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1 From the Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan (KN, YO, YT, and MY); the Department of Nursing, School of Health Sciences, Niigata University, Niigata City, Japan (KU and TN); and the Department of Health and Nutrition, Niigata University of Health and Welfare, Niigata City, Japan (TS)
2 Supported by a grant from The National Dairy Promotion and Research Association of Japan, 2002; a grant from the Japan Osteoporosis Foundation; and a Grant-in-Aid for Scientific Research (C) no.15590540 from The Ministry of Education, Culture, Sports, Science and Technology of Japan. The standard 25(OH)D3 was a gift from F Hoffmann-La Roche Ltd (Basel, Switzerland). 3 Address reprint requests to K Nakamura, Division of Social and Environmental Medicine, Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata City 951-8510, Japan. E-mail: kazun{at}med.niigata-u.ac.jp.
ABSTRACT
Background: The adverse effects of poor nutrition on the bones of young Asian women have not been fully elucidated.
Objective: The purpose of this study was to investigate possible associations of vitamin D nutrition, calcium intake, and other nutrients with bone metabolism and bone mass in young Japanese women.
Design: The subjects were 108 female college students aged 19-25 y. Dietary nutrients were measured by using the duplicate sampling method on 3 weekdays. Serum 25-hydroxyvitamin D [25(OH)D], intact parathyroid hormone, and bone turnover markers were also measured. Bone mineral density (BMD) of the spine and femur was measured by dual-energy X-ray absorptiometry.
Results: The proportions of the subjects with low 25(OH)D (<30 nmol/L) and high intact parathyroid hormone (6.9 pmol/L) concentrations were 32.4% and 15.7%, respectively. Serum 25(OH)D concentrations (P = 0.0265) and calcium intake (P = 0.0103) were inversely associated with serum intact parathyroid hormone. In addition to weight and physical activity, the presence of mild hyperparathyroidism was associated with a low BMD of the lumbar spine (P = 0.0062) and the femoral neck (P = 0.0250), and a low calcium intake was associated with a low BMD of the femoral neck (P = 0.0044).
Conclusions: Low calcium intake (based on low BMD of the femoral neck only) and mild hyperparathyroidism (based on low BMD of both the femoral neck and lumbar spine), partly explained by low vitamin D nutrition and a low calcium intake, are important predictors of low BMD in young Japanese women. Effects of poor nutrition and mild hyperparathyroidism on bone peak bone mass in young women should be further investigated in longitudinal studies.
Key Words: Bone density • calcium-to-phosphorus ratio • dietary calcium • 25-hydroxyvitamin D • hyperparathyroidism • Japanese • vitamin D • vitamin K • young women
INTRODUCTION
Poor nutrition is a risk factor for decreased bone mass and osteoporosis in middle and old age. Nutrition is thought to be a factor that affects the maximal peak bone mass of young women, and, because attaining it is an important strategy for preventing future osteoporosis, it is important to clarify the effect of nutritional status on their bone mass.
Vitamin D insufficiency can cause hyperparathyroidism and bone loss. It has typically been diagnosed in elderly people (1), but recent research has suggested that vitamin D insufficiency may also be prevalent in young women. Low serum 25-hydroxyvitamin D [25(OH)D] concentrations have been detected in 20-40% of healthy young women at the cutoff values for vitamin D insufficiency used for elderly people (2-4). Earlier studies (2-4) evaluated associations between low 25(OH)D concentrations and peripheral bone mass, but they have not investigated associations with spinal or femoral bone mass; thus, the adverse effects of low vitamin D concentrations on the bones of young women have not been fully elucidated.
Sufficient calcium intake is needed to maintain zero or positive calcium balance in the body. The contribution of calcium intake to peak bone mass has been well studied in young white populations whose calcium intake is relatively high (5), but peak bone mass in young women with low calcium intakes have not been thoroughly studied. The calcium intakes of young Asians have been reported to be low, ie, a mean daily calcium intake <500 mg/d (6-8). However, no clear association between dietary calcium intake and bone mass in the spine or femur has been established, and other nutrients, such as phosphorus (9), protein (10), and vitamin K (11, 12), may affect bone health. Low intakes of these nutrients by young women may be associated with low bone mass, because young women generally tend to restrict their caloric intake (13). The purpose of this study was to clarify the effects of vitamin D nutrition, calcium intake, and intake of other nutrients on the bone metabolism and bone mass of women with an approximate age of 20 y.
SUBJECTS AND METHODS
Subjects
The target population consisted of second- and third-year female students in the Nursing Course at Niigata University School of Medicine; 112 (75.7%) of the 148 students in that population agreed to participate in the study. The following students were excluded from the analysis: 2 students who had received corticosteroid hormone therapy and 2 students aged 29 and 32 y, respectively, who were considered outliers because of their age. Ultimately, data from 108 women were analyzed. Written informed consent was obtained from all subjects. The protocol of this study was approved by the Ethics Committee of Niigata University School of Medicine. This study included assessment of a wide range of dietary nutrients, blood and urine examinations, and bone mass measurements. It was conducted between October and December 2002.
Dietary assessment
Dietary nutrients were measured by direct analysis of samples of the subjects' meals obtained by the duplicate portion sampling method. The subjects were asked to provide duplicates of all meals, drinks, snacks, etc, that they consumed on 3 consecutive weekdays before the day of the physical examination, which included blood and urine examinations. The following instructions were given: 1) provide all foods and drinks that you consume except tap water and tea without milk; 2) place the foods in a plastic bag; 3) place all daily meal samples in an opaque bag and submit them in an opaque box to prevent them from being seen by others; 4) provide only ordinary meals, not special meals for a special occasion, because unusual meals would not be of benefit to either the subjects or the researchers; however, eating out is not prohibited; and 5) do not change your eating pattern, especially with regard to milk, milk products, and small fish, which are important sources of dietary calcium. The price of all provided meals was paid for by the researchers. The food samples for 3 d were thoroughly mixed with a food blender and dried at 110 °C. Calcium, phosphorus, sodium, potassium, and protein were measured in the dried samples. Calcium, sodium, and potassium were measured with an atomic absorption spectrophotometer, and phosphorus was measured with the spectrophotometric molybdovanadate method. The molar ratio of dietary calcium to phosphorus was calculated. Protein intake was computed on the basis of dietary nitrogen measured by the micro-Kjeldahl method. The intra- and interassay CVs were 1.1% and 2.6%, respectively, for calcium, 1.4% and 0.8% for phosphorus, 1.5% and 1.3% for sodium, 2.8% and 5.2% for potassium, and 1.5% and 0.7% for protein. The total dry weight of a meal was assumed to reflect its total energy content. To validate this assumption, we measured the energy in 10 random samples from the mixed diets and found that the coefficient for the correlation between total dry weight and total energy was 0.995. All nutrients were measured by NAC Co, Ltd (Tokyo, Japan). The detailed procedure of the duplicate portion sampling method and the methods of measuring the dietary nutrients were described elsewhere (14).
Blood examination
A fasting blood specimen was drawn from each subject in the morning between 0730 and 0830. Whole blood was immediately centrifuged at 1613 x g for 10 min at room temperature to obtain the serum, and the serum was stored at –40 °C until analyzed. Serum 25(OH)D3 concentrations were measured by HPLC with the 2-step method (15), and the interassay CV was 5.0%. Serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] concentrations were measured by radioimmunoassay (Immunodiagnostic Systems Ltd, Boldon, United Kingdom; reference value: 48-144 pmol/L; interassay CV: 4.9%). Serum intact parathyroid hormone concentrations were measured by 2-site immunoradiometric assay with an Allegro PTH kit (Nichols Institute Diagnostics, CA; reference value: 1.06-6.90 pmol/L; interassay CV: 10.1%). Subjects whose intact parathyroid hormone concentration exceeded the upper limit of 6.90 pmol/L received a diagnosis of (mild) hyperparathyroidism. The concentrations of 2 of the biochemical indexes of bone turnover, serum osteocalcin (OC) and serum bone alkaline phosphatase (bone ALP), were measured by immunoradiometric assay (Mitsubishi Kagaku Iatron Inc, Tokyo, Japan; interassay CV: 5.9%) and polyacrylamide gel disc electrophoresis (Quantimetrix Corporation, CA; interassay CV: 3.2%), respectively. Serum undercarboxylated OC (ucOC) concentrations were determined by enzyme-linked immunoassay (Takara Bio Inc, Otsu, Shiga, Japan; interassay CV: 5.7%). The ratio of ucOC to OC was used as an indicator of vitamin K nutritional status. Serum calcium concentrations were determined with a Clinical Analyzer 7450 (Hitachi High-Technologies Corporation, Tokyo).
Urine examination
The second urine samples were collected in the morning between 0730 and 1000 and were stored at –40 °C until analyzed for biochemical markers of bone resorption. Enzyme-linked immunoassays were used to measure urinary deoxypyridinoline (Quidel Corporation, San Diego, CA; interassay CV: 5.7%) and urinary type I collagen cross-linked N-telopeptides (NTX-I; Osteomark NTx, NJ; interassay CV: 3.9%). Urinary calcium was determined with a Clinical Biochemistry Analyzer (BioMajesty 12; JEOL Ltd, Tokyo, Japan). All urinary measurements were corrected for the urinary creatinine concentration.
Bone mass measurements
Bone mineral content (BMC) and bone mineral density (BMD) in the lumbar spine and the femoral neck were measured by dual-energy X-ray absorptiometry (QDR-2000; Hologic Inc, Bedford, MA), which was performed by a single trained physician. The CVs for the BMC and BMD measurements were 3.5% and 0.7%, respectively, for the lumbar spine and 1.3% and 0.9%, respectively, for the femoral neck. Long-term CVs for BMC and BMD measurements with the use of the standard phantom were 0.5% and 0.42%, respectively. The height and weight of the subjects in underwear were measured in the afternoon according to the standardized procedure.
Physical activity
Information on age, age at menarche, current physical activity, past exercise habits, past medical history, menstrual cycle, and lifestyle was obtained by interview. Current level of physical activity was evaluated by calculating the metabolic METS (ratio of working metabolic rate to resting metabolic rate), which we refer to as the METS index below, based on the 7-d total activity recall of each subject (16). Past exercise habits in junior high school (12-15 y of age) and high school (15-18 y of age) were estimated on the basis of the frequency of sports-club activities. Subjects who exercised 3 times/wk for >2 y were classified in the high-exercise group.
Statistical analyses
Means and SDs were calculated to characterize continuous variables, and data for all continuous variables were tested for normal distribution. Because data for dietary calcium, the ratio of calcium to phosphorus, dietary protein, intact parathyroid hormone, deoxypyridinoline, NTX-I, urinary calcium, BMC of the lumbar spine and femoral neck, ucOC, ratio of ucOC to OC, and the METS index were skewed toward higher values, they were logarithmically transformed for the statistical tests. Pearson's product-moment correlation coefficient was calculated to test for linear correlations between pairs of continuous variables. Qualitative and continuous variables were tested for associations by the t test. The stepwise method of multiple linear regression analysis was used to identify independent variables that predicted BMC or BMD as an outcome variable. Statistically significant variables in the bivariate analyses were candidates for independent variables in the stepwise method. A P value < 0.05 was considered statistically significant. The SAS system for WINDOWS (release 8.02; SAS Institute Inc, Cary, NC) was used to make the computations.
RESULTS
The subjects' demographic and physical characteristics, nutrient intakes, and bone mass profiles are shown in Table 1. The ages of the subjects ranged from 19 to 25 y. The nutrition data of the 2 subjects who were consuming special meals because of strict dieting during the study period were excluded from the statistical analyses. The serum and urinary biochemical profiles are shown in Table 2.
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TABLE 1. Characteristics of the 108 subjects1
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TABLE 2. Serum and urinary biochemical profiles of the 108 subjects1
The numbers of subjects who regularly engaged in sports-club activities in junior high school and high school were 85 (78.7%) and 38 (35.2%), respectively. There were 5 daily smokers (4.6%), 6 occasional or ex-smokers (5.6%), and 97 nonsmokers (89.8%). There were 65 occasional drinkers (60.2%), 43 nondrinkers (39.8%), and no regular daily drinkers.
There were 35 (32.4%) subjects with a low serum 25(OH)D3 concentration (<30 nmol/L) and 17 (15.7%) with a high serum intact parathyroid hormone concentration (6.9 pmol/L). None of the subjects had a low 1,25(OH)2D3 concentration. A correlation analysis showed a negative association (r = –0.264, P = 0.0057) between serum 25(OH)D3 concentrations and serum intact parathyroid hormone concentrations (Figure 1). Calcium intake (r = –0.297, P = 0.0020) and protein intake (r = –0.280, P = 0.0036) were also correlated with the serum intact parathyroid hormone concentrations. The multiple regression analysis by the stepwise method showed that serum intact parathyroid hormone concentrations were predicted independently by the serum 25(OH)D3 concentrations (R2 = 0.070, P = 0.0265) and calcium intake (R2 = 0.058, P = 0.0103).
FIGURE 1.. Scatterplots of serum intact parathyroid hormone (PTH) concentrations versus serum 25-hydroxyvitamin D3 [25(OH)D3] concentrations. The dotted line is the upper limit (6.9 pmol/L, or 65 pg/dL) of the normal range for intact PTH. The Pearson's correlation coefficient between serum 25(OH)D3 and serum intact PTH concentrations was –0.264 (P = 0.0057). To convert nmol/L to ng/dL for serum 25(OH)D3, multiply the values by 0.4; to convert pmol/L to pg/dL for intact PTH, multiply the values by 9.4.
The correlations between biochemical markers of bone turnover and age, weight, physical characteristics, nutrient intakes, and calciotropic hormones are shown in Table 3. All biochemical markers of bone turnover, except urinary calcium, were significantly negatively correlated with age. Serum bone ALP was positively correlated with weight, and urinary NTX-I was positively correlated with serum intact parathyroid hormone. These associations held after adjustment for age. Urinary calcium was significantly correlated with age at menarche, calcium intake, phosphorus intake, calcium:phosphorus, and serum 25(OH)D3.
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TABLE 3. Pearson's correlation coefficients between age, weight, physical characteristics, nutrition intakes, and calciotropic hormones and markers of bone turnover1
Correlations between bone mass variables and age, weight, physical characteristics, nutrient intakes, and calciotropic hormones are shown in Table 4. Weight was positively correlated with all 4 variables of bone mass, and the correlations were statistically significant. The METS index and grip strength were positively correlated with BMC and BMD in the lumbar spine and BMC in the femoral neck. Calcium intake and calcium:phosphorus were positively correlated with BMC and BMD in the femoral neck. Phosphorus intake was positively correlated with BMD in the femoral neck. Serum intact parathyroid hormone was negatively correlated with all 4 variables of bone mass. Total energy was correlated significantly with none of the 4 variables of bone mass.
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TABLE 4. Pearson's correlation coefficients between age, weight, physical characteristics, nutrition intakes, and calciotropic hormones and bone mineral content (BMC) and bone mineral density (BMD)1
Assessment of the qualitative variables showed that exercise in junior high school was significantly associated with BMC (difference = 0.25 g, P = 0.0325) and BMD (difference = 0.062 g/cm2, P = 0.0077) in the femoral neck. Subjects with a high intact parathyroid hormone concentration (6.9 pmol/L) had lower BMC (difference = –4.00 g, P = 0.0112) and lower BMD (difference = 0.074, P = 0.0037) in the lumbar spine and lower BMC (difference = –0.38 g, P = 0.0035) and lower BMD (difference = 0.083 g/cm2, P = 0.0014) in the femoral neck than did subjects with a low intact parathyroid hormone concentration (< 6.9 pmol/L). Neither exercise in high school nor the presence of vitamin D insufficiency (cutoff concentration: 30 nmol/L) was associated with any of the bone mass variables.
The results of the stepwise multiple linear regression analyses to select independent variables predicting bone mass are shown in Table 5. BMD in the lumbar spine was predicted by weight and the METS index and negatively by presence of high intact parathyroid hormone. BMD in the femoral neck was predicted by calcium intake, weight, and exercise in junior high school and negatively by presence of high intact parathyroid hormone.
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TABLE 5. Multiple linear regression analyses with stepwise method for predictors of bone mineral content (BMC) and bone mineral density (BMD), selected from potential independent variables that were significant in the bivariate analyses1
DISCUSSION
Insufficient serum 25(OH)D concentrations were found in nearly 40% of women in the third decade (on the basis of a cutoff of 30 nmol/L for vitamin D insufficiency in adults; 2, 3) and in 14-19% of pubertal girls (on the basis of a cutoff of 25 nmol/L; 4, 17). In our study, 32.4% of the subjects were found to have a 25(OH)D concentration < 30 nmol/L (17.6% when 25 nmol/L was used as the other cutoff). There has been conflicting evidence about the consequences of low 25(OH)D on bone metabolism in young women. Some researchers have reported finding positive associations between the serum 25(OH)D concentrations of young adults or adolescents and BMD and bone strength at various bone sites (2, 4, 18, 19), whereas others have reported no evidence of such associations (3, 17, 20). Our data did not demonstrate that serum 25(OH)D concentrations alone predict BMD; however, the serum 25(OH)D concentrations of our subjects were negatively associated with their serum intact parathyroid hormone concentrations. An inverse correlation between serum 25(OH)D and serum intact parathyroid hormone concentrations has been established in elderly people by many clinical studies (21), and this study yielded the same finding in young people.
This study showed a negative association between calcium intake and intact parathyroid hormone concentrations, independent of the 25(OH)D concentration. Bonofiglio et al (22) reported a similar finding in Italian adolescent girls, in European countries, who had relatively low calcium intakes (mean calcium intake: 611 mg/d). On the other hand, there was no such association in young Finish populations with high calcium intakes (4, 19). The relation between calcium intake and serum parathyroid hormone may be observed exclusively in populations with low calcium intakes as compensatory hypersecretion of parathyroid hormone. Protein intake is also known to be associated with elevated parathyroid hormone secretion (23). This study found such an association using a correlation analysis but not with a multivariate analysis. This was because a strong correlation (r = 0.74) existed between calcium and protein intakes; thus, we could not determine whether protein intake was independently associated with serum intact parathyroid hormone concentrations.
A serum 25(OH)D concentration of 80 nmol/L is currently thought by researchers to be needed for optimal bone health (24). The serum 25(OH)D concentrations in all of the subjects in this study were below this concentration. Likewise, recently published concentrations of serum 25(OH)D in other young or pubertal populations have also been much lower than this threshold: 30 nmol/L in young adult black American women (25), 34 nmol/L in young Japanese women (2, 3), 39 nmol/L in Finnish female adolescents (4), and 46 nmol/L in 20-y-old Icelandic women (17). Therefore, the appropriateness of this threshold should be confirmed.
It was surprising that 16% of healthy young women had high concentrations of intact parathyroid hormone that exceeded the normal range (mild hyperparathyroidism). Moreover, these high parathyroid hormone concentrations were associated with low BMD. High parathyroid hormone concentrations are known to cause bone loss in elderly women, probably via increased bone turnover (1). An earlier study reported an inverse relation between serum parathyroid hormone concentrations and forearm BMD in adolescent girls (26). Our study, however, was the first to show a negative association between mild hyperparathyroidism and BMD in the spine and femoral neck in young women, and the nonsite-specific effect of the mild hyperparathyroidism observed in our study was noteworthy in confirming that finding. The low BMD in mild hyperparathyroidism can be explained by the high level of bone resorption, because of the negative association between serum intact parathyroid hormone and urinary NTX-I.
The results of our study showed that calcium intake was the primary factor predicting BMD of the femoral neck in young college females with an average calcium intake of 380 mg/d. This is in line with the rationale that sufficient calcium intake is essential for bone mass accrual in this age group. The calcium intake by the college females in this study was clearly low, but it was not exceptionally low, because low calcium intakes (around 400 mg/d) are commonly found in young other Asian populations (6, 8, 27, 28). A few studies have investigated young Asian adults for an association between dietary calcium intake and bone mass (6-8), but neither Ho et al (7) nor Saito et al (8) found significant independent associations between calcium intake and BMD in the lumbar spine or femoral neck. Their studies used a food-frequency questionnaire to estimate dietary calcium intake in relatively small populations, and these methodologic limitations may have been responsible for failing to detect any association. Our study used a more reliable technique to assess calcium intake, and thus the positive association with BMD in the femoral neck was valid.
A body of evidence, for the most part obtained from Western populations, has suggested that a high calcium intake is crucial to achieving maximal peak bone mass (5), and physical activity may play an important role in populations whose dietary calcium intake is much higher than in the present Japanese population (29-33). Although variables of current or past physical activity were significantly associated with bone mass in this study, their effect was small. This finding may be explained by the fact the subjects of this study were not very physically active. Only 12 (11%) of the subjects were currently engaged in regular sports activity (>30 min/d on average).
In contrast with the findings for the femoral neck, calcium intake was not associated with BMD in the lumbar spine. We have no plausible explanation for this finding but speculate that the calcium intakes in this population were too low to have varied sufficiently to allow detection of a significant association, ie, most of the subjects had calcium insufficiency on the basis of their lumbar BMD. This would explain the epidemiologic finding that the incidence of hip fracture in Japan is lower than that in the United States but that the incidence of vertebral fracture is higher (34). Interestingly, on the basis of a review of published studies regarding the positive effects of calcium intake on bone mass values in Western populations, Teegarden et al (35) reported the opposite, ie, that low calcium intakes in young adult white women (
This study enabled us to evaluate several relevant nutrients in relation to bone mass. Dietary sodium, potassium, and the ratio of ucOC to OC (an index of vitamin K nutrition) were not significantly associated with variables of bone mass or bone metabolism, and they seemed to be less relevant than were calcium or vitamin D.
The limitation of this study was its cross-sectional design, which sometimes yields results that are misleading in regard to a true causal relation, especially in regard to the relation between calcium intake and bone mass. A longitudinally designed study is needed to confirm the findings obtained in this study.
In conclusion, a low calcium intake (based on a low BMD of the femoral neck only) and mild hyperparathyroidism (based on a low BMD of both the femoral neck and lumbar spine) partly explained by low vitamin D nutrition and low calcium and protein intakes, are important independent predictors of low BMD in young adult Japanese women. Effects of poor nutrition and mild hyperparathyroidism on bone peak bone mass should be investigated further in young women.
ACKNOWLEDGMENTS
We thank Toyo Medic Inc (Tokyo, Japan) for assisting with the bone mass measurements, F Hoffmann-La Roche Ltd (Basel, Switzerland) for their gift of standard 25(OH)D3, and K Akazawa (Professor of Department of Medical Informatics, Niigata University Medical and Dental Hospital) for his advice concerning the statistical analysis.
KN, KU, and MY designed the study. KU, TN, and TS were responsible for the food analyses. YO was responsible for the bone mass measurements. YT was responsible for the blood and urine analyses. KN and KU were the principal authors of the paper. All authors reviewed the article. The authors had no conflict of interest.
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