp
Manuscript received 7 February 2011. Initial review completed
2 June 2011. Revision accepted 12 July 2011.

508

E Imai, T Tsuji, M Sano, T Fukuwatari and K Shibata

surpassed 40 μmol/L. These findings indicate that theurinary content of α-CEHC in the Japanese is almost below detection, because the average plasma concentrationof α-tocopherol in the Japanese is around 20 μmol/L.13
However, as Schultz et al,10 pointed out in their paper,
their results were obtained with only seven participants,
and therefore, further research is necessary to allow theresults to be applied generally.
In 2003, Morinobu et al,14 reported a straightforward
and reliable method of determining α-CEHC, which waslater modified by Stahl et al15 and Lodge et al16 Morinobu
et al14 also reported that α-CEHC was detected in healthy
adult male Japanese volunteers (n = 14). Therefore, it isprobable that α-CEHC is excreted into urine in Japaneseindividuals who take an ordinary amount of α-tocopherol.
The aim of the present study was to determine the possibility that α-CEHC can be used as a biomarker of αtocopherol status in young Japanese adults. We examined
the association between 24 h urinary α-CEHC levels andthe intakes of α-tocopherol in strictly controlled-living
and in free-living participants.
MATERIALS AND METHODSBoth studies were reviewed and approved by the ethics
committee of The University of Shiga Prefecture. The
purpose and protocol of this study was explained to allparticipants before joining the study, and written informed
consent was obtained from each participant.
Subjects and experimental design
Intervention study groupWe recruited students from a registered dietician department. All subjects (male Japanese college students, n = 10)
were housed in the same facility and given the same diet.
The experimental period was 4 weeks. They did not haveregular use of medications or dietary supplements, or habitual alcohol or cigarette consumption. Age, body weight,
height, and body mass index (mean ± SD) were 22.1 ±2.3 years (18–25 years), 63.6 ± 5.2 kg, 174 ± 5 cm, and21.0 ± 1.6 kg/m2, respectively.
Cross-sectional study group
A total of 102 healthy, free-living Japanese females, aged
18-33 years, voluntarily participated in this study. The
exclusion criteria were: presence of cold or influenza, anduse of multivitamin supplements at least once during theprevious month. In addition, we excluded participants
whose 24-hour urine collection or dietary records wereconsidered as incomplete, with a collection time outside
the 22-26 h range, a urine volume of <250 ml, creatinine
excretion in relation to body weight outside the 10.8-25.2
mg/kg range,17,18 or extremely low or high energy intake(<2,090 or >16,700 kJ/d).19 After these exclusions, 76 of
the 102 female students were found to be eligible andwere enrolled into the group.
Dietary records
Intervention study group
The diet given to the participants consisted of a breakfastof bread, margarine, ham, yoghurt, tomato, lettuce, andmilk; a lunch of rice, toasted and seasoned laver (a typeof seaweed), luncheon meat, boiled egg, raw cabbage,

Table 1. Daily intake of energy and nutrients form thebasal diet in intervention study
Energy and nutrients1
Energy (kJ)Protein (g)Fat (g)Carbohydrates (g)α-Tocopherol (mg)Amount11,100
97.5
86.7
361
8.7 (20.2 μmol)

1

Nutrients were calculated from the Standard Tables of Food
Composition in Japan.20

miso soup, and Japanese tea; and an evening meal of rice,soy sauce, seasonal Pacific saury (a type of fish), tofu
(soybean curd), boiled spinach leaves, kiwi fruit and Japanese tea, with a midnight snack of cheese and jelly fruitmix. The daily intakes of energy and nutrients form thebasal diet are shown in Table 1. Nutrients were calculated
by using Standard Tables of Food Composition in Japan,(fifth revised and enlarged edition).20 The subjects ate thisdiet on days 1-5 of each week over the experimental period of 4 weeks and were free to eat what they wanted ondays 6 and 7. In the latter three weeks, the participants
took α-tocopherol acetate in addition to the diet: 21
μmol/d in week 2, 63 μmol/d in week 3, and 125 μmol/din week 4.
Cross-sectional study group
This group underwent a 4-day dietary assessment inwhich the participants were living freely in the university
and consuming their normal diet. The 4-day assessment
began on a Monday (day 1) and ended on Thursday (day4). All food consumed during the 4-day period was recorded using a weighed food recording method. 21 A digitalcooking scale capable of weighing in 1 g increments(Tanita Inc., Tokyo, Japan), a set of dietary record forms,
a dietary record manual, and a disposable camera were
distributed to the participants in advance. In the dietary
record, the ingested food was described (eg “raw”, “boiled”,“cooked”, “skin present”, “a part of cooking ingredients”,
or “with or without seasoning”), and coded according to
the Standard Tables of Food Composition in Japan (fifth
revised and enlarged edition) as for the intervention
group.20 The participants took photographs with a disposable camera of the dishes before and after eating. Several
experienced dietitians used the photographs to complete
the data, and asked the participants to resolve any discrepancies or to obtain further information when needed.
The food that remained after eating was measured on thedigital scales and was deducted from the dietary record.
Food, nutrient and energy intakes were calculated using
SAS statistical software (version 6.12; SAS Institute,Cary, NC, USA), based on the Standard Tables of Food
Composition in Japan.
Collection of 24 hour urine sample
For the intervention study group, the 24 hour urine samples were collected from the second urination on day 4 tothe first urination after 06:30 hours (wake-up time) on
day 5 in each week.
For the cross-sectional group, a single 24-hour urine
sample was collected on day 4 to measure the α-Correlate of vitamin E intake and urinary CEHC
tocopherol metabolite, α-CEHC. In the morning, participants were asked to discard the first specimen and to record the time on the sheet. The following morning, participants were asked to collect a specimen at the same timeas the discarded specimen from the previous morning andto record the time on the sheet.
After the urine samples were collected, the sample
volumes were measured, and aliquots of the urine werestabilized to avoid destruction of α-CEHC. All treatedurine samples were then stored at –20°C until analysis.
Chemicals
α-CEHC was purchased from Cayman Chemical Co., Ltd(Ann Arbor, Michigan, USA). β-Glucuronidase derived
from Escherichia coli was obtained from Nacalai TasqueCo., Ltd (Kyoto, Japan). All other chemicals used were of
the highest purity available from commercial sources.
Analysis of α-CEHCThe concentrations of α-CEHC in urine were measured
by high performance liquid chromatography with electrochemical detection (HPLC-ECD), as described by Morinobu et al.14 β-Glucuronidase (25,000 units) was dissolved in 2.5 ml of 0.1 mol/L sodium acetate-acetate
buffer (pH 4.5) on ice and used to hydrolyze the conjugate immediately after preparation. Urine (1 ml) was
placed into a tube with 1,000 U (100 μl) of βglucuronidase, 100 μl of 57 mmol/L ascorbic acid, and 20
μl of 0.015% dibuthylhydroxytoluene (BHT). After which
the mixture was incubated for 4 hours at 37oC to achievehydrolysis, 50 μl of 6 mol/L HCl and 2 ml of diethylether
were added to stop the reaction. After mixing by vortex
and separating by centrifugation at 1,800×g for 10 min, 1
ml of the diethylether layer was collected and evaporated
to dryness. The residue was dissolved in 200 μl of
0.015% BHT, and a 20 μl aliquot was injected into the
HPLC-ECD.
Statistics
SPSS software (version 16 for Windows; SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Values

Figure 1. Relationship between urinary excretion of α-CEHC and
the intake of α-tocopherol in strictly controlled participants (intervention study). Individual average intake of α-tocopherol is plottedon the x-axis, and the 24-h urinary excretion of α-CEHC, a catabolite of α-tocopherol, is plotted on the y-axis. In total, 10 healthy
male Japanese college students aged 18-25 years were enrolled.
Values are mean ± SD. A significant correlation (r = 0.99, p =0.0043) was obtained. Regression line: y = 0.0214 ( ± 0.0014) x +0.180 (± 0.122).

509

were presented as means ± SD. The daily measurementsof urinary α-CEHC and the dietary α-tocopherol intakes
were not normally distributed, therefore, the data wereconverted logarithmically. Pearson correlation coefficients were calculated to determine the association between urinary and dietary measurements. The value of theurinary excretory ratio (%) was calculated as follows: [αCEHC excretion in the fourth day (μmol/d)/the average αtocopherol intake during 4-days (μmol/d)]100. An analysis of variance (ANOVA) random effects model wasused to quantify inter- and intra-individual percentage
coefficient of variance (% CV), which was used to estimate the variability in α-tocopherol intake.
RESULTS
Relationship between the intake of α-tocopherol and theurinary excretion of α-CEHCIntervention study group
Figure 1 shows the relationship between the intake of αtocopherol and the urinary excretion of α-CEHC. A
strong significant association was observed (r = 0.99, p =0.0043). The average urinary α-CEHC concentration atbaseline (week 1) was 0.74 μmol/d, which increased to
0.94, 1.89, and 3.34 μmol/d after supplementation at thedoses of 21, 63 and 125 μmol/d, respectively. The excretory ratio of α-CEHC was 3.7 ± 1.3, 2.3 ± 0.7, 2.3 ± 1.1,
and 2.3 ± 0.6% for α-tocopherol intakes of 20, 41, 83 and146 μmol/d, respectively.
Cross-sectional study group
The basic characteristics of the 76 young women are presented in Table 2. Each values were similar to those reported for young adults female aged 18–22 years.19 In
brief, the participants were considered as typical female
university students in Japan, characterized by relatively
low BMI (20.2 kg/m2), and low intake of fat (28.4%).
During the experimental period, all participants were living freely, and none of the participants were drinking or
smoking. Average intake of α-tocopherol in participants
was 13.7 ± 3.8 μmol/d, and daily intake of α-tocopherol

Figure 2. Relationship between urinary excretion of α-CEHCand intake of α-tocopherol in young adults’ female (crosssectional study). Measurements were taken of food intake on 4
consecutive days. The urine samples were collected at day 4.
Individual average intake of α-tocopherol is plotted on the xaxis, and the urinary excretion of α-CEHC on day 4 is plottedon the y-axis. In total, 76 healthy, free-living, female college
students aged 18–33 years were enrolled. A significant correlation (r = 0.29, p = 0.0147) was obtained. Regression line: y =0.0228 ( ± 0.0091) x + 0.137 ( ± 0.130).

510

E Imai, T Tsuji, M Sano, T Fukuwatari and K Shibata

Table 2. Characteristics and dietary intakes of female subjects
Variables
Anthropometric variable
Age (years)
Body height (cm)Body weight (kg)Body mass index (kg/m2)
Dietary mean intake at days 1-4
Total energy (kJ/d)
Protein (% of energy)
Fat (% of energy)
Carbohydrate (% of energy)
α-Tocopherol intake (μmol/d)Inter-individual variations on the vitamin E intake (% CV)1
Intra-individual variations on the vitamin E intake (% CV)1
Urinary α-CEHC (μmol/d)Excretory ratio2 of vitamin E (%)

Young adult female (n = 76)
20.1 ± 2.3
158 ± 550.6 ± 5.4
20.2 ± 1.86950 ± 1260
13.8 ± 2.2
28.4 ± 4.5
56.5 ± 4.9
13.7 ± 3.8
41.5
55.3
0.444 ± 0.292
3.58 ± 3.39

1

% CV, percentage coefficient of variance.
Calculated by the formula [α-CEHC excretion on day 4 (μmol/d) / the average α-tocopherol intake over the 4 days (μmol/d)]  100.

2

Table 3. Mean dietary α-tocopherol intake and 24-hoururinary α-CEHC, excretory ratio, and estimated mean
α-tocopherol intake in young adult females

Mean α-tocopherol intake1(μmol/d)Excretory ratio2 (%)
Estimated mean α-tocopherol
intake3 (μmol/d)% ratio4

Young adult female(n = 76)
13.7 ± 3.8
3.58 ± 3.39
12.4 ± 8.2
911

Calculated by average α-tocopherol intake over the 4 days foreach individual.
2
Calculated by the formula [α-CEHC excretion on day 4 (μmol/d) /the average α-tocopherol intake over the 4 days (μmol/d)]  100.
3
The estimated α-tocopherol intake in young adult was calculatedby the individual value of the 24-h urinary excretion of α-CEHCand the excretory ratio of 3.58 %, and the estimated mean αtocopherol intake was calculated by the resulting estimated αtocopherol intake.
4%Ratio between the mean and the estimated mean α-tocopherol
intake.

was similar to adequate intake for Japan. 20 Urinary excretion of α-CEHC was twofold higher in elderly subjects
than healthy peoples. The value of the excretory ratio ofvitamin E in elderly subjects was 1.4-fold higher than in
healthy individuals. Intra and inter-individual variations in
α-tocopherol intakes, the values were both around 50%(Table 2).
Correlations between 24-hour urinary excretion of αtocopherol metabolite, α-CEHC on day 4 and the average
α-tocopherol intake on 4 consecutive days are shown in
Figure 2. A significant association was observed in young
adult females.
Estimated mean α-tocopherol intake calculated by urinary excretion of α-CEHC and the excretory ratio in the
cross-sectional study
Mean dietary α-tocopherol intake and 24-hour urinary αCEHC, excretory ratio, and estimated mean α-tocopherol
intakes in young women are shown in Table 3. The excre-

tory ratio was determined from the urinary excretion of αCEHC and the average α-tocopherol intake over 4 days.
The individual estimated α-tocopherol intake was calculated by average excretory ratio and the individual urinary
α-CEHC value. The estimated mean α-tocopherol intake
in young women was 91% of the real mean α-tocopherol
intake over 4 days in young adult females.
DISCUSSION
Alpha-CEHC, a urinary metabolite of α-tocopherol, wasdescribed in 1995 by Schultz et al10 Excretion of α-CEHC
is considered to reflect saturation of α-tocopherol in thebody, because it is not a metabolite of the α-tocopherol
consumed for antioxidant defense. In other words, thedetection of α-CEHC in urine generally indicates a better
α-tocopherol nutritional status. Therefore, it is generally
considered that the content of α-CEHC is below the limit
of detection under an ordinary dietary habitant. Morinobu
et al,14 however, reported that α-CEHC was detected inhealthy adult male Japanese volunteers. Therefore, it isprobable that α-CEHC is excreted into urine in Japaneseindividuals who consume an ordinary amount of αtocopherol. But, the data are based on only seven persons.
Further research is necessary to allow the results to be
applied generally.
In the present first study, the intervention study was
performed to determine whether urinary α-CEHC excretion correlates with intake of α-tocopherol. We found asignificant positive correlation between urinary α-CEHCand intake of α-tocopherol in healthy young male Japanese adults who consumed a strictly controlled diet withdoses of α-tocopherol ranging from 20 to 145 μmol/d.
In the second experiment, to determine the usefulnessof urinary α-CEHC as a biomarker for α-tocopherol nutritional status, a cross-sectional study was performed on
free-living subjects. A weak but significant correlation
was found between urinary α-CEHC and the mean αtocopherol intake. These results indicate that α-CEHClevels in 24-h urine reflect dietary α-tocopherol intakes
over the past few days, and suggest that α-tocopherol intake can be estimated from urinary α-CEHC values infree-living subjects.

Correlate of vitamin E intake and urinary CEHCThis phenomenon might be only applicable for theJapanese and East-Asian populations. The subjects ate alot of vegetable and were of relatively low BMI and had
low intake of fat. The requirement of α-tocopherol is dependent on the intakes of polyunsaturated fatty acids. The
average intakes of the polyunsaturated fatty acids werearound 10 g/d. The optimum ratio of α-tocopherol (mg/d)
to polyunsaturated fatty acids (g/d) is reported to be
0.60.22 Its average ratio was about 0.60 in the present subjects. This is a reason why a significant association wasobserved even with low intake of α-tocopherol for theJapanese.
Metabolism of α-CEHC from α-tocopherol is somewhat different between Japanese, America and European
populations. For example, the metabolic activity of αtocopherol to α-CEHC might be higher in the Japanesethan in the Americans and Europeans. The precise regulatory mechanism of post-absorption α-tocopherol elimination is not clear. The current knowledge is only of apathway involving cytochrome P450-mediated ωhydroxy lation of the α-tocopherol phytyl side chain, followed by stepwise removal of two or three carbon molecules such as acetyl-CoA or propionyl-CoA, ultimately
yielding the α-CEHC that is excreted in urine.23 Low fat
intakes in the Japanese might bring about surplus ability
to the β oxidation system, which results in increased activity of α-tocopherol to α-CEHC. Sesamin increases tissue α-tocopherol concentration by inhibiting the αtocopherol oxidation pathway;24 sesame oil, which contains sesamin, is often used in Japanese cooking, whichmight bring about the saving effect of α-tocopherol.
Compared to Caucasian, Asian population had significantly lower plasma vitamin E levels in the same environment.25 However, there are no data regarding its metabolism or immune functions among two populations. In
Asian populations, they may be easily excreted into urine
as α-CEHC compared to Caucasians. This is also a reason
why the significant association was observed even with
low intake of α-tocopherol for the Japanese.
The limiting factor of the transport capacity appears to
be plasma lipid concentration. In general, subjects with a
low total plasma lipid concentration did not accumulate
as high concentration of α-tocopherol, as those with ahigher total lipid content; and they started to excrete αCEHC earlier10. If the urinary excretion of α-CEHC isrelated to the α-tocopherol content of plasma lipid, therange of thresholds of excretion is much wider and thecorrelation between plasma concentration and urinary
excretion becomes more pronounced. In the present
cross-sectional study, we did not measure plasma lipid
concentration, and how plasma lipid affected urinary excretion of α-CEHC is unclear.
In terms of the completeness of the dietary assessmentin the present study, there are several limitations in terms
of using a weighed food record method. To reduce errors
associated with self report, several dietitians reviewed thecollated records along with the photographs. The selection of participants from a dietetics course also contributed to reduce reporting errors, as they had nutritional
knowledge and were well trained. Another limitation exists in the present food composition table developed forJapan. In a dietary assessment of free-living people, po-511

tential errors caused by the quality of this table, such as
defects in food composition, are inevitable. These limitations might cause the relatively low correlation in thefree-living experiment population compared with that of
the intervention study.
In conclusion, a significant relationship between the αtocopherol intake and urinary excretion of α-CEHC wasobserved in young Japanese adults.
ACKNOWLEDGMENTS
We thank all volunteers who participated in the present study.
This study represents a part of the results of “Studies on theconstruction of evidence to revise the dietary reference intake
for Japanese people – elucidation of the balance of micronutrients and major elements” (principal investigator, Katsumi Shibata), which was supported by a research grant for Comprehensive Related Diseases from the Ministry of Health, Labour andWelfare of Japan.
AUTHOR DISCLOSURES
None of the authors had any financial or personal conflicts ofinterest associated with this manuscript.
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513

Original Article

Association between 24 hour urinary α-tocopherolcatabolite, 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6hydroxychroman (α-CEHC) and α-tocopherol intake inintervention and cross-sectional studies
Eri Imai MSc1, Tomiko Tsuji PhD1,2, Mitsue Sano PhD1, Tsutomu Fukuwatari PhD1,
Katsumi Shibata PhD1
1

Department of Food Science and Nutrition, School of Human Cultures, The University of Shiga Prefecture,
Shiga, Japan2
Department of Health and Nutrition, School of Health and Human Life, Nagoya Bunri University, Aichi,Japan

24 小時尿液 α-生育醇代謝產物 2,5,7,8-tertranetgyl-2(2’carboxyethyl)-6-hydroxychroman(α-CEHC)與 α-生育醇攝取之介入及橫斷研究之相關
本研究的目的為評估在介入及橫斷研究中，24 小時尿液的 α-生育醇代謝產物
2,5,7,8-tertranetgyl-2(2’carboxyethyl)-6-hydroxychroman (α-CEHC) 與 α-生育醇攝
取量之相關性。在四週的介入性研究，日本男性 (n=10) 在第一週攝取測試飲
食，後續三周攝取添加不同量 α-生育醇的測試飲食：第二週為 21 μmol/dα-生育
醇、第三週為 63 μmol/dα-生育醇與第四週為 125 μmol/dα-生育醇。在這個嚴格
控制的實驗中，觀察到 α-生育醇的攝取量與尿液中的 α-CEHC 有顯著的相關(r=0.99, p<0.001)。在橫斷性研究，76 名年輕一般研究對象 (18-33 歲) 紀錄連續四天攝取的所有食物。在橫斷性研究這個相關性儘管不強，但是顯著的(r=0.29, p<0.05)。在橫斷性研究中的成年人，以尿液 α-CEHC 計算其平均估計
α-生育醇攝取量，其排泄率為四日平均攝取量的 91%。無論組別平均值或是組
別中個體的排序結果，都顯示尿液中的 α-CEHC 量可反映年輕日本一般成年人
近期的 α-生育醇攝取量，且可以當作過去幾天的攝取量測量方法。
關鍵字：α-生育醇、代謝產物、CEHC、尿液、生物標記doi:10.1017/S1368980010001904

Public Health Nutrition: 14(2), 327–333

Twenty-four-hour urinary water-soluble vitamin levels correlatewith their intakes in free-living Japanese schoolchildren
Tomiko Tsuji1,2, Tsutomu Fukuwatari1, Satoshi Sasaki3 and Katsumi Shibata1,*
1

Department of Food Science and Nutrition, School of Human Cultures, The University of Shiga Prefecture,2500 Hassaka, Hikone, Shiga 522-8533, Japan: 2Department of Health and Nutrition, School of Health andHuman Life, Nagoya Bunri University, Aichi, Japan: 3Department of Social and Preventive Epidemiology,
School of Public Health, The University of Tokyo, Tokyo, Japan
Submitted 15 December 2009: Accepted 10 May 2010: First published online 25 June 2010

Abstract
Objective: To examine the association between 24 h urinary water-soluble vitaminlevels and their intakes in free-living Japanese schoolchildren.
Design: All foods consumed for four consecutive days were recorded accurately
by a weighed food record. A single 24 h urine sample was collected on the fourthday, and the urinary levels of water-soluble vitamins were measured.
Setting: An elementary school in Inazawa City, Japan.
Subjects: A total of 114 healthy, free-living, Japanese elementary-school children
aged 10–12 years.
Results: The urinary level of each water-soluble vitamin was correlated positively
to its mean intake in the past 2–4 d (vitamin B1: r 5 0?42, P , 0?001; vitamin B2:
r 5 0?43, P , 0?001; vitamin B6: r 5 0?49, P , 0?001; niacin: r 5 0?32, P , 0?001;
niacin equivalents: r 5 0?32, P , 0?001; pantothenic acid: r 5 0?32, P , 0?001; folic
acid: r 5 0?27, P , 0?01; vitamin C: r 5 0?39, P , 0.001), except for vitamin B12(r 5 0?10, P 5 NS). Estimated mean intakes of water-soluble vitamins calculatedusing urinary levels and recovery rates were 97–102 % of their 3 d mean intake,except for vitamin B12 (79 %).
Conclusions: The results show that urinary levels of water-soluble vitamins, except
for vitamin B12, reflected their recent intakes in free-living Japanese schoolchildren
and could be used as a potential biomarker to estimate mean vitamin intake.

Since vitamin deficiencies cause various disorders in thegrowth of schoolchildren, a method to evaluate vitaminstatus easily and accurately is desired for early screening
at a primary preventive stage. Methods using biomarkers
for assessing vitamin intakes offer an effective approachto evaluate vitamin status in individuals. Many precedingstudies have investigated urinary excretion as a biomarker
for vitamin intake(1–3). We have also reported recently
that 24 h urinary levels of water-soluble vitamins correlate
highly with their intakes for Japanese college students ina strictly controlled environment(4,5). Performing a study
under a free-living environment without any interventions
is the next step to confirm the applicability of the biomarker method. In the present study, we examined theassociation between 24 h urinary excretion of water-soluble
vitamins and their dietary intakes for free-living schoolchildren to confirm the validity of the findings obtained in
the controlled environment.
To capture dietary intake and calculate nutrients undera free-living environment, we used a weighed foodrecord for four consecutive days. Although a weighed*Corresponding author: Email kshibata@shc.usp.ac.jp

Keywords
Urinary water-soluble vitamin
Biomarker
Free-living
Japanese schoolchildren

food record can provide relatively precise information
regarding dietary intake compared with other dietaryassessment methods(6), it is difficult for schoolchildren
to complete a weighed food record without support.
Few studies have reported this kind of assessment for
free-living schoolchildren(7), while many studies havereported using a 24 h recall(8), a dietary diary(9) or an
FFQ(10). To overcome the difficulty of using a weighed
food record for schoolchildren, we formed a close andcooperative relationship not only with the children but
also their parents and teachers in the target elementary
school before starting the study, through supporting theprolonged dietary education programme provided by
the school board.

Methods
Participants
A total of 132 healthy, free-living schoolchildren aged
10–12 years voluntarily participated in the present study.
r The Authors 2010

328

The purpose and protocol were explained to all participants, as well as their parents, before joining the study, andwritten informed consent was obtained from each parent
because all participants were less than 20 years old. We
excluded participants diagnosed with the common cold or
influenza, and those who had taken multivitamin supplements at least once during the previous month. In addition,
we excluded participants whose 24 h urine collection
or dietary records were considered incomplete, with acollection time outside the range of 22–26 h, urine volume,250 ml, creatinine excretion in relation to body weightoutside the range of 10?8–25?2 mg/kg(11,12) or extremely
low or high energy intake (,2092 or .16 736 kJ/d)(13).
After these screenings, 114 schoolchildren (sixty-sevenboys and forty-seven girls) were found to be eligible.
The study was reviewed and approved by The EthicalCommittee of The University of Shiga Prefecture.
Dietary records
This was a 4 d dietary assessment in which the participants were living freely and consuming their normal diet.
The assessment was performed at one of the elementary
schools in Inazawa City (population .130 000) in Aichi
Prefecture, Japan, in June 2007 and June 2008. The firstday (Monday) of the experimental period was definedas day 1, the second day as day 2, the third day as day 3,
and the fourth day as day 4. All foods consumed during
the 4 d period were recorded using a weighed foodrecord(14). A digital cooking scale (1 g unit; Tanita Inc.,Tokyo, Japan), a set of dietary record forms, a dietary
record manual and a disposable camera were distributed
to the participants in advance. Upon entry in the dietary
record, the status of food at oral intake was identified
as ‘raw’, ‘boiled’, ‘cooked’, ‘the presence of skin’, ‘a part ofcooking ingredients’ or ‘with or without seasoning’, andcoded according to the fifth revised and enlarged editionof the Standard Tables of Food Composition in Japan(15).
The participants with support from their parents took
photographs with the disposable camera of the dishes
before and after eating. Several experienced dietitians
used the photographs to check the records, asking participants or their parents to resolve any discrepancies or
to give further information when needed. The food thatremained after eating was measured with a digital scale
and was deducted from the dietary record. For school
meals, the registered dietitians completed the records onbehalf of the participants. Nutrient and energy intakes
were calculated using the SAS statistical software package
version 6?12 (SAS Institute Inc., Cary, NC, USA), basedon the current Standard Tables of Food Composition in
Japan(15). For vitamins, the intakes of eight water-soluble
vitamins –vitamins B1, B2, B6, B12, niacin, pantothenic
acid, folic acid and vitamin C – were calculated, except
for biotin which is not designated in the current Standard
Tables of Food Composition in Japan. Since niacin is synthesized from tryptophan, the amount of niacin equivalents

T Tsuji et al.

was handled separately from niacin. Since 1 mg nicotinamide is synthesized from 60 mg tryptophan(16), niacin
equivalents was calculated as the sum of niacin and 1/60
tryptophan intakes. For calculating mean vitamin intakes,
the 2 d mean intake corresponds to average intakes ondays 3 and 4. Similarly, the 3 d mean intake corresponds
to average intakes on days 2–4, and the 4 d mean intake
corresponds to average intakes on days 1–4.
24 h urine sampling
A single 24 h urine sample was collected on the fourth day
to measure urinary levels of water-soluble vitamins and their
metabolites. It was collected from the second passage ofurine on the fourth day to the first passage on the fifth day.
The participants were asked to record all the times of urination on the sheet. After the total urine sample was collected, the volume was measured. Aliquots of the urine werestabilized to avoid destruction of water-soluble vitamins andtheir metabolites, and then stored at 2208C until analysis.
Urinalysis
Urinary thiamine was determined by post-HPLC labelled
fluorescence(17). Urinary riboflavin was determined by
HPLC(18). Urinary vitamin B6 metabolite, 4-pyridoxic acid,was determined by HPLC(19). To measure urinary vitamin
B12, urine samples were added to 0?2-mM acetate buffer(pH 4?8), vitamin B12 was converted to cyanocobalaminby boiling for 30 min with 0?0006 % w/w potassium cyanide
at acidic pH, and cyanocobalamin was determined by amicrobioassay using Lactobacillus leichmanii ATCC
7830(20). Urinary N 1-methyl-2-pyridone-5-carboxamide andN 1-methyl-4-pyridone-3-carboxamide(21) and N 1-methylnicotinamide(22) were determined by HPLC, and the sum ofthese compounds was determined as nicotinamide metabolites. Urinary pantothenic acid was determined by amicrobioassay using Lactobacillus plantarum ATCC
8014(23). Urinary folic acid was determined by a microbioassay using Lactobacillus casei ATCC 2733(24). Urinary
reduced and oxidized ascorbic acid and 2,3-diketogluconic
acid were determined by HPLC(25).
Statistical analysis
To exclude extraordinarily abnormal urinary vitamin levels
which might be caused by taking unexpected fortified
foods, participants in the upper 5 % limit in terms ofurinary excretion for each vitamin were removed from the
114 eligible participants, and a total of 108 samples wereidentified to be valid for data analysis for each watersoluble vitamin. Similar to a previous free-living study(2),males and females were not separated for analysis. The
SPSS for Windows statistical software package version
16 (SPSS Inc., Chicago, IL, USA) was used for statisticalanalysis. Values are presented as means and standard
deviations. Since measurements of urinary and dietary
water-soluble vitamins were not distributed normally, thedata were converted logarithmically. Pearson correlation

Correlation of urinary vitamins and intakes

329

Table 1 Characteristics of the participants: 114 eligible Japanese elementary-school children aged 10–12 years
Total (n 114)
Variable
Anthropometric variables
Age (years)
Body height (cm)Body weight (kg)Rohrer index (kg/cm33107)
Obesity index (%)
Dietary intakeTotal energy (kJ/d)
Protein (% of energy)
Fat (% of energy)
Carbohydrate (% of energy)% Energy intakeBreakfast
Lunch
SupperSnacksBoys (n 67)

Girls (n 47)

Mean

SD

Mean

SD

Mean

SD

10?8
144?0
36?7
122?0
24?01

0?7
7?7
8?3
17?9
3?8

10?7
142?2
34?6
119?3
26?5

0?7
7?7
7?2
15?9
13?3

11?0
146?5
39?8
125?7
0?4

0?7
7?0
8?9
20?1
13?8

8489
14?9
29?0
54?8

1298
2?5
5?8
8?7

8665
14?9
29?1
54?7

1409
2?6
6?0
9?3

8238
14?8
28?8
55?1

1086
2?1
5?5
7?7

-

21?3
32?7
31?1
14?8

21?7
32?1
31?4
14?8

20?8
33?6
30?8
14?9

-Dietary intake assessed from the consecutive 4 d dietary records.
-Average starting time of each meal: breakfast, 06.50 hours; lunch, 12.30 hours; supper, 18.40 hours.
-

coefficients were calculated to determine the association
between urinary and dietary measurements, and between
dietary and estimated water-soluble vitamin intakes.
P , 0?05 was considered statistically significant. An ANOVA
random-effects model was used to quantify inter- and intraindividual CV (%CV), which was used to estimate variability
in vitamin intake.

Results
The characteristics of the 114 eligible participants arepresented in Table 1. Since each value was almost thesame as those reported for children aged 10–11 years in
the Dietary Reference Intakes for Japanese in 2005(13), theparticipants were considered as typical elementary-school
children in Japan. During the experimental period, allparticipants were living freely. Inter- and intra-individual
variations in dietary intake of water-soluble vitamins for theconsecutive 4 d period are shown in Table 2. For intraindividual variations, %CV was 25–45 %, except for vitamin
B12 and vitamin C. For inter-individual variations, vitamin
B1, vitamin B12, folic acid and vitamin C exceeded 50 %.
The correlations between 24 h urinary excretion ofwater-soluble vitamins and their intakes are shown in
Table 3. For all vitamins except for vitamin B12, a significant positive correlation was found between urinary
excretion and dietary intake on day 4. For all vitamins
except for pantothenic acid, the correlations on day 4
were higher than those on other days.
To examine the influence of dietary intake during thepast few days on 24 h urinary excretion, we calculated thecorrelations between 24 h urinary excretions and mean
dietary intakes, which are shown in Table 4. For all vitamins
except for B12, niacin equivalents and folic acid, the correlations between the urinary excretion (column 2 in Table 3)

Table 2 Inter- and intra-individual variations in the dietary intake ofwater-soluble vitamins measured for the consecutive 4 d experimental period: eligible Japanese elementary-school children aged
10–12 years%CV (n 108)Vitamin
Vitamin B1
Vitamin B2
Vitamin B6
Vitamin B12
NiacinNiacin equivalents
Pantothenic acid
Folic acid
Vitamin C

Inter-individual
variations

Intra-individual
variations

71?0
28?8
5?7
166?8
30?4
8?8
42?7
87?4
62?2

31?1
29?5
32?1
95?0
33?1
25?2
25?0
45?0
65?5

-A total of 108 samples were valid for data analysis after removing the upper
5 % limit in terms of urinary excretion for each vitamin.

and the 3 d mean intake (column 5 in Table 4) were higher
than those based on daily intake shown in Table 3 (columns
6, 9, 12 and 15). Because the most significant correlations
were found between the urinary excretion and the 3 d mean
intake, recovery rates (column 11 in Table 4) were derived
from the urinary excretions (column 2 in Table 3) and the
3 d mean intakes (column 5 in Table 4), which are also
shown in Table 4. Estimated mean intakes of water-soluble
vitamins (column 13 in Table 4) were calculated using theserecovery rates and urinary excretions. Estimated mean
intakes, except for vitamin B12, niacin equivalents and folicacid, correlated with 3 d mean intakes and were 97–102 % of
the 3 d mean intake, except for vitamin B12 (79 %).
Discussion
In the present study we found a significant positive correlation between the urinary excretion and the dietary

330
Table 3 Measured values for 24 h urinary excretion collected on day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24 h urinary excretion and daily vitaminintake (n 108), among eligible Japanese elementary-school children aged 10–12 years

0?766
0?290
2?36
0?0256
–
65?6
11?6
16?8
161

0?383
0?209
0?92
0?0147
27?6
5?5
6?6
221

Vitamin intake at day 1

Mean

SD

-

SD

Vitamin intake at day 2

r-

Mean

SD

-

Vitamin B1 (mmol/d)
Vitamin B2 (mmol/d)
Vitamin B6 (mmol/d)
Vitamin B12 (nmol/d)Niacin (mmol/d)Niacin equivalents (mmol/d)Pantothenic acid (mmol/d)
Folic acid (nmol/d)
Vitamin C (mmol/d)Mean

Vitamin intake at day 3
r-

Mean

SD

-

Vitamin

Vitamin intake at day 4

r-

Mean

SD

r-

3?13
3?47
5?93
3?15
97?0
214
27?6
575
477

1?01
0?94
1?86
1?97
32?3
56
6?9
170
225

0?41***
0?36***
0?42***
0?18
0?28***
0?28**
0?23*
0?27**
0?35***

2?90
3?75
5?96
4?85
101?7
218
30?1
615
448

0?85
1?13
1?65
5?93
38?2
56
7?4
423
313

0?25**
0?36***
0?32***
0?14
0?11
0?23**
0?20*
0?12
0?23*

2?60
3?59
5?97
4?76
105?3
218
27?0
491
403

0?74
1?00
1?69
4?29
31?3
52
6?3
123
289

0?22*
0?33***
0?36***
20?02
0?21*
0?16
0?31***
0?18
0?26**

2?75
3?60
6?00
4?64
101?4
218
28?7
532
445

0?92
1?17
2?41
3?37
32?5
56
7?8
164
328

0?07
0?23*
0?17
0?11
0?23*
0?25**
0?25**
0?24*
0?18

-

24 h urinary vitamin excretion--Urinary excretion for each vitamin corresponds to: thiamin for vitamin B1; riboflavin for vitamin B2; 4-pyridoxic acid for vitamin B6; the sum of nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide
and N1-methyl-4-pyridone-3-carboxamide for niacin equivalents; the sum of reduced and oxidized ascorbic acid and 2,3-diketogluconic acid for vitamin C.
-r indicates the correlation between urinary excretion and dietary intake of the vitamin; significance of the correlation: *P , 0?05, **P , 0?01, ***P , 0?001.
-

Table 4 Summary of values derived from measured values (daily vitamin intake and 24 h urinary excretion in Table 3), i.e. mean dietary intakes and their correlations with 24 h urinary excretion,
recovery rates and estimated mean intakes (n 108), among eligible Japanese elementary-school children aged 10–12 years

Vitamin

Mean

Vitamin B1 (mmol/d)
Vitamin B2 (mmol/d)
Vitamin B6 (mmol/d)
Vitamin B12 (nmol/d)Niacin (mmol/d)Niacin equivalents (mmol/d)Pantothenic acid (mmol/d)
Folic acid (nmol/d)
Vitamin C (mmol/d)

3?02
3?61
5?94
4?00
99?4
216
28?8
595
462

3 d mean vitamin intake(day 2–day 4)

4 d mean vitamin intake(day 1–day 4)% Recovery--

2 d mean vitamin intake(day 3–day 4)

Estimated mean vitamin intakey

SD

rJ

Mean

SD

rJ

Mean

SD

rJ

Mean

SD

Mean

SD

0?77
0?85
1?41
3?14
26?0
48
6?0
236
200

0?42***
0?41***
0?45***
0?19*
0?24*
0?29**
0?26**
0?23*
0?39***

2?88
3?60
5?95
4?25
101?3
217
28?2
560
442

0?63
0?79
1?29
2?55
21?7
43
5?6
174
183

0?42***
0?43***
0?49***
0?10
0?29**
0?29**
0?32***
0?24*
0?39***

2?85
3?60
5?96
4?35
101?4
217
28?3
553
443

0?58
0?78
1?35
2?10
20?4
39
5?7
147
170

0?35***
0?42***
0?43***
0?10
0?32***
0?32***
0?32***
0?27**
0?39***

27?6
7?9
39?8
0?7
–
30?7
41?4
3?1
36?4

12?2
5?2
14?0
0?6

2?83
3?66
5?90
3?72
–
215
28?1
536
447

1?42
2?63
2?30
2?14
91
13?3
211
613

% Ratio--0?37***
0?26**
0?41***
0?06
–
0?20*
0?27**
0?09
0?39***

100
102
100
79
–
99
99
97
100

T Tsuji et al.

-Mean dietary intake was calculated using daily dietary intake (Table 3).
-% Recovery rate was derived from 24 h urinary excretion (Table 3)/3 d mean intake 3100.
yEstimated mean intake was calculated using 24 h urinary excretion (Table 3) and recovery rate.
Jr indicates the correlation between 24 h urinary excretion (Table 3) and mean intake; significance of the correlation: *P , 0?05, **P , 0?01, ***P , 0?001.
zr indicates the correlation between 3 d mean dietary intake and estimated intake; significance of the correlation: *P , 0?05, **P , 0?01, ***P , 0?001.
--% Ratio indicates the ratio between 3 d mean intake and mean estimated intake.

12?6
19?5
1?3
50?3

rz

-

Correlation of urinary vitamins and intakes

intake of seven water-soluble vitamins, except for vitamin
B12, in free-living Japanese schoolchildren aged 10–12
years. The correlation between the urinary excretion andthe dietary intake on the same day as urine collection was
highest, except for pantothenic acid, compared with thecorrelations on other days. Moreover, the correlations
between the urinary excretion and the mean dietaryintakes during the past 2–4 d showed higher correlations,
except for vitamin B12 and folic acid, than those for daily
intakes. These findings show that urinary levels of watersoluble vitamins are affected by not only their dietaryintakes on the same day as urine collection, but also theirintakes over the past few days.
The earlier intervention study showed extremely highpositive correlations between urinary levels of watersoluble vitamins and their intakes(4). In the earlier study,
participants comprised college students and they consumed exactly the same defined diets, with or without
synthesized water-soluble vitamin mixtures, for 4 weeks.
In the present study, the dietary assessment for schoolchildren using a weighed food record was performed for
four consecutive days without intervention. Assuming the
dietary assessment protocol in the present study contributed best to reduce the errors in the dietary records,
the similar results from the different groups and protocols
indicate that the urinary levels of water-soluble vitamins
are closely associated with vitamin intakes, and that this istrue even for free-living schoolchildren.
Correlation coefficients between the urinary excretions
and the 3 d mean intakes ranged from 0?24 to 0?49 with amean of 0?36, except for vitamin B12, which showed a lower
level than reported in our earlier study(4). The considerable
inter- and intra-individual variability for vitamin intakes in afree-living environment might affect these modest correlations. In addition, several factors are also known to affectwater-soluble vitamin metabolism. For example, carbohydrate and physical activity are known to affect vitamin B1
metabolism(26–28), the bioavailability of pantothenic acid infood is half that of free pantothenic acid(29), and the singlenucleotide polymorphism of the methylenetetrahydrofolate
reductase gene affects folic acid metabolism(30). These
factors might also affect the modest correlations.
The dietary habits of the schoolchildren who participatedin this study were well disciplined. They had regularbreakfast (before 07.00 hours), school lunch (around 12.30
hours) and supper (around 18.40 hours), with few snacks.
The daily distributions of energy intakes were 21 % at
breakfast, 33 % at lunch, 31 % at supper and 15 % forsnacks, which is thought to be well balanced comparedwith that reported in a previous study: 24 % at breakfast,
30 % at lunch, 23 % at supper and 23 % for snacks(31). Fiftyfive per cent of energy intake was obtained from carbohydrates, 30 % from fats and 15 % from protein, which fitswith the Dietary Reference Intakes for Japanese(13). These
data show that the participants had regular dietary habitswith well-balanced nutrition.

331

In terms of the completeness of the dietary assessmentin the present study, there are several limitations of using
a weighed food record method. One of the limitations
is the reliance on self-report. In the present study, toreduce errors associated with self-report, several dietitians
reviewed the collated records along with the photos.
Another limitation exists in the present food composition table in Japan. In a dietary assessment for free-living
people, potential errors caused by the quality of the foodcomposition table are inevitable, such as defects in foodcomposition. For example, the composition of Japanese
tea may vary depending on whether the extract of tea wasmade personally or whether it was a bottled tea beverage,
because the present Japanese food composition tablecannot differentiate such products. Such restrictions may
lower the accuracy of the data obtained from a weighed
food record. However, identifying the food status at oral
intake and coding the intake according to the food composition table should contribute to increase the accuracy of
the records.
In terms of completeness of 24 h urine collection, weused the INTERMAP criteria(11) as already described.
Because the p-aminobenzoic acid (PABA) method
requires intervention by taking PABA tablets orally andwould be difficult for schoolchildren, we did not use thatmethod to avoid any interventions. Because the participants in the present study were well motivated for the
study, the proportion of them with incomplete urine
samples was presumed to be small(32).
We have recently reported the intra-individual variations of urinary water-soluble vitamins in young Japanese, and our intervention study showed that thecollection of 24 h urine samples for 1–5 d was required toestimate those values within 20 % of the true mean(33).
Indeed, correlation between the 30 d mean urinary thiamin excretion and 30 d mean thiamin intake was higher
than that between daily excretion and daily intake(1). In
the present study, urinary water-soluble vitamins were
measured based on a single 24 h urine sample. Thus theurinary vitamin contents have potential for data inaccuracy from variability, and the results should be interpreted
cautiously. However, recent findings also suggest thatusing several days of 24 h urine sample would improve
the relationships between urinary excretion and intake ofwater-soluble vitamins.
A significant correlation was not found between urinary
vitamin B12 and dietary intake in this or a previous study(4).
This is consistent with studies showing that urinary vitamin
B12 increased by only 1?5 to 2 times when 1 mg of vitamin
B12, which is 300 times higher than usual intake, wasadministered orally, and by 2–3 times when 0?45 mg was
injected intramuscularly(34,35). Foods including vitamin B12
were so limited that its intake showed an extremely highinter- and intra-individual variation in the present study.
Estimated mean intakes of water-soluble vitamins calculated using the urinary levels and recovery rates correlated

332

well with the 3 d mean intakes, except for vitamin B12
and folic acid, and the estimated mean intakes agreed
exactly with the 3 d mean intakes. These findings suggestthat urinary levels of water-soluble vitamins can be used
as a biomarker to assess their estimated mean intakes.
As training schoolchildren to collect urine samples iseasier than completing weighed food records, a nutritional assessment for water-soluble vitamins using urine
samples and recovery rates is expected to be one of theapplications of the present study.
In conclusion, for free-living Japanese schoolchildren
aged 10–12 years, we found that 24 h urinary levels ofwater-soluble vitamins, except for vitamin B12, correlatedwith their recent intakes, and can be used as a biomarker
to assess, compare and validate estimated mean intakes ofwater-soluble vitamins.
Acknowledgements
Source of funding: This study represents the results of‘Studies on the construction of evidence to revise theDietary Reference Intake for Japanese people – Elucidation of the balance of micronutrients and major elements’(Principal Investigator: Katsumi Shibata), which was
supported by a research grant for Comprehensive RelatedDiseases from the Ministry of Health, Labour and Welfareof Japan. Conflict of interest: The authors have no conflictof interest to declare. Author responsibilities: T.T.
designed the study, performed experiments, completed
the statistical analysis and prepared the manuscript. T.F.
helped design the study, performed experiments andassisted with data analysis. S.S. reviewed the study andassisted with data analysis. K.S. contributed to the study
design and supervised the study. All authors criticallyreviewed the manuscript. Acknowledgements: We thankall the schoolchildren and their families who supported
this assessment. We also thank the teachers in Orizu
Elementary School and the staff of the school board in
Inazawa City, who expressed understanding and cooperated
with this assessment.
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Vol.3, No.11, 672-676 (2011)

Healthdoi:10.4236/health.2011.311113

Estimation of mineral and trace element intake in vegans
living in Japan by chemical analysis of duplicate diets
Munehiro Yoshida*, Noriko Ôgi, Yuki Iwashita
Laboratory of Food and Nutritional Sciences, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Japan;*
Corresponding Author: hanmyou4@kansai-u.ac.jp
Received 10 September 2011; Revised 5 October 2011; Accepted 21 October 2011.

ABSTRACT
Thirty-six daily duplicate diet samples were collected from 12 healthy female Japanese vegans
and sodium, potassium, calcium, magnesium,
phosphorus, iron, zinc, copper, manganese,
iodine, selenium, chromium and molybdenum inthe diets were measured to estimate mineraland trace element intake by Japanese vegans.
Significantly higher intake of potassium, magnesium, phosphorus, iron, copper, manganeseand molybdenum was observed in vegans thanin general Japanese women, but no difference
was observed in sodium, iodine, selenium andchromium intake. Vegan calcium intake tended
to be low compared to that of general women
but the difference was not significant. Sincehigh potassium, magnesium and iron intakescannot be achieved by general Japanese dietsand high intake of potassium and magnesium
may prevent hyperextension and cardiovasculardisease in vegans, there are few problems withJapanese vegan diets regarding mineral andtrace element intake, except for calcium intake,which is low as it is in the general Japanesepeople.
Keywords: Vegan; Mineral intake; Trace Element
Intake; Duplicate Diets; Japan

increases the risk of several micronutrient deficiencies,
including vitamin B12, vitamin D and n-3 fatty acids [2].
Regarding the intake of minerals and trace elements,
vegetarians, including vegans, show low intakes of calcium, zinc and selenium because the main sources ofthese micronutrients are animal foods in Western diets[3,4].
Traditional Asian diets are predominately plant-based,differing from Western diets. In Japan, although theconsumption of meat and dairy products has increased
along with the Westernization of society, more than
three quarters of the energy intake still depends on plantfoods [5]. Accordingly, it is thought that the effect of
adopting a vegan diet on the nutrient intake pattern isdifferent between the West and Japan. However, little
research has examined the nutrient intake of vegetarians
in Japan [6], and research on the intake of minerals andtrace elements by Japanese vegans is scarce. In the present study, to evaluate mineral and trace element intake
by Japanese vegans, duplicate diet samples were collected from Japanese vegans, and concentrations of sodium, potassium, calcium, magnesium, phosphorus, iron,zinc, copper, manganese, iodine, selenium, chromium
and molybdenum were measured.

2. SUBJECTS AND METHODS
2.1. Subjects and Duplicate Diet
Sampling

Vegetarian diets, essentially excluding animal foods,
have become increasingly popular in developed countries [1]. These diets are classified according to the typesof animal foods consumed, and strict vegetarians consuming no foods of animal origin are known as vegans.
Although vegan diets cause lower serum cholesterol,
lower blood pressure and a reduced risk of cardiovascular diseases, eliminating all animal foods from the dietIn the present study, vegans were defined as people
eating food of plant origin only. Twelve healthy femalevegans were recruited through a vegetarian food shop
located in Chiba Prefecture, Japan. The characteristics ofthe subjects are described in Table 1. Duplicate meals,
beverages and between-meal snacks were collected over
24 h period; 36 duplicate diets from 12 subjects were
sampled for 3 consecutive days between September andNovember 2010. All subjects gave informed consent for
the use of their personal information in this study.

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1. INTRODUCTION

M. Yoshida et al. / Health 3 (2011) 672-676
Table 1. Characteristics of vegan subjects (n = 12).
Mean ± SD

MedianAge (y)48.4 ± 12.9

47.5

Duration of vegan diet (y)20.7 ± 14.5

12.0

Height (cm)156.4 ± 7.7

157.0

Weight (kg)49.1 ± 8.9

48.5

Body mass index (kg/m2)

19.9 ± 2.4

19.7

2.2. Treatment of Samples
The daily duplicate diet sample was freeze-dried, homogenized and milled. Approximately 1 g of the driedsample was mixed with 200 mL of 1% HCl, shaken for
30 min and centrifuged. The supernatant was filtrated
with 0.45-µm membrane filter. Filtrate thus obtained
was used for the determination of sodium and potassium.
Another 1 g of the dried sample was heated with 10 mL
metal-free HNO3 until the disappearance of insolublecomponents, and then, 2 mL metal-free HClO4 wasadded to the digestion mixture, which was further heateduntil the appearance of white vapor of HClO4. The volume of the digest was made up to 10 mL with pure water.
Diluted digest thus obtained was used for the determination of calcium, magnesium, phosphorus, iron, zinc,copper, manganese, selenium and molybdenum. For the
analysis of chromium, approximately 1 g of the driedsample was heated in an electric furnace (F-B1414M; As
One, Osaka, Japan) at 550˚C for 16 h [7]. After dry incineration, the remaining ash was dissolved in 10 mL of
0.1 M HNO3. Iodine in the dried samples was extractedwith 0.5% tetramethylammonium hydroxide (TMAH)[8]. Two hundred milligrams of the dried samples was
mixed with 40 mL of 0.5% TMAH and left overnight.
The mixture was heated at 60˚C for 6 h and centrifuged.
The supernatant was filtrated through a 0.45-µm membrane filter.

673
673

analysis, pulse; isotopes monitored, 52Cr, 82Se, 95Mo,
97
Mo, 98Mo and 127I. Rhodium (103Rh) and tellurium
126( Te, 128Te and 130Te) were used as internal standards.
Phosphorus was determined with vanadomolybdate absorption spectrometry [9]. Protein, total lipid and energy
were analyzed by a commercial service system (Japan
Functional Food Analysis and Research Center, Fukuoka,
Japan).

2.4. Statistical Analysis
For each subject, mean daily intake was calculated
from the analytical results of duplicate diet samples from
3 consecutive days. The mean and median of the daily
intake for 12 subjects were then calculated. For iodine,
the mean and median were also calculated when eachvalue was logarithmically transformed because values
highly varied. Mean daily intake for 12 subjects was
statistically compared with the mean daily intake bygeneral Japanese women aged 30 to 49 y described in
the National Health and Nutritional Survey in Japan(NHNSJ) [10] by calculation of the Z-score; in which
women aged 30 to 49 y in NHNSJ, 2008 (n = 1053)
were regarded as a population.

3. RESULTS AND DISCUSSION

Sodium, potassium, calcium, magnesium, iron, zinc,copper and manganese were measured using atomic absorption spectrometer (AA-6300; Shimadzu, Kyoto, Japan). Iodine, selenium, chromium and molybdenum were
determined by inductively coupled plasma mass spectrometry (ICPMS) with direct nebulization. The ICPMS
operating conditions were as follows: instrument, ICPM8500 (Shimadzu); forward power, 1200 W; coolant gas
flow rate, 7.0 L/min; auxiliary gas flow rate, 1.5 L/min;nebulizer gas flow rate, 0.58 L/min; sampling depth, 5.0mm; integration time, 2.0 s; number of run, 20; mode of

In Table 2, daily intake of major nutrients, minerals
and trace elements by 12 Japanese female vegans was
summarized and compared with those by general Japanese women and several criteria in the Dietary Reference
Intakes for Japanese (DRIJ) [11]. For the intake of energy, protein and total lipids, no difference was observed
between vegans and general women.
Among major mineral intake, calcium intake by vegans was below the estimated average requirement(EAR) and tended to be low compared to that by thegeneral population. In several Western researches, calcium intake by vegans was markedly lower than that byomnivores [12] and lacto-vegetarians [13]. In the present
analysis, vegan calcium intake was somewhat low but
was not significantly lower than in the general Japanese
calcium intake. Since calcium intake by general Japanese people is always low due to the low consumption of
dairy products, the low calcium intake of Japanese vegans may be inconspicuous.
Phosphorus intake by vegans was markedly higher
than by general women. In Western research, a vegandiet contains low phosphorus and is appropriate for patients with renal failure [14]. In the West, because themajor source of phosphorus in general diets is dairy
products, vegan phosphorus intake is comparatively low;
however, Japanese people ingest phosphorus mainly
from plant foods [5]. The difference in the source of

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2.3. Analysis

674

M. Yoshida et al. / Health 3 (2011) 672-676

Table 2. Intake of energy, protein, lipids, minerals and trace elements in Japanese vegans.
NHNSJ, 20081)

Vegans (n = 12)

DRIJ, 20102)

Mean ± SD

MedianMean ± SD

Median

EAR

RDA

AI

DG

UL

Energy (kcal)

1847 ± 141

1840

1682 ± 469

1645

1750

-

-

-

-

Protein (g)56.2 ± 8.1

58.4

60.2 ± 19.0

58.7

40

50

-

-

-

Lipids (% energy)

20.8 ± 7.3

21.0

24.5 ± 14.1

22.6

-

-

-

20 - 25-

Sodium (mg)3649 ± 1719

3029

3696 ± 14153)

3538 3)590 3)--<29503)-

Potassium (mg)3610 ± 1272*

3217

1983 ± 777

1891

-

-

2000

2800

-

Calcium (mg)361 ± 122389

440 ± 224

406

550

650

-

-

2300

Magnesium (mg)494 ± 112*462

214 ± 80

204

240

290

-

-

-

Phosphorus (mg)1225 ± 311*

1197

854 ± 284

830

-

-

-

-

3000

Iron (mg)13.0 ± 2.4*

12.2

6.9 ± 3.0

6.5

9.0

11.0

900

-

40

Zinc (mg)8.3 ± 1.6

9.1

7.1 ± 2.4

6.9

8

9

-

-

35

Copper (mg)1.75 ± 0.37*

1.66

1.00 ± 0.35

0.96

0.6

0.7

-

-

10

7.5 ± 2.2

7.9

-

-

-

-

3.5

-

11

1158

-

-

95

130

-

-

2200

Manganese (mg)
Iodine (µg)

1865 ± 1934
4)

5)

788 (255 - 2441)

746

Selenium (µg)

87 ± 34

76

-

-

20

25

-

-

230

Chromium (µg)

27 ± 8

28

-

-

25

30

-

-

-

Molybdenum (µg)

540 ± 207

563

-

-

20

25

-

-

500

1)

*Significant difference from NHNSJ data was observed at p < 0.001 by calculation of Z-score; Values for general Japanese women aged 30 to 49 y (n = 1053)
quoted from the National Health and Nutrition Survey in Japan, 2008 [10]; 2)Criteria for Japanese women aged 30 to 49 y in Dietary Reference Intakes forJapanese, 2010 [11]; EAR, estimated average requirement; RDA, recommended dietary allowance; AI, adequate intake; DG, tentative dietary goal for preventing lifestyle-related diseases; UL, tolerable upper intake level; 3)Calculated from the values for salt; 4)Geometrical mean with SD range in parentheses; 5)Mediancalculated after logarithmic transformation of data for each daily duplicate diet sample.

phosphorus may contribute to the difference in phosphorus intake between Western and Japanese vegans. In
addition, phytate may contribute to the high phosphorus
intake in vegans because whole grains and beans containit at a high level.
No difference was observed between vegans and general women in sodium intake. On the other hand, veganpotassium intake was markedly higher than by general
women and far exceeded the tentative dietary goal for
preventing lifestyle-related diseases (DG) in DRIJ.
Similarly, markedly higher magnesium intake was observed in vegans than in general women. This high intake of potassium and magnesium is probably due to thehigh consumption of vegetables and fruit.
Among trace element intake, significantly higher ironand copper intake was observed in vegans than in general women. Similarly, manganese and molybdenum
intake by vegans was markedly higher than by general
Japanese, as described in several reports [15,16]. Intakeof these four trace elements far exceeded the recom-

mended dietary allowance (RDA) or the adequate intake(AI) in DRIJ. High intake of copper and manganese isalso reported in Western researches [17], probably, because the high consumption of whole grains and beans
results in high intake of these trace elements. The mean
and median of vegan molybdenum intake exceeded thetolerable upper intake level of this element in DRIJ. Thisis also caused by high consumption of cereals and beans
since they particularly soybean, contain molybdenum at
a high level [16].
Although vegan zinc intake has been reported to be
low [12], there was no difference between vegans andgeneral women; however, because it has been reported
that the serum zinc level in Japanese vegetarians tends tobe low [18], it is necessary to examine whether phytate
and/or dietary fiber, which are contained in whole grains
and beans at a high level, decrease the bioavailability ofzinc in Japanese vegan diets.
Since the main sources of selenium in general Japanese diets are fish, meats and eggs [19], the low sele-

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nium intake by Japanese vegans is concern; however,selenium intake by Japanese vegans was comparable tothat by general Japanese described in several previous
reports [19-21]. Japanese vegans may ingest selenium
from imported wheat and soybeans, which contain selenium at a high level [22]. Similarly to selenium intake,iodine and chromium intake by vegans was also comparable to general Japanese people described in the literature [20,23].
In conclusion, Japanese vegans are estimated to ingest
high potassium, magnesium, phosphorus, iron, copper,
manganese and molybdenum compared to general Japanese people. In particular, high potassium, magnesium
and iron intake cannot be achieved by ingesting general
Japanese diets. High intake of potassium and magnesium
may lead to the preventing of hyperextension and cardiovascular disease in vegans [24]. Accordingly, there
are few problems with Japanese vegan diets regarding
mineral and trace element intake, except for calcium
intake, which is low as it is in general Japanese people.

4. ACKNOWLEDGEMENTS
This study was supported by a grant for comprehensive research on
cardiovascular and lifestyle disease from the Ministry of Health, Labour and Welfare of Japan.

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and Giovannetti, S. (1996) A low-nitrogen low-phosphorus vegan diet for patients with chronic renal failure.
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Openly accessible at http://www.scirp.org/journal/HEALTH/Asia Pac J Clin Nutr 2011;20 (1):56-61

56

Original Article

High prevalence of hypovitaminosis D and K in patientswith hip fracture
Tetsuo Nakano MD1, Naoko Tsugawa PhD2, Akiko Kuwabara PhD3,4, Maya Kamao MSc2,
Kiyoshi Tanaka MD4, Toshio Okano PhD2
1

Department of Orthopedics, Tamana Central Hospital, Tamana, Kumamoto, Japan
Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan3
Department of Health and Nutrition, Osaka Shoin Women’s University
4
Department of Food and Nutrition, Kyoto Women's University, Kyoto, Japan
2

Although hip fracture is considered to be associated with hypovitaminosis D and K, few reports have previously
studied both of them. We have studied the vitamin D- and K-status as well as the general nutritional status in ninety-nine patients with hip fracture. Mean serum concentration of 25hydroxy-vitamin D (25OH-D) in female
fractured patients was only approximately 9 ng/mL, suggesting severe vitamin D deficiency. There was no significant difference between the two groups in serum concentration of intact parathyroid hormone in both genders
and serum 25OH-D levels in the male subjects. Plasma concentrations of phylloquinone (vitamin K1; PK) andmenaquinone-7 (MK-7) were significantly lower in the fractured group than in the control group in both genders.
Logistic regression analysis indicated that circulating concentrations of albumin, PK and 25OH-D were the significant and independent determinants of fracture risk, with their higher concentrations associated with decreased
fracture risk. Finally, principal component analysis (PCA) was performed to summarize the clinical parameters
into smaller numbers of independent components. Three components were obtained, each representing the overall nutritional status, the vitamin D status, and the vitamin K status. In conclusion, our study has shown that patients with hip fracture have vitamin D and K deficiency independent of general malnutrition.

Key Words: hypovitaminosis D, hypovitaminosis K, patients with hip fracture, general malnutrition, principal component analysis

INTRODUCTION
Hip fracture is the most serious consequence of osteoporosis. In addition to the high mortality rates after fracture,even the survivors suffer from functional impairment andlimited daily activities.1 With increased percentage of theelderly in the society, the incidence of hip fracture is constantly increasing in Japan, as in other countries.2 Hip
fracture is also considered to be a great burden to the society because of costly medical expenditure.3
Among the various risk factors of hip fracture so far
reported are the nutritional ones including poor vitamin D
and K status. “Vitamin deficiency” causes various disorders
with phenotypic abnormalities, such as osteomalacia andrickets by vitamin D deficiency, and clotting abnormality
by vitamin K deficiency. Recently, however, it is known
that inadequate supply of vitamins, even in the milder
form, causes increased susceptibility to various diseases,
and is called vitamin insufficiency.4 For example, vitamin
D insufficiency, through decreased calcium absorption
and negative calcium balance, is associated with decreased bone mineral density (BMD) and increased riskof fracture. The prevalence of hypovitaminosis D has
been reported to be quite high in patients with hip fracture
in various countries.5-7
The most essential role of vitamin K is to act as thecoenzyme in the γ-carboxylation of glutamic acid residue(glu) to γ-carboxyglutamin acid (gla) residue, throughwhich four of the clotting factors acquire calcium binding
capacity. It has long been held that the sole physiologicalaction of vitamin K is the γ-carboxylation of these clotting factors in the liver. Recently, however, extrahepatic
action of vitamin K has come to receive much attention.8
For example, mice devoid of the matrix gla protein (MGP)
gene, which is a gla-containing protein present in theskeleton and vasculature, died of severe arterial calcification.9 Although mice lacking the osteocalcin gene had
apparently higher bone mineral density than the control
ones, they were more susceptible to bone loss after ovariectomy than their normal littermates, suggesting thecompromised bone quality in these mice.10 There also
have been clinical observations to show the association
between vitamin K inadequacy and hip fracture. For example, high intake of vitamin K was associated with

Corresponding Author: Dr Akiko Kuwabara, Department of
Health and Nutrition, Osaka Shoin Women’s University, 4-2-26
Hishiyanishi, Higashiosaka-shi, Osaka 577-8550 Japan.
Tel: +81-6-6723-8181; Fax: +81-6-6723-8348
Email: kuwabara.akiko@osaka-shoin.ac.jp
Manuscript received 2 August 2010. Initial review completed 8
November 2010. Revision accepted 27 January 2011.

T Nakano, N Tsugawa, A Kuwabara, M Kamao, K Tanaka and T Okano
decreased risk of hip fracture,11 and high serum concentration of undercarboxyled osteocalcin (ucOC), which is asensitive indicator of insufficient vitamin K action in theskeleton, was a significant risk factor of hip fracture independent of BMD.12
Despite these observations, there have been few reports
to evaluate the status of these two bone-active vitamins in
hip fractured patients.13 Thus in the current study, we
have studied the serum concentration of these two boneactive vitamins in patients with hip fracture and agematched controls.
MATERIALS AND METHODS
Subjects
Consecutive patients with hip fracture transferred to Tamana Central Hospital were studied. The duration of theenrollment was 6 months. Written informed consent was
obtained in 99 cases from the patients or a family member
when obtaining the patients’ approval was practically
impossible because of their poor general condition. Agematched nursing home residents in close proximity to thehospital in Tamana City served as the control. Those
without severe liver or kidney dysfunction, or those receiving bone-active drugs or supplementation with vitamin D or K, were encouraged to participate in the study,
and the consent was obtained in 48 cases.
Informed consent was similarly obtained in 48 cases.
Their background profiles are shown in Table 1. The study
protocol was approved by the Ethical Committee of Tamana Central Hospital.
Laboratory data
Blood was drawn within 24 hours following the fracture.
After centrifugation, plasma and serum were stored underdark condition at –30C until assay. Serum concentration57

of 25 hydroxy-vitamin D (25OH-D) was measured byradioimmunoassay (RIA) (DiaSorin, Stillwater, MN, USA).
Serum level of intact parathyroid hormone (PTH) was
measured by electro chemiluminescent immunoassay(ECLIA) (Roche Diagnostics, Mannheim, Germany).
Plasma vitamin K1 (phylloquinone; PK), and K2 (menaquinone-7; MK-7) levels were determined by highperformance liquid chromatography-tandem mass-mass
spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS) using a HPLC system (Shimadzu, Kyoto, Japan) and API3000 LC-MS/MS System(Applied Biosystems, Foster City, CA) with 18O-labeled
vitamin K as the internal standard.14
Statistical analyses
Statistical analyses were done with SPSS 17.0J. Comparison of two independent groups was done with Student’s ttest or Mann-Whitney test depending on normality. The
association between vitamin status and the occurrence of
hip fracture was analyzed by logistic regression analysis.
The relationship between various nutritional indices andcirculating vitamin D- and K-levels was analyzed withprincipal component analysis (PCA) as previously described.15
RESULTS
Blood tests
Baseline characteristics and data from blood examination
are shown in Table 1. Serum albumin concentration was
significantly lower in the fractured group in both genders,and serum cholesterol concentration and blood hemoglobin level were significantly lower in female patients withfracture. In Table 2 shows the blood concentrations ofvitamin D, vitamin K and related molecules. Mean serum
concentration of 25OH-D, which most reliably represents

Table 1. Patients’ profiles
Male
Age
Serum albumin (g/dL)
Serum cholesterol (mg/dL)
Serum BUN (mg/dL)
Hemoglobin (g/dL)
Serum GOT (U/L)
Serum GPT (U/L)

Control (n=13)82.2±9.3
4.3±0.5
175.4±41.9
24.1±2.2
12.4±2.2
26.2±20.4
19.3±16.2

FemaleFracture (n=27)82.6±7.6
3.5±0.5**
156.1±36.6
29.5±26.1
11.9±1.932.1±9.4
22.8±21.1

Control (n=35)84.1±7.8
4.4±0.2
232.3±37.0
20.6±7.4
12.5±1.1
23.9±7.2
13.7±8.6

Fracture (n=72)85.5±7.0
3.6±0.4**
179.4±39.4**
20.6±10.2
10.8±1.8**
20.9±7.5
14.0±8.4

Data are shown as mean ± SD. The asterisk (**) denotes that the value in fracture group is significantly different from that in control group(p<0.01) by Student’s t-test. BUN, GOT, and GPT are abbreviations for blood urea nitrogen, glutamyl oxaloacetic transaminase, glutamyl
pyruvate transaminase, respectively.

Table 2. Serum concentrations of vitamin D, vitamin K and related molecules
Male
Serum 25OH-D (ng/mL)
Serum intact PTH (pg/mL)
Plasma PK (ng/mL)
Plasma MK-7 (ng/mL)

Control (n=13)20.7±7.364.3±53.7
0.55±0.31
4.28±3.75

FemaleFracture (n=27)19.0±13.0
61.4±34.4
0.31±0.24*
1.60±1.60**

Control (n=35)
18.6±6.3
56.0±23.2
0.77±0.36
10.8±7.01

Fracture (n=72)
9.1±4.6**67.8±33.9
0.46±0.36**
2.67±4.13**

Data are shown as mean ± SD. The asterisk denotes that the value in fracture group is significantly different from that in control group (*;p<0.05, **; p<0.01) by Student’s t-test. 25OH-D, PK , and MK-7 are the abbreviations for 25 hydroxy-vitamin D, phylloquinone, and menaquinone-7, respectively.

58

Hypovitaminosis D and K in hip fractured patients

Table 3. Logistic regression analysis
Odds ratio (95%CI)
0.246 (0.090-0.673)
0.072 (0.009-0.612)
0.003 (0.000-0.054)
0.867 (0.747-1.006)
1.482 (0.891-2.465)
2.464 (0.381-15.95)

Serum 25OH-D (per 10ng/mL increase)
Plasma PK (per 1ng/mL increase)
Albumin (per 1g/dL increase)MK-7 (per 1ng/mL increase)
Hemoglobin (per 1g/dL increase)
Sex (1; Male, 2; Female)

p value<0.001
0.016<0.001
0.061
0.129
0.344

Logistic regression analysis with stepwise method was done. Sex, circulating concentrations of albumin, hemoglobin, 25OH-D, PK, and MK7 were included for analysis.

Table 4. Principal component analysis of nutrition indicesSerum Albumin
Serum total Cholesterol
Hemoglobin
Serum 25OH-D
Plasma PK
Plasma MK-7

Component 1†
0.744
0.824†
0.538†
0.035
0.191
0.773†

Component 2
0.481†
0.098
0.589†
0.902†
0.109
0.009

Component 3-0.0280.157-0.2690.228
0.922†
0.210†

Factor loadings to three components after varimax rotation are shown. Loadings greater than 0.35

the vitamin D status, was approximately 20 ng/mL in allgroups, except for the female fracture group where it wasapproximately 9 ng/mL. In both genders, serum 25OH-D
levels were lower than 20 ng/mL in 90% and 61% of subjects, in the fracture and control groups, respectively. It
was below 10 ng/mL in 50% and 7% of subjects in thefracture and control group, respectively. Serum concentration of intact PTH, which is a sensitive indicator ofvitamin D insufficiency; hence secondary hyperparathyroidism, was not different between control and fracturegroups in males. It was slightly higher in the fractured
group than in the control group in female, which, however, did not reach statistical significance (p=0.07).
Serum concentrations of PK and MK-7 were significantly lower in the fracture group than in the control
group in both genders.
Logistic regression analysis for variables associatedwith hip fracture
In order to evaluate whether the above-mentioned vitamin
insufficiency is related to the occurrence of hip fracture,logistic regression analysis was performed. Of the factors
subjected for analysis, circulating concentrations of albumin, PK and 25OH-D were the significant determinants, whereas MK-7, gender or hemoglobin level wasnot (Table 3). The odds ratio for fracture markedly decreased in accordance with increased concentrations ofalbumin, PK and 25OH-D.
Principal component analysis (PCA)
Since patients with hip fracture are generally malnourished, we considered it to be important whether the low
vitamin D- and K-status as described above simply reflects
overall malnutrition. Then PCA was performed with parameters included for analysis being: serum albumin andcholesterol concentrations, blood hemoglobin levels, andplasma 25OH-D, PK and MK-7. Three components wereobtained as shown in Table 4. The first component wascontributed by high serum albumin, total cholesterol,
blood hemoglobin and plasma MK-7. The second component consisted of high serum albumin, blood hemoglobin and serum 25OH-D. The third component was composite of high plasma PK. Each component was interpreted as follows; the first, second, and third component
representing overall nutritional status, vitamin D status,
and vitamin K1 status, respectively.
DISCUSSION
In the present study, we have studied the blood concentration of 25OH-D, PTH, PK, MK-7 and other nutritional
indices. In 90% of patients with hip fracture, serum 25OHD level was lower than 20 ng/mL which is a generally
accepted cut-off for hypovitaminosis D. In half of thepatients, serum 25OH-D concentration fell into the severe
hypovitaminosis D range of below 10 ng/mL. Nurmi et al.
reported that serum 25OH-D level was lower than 15
ng/mL and 8 ng/mL in 53% and 9%, respectively, of thepatients with hip fracture in Finland.16 In a study on Japanese patients with hip fracture, Sakuma et al. reported
that 62% of the patients had their serum 25OH-D level
below 20 ng/ml.7 Thus, the prevalence of hypovitaminosis D in the present study was compatible with the previous studies, but was even higher.
Serum concentration of 25OH-D in the fracture groupwas significantly lower than that in the control group in
women, but not in men. There have been some reports toshow that elderly women are more prone to vitamin D
deficiency than elderly men. Hirani et al. reported thathypovitaminosis D was more prevalent in women thanmen with a odds ratio of 2.1.17 Maggio et al. reported thatage-related decline of serum 25OH-D was already evident
shortly after age 50 in women, whereas in men it started
only after age 70.18 Thus there seems to be a gender dif-T Nakano, N Tsugawa, A Kuwabara, M Kamao, K Tanaka and T Okano

ference that women are more prone to vitamin D inadequacy, for which there is no clear explanation at present.
Lack of significant difference in serum PTH level between fracture and control groups is most likely due tothe large standard deviation in serum PTH concentration.
However, there still can be alternative explanations. There
have been some reports describing the absence of PTH
elevation in face of hypovitaminosis D in patients withhip fracture.19-22 Sahota et al. studied the vitamin D status
in the post-hip fracture patients. They found that only halfof them had elevated serum PTH levels, the rest had normal to low serum PTH levels in face of hypovitaminosis
D.19 As an explanation for this apparently paradoxical
observation, they postulated magnesium deficiency as theunderlying cause since magnesium deficiency is known to
be associated with impaired PTH secretion.20 Thus thequestion has now come to our attention whether skeletal
impairment in hypovitaminosis D can be explained bysecondary hyperparathyroidism alone. A recent paper from
Finland also reported that serum PTH level was within
the reference range despite hypovitaminosis D in 74.8%
of the bedridden geriatric patients.21 Patients in the lowest
quartile of serum PTH level were associated with the history of hip fracture (odds ratio 2.9). Thus it is obviousthat hypovitaminosis D is associated with increased riskof hip fracture, although further studies are required todetermine whether it is mediated by secondary hyperparathyroidism or due to hypovitaminosis per se.
Compared to vitamin D, far smaller number of papers
has been published on the relationship of vitamin K withhip fracture. Epidemiological studied have shown thathigher intake of vitamin K is associated with lower risk of
hip fracture.11,23 Among the two vitamin K analogs studied here, PK seems to best represent the vitamin K statusof these subjects. Kaneki et al. reported that there is alarge geographic difference in serum MK-7 concentrationin Japan, which could be accounted for by the frequency
of consuming natto, which contains extraordinary amountof MK-7.24 Blood concentrations of PK and MK-7 wereconsistently lower in fractured patients than control subjects in both genders.
Kawana et al. reported that there was no significant alteration in the circulating concentrations of PK and MK-7
in hip fractured patients.25 In their paper, these concentrations were below the detection limit in the substantial
number of subjects. Blood vitamin K levels were reported
to be below the detection limit in other papers also.24,26 In
our data using newly developed LC-APCI-MS/MS method for the determination of circulating vitamin K levels,
serum concentrations of PK and MK-7 were detectable inalmost all subjects.14 Thus, previous reports using lesssensitive assay methods should be interpreted with caution.
In fractured subjects, serum albumin concentration
was significantly lower in both genders, and hemoglobin
level and serum cholesterol concentration was significantly lower in the females. Thus patients with hip fracture
are malnourished. Then it was considered mandatory toanalyze the relationship between the overall malnutritionand decreased levels of circulating these vitamins. We
have studied it with two analytical procedures; logistic
regression analysis and principal component analysis(PCA). Logistic regression analysis revealed that serum

59

concentrations of 25OH-D, PK and albumin were significant contributing factors for fracture risk, and suggested
that circulating 25OH-D and PK levels contributed to theincreased risk of fracture independent of general malnutrition.
Finally PCA was done. Three components were obtained, representing overall nutritional status, vitamin D
status, and vitamin K status, respectively. Since thesecomponents are, by their definition, independent of eachother, these results strongly suggest that hypovitaminosis
D and K in patients with hip fracture is not merely a manifestation of general malnutrition. At present, the reason
for the association of MK-7 with the first component,
representing the overall nutritional status is not known.
We have also recently reported that institutionalized elderly subjects had high prevalence of hypovitaminosis D
and K, which is independent of general malnutrition by
PCA.15
One of the limitations of the current work is that it is acase control study, but not a prospective one. Since theassociation of hip fracture with the insufficiency of twobone-active vitamins; vitamin D and vitamin K has been
scarce, we have done this study as the initial step.
Another limitation is that the nursing home residents
adjacent to the hospital were the control subjects. It is
unclear whether the control subjects represent the average
Japanese elderly population or not. However, it is quite
unlikely the nursing home residents have nutritional status far better than the average Japanese elderly. Rather,
they are likely to be equal to or worse than the average.
Thus, we believe that our finding that the blood levels ofthese vitamins in fractured patients were even lower thanthat in nursing home residents has clinical implications.
In summary, patients with hip fracture had lower serum concentration of vitamin K in both genders, and lower serum concentration of vitamin D in female subjects.
Since blood samples were obtained within 24 hours after
fracture, these data is likely to represent the patients’ status before fracture. Lower serum albumin concentrationin fractured patients suggests that these subjects are also
generally malnourished. Insufficiency of these vitamins
as well as the overall malnutrition is likely to predisposeelderly people to hip fracture, and intervention study tocorrect these abnormalities is needed.
ACKNOWLEDGMENTS
This study was supported by Health and Labor Science Research Grant from the Ministry of Health, Labor and Welfare,Japan, and Grant-in-Aid for Scientific Research from Japan
Society for the Promotion of Science (JSPS).
AUTHOR DISCLOSURES
None of the authors have any conflicts of interest.
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3. Kanis JA, Brazier JE, Stevenson M, Calvert NW, Lloyd
Jones M. Treatment of established osteoporosis: a system-

60

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6. Nurmi I, Kaukonen JP, Luthje P, Naboulsi H, Tanninen S,
Kataja M, Kallio ML, Leppilampi M. Half of the patientswith an acute hip fracture suffer from hypovitaminosis D: aprospective study in southeastern Finland. Osteoporos Int.
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7. Sakuma M, Endo N, Oinuma T, Hayami T, Endo E, Yazawa
T, Watanabe K, Watanabe S. Vitamin D and intact PTH status in patients with hip fracture. Osteoporos Int. 2006;17:
1608-14.
8. Vermeer C, Shearer MJ, Zittermann A, Bolton-Smith C,
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9. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer
RR, Karsenty G. Spontaneous calcification of arteries andcartilage in mice lacking matrix GLA protein. Nature.
1997;386:78-81.
10. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C
et al. Increased bone formation in osteocalcin-deficient mice.
Nature. 1996;382:448-52.
11. Booth SL, Tucker KL, Chen H, Hannan MT, Gagnon DR,
Cupples LA et al. Dietary vitamin K intakes are associatedwith hip fracture but not with bone mineral density in elderly men and women .Am J Clin Nutr. 2000;71:1201-8.
12. Seibel MJ, Robins SP, Bilezikian JP. Serum undercarboxylated osteocalcin and the risk of hip fracture. J Clin Endocrinol Metab. 1997;82:717-8.
13. Roberts NB, Holding JD, Walsh HP, Klenerman L, Helliwell T, King D, Shearer M. Serial changes in serum vitamin
K1, triglyceride, cholesterol, osteocalcin and 25hydroxyvitamin D3 in patients after hip replacement for
fractured neck of femur or osteoarthritis. Eur J Clin Invest.
1996;26:24-9.
14. Suhara Y, Kamao M, Tsugawa N, Okano T. Methods for thedetermination of vitamin K homologues in human plasmausing high-performance liquid chromatography-tandem
mass spectrometry. Anal Chem. 2005;77:757-63.
15. Kuwabara A, Himeno M, Tsugawa N, Kamao M, Fujii M,
Kawai N et al. Hypovitaminosis D and K are highly preva-

lent and independent of overall malnutrition in the institutionalized elderly. Asia Pac J Clin Nutr. 2010;19:49-56.
16. Nurmi I, Kaukonen JP, Lüthje P, Naboulsi H, Tanninen S,
Kataja M, Kallio ML, Leppilampi M. Half of the patientswith an acute hip fracture suffer from hypovitaminosis D: aprospective study in southeastern Finland. Osteoporos Int.
2005;16:2018-24.
17. Hirani V, Primatesta P. Vitamin D concentrations amongpeople aged 65 years and over living in private households
and institutions in England: population survey. Age Ageing.
2005;34:485-91.
18. Maggio D, Cherubini A, Lauretani F, Russo RC, Bartali B,
Pierandrei M et al. 25(OH)D Serum levels decline with age
earlier in women than in men and less efficiently prevent
compensatory hyperparathyroidism in older adults. J Gerontol A Biol Sci Med Sci. 2005;60:1414-9.
19. Sahota O, Gaynor K, Harwood RH, Hosking DJ. Hypovitaminosis D and “functional hypoparathyroidism” –the NoNoF (Nottingham Neck of Femur) study. Age Ageing. 2001;
30:467-72.
20. Sahota O, Mundey MK, San P, Godber IM, hosking DJ.
Vitamin D insufficiency and the blunted PTH response inestablished osteoporosis: the role of magnesium deficiency.
Osteoporos Int. 2006;17:1013-21.
21. Björkman MP, Sorva AJ, Risteli J, Tilvis RS. Low parathyroid hormone levels in bedridden geriatric patients with vitamin D deficience. J Am Geriatr Soc. 2009;57:1045-50.
22. Fisher A, Srikusalanukul L, Davis M, Smith P. Hip fracture
type: Important role of parathyroid hormone (PTH) response
to hypovitaminosis D. Bone. 2010;47:400-7.
23. Feskanich D, Weber P, Willet WC, Rockett H, Booth SL,
Colditz GA. Vitamin K intake and hip fracture: a prospective study. Am J Clin Nutr. 1999;69:74-9.
24. Kaneki M, Hodges SJ, Hosoi T, Fujiwara S, Lyons A, CreanSJ et al. Japanese fermented soybean food as the major determinant of the large geographic difference in circulatinglevels of vitamin K2: possible implications for hip-fracturerisk. Nutrition. 2001;17:315-21.
25. Kawana K, Takahashi M, Hoshino H, Kushida K. Circulating levels of vitamin K1, menaquinone-4, and menaquinone-7 in healthy elderly Japanese women and patients withvertebral fractures and patients with hip fractures. Endocr
Res. 2001;27:337-43.
26. Hodges SJ, Akesson K, Vergnaud P, Obrant K, Delmas PD.
Circulating levels of vitamins K1 and K2 decreased in elderly women with hip fracture. J Bone Miner Res. 1993;
8:1241-5.

T Nakano, N Tsugawa, A Kuwabara, M Kamao, K Tanaka and T Okano

61

Original Article

High prevalence of hypovitaminosis D and K in patientswith hip fracture
Tetsuo Nakano MD1, Naoko Tsugawa PhD2, Akiko Kuwabara PhD3,4, Maya Kamao MSc2,
Kiyoshi Tanaka MD4, Toshio Okano PhD2
1

Department of Orthopedics, Tamana Central Hospital, Tamana, Kumamoto, Japan
Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan3
Department of Health and Nutrition, Osaka Shoin Women’s University
4
Department of Food and Nutrition, Kyoto Women's University, Kyoto, Japan2

髖部骨折病患維生素 D 與 K 不足之高盛行率
過去研究顯示髖部骨折與維生素 D 及維生素 K 不足有關，但較少研究將兩者共同納入探討。本研究之對象為 99 位有髖部骨折的病患，檢測其整體營養及體內
維生素 D 與維生素 K 的狀態。女性患者血清 25-羥化維生素 D(25OH-D)濃度平
均只有約 9 ng/mL，顯示女性患者有嚴重維生素 D 缺乏。男女性患者血清中副甲狀腺素及男性血清 25OH-D 平均濃度與對照組皆沒有顯著差異。然而在男女性髖
部骨折患者，其血漿維生素 K1 及維生素 K2 濃度都顯著較對照組低。以羅吉斯回歸分析發現，體內白蛋白、維生素 K1 及 25OH-D 濃度皆為骨折發生風險之顯著
獨立預測因子，具呈負相關。最後以主成份分析進行臨床參數統整後，獲得三項代表參數，分別代表整體營養狀態、維生素 D 營養狀態及維生素 K 狀態。總而
言之，本研究顯示髖部骨折患者易出現維生素 D 及維生素 K 缺乏，且與整體營
養不良無關。
關鍵字：維生素 D 缺乏、維生素 K 缺乏、髖部骨折病患、整體營養不良、主成
分分析

Asia Pac J Clin Nutr 2011;20 (1):50-55

50

Original Article

Bone is more susceptible to vitamin K deficiency thanliver in the institutionalized elderly
Akiko Kuwabara PhD1,4, Minori Fujii RD2, Nobuko Kawai CSW2, Kunihiko Tozawa SE3,
Shoko Kido PhD4, Kiyoshi Tanaka MD4
1

Department of Health and Nutrition, Osaka Shoin Women’s University, Osaka, Japan
Nursing Home Kayu-Shirakawa, Kyoto, Japan
3
Sanko Junyaku Co., Ltd., Tokyo, Japan
4
Department of Food and Nutrition, Kyoto Women’s University, Kyoto, Japan
2

In Japan, γ-carboxylation of blood coagulation factors is the basis for determining adequate intake (AI) for vitamin K in Dietary Reference Intakes (DRIs) issued in 2010. Recently, vitamin K is also known to be essential for
preventing fracture. In this study, relative susceptibility of liver and bone to vitamin K deficiency was studied.
Thirty-seven elderly institutionalized subjects were evaluated for vitamin K status by measuring serum PIVKA(protein induced by vitamin K absence) –II and ucOC (undercarboxylated osteocalcin) levels, as sensitivemarkers for hepatic and skeletal vitamin K deficiency, respectively. Serum PIVKA-II and ucOC levels, with
their cut-off values in the parentheses, were 20.2±8.9 mAUmL (28 mAU/mL) and 4.7±3.0 ng/mL (4.5 ng/mL),
respectively. Median vitamin K intake was approximately 200 μg/day, which is more than 3 times higher thanthe current Japanese AI. Vitamin K intake was significantly correlated with serum PIVKA-II and ucOC/OC levels, but not with serum ucOC level. Although serum ucOC level is generally a good indicator for vitamin K
status, multiple regression analysis revealed that elevated bone turnover marker significantly contributed to serum ucOC level. All subjects had vitamin K intake exceeding AI for vitamin K. Nevertheless, serum PIVKA-II
and ucOC concentrations exceeded the cut-off value in 14% and 43% of subjects, respectively. The present findings suggest that vitamin K intake greater than the current AI is required for the skeletal health in the institutionalized elderly.

Key Words: vitamin K, adequate intake, γ-carboxylation, ucOC, PIVKA-II

INTRODUCTION
Gamma-glutamyl carboxylase (GGCX) catalyzes the conversion of glutamyl (Glu) residue into γ-carboxyglutamyl(Gla) residue in certain proteins. The most fundamentalrole of vitamin K is the one as a cofactor of GGCX.1 Although GGCX is present in various tissues, its role in theliver has received most attention until recently. In theliver, conversion of Glu residue to Gla residue takes placein four of the blood coagulation factors (II, VII, IX, andX), by which they acquire calcium-binding ability and are
activated.1 Recently, attention have been focused on thephysiological roles of vitamin K-dependent proteins in
extrahepatic tissues such as bone and blood vessel.2,3 Osteocalcin is produced by osteoblasts, the most abundantnon-collagenous protein in the bone matrix. Through γcarboxylation, osteocalcin gains hydroxyapatite-binding
ability, and regulates bone mineralization.2 Recent evidences strongly suggest that skeletal vitamin K deficiency
increases the risk of hip fracture.4 Matrix Gla protein(MGP); another vitamin K-dependent protein, is an inhibitor of vascular calcification.5-7
In the current Japanese Dietary Reference Intakes(DRIs) issued in 2010, Adequate Intake (AI) for vitamin
K in the adult is uniformly 75 μg/day for men and 65
μg/day for women. These values however, carries someproblems when applied to the study population.8 First,
they are based on data from America or Europe. Since
nutrients intake is greatly dependent on nationality or
dietary patterns, vitamin K status in the Japanese must be
studied. Second, they are from healthy young volunteers,
not from the elderly who are likely to have nutrients malabsorption. This is especially the case with fat-soluble
vitamins including vitamin K due to various factors suchas decreased secretion of bile acids and pancreatic juice,
and reduced dietary fat intake.8 Finally, AI for vitamin K
was determined as the dose sufficient to maintain normal
blood coagulation with little mentioning to bone.8 Serumlevels of protein induced by vitamin K absence-II (PIVKAII) and undercarboxylated osteocalcin (ucOC) are sensitive markers for vitamin K deficiency in the liver andbone, respectively. Vitamin K status in the liver and bone

Corresponding Author: Dr Kiyoshi Tanaka, Department ofFood and Nutrition, Kyoto Women’s University, 35, Imakumano-kitahiyoshicho, Higashiyama 605-8501 Japan.
Tel: +81-75-531-7125; Fax: +81-75-531-7153
Email: tanakak@kyoto-wu.ac.jp
Manuscript received 30 May 2010. Initial review completed 4
October 2010. Revision accepted November 2010.

A Kuwabara, M Fujii, N Kawai, K Tozawa, S Kido and K Tanaka

can be separately evaluated by measuring these markers.
By employing such methodology, previous studies haveshown that much higher doses of vitamin K are needed
for the γ-carboxylation of osteocalcin than for that of blood
coagulation factors.9,10
Thus it is possible that an elderly judged to be vitamin
K sufficient based on the current AI has skeletal vitamin
K deficiency and increased fracture risk. In this paper, wehave measured serum PIVKA-II and ucOC levels, assessedvitamin K intake, and studied the prevalence of vitamin K
deficiency in the liver and bone in the institutionalized
elderly.
MATERIALS AND METHODS
Subjects
The study subjects were 37 institutionalized elderly (male8, female 29) in a nursing home, Kayu-Shirakawa. Exclusion criteria were routine medication that has potential
interference with bone metabolism and vitamin K status
such as warfarin. None had history of hepatic diseases.
Detailed information about this study was given and written consent was obtained from the subject or the proxy.
The study protocol was approved by the ethical committee in Kyoto Women’s University.
Laboratory data
Blood was obtained after overnight fasting. After centrifugation, serum was kept frozen at –30°C until analysis.
Serum PIVKA-II and ucOC levels were measured byelectro chemiluminescence immunoassay (ECLIA) (Sanko Junyaku, Co, Ltd, Tokyo, Japan) as the markers ofhepatic and skeletal vitamin K deficiency, respectively.
Serum intact osteocalcin (intact OC) was measured byenzyme immunoassay (EIA) (Mitsubishi Yuka, Tokyo,
Japan). The ucOC/OC was calculated as the ratio of
ucOC to intact OC. Serum levels of tartrate-resistant acidphosphatase-5b (TRACP-5b) and bone specific alkaline
phosphatase (BAP) were measured by EIA (DS Pharma
Biomedical, Osaka, Japan) and chemiluminescence enzyme immunoassay (CLEIA) (Beckman Coulter Inc, Tokyo, Japan), respectively. TRACP-5b and BAP are markers of bone resorption and bone formation, respectively.
The reference range of serum TRACP-5b was 170-590
mU/dL in male and 120-420 mU/dL in female, and that ofserum BAP was 3.7-20.9 μg/L in male and 3.8-22.6 μg/L
in female.
Nutrition intake study
Nutrient intake was assessed by food record method. The
intake of vitamin K was calculated by multiplying theamount of vitamin K supplied from the institution withthe average percentage intake. Based on these records,
their intake of vitamin K was calculated using the software (Healthy Maker Pro 501, Mushroom Software Corp,
Okayama, Japan). Vitamin K intake/kg body weight wasalso calculated, since 1 μg/kg of vitamin K is considered
to be sufficient for maintaining normal coagulation in theadult according to the Japanese DRI 2010.8
Statistical analyses
Statistical analyses were performed using the SPSS 17.0 J
for Windows (SPSS. Japan Inc, Tokyo, Japan). Associa-

51

tion between variables was analyzed by Pearson’s orSpearman rank correlation coefficient. Multiple regression
analyses with stepwise method were performed to determine independent determinants for serum ucOC anducOC/OC. Chi-square test was employed for categoricaldata.
RESULTS
Background profiles of the study subjects
The background profiles and biochemical data are shownin Table 1. Care level is a 5-grade score which is commonly used in the long-term care insurance in Japan withhigher number indicating more intensive care needed. It
was higher than grade 3 in 78% of subjects, indicatingthat they had low physical activity level. For example,
most of the present subjects required wheelchair fortransportation. In 27% of subjects, serum albumin level
was lower than 3.5 g/dL, which is a generally accepted
cut-off for malnutrition. Overall, nutritional parameters
including the biochemical indicators and body mass index(BMI) remained within the reference range for most of
the subjects. Thus, despite the elderly population and highlevel of care needed, the subjects’ nutritional status wasconsidered to be generally preserved. Although average
serum TRACP-5b and BAP levels were within the reference range as a whole, 20% and 32% of subjects had serum BAP and TRACP-5b level above upper reference
range, respectively. Serum PIVKA-II and ucOC levels
were 20.2±8.9 mAU/mL and 4.7±3.0 ng/mL, respectively.
All subjects were on orally consumed their meals. Although energy intakes were lower than estimated energy
requirement (EER) of DRI in all men and 93% of women,
the intake of macronutrients such as protein, fat and carbohydrates appeared appropriate for their age and sex.
Average vitamin K intake was 194±51 (median; 197)

Table 1. Baseline data of the study subjects
Age (y)Care level
Body weight (kg)Height (cm)BMI (kg/m2)
Serum Albumin (g/dL)
Serum triglyceride (mg/dL)
Serum total cholesterol (mg/dL)
eGFR (ml/min./1.73m2)
Serum BAP (μg/L)
Serum TRACP-5b (mU/dL)
Serum ucOC (ng/mL)
Serum total OC (ng/mL)
ucOC / intact OC
Serum PIVKA-II (mAU/mL)
Energy intake (kcal)
Protein intake (g)Fat intake (g)Carbohydrates intake (g)Vitamin K intake (μg/day)
Vitamin K intake/BW (μg/BW
kg/day)(M/F; 8/29, n=37)85.1±8.2（87.0）
Median; 3 (min-max; 1-5)45.9±6.1（46.1）
149.3±9.7（145.3）
20.6±2.5（20.0）
3.7±0.3（3.8）
119±41（118）198±49（191）65.4±15.8（63.3）
18.4±9.6（17.6）
365.2±124.9（372.0）
4.7±3.0 (3.8)
6.1±3.1 (5.4)
0.81±0.36 (0.80)
20.2±8.9 (18.0)1346±129 (1401)
53.2±5.2 (55.4)
35.6±3.6 (36.9)
193.8±18.7 (199.4)
194±51 (197)3.5±1.1 (3.4)

Data are expressed as mean±SD with the values in parentheses
showing the median.

52

Bone is more susceptible to vitamin K deficiency

μg/day in the study population, 166±50 (median; 159)
μg/day in males and 202±49 (median; 224) μg/day infemales. It was approximately 220% and 310% of the AI
in DRI in male and female subjects, respectively. All subjects had vitamin K intake exceeding AI. In addition, thevitamin K intake/kg body weight was 3.5±1.1 μg/day inthe present study subjects, far exceeding 1μg/kg.
Correlations among vitamin K intake and serum PIVKA-II, OCs
Table 2 shows that vitamin K intake was significantly
correlated with serum PIVKA-II and ucOC/OC levels,
but not with serum ucOC concentrations. (Table 2)
Correlations among serum OCs and bone turnovermarkers
Serum TRACP-5b and BAP levels were significantly
correlated with serum ucOC concentration, but not with
ucOC/OC ratio. (Table 3)Multiple regression analyses for serum OCs levels
Multiple regression analyses revealed that serum
TRACP-5b level was a significant determinant of serum
ucOC concentration. Vitamin K intake was a significant
predictor for ucOC/OC. (Table 4)
Relative susceptibility of liver and bone to vitamin K
deficiency
Serum PIVKA-II level exceeded the cut-off level (28mAU/mL) in only 14% of the subjects, whereas serum
ucOC concentration was above the cut-off value (4.5
ng/mL) in 43% of subjects, which was significantly different by chi-square test (p<0.001). (Table 5)
DISCUSSION
Vitamin status could be evaluated by several ways suchas measuring its blood concentration or measuring themarkers representing the vitamin status. Recently, we
have reported that the prevalence of vitamin D- and Kdeficiency is quite high in the institutionalized elderly bymeasuring plasma levels of 25 hydroxy-vitamin D concentration which is the best indicator of vitamin D status,and plasma vitamin K concentration.11 Plasma vitamin K
concentrations, however, only reflect the vitamin K status
as a whole, and do not provide us with information regarding the vitamin K status in various tissues individually. Thus, in this study, we have evaluated the subjects’vitamin K status by measuring their serum levels of PIVKA- II and ucOC rather than their plasma vitamin K levels.
First, we have studied the association between serum
levels of PIVKA-II and ucOC, and vitamin K intake. Vitamin K intake was significantly correlated with PIVKAII and ucOC/OC, but not with ucOC. Similar findings
were also reported by Booth et al that circulating levels of
PIVKA-II and ucOC/OC ratio reflected dietary vitamin K
intake, whereas serum ucOC levels did not.9 Two mechanisms were considered to be responsible for these find-Table 2. The correlation between vitamin K intake and serum levels of PIVKA-II and ucOC
ucOC
Vitamin K intake

r
0.092

p-value
0.588

r-0.416

ucOC/OCp-value
0.010

PIVKA-IIr
p-value-0.362
0.028

Correlations of vitamin K intake with markers for vitamin K deficiency were analyzed by Spearman rank correlation.

Table 3. The correlation of serum ucOC and uc/OC ration and bone turnover markers
ucOC
Serum TRACP-5b
Serum BAP

r
0.425
0.517

ucOC/OCp-value
0.009
0.001

r
0.014
0.243

p-value
0.935
0.147

Correlations of serum OCs with bone turnover markers were analyzed by Spearman rank correlation.

Table 4. Multiple regression analyses for serum ucOC level and ucOC/OC ratio
Dependent variable
ucOC
ucOC/OC

R2
0.206**
0.134*

Independent variable
Serum TRACP-5b
Vitamin K

β0.454-0.366p-value
0.005
0.026

The abbreviations are β for β coefficient. Independent predictor(s) for serum OCs levels were analyzed by multiple regression analyses withstepwise method. Sex, serum TRACP-5b, and vitamin K intake (μg) were included in all analyses.
*; p< 0.05, **; p< 0.01

Table 5. Number of subjects with vitamin K sufficiency and deficiency in the liver and boneIn the bone (serum ucOC concentration)
In the liver (serum PIVKA-II concentration)

Vitamin K sufficiency
21 (57%)32 (86%)

Vitamin K deficiency
16 (43%)5 (14%)

Values represent number of subjects, with percentage of subjects in the parentheses. Vitamin K status in the bone and that in the liver weresignificantly different by chi-square test (p<0.001).

A Kuwabara, M Fujii, N Kawai, K Tozawa, S Kido and K Tanaka

ings. The first is the different bioavailability of phylloquinone (PK; vitamin K1) and menaquinones (MKs; vitamin K2). In the present study, PK was the major form ofvitamin K taken as in America or Europe,12,13 since thesubjects had no intake of natto which contains large
amount of MK-7 during the study.14 Recent studies haveshown that PK can be utilized for γ-carboxylation in theliver, but can only be utilized in extrahepatic tissues afterconversion into MK-4.15,16
Second issue is the association of serum ucOC levelwith bone turnover. Serum levels of BAP and TRACP-5b
reflect osteoblastic bone formation and osteoclastic bone
resorption, respectively, and are elevated in the high turnover state. Since osteocalcin is produced in osteoblasts,17
it is conceivable that serum concentration of osteocalcin
as well as its subfraction, ucOC level is increased withhigh turnover. Thus, it is currently under debate whether
ucOC alone is satisfactory or measurement of ucOC as
well as ucOC/OC is a better indicator of vitamin K status.
In the present study, vitamin K intake was a significant
predictor for ucOC/OC, but not with ucOC. Therefore,
there is a possibility that ucOC/OC is a better index forvitamin K status than serum ucOC concentration. Unfortunately, however, there is no cut-off value publishedregarding ucOC/OC ratio, while the clinical usefulness ofserum ucOC measurement is increasingly acknowledged.
Thus, analysis using ucOC/OC could not be done as serum ucOC level in Table 5.
The cut-off value of 4.5 ng/mL for serum ucOC was
validated by Shiraki by simultaneously evaluating the
subjects’ dietary intake of vitamin K, blood levels of vitamin K and ucOC.18 They also reported that serum ucOC
concentration exceeding 5.5 ng/mL was associated withincreased risk of fracture. The clinical usefulness of ucOC
measurement was previously reported, although with different assay procedure of hydroxy-appatite binding assay.
In the European epidemiological study, Vergnaud et alreported that subjects in the lowest quartile of femoral
neck bone mineral density (BMD) and those in the highest quartile of ucOC had increased hip fracture risk withan odds ratio of 2.4 and 1.9, respectively. These two risk
factors were independent of each other, and those withboth conditions had a even higher odds ratio of 5.5.19
Thus, serum ucOC concentration is shown to be a good
indicator of skeletal vitamin K deficiency, and a predictorof fracture risk.
In the current study subjects with vitamin K intake far
exceeding AI, serum concentration of PIVKA-II anducOC were within the reference range in 86% and 57% of
the subjects respectively, which was significantly different. Thus, their vitamin K intake is sufficient for γcarboxylation in the liver, but not in the bone, and bone ismuch more susceptible to vitamin K deficiency than liver.
Such difference is likely to arise from the anatomical basis that vitamin K absorbed from the intestine is firsttransported to liver and preferentially used there, thenutilized in extrahepatic organs.9,10
Booth et al in their depletion-repletion studies, reported that the γ-carboxylation of prothrombin was restored at 200 μg/day of PK, whereas that of osteocalcin
was not even at 450 μg/day of PK.9 Schurgers et al also
reported that undercarboxylated prothrombin concentra-

53

tion was significantly decreased at supplementary intakeof 100 μg/day of PK, whereas ucOC level did not decrease below 300 μg/day of PK.10 Furthermore, Binkley
et al reported that supplementation with 1,000 μg/day ofvitamin K was optimal for the maximal γ-carboxylationof osteocalcin.20 These results suggest that at least 300500 μg g/day of vitamin K intake is required for the sufficient γ-carboxylation in the bone. Our results in the Japanese elderly are compatible with these results from Caucasians, and have additionally provided data on the prevalence of hepatic and skeletal vitamin K deficiency.
We believe that this paper is of importance in considering the AI for vitamin K. The current DRI states that
the AI for vitamin K was determined based on its requirement for the γ-carboxylation of blood coagulation
factors. The present findings suggest that vitamin K intake greater than the current AI is required for the skeletal
health in the institutionalized elderly. Further studies withlager number of subjects and intervention studies are necessary to define the amount of vitamin K necessary forthe elderly.
AUTHOR DISCLOSURES
None of the authors have any conflicts of interest.
REFERENCES
1. Vermeer C. γ-Carboxyglutamate-containing proteins and thevitamin K-dependent carboxylase. Biochem J. 1990;266:
625-36.
2. Booth SL. Skeletal functions of vitamin K-dependent proteins: not just for clotting anymore. Nutr Rev. 1997;55:2824.
3. Vermeer C, Shearer MJ, Zittermann A, Bolton-Smith C,
Szulc P, Hodges S, Walter P, Rambeck W, Stöcklin E, Weber P. Beyond deficiency: potential benefits of increased intakes of vitamin K for bone and vascular health. Eur J Nutr.
2004;43:325-35.
4. Booth SL, Tucker KL, Chen H, Hannan MT, Gagnon DR,
Cupples LA et al. Dietary vitamin K intakes are associatedwith hip fracture but not with bone mineral density in elderly men and women. Am J Clin Nutr. 2000;71:1201-8.
5. Danziger J. Vitamin K-deoendent proteins, warfarin, andvascular calcification. Clin J Am Soc Nephrol. 2008;3:150410.
6. Proudfot D, ShanahanCM. Molecular mechanisms mediating vascular calcification: role of matrix Gla protein. Nephrology. 2006;11:455-61.
7. Shanahan CM, Proudfoot D, Farzaneh-Far A, Weissberg PL.
The role of Gla proteins in vascular calcification. Crit Rev
Eukaryot Gene Expr. 1998;8: 357-75.
8. Ministry of Health, Labour, and Welfare, Japan. Dietaryreference intakes for Japanese 2010. Tokyo: DaiichiShuppan; 2010 (in Japanese).
9. Booth SL, Martini L, Peterson JW, Saltzman E, Dallal GE,
Wood RJ. Dietary phylloquinone depletion and repletion inolder women. J Nutr. 2003;133:2565-9.
10. Schurgers LJ, Shearer MJ, Hamulyák K, Stöcklin E, Vermeer C. Effect of vitamin K intake on the stability of oral
anticoagulant treatment: dose-response relationships inhealthy subjects. Blood. 2004;104:2682-9.
11. Kuwabara A, Himeno M, Tsugawa N, Kamao M, Fujii M,
Kawai N et al. Hypovitaminosis D and K are highly prevalent and independent of overall malnutrition in the institutionalized elderly. Asia Pac J Clin Nutr. 2010;19:49-56.

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Bone is more susceptible to vitamin K deficiency

12. Kamao M, Suhara Y, Tsugawa N, Uwano M, Yamaguchi N,
Uenishi K, Ishida H, Sasaki S, Okano T. Vitamin K content
of foods and dietary vitamin K intake in Japanese young
women. J Nutr Sci Vitaminol. 2007;53:464-70.
13. Booth SL, Suttie JW. Dietary intake and adequacy of vitamin K. J Nutr. 1998;128:785-8.
14. Kaneki M, Hodges SJ, Hosoi T, Fujiwara S, Lyons A, CreanSJ et al. Japanese fermented soybean food as the major determination of the large geographic difference in circulatinglevels of vitamin K2: possible implications for hip-fracturerisk. Nutrition. 2001;17:315-21.
15. Okano T, Shimomura Y, Yamane M, Suhara Y, Kamao M,
Sugiura M, Nakagawa K. Conversion of phylloquinone (Vitamin K1) into menaquinone-4 (Vitamin K2) in mice: twopossible routes for menaquinone-4 accumulation in cerebraof mice. J Biol Chem. 2008;283:11270-9.
16. Thijssen HH, Vervoort LM, Schurgers LJ, Shearer MJ. Menadione is a metabolite of oral vitamin K. Br J Nutr. 2006;
95:260-6.

17. Price PA, Otsuka AA, Poser JW, Kristaponis J, Raman N.
Characterization of a gamma-carboxyglutamic acid-containing
protein from bone. Proc Natl Acad Sci U S A. 1976;73:
1447-51.
18. Shiraki M, Aoki C, Yamazaki N, Ito Y, Tsugawa N, Suhara
Y, Okano T. Clinical assessment of undercarboxylated osteocalcin measurement in serum using an electrochemiluminescence immunoassay: Establishments of cut-off values todetermine vitamin K insufficiency in bone and to predict
fracture leading to clinical use of vitamin K2. Iyaku to yakugaku. 2007;57:537-46. (in Japanese)
19. Vergnaud P, Garnero P, Meunier PJ, Bréart G, Kamihagi K,
Delmas PD. Undercarboxylated osteocalcin measured with a
specific immunoassay predicts hip fracture in elderly women: the EPIDOS Study. J Clin Endocrinol Metab. 1997;82:719-24.
20. Binkley NC, Krueger DC, Kawahara TN, Engelke JA,
Chappell RJ, Suttie JW. A high phylloquinone intake is required to achieve maximal osteocalcin γ-carboxylation. Am
J Clin Nutr. 2002;76:1055-60.

A Kuwabara, M Fujii, N Kawai, K Tozawa, S Kido and K Tanaka

55

Original Article

Bone is more susceptible to vitamin K deficiency thanliver in the institutionalized elderly
Akiko Kuwabara PhD1,4, Minori Fujii RD2, Nobuko Kawai CSW2, Kunihiko Tozawa SE3,
Shoko Kido PhD4, Kiyoshi Tanaka MD4
1

Department of Health and Nutrition, Osaka Shoin Women’s University, Osaka, Japan
Nursing Home Kayu-Shirakawa, Kyoto, Japan
3
Sanko Junyaku Co., Ltd., Tokyo, Japan
4
Department of Food and Nutrition, Kyoto Women’s University, Kyoto, Japan2

居住機構老人骨骼比肝臟易受維生素 K 缺乏影響
日本 2010 年發佈的膳食營養素參考攝取量(DRI)中，維生素 K 的足夠攝取量是根
據凝血因子的 γ-羧化作用而訂定的。近來，維生素 K 也被視為預防骨折不可或缺的角色。本研究在於比較肝和骨骼對維生素 K 缺乏的敏感性。評估 37 位居住
機構的老人之維生素 K 狀況─測量血清 PIVKA–II (因維生素 K 缺乏所產生的蛋白質)和 ucOC (未羧化的骨鈣素)濃度，兩者分別為肝和骨骼在維生素 K 缺乏時的敏感指標。受試者血清 PIVKA-II 和 ucOC 濃度分別為 20.2±8.9 mAU/mL (臨界值28 mAU/mL)和 4.7±3.0 ng/mL (臨界值 4.5 ng/mL)。維生素 K 攝取量中位數約為
200 μg/day，超過了日本目前所建議的足夠攝取量 3 倍。維生素 K 攝取量與血清
PIVKA-II 和 ucOC/OC 濃度顯著相關，但與血清 ucOC 濃度無相關。雖然血清ucOC 濃度是體內維生素 K 狀況很好的指標，但複迴歸分析顯示骨骼轉換標記增
加，也會影響血清 ucOC 濃度。所有的受試者維生素 K 攝取量皆超過足夠攝取
量。然而，分別有 14%和 43%受試者的血清 PIVKA-II 和 ucOC 濃度超過臨界
值。本研究結果建議，對於住在機構的老人，為維持骨骼健康，維生素 K 攝取
量應超過目前建議的足夠攝取量。
關鍵字：維生素 K、足夠攝取量、γ-羧化作用、未羧化骨鈣素、PIVKA-IIAuthor's personal copy

Medical Hypotheses 76 (2011) 336–338

Contents lists available at ScienceDirect

Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

Body weight divided by squared knee height as an alternative
to body mass index
Akiko Kuwabara a,b, Yoko Ogawa-Shimokawa a, Kiyoshi Tanaka a,⇑ab

Department of Food and Nutrition, Kyoto Women’s University, Kyoto, Japan
Department of Health and Nutrition, Osaka Shoin University, Osaka, Japana r t i c l e

i n f oArticle history:
Received 16 September 2010
Accepted 22 October 2010a b s t r a c t
Weight/height2 (Quetelet’s index) is the basis for deﬁning both underweight and obesity. Height, however, is often not precisely measurable in the elderly due to involutional changes such as spinal deformity. Body volume or body surface area are not proportionately decreased even with height loss.
Previous reports have shown that Quetelet’s index is overestimated in the elderly with height loss. Then
we have made a hypothesis described below.
Maximal height or height at youth would better represent the subjects’ nutritional or clinical status.
The distinction of these two heights has not been mentioned before. There have been many publications
showing the equations to estimate height from the surrogate parameter(s) such as knee height (KH).
Most equations published so far are expressed as estimated height = a + b  KH c  age, where a, b,
and c are constants. Negative correction by age is unexceptionally far greater in women than in men.
Apparently, previous researchers have estimated current height by their equations.
Maximal height cannot be measurable. It, however, is unaffected by age by its deﬁnition. Therefore,
maximal height does not have to be corrected by age, and would be almost proportional to KH. Then
weight/KH2 could be a better alternative to the most commonly used weight-height ratio; weight/
height2; the Quetelet’s index.
Height is the basis for various clinically important indices such as body surface area (BSA) and energy
requirement. Employing current height could lead to the underestimation of BSA or energy requirementin the elderly with height loss. Our hypothesis described here would yield a novel and better indices forthe clinical assessment of the elderly.
Ó 2010 Elsevier Ltd. All rights reserved.

Introduction
Body weight is one of the most fundamental indices in the clinical evaluation of the subjects. Longitudinal and serial observationof body weight provides us with valuable information concerning
the alteration in the subjects’ clinical status. In the cross-sectionalsettings, however, the usefulness of body weight for the clinicalevaluation is seriously limited by the fact that it is inﬂuenced by
the body size. Therefore, weight must be corrected by some parameter(s) representing the body size. For such purpose, body weight
divided by squared height (weight/height2) is commonly employed
and called body mass index (BMI) [1]. High BMI and low BMI are
the basis for the diagnosis of obesity and emaciation, respectively.
Why is the weight/height2 the standard weight-height ratio?
Since human body is three-dimensionally structured, body volume
may be proportional to height3. In that case, body weight would be⇑ Corresponding author. Address: Department of Food and Nutrition, Kyoto
Wome’s University, 35, Imakumano-kitahiyoshicho, Higashiyama, Kyoto 605-8501
Japan. Tel.: +81 75 531 7125; fax: +81 75 531 7153.
E-mail address: tanakak@kyoto-wu.ac.jp (K. Tanaka).
0306-9877/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.mehy.2010.10.033

proportional to height3. Then one could argue for weight/height3 as
another weight-height ratio. Indeed, weight/height2 is not the only
weight-height ratio, and might be more properly called Quetelet’s
index (hereafter abbreviated as QI) [1,2].
Other weight-height ratios have been reported, such as weight/
height ratio (weight/height), Khosla-Lowe index (weight/height3),
Ponderal index (weight/height1/3), Benn’s idex (weight/heightp)
where p is a population-speciﬁc exponent [3]. Of these, QI is considered to be the most appropriate weight-height ratio, since it fulﬁlls the following requirements.
Thus, a preferred weight-height ratio must be maximally correlated with body mass and minimally correlated with stature [4].
Hereafter in this paper, weight/height2 will be designated as QI
rather than BMI for clarity. The idea of body weight divided bysquared height (weight/height2) as the weight-height ratio was
originally developed by a Belgian mathematician, Adolphe Quetelet in the 19th century [2]. World Health Organization (WHO) deﬁned overweight as well as underweight based on QI [5]. In otherwords, QI is not the theoretically derived standard weight-heightratio a priori, but has become the de facto standard because of its
clinical usefulness.

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A. Kuwabara et al. / Medical Hypotheses 76 (2011) 336–338

Quetelet’s index in the elderly

Hypotheses

The curve showing the relationship between QI and mortality
are considered to be U-shaped. Thus, both overweight or obesity,and underweight are associated with increased mortality. There
have been numerous publications, however, to cast doubt thatthe above theory holds true in the elderly [4,6–8]. Two examples
will be given below.
First, many studies have indicated that the optimal QI associated with the lowest mortality differs in the elderly and in theyounger generations. Andres reported that QI with the lowest mortality was 22.9 kg/m2, 25.8 kg/m2, and, 26.6 kg/m2 in men aged
40–49 years, 50–59 years, and 60–69 years, respectively. In
women, it was 23.2 kg/m2, 25.2 kg/m2, and 27.3 kg/m2, respectively [4]. Matsuo also reported that QI with the lowest mortality
was 22.5 kg/m2 and 24.8 kg/m2 in men aged 40–59 years and
60–79 years old, and it was 21.9 kg/m2 and 23.3 kg/m2 in women
aged 40–59 years and 60–79 years old [6]. Thus, it has consistently
been demonstrated that QI with the lowest mortality is higher thanin the younger generations.
Second, the association between the underweight and the allcause mortality is much more debated. Excess mortality associatedwith underweight has been reported to be lower, higher, or unaffected by aging [6].

Height generally means the distance between the top and bottom of the body. Height loss means the shortening of this distance.
We believe that two types of height should be distinguished in theclinical evaluation of the elderly; height A and height B in Fig. 1.
Height A is the current height. Height B would be quite close to
the height at youth or maximal height. A woman in Fig. 1 hasspinal deformity and height loss, but her body volume or body surface area is not proportionally diminished. Then, it is obvious that
calculating QI using the current height (height A in Fig. 1) would
lead to the signiﬁcant overestimation.
Although previous authors on establishing the equation to predict height from KH do not seem to have considered the distinctionof these two heights, but much greater negative correction by age
in women strongly indicate that they meant current height in theirequations.
Then we have come to an idea that employing maximal height
would yield a better estimate of weight–height ratio in the elderly.
Since maximal height cannot be measured in the elderly, it must be
estimated. Unlike the current height, maximal height is independent of age by its deﬁnition. Therefore, the correction by age would
be unnecessary for the estimation of maximal height. KH is little
affected by age and would be almost proportional to maximalheight.
Then we have made a hypothesis that weight divided by squared
KH (BMI–KH) could be a good alternative to usual QI in the clinicalevaluation of the elderly. Malnutrition in the elderly is a major healthproblem, and the signiﬁcance of nutritional assessment has beenstressed [12,13]. Despite many parameters currently available, suchas anthropometric and laboratory ones, there is no consensus onwhat parameter would best predict the nutritional status of the elderly. We believe that BMI–KH could be a promising alternative tousual BMI; Quetelet’s index in the elderly.

Height loss in the elderly
Aging is almost inevitably associated with height loss. Various
factors contribute to the involutional height loss, the most important cause of which would be the vertebral compression fracturecaused by osteoporosis. Osteoporosis is a condition that renders
the patients susceptible to fragility fractures, such as spinal, hip,and wrist fractures [9]. Spinal fracture, which is the most common
osteoporosis-related fracture, is a compression one in its nature.
Thus it causes spinal deformity and height loss. Since post-menopausal estrogen deﬁciency is the most important cause of osteoporosis, it is quite conceivable that women are at much greater riskfor height loss than men. Prospective study with repeated heightmeasurement has conﬁrmed that women indeed lost more height
than men [10]. Sorkin et al. have reported that average cumulative
height loss from age 30 to 70 was 3 cm for men and 5 cm for women, and that from age 30 to 80 was 5 cm for men and 8 cm for
women. Such involutional height loss poses serious problem upon
the validity of QI as the weight-height ratio in the elderly. It is already pointed out that there will be an overestimation of QI in theelderly because of shortened height [10]. As described above, anideal weight-height ratio should be minimally correlated with stature. QI is generally considered to fulﬁll this requirement, which,however, may not always hold true in the elderly.

Clinical implications of BMI–KH
We believe that taking the distinction of two heights could yielda solution to the above-mentioned apparent paradox regarding the
QI in the elderly.

Which is height?
(A)

(B)

Estimation of height using surrogate measurement
Height cannot be exactly measured in the elderly too often, due
to various reasons such as vertebral fracture, disc degeneration andfrailty. Then height is estimated from the surrogate parameterssuch as arm span and knee height (KH) [1]. Of these, KH is the mostfrequently used, since it could be easily measured even in the elderly and minimally affected by the involutional changes. Manyequations have been so far published to predict height from KH,
the most well known of which is Chumlea’s one; height = 64.19 +2.02  KH 0.04  age for men, and 84.88 + 1.83  KH 0.24  age
for women [11]. In most equations hitherto published, negative
height correction by age is much greater in females than in males,
the reason of which, however, has not been described in the previous publications.

Fig. 1. Two heights are shown. Height (A) is the distance between the ground andthe top of the body, and the current height. Height (B) corresponds to the maximalheight or height at youth.

Author's personal copy

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A. Kuwabara et al. / Medical Hypotheses 76 (2011) 336–338

As the possible causes of inconsistency in the association between obesity and underweight and mortality in the elderly, Nagai
et al. have mentioned various possibilities, such as history of cancer
and cardiovascular diseases, inadequate adjustment for several confounders including smoking, alcohol consumption, physical activity,and socioeconomic status [8]. Another factor of importance notmentioned by them is the validity of height measurement. Given
the QI calculated with current height, thus overestimated, the relationship between QI and mortality would be obscured. Althoughhigher QI has been reported to be associated with the lowest mortality in the elderly [4,6], ‘‘true’’ QI may not be high.
In a large-scale and long-term cohort study in Japan, multivariate-adjusted relative risk (RR) for all-cause mortality was greatly
dependent on the age of the study subjects in women [6]. In those
40–59 years old, RR in the group with QI exceeding 30 kg/m2 was
2.23 (95% conﬁdence interval; CI 1.46–3.42) compared to that withQI 21.0–22.9 kg/m2, whereas it was only 1.39 (95% CI 1.14–1.69)
60–79 years old. In contrast, RR in men was not different between
age groups. Although the reason for this gender difference is notdiscussed by the authors, we believe that our hypothesis could
yield a possible explanation. Since women are much more likely
to lose their height, their QI is quite prone to be overestimated.
Thus it is possible that the subjects with QI higher than 30 kg/m2
in the above-mentioned study is actually not obese, thus was not
associated with higher mortality.
Other clinical implications
Height is an essential anthropometric parameter in the patients’evaluation. In addition to BMI, It is also a prerequisite for the calculation of such important indices as body surface area (BSA),
and resting energy expenditure (REE) [14,15]. BSA is considered
to be superior to body weight as an index of metabolically active
mass, since it is less affected by adiposity. BSA could be calculated
by various equations such the one by Dubois and Dubois; BSA(m2) = 0.007184  weight (kg)0.425  height (cm)0.725 [14]. BSA
has various clinical applications with some examples given below.
Glomerular ﬁltration rate (GFR) is corrected by BSA [16]. Furthermore, dosage of some therapeutic drugs is determined in termsof BSA [17].
Furthermore, height is needed for determining the patients’ energy expenditure. REE is usually calculated using various equations, the well-known of which is the Harris-Benedict equation;
BMR = 66 + 13.7  weight(kg) + 5  height(cm) 6.76  age(years) for men and BMR = 655 + 9.6  weight (kg) + 1.8  height(cm) 4.7  age (years) for women [15]. Patients’ total energy
expenditure (TEE) is then calculated as TEE = REE  activity factor  stress factor. Thus height is necessary in deciding how muchenergy the patients’ need, and essential in the medical nutritional
therapy. Then, estimating height using the above-mentioned equations can be problematic. For example, if a dietitian estimatesheight from KH using Chumlea’s equation, calculate REE with Harris-Benedict equation using the height estimated from KH, anddetermine the TEE, i.e. the energy intake necessary for the elderly

subjects, it could be a substantial underestimate. Then the elderly
subjects may experience malnutrition due to insufﬁcient energy
supplied. Epidemiological studies, favorably the cohort ones, withmortality and morbidity as the clinical outcomes, are to be performed for the comparison of QI and BMI–KH as the clinical usefulweight-height ratio in the elderly.
Although detailed consideration on the possible roles of KH inestimating BSA or BEE is beyond the scope of this manuscript, we
believe that there is a possibility that these parameters are better
predicted by maximal height or KH than the current height.

Conﬂict of interest
None of the authors have any conﬂict of interest.
Acknowledgement
We would like appreciate Kiyoaki Tanaka for preparing Figure 1.
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Nutritional Assessment. 2 nd. Oxford University Press, 2005. p. 245–271.
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Hindawi Publishing Corporation
UlcersVolume 2011, Article ID 594532, 5 pages
doi:10.1155/2011/594532

Clinical Study
Fat Restriction Is Associated with Impaired Quality of Life inPatients with Ulcerative Colitis and Crohn’s Disease
A. Kuwabara,1, 2 H. Nakase,3 H. Tsuji,4 K. Shide,4 T. Chiba,3 N. Inagaki,4 and K. Tanaka2
1 Department of Health and Nutrition, Osaka Shoin Women’s University, 4-2-26 Hishiyanishi, Higashiosaka-shi,

Osaka 577-8550, Japan
2 Department of Food and Nutrition, Kyoto Women’s University, 35 Imakumano-kitahiyoshi-cho, Higashiyama-ku,

Kyoto 605-8501, Japan
3 Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Shogoin kawahara-cho,

Sakyo-ku Kyoto, 606-8507, Japan
4 Department of Diabetes and Clinical Nutrition, Kyoto University Graduate School of Medicine, 54 Shogoin kawahara-cho,

Sakyo-ku Kyoto, 606-8507, Japan
Correspondence should be addressed to K. Tanaka, ktanaka@zeus.eonet.ne.jp
Received 9 August 2010; Accepted 21 September 2010
Academic Editor: Gyula Mozsik
Copyright © 2011 A. Kuwabara et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn’s disease, is reported to be associated with impaired healthrelated quality of life (QOL). Although decreased QOL in these subjects has been reported to be associated with various factors, theeﬀect of nutritional therapy, especially nutrients intake on QOL has received less attention. In this study, we evaluated the various
factors including nutrients intake on QOL using SF-8 in 64 patients with IBD. Patients with IBD seem to have decreased QOL
especially in the mental aspects. The percentage energy intake from fat of total energy fat intake (% energy) of the whole subjects,
was lower than those of the annual National Nutrition Survey in Japan. Multiple regression analyses revealed that fat intake (%energy) was a significant predictor for mental component summary. In conclusion, fat restriction contributes to impaired QOL
especially in the mental aspects in IBD patients.

1. Introduction
Inflammatory bowel disease (IBD); ulcerative colitis (UC)and Crohn’s disease, is reported to be associated with
impaired health-related quality of life (HR-QOL). In thispaper, HR-QOL will be simply designated as QOL. Decreased
QOL in these subjects has been reported to be related tovarious factors such as age, gender [1, 2], treatment eﬀects[3], disease activity, and social environment [4]. However,
the eﬀect of nutritional therapy on the QOL of IBD patientshas received less attention, most of which is devoted to theparenteral nutrition therapy, not the nutritional therapy ingeneral [5, 6].
Since excessive fat intake is considered to worsen the
inflammation in the intestine, its restriction has traditionallybeen employed in Japan as the oral nutritional therapy for

IBD patients, especially for those with CD, which, however,has its own pros and cons.
Recently, we have studied the possible involvement ofhypovitaminosis D and K in the development of osteoporosis
in IBD patients [7]. In face of apparently suﬃcient intake ofthese vitamins, their plasma levels were quite low in thesepatients. Paradoxically, plasma concentrations of vitamin D
and K were correlated with the fat intake but not with theirintake of these vitamins. These results were more prominent
in patients with CD than those with UC. Then it was
concluded that fat-soluble substances such as vitamin D and
K were not eﬀectively absorbed from the intestine without
concomitant intake of enough fat.
Through this paper, we were interested in what fat
restriction means from the patients’ perspectives and studied

2the eﬀect of fat restriction on the QOL of IBD subjects in thispaper.

2. Subjects and Methods
2.1. Subjects. Study subjects were 64 patients with IBD attending the gastroenterology clinic at the Kyoto University
Hospital; 33 with CD (19 men/14 women) and 31 withUC (20 men/11 women). Detailed information was given
and written consent was obtained. The study protocol wasapproved by the ethical committee of the Kyoto Women’s
University. Almost all patients (27/33 in CD and 28/33 inUC) were receiving 5-aminosalicylic acid. Glucocorticoid
therapy was given to four and two patients with CD
and UC, respectively. Immunosuppressive drug therapy
was performed in 25 and 4 patients with CD and UC,
respectively. Eight patients with CD, but none with UC, wereon combined therapy of infliximab, synthetic glucocorticoid,and immunosuppressive drug. Fifteen patients with CD and
one with UC were on enteral or total parenteral nutrition
therapy, respectively.
2.2. Methods
2.2.1. Dietary Information. Dietary information was obtained from food intake records in 2 weekdays by the patients.
By calculating these records, their energy and nutrients intakes were obtained by computer software program (Healthy
Maker Pro 501, Mushroom soft Corp.).
2.2.2. QOL Measurement. QOL was assessed using theJapanese Short Form Health Survey (SF-8), a widely usedgeneric questionnaire [8]. Eight subscales are obtained;
physical function (PF), role physical (RP), bodily pain (BP),general health (GH), vitality (VT), social function (SF), roleemotional (RE), and mental health (MH). RP and RE refer tothe limitations due to physical or emotional reasons, respectively. They are also summarized into two summary scores:
physical component summary (PCS) and mental component
summary (MCS). Data are transformed to deviation scoresbased on Japanese norms [8]. Higher scores indicate better
QOL, with 50 corresponding to the national norms.
2.2.3. Statistical Analyses. Statistical analyses were performed
using SPSS 17.0 J for Windows (SPSS. Japan Inc., Tokyo,
Japan). Comparison of data from IBD patients with Japanese
norms was done by one-sample t test. The diﬀerence between
two independent groups was analyzed by unpaired t test or
Mann-Whitney test depending on normality. Correlations
between two independent variables were analyzed by Pearson’s or Spearman’s correlations. Multiple regression analysis
was performed to determine independent factors for QOL
scores in IBD patients.

3. Result3.1. Background Profiles and Biochemical Indices. The baseline characteristics of the patients are shown in Table 1.

UlcersTable 1: Background profiles and results from blood tests inpatients with CD and UC.
CDUC

P valueAge (y)35.6 ± 7.3

41.7 ± 17.3

.343a

Sex (F/M)

19/14

20/11

—

Disease duration (y)13.7 ± 7.4

6.8 ± 4.8<.001bBody mass index(kg/m2 )Disease location(involving small
bowel/not involving
small bowel)

19.5 ± 2.3

21.1 ± 3.3

.025b

30/2

0/31

—

Glucocorticoid
therpyImmunosuppressive
therapy
Immunopotentiating
therapy (TNF-α)

4

2

—

25

4

—

8

0

—

Enteral or total
parenteral nutrition
therapy

15

1—

C-reactive protein(g/dl)

0.6 ± 1.0

0.3 ± 0.6.135bAlbumin (g/dl)
Total cholesterol(mg/dl)3.9 ± 0.4
126.9 ±
25.0

4.3 ± 0.3<.001b177.1 ± 40.3<.001bValues represent mean ± SD. Comparison of indices between patients with
CD and those with UC was done by unpaired t testa or Mann-Whitney testb
depending on normality.

CD patients had significantly longer disease duration andlower BMI than UC patients. While nutritional indices suchas serum albumin and total cholesterol were lower in CD
subjects, there was no significant diﬀerence in C-reactive
protein which is an inflammatory parameter between thesegroups. Most of patients were in remission.
3.2. Energy and Nutrients Intake in CD and UC Patients.
Food intake could be evaluated in 62 patients (31 with
CD and 31 with UC). Energy and nutrients intake in thesepatients is shown in Table 2. Fourteen patients with CD wereon enteral nutrition, and each one of subjects with CD and
UC was on total parental nutrition. Although the energy
intake was not significantly diﬀerent between the two groups,
fat intake was significantly lower in CD patients than UC
subjects. The annual National Nutrition Survey in Japan(NNS-J) in 2008 showed that in subjects of 30–39 or 40–49,
years of age including both genders [9], the daily fat intake(% energy) was 26.5% or 25.6%, respectively. These weresignificantly higher than those of IBD subjects in this study(P = .001; data not shown). Subjects with enteral or parental
nutrition had fat intake only approximately half of that in
subjects with oral intake (data not shown). The percentage
energy intake from protein, fat, and carbohydrates was
significantly diﬀerent between CD and UC subjects.

Ulcers3
Table 2: Comparison of nutrient intakes in CD and UC patients.

EnergyIntake (kcal)

ProteinFat

Intake (g)Intake (g)Carbohydrates
Protein (% energy)

Intake (g)Fat (% energy)
Carbohydrates (% energy)

IBD (n = 62)

CD (n = 31)

UC (n = 31)

P value

1816 ± 465 (1804)
66.0 ± 21.8 (63.5)
44.7 ± 21.6 (43.0)

1847 ± 392 (1842)
71.0 ± 20.6 (67.2)
38.7 ± 17.6 (37.4)

1785 ± 533 (1764)
60.9 ± 22.0 (61.6)
50.6 ± 23.6 (48.1)

NSNS
P < .05275.4 ± 91.6 (268.6)
14.4 ± 2.7 (14.2)

298.3 ± 93.1 (275.7)
15.0 ± 2.2 (15.6)

252.4 ± 85.4 (254.9)
13.5 ± 2.9 (13.6)

P < .05
P < .001

22.4 ± 9.6 (24.6)
63.2 ± 9.6 (62.4)

19.5 ± 8.9 (18.9)
65.2 ± 8.6 (64.0)

25.2 ± 9.5 (26.8)
56.5±9.5 (60.5)

P < .001
P < .001

Data are expressed as mean ± SD with the values in parentheses showing the median. Comparison of indices between patients with CD and those with UC
was done by unpaired t test

Table 3: Dimensional SF-8 scores in patients with CD and UC.

PF
RP
BP
GH
VT
SF
RE
MH
PCS
MCS

IBD (n = 64)

CD (n = 33)

UC (n = 31)

50.1 ± 4.7 (53.6)

50.1 ± 4.5 (53.6)

50.0 ± 5.0 (53.6)∗

48.2 ± 6.8 (48.5)

48.7 ± 5.3 (48.5)

47.7 ± 8.1 (48.5)

50.8 ± 7.6 (51.8)

50.5 ± 6.8 (51.8)

51.2 ± 8.5 (51.8)∗

47.8 ± 7.5 (50.7)∗

47.7 ± 6.5 (50.7)

47.8 ± 8.5 (50.7)

49.6 ± 6.5 (54.5)

48.4 ± 5.7 (45.3)

51.0 ± 7.1 (54.5)

46.2 ± 8.3 (45.2)∗

46.9 ± 7.2 (45.2)∗

48.3 ± 6.4 (49.1)

48.0 ± 6.5 (49.1)

48.6 ± 6.5 (49.1 )∗46.8 ± 7.5 (45.0)∗

49.2 ± 5.4 (49.0)

48.9 ± 7.9 (50.0)

∗∗
∗

∗∗

47.3 ± 6.5 (45.0)

49.0 ± 6.7 (49.1)∗∗∗

45.5 ± 9.4 (45.2)
47.8 ± 5.4 (50.3)

46.1 ± 6.6 (46.5) ∗∗ 45.7 ± 7.1 (46.6) ∗∗ 46.6 ± 6.0 (46.5)

Data are expressed as mean ±SD with median in the parentheses. Onesample t test was used for comparison between Japanese norms and scoresof CD or UC patients. The asterisk denotes the significant diﬀerence (∗ P >.05; ∗∗ P > .01; ∗∗∗ P > .001).

3.3. QOL Assessment. In Table 3 is shown the eight subscales
and two summary scores of SF-8 in subjects with IBD
patients. Since data are expressed as the deviation values
normalized by the Japanese normative values, the value “50”correponds to Japanese norm. Subscales such as RP, GH, SF,
MH, and MCS were significantly lower than the Japanese
norms.
Table 3 shows the comparison between CD and UC
subjects. There were no significant diﬀerences in the eightsubscales and two summary scores except for lower VT in
CD patients than in those with UC.
3.4. Correlations between PCS/MCS Scores and Clinical Characteristics, Biochemical Markers, and Nutritients Intakes. We
analyzed the correlation between these summary scores andbiochemical indices, fat intake expressed as the percentage
energy intake from fat of total energy, fat intake (% energy)(Table 4). Fat intake (% energy) was significantly correlatedwith MCS in CD patients. There was significant but weak,correlation between PCS and serum albumin and MCS
and BMI in UC patients. In the whole subjects, BMI was

significantly correlated with PCS, and fat intake (% energy)
was associated with MCS.
3.5. Multiple Regression Analysis for Variable Associated with
PCS/MCS Scores. Then multiple regression analyses weredone to study the determinant(s) of the subjects’ PCS andMCS (Table 5). Variables included in the analysis were typesof disease (CD/UC), BMI, serum concentrations of Alb, andfat intake (% energy). BMI was the significant predictor of
PCS score (βcoeﬃcient 0.29, P = .023) whereas fat intake was
the only significant determinant of MCS score (βcoeﬃcient
0.29, P = .027).

4. Discussion
Recently, various questionnaires have been developed for
QOL evaluation, both generic and disease targeted [10].
Generic ones, by their definition, only consist of questions
related to the subjects’ general status and do not include
the questions related to the features which are specific to acertain disease. Therefore, they are applicable to such studies
as comparing the impact on QOL by various diseases or evento the evaluation of healthy subjects. In contrast, diseasetargeted ones include items specific to a certain disease. They
can be more sensitive than the generic ones in detecting
the QOL impairment closely related to a certain diseasestate but are not applicable to the evaluation of patientswith other diseases. Various disease-targeted questionnaires
have been developed for IBD subjects; the most well knownof which would be IBDQ (inflammatory bowel diseasequestionnaire) including many items related to the patients’gastroenterological problems [11]. Since the purpose of ourcurrent work was to study the eﬀects of nutritional therapy
on the patients’ QOL, we considered it more appropriate toevaluate the patients’ QOL using the generic questionnaire.
SF-36 is one of the most commonly used genericquestionnaires, and SF-8, used in this study, is the shortened
one. Eight subscales, two summary scores are obtained, andexpressed as the deviation values, which are normalized by
the nations’ normative value. Many previous papers on the
QOL of IBD patients using SF-36 seem to have handled thedata improperly [2, 4]. For example, Bernklev and Andersson
expressed their data as the 0–100 scale scores [2, 4], which

4

UlcersTable 4: Correlations between PCS/MCS scale scores and clinical characteristics, biochemical markers, and fat intake as proportion of totalenergy intake.
IBD (n = 64)
Diseaae duration (y)Body mass index(kg/m2 )C-reactive protein(g/dl)
Albumin (g/dl)
Total cholesterol(mg/dl)Fat intake(% energy)

CD (n = 33)r

MCS−0.175

PCS
0.070

MCS
−0.221PCS−0.085MCS−0.066r

0.261∗

0.088

0.144

−0.0750.248

0.415∗

r−0.0830.075−0.0580.196−0.116−0.045

∗

0.059

r

0.235

0.082

0.092

0.064

0.424

r

0.033

0.196

−0.132

0.169

0.174

0.249

0.175

∗

0.238

0.109

r

The asterisk denotes the value is significant correlation (∗ P

0.287
< .05, ∗∗ P

PCS scoreβ
CD/UC(1;CD, 2;UC)

0.146

0.458

∗∗∗

< .01, ∗∗∗ P < .001) by Pearson’s correlation or Spearman’s correlation.

Table 5: Multiple regression analyses for the predictor(s) of PCS
and MCS scores in IBD patients.

r 2 = 0.086

UC (n = 31)

PCS
0.012

MCS scoreP = .023

r 2 = 0.081

P = .027

P

βP−0.141.283−0.059

.657

BMI

0.293

.023

0.069

.594

Alb

0.141

.309

0.024

.855

Fat intake (%total energy)

0.121

.347

0.285

.027

Abbreviations are as follow: β for β coeﬃcient and P for P value. Determinants of independent predictors for PCS/MCS scores were analyzed
by multivariate analysis with stepwise method. Variables included were
CD/UC, BMI, serum albumin concentration, and fat intake (% totalenergy)can be misleading [12]. In the present paper, data wereanalyzed according to the authorized instruction.
In this study, subscales such as RP, GH, SF, RE, MH,
and MCS were significantly lower than the Japanese norms.
Decreased RP in face of normal PF is conceivable consideringthat the patients do not have severe physical impairment
but have some limitation in their daily activities by reasons
such as the bowel habit problem. Impaired SF would be also
conceivable from the similar viewpoint. As a whole, patientswith IBD seem to have decreased QOL especially in themental aspects.
Then, we have analyzed variables associated with PCS
and MCS. There were substantial diﬀerences in the objective
clinical features of patients with CD and UC. For example,
CD patients had longer disease duration and lower nutritional status than those of UC subjects. Nevertheless, there
were no significant diﬀerences in 7 out of 8 dimensions
between the two conditions. Namely, QOL which represents
the patients’ subjective evaluation of their health states seems
to be impaired in both CD and UC patients.
Then, we have studied the determinants for PCS andMCS. PCS score was correlated with indices representing

their nutritional status such as BMI (r = 0.261, P < .05)
and albumin with marginal significance (r = 0.235, P =.066). In contrast, none of these factors were significantly
correlated with MCS. Thus, it was considered unlikelythat disease activities or other clinical features alone could
account for the impaired mental aspects of QOL in thesesubjects. The association of QOL with mental aspects of thesubjects has been previously reported. Boye et al. reported
that neuroticism was a significant predictor for mental andvitality subscales of SF-36 in IBD patients using multiple
regression analyses controlled for gender, age, and clinicaldisease activity [13]. Martin also reported that QOL was not
closely correlated with the clinical features in CD patients[14]. These results, together with our current findings,
suggest that mental aspects can more strongly aﬀect QOL
than clinical ones in IBD patients.
Theoretically, it is well known that the QOL scores insubjects with disabilities are higher than those anticipatedfrom their objective physical impairment (disability paradox) [15]. This phenomenon is because subjects with longterm disabilities change their internal standard and make theadaptation to their actual status (response shift) [16].
Next, we have made a hypothesis that nutrients intake
such as fat restriction may contribute to the impairmentof mental aspects of QOL in these subjects. Although CD
patients had lower fat intake than UC subjects, fat intake (%energy) of the whole subjects was significantly lower thanthose of the NNS-J.
Then, we have analyzed the association between thesesummary scores and their fat intake (% energy). Fat intake(% energy) was significantly associated with MCS, but notwith PCS in patients with IBD. When CD and UC patientswere separately analyzed, the correlation coeﬃcients wasalmost the same, but not statistically significant anymore,probably due to the smaller number of study subjects. We
then have performed the multivariate analysis. Of the various
factors included for analysis types of disease (CD/UC), BMI,
serum albumin, fat intake (% energy), BMI, and fat intake(% energy) were the only significant determinants of PCS
and MCS, respectively. Since many IBD patients are young,they are quite likely to favor foods rich in fat. Nevertheless, fatUlcers
restriction is the common practice in the nutritional therapy
for IBD. It is quite conceivable that fat restriction impairs themental and social aspects of QOL, and enteral nutrition will
make the matter even worse. Of interest, but not apparently
compatible with our findings, is the report by Kuriyama
et al. They reported that enteral nutrition improved thehealth-related quality of life of Crohn’s disease patientswith long-term disease duration, and enteral nutrition was
an independent factor for bowel symptoms and systemic
symptoms [17]. In their study, IBDQ was employed for theassessment of QOL, which is an IBD-targeted questionnairewith many items related to the patients’ gastroenterologicalproblems. Thus it is likely that only the physical aspects of
QOL were detected, and mental aspects were overlooked intheir study.
Two additional considerations might be added to thecurrent finding: decreased QOL in IBD patients and its association with fat restriction. First, considering the response
shift, actual detrimental eﬀect of fat restriction on the mental
aspects of QOL might be even greater. Second, the adaptation
process seems to be only partial. Chronic pain is known to be
associated with response shift [18]. However, the associationof fat restriction with impaired mental aspects of QOL was
obvious in the current study. Since food intake is one of themost fundamental requirements, it is likely that subjects with
fat restriction cannot easily adapt to a situation with longterm fat-restricted diet.
In conclusion, fat restriction exerts undesirable eﬀects
on IBD patients in two diﬀerent ways: decreased intestinal
absorption of fat-soluble substances such as vitamin D and
K and impaired QOL especially in the mental aspects.

Conflict of interests
None of the authors have any conflict of interests.

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ORIGINAL ARTICLE

Relationship of homocysteine and homocysteinerelated vitamins to bone mineral density inJapanese patients with type 2 diabetes
Chizumi Yamada1, Shimpei Fujimoto1*, Kaori Ikeda1, Yuki Nomura2, Ami Matsubara2, Miwako Kanno2, Kenichiro Shide2,
Kiyoshi Tanaka3, Eri Imai4, Tsutomu Fukuwatari4, Katsumi Shibata4, Nobuya Inagaki1
ABSTRACT
Aims/Introduction: To estimate nutritional risk factors for osteoporosis in patients with type 2 diabetes, bone mineral density,
homocysteine level, and intakes and levels of Hcy-related vitamins including folate, vitamin B6 and vitamin B12 were analyzed in across-sectional study.
Materials and Methods: Lumbar spine and femoral neck bone mineral density, serum concentrations of vitamin B6, vitamin B12,and folate and plasma homocysteine levels were measured in 125 Japanese patients with type 2 diabetes. Nutrient intake values
were evaluated using a food frequency questionnaire.
Results: Homocysteine was inversely correlated with bone mineral density, and with both dietary intake and serum concentrationof folate. Intake of green vegetables was correlated with intake and level of folate and homocysteine levels. When the population
was analyzed across the quartiles, bone mineral density, serum folate concentration, folate intake and intake of green vegetables
were lowest in the highest homocysteine group.
Conclusions: In patients with type 2 diabetes, the nutritional status of folate might affect the homocysteine level, a putative risk
factor for osteoporosis. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00088.x, 2011)
KEY WORDS: Osteoporosis, Homocysteine, Folate

INTRODUCTION
Diabetes is becoming increasingly recognized as a risk factor for
osteoporotic fracture. Although fracture risk in patients with
type 2 diabetes is increased compared with normal subjects, notonly in those with low bone mineral density (BMD) but also in
those with normal or high BMD1–3, decreased BMD is a major
determinant of fragility fracture.
Patients with type 2 diabetes often follow a calorie-restricted
diet, but few studies have investigated the sufﬁciency of thesenutrients for the maintenance of skeletal health. Generally,
nutrient intake increases along with energy intake. Ad libitum
food intake values obtained from a longitudinal study in institutionalized elderly found that intake values of vitamins increased
along with increased energy intake4. In contrast, implementation of a low-fat, low-energy diet (1000 or 1500 kcal/day) inpatients with overweight and hyperlipidemia has been shown to
Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto
University, 2Department of Metabolism and Clinical Nutrition, Kyoto University Hospital,
Kyoto, 3School of Human Culture, The University of Shiga Prefecture, Hikone, and4
Department of Food and Nutrition, Kyoto Women’s University, Kyoto, Japan*Corresponding author. Shimpei Fujimoto Tel.: +81-75-751-3560
Fax: +81-75-751-4244 E-mail address: fujimoto@metab.kuhp.kyoto-u.ac.jp
Received 28 July 2010; revised 8 November 2010; accepted 9 November 2010

result in a decrease of the intake of certain nutrients, including
B-vitamins5.
Folate, vitamin B6 and vitamin B12 are important enzymatic
cofactors in the synthesis of methionine from homocysteine(Hcy), and an elevation of Hcy can be caused by insufﬁciency of
folate, vitamin B6 or vitamin B12. Numerous studies have linked
high circulating Hcy levels and low concentrations of folate orvitamin B12 with increased risk of low BMD in non-diabetic subjects6–14. The possibility that elevated Hcy is a risk factor for osteoporosis is suggested by studies of patients with homocystinuria,
a rare autosomal recessive disease characterized by markedly elevated levels of plasma Hcy, in which early onset of generalized
osteoporosis has occurred15,16. The underlying pathophysiologicalmechanism of osteoporosis in patients with elevated Hcy is notcompletely understood. Hcy has been reported to interfere withcross-links of newly formed collagen17,18, and consequently withbone mineralization and strength19, as well as to stimulate osteoclast formation and activity20,21. However, there has been noreport on the association of Hcy and Hcy-related vitamins withosteoporosis in patients with diabetes. Furthermore, vitamin
insufﬁciency was evaluated only by serum vitamin concentrations
in most of these studies, and there has been no comprehensive
investigation of the relationship of dietary intake of nutrients andª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Journal of Diabetes Investigation Volume 2 Issue 3 June 2011

1233

Yamada et al.

serum vitamin concentrations with Hcy and BMD among subjects in the same study.
In the present study, to evaluate nutritional risk factors forosteoporosis in patients with type 2 diabetes, BMD, Hcy level,
and intakes and levels of Hcy-related vitamins including folate,vitamin B6 and vitamin B12 were analyzed.

MATERIALS AND METHODS
Study Population

A total of 125 Japanese patients with type 2 diabetes admitted
between December 2008 and June 2009 to Kyoto University
Hospital were sequentially enrolled in the study. Lateral lumbar
X-ray was carried out to exclude those with scoliosis, compression fractures and ectopic calciﬁcations. Subjects with bilateral
hip fractures or prosthesis and other diseases that might inﬂuence bone metabolism including liver disease, renal dysfunction(serum creatinine above 2 mg/dL), hyperthyroidism, hyperparathyroidism, hypercorticoidism, and hypogonadism were
excluded. All subjects were free of drugs that inﬂuence bone
and calcium metabolism including glucocorticoids, bisphosphonates, calcitonin injection, estrogens, selective estrogen receptor
modulators, vitamin D, vitamin K, thiazide diuretics, heparinand anticonvulsants. The number of patients treated with thiazolidinedione and metformin was 7 and 28, respectively. The
present study was cross-sectional in design, and was approvedby The Ethical Committee of Kyoto University Hospital andcomplies with the Helsinki Declaration. Written informed consent was obtained from all participants.
Measurement of Bone Mineral Density

BMD was measured by dual-energy X-ray absorptiometry(DXA; Discovery; Hologic, Waltham, MA, USA) at the lumbar
spine (L1-L4) and femoral neck. The coefﬁcient of variation of
the measurements of BMD was 0.39%. BMD (g/cm2) was
expressed as Z-score calculated on the basis of the normal reference values of the age- and sex-matched Japanese group provided by the DXA system manufacturer. Because male andfemale patients of different ages were included in the study,
comparison of BMD was made based on Z-scores. Fat mass andlean body mass (without bone mineral content) were measured
by DXA (Hologic Discovery; Hologic) using whole-body absorptiometry software, and each value was expressed in kilograms.
Biochemical Measurements

Blood samples were obtained after overnight fasting immediately
after admission. Glycosylated hemoglobin (HbA1c) was measured by high performance liquid chromatography (HPLC). The
value for HbA1c (%) is estimated as a National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) calculated by the formula HbA1c (%) = HbA1c [Japan Diabetes
Society (JDS); %] +0.4%, considering the relational expression of
HbA1c (JDS; %) measured by the previous Japanese standard
substance and measurement methods and HbA1c (NGSP)22.
Fasting serum C-peptide was measured by ELISA (ST AIA-

234

Journal of Diabetes Investigation Volume 2 Issue 3 June 2011

PACK C-Peptide; Toso Corporation, Tokyo, Japan). Bonespeciﬁc alkaline phosphatase (BAP) was measured by enzyme
immunoassay (Osteolinks BAP; DS Pharma Biomedical, Suita,
Japan), and urine N-terminal cross-linked telopeptide of type-I
collagen (uNTx) was measured by ELISA (Osteomark NTx
ELISA Urine; Inverness Medical, Waltham, MA, USA). Plasma
Hcy levels were determined by HPLC using a thiol-speciﬁc ﬂuorogenic reagent, ammonium 7-ﬂuorobenzo-2-oxa-1,3-diazole4-sulfate23, and the upper limit of Hcy was 13.5 nmol/L. As pyridoxal 5¢-phosphate (PLP) is the predominant circulating formof vitamin B6, serum PLP concentrations were measured by
HPLC24,25 for evaluation of vitamin B6 status. For vitamin B12
measurement, 0.2 mmol/L acetate buffer (pH 4.8) was added tothe serum samples, and the vitamin B12 was converted to cyanocobalamin by boiling with 0.0006% potassium cyanide at acidic
pH. Cyanocobalamin was determined by the microbioassay
method using Lactobacillus leichmanii, ATCC 783024,25. Serum
folate was determined by the microbioassay method using
Lactobacillus casei ATCC 273324,25.
Evaluation of Dietary Nutrient Intake

A food frequency questionnaire (FFQ) validated by Takahashi
et al.26,27 was used to calculate nutrient intakes. The FFQ usedin the present study included questions on the consumption ofvarious food items over the previous 1 or 2 months. Daily nutrient intake was calculated by multiplying the frequency of consumption of each food by the nutrient content of the portion
size and summing the products for all food items. The FFQ isvalidated against 7-day dietary records and the FFQ-estimatednutrient intake values are 72–121% of those of 7-day dietary
records26. The reproducibility of the FFQ at intervals of
1–2 months is 93–119% for each nutrient26. Correlations of dietary folate intake, serum folate concentration, and plasma Hcylevel with intakes of various food groups including grain/rice,
potato, green vegetables, other vegetables, fruits, seaweeds,beans/soy products, seafood, meats, egg, milk products and oil/
fat were evaluated.
Statistical Analysis

Data were expressed as mean ± SD. SPSS statistical software (version 13.0; SPSS, Chicago, IL, USA) was used for all statisticalanalyses. Pearson’s correlation coefﬁcient was calculated as ameasure of association by adjusting for age and sex whereappropriate. Stepwise multiple linear regression analyses werecarried out to determine independent factors for plasma Hcylevels including (i) dietary vitamin B6, vitamin B12 and folate
intake values; and (ii) serum PLP, vitamin B12 and folate concentrations as independent variables. The relationship between
BMD with Hcy and Hcy-related vitamins was further exploredusing a quartile-based analysis. Statistical differences among thegroups were evaluated using analysis of covariance (ANCOVA)
adjusted for age and sex, and Dunnett’s multiple comparison
tests by comparison with the highest Hcy group. P < 0.05 wasconsidered signiﬁcant.

ª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Homocysteine and bone in diabetes

RESULTSClinical characteristics, laboratory data and nutrient intake ofsubjects are shown in Table 1. The average serum vitamin B12
concentration was 1.45 ± 0.45 pmol/mL (Table 1) and there
was no difference between patients taking metformin (1.52 ±0.49 pmol/mL, n = 97) and those without (1.43 ± 0.49 pmol/
mL, n = 28). Nutrient intake values were signiﬁcantly positively
correlated with total energy intake (Table 2). Dietary vitamin
B6, vitamin B12 and folate intake values were positively correlated with serum vitamin B6, vitamin B12 and folate levels,
respectively (Table 2). Plasma Hcy levels were negatively correlated with both dietary intake and serum concentration of folate(Table 2). Only vitamin B6 intake and not vitamin B6 concentration showed a weak negative correlation with Hcy; the inﬂuence of vitamin B12 on Hcy elevation was unclear (Table 2).
Stepwise multiple linear regression analyses were carried out toTable 2 | Correlations among dietary nutrient intake values, serum
concentrations and plasma homocysteine levels adjusted for age and sex

Table 1 | Background characteristics of the study subjects

Hcy, homocysteine; PLP, pyridoxal 5¢-phosphate.

r
Correlations of total energy intake with various nutrient intakes
0.521
Vitamin B6 (mg)0.253
Vitamin B12 (lg)
Folate (lg)0.331
Correlations of intake values with serum concentrations
Vitamin B6
0.192
0.336
Vitamin B12
Folate0.400
Correlations of plasma Hcy levels with B vitamins
Vitamin B6 intake (mg))0.207
Vitamin B12 intake (lg))0.001
Folate intake (lg))0.328Serum PLP concentration (pmol/mL)
0.002
Serum B12 concentration (pmol/mL)
0.001
Serum folate concentration (pmol/mL))0.369P<0.001
0.005<0.001
0.034<0.001<0.001
0.022
0.988<0.001
0.982
0.993<0.001

Characteristic
No. subjects
Male/female
Age (years)
Duration of diabetes (years)
Diabetes treatment(diet/OHA/Ins/Ins + OHA)BMI (kg/m2)
Fat mass (kg)Lean body mass (kg)
Fasting plasma glucose (mg/dL)
HbA1c (%)
Fasting serum C-peptide (ng/mL)
Serum BAP (U/L)
uNTx (nMBCE/mmol Cr)
Energy intake (kcal/day)Protein/fat/carbohydrate
intake (g/day)
Calcium intake (mg/day)
Vitamin D intake (lg/day)
Vitamin B6 intake (mg/day)
Vitamin B12 intake (lg/day)
Folate intake (lg/day)Serum PLP concentration(pmol/mL)
Serum vitamin B12
concentration (pmol/mL)
Serum folate concentration(pmol/mL)
Plasma homocysteine
concentration (nmol/mL)

125
79 (63.2%)/46 (36.8%)
61.2 ± 12.4
11.2 ± 9.4
27 (21.6%)/62 (49.6%)/
28 (22.4%)/8 (6.4%)
24.9 ± 4.9
16.5 ± 9.8
45.9 ± 9.3
160.2 ± 48.6
9.6 ± 2.2
1.71 ± 0.89
23.5 ± 8.7
35.6 ± 19.8
2073.2 ± 582.5
73.6 ± 19.7/64.4 ± 23.7/
278.7 ± 80.2
596.0 ± 213.6
9.21 ± 4.48
1.22 ± 0.34
8.81 ± 4.65
287.4 ± 100.5
61.3 ± 29.1

Data are number of patients (categorized data) or mean ± SD
(quantitative data).
BAP, bone-speciﬁc alkaline phosphatase; BMI, body mass index;
Ins, insulin; OHA, oral hypoglycemic agents; PLP, pyridoxal 5¢-phosphate;uNTX, urine N-terminal cross-linked telopeptide of type-I collagen.

determine independent factors for plasma Hcy levels. Dietary
folate intake was a signiﬁcant predictor of Hcy when dietary
vitamin B6, vitamin B12 and folate intake values were included
as independent variables (R2 = 0.088, b-coefﬁcient = )0.297,
P < 0.001), and serum folate concentration also was a signiﬁcantpredictor of Hcy when serum PLP, vitamin B12 and folate concentrations were included as independent variables (R2 = 0.121,
b-coefﬁcient = )0.347, P < 0.001). We then evaluated the correlations of folate intake and the concentrations of folate and Hcy
with intake of the various food groups determined by FFQ.
Dietary folate intake and serum folate concentration were significantly associated with intakes of certain food groups including
potato, green vegetables, other vegetables and fruits. Only intake
of green vegetables was signiﬁcantly correlated with the plasma
Hcy level (Table 3).
Bone mineral density of lumbar spine (SP-BMD) and femoral
neck (FN-BMD) were positively correlated with body massindex (BMI) and fat mass, although no signiﬁcant correlations
were found in diabetes-related parameters including fasting
plasma glucose, HbA1c and diabetes duration (Table 4). Both
SP-BMD and FN-BMD were positively correlated with fasting
serum C-peptide, but these correlations were cancelled when
adjusted for BMI. Urinary NTx, a marker of bone resorption,
was negatively correlated with FN-BMD. As nutrient intake signiﬁcantly increases with energy intake, nutrition intakes werealso evaluated by adjusting for calories. As a result, calorieadjusted folate intake was positively correlated with SP-BMD,
although the association between calorie-adjusted folate and
FN-BMD did not reach statistical signiﬁcance. There were no
signiﬁcant associations between BMD of both sites and serum
concentrations of vitamin B6, vitamin B12 and folate. The
plasma Hcy concentration was negatively correlated with bothª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Journal of Diabetes Investigation Volume 2 Issue 3 June 2011

1.45 ± 0.45
27.5 ± 10.3
11.2 ± 5.1

235

Yamada et al.

Table 3 | Correlations of dietary folate intake, serum folate concentrationand plasma homocysteine level with various food groups
Dietary folate
intakeSerum folate
concentration

Plasma Hcy
level

r

r

r

P

P

P

Grain/rice)0.076 0.399 )0.086 0.341 )0.056 0.538
Potato0.470 <0.001
0.220 0.014
0.012 0.895
Green vegetables
0.843 <0.001
0.361 <0.001 )0.207 0.020
Other vegetables
0.620 <0.001
0.197 0.027
0.077 0.390
Fruits
0.338 <0.001
0.206 0.021
0.018 0.839
Seaweeds
0.322 <0.001
0.072 0.426
0.071 0.435
Beans/soy products
0.390 <0.001
0.156 0.083
0.016 0.856
Seafood
0.313 <0.001
0.075 0.407 )0.017 0.848
Meats
0.065 0.474
0.042 0.643 )0.070 0.435
Egg
0.278 0.002
0.068 0.450 )0.056 0.538
Milk products
0.108 0.230
0.113 0.208 )0.035 0.698
Oil/fat0.145 0.107
0.161 0.073 )0.112 0.214
Hcy, homocysteine.
Table 4 | Correlations of bone mineral density of lumbar spine and femoral neck with diabetes-related parameters, bone turnover markers andB vitamin status
SP-BMD2

BMI (kg/m )Fasting plasma glucose (mg/dL)
HbA1c (%)
Diabetes duration (years)
Fasting serum C-peptide (ng/mL)
BAP (U/L)
uNTx (nMBCE/mmol Cr)
Vitamin B6 intake (mg)
Vitamin B6 intake (mg/100 kcal)
Vitamin B12 intake (lg)
Vitamin B12 intake (lg/1000 kcal)
Folate intake (lg)
Folate intake (lg/1000 kcal)
Serum PLP concentration(pmol/mL)
Serum B12 concentration(pmol/mL)
Serum folate concentration(pmol/mL)
Plasma Hcy concentration(nmol/mL)6.00

r = –0.278, P = 0.002

4.00

2.00

0.00

–2.00

–4.00
0.00

10.00

20.00
Hcy (nmol/mL)

30.00

40.00

FN-BMD (Z score)(b)r = –0.201, P = 0.025
4.00

FN-BMD

r

P

r

P

0.288
)0.149
0.098
0.082
0.182
0.112
)0.138)0.032
0.113
0.012
0.054
0.103
0.198
)0.062

0.001
0.098
0.194
0.366
0.045
0.218
0.084
0.727
0.211
0.899
0.554
0.256
0.027
0.497

0.463
)0.113
0.053
0.057
0.285
)0.061)0.183)0.053
0.005
0.166
0.148
0.112
0.153
)0.007<0.001
0.210
0.556
0.528
0.001
0.499
0.042
0.559
0.959
0.065
0.102
0.216
0.090
0.936

0.023

0.799

0.058

0.524

0.104

0.248

0.114

0.205

)0.2780.002)0.201

0.025

BAP, bone-speciﬁc alkaline phosphatase; BMI, body mass index; FN-BMD
bone mineral density of femoral neck; Hcy, homocysteine; PLP, pyridoxal
5¢-phosphate; SP-BMD, bone mineral density of lumbar spine; uNTX,
urine N-terminal cross-linked telopeptide of type-I collagen.

SP-BMD and FN-BMD, showing that hyperhomocysteinemia isclearly associated with low BMD in patients with type 2 diabetes(Figure 1).

236

SP-BMD (Z score)(a)Journal of Diabetes Investigation Volume 2 Issue 3 June 2011

2.00

0.00

–2.00
0.00

10.00

20.00
Hcy (nmol/mL)

30.00

40.00

Figure 1 | The relationship between homocysteine (Hcy) and bonemineral density of lumbar spine (SP-BMD) and femoral neck (FN-BMD).

As hyperhomocysteinemia derived from folate insufﬁciency
has been suggested to be involved in low BMD, we comparedclinical characteristics of the study population across the quartiles of Hcy (quartile 1, n = 31, Hcy < 8.3 nmol/mL; quartile 2,n = 32, Hcy 8.3 to <9.9 nmol/mL; quartile 3, n = 32, Hcy 9.9
to <12.8 nmol/mL; quartile 4, n = 30, Hcy > 12.8 nmol/mL).
There were no signiﬁcant differences across the quartiles in general clinical characteristics including age, BMI, diabetes-relatedparameters, energy intake, and vitamin B6 and vitamin B12 status (Table 5). However, SP-BMD and FN-BMD were signiﬁcantly lower in patients in the highest quartile of Hcy thanª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Homocysteine and bone in diabetes

Table 5 | Comparison of clinical characteristics according to homocysteine quartiles adjusted for age and sex
Hcy concentration (nmol/mL)
Male/female
Age (years)
BMI (kg/m2)
Fasting plasma glucose (mg/dL)
HbA1c (%)
Diabetes duration (years)
SP-BMD (Z score)
FN-BMD (Z score)
Energy intake (kcal/day)
Vitamin B6 intake (mg)
Vitamin B12 intake (lg)
Folate intake (lg)Intake of green vegetables (g/day)Serum PLP concentration (pmol/mL)
Serum B12 concentration (pmol/mL)
Serum folate concentration (pmol/mL)
Plasma Hcy concentration (nmol/mL)

Quartile 1(4.9–8.0)

Quartile 2(8.1–9.9)

Quartile 3(10.0–12.8)

Quartile 4(12.8–35.7)

17/14
59.3 ± 13.8
25.0 ± 4.4
158.8 ± 44.8
10.1 ± 2.3
9.5 ± 8.4
1.34 ± 1.43*
0.45 ± 0.99**2161 ± 543
1.31 ± 0.35
8.59 ± 3.44
323.5 ± 92.2**
101.9 ± 65.3*
65.0 ± 33.1
2.39 ± 0.88
33.6 ± 11.5**
6.9 ± 0.9**

21/11
58.1 ± 12.6
25.8 ± 5.0
162.0 ± 45.8
9.9 ± 2.5
10.2 ± 9.7
1.24 ± 1.38*
0.32 ± 1.23*
2145 ± 565
1.26 ± 0.36
8.86 ± 4.64
287.2 ± 108.0*
86.1 ± 60.6
60.0 ± 24.6
2.90 ± 1.61
26.9 ± 7.6*
9.1 ± 0.5**

23/9
63.9 ± 8.7
25.0 ± 5.6
155.6 ± 50.0
9.1 ± 1.8
12.6 ± 8.6
1.39 ± 1.24*
0.26 ± 0.96*
2069 ± 563
1.21 ± 0.32
9.49 ± 5.24
305.0 ± 91.8**
89.3 ± 47.5
58.9 ± 32.9
2.50 ± 0.73
26.9 ± 9.0*
11.3 ± 0.8**

18/12
64.0 ± 13.4
23.8 ± 4.5
164.6 ± 55.4
9.4 ± 2.1
12.4 ± 9.0
0.50 ± 1.18
)0.27 ± 1.03
1910 ± 650
1.09 ± 0.29
8.27 ± 5.21
231.7 ± 89.1
68.9 ± 49.2
61.4 ± 26.2
2.53 ± 0.92
21.7 ± 8.7
17.8 ± 6.1

ANCOVA

P
0.212
0.461
0.721
0.378
0.183
0.037
<0.001
0.260
0.136
0.798
0.001
0.043
0.943
0.419
<0.001<0.001

BMI, body mass index; FN-BMD bone mineral density of femoral neck; Hcy, homocysteine; PLP, pyridoxal 5¢-phosphate; SP-BMD, bone mineraldensity of lumbar spine. Mean ± SD, *P < 0.05, **P < 0.01 relative to the highest homocysteine quartile group.

those in patients in the other quartiles. Furthermore, patients in
the highest Hcy quartile showed signiﬁcantly decreased dietary
folate intake, serum folate concentration and intake of green
vegetables compared with those in the lower Hcy quartiles.
Because the caloric intake was similar across the quartiles, thequality of the diet might be poor in the highest Hcy group.
Quartile analysis revealed that the highest Hcy group showed
the lowest BMD, the lowest serum folate concentration, the lowest folate intake and the lowest intake of green vegetables.

DISCUSSION
In the present study, hyperhomocysteinemia was found to beclearly associated with low BMD in type 2 diabetes patients, as
it has been reported to be in non-diabetic subjects6–14. Furthermore, folate insufﬁciency might be one of the important factors
in hyperhomocysteinemia, as plasma Hcy levels were negatively
correlated with both dietary intake and serum concentration of
folate.
Osteoporosis is a multifactorial disease, a major health problem characterized by low BMD, deterioration of bone microarchitecture and increased risk of fracture. Elevation of Hcy is oneof the important risk factors for osteoporosis28,29, and can becaused by insufﬁciency of Hcy-related vitamins, such as folate,vitamin B6 and vitamin B126–14. Because dietary risk factors canbe improved when recognized, sufﬁciency of Hcy-related vitamins and its relationship to osteoporosis in patients with type 2
diabetes is of primary concern.
Elevation of Hcy can be caused by insufﬁciency of folate, vitamin B6 or vitamin B12, and the plasma Hcy level is considered
to be a fairly sensitive index of folate metabolic status comparedwith that of the other factors in non-diabetic subjects. Previous
studies reported hyperhomocysteinemia was observed in 86% ofsubjects with clinically expressed folate deﬁciency30; folate is amajor determinant of Hcy levels in healthy people31,32 and vitamin B12 inﬂuences Hcy levels less than folate does33,34. Folate,vitamin B6 and vitamin B12 are water-soluble vitamins, which
are in general not readily stored and consistent daily intake isimportant. Usually, folate and vitamin B6 deﬁciency develops
within a month of insufﬁcient intake. In contrast, it is known
that patients with complete loss of intrinsic factor require
3–5 years to become overtly vitamin B12 deﬁcient35. Vitamin
B12 is a unique water-soluble vitamin, and because 80% of the
2.5 mg average whole body stock of vitamin B12 is reserved in
the liver and vitamin B12 excreted in the bile and is effectively
reabsorbed in the intestine, clinical signs of vitamin B12 deﬁciency take a long time to appear and progress slowly36. Somepatients in the present study were taking metformin, which is
known to inhibit absorption of vitamin B1237, but there was nodifference between the patients taking metformin and those nottaking metformin. As to vitamin B6, only a weak negative correlation between vitamin B6 intake and Hcy was not enough toconclude that vitamin B6 is a nutritional risk factor for osteoporosis, and there have been no other studies showing the effectof vitamin B6 on BMD.
Leafy green vegetables, such as spinach and broccoli, are richsources of folate. Folate is also contained in a variety of foods
including fruits, beans, seaweeds, liver and egg yolk. To investigate the cause of folate insufﬁciency, we focused particularly on
dietary sources of folate. We evaluated the association of dietary
folate intake, serum folate concentration, and plasma Hcy levelª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Journal of Diabetes Investigation Volume 2 Issue 3 June 2011237

Yamada et al.

with various food groups, and found that intake of green vegetables correlated well with folate status and Hcy levels. Furthermore, it was revealed by the quartile analysis that the highest
Hcy group showed the lowest BMD, the lowest serum folate
concentration, the lowest folate intake and the lowest intake ofgreen vegetables. This analysis suggests that insufﬁcient intake ofgreen vegetables, but not insufﬁcient caloric intake, causes folate
insufﬁciency in the group with the highest Hcy.
The strength of the present study is that it is the ﬁrst study
to show that nutritional status of folate might affect the homocysteine level, a putative risk factor for osteoporosis, in Japanesepatients with type 2 diabetes. The present study is also meaningful in promoting awareness of the importance of diet quality,
because patients with diabetes are at high risk of developing
osteoporosis. In contrast, the present study has some limitations. First, the sample size was not large enough for conclusions regarding marginal insigniﬁcant P-values. We estimatedsample size using a correlation coefﬁcient obtained from a previous cross-sectional study assessing the relationship between
BMD and plasma Hcy8. The correlation coefﬁcient of femoral
BMD with Hcy was )0.18 and the sample size was estimatedto be n = 153 (two-sided a = 0.1, b = 0.2), while we analyzed
125 patients. Second, we only analyzed patients with type 2 diabetes and comparison with non-diabetic subjects is necessary.
An unanswered question is whether diabetes modulates theeffects of nutritional state of folate on Hcy metabolism, and theeffects of Hcy levels on BMD. Finally, a longitudinal study isrequired to examine the effects of Hcy on rate of BMD loss
and risk of fracture for a longer duration in patients with type 2
diabetes. It is also necessary to determine whether encouraging
patients with higher Hcy levels to eat more green vegetables
is useful as a dietary intervention to improve Hcy levels
and BMD.
In conclusion, the present study shows that BMD inversely
correlates to plasma Hcy levels in Japanese patients with type 2
diabetes, and that dietary intake and the serum concentration of
folate are determinant factors of Hcy levels. When our group
was analyzed across quartiles, BMD, serum folate concentration,
folate intake and intake of green vegetables were lowest in thehighest Hcy group. Taken together, in Japanese patients with
type 2 diabetes, a diet low in green vegetables rather than a calorie-restricted diet might be the more important factor in the
declining nutritional status of folate that increases the Hcy level,a putative risk factor for osteoporosis.

ACKNOWLEDGEMENTS
This study was supported in part by Diabetes Masters Conference and Japan Diabetes Foundation. The authors declare no
conﬂicts of interest.
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3. Adami S. Bone health in diabetes: considerations for clinicalmanagement. Curr Med Res Opin 2009; 25: 1057–1072.
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5. Grzybek A, Klosiewicz-Latoszek L, Targosz U. Changes in theintake of vitamins and minerals by men and women withhyperlipidemia and overweight during dietetic treatment.
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6. Gjesdal CG, Vollset SE, Ueland PM, et al. Plasma total homocysteine level and bone mineral density: the Hordaland
Homocysteine Study. Arch Intern Med 2006; 166: 88–94.
7. Baines M, Kredan MB, Usher J, et al. The association of homocysteine and its determinants MTHFR genotype, folate, vitamin B12 and vitamin B6 with bone mineral density inpostmenopausal British women. Bone 2007; 40: 730–736.
8. Golbahar J, Hamidi A, Aminzadeh MA, et al. Association ofplasma folate, plasma total homocysteine, but not methylenetetrahydrofolate reductase C667T polymorphism, withbone mineral density in postmenopausal Iranian women:a cross-sectional study. Bone 2004; 35: 760–765.
9. Golbahar J, Aminzadeh MA, Hamidi SA, et al. Association of
red blood cell 5-methyltetrahydrofolate folate with bonemineral density in postmenopausal Iranian women. Osteoporos Int 2005; 16: 1894–1898.
10. Morris MS, Jacques PF, Selhub J. Relation between homocysteine and B-vitamin status indicators and bone mineraldensity in older Americans. Bone 2005; 37: 234–242.
11. Cagnacci A, Baldassari F, Rivolta G, et al. Relation of homocysteine, folate, and vitamin B12 to bone mineral density of
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12. Tucker KL, Hannan MT, Qiao N, et al. Low plasma vitamin
B12 is associated with lower BMD: the Framingham Osteoporosis Study. J Bone Miner Res 2005; 20: 152–158.
13. Dhonukshe-Rutten RA, Pluijm SM, de Groot LC, et al. Homocysteine and vitamin B12 status relate to bone turnovermarkers, broadband ultrasound attenuation, and fractures inhealthy elderly people. J Bone Miner Res 2005; 20: 921–929.
14. Dhonukshe-Rutten RA, Lips M, de Jong N, et al. Vitamin B-12
status is associated with bone mineral content and bonemineral density in frail elderly women but not in men. J Nutr
2003; 133: 801–807.
15. Harpey JP, Rosenblatt DS, Cooper BA, et al. Homocystinuria
caused by 5,10-methylenetetrahydrofolate reductase deﬁciency: a case in an infant responding to methionine, folinicacid, pyridoxine, and vitamin B12 therapy. J Pediatr 1981; 98:
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16. Mudd SH, Skovby F, Levy HL, et al. The natural historyof homocystinuria due to cystathionine beta-synthasedeﬁciency. Am J Hum Genet 1985; 37: 1–31.

ª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Homocysteine and bone in diabetes

17. Lubec B, Fang-Kircher S, Lubec T, et al. Evidence for McKusick’s hypothesis of deﬁcient collagen cross-linking inpatients with homocystinuria. Biochim Biophys Acta 1996;
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23. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with ﬂuorescence detection. J Chromatogr 1987;
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24. Shibata K, Fukuwatari T, Watanabe T, et al. Intra- and interindividual variations of blood and urinary water-soluble vitamins in Japanese young adults consuming a semi-puriﬁed
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25. Fukuwatari T, Yoshida E, Takahashi K, et al. Effect of fastingon the urinary excretion of water-soluble vitamins inhumans and rats. J Nutr Sci Vitaminol (Tokyo) 2010; 56:
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26. Takahashi K, Yoshimura Y, Kaimoto T, et al. Validation of afood frequency questionnaire based on food groups for
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221–232.

27. Miyaki K, Tohyama S, Murata M, et al. Salt intake affects therelation between hypertension and the T-786C polymorphism in the endothelial nitric oxide synthase gene. Am J
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28. van Meurs JB, Dhonukshe-Rutten RA, Pluijm SM, et al. Homocysteine levels and the risk of osteoporotic fracture. N Engl J
Med 2004; 350: 2033–2041.
29. Herrmann M, Peter Schmidt J, Umanskaya N, et al. The roleof hyperhomocysteinemia as well as folate, vitamin B6 andB12 deﬁciencies in osteoporosis: a systematic review. Clin
Chem Lab Med 2007; 45: 1621–1632.
30. Savage DG, Lindenbaum J, Stabler SP, et al. Sensitivity ofserum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deﬁciencies.
Am J Med 1994; 96: 239–246.
31. Selhub J, Jacques PF, Wilson PW, et al. Vitamin status andintake as primary determinants of homocysteinemia in anelderly population. JAMA 1993; 270: 2693–2698.
32. Selhub J, Jacques PF, Rosenberg IH, et al. Serum total homocysteine concentrations in the third National Health andNutrition Examination Survey (1991–1994): population reference ranges and contribution of vitamin status to highserum concentrations. Ann Intern Med 1999; 131: 331–339.
33. Lindenbaum J, Rosenberg IH, Wilson PW, et al. Prevalence of
cobalamin deﬁciency in the Framingham elderly population.
Am J Clin Nutr 1994; 60: 2–11.
34. Carmel R. Cobalamin Deﬁciency, Homocysteine in Health andDisease. Cambridge University Press, Cambridge, 2001.
35. Chanarin I. The Megaloblastic Anemias, 2nd edn. Blackwell
Scientiﬁc, Oxford, 1979.
36. Food and Nutrition Board, Institute of Medicine. The B vitamins and choline: overview and methods. In: Institute ofMedicine. Dietary Reference Intakes: For Thiamine, Riboﬂavin,
Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. National Academy Press, Washington DC,
1998; 306–356.
37. Adams JF, Clark JS, Ireland JT, et al. Malabsorption of vitamin
B12 and intrinsic factor secretion during biguanide therapy.
Diabetologia 1983; 24: 16–18.

ª 2010 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd

Journal of Diabetes Investigation Volume 2 Issue 3 June 2011239

Author's personal copy

Clinical Nutrition 30 (2011) 261

Contents lists available at ScienceDirect

Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu

Correspondence

Overestimated serum albumin levels in patients with hip fracture
Hip fracture is associated with high mortality and morbidity.
Malnutrition has been reported to an important predictor of clinicaloutcomes. Serum albumin level and blood total lymphocyte count(TLC) are considered to be the simple, but reliable markers indicating the patients’ nutritional status, and often employed invarious clinical settings including hip fracture.1 Recently O’Daly
et al.2 in the recent issue of Clinical Nutrition, studied the signiﬁcance of these two markers as the predictors of outcome in hip fractured patients. They deﬁned protein energy malnutrition (PEM) as
serum albumin level below 3.5 g/dl and TLC below 1500/mm3.
Patients with both parameters below the cut-off had higher one
year mortality than those with both values above the cut-off(odds ratio; 4.6). Cox regression analysis revealed that serum
albumin level (hazard ratio; 0.932) and age were independent
prognostic factors of mortality. Lee et al.3 also reported that serum
albumin was associated with increased post-operative complications (odds ratio; 6.23) after adjusting for various confounders.
Although these ﬁndings are of clinical interest, measurement
method for serum albumin is not described. Serum albumin levelcan be measured with various ways, each having its own pros
and cons. BCG (bromocresol green) binding assay is the mostfrequently used one, but has the disadvantage that BCG also binds
to a1 and a2 globulins, which are acute phase proteins andincreased in inﬂammation. Thus, serum albumin level can be overestimated with BCG assay. Then, we have measured serum albumin
level in 86 patients with hip fracture by BCG assay (albumin-BCG)
and nepherometry (albumin-N), which is based on antigen–antibody interaction and free from the interference by globulins.
Written consent was obtained from the subjects or the proxy.
Albumin-BCG (3.86  0.42 g/dl) was signiﬁcantly higher thanalbumin-N (3.51  0.43 g/dl) by paired t-test (p < 0.001). Next,
we have compared the number of subjects with their serum
albumin level below or above the cut-off of 3.5 g/dl (Table 1). All
subjects with albumin-BCG below 3.5 g/dl had also albumin-N
below 3.5 g/dl. In contrast, approximately half of the subjects
with albumin-BCG above 3.5 g/dL had albumin-N below 3.5 g/dl.
Our results show that the measurement of serum albumin levelby BCG assay could overestimate the serum albumin level, andunderestimate the percentage of subjects with PEM in hip fractured
patients. Thus, the method of serum albumin measurement mustbe speciﬁed, and the positive interference by globulins in the

Table 1Comparison of albumin-BCG and albumin-N.
Albumin-N

Albumin-BCG<3.5 g/dl
3.5 g/dl<3.5 g/dl

3.5 g/dl

13
36

0
37

Albumin-BCG and Albumin-N indicate the serum albumin level determined by BCG
assay, and that by nepherometry, respectively. Data were analyzed by chi-square
test (p ¼ 0.002).

bye-binding assay should be taken into account in the nutritional
assessment of the hip fractured subjects.
Conﬂict of interestv statement
None of the authors have any conﬂicts of interest.
References
1. Gibson RS. Assessment of protein status. In: Gibson RS, editor. Principles of nutritional assessment. 2nd ed. New York: Oxford University Press; 2005. p. 403–42.
2. O’Daly BJ, Walsh JC, Quinlan JF, Falk GA, Stapleton R, Quinlan WR, et al. Serum
albumin and total lymphocyte count as predictors of outcomes in hip fractures.
Clin Nutr 2010;29:89–93.
3. Lee HP, Chang YY, Jean YH, Wang HC. Importance of serum albumin level in the
preoperative tests conducted in elderly patients with hip fracture. Injury 2009;40:756–9.

Tetsuo Nakano
Department of Orthopedics, Tamana Central Hospital, Japan
Akiko Kuwabara*
Department of Health and Nutrition,
Osaka Shoin Women's University, 4-2-26 Hishiyanishi,
Higashiosaka-shi, Osaka 577-8550, Japan*Tel.: þ81 6 6723 8181;
fax: þ81 6 6723 8348.
E-mail address: kuwabara.akiko@osaka-shoin.ac.jp.
Kiyoshi Tanaka
Department of Food and Nutrition, Kyoto Women's University, Japan

0261-5614/$ – see front matter Ó 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
doi:10.1016/j.clnu.2010.09.005

29 July 2010608

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日本人の食事摂取基準における目安量は健康人の摂取の中央値 でよいのか ?
Adequate intakeは D fOr宙 tamin D;how is it to be determined?

本稿 は トビックスの原稿 として書 い た ものであ るが最近 の話題 を取 り上 げて概説 す るとい う本来 の トピ ック
スではない 著者 らがふだん疑 間に感 じてい る点を述 べ

D不 足の意義が考慮 されるようになつて きた と思 われる
例 えば,第 六次改訂 日本人の栄養所要量 にお い ては,「 20〜 46歳 の 人で 1 7μ g(681U)ノ 日以下の ビタミン D摂 取て, ビタ ミン学会会員諸先 生の ご意見 を承 りた い とい う
意図で書 い た ものであ り,い わば著者 らの個人的見解 を述べ たもので あ ることを,最 初 にお ことわ りしてお くを数 年間続 けると骨軟化症が認 め られるようにな り,25
μg(1001U)/日 では発生 はみ られなかつた との報告があるので,2 5μ g(1001U)/日 とした」と述べ られてお り, この記述 はクル病 骨軟化症防止 を念頭 にお い た,欠 乏症
対策 と理解 される しか し,現行 の 2010年 版 におい ては「成 人, と くに高齢者 にお いて, ビタミン D欠 乏 とはい,

ビタミンの欠乏 によ り,古 典的欠乏症が起 こる

ビタ

ミン Bl欠 乏による脚気 ウェルニ ッケ脳症,ナ イアシン欠乏 によるペ ラグラ, ビタミン C欠 乏 による壊血病, ビ
タミン K欠 乏 による出血傾向な ど 多数 の有名な例がある 少な くともわが国にお い ては, これ らの 多 くはほぼ克服 された といって よいが,近 年 ビタミンの新たな意義が注 目されてい る
古典的な欠乏症 を起 こす ほ どの ビ タミン欠乏 (dendc̲
り軽 度 の ビ タ ミ ン不足 (insumcicncy)で あ って も,")よ
疾患 の リス クが増加す る
ビタミン Dを 例 に述 べ る と,骨 は タ ンパ ク質 (主 に コ
ラーゲ ン)の 枠組み の上 に, リン酸 カルシウムが 沈着 して(石 灰化)形 成 される ビタミン Dの 最 も基本的な作用は,

腸管か らの カルシウム リン吸J又 促進 なので, ビタミンD欠 乏の結果 ,石 灰 化障害が起 こるのが クル病 骨軟化症である
一方, よ り軽度の不足 の場合,石 灰化障害 は起 こ らないが,骨 粗豚症 骨折 の リス ク増加が起 こる 副甲1大 腺ホル
モ ン (PIH)と ビ タミン Dは 血清 カルシウム濃度 を維持するのが重要 な役割であ り,PTHは ビタ ミン Dの 活性化 を促進 し,活 性型 ビ タミンDは PrH分 泌 を抑制す るとい う
形で,両 者 は協調 して,血 清 カルシウム濃度を維持するのに作用す るが,作 用が過剰 となって,高 カルシウム血症を起 こさない ように, フイー ドバ ック調節 を受けている し
たが って, ビ タミ ン D不 足状態 にお い ては,PrH分 泌 が克進 し,そ れによって骨吸収が尤進 して,骨 粗穆症 をきた
す なお, ビ タミン D栄 養状態 の最 もよい指標 は:血 中
25ヒ ドロキシ ビタミン D(250HD)濃 度 であ る
わが 国にお い て,残 念 なが らまだまだ ビタミ ン不足の重要性が十分認識 され て い るとは言 いがた い状況 で あるが,そ の重要性が わか りに くいの も事実 であ る 脚気
クル病 壊 血病 と列挙す れば明 らかなよ うに,古 典的欠乏症 は外 見上 の異常 を伴 うので,個 々の人 ごとにその有無が判定 で きる しか し, ビタミ ン D不 足 の場合 ,外 見
上 は何 の異常 もな く,疫 学調査 によ り初 めて疾患 リスク
が1曽 加 してい ることが 明 らかになる
食事摂取基準 にお いて も,改 訂 の度 に徐 々 に ビタミン,

えな い ものの ビタミン D不 足の状態が長期 にわたって続くと,血 中副 甲状腺 ホルモ ン濃度が上昇 し,骨 密度が低下する したが って,正 常なカルシウム利用能が保持 され血中副甲状腺 ホルモ ン濃度が上昇 しない血中 25‑ヒ ドロキ
シビタミン D濃 度を維持す るのに必要 な量の ビ タミン D,を摂 取す ることが.骨 折や骨粗修症 などの予防の観点か
ら重要 と考 え られる しか し,そ の血 中濃度 を与 える ビ
タミン D摂 取量 に関す る根拠 は乏 しいため,そ の血 中濃度 を維持 してい る と考 えられる集 団 のビタミン D摂 取量の中央値 を目安量 と したJと の記述が み られ, これは明 ら
かに, ビタ ミン D不 足 による骨折 リス ク増加対策 をも意識 した ものである この ように,不 足 をも考慮す る時代になる と, 目安量の策定 にお い て,欠 乏対策だけを考 え
てい た 時代 にはなかった,新 た な問題点 を生 じて きたのではないか とい うのが ,著 者 らが最近考 えてい ることである
目安量の策定理論 に関 して,2010年 版 の記述 を引用す
ると,「 特定 の集団にお いて,生 体指標等 を用 い た健 康状
態 の確認 と当該栄養素摂取量 の調査 を同時 に行 い。そ の
結果か ら不足状態 を示す者力ヽまとん ど存在 しない摂取量を推測 し,そ の値 を用 い る 対象集団 で不足状態 を示 す
者が ほ とん ど存在 しない場合 には栄養素摂取量 の 中央値を用 い る 」とされてい るビ タミン Dに 関す る具体的な数字 として,「 成人にお いて血 中副 甲状腺 ホルモ ン濃度 の上 昇 を抑制 し,骨 密度 の低下 を予防す るのに最低 限必要 な血中 25‐ ヒ ドロキシ ビタ
ミン D濃 度 は 50 nmol几 前後であると考えられる」と書かれてい る
表 1は ,食 事摂取基準 2010年 版 の ビ タ ミン Dの 項 に示 されて い る表 を改変引用 した ものであ る 確 かにここで 引用 されて い る論文 にお い て,50〜 69歳 の 集団 における平均 250HD濃 度 は 50 nmo1/L(20 ng/mL)を 越 えて いるが,平 均値が 50 nlnol圧 (20nノ mL)を 越 えてい るか らとい って,「 特定 の集団において不足状態 を示 す人 がほ とんH号 (11月 )2011〕

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表 1 日本人女性 を対象 として血清 25‐ ヒ ドロキシビタミンD濃 度を測定 した報告
文献

人数

7

117

調査地域

m清 25ヒ ドロ キ シ ビ タ ミ ン D濃 度

50 nmo1/L(nll101 L)

未 満 者 の 割合

新潟 (9月 )83■ 2267%

新潟 (2月 )547± 94309%

長野 (限 定 せ ず )501± 136

新潟 (9月 )80± 16新潟 (9月 )フ86± 182

497%
30%
58%

新潟 (2月 )597± 171新潟 (2月 )599± 170

新潟 (2月 )591± 161

285%
280%
286%

新潟 (11月 )556± 146351%

対応す る年齢 階級 の ビタ ミン D
摂取量 中央値 (μ ゾ 日)

日本人の食事摂取基準 2010年 版 1よ り改変引用 した「50 nmo1/L未 満者の割合」は 正規分布するものと仮定して,平 均値 標準偏差に基づ き 著者らが計算 したものである

仮 に 250HD濃の度が正規分布す るもの として,摂 取基準 表 に示 されてい る平均 ±標準偏差 の値 に基 づ き,50 nlnol厄 を下 回る
対象者 の 割合 を概算 した結果 を,「 50 nlllol几 未満者 の 割
合 Jと して加筆 した 一見 して明 らかに,多 くの文献 におい て,50 nmol几 未満者 の割合 はかな り高 い値 で あ った
す るとこれ らの集団 は ビタミン Dが 充足 してい る集団でど観 察 されない」と言 えるのであろうか

あるか ら,そ れに対応す る性 年齢階級 におけるlI取 の中央値 をもって 目安量 として もよいのだろ うか とい う疑間が生 じて くる
そうする と, ビタミン Dの 摂取基準策定 にどうい う方
法論 を用 い ることがで きるので あ ろ うか 2010年 カルシ
ウム ビ タミン Dに つい て,ア メ リカ カナダの食事摂取基準が全面改訂 された 方法論 か らみ ると, 目安量で
はな く,推 定平均必要量 (E思 )推 奨量 (RDA)に 変 わったのが大 きな変化である これ らは欠乏 充足実験 によって定 め られるの力'原 則 であ り,EARは 当該集団にお い て50%の 人が必要量 を満 たす量 ,RDAは ほ とん どの人 (97〜 98%)が 満 たす量 として定 め られる このアメリカ カ

ナダの食事摂取基 準 にお い ては,骨 の健康 を維持す るための血 中 250HD濃 度 (50 nmol L)を 基 に, ビ タ ミン Dの摂取量が算定 されてお り,RDAは ,血 中 250HD濃 度 と摂取量 の容量依存性試験 の結果か ら定め られてい る ア
メリカ カナダの食事摂取基準 における RDAは ,1歳 から 70歳 まで 6001U/日 となってい る カ ー方 わが 国の平成のでは な く,従 来 の 目安量の概念 に従 って策定す るな らば,現 行 の摂取基準 の よ うな定 め方 にな らざるを得 ないであろう しか し,栄 養素 の不足 をも考慮 した場合の摂取基準策定 にお い て,そ の摂取 の 中央値 をもって 目安量とす ることがで きるような健常 人 とい うものはそ もそ も存在す るので あ ろ うか 一 見 して外見上 の異常 を伴 わ ない対象者であ って も,不 足 の リスクの低 い 人か ら高 い 人
まで連続 的 に分布する と考 える方が よ り摂取基準 の精神に合致す るように思 われてな らない
ビタミン不足 をも考慮 しなけれ ば な らない時代 にお いては, 目安量策定 の方法論 に関 して,再 検討 の時期 が 来
てい るのではない だろ うかκ″ Zο ras:Dtta″ rcfcrcncc llltakcs,adcquatc intakc,宙 ta‐

mln D,mcdian,ostcopOrosis
lDcpartlllcnt ofFood and Nu血

Univcrsi″

KiyOshi Tanakal Akiko Kun abara2

1京都女子大学家政学部食物栄養学科

2大 阪樟蔭女子大学学芸学部人間栄養学科

田中

清 1,乗 原晶子 219年 国民健康栄養調査 の結果 を見ると,成 人で 6001U日
摂取で きてい るのは,集 団 の上 位 10%に 限 られて い るのが実際であ り', この数字 を日本 に適用で きるのか どうかについ ては,多 くの議論が必要 で あ るが , ビタ ミン不足をも考慮 した場合, この策定 の方法論 は注 目す べ きものと思われる
誤解 の ない よ うに一言述べ てお くと,著 者 らは 2010年
版 に定め られた 目安量 の値 が 不適切 であると述 べ ているК,oto WOmen'sU面 verslサ2Dcpartmcnt of Hcalth and Nutrition,Osakàon
Shoinヽ、mcn s

参考文献
1)第 一 出 版 編 集 部 編 :厚 生 労働 省 策 定 日本 人 の 食事 摂 取 基 準(2010年 版 )第 一 出版

東京 20092)Ins● ●lte of Medicin 201l Dieta,reFerence intakes for calcitl n andvitamin D WashingtOn,DC:Tlle National Academies Press3)健 康

栄 養情報研 究会 :国 民健康

生労働省 国民健康

栄 養 の現状― 平成 19年 厚栄養調査 報告― ,第 一 出版

東京 2009

Nutrients 2010, 2, 752-761; doi:10.3390/nu2070752
OPEN ACCESS

nutrients
ISSN 2072-6643
www.mdpi.com/journal/nutrients
ArticleFractional Absorption of Active Absorbable Algal Calcium(AAACa) and Calcium Carbonate Measured by a Dual
Stable-Isotope Method
Kazuhiro Uenishi 1,*, Takuo Fujita 2, Hiromi Ishida 3, Yoshio Fujii 4, Mutsumi Ohue 2,
Hiroshi Kaji 5, Midori Hirai 6, Mikio Kakumoto 6 and Steven A. Abrams 7
1

2

3

4

5

6

7

Laboratory of Physiological Nutrition, Kagawa Nutrition University. 3-9-21 Chiyoda, Sakado,
Saitama 350-0288, Japan
Katsuragi Hospital, 250-1 Makamicho, Kishiwada, Osaka 596-0842, Japan;
E-Mail: fujita@katsuragi-hosp.or.jp (T.F.)
Laboratory of Administrative Dietetics, Kagawa Nutrition University. 3-9-21 Chiyoda, Sakado,
Saitama 350-0288, Japan; E-Mail: ishida@eiyo.ac.jp
Fujii Clinic, 4-18-1 Tsutsujigaoka, Tarumi-ku, Kobe, Hyogo 650-0853, Japan;
E-Mail: fujii-naika-clinic@crocus.ocn.ne.jp
Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Kobe
University Graduate School of Medicine, 7-5-1 Kusunokichou, Chuo-ku, Kobe, Hyogo 650-0017,
Japan; E-Mail: hiroshik@med.kobe-u.ac.jp
Department of Hospital Pharmacy and Pharmacodynamics, Kobe University Graduate School of
Medicine, 7-5-1 Kusunokichou, Chuo-ku, Kobe, Hyogo 650-0017, Japan;
E-Mail: midorih@med.kobe-u.ac.jp (M.K.)
Department of Agriculture, Agricultural Research Service, Children’s Nutrition Research Center,
Department of Pediatrics, Baylor College of Medicine and Texas Children’s Hospital, Houston,
Texas 77030, USA; E-Mail: sabrams@bcm.tmc.edu* Author to whom correspondence should be addressed: E-Mail: uenishi@eiyo.ac.jp;
Tel.: 81-492-843-895; Fax: 81-492-843-895.
Received: 10 June 2010; in revised form: 2 July 2010 / Accepted: 8 July 2010 /Published: 12 July 2010

Abstract: With the use of stable isotopes, this study aimed to compare the bioavailabilityof active absorbable algal calcium (AAACa), obtained from oyster shell powder heated to ahigh temperature, with an additional heated seaweed component (Heated Algal Ingredient,Nutrients 2010, 2

753

HAI), with that of calcium carbonate. In 10 postmenopausal women volunteers aged 59 to
77 years (mean ± S.D., 67 ± 5.3), the fractional calcium absorption of AAACa and CaCO3
was measured by a dual stable isotope method. 44Ca-enriched CaCO3 and AAACa wereadministered in all subjects one month apart. After a fixed-menu breakfast and pre-test
urine collection (Urine 0), 42Ca-enriched CaCl2 was intravenously injected, followed byoral administration of 44Ca-enriched CaCO3 without carrier 15 minutes later, and complete
urine collection for the next 24 hours (Urine 24). The fractional calcium absorption was
calculated as the ratio of Augmentation of 44Ca from Urine 0 to Urine 24/ augmentation of42
Ca from Urine 0 to Urine 24. Differences and changes of 44Ca and 42Ca were corrected bycomparing each with 43Ca. Fractional absorption of AAACa (mean ± S.D., 23.1 ± 6.4), wasdistinctly and significantly higher than that of CaCO3 (14.7 ± 6.4; p = 0.0060 by paired ttest). The mean fractional absorption was approximately 1.57-times higher for AAACa
than for CaCO3. The serum 25(OH) vitamin D level was low (mean ± S.D., 14.2 ± 4.95
ng/ml), as is common in this age group in Japan. Among the parameters of the bone andmineral metabolism measured, none displayed a significant correlation with the fractional
absorption of CaCO3 and AAACa. Higher fractional absorption of AAACa compared with
CaCO3 supports previous reports on the more beneficial effect of AAACa than CaCO3
for osteoporosis.
Keywords: active absorbable algal calcium (AAACa); calcium carbonate; dual stable Ca
isotope method; fractional absorption (FA); parathyroid hormone (PTH)

1. Introduction
Active absorbable algal calcium (AAACa) prepared from heated oyster shell and seaweed is aunique calcium supplement with high bioavailability, with a characteristic lamellar crystalline structure
quite unlike that of calcium oxide and calcium carbonate (CaCO3) [1]. In the Katsuragi Calcium study,
a prospective, randomized, double blind and placebo-controlled study compared the effect of AAACa
on osteoporosis with that of CaCO3 in hospitalized women with a mean age of 80 years. It was foundthat AAACa alone increased spinal bone mineral density significantly over the level in subjects given aplacebo, whereas CaCO3 did not [2,3]. Fracture occurrence over the two year test period from among
58 subjects was 0 of 5 in the AAACa Group, 2 of 7 in the CaCO3 Group and 3 of 5 in the PlaceboGroup, on evaluation of all X-rays available at the beginning and end of the test period. The AAACaGroup exhibited a significantly lower rate of fracture occurrence than the placebo group, but the
CaCO3 Group showed no significant difference from placebo group. Serum parathyroid hormone(PTH) was also suppressed more efficiently by AAACa than CaCO3.
Despite all these indirect lines of evidence indicating a high bioavailability of AAACa, a directabsorption test by a dual isotope method has not been conducted to date. We have therefore attempted
to measure the fractional absorption of AAACa by using the dual stable-isotope method [4,5] toNutrients 2010, 2754

compare it with CaCO3 in subjects in the age group most likely to need effective calcium
supplementation to maintain their bone health: postmenopausal women.
2. Experimental Section
2.1. Subjects
Ten postmenopausal women between 59 and 77 years of age (mean ± SD, 67 ± 5.3 years) leading anormal healthy daily life without any known disease possibly affecting bone and mineral metabolism
volunteered to participate as test subjects in the present study by providing written consent (Table 1). One
subject, shown in parenthesis in Tables 1 and 2, was dropped from analysis because of a measured
fractional absorption (FA) value of 0% on giving CaCO3. The Institutional Review Board of the Fujii
Medical Clinic approved the study.
Table 1. Background of the test subjects.
No.

Age

Heigh
t
(cm)
154
147
157
148
(153)
152
151
150
145
148
150

Weigh
t
(kg)
54
50
63
43
(60)
58
56
48
50
48
52

Systolic blood pressure(mmHg)

Diastolic blood pressure(mmHg)

68
72
65
65
(59)
59
65
77
64
71
67

Years
after
menopause
19
23
15
13
(9)
8
13
28
15
19
17

1
2
3
4
(5) *
6
7
8
9
10
Mea
n
SD

138
142
148
125
(133)
152
150
142
140
122
139

80
62
70
70
(88)
85
85
80
90
70
76

5.3

6.0

3.7

6.1

10.4

9.3

* Case No. 5 was not included in the statistical analysis.

2.2. Background Data of the Test Subjects
In order to assess the metabolic background of the test subjects, serum Ca, P, albumin, creatinine,
BUN, 25(OH)vitamin D, intact parathyroid hormone (PTH), bone specific alkaline phosphatase (BAP),
urinary N-terminal type I collagen fragments (NTx) and urinary calcium/ creatinine ratio(UCa/ Cr) were measured prior to the test. The laboratory tests related to bone and calcium metabolism
gave results approximately within the normal range, as shown in Table 2, except for one subject, who
had a serum 25(OH) vitamin D level in the insufficiency range (7.6 ng/mL). This subject was without
symptoms and signs of vitamin D insufficiency such as hypocalcemia, hypophosphatemia, highalkaline phosphatase, muscle weakness and bone pain.

Nutrients 2010, 2

755

Table 2. Parameters of mineral and bone metabolism of the test subjects.

No.

Serum

Serum

Serum

Serum

Ca

P

albumincreatinine

mg/dL

mg/dL

g/dL

mg/dL

BUN
mg/dL

25(OH)

Intactvitamin D

PTH

ng/dL

pg/dL

BAP
U/L

Urine

Urine

NTx/CrCa/CrnMBCE/

mg/mg

mMCr
1

9.7

3.9

4.4

0.83

11.0

16.8

48

15.2

32.2

0.03

2

9.5

4.5

4.0

0.80

21.9

16.9

31

15.1

16.0

0.06

3

9.7

3.1

4.6

0.71

12.1

11.6

40

35.8

31.1

0.45

4

10.3

3.5

5.1

0.49

12.5

11.7

44

32.4

35.9

0.36

5*

(9.5)

(3.4)

(4.5)

(0.74)(14.2)(21.8)(61)(21.6)(29.0)(0.08)

6

9.8

3.5

4.7

0.74

17.8

15.4

44

19.1

23.0

0.12

7

9.3

4.4

4.4

0.60

17.8

24.7

42

17.0

42.2

0.22

8

9.9

2.9

4.6

0.59

13.5

7.6

50

34.9

34.5

0.17

9

9.8

3.7

4.4

0.74

13.7

12.7

34

27.5

16.3

0.20

10

9.5

3.3

4.2

0.54

13.6

10.8

34

45.9

21.9

0.30

Mean

9.7

3.6

4.5

0.67

14.9

14.2

41

27.0

28.1

0.21

SD

0.29

0.55

0.31

0.119

3.53

4.95

6.59

11.02

9.20

0.138

Ca: calcium; P: phosphorus; BUN: Blood urea nitrogen; PTH: parathyroid hormone; BAP: Bone specific alkaline
phosphatase; BCE: Bone collagen equivalent.
* Case No. 5 was not included in the statistical analysis

2.3. Materials
The first part of the test was performed on March 9, 2009, using 44Ca-enriched CaCO3 for oral load
and 42Ca in the form of CaCl2, for intravenous injection (Table 3). On April 13, 2009, after one month,
exactly the same procedure was repeated on the same test subjects, except for the use of 44Ca-enriched
AAACa in the place of CaCO3 to ensure the stable isotope constituent of the body reached equilibrium.
Intrinsic labeling is no doubt ideal, but it is impossible to label the shell of oysters abiding in the ocean,so an extrinsic labeling was adopted as the best substitute for it. The material for AAACa was obtainedby heating oyster shell to 1,000 oC, resulting mostly in CaO powder after losing much of the organic
components. To 5,082 mg of this CaO powder, 450.4 mg CaO Ca fraction was added that consisted of
95.9 ± 0.3% 44Ca supplied by TRACE SCIENCES INTERNATIONAL (Ontario, Canada), and wasthoroughly mixed in a melting pot. Aqueous solution of a small amount of algal component was preheated at a high temperature in a manner similar to the oyster shell to start a chemical reaction lasting
for about 10 minutes. After sufficient stirring, it was divided into small portions for actual use andpreserved in vacuum. The final product mostly consisted of Ca(OH)2.

Nutrients 2010, 2

756

CaCO3 labeled with 44Ca was also obtained from the same source (the Ca fraction consisting of
95.9 ± 0.3% 44Ca). To 781.6 mg of this material, 9,075 mg CaCO3 (Japanese Pharmacopeia) was
added, thoroughly mixed, and divided into small proportions and stored.
AAACa particle mean size was 5.8 microns; maximum size was 75 microns and CaCO3 particle
size ranged from 10 to 20 microns. As these values are based on different occasions of measurements
they may not be directly comparable, but appears to lie over a similar range. If anything, a larger size iscompatible with slower absorption.
For two subjects, part of the first urine sample was lost; in these cases, both parts of the test wererepeated on July 30 and August 27, and the data from the uneventfully performed second set of tests
were used to replace those of the first set.
The safety of the intravenous injection of CaCl2 was verified before the study by the absence of any
signs of toxicity such as chills, fever, neuromuscular irritability, skin eruptions, disturbance of
consciousness, etc.
Table 3. Amount of isotope Ca (mg) per subject in 1 study.
Isotope
42

Ca
Ca
44
Ca
43

Oral
CaX
Supplied
0.01
0.01
25.38

IV
Added
1.79
0.39
5.52

Total
1.80
0.40
30.90

CaY
Supplied
0.00
0.00
25.38

Added
1.79
0.39
5.53

Total
1.79
0.39
30.91

Total
3.192
0.00370.0334The contents of Ca isotopes in the material used for the preparation of CaX and CaY on arrival fromthe supplier (Supplied), their contents in the material added to prepare samples for administration(Added) and the final total (Total) are indicated in Table 3.
A total of approximately 300 mg of Ca containing approximately 30 mg 44Ca isotope (25 + 5) was
orally administered to each subject and about 3 mg 42Ca isotope was injected before the study and nosymptoms and signs of toxicity were reported.
2.4. Test Procedure
After taking a fixed menu breakfast consisting of fruit juice, toast, eggs and coffee, a pre-test urine
sample was collected (Urine 0) and 44Ca-enriched CaCO3 was orally administered followed by the
intravenous injection of 42Ca-enriched CaCl2 15 minutes later. A complete collection of 24 h urine
followed (Urine 24). After one month to ensure clearance of the enriched isotope, exactly the sameprocedure, except for the use of 44Ca-enriched AAACa instead of 44Ca-enriched CaCO3, was repeated.
2.4.1. Measurement of the Stable Isotope
Sample preparation for isotope enrichment measurement was conducted according to the method ofPatterson et al. [6]. By using the inductively coupled plasma mass spectrometry (ICP-MS, Agilent
7500 cs, Agilent Technologies, Inc., Tokyo), 42Ca, 43Ca, 43Ca and other measurable stable Ca isotopes
were measured in both Urine 0 and Urine 24. Utilizing 43Ca as an internal standard of the stable CaNutrients 2010, 2757

isotopes, the ratio of each stable Ca isotope to 43Ca was calculated. The increase of the 42Ca/43Ca and44
Ca/43Ca in Urine 24 above the pretest natural abundance level for each test subject over thecorresponding value in Urine 0 was then obtained. By dividing the ratio of the actual amount of theenrichment of 44Ca by the corresponding amount of the enrichment of 42Ca from Urine 0 to Urine 24,
the FA of the 44Ca-enriched material was obtained; for CaCO3 in the first part of the test and AAACain the second part (Table 3).
2.5. Statistical Analysis
The Excel Statistical Package was used to compare the FA of CaCO3 and AAACa by paired t-test.
A correlation matrix among the FA data, age and parameters of bone and mineral metabolism wasconstructed and evaluated by the Spearman method in view of the inclusion of variables with uncertain
distribution. The p values < 0.05 were considered significant.
3. Results and Discussion
As shown in Table 4, the mean Fractional absorption (FA) of AAACa, 23.1 ± 6.4%, was 1.57times higher than the corresponding value of CaCO3, 14.7 ± 6.4%, with a significant difference at
p = 0.0060 determined using paired t-test.
Table 4. Fractional absorption (FA) of CaCO3 and AAACa by dual stable isotope method.
Subject
1
2
3
4
(5) *
6
7
8
9
10
Mean
SD

FA CaCO3
7.5
20.0
21.9
19.6
(0.0)
6.1
14.3
11.7
8.7
22.2
14.7
6.4

FA AAACa
21.1
29.7
34.7
20.4
(18.8)
22.7
24.9
11.9
22.1
20.4
23.1
6.4

Paired comparison between FA CaCO3 and FA AAACa
p = 0.0060, t = 3.708 (paired t-Test)* Subject 5 was not included in the statistical analysis.

According to the evaluation by means of the correlation coefficient matrix (Spearman) (Table 5)among the parameters of bone and mineral metabolism summarized in Table 2, no significant
correlation was found between the FA of either calcium carbonate or AAACa and each parameter. In
the subject with the lowest serum 25(OH) vitamin D of 7.6 ng/mL, the FA of CaCO3 value wasmedium in the group, i.e., 11.7%, fifth from the lowest, and the FA of AAACa, 11.9%, was the lowest
in the group.

Nutrients 2010, 2

758

Until the advent of the dual isotope method, the true FA of calcium was extremely difficult tomeasure due to the complex behavior of calcium in living organisms, such as the rapid exchange
through multiple Ca pools and various pathways of exit and reentrance [7,8]. Utilizing the presence ofmultiple stable isotopes in nature, the dual stable isotope method was developed to circumvent thiscomplexity, and it is the only method of directly measuring the fractional intestinal Ca absorption.
Abrams and coworkers as well as other investigators [12-23] have used this method extensively toestimate calcium absorption, establishing it as the gold standard for calcium absorption. Since calcium
absorption is influenced by age and the state of bone, as well as mineral metabolism, a correlation
matrix was constructed and evaluated by Spearman’s method (Table 5). None of the metabolic
parameters tested exhibited significant correlation with FA of the calcium compounds. Absence ofsignificant correlation between FA of calcium compound and age was expected because of the narrow
age range of this group.
The FA of Ca compounds obtained in this study of postmenopausal women, with a mean age of 66
years and with a tendency of low 25(OH) vitamin D, appears to be much lower than those observed inchildren and younger subjects: FA; 54.8–63.1% [21], 58.2–64.3% [22], and also younger
postmenopausal women with mean age of 56: FA; 34.6–39.1% [23]. In healthy volunteers between 25
to 45 years much lower values, yet still higher than the results in the present study, were reported: FA;
26–31% [24]. The reduced FA in the current study subjects could also be due to reduced estrogen level
after menopause. FA is, thus, markedly influenced by age. The age range of the test subjects was quite
narrow in this group of subjects, unsuitable for the assessment of the age-FA correlation. Statistically,
the tendency of age-FA correlation was non-significant.
Table 5. Spearman’s correlation matrix and correlation coefficients among fractional
absorption (FA) and parameters of bone and mineral metabolism.

FA

FA

FA

CaCO3

AAACa

1.00000.1423

Age

SCa

SP

Salb

Cre

BUN

25D

PTH

BAP

UNTx

UCa/Cr

0.4238−0.388−0.192−0.340−0.485−0.0335−0.3167−0.5630−0.4833−0.1333

0.63332

5

5

4−0.349−0.5290.4328−0.2130.5042

0.34450.5690−0.5232−0.3766−0.27620.0418

0

7

CaCO3
FA

1.0000

AAACa

7

SCa: serum calcium; SP: serum phosphate; Salb: serum albumin; Cre: creatinine; BUN: blood urea nitrogen; 25D:
25(OH)vitaminD; PTH: parathyroid hormone; BAP: bone specific alkaline phosphatase; UNtx: urine N-terminal type I
collagen fragments; UCa/Cr: urinary Ca/creatinine ratio

Although these subjects are reasonably homogeneous and apparently free of any comorbidity, which
could potentially influence the test results, the present study is limited by the small number of test
subjects. Unlike similar studies conducted in this field in the past, post-menopausal women – who needcalcium supplementation most because of high risk of osteoporosis – were asked to participate. A
rather low intra-group variation was encouraging, and a clear-cut difference in FA between the two test
materials may also add to the credibility of the conclusion.
It is possible that the difference in molecular weight and physicochemical properties of the
44
Ca-enriched CaCO3 and AAACa, mostly consisting of Ca(OH)2 as the result of oxidation of CaCO3

Nutrients 2010, 2759

obtained from oyster shell, cannot be completely ruled out. The similar molecular size and comparable
particle size actually measured as 5.8 to 75 for AAACa and 10 to 20 for CaCO3 and this is a limitationof the result but should not have affected the primary outcome. In view of the similar molecular sizeand physicochemical properties between CaCO3 and AAACa, both much smaller than organic Ca salts,
however, confounding effect exerted on the calculation of the absorptive rate is rather unlikely and theconclusion of difference in the absorption rate between the two compounds should bereasonably supported.
4. Conclusions
This study aimed to compare the bioavailability of active absorbable algal calcium (AAACa), oyster
shell powder heated to a high temperature, with an additional heated seaweed component (Heated
Algal Ingredient, HAI), with that of calcium carbonate. The Fractional absorption of AAACa, (mean ±S.D.; 23.1 ± 6.4) was distinctly and significantly higher than that of CaCO3 (14.7 ± 6.4;
p = 0.0060 by paired t-test). The mean was approximately 1.57-times higher for AAACa than CaCO3.
Higher fractional absorption of AAACa compared with CaCO3 supports previous reports on the more
beneficial effect of AAACa than CaCO3 on osteoporosis.
Acknowledgements
Kazuo Chihara MD, former Dean, Kobe University School of Medicine, encouraged and supported
the study. Fujix Corporation provided 42Ca and 44Ca enriched compounds, imported by Taiyo Nippon
Sanso Corporation and Kawaguchi Liquefaction Chemical Corporation, clearing up all the difficult
official procedures.
The authors have no conflict of interest.
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Yergey, A.L.; Vieira, N.E.; Couell, D.G. Direct measurement of dietary fractional absorption
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8. Hillman, L.S.; Tack, E.; Covell, D.G.; Vieina, N.E.; Yergey, A.L. Measurement of true calcium
absorption in premature infants using intravenous 46Ca and oral 44Ca. Pediatr. Res. 1988, 23,589-594.
9. Abrams, S.A. Assessing mineral metabolism in children using stable isotopes. Pediatr. Blood
Cancer 2008, 50, 438-441.
10. Abrams, S.A.: Griffin, I.J.; Hawthorne, K.M. Chen, Z.; Gunn, S.K. Wilde, M.; Darlington, G.;
Shypailo, R.J.; Ellis, K.J. Vitamin D receptor Fok1 polymorphisms affect calcium absorption,
kinetics, and bone mineralization rates during puberty. J. Bone. Miner. Res. 2005, 20, 945-953.
11. Abrams, S.A. Using stable isotopes to assess the bioavaila bility of minerals in food-fortification
programs. Forum Nutr. 2003, 56, 312-313.
12. Vargas Zapata, C.L.; Donangelo, C.M.; Woodhouse, L.R.; Abrams, S.A.; Spencer, E.M. King, J.C.
Calcium homeostasis during pregnancy and lactation in Brazilian women with low calcium
intakes: a longitudinal study. Am. J. Clin. Nutr. 2004, 80, 417-422.
13. Abrams, S.A.; Griffine, I.J. Hicks, P.D. Gunn, S.K. Pubertal girls only partially adapt to low
dietary calcium intakes. J. Bone Miner. Res. 2004, 19, 757-763.
14. Abrams, S.A. Griffin, I.J.; Herman, S. Using stable isotopes to assess the bioauailabilty of mineralin food fortification programs. Food Nutr. Bull. 2002, 23, 158-165.
15. Abrams, S.A. Calcium turnover and nutrition through the life cycle. Proc. Nutr. Soc. 2001, 60,
283-289.
16. Abrams, S.A. Using stable isotope to assess mineral absorption and utilization by children. Am. J.
Clin. Nutr. 1999, 70, 955-964.
17. Miller, J.Z.; Smith, D.L.; Flora, L.; Peacock, M.; Johnston, C.C., Jr. Calcium absorption inchildren estimated from single and double stable calcium isotope techniques. Clin. Chim. Acta.
1989, 183, 107-113.
18. Ames, S.K.; Ellis, K.J.; Gunn, S.K.; Copeland, K.C.; Abrams, S.A. Vitamin D receptor gene Fok1
polymorphism predicts calcium absorption and bone mineral density in children. Bone Miner. Res.
1999, 14, 740-746.
19. Eastell, R.; Vieira, N.E.; Yergey, A.L.; Riggs, B.L. One day test using stable isotopes to measuretrue fractional calcium absorption. J. Bone Miner. Res. 1989, 4, 463-468.
20. Fairweather – Tait, S.; Johnson, A.; Eagles, J.; Ganatra, S.; Kennedy, H.; Gurr, MI. Studies on
calcium absorption from milk using a double label stable isotope technic. Br. J. Nutr. 1998, 62,379-388.
21. Lee, W.T.; Leung, S.S.; Fairweather-Tait, S.J.; Leung, D.M.; Tsang, H.S.; Eagles, J.; Fox, T.;
Wang, S.H.; Xu, Y.C.; Zeng, W.P. Ture fractional calcium absorption in Chinese children
measured with stable isotope( 42Ca and 44Ca ). Br. J. Nutr. 1994, 72, 883-897.
22. Lee, W.; Leung, S.S.; Xu, Y.C.; Wang, S.H.; Zeng, W.P.; Lau, J.; Fairweather-Tait, S.J. Effects ofdouble-blind controlled calcium supplementation on calcium absorption in Chinese children
measured with stable isotopes (42Ca and 44Ca). Br. J. Nutr. 1995, 73, 311-321.

Nutrients 2010, 2761

23. Tanimoto, H.; Fox, T.; Eagles, J.; Satoh, H.; Nazawa, H.; Okiyama.; Morinaga, Y.; Fairweather–
Tait, S. Acute effects of poly-gamma-glutamic avid on calcium absorption in postmenopausal
women. J. Am. Coll. Nutr. 2007, 26, 645-649.
24. van der Hee, R.M.; Miret, S.; Slettenaar, M.; Duchateau, G.S.; Rietveld, A.G.; Wikinson, J.E.;
Quail, P.J.; Berry, M.J.; Dainty, J.R.; Teucher, B.; Fairweather-Tait, S.J. Calcium absorption fromfortified ice cream formulations compared with calcium absorption from milk. J. Am. Diet Assoc.
2009, 109, 830-835.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article
distributed under the terms and conditions of the Creative Commons Attribution license(http://creativecommons.org/licenses/by/3.0/).

Asia Pac J Clin Nutr 2010;19 (1):49-56

49

Original Article

Hypovitaminosis D and K are highly prevalent andindependent of overall malnutrition in theinstitutionalized elderly
Akiko Kuwabara PhD1, Masako Himeno MSc1, Naoko Tsugawa PhD2,
Maya Kamao MSc2, Minori Fujii RD3, Nobuko Kawai CSW3, Miyuki Fukuda MSc1,
Yoko Ogawa MSc1, Shoko Kido PhD1, Toshio Okano PhD2, Kiyoshi Tanaka MD1
1

Department of Food and Nutrition, Kyoto Women’s University, Higashiyama, Japan
Department of Hygienic Sciences, Kobe Pharmaceutical University, Higashiyama, Japan
3
Nursing Home Kayu-Shirakawa, Higashiyama, Japan
2

There have been methodological problems for studying hypovitaminosis D and K in the elderly. First, studies
were done either by evaluating food intake or measuring their circulating levels, but rarely by both in Japan. In
this paper, vitamin D and K intakes and their circulating levels were simultaneously determined. Second issue iswhether hypovitaminosis D and K are independent of general malnutrition, prevalent in the elderly. We tried tostatistically discriminate them by principal component analysis (PCA). Fifty institutionalized elderly were evaluated for their circulating 25 hydroxy-vitamin D (25OH-D), intact parathyroid hormone (PTH), phylloquinone(PK), menaquinone-7 (MK-7) levels, and their food intake. Although average vitamin D intake (7.0 μg/day) exceeded the Japanese Adequate Intake (AI) of 5.0 μg/day, average serum 25OH-D concentration was in the hypovitaminosis D range (11.1 ng/mL). Median vitamin K intake was 168 μg/day, approximately 2.5 times as highas AI for vitamin K. Nevertheless, plasma PK and MK-7 concentrations were far lower than those of healthy
Japanese elderly over 70 years old. PCA yielded four components; each representing overall nutritional, vitamin
K2, vitamin D, and vitamin K1 status, respectively. Since these components are independent of each other, vitamin D- and K-deficiency in these subjects could not be explained by overall malnutrition alone. In summary, institutionalized elderly had a high prevalence of hypovitaminosis D and K, and the simultaneous determination of
their circulating level and dietary intake is mandatory in such studies. PCA would yield fruitful results for eliminating the interference by confounders in a cross-sectional study.

Key Words: hypovitaminosis D, hypovitaminosis K, principal component analysis, adequate intake, institutionalized
elderly

INTRODUCTION
Vitamin D is of utmost importance in enhancing the intestinal absorption of calcium and phosphorus,1,2 with its
deficiency causing skeletal mineralization defect; rickets
and osteomalacia. Recently, it has come to the generalattention that inadequate supply of vitamin D, even in its
milder form (vitamin D insufficiency), is associated withincreased risk of fracture through negative calcium balance, hence secondary hyperparathyroidism.1,2 Vitamin D
insufficiency is also reported to be associated with muscle
weakness. Recent clinical studies have indicated that intervention with vitamin D supplementation reduced theincidence of falling in elderly subjects.3 Clinically important non-vertebral fractures, such as hip and wrist fractures are triggered by falling. Thus, vitamin D insufficiency would render the elderly subjects more prone tofracture through its effects both on the skeleton and muscle. Recently, lower serum level of 25 hydroxy-vitamin D(25OH-D) was reported to be a significant risk factor
even for mortality.4

Vitamin D insufficiency is quite common in the elderly population,5,6 and institutionalized elderly are at evenhigher risk for vitamin D insufficiency.7-10 Factors hitherto postulated to be responsible include low dietary vitamin D intake,7,9 reduced dermal capacity to produce
vitamin D with aging and minimal sun exposure.11,12
In contrast to vitamin D, the skeletal action of vitamin
K has called our attention only quite recently. The only
biological action of vitamin K has been considered to be
its role as the coenzyme of γ-glutamyl carboxylase(GGCX) in the liver, by which additional carboxyl groupis introduced into the glutamic acid residue in four of theCorresponding Author: Dr Kiyoshi Tanaka, Department ofFood and Nutrition, Kyoto Women’s University, 35, Imakumanokitahiyoshicho, Higashiyama 605-8501 Japan.
Tel: +81-75-531-7125; Fax: +81-75-531-7153
Email: tanakak@kyoto-wu.ac.jp
Manuscript received 8 July 2009. Initial review completed 13
October 2009. Revision accepted 23 November 2009.

A Kuwabara, M Himeno, N Tsugawa, M Kamao, M Fujii, N Kawai, et al

50

Table 1. Background profiles and results from blood tests of the study subjectsnAge (y)Level of care needed
Body height (cm)Body weight (kg)Body mass index (kg/m2)
Serum albumin (g/dL)
Serum total cholesterol (mg/dL)
Serum triglyceride (mg/dL)
Serum aminotransferase (U/L)
Serum alanine aminotransferase (U/L)
eGFR (mL/min./1.73m2)
Serum 25-hydroxyvitamin D (ng/mL)
Serum parathyroid hormone (pg/mL)
Plasma phylloquinone (ng/mL)
Plasma menaquinone-7 (ng/mL)

Total
50
87.6±8.0 (88.5)
3.6±1.1 (4.0)
144.0±11.6 (142.0)
43.6±9.3 (43.2)
21.0±3.8 (20.1)
3.7±0.4 (3.7)
184±37 (184)98±41 (92)22±11 (19)16±10 (13)61±20 (60)11.1±3.1 (11.2)
30.8±11.8 (30.0)
0.73±0.70 (0.58)
0.53±0.37 (0.45)Male15
84.9±7.9 (83.0)
3.3±1.0 (3.0)
157.0±7.8 (159.0)
50.3±7.9 (49.9)
20.5±3.4 (19.6)
3.8±0.4 (3.9)
186±26 (195)
96±47 (75)20±7 (17)16±7 (13)67±19 (67)10.3±3.5 (9.3)
29.9±11.1 (31.0)
0.62±0.29 (0.60)
0.59±0.47 (0.47)

Female3588.7±7.8 (90.0)
3.7±1.2 (4.0)
138.4±7.8 (139.0)
40.7±8.3 (38.1)
21.3±4.0 (20.2)
3.6±0.4 (3.6)
183±41 (183)98±39 (93)22±12 (19)16±12 (12)59±21 (57)11.5±3.0 (11.6)
31.3±12.2 (30.0)
0.77±0.82 (0.53)
0.51±0.32 (0.44)

p value
0.133
0.228
<0.01
0.001
0.476
0.136
0.828
0.403
0.603
0.235
0.208
0.274
0.736
0.992
0.849

Data are expressed as mean±SD with the values in parentheses showing the median.
Comparison of indices between males and females were done by unpaired t test or Mann-Whitney test depending on normality.
eGFR; estimated Glomerular Filtration Rate.

Table 2. Daily dietary intakes of the study subjects
Energy (kcal)
Protein (g)Fat (g)Carbohydrates (g)Calcium (mg)
Vitamin D (μg)
Vitamin K (μg)

Total1322±159 (1387)
51.0±5.8 (53.3)
32.8±3.9 (34.6)
178±20 (186)494±53 (504)7.0±1.4 (7.7)
155±30 (168)Male
1374±96 (1416)
53.1±3.6 (54.6)
34.2±2.4 (35.3)
185±12 (189.7)
503±50 (506)7.4±0.9 (7.8)
164±19 (172)

Female1300±175 (1386)
50.2±6.3 (53.5)
32.2±4.3 (34.5)
175±21 (186)490±54 (502)6.9±1.5 (7.6)
151±33 (168)p value
0.160
0.091
0.095
0.093
0.157
0.107
0.107

Data are expressed as mean±SD with the values in parentheses showing the median. Comparison of indices between male and women weredone by unpaired t test or Mann-Whitney test depending on normality.

blood coagulation factors (II, VII, IX, X) to yield γglutamic carboxyl (Gla) residue.13 Other extrahepatic proteins are also γ-carboxylated by GGCX, such as osteocalcin (bone Gla protein; BGP) and matrix gla protein(MGP).14 Recent evidences suggest that vitamin K deficiency is associated with increased risk of fracture. Whensubjects were categorized into quartiles according to theirvitamin K intake, fracture risk in the lowest quartile wastwice as high as that in the highest quartile.15 The ageadjusted incidence of vertebral fracture was significantly
higher in subjects with low plasma phylloquinone levels
than those with high plasma levels in Japanese women.16
In addition, the association of circulating vitamin K leveland bone mineral density (BMD) has also been reported.
For example, low plasma phylloquinone concentration
was associated with low BMD at the femoral neck in men,and lower spine BMD in postmenopausal women without
estrogen replacements.17 High serum concentration ofundercarboxyled osteocalcin (ucOC), which is a sensitive
indicator of skeletal vitamin K insufficiency, was a significant risk factor of hip fracture independent of BMD.18,19
Plasma phylloquinone level is subject to alteration by
aging,20,21 and elderly subjects have been reported to have
low plasma phylloquinone concentrations.22 Of note is thereport that elderly nursing home residents generally had apoor dietary vitamin K intake compared to the ambulatory elderly.23

Studies on the role of hypovitaminosis D and K in theelderly, especially the institutionalized ones are greatly
hampered by the fact that they are also generally malnourished. Arguments against the significance of thesevitamins have been made that decreased serum concentrations of these vitamins is merely a reflection of overallmalnutrition. In this paper, we have tried to statisticallydiscriminate hypovitaminosis D and K from general malnutrition by using principal component analysis (PCA),
which has been employed in clinical nutrition for the
analyses of dietary pattern.24,25
MATERIALS AND METHODS
Subjects
The study subjects were 50 institutionalized elderly (male15, female 35) in a nursing home, Kayu-Shirakawa. Exclusion criteria were routine medication that has potential
interference with vitamin D or vitamin K status. Detailed
information about this study was given and written consent was obtained from the subject or the proxy. The
study protocol was approved by the ethical committee in
Kyoto Women’s University.
Laboratory data
Blood was obtained after overnight fasting. After centrifugation, serum was kept frozen at –30°C until analysis.
Serum concentration of 25OH-D was measured by radioimmunoassay (RIA) (DiaSorin, Stillwater, MN, USA).

Hypovitaminosis D and K in the elderly

Circulating level of intact parathyroid hormone (PTH)
was measured by electro chemiluminescent immunoassay(ECLIA) (Roche Diagnostics, Mannheim, Germany).
Plasma vitamin K1 (phylloquinone; PK), and menaquinone-7(MK-7) levels were determined by high-performanceliquid chromatography-tandem mass-mass spectrometry
with atmospheric pressure chemical ionization (LCAPCI-MS/MS) using a HPLC system (Shimadzu, Kyoto,
Japan) and API3000 LC-MS/MS System (Applied Biosystems, Foster City, CA) with 18O-labeled vitamin K as
the internal standard.26
Nutrition intake study
Since the subjects were institutionalized and their dietwas supplied from the institution, their nutrients and energy intake were calculated by multiplying the supplied
nutrients on the basis of the Standard Tables of Food
Composition in Japan, 5th ed. with the average percentage
intake in a preceding month by the staff.27 Percentage
intake was assessed for each subject at every meal, andthe monthly average percentage intake was calculated.
Based on these records, their intake of energy and nutrients was calculated using software (Healthy Maker Pro
501, Mushroom Software Corp, Okayama, Japan).
Statistical analyses
Statistical analyses were performed with SPSS 15.0J(SPSS Japan Inc., Tokyo, Japan). Comparison of two
independent groups was made with Student’s t-test or
Mann-Whitney test depending on normality. Multiple
regression analyses by stepwise method were performed
to determine independent factors for circulating levels ofvitamin D and K levels. The relationship between various
nutritional indices and circulating vitamin D- and K- levels was analyzed with principal component analysis(PCA), which is a statistical method to summarize thevarious parameters into a small number of summary factors (components). These components are obtained insuch a way that the first component is extracted from theinitial raw data with the maximal amount of information(eigenvalue), and the second one is extracted from theremaining information. Therefore, each component ismutually independent. Components with the eigenvalue
greater than 1 were adopted, as in usual practice.
RESULTS
Biochemical markers and circulating concentrations ofvitamin D and K
Baseline characteristics and data from blood examination
are shown in Table 1. There was no gender difference inthe age and level of care needed, which is a 5-grade scorein the long-term care insurance in Japan with a higher
number indicating the need for more intensive care. The
level of care needed was higher than grade 3 in 78% ofsubjects. Most of the present subjects required wheelchairfor transportation. Body height and body weight weresignificantly higher in males than in females. Body massindex (BMI), or serum albumin, total cholesterol andtriglyceride concentrations did not significantly differ
between the two groups. Generally, serum albumin level
less than 3.5 g/dL is considered to indicate malnutrition.
Serum albumin level was below this value in 26% of sub-51

jects. Inasmuch as the advanced age and high level ofcare needed, nutritional parameters remained within thereference range in most of the subjects. None of the study
subjects had severe hepatic or renal dysfunction. There isa general consensus that a serum 25OH-D concentration
less than 20 ng/mL indicates hypovitaminosis D.2 Serum
25OH-D concentration was <10 ng/mL in 40% of subjects, 10-20 ng/mL in 58%, and ≥20 ng/mL in only one
subject. None of the subjects had a serum PTH level
above the cut-off value (65 pg/mL). Plasma PK and MK7 concentrations in all of the subjects were 0.73±0.70
ng/mL and 0.53±0.37 ng/mL, respectively. In the present
study, serum PK was less than 1 ng/ml and serum MK-7
was less than 1 ng/ml, in 85% and 90% of the subjects,
respectively. The interpretation for these values will begiven in the “Discussion” section. There were no genderdifferences in plasma vitamin K levels, serum 25OH-D or
PTH.
Nutritional intake in the study subjects
The nutrients intake in the males and females were notstatistically different as shown in Table 2. During the
preparation of this paper, Dietary Reference Intake (DRI)
for Japanese 2010 (DRI 2010) was released on May 29,
2009.28 Since this work was done in 2006, however, consideration is made basically according to DRI 2005.29 The
intake of macronutrients such as protein, fat and carbohydrates appeared appropriate for their age and sex. The
adequate intakes (AI) for calcium in Japan are 750 mg formen and 650 mg for women over 70 years. The AI for
vitamin D is 5 μg/day, and that for vitamin K is 75
μg/day for men and 65 μg/day for women respectively.
Although average calcium intakes in both groups werelower than the AI in DRI 2005, the average daily vitamin
D intake was 7.0 μg, which is 140% of the AI in DRI
2005. The average daily intake of vitamin K in wholesubjects was 155 μg, which is more than twice the AI for
each gender. Thus, apparently these subjects had sufficient intakes of vitamin D and K based on AI in DRI
2005.
Multiple regression analyses for the determination of
independent factor for circulating vitamin D, K concentrations.
In multiple regression analyses, vitamin D intake was asignificant determinant of serum 25OH-D level, although
the R2 was low. Serum triglyceride level was the only
significant predictor for plasma MK-7 concentration, andvitamin K intake and serum triglyceride concentrations
significantly contributed to plasma PK level (Table 3).
Principal component analysis (PCA)
Since institutionalized elderly are generally malnourished,
it is quite important to determine whether the low vitaminD - and K -status is independent of overall malnutrition ornot. Then PCA was performed with the parameters included for analysis being serum albumin, triglyceride,cholesterol, 25OH-D, PTH levels and plasma PK, MK-7
concentrations. Four components were obtained and explained 82% of the variance. The first component wascomposite of high albumin, total cholesterol and 25OH-D,and second component consisted of high triglyceride, low52

A Kuwabara, M Himeno, N Tsugawa, M Kamao, M Fujii, N Kawai, et al

Table 3. Multiple regression analyses for the determination of independent factors for circulating vitamin D, K concentrations
Serum 25OH-D

R2
0.095

p value
0.033

Variable
Vitamin D intake

β
0.309

p value
0.033

Plasma PK

0.181

0.011

Vitamin K intake
Triglyceride

0.290
0.380

0.042
0.009

Plasma MK-7

0.255

<0.001

Triglyceride

0.505<0.001

Only significant predictors are shown. The abbreviations are β for β coefficient, and p for p value. Independent predictor for serum 25OH-D
or plasma PK, MK-7 concentrations was analyzed by multivariate analysis with stepwise regression. Age, level of care needed and serum
triglyceride and total cholesterol concentrations were included in all analyses. Vitamin D intake was additionally included in the analysis
for plasma 25OH-D concentration. For plasma PK and MK-7, vitamin K intake was additionally included.

Table 4. Principal component analysis of nutrition indicesSerum Albumin
Serum triglyceride
Serum total cholesterol
Serum 25OH-D
Serum PTH
Plasma PK
Plasma MK-7

Component 1
0.880
0.229
0.800
0.434
0.156
-0.0140.117

Component 2
0.004
0.734
0.320
-0.457-0.2730.030
0.832

Component 3
0.047
0.119
-0.046-0.6580.877-0.071-0.238Component 4
0.059
0.380
-0.060-0.033-0.0900.986-0.152

Factor loadings to four components after varimax rotation are shown. Loadings greater than 0.35 are shown in bold
Four components thus obtained were considered to represent the following nutritional status; component 1: overall nutritional status, component 2: vitamin K2 status, component 3: vitamin D status, and component 4: vitamin K1 status.

25OH-D, and high MK-7. The third component wascomposite of low 25OH-D and high PTH, and the fourthcomponent was composed of high triglyceride and high
PK. The interpretation of each component was made as
follows; the first component representing overall nutritional status, the second component, vitamin K2 status,
the third component, vitamin D status, and the fourthcomponent representing vitamin K1 status (Table 4).
DISCUSSION
Nutritional status would be adequately assessed by both
evaluating the subjects’ food intake and measuring theircirculating or urinary markers. This principle would holdtrue especially in the elderly, since they are at high risk
for malabsorption or utilization defects of nutrients. Unfortunately in Japan, vitamin D and K status in the elderly
has been studied either by evaluating their food intake, as
in the annual National Nutrition Survey Japan (NNS-J) or
by measuring circulating level of these vitamins,21,30-33
but rarely by both.12,34
Institutionalized elderly have been our special concern,
since they are much more susceptible to hypovitaminosis
D and K deficiency than the healthy elderly. The NNS-J
in 2006 showed that subjects over 70 years of age, including both genders, had the following daily nutrients intakes:
energy 1761 kcal, calcium 551 mg, vitamin D 9.0 μg,
vitamin K 273 μg,35 which were higher than those of thesubjects in the present study. Gastrointestinal absorptionof nutrients in the present study subjects would be impaired also. These considerations led us to simultaneously
evaluate both vitamin D and K intakes and its circulatinglevels in the present study.

Before the interpretation of our data, determination
procedure for vitamin K deserves some discussion. There
have been discrepancies on the plasma concentration ofvitamin K in the previous literature, which is at least
partly due to the different determination procedure employed. Recently we have developed a novel procedure
for the determination of vitamin K analogs with high sensitivity and specificity, based on high-performance liquid
chromatography-tandem mass-mass spectrometry withatmospheric pressure chemical ionization (LC-APCIMS/MS).26 With this procedure, plasma concentrations of
PK and MK-7 were 0.73±0.70 ng/mL (median 0.58
ng/mL) and 0.53±0.37 ng/mL (median 0.45 ng/mL), respectively in the current study. In our recent study,
plasma concentrations for PK and MK-7 were 1.29±1.09
ng/mL (median 0.94 ng/mL) and 4.21±6.81 ng/mL (median 2.14ng/mL), respectively in the healthy Japaneseelderly over 70 years old using the same assay procedure.
21
In the same study, lowest concentration of plasma vitamin K level to avoid the elevation of serum ucOC concentration was 2.5 ng/ml for PK and 6.4 ng/ml for MK7.21 Since serum ucOC level is a sensitive indicator ofskeletal vitamin K insufficiency, these figures can yield arough estimate of circulating vitamin K levels needed by
the skeleton.
The median intake of vitamin K in the current subjects
was 168 μg, which was more than twice the AI in DRI
2005. The AI for vitamin K was not altered in DRI 2010.
Dietary vitamin K intake has been identified as an important determinant of plasma phylloquinone concentrationin previous studies.36,37 In the present study, vitamin K
intake was also significantly associated with plasma PK,
but not with plasma MK-7. Since they were not supplied

Hypovitaminosis D and K in the elderlywith fermented soybean; natto, which contains exraordinary amount of MK-7,38 phylloquinone from green vegetables is likely to be the major contributors to the total
vitamin K intake in our subjects. Thus plasma PK alone
correlated with total vitamin K intake, adjusted by serum
triglyceride. These data strongly suggest that these subjects are vitamin K-deficient in spite of the fact that theirdietary intake is far above the AI in according to DRI
2005, and increased vitamin K intake would be effectivein improving plasma PK levels in institutionalized elderly
in present study.
As in the case of vitamin K, average dietary intake ofvitamin D was around 7 μg/day, which is approximately
140% of the AI in subjects in the present study. Nevertheless, the average serum 25OH-D concentration was only
11.1 ng/mL. Thus, most subjects in the present study had
hypovitaminosis D in spite of apparently sufficient vitamin D intake.
Although the multiple regression analysis has identified vitamin D intake as the significant contributor to serum 25OH-D concentration, the R2 value was low, whichindicates that the current model could explain only asmall portion of variation. Several factors could be responsible for the above results. First, because of walking
disability and other physical dysfunction, the chance ofsun exposure was minimal in most of the current study
subjects, but it was not null. Thus, sun exposure may also
partly explain the above results. Unfortunately, however,detailed information about sun exposure was unavailable.
Furthermore, ADL itself has been reported to be related
to serum 25OH-D levels,39 on which detailed information
is not available in the current study. Secondly, the intestinal absorption of vitamin D is likely to decrease due tofactors such as compromised intestinal ability for nutrients absorption and limited fat intake.40 Nevertheless, oralvitamin D intake seems to be of value in the institutionalized elderly for improving their vitamin D status. Cashman et al. reported dose-dependent increase in serum
25OH-D concentration after incremental supplementation
with vitamin D3 in free-living adults over 64 years ofage.41 Although AI for vitamin D slightly increased to 5.5
μg/day in recently issued DRI 2010, the elderly subjects
are likely to require much more vitamin D intake to avoid
hypovitaminosis D considering the various problems tointerfere with absorption and utilization as discussed
above. A second issue with regard to the above discussion;
disturbed intestinal absorption and limited fat intake, will
also apply to the discrepant intake and circulating level ofvitamin K.
Although serum 25OH-D level was extremely low, average serum PTH level was within the reference range.
Circulating 25OH-D concentrations showed significant
negative correlation with serum PTH levels (r= -0.293, p=
0.041; data not shown), which suggests that the negative
feedback regulation of PTH secretion by vitamin D is not
impaired in the current population. Kuchuk et al. reported
that the elevation of serum PTH concentration by vitamin
D deficiency is moderate in its magnitude, and usually
fell into the reference range.42 Thus they stressed the importance of serum 25OH-D level, and argued that forbone health maintenance and physical performance in the53

elderly, serum 25OH-D concentration above 50-60
nmol/L (20-24 ng/mL) was required.
Although the institutionalized elderly are considered tobe generally malnourished,43-45 nutritional status appeared
rather satisfactory in the present study subjects in face ofhypovitaminosis D and K. Then we analyzed the relationship between the overall nutrition and circulating levelsof vitamin D and K by PCA. The PCA have yielded four
components representing: overall nutritional status, vitamin D status, vitamin K2 status, and vitamin K1 statusrespectively. Serum 25OH-D also exhibited some association with the first component, representing the overall
nutritional status. One of the reasons for the above results
would be that 25OH-D is bound to vitamin D-binding
protein (DBP) and albumin during its transport in circulation.46 Since these components are independent of eachother by their definition, these results suggest that hypovitaminosis D and K in the institutionalized elderly do not
merely reflect general malnutrition, and have their own
role. Confounders are serious challenge in the clinicalstudies. In the intervention studies, randomization would
eliminate the interference by the confounders. It would beless problematic in the case of cohort studies. Adjustment
for confounders is quite difficult in the cross-sectional
studies like the current one. Multivariate analyses such as
PCA would be of help in eliminating the interference byconfounders in this type of studies.
In conclusion, institutionalized elderly had high prevalence of hypovitaminosis D and K in spite of their dietaryintake exceeding the AI in DRI 2005 in Japan, whichsuggests that the requirement for these vitamins would behigher in these subjects. Additionally, hypovitaminosis D
and K were shown to be independent of general malnutrition by PCA, which would be a useful analytical procedure for eliminating the interference by confounders incross sectional studies.
ACKNOWLEDGMENTS
This study was supported by Health and Labor Science Research Grant entitled “Studies on the Dietary Reference Intakes(Recommended Dietary Allowance) for Japanese” from the
Ministry of Health, Labor and Welfare, Japan.
AUTHOR DISCLOSURES
None of the authors have any conflicts of interest.

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1986;63:954-9.

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A Kuwabara, M Himeno, N Tsugawa, M Kamao, M Fujii, N Kawai, et al

Original Article

Hypovitaminosis D and K are highly prevalent andindependent of overall malnutrition in theinstitutionalized elderly
Akiko Kuwabara PhD1, Masako Himeno MSc1, Naoko Tsugawa PhD2,
Maya Kamao MSc2, Minori Fujii RD3, Nobuko Kawai CSW3, Miyuki Fukuda MSc1,
Yoko Ogawa MSc1, Shoko Kido PhD1, Toshio Okano PhD2, Kiyoshi Tanaka MD1
1

Department of Food and Nutrition, Kyoto Women’s University, Higashiyama, Japan
Department of Hygienic Sciences, Kobe Pharmaceutical University, Higashiyama, Japan
3
Nursing Home Kayu-Shirakawa, Higashiyama, Japan2

居住機構中的老年人有高盛行率的維生素 D 及維生素K 缺乏症且與整體的營養不良無相關
研究老年人的維生素 D 及維生素 K 缺乏症有許多方法學上的問題。首先，大多研究是藉由評估食物的攝取或是測量血中的濃度來進行的，但在日本很少
同時利用這兩種方法。在本篇文章中，維生素 D 及維生素 K 的攝取以及老年
人的血中濃度是同步測量的。第二個議題是維生素 D 及維生素 K 缺乏症是否
與盛行於老年人的一般營養不良情形相關。我們試著藉由統計的主成份分析
方法去分辨。評估 50 位機構中的老年人血中的 25-羥化維生素 D、副甲狀腺素、維生素 K1、維生素 K 2 濃度，以及食物攝取。雖然平均維生素 D 攝取量(每天 7 克)超過日本所訂定的足夠攝取量(每天 5 克)，但平均血清中 25-羥化維
生素 D 濃度(11.1 ng/mL)卻屬維生素 D 缺乏的範圍。維生素 K 攝取量的中位數
為每天 168 克，這幾乎是維生素 K 的足夠攝取量的 2.5 倍。但是，血漿中維生素 K1 及維生素 K 2 濃度是遠低於 70 歲以上健康的日本老人。應用主成份分析法，結果產生 4 個成份，分別代表整體營養狀況、維生素 K 2、維生素 D 及維生素 K1 的營養狀況。既然每個成份都各自獨立，則這些老人的維生素 D 及維生素 K 缺乏不能用整體營養不良加以解釋。總之，在這些機構中的老年人具有高盛行率的維生素 D 及維生素 K 缺乏；爾後這類研究應該同時測量血中濃度及飲食攝取。主成份分析法，可排除橫斷性研究中其他干擾因子的作用，
而得到有效的結果。
關鍵字：維生素 D 缺乏、維生素 K 缺乏、主成份分析、足夠攝取量、機構中的老年人128

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〔ビタミン 84 巻

ビタミン D による骨折予防効果の意義：医療経済の視点から
Fracture Prevention by Vitamin D: from the Perspectives of Health Economy
ビタミン研究において，基礎研究，疫学・臨床研究は，お互いに合い補いあう関係であろうが，もう一つの研究
分野は，社会におけるビタミンの役割である．しかし，このような研究はわが国では従来ほぼ皆無なので紹介したい．ほとんどの読者にとって，ふだん聞きなれない用語・概念がでてくることや一部に私見が入っている点はご容
赦願いたい．なお，筆者は骨粗鬆症を主な研究分野としており，以下ビタミン D による骨折抑制を例として述べる．
本稿における骨折の記述は，大腿骨近位部骨折が主となるので，最初に簡単に説明しておく．この骨折はいわゆる「足の付け根」が折れるもので，高齢者に多い．受傷
後 1 年以内の死亡率が高いだけではなく，死亡をまぬがれても，生活レベルが低下するため，要介護の原因となる例も少なくない．このことは患者本人にとって，大きな不幸であることは言うまでもないが，医療費・介護に要する費用も莫大であり，社会に及ぼす影響も大きい 1）．
従って，大腿骨近位部骨折の予防は医学的にも，社会的にも緊急の課題となっている．
近年種々の骨粗鬆症治療薬が臨床現場で用いられているが，主に投与されているのは骨吸収抑制薬である．その代表である bisphosphonate 系薬剤は，強力な骨吸収抑制薬であり，骨折の発生率を半減させるほどの効果を示
す．最近一部の国と地域では，人口の高齢化にも関わらず，
大腿骨近位部骨折の発生率が減少しており，その減少は骨密度測定の普及や bisphosphonate のような新薬の処方との関連が考えられている 2）．これに勝るとも劣らない
新薬が近年さらに次々と開発されている．このような画期的新薬と比べると，ビタミンをはじめとする栄養療法の効果は小さい．
最近欧米においては，医療行為は有効であることを証明しただけでは不十分で，かけた費用に見合うだけの効果を挙げることを示す必要があると考えられている 3）．
この費用に対する効果（費用対効果）の視点から見ると，ビタミンを含む栄養療法には，新たな側面が見えてくる．
疾患の予防は，一次予防，二次予防，三次予防に分類される．三次予防はリハビリテーションのように，疾患に罹った後の対策，二次予防は疾患の早期診断と早期治療，一次予防は生活習慣の改善などによって，疾患を未然に防ぐことを目指すものである．また，予防に関する
ア プ ロ ー チ の 方 法 か ら，high risk approach と population
approach に分けられる．前者はその名前の通り，疾患発生のリスクの高い群に対して集中的に介入を行うものである．疾患発生リスクが高く，その薬剤の予防効果が大
きいほど費用対効果はよい値となる．従って，骨折リス
クの非常に高い群に対して bisphosphonate などを用い，薬剤治療介入を行うことは費用対効果に優れた医療行為である．
しかし，中～低リスク集団に対してもこのような治療介入を行うことは，要する費用や発生しうる副作用の可能性を考えると，とうてい実行不可能である．例えば，
50 歳以上の全女性に，bisphosphonate を服用させるというのは，あり得ない選択である．このような場合には，集団のリスクを低い方に平行移動させる population approach が行われる．その場合，多数かつ多様な対象者に対して行いうることが必須であるから，安全かつ安価なものでなければならず，栄養，運動などの生活習慣改善が中心となる．
言い換えると，ビタミンなどの栄養療法は絶対的な効
果は新薬より小さいが，かかる費用は圧倒的に低いので，費用対効果には優れており，中～低リスク集団に対する一次予防，population approach には非常に適しているということである．わが国ではこのような研究は皆無だが，海外では医療行為に対する経済評価は盛んに行われており，ビタミン D の骨折予防効果を扱ったものもいくつか
ある 4）-8）．
一例を示すと，Decalyos Study は，フランスで行われた臨床研究であり，3,270 名の高齢女性を 2 群に分け，一方にはビタミン D（800
IU/ 日）＋カルシウム（1,200 mg/ 日）
3の投与が，他方にはカルシウム（1,200 mg/ 日）のみの投与が，36 ヶ月行われた．大腿骨近位部骨折は，Ca のみ群では 1,127 名中 184 名に起こったのに対し，D 群では 1,176
名中 138 名であった．すなわち，1,000 名あたり 46 件の大腿骨近位部骨折が予防できたことになる．この骨折は非常に医療費のかかる骨折であるので，もし施設入居高齢者全員にビタミン D ＋カルシウムの補充を行うならば，それにより，1 億 5 千万フランの医療費が削減できるものと推測されている 4）．
骨折の危険因子として低骨密度はもちろん重要だが，大腿骨近位部骨折など非椎体骨折の場合，転倒がきっかけになる例が非常に多い．最近ビタミン D の転倒防止効
果も注目されていることから，ビタミン D は骨密度低下を防ぐ作用に加えて，複数の作用点から骨折予防に役立つ可能性がある．なお上記の研究は，大腿骨近位部骨折
のみに注目したものであるが，実際にはビタミン D 欠乏（不足）の改善によって，それ以外の骨折についても予防3 号（3 月）2010〕

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効果が見込まれるので，実際の経済効果はさらに大きく，
上記の推定はおそらく過小評価である．
最近海外では，400IU/ 日のビタミン D では骨折を防ぐ
ことができず，最低 800IU/ 日程度を要するという大規模研究やメタアナリシスがあいついで発表され 9）10），ビタ
ミン D による骨折予防の社会的側面にも言及されている．
予防効果を評価するのに，NNT（number needed to treat）という指標がよく用いられる．これはその疾患（ここでは骨折）の発生を 1 件防ぐためには，何人に対して介入する必要があるかという数字であり，NNT が小さい方が効率のよい予防介入である．当然，疾患発生リスクの高い集団に対して介入した方が NNT はよい値となる．Tang らは，
29 の RCT（randomized controlled trial），63,897 例のデータのメタアナリシス結果から，ビタミン D の有効性を報告しているが，NNT = 63 であったと述べている 9）．この研究は 50 歳以上の女性が対象であり，決して骨折リスクが非常に高い集団だけを選んだものではないことを考えると，NNT = 63 は非常によい数字であり，循環器領域でのNNT と比べても遜色ない 11）．
ビタミン D の骨折予防効果に関しては，近年の海外での進歩に比べて，日本での臨床研究が非常に遅れている．
多数の介入試験が海外で発表されてきたが，これまでわが国ではほぼ皆無であった．最近，我々は施設入所高齢者に対する，ビタミン D3 による介入試験を行った 12）13）．
血液中の 25-hydroxyvitamin D 濃度が最もよいビタミン D
栄養状態の指標だが，日本人の食事摂取基準 2005 年版の目安量である，200 IU/ 日では極めて不十分な効果しか得られず，少なくとも 800 IU/ 日を要することを報告した．
しかし，これはまだ血液中のビタミン D 濃度を指標としたのにとどまっており，骨折を指標したものではない．
このようなわが国の現状では，本稿に述べたような医療
経済からみたビタミンの評価というような話題は時期尚早なのかもしれない．しかし，このようなビタミンの評
価は社会と行政に対してビタミンの重要性を訴えていく，
一つの有力な視点となるものであり，今後わが国でも研究が行われるべき分野であると考え，あえて紹介した．

13）Kuwabara A, Tsugawa N, Tanaka K, Fujii M, Kawai N, Mukae S,

Key Words：vitamin D, fracture prevention, health economics,

T, Kido S, Okano T (2009) Improvement of vitamin D status in Japa-

文献1）田中清，萩野浩，原田敦他（2007）骨粗鬆症における薬物療法
に関する医療経済評価．Osteoporos Jpn 15, 51-55
2）Guilley E, Chevalley T, Herrmann F, Baccino D, Hoffmeyer P, Rapin
CH, Rizzoli R (2008) Reversal of the hip fracture secular trend is related to a decrease in the incidence in institution-dwelling elderly
women. Osteoporos Int 19, 1741-1747
3）田中清，武田英二，門脇孝，恩地森一，立川倶子，清野裕（2006）
生活習慣病の治療・予防における栄養療法の意義と医療経済評
価．日本病態栄養学会誌 9, 3-94）Lilliu H, Pamphile P, Chapuy M-C, Schulten J, Arlot M, Meunier PJ(2003) Calcium-vitamin D3 supplementation is cost-effective in hip
fracture prevention. Maturitas 44, 299-305
5）Kanis JA, Dawson A, Oden A, Johnell O, de Laet C, Jonsson B (2001)
Cost-effectiveness of preventing hip fracture in the general female
population. Osteoporos Int 12, 356-361
6）Torgenson DJ, Kanis JA (1995) Cost-effectiveness of preventing hip
fracture in the elderly population using vitamin D and calcium. QJM
88, 135-139
7）Willis MS (2002) The health economics of calcium and vitamin D3
for the prevention of osteporotic hip fractures in Sweden. Int J Technol Assess Health Care 18, 791-807
8）Vestergaard P, Rejnmark L, Mosekilde L (2001) Hip fracture prevention: cost-effectiveness strategies. Pharmacoeconomics 19, 449-468
9）Tang BM, Eslick GD, Nowson C, Smith C, Bensoussan A (2007) Useof calcium or calcium in combination with vitamin D supplementation to prevent fractures and bone loss in people aged 50 years andolder: a meta-analysis. Lancet 370, 657-666
10）Bischoff-Ferrari HA, Willett WC, Wong JB, Giovannucci E, Dietrich
T, Dawson-Hughes B (2005) Fracture prevention with vitamin D supplementation: a meta-analysis of randomized controlled trials. JAMA
293, 2257-2264
11）Reginster J-Y (2007) Calcium and vitamin D for osteoporotic fracturerisk. Lancet 370, 632-634
12）Himeno M, Tsugawa N, Kuwabara A, Fujii M, Kawai N, Kato Y, Kihara N, Toyoda T, Kishimoto M, Ogawa Y, Kido S, Noike T, Okano T,
Tanaka K (2009) Effect of vitamin D supplementation in the institutionalized elderly. J Bone Miner Metab 27, 733-737
Kato Y, Kojima Y, Takahashi K, Omura K, Kagawa R, Inoue A, Noike

primary prevention, population approach
Department of Food and Nutrition, Kyoto Women’s University
Kiyoshi Tanaka, Akiko Kuwabara
京都女子大学家政学部食物栄養学科
田中清，桒原 晶子

nese institutionalized elderly by supplementation with of 800 IU vitamin D3. J Nutr Sci Vitaminol 55, 453-458In: Vitamin D: Nutrition, Side Effects and Symptoms
Editor: Stephanie R. Malone

ISBN: 978-1-61728-601-8© 2009 Nova Science Publishers, Inc.

Chapter 8

PATIENTS WITH CROHN’S DISEASE HAVEHYPOVITAMINOSIS D AND K, WHICH IS
INDEPENDENT OF GENERAL MALNUTRITION
Akiko Kuwabara1, Naoko Tsugawa2, Maya Kamao2, Hiroshi
Nakase3, Tsutomu Chiba3, Toshio Okano2and Kiyoshi Tanaka1*
1

Department of Food and Nutrition, Kyoto Women's University, Kyoto, Japan2
Department of Hygienic Sciences3
Department of Gastroenterology, Kyoto University Graduate School of Medicine

INTRODUCTION
Inflammatory bowel disease (IBD) consisting of Crohn’s disease (CD) and ulcerative
colitis (UC) is an inflammatory disease affecting the gastrointestinal tract. As its nameimplies, UC mainly affects large intestine. In contrast, CD can affect all part of thegastrointestinal tract from mouth to anus, but in most cases with CD, small intestine isinvolved. Nutritional complications are common in patients with IBD, especially those with
CD[1,2]. Deficiencies of proteins, calories, and vitamins are quite prevalent in subjects withCD, which may be caused by such factors as inadequate dietary intake, intestinal loss ofprotein, or malabsorption[3-6]. Previous studies have reported the high prevalence ofhypovitaminosis D or K in patients with IBD [7-11].
Vitamin D, amongst its diversity of actions, enhances intestinal absorption of calcium andphosphorus as its fundamental action [12]. Its deficiency causes mineralization defect; rickets
and osteomalacia[13]. Its inadequacy, even in its milder form (insufficiency) increases therisk of fracture through negative calcium balance and secondary hyperparathyroidism[14].
Recently, the skeletal action of vitamin K has come to our attention, although its sole actionof clinical significance has long been considered to be the one as the co-factor for γ*

Corresponding author: Department of Food and Nutrition, Kyoto Women’s University, 35, Imakumanokitahiyoshicho, Higashiyama, Kyoto 605-8501, Japan, Phone +81-75-531-7125, Fax +81-75-531-7153, E-mailtanakak@kyoto-wu.ac.jp

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Akiko Kuwabara, Naoko Tsugawa, Maya Kamao et al.

carboxylation of four of the clotting factors in the liver [15]. Skeletal vitamin K insufficiency
has been demonstrated to be a risk factor for fracture [16,17].
Osteoporosis is a common complication of IBD [18-22]. It has also been reported that theincidence of fracture among patients with IBD was greater than that in general population[23]. Thus, hypovitaminosis D and K is likely to be of great importance in the evaluation andpossibly the treatment of osteoporosis associated with IBD.
In our recent report [24], CD patients had significantly lower plasma vitamin K and Dconcentrations, and significantly higher serum levels of markers for the inadequacy of thesevitamins; parathyroid hormone (PTH), protein induced by vitamin K absence (PIVKA)-Ⅱ
and undercarboxylated osteocalcin (ucOC), which are indicators for vitamin D insufficiency,
hepatic vitamin K insufficiency, and skeletal vitamin K insufficiency, respectively. Subjects
with CD had significantly lower BMD scores at almost all measurement sites than those with
UC. Plasma levels of vitamin D and K correlated with BMD and the patients’ fat intake, but
not with their intake of these vitamins. Multiple regression analysis revealed that low plasma
concentrations of vitamin D and K were independent risk factors for low BMD at radius.
These results suggested that maintaining vitamin D- and K-adequacy is necessary for thebone health. The insufficiency of these vitamins is likely to arise from their malabsorption
probably due to restricted fat intake as well as the compromised intestinal ability to absorb thenutrients.
However, nutritional indices such as serum albumin and total cholesterol levels were also
lower in patients with CD than those with UC. Thus one could argue against the role of thesevitamins by stating that decreased blood concentrations of these vitamins merely reflect theoverall malnutrition.
In this paper, we have attempted to demonstrate that decreased blood concentrations ofvitamin D and K in IBD patients are independent of general malnutrition by use of one of themultivariate analyses; principal component analysis (PCA).

SUBJECTS AND METHODS
Study Subjects
The study subjects were 128 patients with IBD (male:78, female:50) consisting of 60
patients with CD (male:36, female:24) and 68 patients with UC (male:42, female:26) visiting
the Gastroenterology Clinic of Kyoto University Hospital. Consent to participate in this study
was obtained after explanation of the objective and protocol of this study.

Bone Mineral Density (BMD) Measurement
BMD was measured at various skeletal sites; lumbar spine, femoral neck, total hip, distal
one-third of radius, ultradistal radius with dual energy X-ray absorptiometry (DXA) using
Hologic QDR-2000 (Bedford, MA). Data were expressed as z-value, which shows a standard
deviation from age- and sex-adjusted average based on the Japanese reference database.

Patients with Crohn’s Disease Have Hypovitaminosis D and K which is Independent… 3

Blood Examination
Blood was obtained after overnight fasting. After centrifugation, plasma or serum
samples were stored at –30℃ with protection from light until analysis. Serum concentrationof 25-hydroxyvitamin D (25OH-D) was measured by radioimmunoassay (RIA) (DiaSorin,
Stillwater, MN, USA). Plasma vitamin K1 (phylloquinone; PK), and menaquinone-7 (MK-7)
levels were determined by high-performance liquid chromatography-tandem mass-mass
spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS), HPLC
analyses were conducted with a HPLC system (Shimadzu, Kyoto, Japan), Mass spectrometry
was performed with API3000 LC-MS/MS System (Applied Biosystems, Foster City, CA).
18
O-labeled vitamin K was the internal standard in the analysis of plasma vitamin K [25].
Serum intact-PTH was measured by an electro immunoradiometric assay (IRMA)(Scantibodies Laboratory, Santee, CA, USA), with 14-66 pg/ml as the reference range.
Other biochemical markers were measured by auto-analyzer. Approximately half ofserum calcium is bound to albumin, and its serum concentration is affected by alteration inserum albumin level. Therefore, serum calcium concentration was corrected as follows;
corrected calcium=calcium – (albumin – 4).

Statistical Analysis
Statistical analyses were done with SPSS 17.0J (SPSS Japan, Tokyo). Comparison of
independent two groups was done with Student’s t-test or Mann-Whitney test depending on
normality. Multiple regression analyses were performed to investigate the determinants for
BMD at each site. Data on various nutritional indices and circulating vitamin D- and K-levels
were analyzed with principal component analysis (PCA), which is a statistical method tosummarize the various parameters into small number of summary factors (components).
These components are obtained in such a way that first component is extracted from theinitial raw data with the maximal amount of information (eigenvalue), and the second one isextracted from the remaining information. Thus, they are independent of each other.
Components with the eigenvalue greater than 1 were adopted as is used in the usual practice.

RESULTS
Biochemical Markers
The baseline characteristics of the study subjects are shown in Table 1. Patients with CD
were significantly younger than those with UC. CD patients had higher CRP level, and lower
nutritional indices such as BMI, hemoglobin, serum albumin and cholesterol concentrations
than those with UC. Other indices were not significantly different between two groups, andlargely fell into the reference ranges.

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Akiko Kuwabara, Naoko Tsugawa, Maya Kamao et al.
Table 1. Baseline characteristics of the study subjects
IBD (n=128)
Age (years)
Sex (Male/Female)
Body mass index (kg/m2)
C-reactive protein (mg/dl)
White blood cell (x103)
Red blood cell (Hemoglobin (g/dL)
Total protein (g/dL)
Albumin (g/dl)
Triglyceride (mg/dL)
Total cholesterol (mg/dl)
Alkaline phosphatase
Corrected calcium (mg/dl)
Phosphorus

CD (n=60)
34.4+/-7.5
36/24
19.2+/-2.7
0.7+/-1.0
6.1+/-1.9
4.3+/-0.5
11.8+/-1.9
6.8+/-0.7
3.9+/-0.4
95.4+/-40.7
131.7+/-25.2
228.9+/-72.3
8.8+/-0.3
3.3+/-0.7

UC (n=68)
41.7+/-16.4
42/26
21.1+/-3.0
0.3+/-0.6
5.9+/-1.9
4.5+/-0.6
13.1+/-2.0
7.1+/-0.4
4.3+/-0.4
101.3+/-77.6
176.5+/-36.6
227.5+/-95.8
8.8+/-0.3
3.4+/-0.4

p value
<0.01
<0.01
0.01b
NS
NS
<0.01
0.01
<0.01
NS
<0.01
NS
NS
NS

Data are expressed as mean+/-SD.
The p value indicates the statistical difference between CD and UC patients based on Student’s t-test or
Mann-whitney test depending on normality.

Table 2. Serum concentrations of 25(OH)D, PTH, and vitamin K in IBD patientsIBD (n=128)
25OH-D (ng/ml)
PTH (pg/ml)
PK (ng/ml)
MK-7 (ng/ml)

CD (n=60)
14.1+/-8.5 (12.6)
30.6+/-12.4 (28.0)
0.60+/-0.72 (0.45)
2.70+/-7.49 (0.45)

UC (n=68)
18.5+/-7.2 (18.2)
26.0+/-9.7 (25.5)
0.86+/-0.54 (0.78)
6.43+/-10.1 (1.67)

p value
<0.01
0.04
<0.01
<0.01

Data are expressed as mean+/-SD with the values in parentheses showing the median.
The p value indicates the statistical difference between CD and UC patients based on Student’s t-test or
Mann-whitney test depending on normality.

Circulating Levels of Vitamins D and K
Serum 25OH-D concentration was significantly lower and PTH level was significantly
higher in CD patients than UC subjects. There is a general consensus that serum 25OH-D
concentration less than 20 ng/mL indicates hypovitaminosis D. Serum 25OH-D level was
below 20 ng/ml in 82% of patients with CD and 57% of patients with UC. Most of thesubjects had serum PTH level within the reference range (≤66 pg/mL). Serum concentrationsof PK and MK-7 were significantly lower in patients with CD than those with UC (Table 2).
In our recent report using the same assay procedure, mean plasma PK level was 1.74 ng/ml,
and plasma MK-7 level was 4.96 ng/ml in healthy Japanese women aged 30-49 [26].
Therefore, circulating levels of vitamin K were considered to be extremely lower in IBD
patients than that in the healthy subjects.

Patients with Crohn’s Disease Have Hypovitaminosis D and K which is Independent… 5
Table 3. BMD in patients with CD and UC

Lumbar spine (L1-4)
Femoral neckTotal hipDistal one-third of radius
Ultra distal radius

CD (n=27)-0.52+/-0.64**-1.02+/-0.85**-0.87+/-0.81**-1.85+/-1.15**-1.87+/-0.86**

UC (n=31)-0.45+/-1.15*-0.45+/-1.12-0.27+/-1.08-1.79+/-1.51**-1.20+/-1.36**

p value
NS
NS
0.03
NS
NS

Data are shown as the z-score normalized by gender and age and expressed as mean +/- SD. The pvalue denotes the statistical significance in BMD in CD and UC subjects. The asterisks show thestatistically significant difference from zero (*: p<0.05, **<0.01).

BMD Measurement
In Table 3 are shown the data on BMD. Although patients with CD had z-scoresignificantly lower than zero at all measurement sites, UC subjects had z-score below zero at
lumbar spine, distal one-third and ultradistal radius. BMD in CD patients was significantly
lower at total hip

Principal Component Analysis (PCA)
PCA was performed with the parameters included for analysis being hemoglobin serum
albumin, total cholesterol, 25OH-D level and plasma PK, MK-7 concentrations (Table 4).
Two components were obtained and explained 56.7% of the variance. The first component
was composite of high albumin, total cholesterol and hemoglobin, and second component
consisted of high 25OH-D, PK and MK-7. The interpretation of each component was made as
follows; the first component representing overall nutritional status, the second component,
vitamin D and K status. When CD and UC subjects were analyzed separately, vitamin D andK status was also independent of overall nutritional status (data not shown).
Table 4. Principal component analysis of nutrition indicesSerum Albumin
Serum total cholesterol
Hemoglobin
Serum 25OH-D
Plasma PK
Plasma MK-7

Component 1
0.828
0.675
0.786
0.309
-0.1250.130

Component 2
0.040
0.056
0.241
0.730
0.820
0.498

Factor loadings to four components after varimax rotation are shown. Loadings greater than 0.35 areshown in bold
Two components thus obtained were considered to represent the following nutritional status;
component 1 overall nutritional status, component 2 vitamin status

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Akiko Kuwabara, Naoko Tsugawa, Maya Kamao et al.
Table 5. Comparison of two parameters obtained from PCA.

First Component(Overall Nutritional Status)
Second Component(Vitamin D and K Status)

CD (n=60)-0.54+/-0.87

UC (n=68)
0.47+/-0.86

P value<0.001-0.25+/-1.08

0.22+/-0.87

0.006

The p value indicates the statistical difference between CD and UC patients based on Student’s t-test.

Next, these two summary scores were compared between CD and UC subjects. Both ofthem were significantly lower in CD patients than UC subjects, which suggests that patientswith CD are both under-nourished and in vitamin D- and K-insufficiency (Table 5).

Multiple Regression Analyses for Factors Associated with BMD Z Scores At
Various Site
Multiple regression analyses were done to study to what extent the BMD (z-score) could
be explained by these summary scores (Table 6). BMD at all measurement sites except
lumbar spine and distal one-third radius were significantly contributed by these two summaryscores, with first component (overall nutritional status) contributing to BMD at ultradistal
radius, and second component (vitamin status) significantly contributed to BMD at femoral
neck, total hip, and ultradistal radius.
Table 6. Multiple Regression Analyses for BMD.
Sites
Lumbar spine

R2
0.06

p value
0.07

Femoral neck

0.21

<0.01

Total hip

0.18

<0.01

Distal one-third of radius

0.02

0.65

Ultradistal radius

0.14

<0.01

Variable
Overall Nutritional Status
Vitamin D and K Status
Overall Nutritional Status
Vitamin D and K Status
Overall Nutritional Status
Vitamin D and K Status
Overall Nutritional Status
Vitamin D and K Status
Overall Nutritional Status
Vitamin D and K Status

β
0.10
0.27
0.11
0.47
0.22
0.36
0.10
0.07
0.28
0.26

p value
0.44
0.04
0.45
<0.01
0.09
<0.01
0.51
0.63
0.04
0.05

Abbreviations are as follow; β for β coefficient and p for p value. Determinants of independent
predictors for BMD at each site were analyzed by multivariate analysis with stepwise method.
Variables included were two parameters obtained from PCA.

Patients with Crohn’s Disease Have Hypovitaminosis D and K which is Independent… 7

DISCUSSION
The insufficiency of vitamin D and K are associated with increased risk offracture[14,27-29]. However, the importance of vitamin insufficiency is often overlooked.
Classical vitamin deficiency diseases are easily diagnosed, since they are accompanied byphenotypic abnormalities such as rickets or osteomalacia in vitamin D deficiency, andbleeding tendency in vitamin K deficiency. In contrast, vitamin insufficiency could not bediagnosed for the individual subjects phenotypically, and the increased risk of chronic
diseases due to vitamin insufficiency becomes apparent only by the epidemiological studies.
Thus each subject could only be evaluated by the surrogate markers. Serum concentrationof 25OH-D best reflects the vitamin D status. It is a general consensus that its concentration
below 20 ng/mL is associated with increased fracture risk, although recent evidences indicatethat higher concentration around 30 ng/mL is necessary for fracture prevention[30,31]. In thecase of vitamin K, there are some methodological problems. First, vitamin K consists ofphylloquinone (vitamin K1) and menaquinones (vitamin K2), the latter further composed ofseveral analogs with different length of side chain. Therefore, vitamin K status cannot be
evaluated by a single measurement of blood level of one of the vitamin K analogs. In
addition, although circulating vitamin K levels have been measured with various methods,
results are different between assay procedures. The present data were obtained with our newly
developed LC-APCI-MS/MS procedure with stable isotope-labeled internal standard yielding
high sensitivity and specificity [25]. Thus, measurement of circulating levels of thesevitamins aids us to evaluate the subjects’ vitamin status, especially vitamin insufficiency.
In the present study, patients with IBD had hypovitaminosis D and K, especially thosewith CD. Patients with CD had also lower overall nutritional status than those with UC. We
have recently reported that hypovitaminosis D and K were associated with decreased BMD in
IBD subjects[24]. However, osteoporosis is a common complication of IBD, for which manyfactors have been reported to be responsible; low body weight, inflammatory process, andtherapeutic glucocorticoid use[18-22].
Thus, it must be decided whether the decreased concentrations of vitamin D and K are
merely the reflection of overall malnutrition or not. Confounders are serious challenges in theclinical and epidemiological studies. In the intervention studies, randomized controlled trial(RCT) would largely eliminate the interference by confounders. Of the observational studies,cohort study would be less sensitive to the confounding variables. In the cross-sectional
studies, eliminating the interference by confounders is a tough methodological challenge.
The main strategies to control for confounding in observational epidemiologicalinvestigations and, in particular, in case-control studies, are restriction, matching,stratification and fitting of regression models. In the present study, we have attempted todiscriminate hypovitaminosis D and K from general malnutrition by use of another statisticalmethod; principal component analysis (PCA). It is a useful statistical procedure to summarize
the complex and diverse data into the small number of independent components (summaryscore). In the epidemiological or clinical studies, PCA has been employed in the dietary
pattern analysis [32,33].
Two components or the summary scores obtained in the current study were considered torepresent the overall nutritional status, and vitamin D- and K-status, respectively. Both ofthem were lower in patients with CD than those with UC. Since they are independent of each

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Akiko Kuwabara, Naoko Tsugawa, Maya Kamao et al.

other, these results suggested that subjects with CD had both hypovitaminosis D and K, andgeneral malnutrition, but the former is not merely a reflection of the latter. The results bymultiple regression analyses indicated that insufficiency of these vitamins would negatively
affect BMD at various skeletal sites.
We believe that our current report carries significance both clinically andmethodologically, it is not free from limitation. Although the multiple regression analysis has
identified vitamin D and K status as the significant contributor to BMD at femur and radius,
the R2 value was low, which indicates that the current model could explain only a small
portion of variation. Further studies are required for the additional determinants for BMD in
IBD.
In conclusion, patients with CD have hypovitaminosis D and K independent of generalmalnutrition, and analyzing data from cross-sectional studies with PCA would be of help in
eliminating the interference by confounding variables.

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