１．発表論文

H 22 年度（2010 年度）

1.

Shibata K, Suzuki S, Sato J, Ohsawa I, Goto S, Hashiguchi M and Tokudome S. Abdominal
circumference should not be required criterion for the diagnosis of metabolic syndrome. Environ
Health Prev Med. 2010; 15: 229-235.

2.

Yamada T, Fukatsu M, Suzuki S, Yoshida T, Tokudome S, and Joh T. Alcohol drinking may not
be a major risk factor for fatty liver in Japanese undergoing a health checkup. Dig Dis Sci 2010;
55: 176-182.

3.

佐々木敏. 特集：日本人の食事摂取基準（2010年版）策定の考え方. 日本人の食事摂取基準（2010年版）の策定の概要. 静脈経腸栄養 2010; 25: 763-768.

4.

Murakami K, Sasaki S, Takahashi Y, Uenishi K; Japan Dietetic Students' Studyfor Nutrition and Biomarkers Group. Neighborhood socioeconomic status in relation to dietary intake and insulin resistance syndrome in female Japanese dietetic students. Nutrition. 2010; 26(5):508-514.

5.

森田明美. ライフステージ別の栄養 高齢者. 食品保健の科学. 2010; 145-149.

6.

Uenishi K, Nakamura K. Intake of dairy products and bone ultrasound measurement in lateadolescents: a nationwide cross-sectional study in Japan. Asia Pac J Clin Nutr. 2010; 19(3):432-439.

7.

Uenishi K, Fujita T, Ishida H (他6人). Fractional absorption of active absorbable algal calcium(AAACa) and calcium carbonate measured by a dual stable-isotope method. Nutrients. 2010; 2:752-761.

8.

Tsubota-Utsugi M, Ohkubo T, Kikuya M (他９人). High Fruit Intake is associated with Lower
Risk of Future Hypertension determined by Home Blood Pressure Measurement: The OHASAMA
study. J Hum hypertens. 2001; Mar;25(3):164-171

9.

野末 みほ, Jun Kyungyul, 石原 洋子, 武田 安子, 永井 成美, 由田 克士, 石田 裕美. 小学5年生の学校給食のある日とない日の食事摂取量と食事区分別の比較. 栄養学雑誌. 2010;
68(5): 298-308.

10. 冨澤 真美, 須藤 美智子, 木村 誠知子, 平野 美知, 石川 俊次, 石田 裕美. 事業所給食施設におけるヘルシーメニューの給与エネルギー量. 栄養学雑誌. 2010; 68(4): 270-275.
11. Kuwabara A, Himeno M, Tsugawa N, Kamao M, Fujii M, Kawai N, Fukuda M, Ogawa Y, Kido S,
Okano T, Tanaka K. Hypovitaminosis D and K are highly prevalent and independent of overallmalnutrition in the institutionalized elderly. Asia Pac J Clin Nutr. 2010; 19(1): 49-56.
12. Kuwabara A, Fujii M, Kawai N, Tozawa K, Kido S, Tanaka K. Bone is more susceptible to vitamin
K deficiency than liver in the institutionalized elderly. Asia Pac J Clin Nutr. 2011; 20(1): 50-55.
13. 田中清, 桒原晶子. ビタミン K. 総合臨床. 2010; 59: 594-598.
14. Yoshida M, Hatakeyama E, Hosomi R, Kanda S, Nishiyama T, Fukunaga K. Tissue accumulation
and urinary excretion of chromium in rats fed diets containing graded levels of chromium chloride
or chromium picolinate. J Toxicol Sci. 2010; 35(4): 485-491.
15. 吉田宗弘. 日本人の微量ミネラルの食事摂取基準（2010）. ビタミン. 2010; 84: 240-245.

２．学会発表

H 22 年度（2010 年度）

上西一弘他、ダブルアイソトープ法による閉経後女性のカルシウム吸収率の検討．2010 年 10 月 23
日．第 12 回日本骨粗鬆症学会（大阪）432

Asia Pac J Clin Nutr 2010;19 (3):432-439

Original Article

Intake of dairy products and bone ultrasoundmeasurement in late adolescents: a nationwide crosssectional study in Japan
Kazuhiro Uenishi PhD1, Kazutoshi Nakamura MD2
1

Laboratory of Physiological Nutrition, Kagawa Nutrition University, Saitama, Japan
Department of Community Preventive Medicine, Niigata University, Graduate School of Medical and Dental
Sciences, Niigata, Japan

2

Introduction: There is little evidence regarding the effects of dairy product intake on bone mineralization amonglate adolescents, especially in Asians. The aim of this study was to determine the association between dairy
product intake and bone strength as measured by quantitative ultrasound (QUS) in a large Japanese population.
Methods: Subjects were 38,719 high school students (14,996 males and 23,723 females) across 33 prefectures inJapan. Bone stiffness of the calcaneus was measured by QUS densitometry (AOS-100, Aloka). Subjects were
given a self-administered questionnaire, which included questions on gender, age, height, weight, consumptionof dairy products, and levels of physical activity. Intake of milk and yogurt were classified as none, 1-99, 100199, 200-399, and ≥400 ml/day. Results: The proportion of subjects who consumed milk 400 ml/day or morewas 21% in males and 7.3% in females, while 24% of males and 41.1% of females did not consume milk. After
adjusting for physical activity, weight, gender, age, and area of residence, milk intake (R2=2.8%, p<0.001) andyogurt intake (R2=0.1%, p<0.001) were independently associated with the QUS measurement. Similar associations were found in males and females when a gender-stratified analysis was conducted. Conclusion: We found apositive dose-effect relationship between milk intake and bone strength in late adolescents, to whom we recommend milk intake of 400 ml/day or more to obtain greater bone mass.

Key Words: adolescent, bone density, calcium, dairy products, quantitative ultrasound measurement

INTRODUCTION
Dairy products, especially milk from cows, are an important source of calcium (Ca), which is essential for maintaining normal skeletal growth in children and adolescents. Many observational and interventional studies haveshown that adequate Ca intake is required to attain maximal peak bone mass.1,2 In addition, several researchers
have demonstrated a favorable effect of increased milk
and other dairy intake on bone parameters.3-8 However,
Lanou et al.9 conducted a systematic review that revealed
insufficient evidence to support the idea that increased
intake of milk or other dairy products has a favorable
effect on promoting bone mineralization in children andadolescents. They also pointed out that such evidence isscarce in non-Caucasian children and adolescents.
Dairy products are not popular in Japan. However, almost all elementary schools and most junior high schools
provide students with a bottle of milk (200 ml) as part ofthe school lunches, which is supervised by a registereddietician. This enables students to maintain a relatively
high Ca intake. The median Ca intake of students in theage groups 6-8, 9-11, and 12-14 years old were 614, 706,
and 788 mg/day in males and 586, 659, and 656 mg/day
for females, respectively.10 However, high school students
are rarely provided with school lunch (i.e., milk) and,
therefore, their Ca intake is lower. The median Ca intake

of students 15-17 years old was 570 mg/day in males and467 mg/day for females.10
Despite ample evidence regarding the positive effectof increased Ca intake on bone mass in pre- and peripubertal children, less is known about the influence of Ca
intake on bone mineral accretion in the late teenage
years.11 We hypothesized that increased Ca intake fromdairy products is important for Japanese high school students whose Ca intake is low. The aim of this study was
to determine the association between intake of dairy
products and bone strength, as measured by quantitative
ultrasound (QUS) in a large population of late Japaneseadolescents.
MATERIALS AND METHODS
Subjects
We targeted high school students aged 15-18 years. We

Corresponding Author: Dr Kazuhiro Uenishi, Laboratory ofPhysiological Nutrition, Kagawa Nutrition University, 3-9-21
Chiyoda, Sakado City, Saitama 350-0288, Japan.
Tel: +81-49-284-3895; Fax: +81-49-284-3895
Email: uenishi@eiyo.ac.jp
Manuscript received 6 February 2010. Initial review completed
22 April 2010. Revision accepted 10 June 2010.

K Uenishi and K Nakamura
contacted 33 prefectures (of 47 prefectures) in Japan andinvited high schools to join our study. In total, 236 highschools participated on a voluntary basis. Among the targeted 40,111 students, 38,719 students (96.5%, 14,996
males and 23,723 females) agreed to participate in thisstudy. This study was conducted according to the guidelines established by the Declaration of Helsinki and allprocedures involving human subjects were approved by
the Ethics Committee of Kagawa Nutrition University.
Written informed consent was obtained from all subjects.
QUS measurements and surveys using self-administeredquestionnaires were conducted in each high school fromsummer of 2006 to fall of 2007.
QUS Measurement
The strength of the calcaneus, or heel bone, of the rightfoot was evaluated with QUS densitometry using AOS100 (Aloka, Tokyo). A QUS device measures elasticity
and bone strength, which is dependent on both bone massand bone architecture. The AOS-100 measures the speedof sound (SOS; in m/second) and the transmission index(TI). TI values are related to frequency-dependent attenuation. SOS and TI values measured with the AOS100 are highly correlated with the SOS (r=0.89) and433

broadband ultrasound attenuation (BUA) (r=0.88) measured with a conventional QUS device (UBA575+, HologicInc.).12 Osteo sono-assessment index (OSI), which is an
estimate of elastic modules equal to the product of physical density and the square of SOS, was calculated as a
combined parameter of SOS and TI by multiplying TI by
the square of SOS.12 OSI showed better reproducibility
than BUA. The validity of the present QUS measurement
has been previously published and coefficients of variation of SOS, TI, and OSI measurements were 0.3%, 1.2%,
and 1.6%, respectively, 12 which is within the standard
value provided by the manufacturer.
Demographic and Lifestyle Characteristics
The self-administered questionnaire included questions
regarding the subject’s gender, age, height, weight, current
consumption of dairy products, and current physical activity level. Subjects reported their height and weight tothe nearest 1 cm and 1 kg, respectively, and body massindex (BMI) was calculated by dividing weight (kg) by
the square of height (m2). Intake of milk and yogurt wasclassified as none, 1-99 ml/day, 100-199 ml/day, 200-399
ml/day, and ≥400 ml/day; the intake of cheese was classified as none, 1-19 g/day, 20-39 g/day, 40-59 g/day, andTable 1. Subject characteristics
Mean or Proportion
Male (n=14,996)

Female (n=23,723)

p value

16.3 (SD 0.9)
170.2 (SD 5.9)
61.4 (SD 10.6)
21.2 (SD 3.2)
1577 (SD 174)
3.06 (SD 0.40)

16.4 (SD 0.9)
157.8 (SD 5.4)
51.5 (SD 7.7)
20.7 (SD 2.8)
1592 (SD 164)2.88 (SD 0.35)<0.001<0.001<0.001<0.001<0.001<0.001

24.0%
14.4%
14.8%
25.8%
21.0%

41.1%
19.1%
15.4%
17.2%
7.3%

<0.001χ2=2597

43.0%
35.0%
14.2%
5.8%
2.0%

37.7%
38.7%
17.2%
5.0%
1.3%

<0.001χ2=193

62.0%
19.9%
9.6%

61.8%
21.3%
10.4%

<0.001
χ2=78≥40 to <60

4.1%

3.5%

≥60

4.4%

3.0%

36.1%
10.6%
10.3%
43.0%

61.4%
11.3%
6.3%
21.0%

Age (years)
Height (cm)Weight (kg)Body mass index (kg/m2)
Speed of sound (m/sec)
Osteo sono-assessment index (106)
Milk intake (ml/day)0>0 to <100≥100 to <200≥200 to <400
≥400
Yogurt intake (ml/day)0>0 to <100≥100 to <200≥200 to <400
≥400
Cheese intake (g/day)0>0 to <20
≥20 to <40

Frequency of exercise (times/week)
≤1
2- 3
4- 5
≥6

<0.001χ2=2836

Dairy products and bone health in adolescents434≥60 g/day. These classifications are easy to understandfor most Japanese because a bottle or a pack of milk has
historically corresponded to approximately 200 ml, a cupof yogurt has corresponded to approximately 100 ml, anda standard serving size of cheese corresponds to 20 g. As
for physical activity, subjects were asked to report thefrequency of their current, weekly exercise regime.
Statistical analysis
All continuous variables were checked for normality.
Weight and BMI were skewed to higher values, and weretransformed logarithmically prior to conducting statisticaltests. Student’s t-test was used to test for differences between two groups. The chi-square test was used to test forindependence of categorical data. Regression analyses
were used to assess the linear association between predictor
variables and the QUS value. In multiple regression analyses, exercise habit, body statue, gender, age, and area ofresidence (either in Tohoku, Kanto, Chubu, Kinki, Chugoku, Shikoku, or Kyushu area in Japan) were considered
as covariates. The parameter “area” was treated as adummy variable. SAS statistical package (version 9.13,
SAS Institute, Cary, NC, USA) was used for all analyses.

A p-value <0.05 was considered significant.
RESULTS
Table 1 shows the characteristics of all subjects. Meanvalues of age, height, weight, BMI, and OSI were significantly different between males and females. Males had
higher milk intake and exercised more frequently thanfemales. There was a slight but significant difference inyogurt and cheese intake between males and females.
Table 2 shows the results of simple linear regression
analyses with OSI as the outcome. All predictor variables
were significantly associated with OSI. Weight was thestrongest predictor of OSI, followed by exercise frequency, BMI, gender, milk intake, and age. Table 2 also
shows results according to gender. Figure 1 illustrates
mean OSI by levels of milk intake. The mean OSI of themilk intake groups were higher than the no milk intake
group, by 0.7% in the 1-99ml-group, 2% in the 100199ml-group, 3.8% in the 200-399ml-group, and 6.7% inthe ≥400ml-group. Figure 2 illustrates mean OSI by levels of milk intake according to gender. The pattern ofassociation between OSI and milk intake was similar between genders.

Table 2. Simple linear regression analyses with OSI as the dependent variable by gender
Regression coefficient (b)R2 (%)

p-value

0.044

2.8

<0.001

Yogurt intake*

0.025

0.4

<0.001†

Cheese intake

0.01..

0.1<0.001Frequency of exercise‡

0.075

6.8

<0.001

Weight (kg)§

0.746

11.2

<0.001

Body mass index (kg/m2)§

0.709

6

<0.001

Gender (1, male; 0, female)

0.184

5.6

<0.001Age (years)

0.043

1.1

<0.001

Milk intake*

0.034

1.6

<0.001

Yogurt intake*

0.028

0.5

<0.001†

Cheese intake

0.009

0.1

0.003

Frequency of exercise‡

0.048

2.7

<0.001

Predictor variable
Total
Milk intake*

Males

Weight (kg)§

0.587

5.6

<0.001

Body mass index (kg/m2)§

0.64..

5.1<0.001Age (years)

0.074

3.1

<0.001

Females
Milk intake*

0.027

1.1

<0.0010.026

0.5<0.0010.008

0.1<0.001

Yogurt intake*†Cheese intake‡*

Frequency of exercise

0.071

6.2

<0.001

Weight (kg)§

0.689

7.6

<0.001

Body mass index (kg/m2)§

0.676

5.8

<0.001Age (years)

0.029

0.6

<0.001

Coded as 1: 0 ml/day; 2: >0 to <100 ml/day; 3: ≥100 to <200 ml/day; 4: ≥200 to <400 ml/day; 5: ≥400 ml/day
Coded as 1: 0 g/day; 2: >0 to <20 g/day; 3: ≥20 to <40 g/day; 4: ≥40 to <60 g/day; 5: ≥60 g/day‡
Coded as 1: ≤1 time/week; 2: 2-3 times/week; 3: 4-5 times/week; 4: ≥6 times/week§
Logarithmically transformed†

K Uenishi and K Nakamura

435

Osteo sono-assessment index (106)

3.6
3.4
3.2
3.0
2.8
2.6
2.4
0

1-99

100-199

200-399

400-

Milk intake (ml/day)

Osteo sono-assessment index (106)Figure 1. Mean osteo sono-assessment index (OSI) by levels of milk intake. Mean OSIs in milk intake groups were higher than the nomilk intake group.

3.6

3.6

3.4

3.4

3.2

3.2

3.0

3.0

2.8

2.8

2.6

2.6

2.4

0

1-99

100-199

200-399

400-

Milk intake (ml/day)

2.4

0

1-99

100-199

200-399

400-

Milk intake (ml/day)Figure 2. Mean osteo sono-assessment index (OSI) by levels of milk intake in males (left) and females (right). The pattern of association
between OSI and milk intake was similar between genders, with R2 vales of milk intake being 1.6 % for males and 1.1% for females.

Table 3 shows the results of multiple regression analyses. In this multivariate model, we included weight as theindex of body statue, but did not include BMI because
BMI was highly correlated with weight (r=0.821). After
adjusting for exercise frequency, weight, gender, age, andarea of residence, regression analyses indicated that milk
and yogurt intake were independently associated with
OSI, while cheese intake was not. The interaction of milk
intake and exercise frequency was not significant(p=0.424) when the interaction term was added to themultivariate model. Table 3 shows these results according
to gender. R2 values of milk intake in males (1.6%) andfemales (1.1%) were lower than that in males and females
combined (2.8%).
DISCUSSION
The relationship between Ca intake and bone health inchildren and adolescents is an area of interest for clinicians and epidemiologists. Most recently, Winzenberg etal.13 demonstrated in their meta-analysis of randomized
clinical trials that Ca supplementation has a small effecton the bone mineral density (BMD) of upper arm limbs
and total body, but no effect on hip or vertebral BMD.
Their study, however, has several limitations. The base-line Ca intake in most studies enrolled in the metaanalysis ranged from moderate to high. Another metaanalysis has revealed that Ca supplementation among
subjects with low Ca intake has a positive effect on bonemineral content (BMC), much larger than that seenamong children with normal Ca intake.14 Therefore, it isimportant to demonstrate this association in low-Caintake populations. The present study did not evaluate
total Ca intake. However, the results of the National
Health and Nutrition Survey can be applied to the present
study because we used a nationwide sample. The 2004
National Health and Nutrition Survey reported that themedian Ca intake among 15-17 year old adolescents was
570 mg/day in males and 467 mg/day in females, which
are both lower than the recommended dietary reference
intakes for Japanese of 1100 mg/day and 850 mg/day,
respectively.15 The subjects in the present study represent
a low Ca intake population.
The present study demonstrated a clear dose-effect relationship between milk intake and calcaneal QUS values
in adolescents aged 15-18 years, thereby demonstrating
the importance of milk for bone health in Japanese adolescents. Specifically, milk intake of 400 ml/day or moreis recommended to attain maximal peak bone mass. Sev-

Dairy products and bone health in adolescents

436

Table 3. Multiple linear regression analyses with OSI as the dependent variable by gender
Predictor variable*

Regression coefficient (b)R2 (%)

p-value2

Total

Overall R =20.0†

Milk intake

0.016

2.8

<0.001

Yogurt intake†

0.011

0.1

<0.0010.00005

0

0.978

0.064

5.3

<0.001

Weight (kg)

0.599

8.1

<0.001

Gender (1, male; 0, female)

0.035

0.1

<0.001Age (years)

0.052

1.7

<0.001

Cheese intake‡
Frequency of exercise

§

¶

2

Males

Overall R =15.2

Milk intake†

0.02

1.6

<0.001†

Yogurt intake

0.01

0.2

0.002

Cheese intake‡-0.003

0

0.372

0.059

1.9

<0.001

Weight (kg)

0.512

5.1

<0.001Age (years)

0.082

3.7

<0.001§Frequency of exercise¶

2

Overall R =16.4

Females
Milk intake†

0.013

1.1

<0.001†

Yogurt intake

0.012

0.2

<0.001

Cheese intake‡

0.002

0

0.453

0.07

5.5

<0.001§Frequency of exercise¶

Weight (kg)

0.659

7

<0.001Age (years)

0.033

0.9

<0.001*Dummy variable of “area of residence” is also included in the multivariate model in addition to the variables shown in this table†
Coded as 1: 0 ml/day; 2: >0 to <100 ml/day; 3: ≥100 to <200 ml/day; 4: ≥200 to <400 ml/day; 5: ≥400 ml/day‡
Coded as 1: 0 g/day; 2: >0 to <20 g/day; 3: ≥20 to <40 g/day; 4: ≥40 to, <60 g/day; 5: ≥60 g/day§
Coded as 1: ≤1 time/week; 2: 2-3 times/week; 3: 4-5 times/week; 4: ≥6 times/week¶
Logarithmically transformed

eral studies have tried to determine the association between milk intake and bone mass in adolescents. In one
study, dairy food supplements, including 300-400 mg/dayof Ca, increased the bone mass of various bone sites
among Caucasian adolescents.3,5 Matkovic et al.16 showedthat Ca and dairy products influence bone mass acquisition in 15-18 year old females, leading to a higher peakbone mass. They also determined that Ca exerts its action
on bone accretion during growth, primarily by influencing volumetric bone mineral density, while dairy products
may have an additional impact on bone growth and periosteal bone expansion. One study has specifically been
conducted among East Asians, and showed that the highmilk intake group (128 mg/day of milk) had significantly
higher BMC at the distal radius than the no-milk intake
group of Chinese girls aged 12-14 years.6 These previous
studies are all in line with the results of our study.
Studies conducted on the association between Ca intake and bone mass in late teens are much fewer than in
pre- and peripubertal children.11,13,14 Two interventional
studies targeting late adolescents showed that Ca supplementation was effective for bone mass accrual.17,18 An
observational study using a QUS device showed that Ca
intake is associated with calcaneal QUS values in American girls aged 14-18 years.19 All of these studies, includ-ing the present study, suggest that a favorable effect of Ca
and dairy foods on bone mass or bone growth is consistently observed in late adolescents.
Yogurt intake was also associated with QUS values
independently, but its contribution was small (R2=0.1%),
while cheese intake was not associated with QUS. Thisfinding can be explained partly by the fact that intake of
yogurt and cheese was less than that of milk. In general,
dairy products do not appear to be common foods in the
dietary habits of the Japanese. Yogurt was not consumed
by 40% of the subjects in this study, and cheese was notconsumed by 60%. Furthermore, milk was not consumed
in 40% of female subjects. The National Health and Nutrition Survey demonstrated that dairy products only account for 30% of total Ca intake. 10 Low Ca intake due tolow dairy product intake is common in Asians.6,20 Therefore, increased intake of dairy products may be an effective strategy to maximize peak bone mass in late Asianadolescents.
The prevalence of lactose intolerance in the Japanese,like that in other Eastern Asians,6 is high; the prevalencein Japanese high school students is reported to be 33.8%.21
As the relatively lower consumption of dairy products inJapanese girls may be due in part to the symptoms of lactose intolerance, strategies other than simply encouraging

K Uenishi and K Nakamura
the consumption of dairy products may be needed to increase their calcium intake.
Physical activity has been shown to be another key
factor associated with bone mass in late adolescents.19,22,23
In the present study, physical activity was found to be amore influential factor on QUS values than milk intake,especially for females. This finding was partly explainedby the fact that many subjects exercised frequently; 43%of males and 21% of females reported exercising six ormore times a week. Many high school students in Japanenthusiastically participate in school sports club activities,
and thus both the frequency and intensity of their physicalactivity is considered to be high.
This study has many strengths. The sample size of thisstudy was large, such that we had sufficient statisticalpower to detect a possible association between dairy
product intake and QUS values. Furthermore, subjects
were recruited from various regions of Japan, whichmakes the results generalizable to all Japanese late adolescents. This study also has several limitations. First, wemeasured bone stiffness of only the calcaneus using a
QUS device. Although this technique is useful in estimating bone mass or bone growth in adolescents,19,24,25 we
did not obtain information on other bone sites of interest,
such as the proximal femur and vertebrae. The effect ofdairy products on these bones may not be the same as thatseen with the calcaneus, and thus this needs to be clarified
in future studies. Second, we did not evaluate pubertal
status, which would have had an effect on bone status,
especially for younger subjects (only a few years after
puberty onset). Third, information on consumption of
dairy products was self-reported. One may drink milk byfamily- or economic-packs rather than from individual
packages or bottles. This method of evaluation is associated with potential measurement bias, which may haveattenuated the association between milk intake and QUS
values. Finally, the design of this study was crosssectional, which does not necessarily indicate causal relationships. Intervention studies are needed to remedy these
problems.
The present study demonstrated a dose-effect relationship between milk intake and bone strength in late adolescents, to whom we recommend an intake of 400
ml/day or more to obtain greater bone mass. These findings may be generalizable to populations whose dairy
product intake is generally low.
ACKNOWLEDGEMENTS
We would like to thank all students and their schools for participating in this study. This study was financially supported by
the Japan Dairy Association. The authors have no conflict ofinterest. KU was responsible for QUS measurement and datacollection. KN was responsible for statistical analysis. KU andKN designed the study and wrote the paper.
AUTHOR DISCLOSURESThe authors have no conflicts of interest.
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9. Lanou AJ, Berkow SE, Barnard ND. Calcium, dairy products, and bone health in children and young adults: a reevaluation of the evidence. Pediatrics. 2005;115:736-43.
10. Society for Information on Health and Nutrition. The National Health and Nutrition Survey in Japan, 2004. Daiichi
Shuppan, Tokyo; 2006.Japanese.
11. Prentice A, Ginty F, Stear SJ, Jones SC, Laskey MA, Cole
TJ. Calcium supplementation increases stature and bonemineral mass of 16- to 18-year-old boys. J Clin Endocrinol
Metab. 2005;90:3153-61.
12. Tsuda-Futami E, Hans D, Njeh CF, Fuerst T, Fan B, Li J,
He YQ, Genant HK. An evaluation of a new gel-coupled ultrasound device for the quantitative assessment of bone. Br J
Radiol. 1999;72:691-700.
13. Winzenberg T, Shaw K, Fryer J, Jones G. Effects of calcium
supplementation on bone density in healthy children: metaanalysis of randomised controlled trials. BMJ. 2006;333:775.
14. Huncharek M, Muscat J, Kupelnick B. Impact of dairy
products and dietary calcium on bone-mineral content inchildren: results of a meta-analysis. Bone. 2008;43:312-21.
15. Ministry of Health, Labour, and Welfare, Japan. Dietary
Reference Intakes for Japanese, 2005. Ministry of Health,Labour, and Welfare, Japan, Tokyo; 2004. (In Japanese).
16. Matkovic V, Landoll JD, Badenhop-Stevens NE, Ha EY,
Crncevic-Orlic Z, Li B, Goel P. Nutrition influences skeletal
development from childhood to adulthood: a study of hip,spine, and forearm in adolescent females. J Nutr. 2004;134:
701S-5S.
17. Stear SJ, Prentice A, Jones SC, Cole TJ. Effect of a calcium
and exercise intervention on the bone mineral status of 1618-y-old adolescent girls. Am J Clin Nutr. 2003;77:985-92.
18. Ho SC, Guldan GS, Woo J, Yu R, Tse MM, Sham A, Cheng
J. A prospective study of the effects of 1-year calciumfortified soy milk supplementation on dietary calcium intake
and bone health in Chinese adolescent girls aged 14 to 16.
Osteoporos Int. 2005;16:1907-16.
19. Robinson ML, Winters-Stone K, Gabel K, Dolny D. Modifiable lifestyle factors affecting bone health using calcaneus
quantitative ultrasound in adolescent girls. Osteoporos Int.
2007;18:1101-7.
20. Novotny R, Boushey C, Bock MA, Peck L, Auld G, Bruhn
CM et al. Calcium intake of Asian, Hispanic and whiteyouth. J Am Coll Nutr. 2003;22:64-70.

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21. Kosuge N, Yoshimatsu M, Tsukada K. Investigation into
lactose absorption in Japanese children and adults: relation
to intake of milk and dairy products. J Jpn Pediatr Soc.
1998;102:1090-7. (in Japanese)
22. French SA, Fulkerson JA, Story M. Increasing weightbearing physical activity and calcium intake for bone mass
growth in children and adolescents: a review of intervention
trials. Prev Med. 2000;31:722-31.
23. Foo LH, Zhang Q, Zhu K, Ma G, Greenfield H, Fraser DR.
Influence of body composition, muscle strength, diet andphysical activity on total body and forearm bone mass in
Chinese adolescent girls. Br J Nutr. 2007;98:1281-7.
24. Cvijetić S, Barić IC, Bolanca S, Juresa V, Ozegović DD.
Ultrasound bone measurement in children and adolescents.
Correlation with nutrition, puberty, anthropometry, andphysical activity. J Clin Epidemiol. 2003;56:591-7.
25. Babaroutsi E, Magkos F, Manios Y, Sidossis LS. Lifestylefactors affecting heel ultrasound in Greek females acrossdifferent life stages. Osteoporos Int. 2005;16:552-61.

K Uenishi and K Nakamura

439

Original Article

Intake of dairy products and bone ultrasoundmeasurement in late adolescents: a nationwide crosssectional study in Japan
Kazuhiro Uenishi PhD1, Kazutoshi Nakamura MD2
1

Laboratory of Physiological Nutrition, Kagawa Nutrition University, Saitama, Japan
Department of Community Preventive Medicine, Niigata University, Graduate School of Medical and
Dental Sciences, Niigata, Japan2

乳製品攝取與青少年晚期骨骼超音波測量：日本全國
橫斷性研究
前言：目前很少，特別是對亞洲人，關於乳製品的攝取與青少年晚期骨骼礦
化影響的實證資料。本篇研究之目的在於評估日本大型族群乳製品的攝取與
定量超音波儀(QUS)測量之骨骼強度間的關係。方法：共有遍及 33 縣的
38,719 位高級中學的學生為個案(14,996 位男生及 23,723 位女生)。跟骨硬度
以定量超音波儀(AOS-100，Aloka)測量。給予個案一份自評問卷，包含性
別、年齡、身高、體重、乳製品攝取及體能活動情形。鮮奶及酸奶的攝取被分類為無攝取、每天攝取 1-99、100-199、200-399 及≥400 mL。結果：鮮奶
每天攝取≥400 mL 的比例在男生為 21%，女生為 7.3%；另外有 24%的男生及41.1%的女生無攝取鮮奶。在校正體能活動、體重、性別、年齡及居住區域
後 ， QUS 測 量 值 與 鮮 奶 攝 取 (R2=2.8% ， p<0.001) 及 酸 奶 攝 取 (R2=0.1% ，
p<0.001)分別有相關。將男生與女生按性別分層後，仍有類似的結果。結論：青少年晚期的鮮奶攝取量與骨骼強度之間呈現正向的劑量效應，因此我
們建議，此段年齡的青少年每天至少攝取鮮奶 400 mL，以獲得較好的骨質。
關鍵字：青少年、骨密度、鈣、乳製品、定量超音波測量

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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Journal of Human Hypertension (2011) 25, 164–171& 2011 Macmillan Publishers Limited All rights reserved 0950-9240/11
www.nature.com/jhh

ORIGINAL ARTICLE

High fruit intake is associated with a lower
risk of future hypertension determined byhome blood pressure measurement: the
OHASAMA study
M Tsubota-Utsugi1, T Ohkubo2,3, M Kikuya2, H Metoki2, A Kurimoto4, K Suzuki4,
N Fukushima2,5, A Hara2, K Asayama3, H Satoh5, Y Tsubono6 and Y Imai2
1

Nutritional Epidemiology Program, National Institute of Health and Nutrition, Tokyo, Japan; 2Department ofClinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences
and Medicine, Sendai, Japan; 3Department of Planning for Drug Development and Clinical Evaluation,
Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan; 4Division of Community
Health Nursing, School of Health Sciences, Faculty of Medicine, Tohoku University, Sendai, Japan;
5
Department of Environmental Health Sciences, Tohoku University Graduate School of Medicine,
Sendai, Japan and 6School of Public Policy, Tohoku University, Sendai, Japan

We investigate associations of fruit and vegetable intake
with the risk of future hypertension using home bloodpressure in a general population from Ohasama, Japan.
We obtained data from 745 residents aged X35 years
without home hypertension at baseline. Dietary intake
was measured using a validated 141-item food frequency questionnaire, and subjects were then divided
into quartiles according to the fruit and vegetable intake.
Home hypertension was defined as home systolic/diastolic blood pressure of X135/85 mm Hg and/or
the use of antihypertensive medication. During a 4-year

follow-up period, we identified 222 incident cases ofhome hypertension. After adjustment for all putative
confounding factors, the highest quartile of fruit intake
was associated with a significantly lower risk of futurehome hypertension (odds ratio 0.40, 95% confidence
interval 0.22–0.74, P ¼ 0.004). In conclusion, this study,
based on home blood pressure measurement, suggeststhat higher intake of fruit is associated with a lower
risk of future home hypertension.
Journal of Human Hypertension (2011) 25, 164–171;
doi:10.1038/jhh.2010.48; published online 6 May 2010

Keywords: fruit intake; vegetable intake; nutrition; home blood pressure; home hypertension; healthy community

resident

Introduction
Hypertension is a major cause of morbidity andmortality,1 with many studies indicating it to besignificantly associated with an increased risk ofcardiovascular disease (CVD) events.2,3For several decades, researchers have mainly
focused on the potentially adverse or preventive
effects of various dietary factors.4–16 Among these,
fruit and vegetable intake has a especially powerful
association with lower blood pressure (BP), andwas found to reduce the risk of hypertension.4–7 Our
previous study using self-measured BP at home
Correspondence: Dr M Tsubota-Utsugi, Scientific Evaluation of
Dietary Reference Intakes’ Project, Nutritional Epidemiology
Program, National Institute of Health and Nutrition, Toyama
1-23-1, Shinjuku-ku, Tokyo 162-8636, Japan.
E-mail: mutsugky@nih.go.jp
Received 6 September 2009; revised 10 March 2010; accepted 3
April 2010; published online 6 May 2010

(home BP) found a significant cross-sectional association between intake of fruit and risk of hypertension.17 However, although most studies exclude
subjects who report a dietary change resulting
from a diagnosis, the cross-sectional studies cannot
remove subjects who change their diet after the
diagnosis of hypertension.
Furthermore, in the majority of these studies, thedefinition of hypertension was based on conventional BP measurements. Because of the white-coat
effect, a condition characterized by an elevated BP
reading in a medical setting, these studies often
overestimate the risk of high BP.18 On the other
hand, home BP measurements enable researchers to
obtain multiple measurements over a long observation period under relatively controlled conditions.19–22 The main strength of home BP is that it
is not influenced by observer and regression dilution biases or the white-coat effect. Because of
these benefits, home BP measurements are nowFruit intake and risk of future home hypertension
M Tsubota-Utsugi et al165

considered a more accurate and reliable way ofreflecting target organ damage and the prognosis
of CVD when compared with conventional BP
measurement taken in a medical setting,19–22 andare also recommended in several general hypertension guidelines.23,24
Moreover, as there are geographical differencesin the types of food intake and risk factors among
countries,25,26 it is important to confirm the reproducibility of previous findings of associations
between BP and food and nutrient components ineach population.
The aim of this study was to examine theassociation of fruit and vegetable intake with therisk of hypertension diagnosed by home BP during 4
years of follow-up in a Japanese general population.

Subjects and methods
DesignThis study was part of the Ohasama study, alongitudinal community-based observational studyof individuals who have participated in a home BP
measurement project in Ohasama, Iwate prefecture,
Japan. The geographic and demographic characteristics of the study subjects have been reported
previously.19,27
This study was approved by the institutional
review board of Tohoku University School of
Medicine and by the Department of Health of the
Ohasama Town Government. Subjects provided
written informed consent to participate.

Study population

In 1998, there were 5081 individuals aged X35 years
in Ohasama. Of the 4628 who answered thequestionnaire (response rate 91.1%), 1820 subjects
took part in home BP measurement; these individuals collected their own BP data on at least 3 days
during the 4-week measurement period in 1998.
Among those, people who had home hypertension
at baseline (n ¼ 394) and those who died (n ¼ 43) or
moved away from the town (n ¼ 5) before the followup measurements were excluded from the study.
Of the remaining 1378 eligible individuals, 805
subjects (58%) took part in the follow-up home BP
measurements.
In addition, 60 subjects were excluded for thefollowing reasons: those who took o3 home BP
measurements at follow-up (n ¼ 20) and those who
had extreme levels of energy intake (in the upper orlower 2.5% of the range for all subjects: n ¼ 40).
Finally, data from 745 subjects who were normotensive on baseline home BP (274 men and 471
women) were analysed. Compared with those who
were ultimately excluded based on the exclusion
criteria of the 1378 eligible individuals, the 745
participants who completely fulfilled the study
criteria were more likely to be men, and of older age.

Home BP measurement

Baseline home BP was measured using theHEM701C monitor (Omron Healthcare Co., Ltd,
Kyoto, Japan), a semiautomatic device based on the
cuff-oscillometric method, which generates a digital
display of both systolic BP and diastolic BP. We
used HEM747ICN devices (Omron) for follow-up
measurements. Both devices have been validated28
and satisfy the criteria of the Association for
the Advancement of Medical Instrumentation(the HEM747ICN is exactly same as the Omron
HEM735C except that the latter does not incorporate
an integrated circuit memory). As the circumferenceof the arm was o34 cm in most cases, we used astandard arm cuff in all cases.27 In this study, home
BP was defined as the mean of all first measurements recorded during the 4-week period. The mean(±s.d.) number of home BP measurements was
23±6. Hypertension was defined as use of antihypertensive medication and/or home BP values of
X135/85 mm Hg at follow-up measurement.20–24
Food frequency questionnaire

Standardized methodology was used to calculate
fruit and vegetable intake from data obtained in a
Japanese version of a food frequency questionnaire.
The reproducibility and validity of this questionnaire were previously reported in detail.29,30 The
questionnaire asked about the average frequency of
intake of each food during the previous year
according to nine frequency categories ranging fromno consumption to X7 times per day. A standard
portion size of one serving was specified for each
food, and respondents were asked whether theirusual portion was larger (41.5 times), the sameor smaller (o0.5) than the standard. In this study,
we took into account energy from food sources ofalcohol; for example, seasonings that include alcohol. However, we did not consider alcohol derived
from alcoholic drinks such as beer and wine in the
total energy count because we treated such alcohol
intake as a separate variable. Nutritional supplements were not taken into account because there
were few supplement users.
All food and nutrient intakes were adjusted for
total energy intake using the residual method,31–33
and separate regression models were performed toobtain the residuals for men and women. Following
this procedure, subjects were divided into quartiles
according to the intake of fruit and vegetables.
In this study, the lowest quartiles were used as
reference categories.
Statistical analysis

To examine how the intake of fruit or vegetables was
associated with the risk of future home hypertension
defined on the basis of home BP measurement, weused multiple logistic regression analyses after
adjustment for other putative confounding factors
Journal of Human Hypertension

Fruit intake and risk of future home hypertension
M Tsubota-Utsugi et al166

related to hypertension. These were gender (men/women), age (continuous), body mass index (BMI;
o25/X25 kg m–2), frequency of exercise (rarely ornever, 1 or 2 h per week and 43 h per week),smoking status (never, past or current smoker),
alcohol consumption (rarely or never, o540 ml ofsake per day and X540 ml of sake per day: 540 ml ofsake ¼ 81 g of alcohol), energy-adjusted fat intake
and sodium intake (continuous), baseline systolic
home BP (continuous) and a history of diabetes,
hypercholesterolaemia and CVD (yes/no).
Moreover, we stratified the analysis by lifestylefactors, such as overweight (BMI ¼ 25 kg/m–2 ascutoff), frequency of exercise, smoking status andalcohol consumption, to explore associations
related to these factors. We tested interactions byintroducing a multiplicative term into the main
effect models. We also examined the combined
effects of risk factors and fruit or vegetable intakes.
For all analyses, statistical significance was defined
as a two-tailed P-value of o0.05. All analyses wereconducted using SPSS software version 14 forWindows (SPSS Inc., Chicago, IL, USA).

Results
At the time of the follow-up measurements, 222
subjects (29.8%) had developed home hypertension(mean duration of follow-up: 4.1 years). Amongthese, 70 were defined as having home hypertension
because they had started treatment with antihypertensive medication.
The distributions of characteristics across quartileof each fruit and vegetable intake at baseline areshown in Table 1. In the study, the most commonly
consumed type of fruit was citrus fruit (18.5 g day–1),
followed by apple (8.6 g day–1), grape (5.9 g day–1)
and watermelon (5.6 g day–1). Compared with thosein the highest quartile of fruit intake, those in thelowest quartile were more likely to be men, ofyounger age, current smokers, heavier drinkers andwith lower diastolic home BP. Subjects with thehighest quartile of fruit intake tended to consumeless energy and carbohydrate and more sodium, andfruit- and vegetable-related nutrients (that is, potassium, magnesium, b-carotene, folate, vitamin C andtotal dietary fibre) than subjects in the lowest
quartile. As for food intake, the highest intake offruit was associated with low intakes of rice, breadand noodles, and with high intakes of vegetables
and seaweeds. Compared with those with highest
quartile of fruit intake, subjects with quartile 3 offruit intake reported more fat and less protein andcalcium intake (table not shown, intake in quartile 3;fat, 42.7±0.8 g; protein, 61.6±0.6 g; and calcium630±14 mg). We observed similar tendencies forvegetable intake. In each category, the frequencyof exercise and incidence of home hypertension at
follow-up did not differ.
Journal of Human Hypertension

Table 2 shows the association between fruit andvegetable intake and the risk of future home
hypertension. In the sex- and BMI-adjusted analysis,
the highest quartile of fruit intake was associatedwith a significantly lower risk for future home
hypertension (odds ratio compared with the lowest
quartile for fruit intake: 0.44; P ¼ 0.005), whereas no
association was observed for vegetable intake. After
adjustment for putative confounding factors, theseassociations did not change. Compared with thelowest quartile for intake of fruit, a 60.0% lower riskof hypertension was found in those with the highest
quartile of fruit intake (P ¼ 0.004). Further adjustment for putative confounding factors, vegetables
and related nutrients (potassium, b-carotene, folate,vitamin C and total fibre) attenuated these results,
but there was still a significantly lower risk of futurehome hypertension in the highest quartile of fruit
intake (odds ratio ¼ 0.45; P ¼ 0.025).
Regarding joint classification of quartiles of fruit
intake and risk factors for home hypertension,
Figure 1 shows the risk associated with BMI status
at each quartile of fruit intake. The odds ratio for thecomparison of overweight with highest quartileof fruit intake to overweight with lowest quartile offruit intake was 0.21 (P ¼ 0.005). There was no
significant interaction between BMI and fruit intake(P40.10). When we adjusted for baseline diastolic
home BP instead of systolic home BP, the results
were almost the same.

Discussion
This study indicated that high fruit intake isstrongly associated with a lower risk of future home
hypertension. The inverse association between fruit
intake and future home hypertension was persistentamong subgroups of overweight and normal-weightindividuals.
Our study has several strengths. It is the first to
examine whether fruit and vegetable intake predicts
hypertension measured by home BP. Measuring BP
at home can eliminate several biases, such as thewhite-coat effect,20–22 and therefore the results mightmore accurately determine the relationship between
BP and fruit and vegetable intake. Because of theprospective design and exclusion of hypertensive
subjects at baseline, we believe we could minimize
the number of subjects who changed their dietbecause of a diagnosis of high BP.
The second strength is that, to the best of ourknowledge, this is the first study to clarify the
association between fruit and vegetable intake andfuture hypertension in Asian subjects.

Fruit/vegetable intake and future home hypertension

We found that high fruit intake was linked to a lower
risk of future home hypertension, whereas no
association was observed for high vegetable intake.

Fruit intake and risk of future home hypertension
M Tsubota-Utsugi et al
167

Table 1 Distribution of characteristics across quartiles of fruit and vegetable intake (n ¼ 745)
Quartile of fruit consumption

Baseline
Gender (men %)Age
Alcohol consumption (%)
Rarely or nevero540 ml of sake per day
X540 ml of sake per day
Current smokers (%)
Exercise (rarely or never %)Body mass index (kg m–2, X25%)
Home BP (mm Hg)
Systolic
DiastolicMean intakes of food and nutrientsb
Rice, bread and noodles (g)Sugar (g)Nuts (g)Pulses (g)Vegetables (g)Seaweeds (g)Fish and shellfish (g)Meats (g)Eggs (g)Dairy products (g)Energy (kcal)
Protein (g)Fat (g)Carbohydrates (g)Sodium (mg)
Potassium (mg)Calcium (mg)Magnesium (mg)b-carotene (mg)
Folate (mg)
Vitamin C (mg)
Total dietary fibre (g)Follow-upc
Antihypertensive medication (%)
Home BP (mm Hg)
Systolic
DiastolicHome hypertension (%)d1 (n ¼ 187)

4 (n ¼ 186)51.3
55.3±0.826.9
57.4±0.8

65.8
28.3
5.9
27.8
76.5
21.9

82.3
16.1
1.6
13.4
83.3
24.2

115.8±0.7
73.2±0.5566±56.6±0.4
101.7±4.7
2.7±0.5
198.8±8.4
18.2±1.1
65.1±3.1
20.5±129.5±1.2
220±132163±4264.1±0.638.8±0.9
299.4±0.7
4264±151
2396±40631±14287±1.52806±138299.5±8.5
59.9±2.3
14.8±0.4

P-value

Quartile of vegetable consumption

P-value

1 (n ¼ 187)

4 (n ¼ 186)62.6
54.2±0.818.3
59.0±0.8a

0.001
0.290
0.925

68.4
27.3
4.3
27.8
79.7
20.9

83.3
15.1
1.6
9.1
80.1
28.0

113.6±0.8
71.3±0.5

0.273
0.014

115.8±0.7
73.6±0.5

114.3±0.8
71.2±0.5a

0.403
0.002

479±5a8.9±0.473.6±4.7
5.0±0.5a249.2±8.3a
22.8±1.1a
58.5±3.1
17.2±1.0
27.8±1.2
238±131952±4162.3±0.640.1±0.8
305.9±0.7a
5181±150a
2797±40a661±14313.7±4.5a
3686±137a
335.3±8.5a
97.7±2.3a
17.7±0.4ao0.0001
o0.0001
0.057
0.008
o0.0001
0.009
0.143
0.052
0.744
0.497
o0.0001
0.029
0.009
o0.0001
o0.0001
o0.0001
0.332
0.001
o0.0001
0.026
o0.0001
o0.0001685±154.3±0.380.7±7.5
2.3±0.390.3±3.7
13.4±1.247.2±413.9±0.922.6±1.2
241±172135±3856.7±134.0±0.9
336.8±3.82959±131
1879±23549±20232.8±5.2
1477±56205.4±6.7
44.2±1.7
10.3±0.3397±10a11.7±0.6a
100.2±4.5a
4.7±0.6a
390.9±8.6a
29.2±1.4a
68.6±2.6a
22.7±1.5a
31.1±1.5a
212±12
1897±51a
68.2±0.9a
45.3±0.9a
278.7±2.8a
6610±191a
3346±48a
747±17a370±5.2a5211±202a
443.3±10.7a
111.4±3.0a
23.1±0.5ao0.0001
o0.0001
0.021
0.001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
0.166
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001
o0.0001

o0.0001
0.057
0.010

9.1

7.0

0.250

4.3

126.4±1.1
75.5±0.7
34.8

123.9±1.1
73.4±0.6
24.2

0.212
0.056
0.076

124.8±1.1
74.9±0.7
31.6

o0.0001
o0.0001
o0.0001

o0.0001
0.643
0.214

11.3

0.037

124.4±1.1
72.8±0.6
26.9

0.835
0.127
0.450

Continuous variables are presented as mean±s.e.
One-way analysis of variance (ANOVA) was used for continuous variables and w2 test for categorical variables, comparing quartiles of each foodgroup.
aStatistical significance was defined as Po0.05 compared with quartile 1 (lowest) using Bonferroni post hoc test.
b
Data were adjusted for total energy by the residual method.
cMean duration of the period between the baseline and the follow-up home blood pressure (home BP) was 4.1±0.7.
dHome hypertension was defined as use of antihypertensive medication and/or home BP values of X135/85 mm Hg at follow-up.

Results of some studies, which examined theassociation between combined fruit and vegetableintake and risk of hypertension, are partially consistent with the present findings,4–6,34 but no studies
have shown significant inverse associations
between intake of fruit alone and the risk of hypertension. Other studies have reported a significant
protective association between intake of fruitand risk of CVD.15,16 Our results are consistent
with this.

Other related nutrients and home hypertension

We also analysed the intakes of potassium, folate,magnesium, vitamin C and b-carotene, which arehighly correlated with fruit and vegetable intake.
However, these dietary factors were not associatedwith the risk of home hypertension. Moreover,adjustment for these dietary factors did not significantly modify the findings. In this study, the mostcommonly consumed type of fruit was citrus fruit,
followed by apple, grape and watermelon. Although
Journal of Human Hypertension

Fruit intake and risk of future home hypertension
M Tsubota-Utsugi et al
168

Table 2 Adjusted odds ratio (95% confidence interval) for the association between fruit and vegetable intake and the risk of future home
hypertension (n ¼ 745)
Quartile 1

Quartile 2

Quartile 3

Quartile 4

P for trend

Fruit (g day–1)a
Adjustedb
Adjustedc

p38.401.00
1.00

38.41–63.80
0.74 (0.44–1.26)
0.64 (0.36–1.15)

63.8–100.02
0.85 (0.80–1.44)
0.70 (0.39–1.26)

100.03p0.44 (0.25–0.78)
0.40 (0.21–0.74)

0.033
0.037

Vegetables (g day–1)a
Adjustedb
Adjustedc

p143.411.00
1.00

143.42–211.55
0.90 (0.52–1.57)
0.96 (0.52–1.75)

211.56–282.75
1.28 (0.74–2.23)
1.11 (0.60–2.05)

282.76p
0.71 (0.40–1.24)
0.75 (0.40–1.38)

0.168
0.597

a

Data were adjusted for total energy by the residual method.
Adjusted for age, gender and body mass index (BMI).
c
Adjusted for age, gender, BMI, frequency of exercise, smoking status, alcohol consumption, energy-adjusted fat and sodium consumption,baseline systolic home BP, and a past history of diabetes, hypercholesterolaemia and cardiovascular disease.
b

0.63

0.60

0.37*

0.50

0.55

(0.18-0.96)

0.57

0.70

0.51
(0.07-0.62)

Odds ratio (95% CI)

0.80(0.23-1.16

0.90

0.17-0.84)(0.26-1.27)

1.00
1.00

0.40
0.30
0.20

0.42*

0.21*

0.10
0.00

BMI <25
Quartile1
Quartile2

BMI >25

Quartile3
Quartile4 (highest)
Quartile of fruit consumption

Figure 1 Multivariate odds ratio of home hypertension according to joint classifications of fruit intake and body mass index (BMI). The
category of lowest fruit intake and the overweight (BMI of X25) was used as reference. Model adjusted for age, gender, frequency of
exercise, smoking status, alcohol consumption, energy-adjusted fat and sodium consumption, baseline systolic home BP and a pasthistory of diabetes, hypercholesterolaemia and cardiovascular disease. *Po0.05.

individual dietary factors, such as vitamin C andfolate, were not associated with risk of home
hypertension, it is possible that the total balanceof these factors in these commonly consumed fruits
might be useful for the prevention of future
hypertension.
Characteristics of fruit intake

These result, however, showed a lack of continuity
in terms of risk of hypertension for each quartile offruit intake, despite the significant association
between the highest quartile of fruit intake and alower risk of hypertension. Subjects in the third
quartile of fruit intake reported higher fat and lower
protein intake than those in the highest quartile, andthus the risk of hypertension might be influenced
by these factors.
Journal of Human Hypertension

We also found no association between the highest
quartile intake of vegetables and risk of hypertension. This study confirmed the findings of ourprevious cross-sectional study,17 in that those who
consumed more fruit and vegetables had higher
sodium and fat intake. This might be attributable toseasonings, including soy sauce and table salt, andmethods of cooking vegetables, such as deep frying.
The higher fat and sodium intake among those who
consumed more fruit might be attributable to theclose correlation between the intake of fruit andvegetables. A number of factors, such as lifestyle,food availability, food culture and dietary habits,
might also be related to BP and risk of hypertension.
In this study, we found significant differences in
dietary characteristics across quartiles of fruit intake
after adjusting for all putative confounding factors.
Compared with subjects with higher intake of fruit,Fruit intake and risk of future home hypertension
M Tsubota-Utsugi et al169

those with the lowest intake of fruit consumed morecarbohydrate-containing foods and meat, and lessvegetables and seaweed. It therefore seems that ahigher intake of fruit was associated with a healthierdiet. As people consume diets consisting of a variety
of foods with complex combinations of nutrients,
the examination of only single foods could result in
the identification of erroneous associations between
dietary factors and disease. Furthermore, the risk ofhypertension could be attributable to other foodgroups. When several nutrients with small BPlowering effects are consumed together, the cumulative effects may be sufficient for detection. The
dietary pattern approach using factor and cluster
analyses35 could provide more information regarding risk of home hypertension in further studies.

detailed information on lifestyle-associated factors
are required to further investigate the association
observed in this study.

Conclusions

The present results from the Ohasama study suggestthat high intake of fruit is potentially associatedwith a lower risk of future home hypertension.
Although the mechanism for BP lowering throughfruit and vegetable intake remains unclear,37,38
selective intake of healthy foods and nutrients
may prevent hypertension. Using home BP ingeneral subjects enable to be considered highly
health consciousness and subsequent early dietaryintervention is expected to prevent hypertension
and CVD.

Study limitations

Several limitations of this study need to be discussed. First, information regarding food andnutrient intake in this study was obtained on thebasis of dietary recall. The correlation between thefood frequency questionnaire and usual diet has
been well established, but there are several problems, for example, limited number of items andminimal information about portion size.
Second, we did not find a significant interaction
between fruit consumption and BMI. However, agradient declining risk of home hypertension
with increasing fruit intake was apparent only forthe overweight subjects, suggesting that such aninteraction may have been present. Therefore, it ispossible that the lack of statistical significance was
because of the small size of the eight subgroups.
Larger studies would be needed to clarify thepresence of a statistically significant interaction
between fruit consumption and BMI.
The possibility of selection bias also needs to beconsidered when generalizing the present findings,
because only 54.1% of those eligible to participate
in the study agreed to take part. As we excluded
those who had home hypertension at baseline, this
could mean that healthy people were more likely to
be followed up. However, although the nonparticipants were older and had higher energy intake thanthose who participated in the study, other lifestylefactors did not differ significantly between participants and nonparticipants. Marked differences
also exist in the epidemiology of home hypertension
between Japan and Western countries;36 thus,
further research in other ethnic and cultural populations is needed to confirm the generalizability of ourfindings.
In this study, a higher intake of fruit was
associated with a healthier lifestyle such as limited
alcohol intake and avoidance of smoking. Therefore,
although we adjusted for these confounding factors,
it is possible that other factors associated withhealthier lifestyle not measured in this study might
confound the findings. Further studies with more

What is known about this topic
K Fruit and vegetable intake has a powerful association
with lower blood pressure and was found to reduce therisk of hypertension.
K But there are geographical differences in the types offood intake and risk factors among countries, and therelationship of diet with blood pressure in Asian
populations has not been fully investigated.
K Furthermore, there is no study to examine the association
between fruit and vegetable intake and the risk of futurehome hypertension determined by home blood pressure
measurement.
What this study adds
K Higher intake of fruit is associated with a lower risk of
future home hypertension.
K Higher intake of fruit is also associated with a healthierdiet.

Conflict of interest
The authors declare no conflict of interest.

Acknowledgements
This study was supported in part by Grants for
Scientific Research (15790293, 16590433, 17790381,
18390192, 18590587, 19590929 and 19790423)
from the Ministry of Education, Culture, Sports,
Science and Technology, Japan; Grants-in-Aid (H17Kenkou-007, H18-Junkankitou (Seishuu)-Ippan-012
and H20-Junkankitou (Seishuu)-Ippan-009, 013)
from the Ministry of Health, Labour and Welfare,
Health and Labor Sciences Research Grants, Japan;
Grants-in-Aid for Japan Society for the Promotionof Science (JSPS) fellows (16.54041, 18.54042,
19.7152, 20.7198, 20.7477 and 20.54043); Health
Science Research Grants and Medical Technology
Evaluation Research Grants from the Ministryof Health, Labour and Welfare, Japan; Japan
Atherosclerosis Prevention Fund; Uehara Memorial
Journal of Human Hypertension

Fruit intake and risk of future home hypertension
M Tsubota-Utsugi et al
170

Foundation; Takeda Medical Research Foundation;
National Cardiovascular Research Grants; and Biomedical Innovation Grants.

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Journal of Human Hypertension49Asia Pac J Clin Nutr 2010;19 (1):49-56

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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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

50

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

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, 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
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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【学会発表 要旨】
第 12 回日本骨粗鬆症学会（大阪）2010 年 10 月 23 日

ダブルアイソトープ法による閉経後女性のカルシウム吸収率の検討
上西一弘、藤田拓男、石田裕美、藤井芳夫、大植睦、梶博史、平井みどり、角本幹夫、Abrams SA【目的】カルシウムは腸管からの吸収率が低い栄養素の１つであり、その吸収率は成人では 25％程度とされている。また、吸収率は個人差が大きく、加齢により減少することも知られている。しかし、個人ごとの腸管からのカルシウム吸収率を測定することは非常に困難であり、ほとんど行われてはいない。カルシウムの安定同位体（アイソトープ）を用いて吸収率を測定する方法がある。経口摂取するカルシウムの一部をアイソトープでラベルして尿中への排泄量を調べるシングルアイソトープ法はこれまでにわが国でも実施されているが、経口摂取と同時に、静脈中にもう１種類の異なるアイソトープを投与し、2 種類のアイソトープを用いて吸収率を測定する方法、ダブルアイソトープ法は、海外では実施されているが日本での実施例はない。今回我々はダブルアイソトープ法を用いて閉経後女性のカルシウム吸収率を測定することを目的に本研究を行った。
【方法】対象者は平均年齢 67±5 歳の閉経後女性 9 名であり、閉経後年数の平均は 17 年であった。44Ca を経口投与、42Ca を静脈投与した。経口投与したカルシウムは炭酸カルシウ
ムの形態で、300mg 中 30mg が 44Ca である。静脈中には 3mg の 42Ca を投与した。24 時間の採尿を行い、尿中の 44Ca 、42Ca を ICP-MS で測定し、腸管からのカルシウム吸収率を計算した。本研究は香川栄養学園、神戸大学、藤井クリニックの各倫理委員会の承認を得て実施した。
【結果】対象者のカルシウム栄養状態、骨代謝マーカーでは１名の血清 25（OH）ビタミン
D 濃度が 7.6ng/dl と低値であった以外は、特に問題はなかった。ダブルアイソトープ法によるカルシウムの吸収率は 14.7±6.4％であり、6.1％から 22.2％の範囲にあった。
【結語】ダブルアイソトープ法により測定した閉経後女性のカルシウムの吸収率は 14.7±
6.4％であり、個人差も大きかった。本法は比較的簡便にカルシウムの吸収率を測定することができ、基礎研究及び臨床研究に役立つものと期待される。