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3 Laboratório de Bioquímica Nutricional e de Alimentos, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 21941-909; 4 Instituto de Nutrição, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil 20550-900; 5 Laboratório de Genética Humana, Departamento de Genética, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil 21040-360; and 6 Sociedade Brasileira de Densitometria Óssea, Rio de Janeiro, Brazil 22050-000
* To whom correspondence should be addressed. E-mail: donangel{at}iq.ufrj.br.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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Besides environmental factors, including calcium intake, physiologic conditions such as pregnancy and, mainly, lactation are also potential factors affecting bone mass acquisition during adolescence (2–6). Daily calcium secretion into breast milk (200–300 mg/d) is known to induce a transient decrease in bone mass in lactating adults with recovery post-menses and after weaning (7,8). A similar pattern of bone loss during lactation and bone recovery after weaning appears to occur in adolescent mothers (9). However, because the variability of bone mass is known to be dependent on genetic factors (10,11), it is important to evaluate the contribution of these factors to the physiologic skeletal response to lactation in these mothers.
Several gene polymorphisms have been associated with bone mineral density (BMD)7 in different life stages (12). The most investigated genes include those encoding for bone matrix proteins, for bone metabolism regulators, and hormone receptors such as the vitamin D receptor (VDR) gene (11,12). The VDR gene mediates 1,25(OH)2D biological actions in several cellular systems, including those involved in the regulation of calcium and bone metabolism (13). Over 20 polymorphisms have been identified in the VDR gene and those recognized by the restriction enzymes Apa1, Bsm1, TaqI, and Fok1 were found to be related to BMD among different populations (14–20), although other studies have reported no significant effect (21–23).
The relationship between BsmI polymorphism of the VDR gene (but not other VDR genotypes) and bone changes during lactation was examined in adult women (24,25). However, VDR genotypes were not investigated in relation to bone mass and calcium homeostasis during lactation in adolescent mothers. The aim of this study was to evaluate the influence of VDR gene polymorphisms recognized by ApaI, BsmI, and TaqI on bone mass, bone- and calcium-related hormones, and breast milk calcium of lactating adolescents with habitually low calcium intake.
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Methods |
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Morning blood samples from fasting (10 mL) were obtained from each subject within 30 min after breast-feeding. Milk samples (20 mL) were obtained by manual expression. Aliquots of total blood, serum, and milk were stored at –20°C until analyzed.
Habitual calcium intake was assessed by 3 24-h recall questionnaires conducted by the same investigator on different days of the study (recruitment, blood collection, and bone measurements). Dietary nutrient analysis was conducted using the program "The Food Processor" (ESHA Research), with the database adapted to Brazilian foods based on published information (26).
Bone measurements. Total body bone mineral content (TBMC), total body BMD (TBMD), and lumbar spine (L2-L4) BMD (LSBMD) were assessed by dual-energy X-ray absorptiometry using the Lunar DPX densitometer with software version 4.6A (Lunar Radiation). Z scores were obtained by comparison with an age- and gender-matched reference as previously described (9). Total body calcium content was calculated as 38% of TBMC (27).
Laboratory analysis. Calcium concentration in milk was measured by inductively coupled plasma optical emission spectrometry using a Plasma-1000 instrument (Perkin Elmer). Serum prolactin was determined by immunoradiometric assay (Diagnostic Products). Serum concentrations of estradiol (Diagnostic Products), insulin-like growth factor-I (IGF1; Nichols Institute Diagnostics), and 25-hydroxyvitamin D (25-OHD; Nichols Institute Diagnostics) were assessed by radioimmunoassay. Serum intact parathyroid hormone (iPTH; Diagnostic Products) was determined by a chemiluminescent enzyme-labeled immunometric assay. All materials used for sample collection, storage, and analysis were either disposable or previously soaked overnight in dilute nitric acid (1:4) and rinsed with deionized water.
VDR genotyping. DNA was extracted from whole blood (GFX Genomic Blood Purification kit, Amersham Biosciences). VDR genotypes for 3 restriction-fragment-length polymorphisms were determined by PCR amplification and enzymatic digestion of the products with ApaI (Amersham Pharmacia Biotech), BsmI (Q-Biogene), and TaqI (Q-Biogene). Identification of gene polymorphisms were conducted using similar methods as described previously (14,28). Alleles having the respective sites for ApaI, BsmI, and TaqI were denoted by a, b, or t and alleles lacking the respective sites denoted by A, B, or T.
Statistical analysis. We evaluated the effect of VDR genotype on bone mass measurements, hormones, and breast milk calcium by ANOVA after adjustment for confounding factors (years since menarche, weeks postpartum, and habitual calcium intake) that were used as covariates. If ANOVA was significant, post-hoc differences between genotype groups were tested using Tukey's test. Associations between variables were examined by Pearson correlation analysis. Values of P < 0.05 were considered significant. The statistical analyses were performed using Statgraphics version 7 for DOS (Manugistics). Values were reported as means ± SD.
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Results |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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40% of that generally recommended by international groups for adolescents (29,30) and similar in adolescents with different genotypes. Dairy products were the primary source of calcium intake, contributing 60% of total daily intake. Serum 25-OHD concentration was 61.3 ± 7.4 nmol/L for the lactating adolescents studied (n = 40). Serum iPTH and 25-OHD were negatively correlated (r = –0.56; P < 0.01). Serum concentrations of 25-OHD did not differ among the genotype subgroups.
In the entire group of adolescents (n = 40), Z scores for TBMD and LSBMD were –0.55 ± 1.01 and –1.15 ± 1.48, respectively, corresponding to a bone deficit of 3% for TBMD and 10% for LSBMD. TBMD and LSBMD Z scores were correlated with postmenarcheal period (r = 0.43 and r = 0.37, respectively; P < 0.02) but were not correlated with weeks postpartum. However, TBMD Z score (but not LSBMD Z score) was lower in lactating adolescents at 9–24 wk postpartum (n = 20) than in those at 2–8 wk postpartum (n = 20) (–0.71 ± 0.87 and –0.08 ± 1.03, respectively; P < 0.05).
In the genotype subgroups, lactating adolescents with the aa genotype had higher LSBMD (21%; P < 0.05) than those with the AA genotype (Table 1), with postmenarcheal period as a significant covariate (P = 0.009). A similar trend was observed for LSBMD Z scores (P = 0.09) (Table 1). Bone measurements did not differ between BsmI genotypes (Table 2). However, calcium concentration in breast milk was 24% higher (P < 0.05) in adolescents with bb compared with those with BB or Bb genotypes. Serum concentration of iPTH was also higher (80%; P < 0.05) in adolescents with bb compared with those with BB genotype. Lactating adolescents with genotype tt had TBMC 23% higher (P < 0.05) than those with TT and Tt genotypes (Table 3), with postmenarcheal period as a significant covariate (P = 0.01). A similar trend was observed for LSBMD Z scores (P = 0.10). Serum concentrations of IGF1 were also higher (50%; P < 0.05) in tt adolescents compared with those with the TT and Tt genotypes, with weeks postpartum tending to be a significant covariate (P = 0.06). Serum concentrations of estradiol and prolactin were not affected by the different genotypes (Table 3).
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Discussion |
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TOPABSTRACTIntroductionMethodsResultsDiscussionLITERATURE CITED |
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500 mg/d, had a deficit in bone mass, particularly at the lumbar spine (9), consistent with lactation-associated bone loss (7–9). Important deficits in bone mass, as observed in this study, were described at early lactation in adolescent mothers with both high (5) and low (9) calcium intakes, associated with hormonal changes, such as PTH and estradiol, which ensure an adequate transfer of calcium to breast milk (5,9). Several factors, including the length of lactation (31,32), milk volume, total milk calcium output (24), and, in the case of adolescent mothers, time since menarche (9), are known to influence maternal bone mass during lactation. However, given the evidence of strong polygenic control of bone mass (33), it is necessary to consider that physiologic bone changes in response to lactation may also be under the influence of specific gene polymorphisms.
The gene encoding the VDR is one of the major genes implicated in the variability of BMD (11,13,14). Most studies, however, have focused on adult population groups, especially pre- and postmenopausal women (10,34). A more limited number of studies evaluating the influence of VDR genotype on bone mass have been conducted in adolescents (16,18,35–40) with conflicting results. In adolescents, VDR gene polymorphisms at the ApaI site were related with BMD at femoral neck and lumbar spine (16) and at distal radius (35), although the genotypes associated with higher BMD were not the same. Influence of BsmI genotypes on lumbar spine BMD of adolescents was also described (16,18), although several studies reported no effect (38–40). The majority of studies evaluating TaqI polymorphisms reported no associations with BMD or bone mineral content in adolescents (37,38), although a strong tendency for a lower LSBMD in postpubertal girls with the TT genotype compared with those with the Tt genotype has been described (18). FokI genotypes appeared to indirectly affect bone mineral accretion during pubertal growth through an effect on calcium absorption (38) and were also associated with variation in BMD at cortical bone sites in Scandinavian boys, but not girls (36).
Only a few studies have evaluated associations between VDR gene polymorphisms and bone status during pregnancy and lactation in adult mothers, but there are no studies to our knowledge of adolescent mothers. In adult mothers, no significant effect of BsmI polymorphism was observed on changes in bone mineral content from 0.5 to 3 mo of lactation at various skeletal sites (24) and on bone mass changes during postpartum amenorrhea or 1 y after resumption of menses (25). Longitudinal changes in LSBMD after 2 successive pregnancies tended to be related with ApaI polymorphisms but not with TaqI and FokI (41). These studies may indicate that the influence of VDR gene polymorphisms on bone mass is not evident in adult women under conditions of strong physiologic regulation such as pregnancy and lactation. However, the genetic contribution to BMD may change with age (34), being more pronounced at younger ages (35,36).
The results of this study indicate that the possible influence of genetic polymorphisms on bone mass appears to be evident in lactating adolescent women. After adjustment for time since menarche, weeks postpartum, and habitual calcium intake, the lactating adolescents with the aa genotype had bone density at the lumbar spine site 21% higher than those with AA genotype, consistent with a previous study in nonpregnant, nonlactating adolescents (16). Also, TBMC was 23% higher in the adolescents with the tt genotype who were also taller and had higher IGF1 levels compared with those with TT and Tt genotypes. These results are consistent with the stimulatory effect of IGF1 on longitudinal bone growth (42).
In our study in lactating adolescents with low calcium intake, serum PTH and breast milk calcium were influenced by BsmI polymorphism, with simultaneously higher serum PTH and breast milk calcium in the adolescent mothers with the bb compared with the other genotypes. Therefore, the lactating adolescent mothers with the bb genotype and habitually low calcium intake appeared to be adapted to ensure calcium transfer into milk, possibly through PTH-mediated processes. This is consistent with results from a previous study in the same population showing higher serum PTH in lactating adolescents compared with lactating adults (43).
Serum PTH in the lactating adolescents studied was inversely related to serum concentrations of 25-OHD, as found in recent studies (38,44). Moreover, 25-OHD did not differ in the subgroups of VDR genotypes.
Although the VDR polymorphisms evaluated in this study may not directly affect VDR function because of the intronic position (ApaI and BsmI) or the silent character (TaqI) of the polymorphisms, it has been suggested that they may affect transcriptional activity, resulting in different mRNA levels of VDR (11,12). It is also possible that these polymorphisms are in linkage disequilibrium with other genetic polymorphisms (12,45) that directly influence bone and calcium metabolism during lactation. These hypothesis need to be better investigated.
This study indicates that, despite its small sample size, that ApaI, BsmI, and TaqI VDR gene polymorphisms are associated with bone mass and/or breast milk calcium in lactating adolescents with habitually low calcium intake. Adolescent mothers with the aa and tt genotypes appeared to have a better bone status and those with the bb genotype had higher breast milk calcium. Further, larger studies relating VDR gene polymorphisms to bone responses during physiological periods of increased calcium demands are warranted.
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FOOTNOTES |
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2 Author disclosures: F. F. Bezerra, G. M. K. Cabello, L. M. C. Mendonça, and C. M. Donangelo, no conflicts of interest.
7 Abbreviations used: BMD, bone mineral density; 25-OHD, 25-hydroxyvitamin D; IGF1, insulin-like growth factor-I; iPTH, intact parathyroid hormone; LSBMD, lumbar spine bone mineral density; TBMC, total body bone mineral content; TBMD, total body bone mineral density; VDR, vitamin D receptor.
Manuscript received 7 September 2007. Initial review completed 8 October 2007. Revision accepted 20 November 2007.
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