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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Recent studies suggest that variations of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD). In this study, we examined the effect of vitamin D3 supplementation on BMD at the femoral neck in relation to VDR genotype. We analyzed 81 women, age 70 years and over, who participated in a placebo-controlled clinical trial on the effect of vitamin D3 supplementation (400 IU daily for at least 2 years) on BMD and fracture incidence. VDR genotype was based on the presence (b) or absence (B) of the BsmI restriction site. Mean BMD of the right and left femoral neck was measured at baseline and after 1 and 2 years. Dietary calcium, body mass index, and years since menopause were assessed at baseline while biochemical markers were measured at baseline and after 1 year. There was no difference among the BB, Bb, and bb genotype for baseline measurements of BMD at the femoral neck (mean and SD, g/cm2: 0.70 (0.10), 0.71 (0.12), and 0.69 (0.10), respectively), nor for any of the biochemical indices. The mean increase of BMD in the vitamin D group relative to the placebo group, expressed as percentage of baseline BMD, was significantly higher (p = 0.03) in the BB (ΔBMD: 4.4%, p = 0.04) and Bb genotype (ΔBMD: 4.2%, p = 0.007) compared with the bb genotype (ΔBMD: −0.3%, p = 0.61). No significant changes were found for any of the other measured parameters. The VDR genotype-dependent effect of vitamin D supplementation in these elderly subjects suggest a functional involvement of VDR gene variants in determining BMD.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bone mineral density is the major determinant of bone strength and decreases with aging, especially in women after menopause. Vitamin D deficiency is common in the elderly and may contribute to bone loss by causing increased activity of the parathyroid glands.1 Studies have shown a beneficial effect of vitamin D supplementation on bone mineral density (BMD) of the femoral neck in elderly people.2,3 However, the effect on BMD may vary according to the degree of vitamin D deficiency and estrogen status.2 Part of this variability may be explained by genetically determined characteristics of the individual. Recent studies have demonstrated an association of vitamin D receptor (VDR) gene variants with bone mass,4–6 although other studies did not find such a relationship.7,8 In keeping with a functional role of VDR gene variants in bone metabolism, the responsiveness of bone to calcium and vitamin D supplementation was reported to be associated with VDR genotype as well.6,9–11 The present study is the first longitudinal study in Caucasians that examined whether the effect of vitamin D supplementation on BMD of the femoral neck depends on the VDR genotype.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Subjects

Participants were 81 women, age 70 years and over (mean and SD; 78 ± 5 years), living in apartment houses and homes for the elderly. Subjects participated in a clinical trial (1989–1994, n = 2578) on the effect of vitamin D supplementation on the incidence of fractures.12 Participants were randomly allocated to the daily use of vitamin D (400 IU) or placebo tablets. Eligible subjects were participants in a subset (n = 348) of the population in which BMD measurements were performed. As reported earlier, vitamin D supplementation resulted in an increase of BMD of 2.2% at the femoral neck, relative to the change in the placebo group.2 Of this subset, 241 subjects were still alive at the time blood samples were collected for this study. Due to ethical, practical, and methodological considerations, blood samples were collected in subjects who were not severely impaired in cognition, were still in the area, and had not suffered a lower extremity fracture during the study period. Immobilization after a lower extremity fracture may lead to substantial bone loss and therefore affect the results. The protocol was approved by the Ethical Review Board of the Vrije Universiteit Hospital, Amsterdam.

Measurements

At baseline, body mass index (BMI, body weight/length2) was measured and daily dietary calcium intake was assessed by means of a questionnaire.13

BMD (g/cm2) was measured using dual-energy X-ray absorptiometry (DEXA, Norland XR-26), at the right and left femoral neck. Precision error, expressed as coefficient of variation (CV), is 1–2%.14 Measurements were done at baseline, after 1 year, and after 2 years. Data were not obtained on the last BMD measurements in three participants.

Blood samples and 2-h morning urine samples were obtained in fasting subjects. Serum alkaline phosphatase, calcium, albumin, and creatinine were measured using standard laboratory methods. Serum calcium was corrected for serum albumin using the following equation: corrected calcium = serum calcium + (40 − albumin [g/l])0.02 mmol/l. Measurements of the vitamin D metabolites were performed with competitive protein binding assays after isolation by gradient high performance liquid chromatography (HPLC). The intra- and interassay CVs were 5 and 6%, respectively, for 25-hydroxyvitamin D (25(OH)D) and 6 and 15% for 1,25-dihydroxyvitamin D (1,25(OH)2D). Serum intact PTH (PTH[1–84]) was measured in plasma, using a two-step immunochemical method, involving amino-terminal immunoextraction, followed by a midregion immunoassay. The interassay precision (CV) of this method is 10.2%.15 Osteocalcin was measured using a commercially available radioimmunoassay (Incstar Corp., Stillwater, MN, U.S.A.). Hydroxyproline was measured by HPLC with an interassay CV below 3.2% and expressed as the hydroxyproline/creatinine ratio (Hp/Cr).

The VDR genotype was determined by a direct haplotyping polymerase chain reaction (PCR) method measuring three restriction fragment length polymorphisms (RFLPs) simultaneously, i.e., for BsmI, ApaI, and TaqI.16 Only BsmI results are presented, in view of the low number of individuals. Absence of the restriction site is indicated by “B” and presence by “b.”

Data analysis

Differences between the VDR genotypes for baseline characteristics were tested using one-way analysis of variance (ANOVA). Since not all eligible subjects participated, selection might have jeopardized the randomization. Differences in baseline characteristics between the vitamin D and placebo users, for each genotype, were checked for by using Student's t-tests. For skewed variables, transformations were done if applicable. The change of BMD at the femoral neck in a 2-year period was calculated by subtracting the value of the first measurement from the measurement after 2 years for both the left and the right femoral neck. Subsequently, the change in BMD at the left and right femoral neck was averaged for each subject. The change in 25(OH)D, 1,25(OH)2D, PTH, and osteocalcin was calculated by subtracting baseline values from measurement values after 1 year. Differences in the change of BMD and other variables among the different genotypes between the placebo and vitamin D group were analyzed using linear regression analysis. When three comparisons were made for the change in BMD (Table 1), the level of significance was adjusted according to the Bonferroni procedure (α/number of comparisons). In case of three comparisons, a p < 0.02 was considered significant.

Table Table 1. Mean and Standard Deviation (SD) and for Skewed Parameters Median and 25th and 75th Percentiles (25–75%) for Baseline Characteristics According to the Vitamin D Receptor (VDR) Genotypes
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Covariates were entered to check for confounding and to enhance precision. The change in BMD for each genotype was also calculated as a percentage of mean baseline BMD in each genotype. For skewed variables, transformations were done if applicable, and median and 25th and 75th percentiles were given instead of mean values and standard deviations.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

VDR genotype frequencies and baseline characteristics for each VDR genotype are shown in Table 1. There was no significant difference at baseline among the VDR genotypes for any of the parameters measured (p > 0.15). Furthermore, no differences were found in baseline characteristics between vitamin D and placebo users for each genotype. Table 2 shows the changes in BMD and biochemical parameters for the vitamin D group and the placebo group for each VDR genotype. Biochemical parameters not shown in Table 2 did not change. The change in BMD in response to vitamin D supplementation was not significantly related to baseline 25(OH)D levels (p = 0.10) and 1,25(OH)2D levels (p = 0.10). Furthermore, the change in BMD was not related to baseline BMD values (p = 0.75). Change in body weight over the 2-year period was significantly related to the change in BMD (p < 0.01), but did not alter the relationship between the use of vitamin D and the change in BMD. To enhance precision, change in body weight was used as a covariate in the relationships between VDR genotypes and BMD. The difference in change between the placebo and the vitamin D group was (mean and SD, g/cm2) 0.031 (0.014) for the BB genotype, 0.030 (0.009) for the Bb genotype, and −0.002 (0.016) for the bb genotype. These differences in change of BMD between the placebo and vitamin D group, expressed as percentage of baseline BMD, were 4.4% for BB, 4.2% for Bb, and −0.3% for the bb genotype (Fig. 1). The difference in change of BMD between the placebo and the vitamin D group was significantly less for the bb group as compared with the Bb and the BB genotypes combined (p = 0.03). Adjustment for age, age at menopause, BMI, and dietary calcium intake did not change the relationship between BMD and the genotypes.

Table Table 2. The Change in Bone Mineral Density (BMD) at the Femoral Neck in a 2-Year Period, and the Change in Biochemical Indices in a 1-Year Period According to VDR Genotype and the Use of Vitamin D (D3) or Placebo (PI) to Which the Participants Were Randomly Allocated
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Figure FIG. 1. The mean change and standard deviation (error bars) in BMD at the femoral neck in a 2-year period, in the placebo ( ) and vitamin D (▪) groups, are shown. The change is expressed as a percentage of the baseline BMD in each VDR genotype. The difference between the change in BMD in the placebo and the vitamin D group was borderline significant for BB (p = 0.04) and significant for Bb (p = 0.007), adjusted for the change in body weight and according to Bonferroni's corrections.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study showed an association of VDR genotype with the magnitude of the vitamin D–induced change of femoral neck BMD. Changes in biochemical markers for bone metabolism were not related to the VDR genotype. The change in BMD in response to vitamin D administration was significantly less in the bb genotype compared with Bb and BB. The contrast in BMD between the vitamin D and the placebo group was partly a consequence of the increase in BMD in the placebo group in the bb genotype. However, the effect on BMD in the vitamin D group in the Bb and BB genotypes is two to three times that of the bb group. Since we do not know a priori whether the change of BMD in the placebo group is VDR genotype dependent, comparing BMD changes in the vitamin D versus the placebo group was the favorable approach. A similar approach was followed in a previous study.9

Not all randomized eligible subjects were involved in this study, therefore, selection may have biased our results. To check for any potential selection, we tested for baseline differences among the vitamin D and placebo users in each genotype. Furthermore, we adjusted for possible confounders. Other confounders, not measured in our study, may still have biased our results. This study involved a limited number of subjects who were unequally distributed among the different groups. The small number of subjects may have had consequences for statistical testing: although the parameters were tested for skewed distributions, outliers may still have biased these results. However, nonparametrical testing did not change the results. The unequal distribution of subjects among the groups to be compared made two-way ANOVA difficult to interpret. Therefore, separate analyses were performed for each genotype.

There was no difference in baseline BMD and biochemical parameters among the different VDR genotypes, which is in contrast with several other studies.5,6 The reason for the lack of association may be the limited number and the advanced age of the subjects. The relationship between BMD and the VDR genotypes is modified by age, such that the difference in BMD between the different genotypes decreases with age.16,17

A previous study of Japanese women demonstrated a VDR genotype-dependent response in BMD at the lumbar spine to supplementation with an active vitamin D metabolite.11 Opposed to our findings, they found the bb genotype associated with a better response. Most studies that reported an association between the VDR gene and BMD showed higher BMD values in those with the bb genotype.4–6 Similar to our study, in another Dutch population, i.e., the Rotterdam Study, a subgroup of the “b” allele was associated with low BMD and the “B” allele weakly with high BMD.16 Furthermore, two recently published studies also showed lower BMD in those with the bb genotype.18–20 Together with the present results on vitamin D supplementation, these results indicate allelic heterogeneity of the VDR locus in relation to bone metabolism between different populations, which may relate to ethnicity but also to other factors influencing bone metabolism, such as nutrition.

Alternatively, since an anonymous RFLP was analyzed, we cannot exclude linkage to another nearby bone metabolism–related gene. This seems, however, less likely in view of the central role of the VDR gene in vitamin D action and hence in its effect on bone mass. In keeping with this hypothesis, a study on short-term effects of calcitriol administration on the vitamin D endocrine system also demonstrated a VDR genotype dependency of the response,10 albeit more marked in the bb group than in the BB group. However, in that study, as in the Japanese study,11 an active vitamin D metabolite was used. In addition, the subjects in those studies were younger and their calcium intake was probably lower than in the present study. Furthermore, other studies have demonstrated VDR genotype to be associated with rates of bone loss and response to calcium supplementation.6,9

Taken together, our results provide evidence for a functional role of VDR gene variants in relation to bone mass. Assessing VDR genotype might help in explaining the variance in response to vitamin D supplementation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank Dr. Qiuju Huang (Erasmus University Medical School, Rotterdam) for technical assistance and Nel van de Kreeke and Els Lommerse (Academisch Ziekenhuis Vrije Universiteit, Amsterdam) for their help in collecting and processing the data. This work was supported by the Praeventiefonds, the Hague, the Netherlands (grants 00 282 4890 and 28–1112–1). Vitamin D and placebo tablets were provided by Solvay-Duphar, Inc., Weesp, The Netherlands.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Lips P, Obrant KJ 1991 The pathogenesis and treatment of hip fractures Osteoporos Int 1:218231.
  • 2
    Ooms ME, Roos JC, Bezemer PD, van der Vijgh WJ, Bouter LM, Lips P 1995 Prevention of bone loss by vitamin D supplementation in elderly women: A randomized double-blind trial J Clin Endocrinol Metab 80:10521058.
  • 3
    Chapuy MC, Arlot ME, Duboeuf F, Brun J, Crouzet B, Arnaud S et al. 1992 Vitamin D3 and calcium to prevent hip fractures in elderly women N Engl J Med 327:16371642.
  • 4
    Morrison NA, Qi JC, Tokita A et al. 1994 Prediction of bone density from vitamin D receptor alleles Nature (London) 367:284287.
  • 5
    Fleet JC, Harris SS, Wood RJ, Dawson-Hughes B 1995 The BsmI vitamin D receptor restriction fragment length polymorphism (BB) predicts low bone density in premenopausal black and white women J Bone Miner Res 10:985990.
  • 6
    Krall EA, Parry P, Lichter JB, Dawson-Hughes B 1995 Vitamin D receptor alleles and rates of bone loss: Influences of years since menopause and calcium intake J Bone Miner Res 10:978984.
  • 7
    Kroger H, Mahonen A, Ryhannen S, Turunen AM, Alhava E, Maenpaa P 1995 Vitamin D receptor genotypes and bone mineral density Lancet 345:1238.
  • 8
    Garnero P, Borel O, Sornay-Rendu E, Delmas P 1995 Vitamin D receptor gene polymorphisms do not predict bone turnover and bone mass in healthy premenopausal women J Bone Miner Res 10:12831288.
  • 9
    Ferrari S, Rizzoli R, Chevalley T, Slosman D, Eisman JA, Bonjour JP 1995 Vitamin-D-receptor-gene polymorphisms and change in lumbar-spine bone mineral density Lancet 345:423424.
  • 10
    Howard G, Nguyen T, Morrison N, Wantanabe T, Sambrook P, Eisman J, Kelly PJ 1995 Genetic influences on bone density: Physiological correlates of vitamin D receptor gene alleles in premenopausal women J Clin Endocrinol Metab 80:28002805.
  • 11
    Matsuyama T, Ishii S, Tokita A, Yabuta K, Yamamori S, Morrison NA, Eisman JA 1995 VDR gene polymorphisms and vitamin D analog treatment in Japanese Lancet 345:12381239.
  • 12
    Lips P, Graafmans WC, Ooms ME, Bezemer PD, Bouter LM 1996 Vitamin D supplementation and fracture incidence in elderly persons: A randomized, placebo-controlled clinical trial Ann Intern Med 124:400406.
  • 13
    Elders PJM, Netelenbos JC, Lips P, Khoe E, van Ginkel FC, Hulshof KFAM, van der Stelt PF 1989 Perimenopausal bone mass and risk factors Bone Miner 7:123132.
  • 14
    Genant HK, Faulkner KG, Gluer CC, Engelke K 1993 Bone densitometry: Current assessment Osteoporos Int 3 (Suppl 1):9197.
  • 15
    Hackeng WHL, Lips P, Netelenbos JC, Lips CJM 1986 Clinical implications of estimation of intact parathyroid hormone (PTH) versus total immunoreactivity PTH in normal subjects and hyperparathyroid subjects J Clin Endocrinol Metab 63:447453.
  • 16
    Uitterlinden AG, Pols HAP, Burger H et al. 1996 A large scale population based study of the association of vitamin D receptor gene polymorphisms and bone mineral density J Bone Miner Res 11:12411248.
  • 17
    Riggs BL, Nguyen T, Melton III LJ et al. 1995 The contribution of vitamin D receptor gene alleles to the determination of bone mineral density in normal and osteoporotic women J Bone Miner Res 10:991996.
  • 18
    Salamone LM, Ferrell R, Black DM, Palermo L, Epstein RS, Petro N, Steadman N, Kuller LH, Cauley JA 1996 The association between vitamin D receptor gene polymorphisms and bone mineral density at the spine, hip and whole-body in premenopausal women Osteoporos Int 6:6368.
  • 19
    Meunier PJ, Lindsay R 1996 Editorial Osteoporos Int 6:187188.
  • 20
    Houston LA, Grant SFA, Reid DM, Ralston SH 1996 Vitamin D receptor polymorphism, bone mineral density, and osteoporotic vertebral fracture: Studies in a UK population Bone 18:249252.