Low calcium intake is associated with high plasma ... - Springer Link

J Bone Miner Metab (2014) 32:317–323 DOI 10.1007/s00774-013-0499-9

ORIGINAL ARTICLE

Low calcium intake is associated with high plasma homocysteine levels in postmenopausal women Shiro Tanaka • Kazuhiro Uenishi • Yasushi Yamazaki Tatsuhiko Kuroda • Masataka Shiraki

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Received: 15 April 2013 / Accepted: 8 July 2013 / Published online: 20 August 2013 Ó The Japanese Society for Bone and Mineral Research and Springer Japan 2013

Abstract Nutritional interventions targeting homocysteine remain controversial, and further nutritional research is warranted. We thus sought to explore the determinants of plasma homocysteine other than B-group vitamins. This cross-sectional study surveyed the nutritional status of 713 Japanese postmenopausal women using a semiquantitative food frequency questionnaire. Associations between total energy, protein, fat, carbohydrate, and vitamin A and K intakes and homocysteine were insignificant. Mean homocysteine in the second (536.1 ± 34.7 mg/day) and third (712.9 ± 115.6 mg/day) tertiles of calcium intake were lower than in the first tertile (379.6 ± 76.6 mg/day) by -0.57 nmol/mL (95 % confidence interval, -1.10 to -0.04, p = 0.04) and -1.18 nmol/mL (-1.76 to -0.60, p \ 0.01), respectively, after adjustment for lifestyle and clinical factors (trend p \ 0.01). Mean homocysteine in those with dietary calcium intake above the median ([536 mg/day) were lower regardless of the folic acid concentration; the differences were -1.59 nmol/mL

(-2.33 to -0.85, p = 0.02) and -0.75 nmol/mL (-1.37 to -0.12, p \ 0.01) for the high (\7.8 ng/mL) and low folic acid groups, respectively. There was no significant association between calcium and folic acid (p = 0.08). In conclusion, further prospective research to confirm our findings is needed for the development of nutritional inventions targeting homocysteine. Keywords Calcium deficiency Cross-sectional study Homocystinuria Nutritional epidemiology Abbreviations Cr Creatinine eGFR Estimated glomerular filtration rate FFQPOP Food frequency questionnaire for the prevention and management of osteoporosis hs-CRP High-sensitive C-reactive protein MTHFR Methylene tetrahydrofolate reductase PTH Serum parathyroid hormone 25(OH)D 25-Hydroxy-vitamin D

S. Tanaka Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan K. Uenishi Division of Nutritional Physiology, Kagawa University, 3-9-21 Chiyoda, Sakaido, Saitama 350-0288, Japan Y. Yamazaki M. Shiraki (&) Department of Internal Medicine, Research Institute and Practice for Involutional Diseases, 1610-1 Meisei, Misato, Azumino, Nagano 399-8101, Japan e-mail: [email protected] T. Kuroda Public Health Research Foundation, 1-1-7 Nishi-Waseda, Shinjyuku-ku, Tokyo 169-0051, Japan

Introduction Osteoporosis and atherosclerosis are major health burdens leading to death and immobilization in an aging society such as that in Japan [1, 2]. These two morbid states frequently coexist, and an elevated plasma homocysteine concentration is known as a common risk factor for fractures in persons with osteoporosis or cardiovascular events in patients with atherosclerosis [3–6]. Mildly elevated plasma homocysteine levels have been recognized in people bearing the TT genotype of the methylene

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tetrahydrofolate reductase (MTHFR) gene and those with an insufficiency of B-group vitamins [7] or an excess methionine load [8]. It is possible to prevent elevations in homocysteine by nutritional interventions; dietary fortified folic acid, vitamin B12 [9], or their supplements [10] have been shown to reduce homocysteine concentrations. With regard to fracture prevention, administration of folate and mecobalamin, which are potent regulators for decreasing plasma homocysteine, was reported to reduce the risk of hip fracture in stroke patients [11]. On the other hand, reduction in homocysteine by administration of vitamin B12 and folate did not prevent cardiovascular diseases, [1, 12] but increased cancer mortality [13]. Taken together, nutritional interventions targeting homocysteine remain controversial, and further nutritional research is warranted. We thus sought to explore the determinants of plasma homocysteine other than B-group vitamins in a cross-sectional study of postmenopausal Japanese women.

Materials and methods Subjects The Nagano Cohort recruited and followed up women who were receiving medical care as outpatients at a medical institute in Nagano Prefecture, Japan, beginning in April 1993 [1–3]. Of the 2,010 subjects registered until April 2011, a dietary survey was conducted in 947 subjects. After excluding premenopausal women and individuals with endocrine disorders such as thyroid dysfunction and hyperparathyroidism, a history of extensive gastrointestinal surgery, chronic renal failure, or steroid use, 713 postmenopausal women were included in this analysis. The Ethics Committee at the Research Institute and Practice for Involutional Diseases approved the study protocol, and written informed consent was obtained from all subjects prior to participation. All protocols were in compliance with the principles laid down in the Declaration of Helsinki. Assessments At the time of registration, data on the participants’ chronological age, age at menarche and menopause, body weight, body height, and body mass index (BMI) were recorded. BMI was calculated by the formula—body weight (in kilograms) divided by the square meter of body height. A questionnaire elicited information on alcohol and tobacco use. Dietary intake was assessed by the use of a semiquantitative food frequency questionnaire (Food Frequency Questionnaire for the Prevention and Management of Osteoporosis; FFQPOP). Details of the FFQPOP,

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including its reliability, were reported elsewhere [14]. In brief, the FFQPOP consists of 28 items on food intake within the past 2–3 months and the correlation coefficients between estimates from the FFQPOP and the conventional diet record method were 0.668 for calcium, 0.475 for sodium, 0.501 for vitamin A, 0.413 for vitamin D, 0.649 for vitamin K and 0.471 for energy [14]. Systolic blood pressure of C140 mmHg, diastolic blood pressure of C90 mmHg, or treatment with antihypertensive medications indicated the presence of hypertension. Participants were considered to have type 2 diabetes if the concentration of HbA1c was C6.5 %, or if they were undergoing treatment for diabetes. Dyslipidemia was defined as fasting triglyceride levels of C150 mg/dL, high-density lipoprotein (HDL) cholesterol levels of \40 mg/dL, or treatment with lipid-lowering agents. Biochemical indices Non-fasting serum, plasma, and urine samples were collected. Routine biochemical examinations included serum creatinine (Cr), total protein, triglycerides, total and HDL cholesterol, HbA1c, serum parathyroid hormone (PTH), serum calcium, high-sensitive C-reactive protein (hs-CRP), and uric acid. Estimated glomerular filtration rate (eGFR) was calculated from the serum level of creatinine as follows—eGFR (mL/min/1.73 m2) = 194 9 Cr - 1.094 9 age - 0.287 9 0.739 [15]. We used the National Glycated Hemoglobin Standardization Program value for HbA1c, calculated as follows—0.25 ? 1.02 9 Japan Diabetes Society value [16]. Serum level of PTH was measured using the intact PTH CLEIA kit (Mitsubishi Chemical Medience Corporation, Tokyo, Japan). Serum level of calcium adjusted for albumin was calculated by the following formula—calcium = serum calcium ? (4 - albumin). Vitamin B12 and folic acid in serum were measured by chemiluminescent methods using Chemilumi-ACS vitamin B12 and folic acid assay kits (Bayer Medical, Tokyo, Japan) at the same laboratory (Mitsubishi Chemical Medience Corporation). Plasma levels of total homocysteine were measured by an HPLC system [17]. Serum 25-Hydroxy-Vitamin D (25(OH)D) was measured using a competitive protein-binding assay after extraction and purification of the samples using HPLC at Teijin Bio Science Laboratories (Hino, Tokyo, Japan). Statistical analysis To explore the determinants of homocysteine, we classified plasma homocysteine concentrations by tertile and compared nutritional intake among the tertiles by general linear models and trend tests adjusted for age, BMI, waist circumference, hypertension, dyslipidemia, type 2 diabetes,


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smoking, alcohol consumption, HbA1c, low-density lipoprotein (LDL) cholesterol, HDL cholesterol, log triglycerides, eGFR, uric acid, and log hs-CRP. Mean homocysteine concentrations from nutritional intake were compared by general linear models adjusted for age, BMI, waist circumference, hypertension, dyslipidemia, type 2 diabetes, smoking, alcohol consumption, 25(OH)D, log vitamin B12, folic acid, HbA1c, LDL cholesterol, HDL cholesterol, log triglycerides, eGFR, uric acid, and log hs-CRP. Vitamin B12, triglycerides and hs-CRP were log-transformed only as adjustment factors and the median and interquartile range of raw values were used for descriptive analysis as shown in Table 2. Effect modification was explored by interaction tests using general linear models with the covariates listed above. Missing data were treated by the multiple imputation method using the adjustment variables. All reported p values are two-sided and the significance level was set at 5 %. All statistical analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA).

Results The mean and standard deviation (SD) of age, dietary calcium, and total energy intake of the 713 postmenopausal women were 65.1 ± 9.8 years, 543 ± 159 mg/day and 1,612 ± 251 kcal/day, respectively. Both dietary calcium intake and total energy intake were almost identical to the average reported in the National Health and Nutrition Survey in Japan (501 mg/day and 1,682 kcal/day) [18], but the calcium intake was approximately 30 % less than that in the Framingham Osteoporosis Study population (757 ± 354 mg/day) [19]. Although we checked for the use of supplements carefully, some participants who

reported taking no vitamin supplements at registration had extremely high levels of serum vitamin B12 (8 women with C3,000 pg/mL), suggesting that these subjects might have been taking vitamin B12 that had been bought over-thecounter or by a doctor’s prescription. Taking into consideration the difficulty in precise estimation of B-group vitamin intake by food frequency questionnaires and the potential influence of atrophic gastritis, which can reduce absorption of vitamin B12 in foods from the digestive tract [20], direct measurements of vitamin B12 and folic acid in serum samples appeared to be the most reliable means of assessing the vitamin B status in this study. Table 1 summarizes the background factors of the 713 postmenopausal women according to tertiles of plasma homocysteine concentrations. As the study population consisted of outpatients in a clinic in Japan, around half had osteoporosis, dyslipidemia or hypertension. We found significant positive associations with age and the prevalence of hypertension, and a significant negative association with smoking rates. Smokers were the most frequent in the third tertile of homocysteine, but the smoking rate was low (3.2 %). There were no clinically significant differences in BMI across tertiles, although we found statistical significance by testing for trends. Table 2 shows laboratory measurements across the tertiles of homocysteine. There were significant decreasing trends in folic acid, HDL cholesterol, and eGFR and increasing trends in hs-CRP and serum calcium across the tertiles. Table 3 shows a comparison of the nutritional intake between tertiles. There were no significant associations between total energy, protein, fat, carbohydrate, and vitamin A and K intakes and homocysteine. In contrast, dietary calcium correlated negatively with homocysteine concentrations. Table 4 shows the differences in plasma homocysteine levels across tertiles of dietary calcium. The mean

Table 1 Characteristics of the 713 postmenopausal women according to tertile of homocysteine at initial examination

Homocysteine (nmol/mL)

Tertile 1 of homocysteine (N = 223)



Mean

SD

Mean

SD

Mean

6.40

0.77

8.33

pA

SD \0.01

0.54

12.34

3.67

Age (years)

61.3

8.8

64.5

9.3

69.1

9.8

\0.01

Body mass index (kg/m2)

22.0

3.0

23.1

3.4

22.8

3.4

\0.01

Waist circumference (cm)

83.9

8.6

86.8

7.9

86.5

8.6

Hypertension

50.7 %

Dyslipidemia

69.1 %

74.5 %

0.02 \0.01

57.4 %

65.0 %

59.5 %

0.22

Type 2 diabetes

9.9 %

12.8 %

11.3 %

0.69

Smoker

1.8 %

0.8 %

3.2 %

0.0499

Alcohol drinker

9.9 %

5.8 %

3.6 %

0.13

SD standard deviation A

Trend test adjusted for age

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Table 2 Laboratory measurements of the 713 postmenopausal women according to tertile of homocysteine





Mean

Mean

Mean

SD

6.40

Serum vitamin B12 (pg/mL)B Serum folic acid (ng/mL) 25(OH)D (ng/mL) HbA1c (%, NSGP)

0.77

SD

8.33

0.54

pA

SD

12.34

3.67

\0.01

632.0

343.0

592.0

341.0

527.0

293.0

0.06

9.7 22.0

4.1 6.6

8.5 21.4

3.7 6.5

7.5 20.9

3.0 5.9

\0.01 0.09

5.77

0.66

5.80

0.70

5.98

1.41

0.15

LDL cholesterol (mg/dL)

123.8

26.2

128.2

29.5

119.5

30.3

0.48

HDL cholesterol (mg/dL)

68.5

17.1

64.9

18.4

60.4

17.1

\0.01

125.0

105.0

123.0

87.0

128.0

103.0

0.18

73.5

18.0

71.7

16.0

64.5

19.2

\0.01

Triglycerides (mg/dL)B eGFR (mL/min/1.73) hs-CRP (mg/dL)B

0.04

PTH (pg/mL)

0.05

35.9

0.05

14.4

0.07

38.7

19.7

0.06 39.4

0.12 17.2

0.01 0.12

Uric acid (mg/dL)

4.21

1.01

4.48

0.99

4.64

1.10

\0.01

Serum calcium (albumin-adjusted, mg/dL)

9.00

0.43

9.08

0.46

9.14

0.47

0.03

SD standard deviation, 25(OH)D 25-hydroxy-vitamin D, eGFR estimated glomerular filtration rate, hs-CRP high sensitive C-reactive protein, PTH parathyroid hormone A

Trend test adjusted for age

B

Median and inter-quartile range

Table 3 Dietary intake of the 713 postmenopausal women according to tertile of homocysteine

Homocysteine (nmol/mL) Total energy intake (kcal/day) Protein intake (g/day) Fat intake (g/day)




Mean

Mean

Mean

6.40

SD 0.77

8.33

SD 0.54

12.34

pA

SD 3.67

–

1,612.2

237.8

1,625.6

259.7

1,596.2

254.1

0.27

73.7 59.1

12.6 11.3

74.3 60.0

15.2 12.3

74.5 58.8

14.5 11.9

0.41 0.22

Carbohydrate intake (g/day)

203.3

37.8

204.3

38.1

199.6

37.4

0.47

Calcium intake (mg/day)

571.4

158.8

550.7

168.8

509.9

141.8

\0.01

Salt intake (g/day) Vitamin A intake (lg/day)

10.9

1.7

11.2

1.9

11.1

1.6

0.69

984.9

379.9

1,101.5

729.0

1,049.8

612.4

0.86

Vitamin D intake (lg/day)

12.4

1.9

12.2

2.3

12.0

2.2

0.02

Vitamin K intake (lg/day)

327.1

137.8

298.7

134.4

302.6

139.2

0.17

SD standard deviation, BMI body mass index, 25(OH)D 25-hydroxy-vitamin D, LDL low-density lipoprotein, HDL high-density lipoprotein, eGFR estimated glomerular filtration rate, hs-CRP high-sensitive C-reactive protein A Trend test adjusted for age, BMI, waist circumference, hypertension, dyslipidemia, type 2 diabetes, smoking, alcohol consumption, HbA1c, LDL cholesterol, HDL cholesterol, log triglycerides, eGFR, uric acid, and log hs-CRP

homocysteine levels in the second and third tertiles of dietary calcium were lower than in the first tertile by -0.57 nmol/mL (p = 0.04) and -1.18 nmol/mL (p \ 0.01), respectively, after adjustment for lifestyle and clinical factors. The decreasing trend was statistically significant. Figure 1 shows box plots of homocysteine concentrations in four categories according to the medians of calcium intake (\536 or C536 mg/day) and serum concentration of folic

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acid (\7.8 or C7.8 ng/mL). The mean homocysteine concentrations in the high calcium groups within each folic acid group were lower than those in the low calcium groups; differences were -1.59 nmol/mL (95 % confidence interval, -2.33 to -0.85, p = 0.02) for the high folic acid groups and -0.75 nmol/mL (-1.37 to -0.12, p \ 0.01) for the low folic acid groups. There was no significant interaction between calcium and folic acid (p = 0.08).


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Table 4 Differences in plasma homocysteine levels between higher tertiles and the first tertile of dietary calcium Tertile 1 of dietary calcium (379.6 ± 76.6, N = 235)

Tertile 2 of dietary calcium (536.1 ± 34.7, N = 242)

Mean

95 %CI

Mean

Unadjusted

9.85

9.43 to 10.26

9.09

8.68 to 9.50

8.42

AdjustedA

9.71

9.32 to 10.10

9.14

8.77 to 9.51

8.53

Difference from T1

–

-0.57

95 %CI

-1.10 to -0.04

Tertile 3 of dietary calcium (712.9 ± 115.6, N = 236) p

0.04

Mean

-1.18

95 %CI

Trend p p

8.00 to 8.83 8.14 to 8.92 -1.76 to -0.60

\0.01

\0.01

CI confidence interval, BMI body mass index, 25(OH)D 25-hydroxy-vitamin D, LDL low-density lipoprotein, HDL high-density lipoprotein, eGFR estimated glomerular filtration rate, hs-CRP high-sensitive C-reactive protein A Adjusted for age, BMI, waist circumference, hypertension, dyslipidemia, type 2 diabetes, smoking, alcohol consumption, 25(OH)D, log vitamin B12, folic acid, HbA1c, LDL cholesterol, HDL cholesterol, log triglycerides, eGFR, uric acid, and log hs-CRP


40

30

20

10

0 high/high

high/low

low/high

low/low

Dietary calcium/folic acid Fig. 1 Plasma homocysteine concentration according to dietary calcium and serum folic acid concentration classified by their median

Discussion Homocysteine is an intermediate metabolite of the methionine cycle, which is regulated by enzymes and the vitamin B group as a co-factor. Accumulation of homocysteine has been known to be a risk factor for atherosclerosis [6], osteoporotic fractures [4, 5], and frailty [21], but its mechanism is not completely understood, although oxidative stress [22] is likely to be involved. In this context, we have reported significant associations between plasma homocysteine concentrations and age, renal function, vitamin B12 and folate values, and gene polymorphism of MTHFR [3]. In this study, low dietary calcium was significantly associated with an elevated plasma homocysteine concentration in addition to these known factors. Until now, the negative association between dietary calcium intake and plasma homocysteine concentration had

been mentioned in only one paper which reported on the OFELY study [23]; however, the paper contained no comments on this phenomenon. The OFELY study and the present study were both cross-sectional, and it is impossible to conclude a causal relationship or any possible mechanism(s) responsible for this observation. A major difference between the two studies is the amount of calcium intake; the average intake in the OFELY study was around 800 mg/day, which exceeded that in our study by approximately 250 mg/day. A reasonable explanation for this may be ethnic differences, given that habitual calcium intake in Japan is generally less than that in Western countries; the possibility of a confounding bias makes it difficult to compare these cohorts directly. Homocysteine is regulated by the B-vitamin group, but the association between dietary calcium and homocysteine concentration was not dependent on folic acid (Fig. 1). Furthermore, this association remained significant even after adjustments for age, BMI, waist circumference, hypertension, dyslipidemia, type 2 diabetes, smoking, alcohol consumption, 25(OH)D, log vitamin B12, folic acid, HbA1c, LDL cholesterol, HDL cholesterol, log triglycerides, eGFR, uric acid, and log hs-CRP, excluding the potential involvement of confounding factors. Calcium is known to have several beneficial metabolic effects on health. It is well established that calcium supplements lower blood pressure, especially in populations with a low habitual calcium intake (i.e., B800 mg/day) [24]. There are biologically plausible mechanisms for the benefits of calcium in the regulation of body weight [25], although systematic reviews of interventional trials [25] found inconsistent results, making firm conclusions difficult. Other potential benefits were reported for type 2 diabetes [26] and metabolic syndrome [27], but evidence is also sparse. However, these reports support the findings of the present study. Nonetheless, the findings of this study should be interpreted in the context of some limitations. First, the study population consisted of patients who visited a primary care institution, leading to potential

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selection bias. However, anthropometric measurements, nutritional background, and frequencies of co-morbidities were almost the same as in the National Health and Nutritional Survey in Japan. Furthermore, the patient characteristics were not markedly different from the general Japanese population at the given age. Another limitation was the study design, which was cross-sectional, making it difficult to conclude any causative relationship between calcium intake and plasma homocysteine regulation; however, the present results may instigate investigations on this relationship. In conclusion, we found a negative association between dietary calcium and plasma homocysteine concentrations. Further prospective studies to confirm our findings are needed for the development of nutritional inventions targeting homocysteine. Acknowledgments This work was supported in part by a Grant-inAid from the Japan Osteoporosis Foundation and Japan Dairy Association. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. The sponsors of the study played no role in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript, and in the decision to submit the manuscript for publication. Conflict of interest The authors declare that they have no conflict of interest to disclose.


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