研究成果の刊行に関する一覧表
雑誌
発表者氏名

論文タイトル名

発表誌名

巻号

ページ

出版年

Kasuga M.

Dietary Reference Intakes for Japanese.
Preface.

J Nutr Sci
Vitaminol

59,
suppl

S1

2013

Tokudome S.

Dietary Reference Intakes for Japanese.
Foreword.

J Nutr Sci
Vitaminol

59,
suppl

S2

2013

Tsuboyama-Kasaoka N,
Tsubota-Utsugi M, Imai E,
Nakade M, Kasuga M.

Historical overview of the establishment of J Nutr Sci
Dietary Reference Intakes for Japanese.
Vitaminol.

59,
suppl

S6-S8

2013

Sasaki S.

Dietary Reference Intakes for Japanese
2010: basic theories for the development.

J Nutr Sci
Vitaminol

59,
suppl

S9-S17

2013

Sasaki S.

Dietary Reference Intakes for Japanese
2010: basic concepts for application.

J Nutr Sci
Vitaminol

59,
suppl

S18-S25

2013

Tabata I, Ebine N, Kawashima
Y, Ishikawa-Takata K, Tanaka
S, Higuchi M, Yoshitake Y.

Dietary Reference Intakes for Japanese
2010: energy.

J Nutr Sci
Vitaminol

59,
suppl

S26-S35

2013

Kido Y, Shizuka F, Shimomura Dietary Reference Intakes for JapaneseY, Sugiyama T.
2010: protein.

J Nutr Sci
Vitaminol

59,
suppl

S36-S43

2013

Ezaki O, Miyake Y, Sato S, Iso Dietary Reference Intakes for JapaneseH.
2010: fat.

J Nutr Sci
Vitaminol

59,
suppl

S44-S52

2013

Yamada K, Tsuboyama-Kasaoka Dietary reference intakes for Japanese 2010: J Nutr Sci
N, Goda T, Saito K,
carbohydrates.
Vitaminol
Yamanouchi T, Yokoyama T,
Chonan O, Imai E, Nakade M,
Aoe S.

59,
suppl

S53-S56

2013

Tanaka K, Terao J, Shidoji Y,
Tamai H, Imai E, Okano T.

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

J Nutr Sci
Vitaminol

59,
suppl

S57-S662013

Shibata K, Fukuwatari T, Imai
E, Hayakawa T, Watanabe F,
Takimoto H , Watanabe T,
Umegaki K.

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

J Nutr Sci
Vitaminol

59,
suppl

S67-S82

2013

Uenishi K, Ishimi Y, Nakamura Dietary Reference Intakes for JapaneseK, Kodama H, Esashi T.
2010: macrominerals.

J Nutr Sci
Vitaminol

59,
suppl

S83-S902013

Yoshida M, Kikunaga S,
Dietary Reference Intakes for Japanese
Yamauchi J, Tsubota-Utsugi M, 2010: microminerals.
Kodama H, Morita A, Esashi T.

J Nutr Sci
Vitaminol

59, S91-S102
suppl

2013

Morita A, Yoshiike N, Takimoto Dietary Reference Intakes for Japanese
J Nutr Sci
59, S103-S10 2013
Vitaminol
suppl
9H, Tsubota-Utsugi M, Kodama 2010: lifestage.
H, Shimizu T, Sugiyama T,
Tabata I, Tamai H, Tsutsumi C,
Hirahara F, Kuzuya M, Gomi I,
Nakamura K, Yoshida H,
Nakamura M.
Nagae A, Kuwabara A, Tozawa Enteral nutrition and antibiotic use increase e-SPEN Journal,
in press
K, Kumode M, Takeuchi Y,
the risk for vitamin K deficiency in patients
Tanaka K.
with severe motor and intellectual
disabilities.
小林奈穂, 村山伸子, 稲村雪 給食施設における「日本人の食事摂取基 栄養学雑誌
子, 久保田恵, 神田知子, 高 準」の活用の現状（第一報）―病院およ橋孝子, 石田裕美.
び介護老人保健施設を対象とした質問
紙調査―.

173

70 記
念号

印刷中

小林奈穂, 村山伸子, 稲村雪 給食施設における「日本人の食事摂取基 栄養学雑誌
子, 久保田恵, 神田知子, 高 準」の活用の現状（第二報）－高齢者施
橋孝子, 金光秀子, 辻ひろみ, 設を対象としたインタビュー調査―.
石田裕美.
孫田みなみ, 笠岡（坪山）宜 政府が策定する食事指針・ガイドにおけ 栄養学雑誌
代, 瀧沢あす香, 坪田（宇津 る食事摂取基準の活用状況.
木）恵, 今井絵理, 岡純 .
Murakami K, Sasaki S, Okubo Characteristics of under- and over-reporters J Nutr Sci
H, the Freshmen in Dieteticof energy intake among young Japanese
Vitaminol
Courses Study II Group.
Women.

印刷中

印刷中

58

253-62

2012

379

e44

2012

Tokudome S, Nishi N, Tanaka
H.

Towards a better National Health andNutrition Survey in Japan.

Shibata K, Imai E, Sano M,
Fukuwatari T.

The urinary excretory ratio of nicotinamide Biosci
catabolites was associated with the
Biotechnol
conversion ratio of tryptophan to
Biochemnicotinamide in growing rats fed a
niacin-free 20% casein diet.

76

186-1882012

Miyazaki A, Sano M,
Fukuwatari T, Shibata K.

Effects of ethanol consumption on the
B-group vitamin contents of liver, bloodand urine in rats.

Br J Nutr

1081034-1041

2012

Food Nutr Sci

3

123-1282012

善方裕美, 渡辺優奈, 上西一 妊娠初期の骨密度とライフスタイル, 栄 Osteoporosis
弘他．
養摂取状態についての検討－SKY Study Japan(Saitama, Kobe, Yokohama Pregnant
Cohort Study) 第 1 報－.

20514-5162012

神田知子, 高橋孝子, 久保田 栄養管理報告書を用いた特定給食施設
恵, 小林奈穂, 村山伸子, 齊 における食事摂取基準の活用に関する
藤陽子, 増田利隆, 河野美穂, 調査.
石田裕美.

栄養学雑誌

70

140-151

2012

吉田宗弘.

日本衛生学雑誌

67485-491

2012

Yoshida M, Fukuwatari T, Sakai Correlation between Mineral Intake andJ, Tsuji T, Shibata K.
Urinary Excretion in Free-living JapaneseYoung Women.

クロムはヒトの栄養にとって必須の微量元素だろうか？

174

Lancet70 記
念号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.

References
1.
2.
3.
4.
5.

6.

Bonjour JP (1979) Vitamins and alcoholism. I. Ascorbic acid.
Int J Vitam Nutr Res 49, 434– 441.
Bonjour JP (1980) Vitamins and alcoholism. II. Folate andvitamin B12. Int J Vitam Nutr Res 50, 96–121.
Bonjour JP (1980) Vitamins and alcoholism. III. Vitamin B6.
Int J Vitam Nutr Res 50, 215– 230.
Bonjour JP (1980) Vitamins and alcoholism. IV. Thiamin.
Int J Vitam Nutr Res 50, 321– 338.
Bonjour JP (1980) Vitamins and alcoholism. V. Riboflavin. VI.
Niacin. VII. Pantothenic acid. VIII. Biotin. Int J Vitam Nutr
Res 50, 425– 440.
Bonjour JP (1981) Vitamins and alcoholism. IX. Vitamin A.
Int J Vitam Nutr Res 51, 166– 177.

1040
7.
8.

9.
10.

11.

12.

British Journal of Nutrition

13.

14.

15.

16.

17.

18.

19.

20.
21.

22.

23.
24.

25.

26.

A. Miyazaki et al.
Bonjour JP (1981) Vitamins and alcoholism. X. Vitamin D, XI.
Vitamin E, XII. Vitamin K. Int J Vitam Nutr Res 51, 307– 318.
Leevy CM, Baker H, TenHove W, et al. (1965) B-Complex
vitamins in liver disease of the alcoholic. Am J Clin Nutr
16, 339– 346.
Leevy CM, Thompson A & Baker H (1970) Vitamins and liver
injury. Am J Clin Nutr 23, 493 – 499.
Baker H, Frank O, Ziffer H, et al. (1964) Effect of hepaticdisease on liver B-complex vitamin titers. Am J Clin Nutr
14, 1 –6.
Subramanian VS, Subramanya SB, Tsukamoto H, et al. (2010)
Effect of chronic alcohol feeding on physiologicaland molecular parameters of renal thiamin transport. Am J
Physiol Renal Physiol 299, F28 – F34.
Subramanya SB, Subramanian VS & Sain HM (2010) Chronic
alcohol consumption and intestinal thiamin absorption:effects on physiological and molecular parameters of theuptake process. Am J Physiol Gastrointest Liver Physiol
299, G23 –G31.
Hamid A & Kaur J (2007) Long-term alcohol ingestion alters
the folate-binding kinetics in intestinal brush border membrane in experimental alcoholism. Alcohol 41, 441 – 446.
Hamid A, Kaur J & Mahmood A (2007) Evaluation of the kinetic properties of the folate transport system in intestinal
absorptive epithelium during experimental ethanol ingestion. Mol Cell Biochem 304, 265 – 271.
Hamid A & Kaur J (2007) Decreased expression of transporters reduces folate uptake across renal absorptive surfaces
in experimental alcoholism. J Membr Biol 220, 69– 77.
Hamid A, Wani NA, Rana S, et al. (2007) Down-regulation of
reduced folate carrier may result in folate malabsorption
across intestinal brush border membrane during experimental alcoholism. FEBS J 274, 6317 – 6328.
Hamid A, Kiran M, Rana S, et al. (2009) Low folate transport
across intestinal basolateral surface is associated withdown-regulation of reduced folate carrier in in vivo modelof folate malabsorption. IUBMB Life 61, 236 – 243.
Hamid A & Kaur J (2009) Role of signaling pathways in
the regulation of folate transport in ethanol-fed rats. J Nutr
Biochem 20, 291 – 297.
Wani NA & Kaur J (2010) Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic
folate malabsorption in chronic alcoholism. J Cell Physiol
226, 579– 587.
Leevy CM, George WS, Ziffer H, et al. (1960) Pantothenic
acid, fatty liver and alcoholism. J Clin Invest 39, 1005.
Sorrell MF, Baker H, Barak AJ, et al. (1974) Release by ethanol of vitamins into rat liver perfusates. Am J Clin Nutr 27,
743–745.
Israel BC & Smith CM (1987) Effects of acute and chronic
ethanol ingestion on pantothenate and CoA status of rats.
J Nutr 117, 443–451.
Reeves PG (1997) Components of the AIN-93 diets asimprovements in the AIN-76A diet. J Nutr 127, 838S– 841S.
Pullman ME & Colowick SP (1954) Preparation of 2- and6-pyridones of N 1-methylnicotinamide. J Biol Chem 206,
121–127.
Shibata K, Kawada T & Iwai K (1988) Simultaneous microdetermination of nicotinamide and its major metabolites,
N 1-methyl-2-pyridone-5-carboxamide and N 1-methyl-3-pyridone-4-carboxamide, by high-performance liquid chromatography. J Chromatogr 424, 23 –28.
Shibata K, Mushiage M, Kondo T, et al. (1995) Effects ofvitamin B6 deficiency on the conversion ratio of tryptophan
to niacin. Biosci Biotechnol Biochem 59, 2060– 2063.

27.

28.

29.

30.

31.
32.

33.

34.

35.

36.

37.

38.
39.

40.

41.

42.

43.

44.
45.

46.

Fukuwatari T, Wada H & Shibata K (2008) Age-related
alterations of B-group vitamin contents in urine, blood andliver from rats. J Nutr Sci Vitaminol 54, 357– 362.
Shibata K, Fukuwatari T, Ohta M, et al. (2005) Values ofwater-soluble vitamins in blood and urine of Japaneseyoung men and women consuming a semi-purified dietbased on the Japanese Dietary Reference Intakes. J Nutr
Sci Vitaminol 51, 319– 328.
Fukuwatari T, Suzuura C, Sasaki R, et al. (2004) Action site ofbisphenol A as metabolic disruptor lies in the tryptophannicotinamide conversion pathway. J Food Hyg Soc Jpn 45,
231– 238.
Ohkawa H, Ohishi N & Yagi K (1982) A simple method
for micro-determination of flavin in human serum andwhole blood by high-performance liquid chromatography.
Biochem Int 4, 187– 194.
AOAC International (1995) Official Methods of Analysis,
16th ed. Arlington, VA: AOAC International.
Watanabe F, Abe K, Katsura H, et al. (1998) Biologicalactivity of hydroxo-vitamin B12 degradation productformed during microwave heating. J Agric Food Chem 46,
5177 –5180.
Skeggs H & Wright LD (1944) The use of Lactobacillus
arabinosus in the microbiological determination of
pantothenic acid. J Biol Chem 156, 21– 26.
Aiso K & Tamura T (1998) Trienzyme treatment for food folate
analysis. Optimal pH and incubation time for a-amylase andprotease treatment. J Nutr Sci Vitaminol 44, 361– 370.
Fukui T, Iinuma K, Oizumi J, et al. (1994) Agar plate method
using Lactobacillus plantarum for biotin determination inserum and urine. J Nutr Sci Vitaminol 40, 491– 498.
Gregory JF 3rd & Kirk JR (1979) Determination of urinary
4-pyridoxic acid using high performance liquid chromatography. Am J Clin Nutr 32, 879– 883.
Thomson AD, Baker H & Leevy CM (1970) Patterns of
35
S-thiamine hydrochloride absorption in the malnourishedalcoholic patients. J Lab Clin Med 76, 34–45.
Leevy CM & Baker H (1968) Vitamins and alcoholism.
Am J Clin Nutr 21, 1325– 1328.
Pearson WN (1967) Blood and urinary vitamin levels asa potential indices of body stores. Am J Clin Nutr 20,
514– 525.
Neal RA & Pearson WN (1964) Studies of thiamine
metabolism in the rat. II. Isolation and identification of
2-methyl-4-amino-5-pyridinecarboxylic acid as a metaboliteof thiamine in rat urine. J Nutr 83, 351– 357.
Suzuoki Z, Tominaga F, Matsuo T, et al. (1968) Metabolismof thiamine and thiamine tetrahydrofurfuryl disulfide to
4-methylthiazole-5-acetic acid in conventional and germfree
rats under various dosing conditions. J Nutr 96, 433–444.
Amos WH & Neal RA (1970) Isolation and identification of
3-(20 -methyl-40 -amino-50 -pyrimidylmethyl)-4-methylthiazole5-acetic acid (thiamine acetic acid) and 2-methyl-4-amino5-formylaminomethylpyrimidine as metabolites of thiaminein the rat. J Biol Chem 245, 5643 – 5648.
Shibata K & Onodera M (1992) Changes in the conversion
rate of tryptophan-nicotinamide according to dietary fat
and protein levels. Biosci Biotechnol Biochem 56,1104 –1108.
Frank O, Baker H & Leevy CM (1964) Vitamin-binding
capacity of experimentally injured liver. Nature 203, 302– 303.
Collins TD, Eisenga BH, Bhandari SD, et al. (1992) Effects ofethanol on tissue folate incorporation and recovery fromfolate deficiency in rats. Alcohol Clin Exp Res 16, 757 –763.
Tamura H & Halsted CH (1983) Folate turnover inchronically alcoholic monkeys. J Lab Clin Med 101, 623– 628.

B-group vitamin contents in ethanol-fed rats
47.

48.
49.

British Journal of Nutrition

50.

McMartin KE (1984) Increased urinary folate excretion anddecreased plasma folate levels in the rat after acute ethanol
treatment. Alcohol Clin Exp Res 8, 172 –178.
Eichner ER & Hillman RS (1973) Effect of alcohol on serum
folate level. J Clin Invest 52, 584 – 591.
Paine CJ, Eichner ER & Dickson V (1973) Concordance ofradioassay and microbiological assay in the study of theethanol-induced fall in serum folate level. Am J Med Sci
266, 134– 138.
McMartin KE & Collins TD (1983) Role of ethanol metabolism in the alcohol-induced increase in urinary folate
excretion in rats. Biochem Pharmacol 32, 2549 –2555.

51.

52.

53.

1041

McMartin KE, Collins TD & Bairnfather L (1986) Cumulative
excess urinary excretion of folate in rats after repeated ethanol treatment. J Nutr 116, 1316– 1325.
McMartin KE, Collins TD, Eisenga BH, et al. (1989) Effects ofchronic ethanol and diet treatment on urinary folate
excretion and development of folate deficiency in the rat.
J Nutr 119, 1490 –1497.
Romanoff RL, Ross DM & McMartin KE (2007) Acute ethanol
exposure inhibits renal folate transport, but repeatedexposure upregulates folate transport proteins in rats andhuman cells. J Nutr 137, 1260 –1265.

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
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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
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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

REFERENCES[1]

W. Wilett, “Nutritional Epidemiology,” 2nd Edition, Oxford University Press, New York, 1998.

[2]

S. A. Bingham, C. Gill, A. Welch, A. Cassidy, S. A.
Runswick, S. Oakes, R. Lubin, D. I. Thurnham, T. J. Key,
L. Roe, K. T. Khaw and N. E. Day, “Validation of Dietary Assessment Methods in the UK Arm of EPIC Using
Weighed Records, and 24-Hour Urinary Nitrogen andPotassium and Serum Vitamin C and Carotenoids as Biomarkers,” International Journal of Epidemiology, Vol. 26,
Suppl. 1, 1997, pp. S137-S151.
doi:10.1093/ije/26.suppl_1.S137

127

Suzuki, “Sensitivity and Specificity of Published Strategies Using Urinary Creatinine to Identify Incomplete 24-h
Urine Collection,” Nutrition, Vol. 24, No. 1, 2008, pp.
16-22. doi:10.1016/j.nut.2007.09.001[13] T. Imai, S. Sasaki, K. Mori, F. Ando, N. Niino and H.
Shimokata, “Nutritional Assessment of 3-Day Dietary
Records in National Institute for Longevity ScienceLongitudinal Study of Aging (NILS-LSA),” Journal of
Epidemiology, Vol. 10, Suppl. 1, 2000, pp. S70-S76.
doi:10.2188/jea.10.1sup_70[14] K. Murakami, H. Okubo and S. Sasaki, “Dietary Intake in
Relation to Self-Reported Constipation among Japanese
Women Aged 18 - 20 Years,” European Journal of Clinical Nutrition, Vol. 60, No. 5, 2006, pp. 650-657.
doi:10.1038/sj.ejcn.1602365[3]

M. B. E. Libingstone and A. E. Black, “Markers of the
Validity of Reported Energy Intake,” Journal of Nutrition,
Vol. 133, Suppl. 3, 2003, pp. 895S-920S.

[4]

S. A. Bingham, “Urine Nitrogen as a Biomarker for the
Validation of Dietary Protein Intake,” Journal of Nutrition, Vol. 133, Suppl. 3, 2003, pp. 921S-924S.

[15] Ministry of Education, Culture, Sport, Science and Technology of Japan, “Standard Tables of Food Compositionin Japan 5th Revised and Enlarged Edition (in Japanese),”National Printing Bureau of Japan, Tokyo, 2007.

[5]

C. Luceri, G. Caderni, M. Lodovici, M. T. Spagnesi, C.
Monserrat, L. Lancioni and P. Dolara, “Urinary Excretionof Sucrose and Fructose as a Predictor of Sucrose Intake
in Dietary Intervention Studies,” Cancer Epidemiology,
Biomarkers and Prevention, Vol. 5, No. 3, 1996, pp. 167171.

[16] Y. Miyazaki, H. Koyama, Y. Sasada, H. Sato, M. Nojiri
and S. Suzuki, “Dietary Habits and Selenium Intake of
Residents in Mountain and Coastal Communities in Japan,” Journal of Nutritional Science and Vitaminology,
Vol. 50, No. 5, 2004, pp. 309-319.
doi:10.3177/jnsv.50.309[6]

N. Tasevska, S. A. Runswick, A. A. Welch, A. McTaggart and S. A. Bingham, “Urinary Sugars Biomarker
Relates Better to Extrinsic than to Intrinsic Sugars Intake
in a Metabolic Study with Volunteers Consuming Their
Normal Diet,” European Journal of Clinical Nutrition,
Vol. 63, No. 5, 2009, pp. 653-659.
doi:10.1038/ejcn.2008.21[17] H. Hattori, A. Ashida, C. Itô and M. Yoshida, “Determination of Molybdenum in Foods and Human Milk, and an
Estimate of Average Molybdenum Intake in the Japanese
population,” Journal of Nutritional Science and Vitaminology, Vol. 50, No. 6, 2004, pp. 404-409.
doi:10.3177/jnsv.50.404[7]

N. Tasevska, S. A. Runswick, A. McTaggart and S. A.
Bingham, “Twenty-Four-Hour Urinary Thiamine as a
Biomarker for the Assessment of Thiamine Intake,” European Journal of Clinical Nutrition, Vol. 62, No. 9, 2008,
pp. 1139-1147. doi:10.1038/sj.ejcn.1602829

[8]

[9]

N. Tasevska, S. A. Runswick and S. A. Bingham, “Urinary Potassium Is as Reliable as Urinary Nitrogen for Use
as a Recovery Biomarker in Dietary Studies of Free Living Individuals,” Journal of Nutrition, Vol. 136, No. 5,
2006, pp. 1334-1340.
D. Ristic-Medic, Z. Piskackova, L. Hooper, J. Ruprich, A.
Casgrain, K. Ashton, M. Pavlovic and M. Glibetic, “Methods of Assessment of Iodine Status in Humans: A Systematic Review,” American Journal of Clinical Nutrition,
Vol. 89, No. 6, 2009, pp. 2052S-2069S.
doi:10.3945/ ajcn.2009. 27230H[10] L. K. Dahl, “Evidence for an Increased Intake of Sodiumin Hypertension Based on Urinary Excretion of Sodium,”
Proceedings of Society of Experimental Biology and Medicine, Vol. 94, No. 1, 1957, pp. 23-26.
[11] Y. Morinaga, T. Tsuchihashi, Y. Ohta and K. Matsumura,“Salt Intake in 3-Year-Old Japanese Children,” Hypertension Research, Vol. 34, No. 7, 2011, pp. 836-839.
doi:10.1038/hr.2011.55[12] K. Murakami, S. Sasaki, Y. Takahashi, K. Uenishi, T.
Watanabe, T. Kohri, M. Yamasaki, R. Watanabe, K. Baba,
K. Shibata, T. Takahashi, K. Hayabuchi, K. Ohki and J.
Copyright © 2012 SciRes.

[18] Ministry of Health, Labour and Welfare of Japan, “TheNational Health and Nutrition Survey in Japan, 2008 (inJapanese),” Dai-Ichi Shuppan, Tokyo, 2011.
[19] S. Horiguchi, K. Teramoto, T. Kurono and K. Ninomiya,“The Arsenic, Copper, Lead, Manganese and Zinc Contents of Daily Foods and Beverages in Japan and the Estimate of Their Daily Intake,” Osaka City Medical Journal, Vol. 24, No. 1, 1978, pp. 131-141.
[20] M. Yoshida, H. Hattori, S. Ota, K. Yoshihara, N. Kodama,Y. Yoshitake and M. Nishimuta, “Molybdenum Balancein Healthy Young Japanese Women,” Journal of Trace
Elements in Medicine and Biology, Vol. 20, No. 4, 2006,
pp. 245-252. doi:10.1016/j.jtemb.2006.07.004[21] M. Yoshida and K. Yasumoto, “Selenium Content of Rice
Grown at Various Sites in Japan,” Journal of Food Composition and Analysis, Vol. 1, No. 1, 1987, pp. 71-75.
doi:10.1016/0889-1575(87)90013-5[22] M. Nishimuta, N. Kodama, E. Morikuni, Y. H. Yoshioka,
H. Takeyama, H. Yamada, H. Kitajima and K. Suzuki,“Balances of Calcium, Magnesium and Phosphorus in Japanese Young Adults,” Journal of Nutritional Scienceand Vitaminology, Vol. 50, No. 1, 2004, pp. 19-25.
doi:10.3177/jnsv.50.19[23] C. A. Swanson, D. C. Reamer, C. Veillon, J. C. King and
O. A. Levander, “Quantitative and Qualitative Aspects ofSelenium Utilization in Pregnant and Nonpregnant Women: An Application of Stable Isotope Methodology,”
American Journal of Clinical Nutrition, Vol. 38, No. 2,
FNS

128

Correlation between Mineral Intake and Urinary Excretion in Free-Living Japanese Young Women
1983, pp. 169-180.

[24] H. J. Robbrecht and H. A. Deelstra, “Selenium in Human
Urine: Concentration Levels and Medical Implications,”
Clinica Chimica Acta, Vol. 136, No. 2-3, 1984, pp. 107120. doi:10.1016/0009-8981(84)90282-1[25] N. M. Lewis, M. S. Marcus, A. R. Behling and J. L. Greger, “Calcium Supplements and Milk: Effects on AcidBase Balance and on Retention of Calcium, Magnesium,and Phosphorus,” American Journal of Clinical Nutrition,
Vol. 49, No. 3, 1989, pp. 527-533.
[26] Food and Nutrition Board Institute of Medicine, “Iron,”
Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc,

Copyright © 2012 SciRes.

National Academy Press, Washington DC, 2001, pp. 290393.
[27] J. R. Turnlund, “Copper,” In: M. E. Shils, J. A. Olson, M.
Shike and A. C. Ross, Eds., Modern Nutrition in Healthand Disease, 9th Edition, Wikkiams & Wilkins, Baltimore, 1999, pp. 241-252.
[28] J. C. King and C. L. Keen, “Zinc,” In: M. E. Shils, J. A.
Olson, M. Shike and A. C. Ross, Eds., Modern Nutrition
in Health and Disease, 9th Edition, Wikkiams & Wilkins,
Baltimore, 1999, pp. 223-239.
[29] T. Hazell, “Minerals in Foods: Dietary Sources, ChemicalForms, Interactions, Bioavailability,” World Review ofNutrition and Dietetics, Vol. 46, 1985, pp. 1-123.

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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

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e

e

e

e

e

・騰帯血採血・頭蓋ろう検査e

図1

SKY（Saitama，

表1

初産・経産での比較

i52名）

o産婦
i108名）

@ρ
表2

e

e

e

・母乳分泌調査

初産婦

e

e
e

e

Kobe，

Y（）kohama

図2

食：物摂取頻度調査

におけるカルシウ
ム不足者の割合●●●●●・檎恥骨密度（DXA）

e

●●

e

e

産後1年

●●

：讐繧タイル調査｝・

粉乳

授乳期

●●

・踵骨骨密度（QUA） ●・静脈血採血
●

産後6ヵ月

産後3ヵ月

産後ーカ月

分娩時

妊娠後期

妊娠中期

妊娠初期

妊娠期

37

Pregnant

Cohort）Studyのデザイン

年齢標準化SOS 総エネルギーi歳）@（m／sec）@（kcay日）

カルシウム

ビタミンD

ビタミンK

im拶日）

iμ拶日）

iμ疹日）

UVなし
叝ﾆ時間
i時間／，月）30．3±3．51536±27．2

1629±322

414±136．89±2．5

205±12。910。9±19．5

R2．8±3．5

P527±23．5

P673±278

S21±144．0

X±2．0

Q04±91．0P5．9±33．8

T．6E－0．5

O．04487

O．59307@0．61899

O．38301

O．61894

O．33364

カルシウム

ビタミンD

ビタミンK

imgt日）

iμ91日）

iμ9／日）

35歳以上・35歳未満での比較

35歳未満

年齢

標準化SOS 総エネルギーi歳）@（m／sec）@（kcay日）

UVなし
叝ﾆ時間
i時間／月）

30．3±2．71533±271602±310

423±1469±2

207±10614．1±22．0

R5歳以．ヒi48名）

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P631±244

S08±134

X±2

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Q．2E－28

O．0284@0．579@0．551

O，875@0．552@0．939i112名）

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

4

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

no．3

Presented by Medical*Online

2012

139（515）38

第22回婦人科骨粗霧症研究会

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vol．20

no．3

Presented by Medical*Online

2012

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