The last decade has yielded a wealth of information on bone health. The increased interest and advanced technology for bone mass measurements have enhanced our understanding of osteoporosis risk factors, its causes, and routes of prevention and managementReference Prentice, Bonjour, Branca, Cooper, Flynn, Garabedian, Muller, Pannemans and Weber1. Nevertheless, osteoporosis remains a complex, multifactorial condition that leads to an increased risk of fractures, with inadequate calcium intake influencing bone loss and playing an important role in its pathogenesisReference Dawson-Hughes, Marcus, Feldman and Kelsey2. The intakes of some nutrients by older women in rural Korea are lower than recommended levelsReference Kim, Kwon, Shin, Kim and Kim3, with calcium being especially significant since Korean national nutrition surveys began in the 1960s4. Interventions for increasing calcium intake are an important public health issue given the ageing population in Korea. However, the health benefits to be expected from calcium depend not only on how much calcium is consumed but also on how much is absorbedReference Wolf, Cauley, Baker, Ferrell, Charron, Caggiula, Salamone, Heaney and Kuller5.
It has been suggested that vitamin D receptor (VDR) gene polymorphisms influence intestinal calcium absorption, with women with B variants exhibiting reduced calcium absorption efficiency when their calcium intake is low and showing lower bone mineral density (BMD) than women with bb variantsReference Dawson-Hughes, Harris and Finneran6. The impact of this genetic difference reduces with higher calcium intakes or increasing ageReference Ferrari, Rizzoli, Slosman and Bonjour7. Therefore, many factors need to be considered when attempting to improve the calcium intake of older women significantly. The present study investigated the association among VDR genotype, calcium intake and bone strength as indicated by the broadband ultrasound attenuation (BUA) in community-dwelling older women with low calcium intake in rural Korea. Quantitative ultrasound was used since this non-invasive method is easy to apply and has been proposed as an alternative to dual-energy X-ray absorptiometry, which is the currently accepted indicator for assessing bone strengthReference Siffert and Kaufman8.
Materials and methods
Subjects
In total, 350 women aged over 65 years were recruited from residents of Asan, Korea, of whom 335 (96 %) completed a 24 h recall interview, survey form, BUA measurement and blood collection. The subjects were aged from 65 to 94 years, with a mean age of 72·4 years. The study was approved by the Institute of Research Board of Soonchunhyang University and all participants provided their written, informed consent.
Data collection and analysis
Information on demographic characteristics (including physical activity on five scales) was obtained by survey and the dietary calcium intake was assessed using a 2 d, 24 h recall method. Food records were converted to nutrient intake using a computerized nutrient analysis program (CAN-pro; The Korean Society of Nutrition, Seoul, Korea). Approximately 10 ml of venous blood were collected and divided into two tubes: 4 ml were used for VDR genotype analysis, and 6 ml were immediately separated for analyses of serum total alkaline phosphatase, osteocalcin, intact parathyroid hormone and 25-hydroxyvitamin D3. Alkaline phosphatase was analysed spectrophotometrically (TBA-40FR biochemical analyser; Hitachi, Tokyo, Japan), and osteocalcin and intact parathyroid hormone were analysed by an immunoradiometric assay at Samkwang Reference Laboratories (College of American Pathologists Accredited Laboratory 69 944-01, Seoul, Korea). 25-Hydroxyvitamin D3 was analysed using HPLC as described previouslyReference Cho, Ryu, Lee, Lim and Huh9. BUA was measured with quantitative ultrasound at the left calcaneus (QUS-2; Metra Biosystems Inc., Mountain View, CA, USA). Intra- and inter-assay CV were all less than 10 %.
DNA analysis
DNA was extracted from leucocytes and amplified using the PCR. The enzyme BsmI endonuclease was used to define the VDR gene allelic polymorphisms, with BB and bb representing the absence and presence of the restriction site on both alleles, respectivelyReference Morrison, Qi, Tokita, Kelly, Crofts, Nguyen, Sambrook and Eisman10.
Statistics
All statistical analyses were performed using SPSS version 11.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined at the P < 0·05 level based on Student's t test. To minimize extraneous errors in estimating dietary calcium intake due to individual differences in total food intake, calcium intake was adjusted for total energy intakesReference Willett, Stampfer and Willett11. Multiple linear regression analysis was used to examine the relation between BUA and VDR after adjusting for age, weight, height, physical activity, calcium intake and 25-hydroxyvitamin D3. The Bb and BB genotypes were combined in a regression model due to a small number of samples of BB type (n 3).
Results
The data on demographic indexes and nutritional, hormonal and bone factors for the different VDR genotypes are listed in Table 1. A total of 309 (92 %) participants were genotype bb, 23 (7 %) were Bb and 3 (1 %) were BB. The mean calcium intake of the total population was 439·6 (sd 279·6) mg/d, which is only 55 % of the recommended intake level of the Dietary Reference Intake for Koreans (which for calcium is 800 mg/d for women older than 65 years). The calcium intake did not differ between VDR genotypes, but 96 % of the participants had a calcium intake lower than the recommended Dietary Reference Intake for Koreans. The BUA was significantly higher in the bb genotype than in the Bb/BB genotype (P < 0·05, Student's t-test), while 25-hydroxyvitamin D3, parathyroid hormone, osteocalcin and total alkaline phosphatase levels did not differ with the genotype. The multiple linear regression analysis also revealed that the VDR genotype was significantly associated with BUA after adjusting for age, weight, height, physical activity, calcium intake and 25-hydroxyvitamin D3 (P = 0·013).
Table 1 Calcium intake, calcaneal broadband ultrasound attenuation (BUA) and bone markers for different vitamin D receptor (VDR) genotypes, and the impact of VDR on BUA as assessed using multiple linear regression analysis
Mean value was significantly different from that of the bb genotype group: *P < 0·05.
† Dependent variable was BUA, and age, weight, height and physical activity were controlled.
‡ VDR genotype was coded as bb = 0 and Bb/BB = 1.
Discussion
We found a significant association between the VDR BsmI genotype and calcaneal quantitative ultrasound results in Korean postmenopausal older women living in rural communities. Other studies involving Korean subjects have shown both the absenceReference Lim, Park, Park, Song, Lee, Kim, Lee and Huh12, Reference Koh, Nam-Goong, Hong, Kim, Kim, Kim and Kim13and presenceReference Chung, Kim and Kim14 of an association between VDR genotype and BMD or osteoporosis, and those involving various ethnic groups have produced contradictory results for the association between BMD and the b alleleReference Liu, Liu, Recker and Deng15, Reference Thakkinstian, D'Este, Eisman, Nguyen and Attia16. One distinguishing feature of the present study is that the calcium intake was lower than the recommended Dietary Reference Intake for Koreans (800 mg/d) in 96 % of the participants. Moreover, more than half (67 %) of them showed an intake of less than 500 mg/d, at which level active calcium transport increases (mediated by 1,25-dihydroxyl vitamin D), whereas passive diffusion accounts for an increasing proportion of the calcium absorbed as the calcium intake increases above 500 mg/dReference Ireland and Fordtran17. Dawson-Hughes et al. Reference Dawson-Hughes, Harris and Finneran6 reported that the calcium absorption efficiency was lower in women with the BB genotype with a functional defect in the intestinal VDR than in those with the bb genotype, when the calcium intake is low. Other studiesReference Wolf, Cauley, Baker, Ferrell, Charron, Caggiula, Salamone, Heaney and Kuller5, Reference Ferrari, Rizzoli, Slosman and Bonjour7 support the calcium absorption being higher in the b allele than in the B allele, although calcium intakes were not specified. The calcium intake of our population being the level at which it might be influenced by VDR may have resulted in the effect of VDR BsmI genotype on bone strength being greater than that in previous studies conducted in KoreaReference Lim, Park, Park, Song, Lee, Kim, Lee and Huh12, Reference Koh, Nam-Goong, Hong, Kim, Kim, Kim and Kim13 and other countriesReference Thakkinstian, D'Este, Eisman, Nguyen and Attia16. Unfortunately, these previous studies did not examine calcium intakes. Therefore, the conflicting results of VDR BsmI genotype effects on BMD may be attributable to the calcium intakes differing between the study populations.
The results of the current cross-sectional study are subject to the following limitations. Firstly, the use of a 24 h recall method may result in underreporting of dietary food intakesReference Johansson, Wiman, Ahren, Hallmans and Johansson18 and average 2 d, 24 h recall values may not reflect usual calcium intakes. However, the probability of technical errors or information bias is expected to be low in the current study since using energy-adjusted nutrient intake values can effectively reduce a between-person varianceReference Willett, Stampfer and Willett11, Reference Willett and Willett19. Moreover, our study population showed a relatively small within-person variance of 34 % CV for actual calcium intake, compared with a value of 49 % CV for American adult womenReference Willett and Willett19. The energy-adjusted calcium intake values rather than crude intake values further reduced the within-person variance to 28 % CV. WillettReference Willett and Willett19 and Beaton et al. Reference Beaton, Milner, McGuire, Feather and Little20 pointed out that within-person variances in nutrient intakes are largely affected by cultural factors, and the Korean elderly population (especially rural residents) – as analysed in the current study – exhibits a relative small day-to-day dietary intake variance4. Whilst these measures may not have avoided all of the problems associated with the 24 h recall method, it is unlikely that the non-significant association of calcium intake and BUA observed in this population is mainly attributable to measurement error. Secondly, BUA as measured by quantitative ultrasound differs from BMD and cannot be compared directly to the BMD as measured by dual-energy X-ray absorptiometry. Although dual-energy X-ray absorptiometry is considered to be the most accurate clinical method for identifying low BMDReference Kanis and Gluer21, in a community field situation such as in the current study, access to axial dual-energy X-ray absorptiometry is limited and screening for low BMD with dual-energy X-ray absorptiometry is not cost-effective22. We therefore used quantitative ultrasound to assess the calcaneal bone status since it has been used for the same purpose for almost two decades and has been shown to be clinically usefulReference Siffert and Kaufman8, Reference Greenspan, Cheng, Miller and Orwoll23.
In conclusion, the results of the present study suggest that the BUA in populations with a low calcium intake is significantly influenced by the VDR genotype but not by the calcium intake, cross-sectionally.
