Zhen-lin ZHANG1, Yue-juan QIN, Qi-ren HUANG, Jin-wei HE, Miao LI, Qi ZHOU, Yun-qiu HU, Yu-juan LIU
Center for Preventing and Treating Osteoporosis, Osteoporosis Research Unit, The Sixth People's Hospital, Shanghai Jiaotong University, Shanghai 200233, China
1 Correspondence to Dr Zhen-lin ZHANG. Phn/Fax 86-21-6408-1474. E-mail ZZL2002@medmail.com.cn
Received 2003-12-01 Accepted 2004-09-16
KEY WORDS bone density; estrogen receptors; calcitriol receptors; polymorphism (genetics)
ABSTRACT
AIM: To investigate the correlation between calcium treatment in postmenopausal women and estrogen receptor-alpha (ER-alpha) Xba I and Pvu II genotype and vitamin D receptor (VDR) Apa I genotype. Methods: One hundred fifteen postmenopausal Chinese women of Han population were enrolled and treated with calcichew-D3 (1000 mg calcium and 400 U vitamin D3) daily for 1 year. At entry and after 1 year treatment, the bone mineral density (BMD), serum and urinary bone turnover biochemical markers were evaluated. ER-alpha and VDR genotype were analyzed using PCR-restriction fragment length polymorphism. Results: After 1 year of calcium supplementation, a significant increase of BMD and a marked reduction in serum ALP and PTH levels, and a significant increase of serum 25-(OH) vitamin D level were observed (P<0.01 or P<0.05). At entry and after 1 year of treatment, no significant association was found between Xba I, Pvu II, and Apa I genotypes and BMD in L1-4, Neck, and Troch, and all bone turnover marker levels. However, the percentage of change (median, QR) in Neck BMD was significantly different in homozygous XX [-4.14 (from -6.54 to -1.34)] in comparison with Xx [1.72 (from -1.12 to 3.20)] (P<0.001) or xx [1.22 (from -1.74 to 3.06)] Xba I ER-alpha genotype (P=0.001). CONCLUSION: Women with ER-a Xba I genotype XX may have a higher risk of relatively fast bone mass loss in femoral neck after menopause and that they may have a poor responsiveness to calcium supplementation. The changes in BMD are not associated with ER-alpha Pvu II genotype and VDR Apa I genotype after 1 year of calcium supplementation.
INTRODUCTION
Osteoporosis is a disease of low bone mineral mass and microarchitectural deterioration of bone, which leads to increased risk of fracture. Postmenopausal osteoporosis depends on both the peak bone mass in early adulthood and the rate of bone loss after menopause. The peak bone mass achieved and the rate of postmenopausal bone loss are under strong genetic influence[1]. The significant association between Bsm I polymorphism of vitamin D receptor (VDR) gene and bone mineral density (BMD) variation in Caucasian women has been reported[2]. In postmenopausal women the rate of bone loss is also associated with VDR and estrogen receptor-alpha (ER-alpha) gene polymorphism[3,4].
Bone mass in the elderly can be maintained in some individuals with calcium and vitamin D supplementation. But, the anti-osteoporotic treatments present variability in terms of BMD. The variability may be due to genetic factors[5]. In fact, Bsm I polymorphism in VDR gene may modify the BMD response to calcium intake, calcium and vitamin D supplementation, and hormone replacement therapy[6,7]. However, the frequencies of distribution of Bsm I polymorphism in VDR gene in Chinese Han population are significantly different to those of Caucasian. Bsm I BB genotype is rare in Chinese population[8]. Moreover, to our knowledge, no data is available in publications regarding the Apa I polymorphism in VDR gene and the different BMD response to calcium supplementation. In addition, postmenopausal women can be classified as fast and slow bone losers, and postmenopausal estrogen deficiency is related closely to the risk of osteoporosis, but the association between ER-alpha genotype and responsiveness to calcium supplementation in postmenopausal women is unclear. As a result, assessing the association between VDR and ER-alpha genotypes and the effect of calcium supplementation in postmenopausal women should be helpful in choosing anti-osteoporosis treatment according to individual genotype[9,10]. In this study, our aim was to investigate the influence of VDR Apa I and ER-alpha Pvu II and Xba I genotypes on bone loss rates and responsiveness to calcium supplementation in postmenopausal Chinese women.
MATERIALS AND METHODS
Study population The protocol was approved by the Ethical Committee of the Sixth People's Hospital, Shanghai Jiaotong University. The study population comprised 125 unrelated postmenopausal Chinese women of Han ethnicity in Shanghai (62.6±5.5 years old) who visited outpatient clinics of the Sixth People's Hospital, Shanghai Jiaotong University. Postmenopausal women with early menopause (before 40 years of age) and those that had undergone ovariectomy were excluded. None had a history of bone disease or drug use that might affect bone turnover. Using 7 d diet record, the participants recorded daily intakes of food and beverages after oral and written instructions. We reviewed the records with the participants and calculated dietary calcium from food composition tables. The dietary calcium intake was 300-500 mg/d in study individuals. The subjects enrolled received oral calcichew-D3 (Nycomed Pharma, AS, Oslo, Norway) daily including 1000 mg calcium and 400 U vitamin D3. Among the 125 postmenopausal women, 115 participants could be genotyped and analyzed for BMD and bone turnover markers changes after 1 year.
Genotyping Genomic DNA was extracted and purified from EDTA blood samples using routine procedure. Genotypic analysis of VDR gene Apa I, ER-alpha gene Pvu II, and Xba I polymorphisms was determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). VDR gene fragment including the Apa I polymorphism site was amplified using primers 5´-CAGAGCATGGACAGGGA-GCAA-3´ and 5´-GCAACTCCTCATGGCTGA-GGTCTC-3´[3] . ER-alpha gene fragment including the Pvu II and Xba I polymorphism sites was amplified using primers 5´- CTGCCACCCTATCTGTATCTTTTC-CTATTCACC-3´ and 5´- TCTTTCTCTGCCACCCTG-GCGTCGATTATCTGA-3´[4] . The PCR was carried out in 30 µL of a buffer solution: Tris-HCl 10 mmol/L, KCl 50 mmol/L, MgCl2 1.5 mmol/L, 200 µmol/L each of the four deoxyribonucleotides (dNTPs), 2.5 U of Taq polymerase, and 0.25 µmol/L of each primer. PCR was performed with the following steps: at 94 ºC for 5 min and then at 94 ºC for 1 min, at 60 ºC for 1 min, at 72 ºC for 1 min, for 30 cycles, and at 72 ºC for 7 min. After amplification, ER-alpha gene fragment was digested with Pvu II and Xba I restriction endonuclease and electrophoresed in 2.0 % agarose gel, respectively. Absence of the Pvu II and Xba I restriction sites were indicated by "P" and "X" and presence by "p" and "x", respectively. VDR gene fragment was digested with Apa I restriction endonuclease and electrophoresed in 1.8 % agarose gel. Absence the restriction site was indicated by "A" and presence by "a".
BMD measurements The BMD of the lumbar spine 1-4 (L1-4) and left proximal femur including femoral neck (Neck), and trochanter (Troch) were measured using dual-energy X-ray absorptiometry (DXA) (Hologic QDR-2000, Hologic corporation, Waltham, MA, USA) before and after 1 years of treatment. The short-term reproducibilities [coefficient of variation (CV) %] of L1-4, Neck, and Troch measurements were 0.97 %, 1.93 %, and 1.48 %, respectively. The long-term reproducibility of our DXA instrument during the trial based on weekly repeated phantom measurements was 0.45 %.
Clinical examinations and biochemical studies During the follow-up each subject visited the outpatient clinic once a year. Fasting venous blood samples and urinary were obtained and body weight, and height were measured. At the entry and 1 year after treatment, the concentrations of serum calcium, phosphate, alkaline phosphatase (ALP), parathyroid hormone (PTH), 25-hydroxy[25-(OH)]vitamin D, osteocalcin (OC), urinary creatinine-corrected free pyridinoline (PYD), and calcium were measured. Serum intact PTH, 25-(OH)vitamin D, and OC were determined by radioimmunoassay (RIA). The intra-assay and inter-assay CVs were both <10 %. Urinary PYD was measured using enzyme-linked immunosorbent assay (ELISA). The intra-assay and inter-assay CVs for urinary PYD were 4.2 % and 7.9 %, respectively.
Statistical analysis The x 2 test was used for Hardy-Weinberg of ER-alph Pvu II and Xba I genotypes and VDR Apa I genotype. One-year after calcium supple-mentation, percent change in bone turnover markers and BMD were not distributed normally and expressed as the median [interquartile range (IQR)]. One-year changes in bone turnover markers or BMD were (One-year BMD or bone turnover marker-baseline BMD or bone turnover marker)×100/baseline BMD or bone turnover marker. In the postmenopausal women completing 1-year treatment as assigned, the values of BMD and bone turnover chemical markers were compared in the baseline and 1-year after calcium treatment using an unpaired t-test. An association between Apa I, Pvu II , and Xba I genotypes and BMD was evaluated by covariance (ANCOVA). The percent changes in BMD and bone turnover markers in each genotype were analyzed using Kruskal-Wallis H test. All statistical analyses were performed with SPSS 9.0 software and P<0.05 was considered statistically significant.
RESULTS
Genotype frequencies of the study population The distribution of Pvu II genotype was as follows: PP 23.5 %, Pp 45.2 %, and pp 31.3 %, respectively. Frequencies of XX, Xx, and xx genotype were 7.8 %, 40.9 %, and 51.3 %, respectively. The distribution of Apa I genotype was as follows: AA 7.8 %, Aa 38.9 %, and aa 53.3 %, respectively. The genotypes distribution of Pvu II, Xba I, and Apa I were compatible with the population in the Hardy-Weinberg equilibrium.
Characteristics of the study population One-year after supplementation for 1000 mg calcium and 400 U vitamin D3 daily, compared with bone turnover markers at the baseline, the concentrations of serum ALP and PTH were significantly decreased (P<0.05 or P<0.01), and serum 25-(OH) vitamin D was markedly increased (P<0.01), but the values of serum OC and urinary PYD and calcium had no significant difference. After 1-year treatment of calcium, the BMD at L1-4, neck, and troch sites was significantly increased compared with that of baseline (P<0.01 or P<0.05) (Tab 1).
Tab 1. Characteristics of postmenopausal women at baseline and 1 year after calcium supplementation. Median changes and 25th to 75th percentile (interquartile) range in bone turnover markers and BMD in comparison with baseline in each postmenopausal woman supplemented with calcium 1000 mg daily for 1 year. n=115. Mean±SD.
| |
Baseline |
After calcium |
Bone turnover markers |
P value |
| |
|
supplementation |
and BMD change (%) |
|
| Age/year |
62¡À5 |
63¡À5 |
- |
0.801 |
| Years
since menopause/year |
13¡À6 |
14¡À6 |
- |
0.709 |
| Height/cm |
154¡À5 |
152¡À6 |
- |
0.638 |
| Weight/kg |
57¡À8 |
58¡À8 |
- |
0.395 |
| ALP/IU¡¤L-1 |
71¡À20 |
67¡À15 |
-7.3 (-20.0~0.9) |
0.019 |
| BGP/mg¡¤L-1 |
13¡À8 |
12¡À5 |
-3.8 (-36.5~46.0) |
0.556 |
| PTH/ng¡¤L-1 |
31¡À9 |
23¡À9 |
-27.8 (-44.0~-7.0) |
0.000 |
| 25-(OH)
vitamin D/mg¡¤L-1 |
10¡À9 |
17¡À9 |
126.7 (34.0~320.0) |
0.000 |
| Urinary
PYD/ nM¡¤mM-1 Cr |
41¡À35 |
41¡À17 |
29.2 (-24.9~93.2) |
0.975 |
| Urinary
Ca/nM¡¤mM-1 Cr |
0.44¡À0.21 |
0.43¡À0.24 |
-0.4 (-33.3~46.8) |
0.708 |
| L1-4
BMD/g¡¤cm-2 |
0.77¡À0.10 |
0.78¡À0.10 |
0.73 (-0.86~2.72) |
0.001 |
| Neck
BMD/g¡¤cm-2 |
0.61¡À0.07 |
0.62¡À0.07 |
1.15 (-1.74~3.05) |
0.000 |
| Troch BMD/g¡¤cm-2 |
0.49¡À0.06 |
0.50¡À0.06 |
1.03 (-0.85~3.17) |
0.018 |
Association between the genotype and BMD and bone turnover markers At baseline and after 1-year treatment, no significant association was found between Pvu II, Xba I, and Apa I genotypes and BMD at L1-4 and any sites of proximal femur adjusted for age, weight, and years since menopause. However, Xba I genotype was significantly associated with percent change in BMD of neck after 1 year of calcium supplementation (Tab 2). The percent change (median, QR) in neck BMD was significantly different in homozygous XX [-4.14 (from -6.54 to -1.37)] in comparison with Xx [1.72 (from -1.12 to 3.20)] (P<0.01) or xx [1.22 (from -1.74 to 3.20)] Xba I ER-alpha genotype (P=0.001). However, the percent change BMD in L1-4 and troch was not statistically different between Xba I genotype. Moreover, no significant association was found between the percent changes in BMD and Pvu II and Apa I genotypes after calcium supplementation.
Tab 2. Characteristics of the subjects according to ER-alpha Xba I genotype. n=115. Mean±SD. cP<0.01 vs Xx or xx genotype.
|
|
XX |
Xx |
xx |
P
value |
| n (%) |
9 (0.078) |
47 (0.409) |
59 (0.513) |
|
| Age/year |
61¡À5 |
63¡À5 |
62¡À5 |
0.432 |
| Years
since menopause /year |
12¡À7 |
14¡À6 |
13¡À6 |
0.080 |
| Height/cm |
155¡À5 |
154¡À6 |
153¡À5 |
0.810 |
| Weight/kg |
57¡À10 |
57¡À6 |
57¡À9 |
0.581 |
| Bone
markers at baseline |
|
|
|
|
| ALP |
67¡À15 |
75¡À23 |
67¡À16 |
0.161 |
| BGP |
9¡À6 |
12¡À6 |
14¡À9 |
0.195 |
| PTH |
36¡À14 |
31¡À8 |
31¡À9 |
0.386 |
| Pyd |
31¡À4 |
31¡À19 |
46¡À21 |
0.239 |
| 25-(OH)
vitamin D |
16¡À7 |
12¡À8 |
11¡À8 |
0.785 |
| Urinary
Ca |
0.61¡À0.21 |
0.39¡À0.20 |
0.44¡À0.21 |
0.061 |
| Bone
markers after 1 year |
|
|
|
|
| ALP |
70¡À17 |
67¡À16 |
66¡À15 |
0.861 |
| BGP |
11¡À4 |
12¡À5 |
12¡À5 |
0.919 |
| PTH |
27¡À8 |
22¡À9 |
24¡À10 |
0.246 |
| Pyd |
42¡À25 |
44¡À19 |
37¡À18 |
0.934 |
| 25-(OH)
vitamin D |
17¡À9 |
16¡À8 |
18¡À10 |
0.767 |
| Urinary
Ca |
0.46¡À0.29 |
0.46¡À0.22 |
0.40¡À0.25 |
0.525 |
| Bone
markers change/% |
|
|
|
|
| ALP |
6.5 (-21.8~21.3) |
-11.5 (-20.7~6.6) |
-3.7 (-19.2~15.8) |
0.202 |
| BGP |
-4.7 (-43.1~26.8) |
5.1 (-20.3~69.1) |
-27.6 (-38.7~13.1) |
0.181 |
| PTH |
-35.1 (-52.1~0.8) |
-26.0 (-46.0~-13.3) |
-27.5 (-44.0~3.3) |
0.701 |
| Pyd |
-3.5 (12.5~27.9) |
12.0 (8.9~109.9) |
-2.4 (-43.1~70.9) |
0.068 |
| 25-(OH)
vitamin D |
98.6 (13.3~326.4) |
312.2 (256.4~384.2) |
133.6 (39.6~277.5) |
0.924 |
| Urinary
Ca |
-3.5 (-12.5~27.9) |
52.0 (8.9~109.9) |
-2.4 (46.9~71.8) |
0.062 |
| BMD
at baseline /g¡¤cm-2 |
|
|
|
|
| L1-4 |
0.79¡À0.12 |
0.77¡À0.10 |
0.77¡À0.10 |
0.773 |
| Neck |
0.62¡À0.10 |
0.62¡À0.06 |
0.60¡À0.08 |
0.340 |
| Troch |
0.50¡À0.07 |
0.50¡À0.06 |
0.48¡À0.06 |
0.210 |
| BMD
after 1 year/g¡¤cm-2 |
|
|
|
|
| L1-4 |
0.80¡À0.13 |
0.78¡À0.11 |
0.78¡À0.10 |
0.839 |
| Neck |
0.59¡À0.10 |
0.63¡À0.07 |
0.61¡À0.08 |
0.347 |
| Troch |
0.49¡À0.07 |
0.50¡À0.06 |
0.49¡À0.06 |
0.382 |
| BMD
change/% |
|
|
|
|
| L1-4 |
0.73 (-0.86~2.72) |
0.73 (-1.29~2.76) |
0.66 (-0.64~2.84) |
0.965 |
| Neck |
-4.14 (-6.54~-1.34)c |
1.72 (-1.12~3.20) |
1.22 (-1.74~3.06) |
0.003 |
| Troch |
0.00 (-2.65~1.06) |
1.03 (-0.66~3.51) |
1.40 (-0.85~3.18) |
0.361 |
At baseline and after 1-year treatment, serum and urinary levels of bone turnover markers was not significantly associated with Xba I, Pvu II, and Apa I genotypes (Tab 2-4). In addition, the percent changes in bone turnover markers were not significantly different neither in ER-alpha, nor VDR gene polymorphisms.
Tab 3. Characteristics of the subjects according to ER-alpha Pvu II genotype. n=115. Mean±SD.
|
|
PP |
Pp |
pp |
P
value |
| n (%) |
27 (0.235) |
52 (0.452) |
36 (0.313) |
|
| Age/year |
61¡À7 |
62¡À5 |
62¡À5 |
0.074 |
| Years
since menopause /year |
12¡À7 |
14¡À6 |
13¡À6 |
0.069 |
| Height/cm |
155¡À5 |
154¡À6 |
153¡À5 |
0.052 |
| Weight/kg |
57¡À10 |
57¡À6 |
57¡À9 |
0.078 |
| Bone
markers at baseline |
|
|
|
|
| ALP |
70¡À24 |
72¡À19 |
71¡À19 |
0.917 |
| BGP |
9¡À6 |
12¡À6 |
14¡À9 |
0.871 |
| PTH |
36¡À14 |
31¡À8 |
31¡À9 |
0.992 |
| Pyd |
45¡À16 |
33¡À15 |
45¡À18 |
0.331 |
| 25-(OH)
vitamin D |
16¡À7 |
12¡À6 |
11¡À6 |
0.770 |
| Urinary
Ca |
0.61¡À0.21 |
0.39¡À0.20 |
0.44¡À0.21 |
0.601 |
| Bone
markers after 1 year |
|
|
|
|
| ALP |
66¡À16 |
67¡À14 |
68¡À17 |
0.542 |
| BGP |
11¡À4 |
13¡À5 |
11¡À5 |
0.406 |
| PTH |
25¡À11 |
22¡À8 |
24¡À10 |
0.616 |
| Pyd |
42¡À23 |
41¡À23 |
41¡À19 |
0.941 |
| 25-(OH)
vitamin D |
16¡À7 |
16¡À8 |
18¡À10 |
0.616 |
| Urinary
Ca |
0.44¡À0.27 |
0.46¡À0.23 |
0.38¡À0.23 |
0.492 |
| Bone
markers change/% |
|
|
|
|
| ALP |
-3.7 (-22.2~10.9) |
-3.2 (-17.5~21.0) |
-11.1 (-20.2~9.8) |
0.832 |
| BGP |
-12.1 (-39.9~40.1) |
8.4 (-27.6~77.1) |
-23.3 (-37.9~5.7) |
0.263 |
| PTH |
-13.0 (-52.9~8.5) |
-31.0 (-41.3~-14.9) |
-28.8 (-44.0~-8.1) |
0.587 |
| Pyd |
6.6 (-23.0~60.8) |
28.7 (-14.0~106.4) |
38.5 (-38.1~116.5) |
0.648 |
| 25-(OH)
vitamin D |
109.9 (20.3~196.1) |
116.7 (14.3~325.6) |
214.0 (43.5~492.4) |
0.329 |
| Urinary
Ca |
10.4 (-46.1~51.9) |
20.9 (-20.8~88.1) |
-16.3 (-41.7~8.6) |
0.107 |
| BMD
at baseline /g•cm-2 |
|
|
|
|
| L1-4 |
0.76¡À0.02 |
0.78¡À0.02 |
0.78¡À0.02 |
0.801 |
| Neck |
0.61¡À0.02 |
0.63¡À0.01 |
0.60¡À0.01 |
0.116 |
| Troch |
0.50¡À0.01 |
0.51¡À0.01 |
0.50¡À0.01 |
0.625 |
| BMD
after 1year /g•cm-2 |
|
|
|
|
| L1-4 |
0.76¡À0.03 |
0.78¡À0.02 |
0.78¡À0.02 |
0.829 |
| Neck |
0.62¡À0.02 |
0.63¡À0.01 |
0.61¡À0.01 |
0.427 |
| Troch |
0.50¡À0.01 |
0.51¡À0.01 |
0.50¡À0.01 |
0.473 |
| BMD
change/% |
|
|
|
|
| L1-4 |
0.421 (-0.86~2.71) |
0.31 (-0.80~3.0) |
0.31 (-1.11~2.02) |
0.625 |
| Neck |
0.37 (-2.18~3.58) |
0.81 (-2.71~2.64) |
1.21 (-1.70~3.06) |
0.615 |
| Troch |
1.06 (-2.65~4.01) |
1.27 (-0.66~3.90) |
0.26 (-0.87~2.06) |
0.305 |
Tab 4. Characteristics of the subjects according to VDR Apa I genotype. n=115. Mean±SD.
|
|
AA |
Aa |
aa |
P
value |
| n (%) |