1. Introduction
Calcium and vitamin D are essential nutrients with a major role in bone health. Calcium is the most abundant mineral in the body and is essential to functions related to vascular contraction, muscle function, nerve transmission, intracellular signaling, and hormonal secretion [
1,
2]. Bone acts as the calcium reservoir to maintain constant calcium homeostasis, with around 99% of the organism’s calcium supply stored in the bones and teeth. Vitamin D also plays a key role in calcium homeostasis, promoting calcium absorption in the gut and resorption in the kidney, as well as stimulating bone formation and remodeling [
3]. Therefore, optimal intakes of calcium and vitamin D are generally regarded as fundamental factors in the prevention and treatment of osteoporosis.
The associations of calcium intake, vitamin D intake, and vitamin D status with bone mineral density (BMD) and the risk of fractures have been studied extensively over the past decades, but mostly in the elderly population [
4,
5,
6]. An extensive amount of human evidence supports the contribution of calcium for bone homeostatic regulation and treatment of osteoporosis. Observational studies as well as randomized controlled trials have generally shown that higher calcium and vitamin D intakes (either from food or supplementation), compared to lower intakes, may have significant benefits to bone health and reduce the risk of osteoporosis [
1,
7,
8,
9].
Parathyroid hormone (PTH), a hormone secreted by the parathyroid glands in response to low serum calcium levels, is also well recognized as a fundamental part of bone homeostasis. PTH triggers the hydroxylation of 25-hydroxyvitamin D (25(OH)D) to the active form, 1,25-dihydroxyvitamin D (1,25(OH)
2D), leading to enhanced intestinal absorption of calcium. Chronic elevated PTH concentrations can have significant negative impacts on BMD and consequently increase the risk of fractures over time [
2,
3,
9]. Low dietary calcium and vitamin D intakes, as well as an inadequate vitamin D status, can be independent contributors to high PTH concentrations [
10,
11]. The negative correlation between PTH and vitamin D, and negative effects of higher PTH on bone health, have been previously demonstrated, but mostly in elderly and/or osteoporotic populations [
11,
12,
13,
14,
15], and therefore there is still limited data on healthy adults, particularly in Latin America and Brazil [
16].
This analysis aimed to investigate the associations of serum 25(OH)D and plasma PTH concentrations, as well as habitual dietary vitamin D and calcium intakes, age, and adiposity with bone health parameters assessed by either peripheral quantitative computed tomography (pQCT) or dual-energy x-ray absorptiometry (DXA) in in two groups of adult Brazilian women living in opposite latitudes.
2. Materials and Methods
A cross-sectional analysis of endocrine status (specifically 25(OH)D and PTH concentrations), calcium and vitamin D intake, age and adiposity, and bone mineral density was conducted in 130 healthy adult Brazilian women living in England (51° N) and healthy adult Brazilian women living in Brazil (16° S), recruited for the D-SOL study (Interaction between Vitamin D Supplementation and Sunlight Exposure in Women Living in Opposite Latitudes—
clinicaltrials.gov as NCT03318029). The D-SOL study recruited adult women aged 20 to 59 years with Brazilian nationality (born in Brazil and having at least one parent born in Brazil).
2.1. Exclusion Criteria
The participants were screened according to exclusion criteria that included potential cofounders likely to affect vitamin D metabolism (osteoporosis therapy, anti-estrogen treatment, antiepileptic drugs, cancer treatment), taking containing vitamin D (if the prospective participants agreed to stop vitamin D supplementation to join the study, a wash-out period of 8 weeks prior to commencing the trial was accepted), being pregnant or planning a pregnancy during the study period, menopause status (defined as the permanent cessation of menstruation), and living in the UK for less than 3 months at the commencement of the study (for England participants only). All participants provided written informed consent at the commencement of the study.
2.2. Data Collection
The dietary intake of participants, particularly vitamin D and calcium, was determined by 4 consecutive days of estimated diet diaries, including one weekend day. Participants were instructed by the research team on how to correctly complete the diary and asked to give as much detail as possible of every meal, including portion size.
Dietary intake data obtained from participants in the England cohort were analyzed using the Nutritics® nutritional analysis software (version 4.0, Dublin, Ireland), (UK Composition of Foods Integrated Dataset (CoFID) including McCance and Widdowson 7th edition (70)) and those collected in Brazil were analyzed via the Dietwin® software (Version 13 (3090), Rio Grande do Sul, Brazil) (Brazilian Food Composition Table (TACO) (113) and the food composition database from the Brazilian Institute of Geography and Statistics (IBGE), as well as the United States Department of Agriculture (USDA) food composition database).
Participants were instructed to wear light clothing on the day of the visits to minimize different additional weights from clothing. For weight measurement, participants were asked to remove shoes, socks, and heavy coats before stepping on the scale. For the England trial, weight to the nearest 0.1 kg and body fat was obtained using a Tanita Body Composition Analyser MC-180MA (Tanita Cooperatives, Tokyo, Japan). For the Brazil trial, weight was measured to the nearest 0.1 kg using a standard weighing scale (Balmak
®, Santa Bárbara d’Oeste, SP, Brazil) and body composition was determined via DXA scan (GE Healthcare LunarTM DPX NT + 152000, GE Medical Systems, Madison, WI, USA). Anthropometric methods were standardized between the different researchers involved in the study performing the measurements in each country to minimize inter-evaluator variations. Standing height was measured using a wall stadiometer to the nearest 0.1 cm, with participants in an upright posture and barefoot with heels close together and as close as possible to the wall. Waist circumference was measured with a non-extendable standard measure tape, at the narrowest point of the torso, to the nearest 0.1 cm. If this point could not be estimated, the level of the belly button was used as a reference point [
17].
An overnight fasted (8 h) blood sample was collected by venipuncture by trained phlebotomists. Processed serum and plasma samples were divided into aliquots and stored at −80 °C at the University of Surrey, prior to analysis. Samples collected in Brazil followed the exact same procedures and were temporarily stored at −80 °C at the Federal University of Goiás, and sent to be stored at the University of Surrey as well, prior to analysis.
2.3. Laboratory Analysis
Serum 25(OH)D concentrations were determined by the HPLC-MS/MS method on a Waters Acuity UPLC (Triple Quadrupole) TQD® System using a Pentafluorophenyl (PFP) column following supported liquid extraction (SLE). Laboratory intra- and inter-assay coefficients of variation (CV) were 5.6% and 7.8%, respectively. Calcium, albumin, and PTH concentrations were measured using Abbott Architect kits. Serum calcium was measured by using an endpoint spectrophotometric reaction based on the o-cresolphthalein complexone methodology, and serum albumin was measured by using an endpoint spectrophotometric reaction based on the bromocresol green solution dye binding methodology. Serum calcium concentrations were adjusted for albumin concentrations. Plasma intact PTH was measured by in vitro chemiluminescent microparticle immunoassay (CMIA). The manufacturer’s quoted inter-assay CV for calcium was <3%, for albumin <3.8%, and for PTH 4%.
2.4. Bone Measurements
For the England trial, a peripheral quantitative computed tomography scan (pQCT; XCT 2000, Stratec Medizintechnik GmbH, Pforzheim, Germany) was performed on the participant’s non-dominant forearm to measure volumetric bone mineral density at the 4% and 66% radial site. pQCT was performed by the same experienced operator to scan all participants’ radii. For total density and trabecular density, these were calculated using previously published reference data for white Caucasian European women [
18] using the equation: Z-score = ((individual value − expected value for age)/reference standard deviation (SD)). For participants in Brazil, whole body bone mineral density, lower spine (L1–L4) density, and left femur bone mineral density were measured via DXA scan (dual-energy X-ray absorptiometry). The same experienced operator performed all DXA scans with all participants. Z-scores of Brazilian participants for the lumbar spine (L1–L4) and femur (femoral neck) were automatically calculated by the DXA scan, which used white Caucasian Women as a reference [
19].
The same cut-off values for Z-score classification (i.e., Z-score ≥ −2.0 defined as normal (low-risk score) and Z-score ≤ −2.0 defined as low bone mineral for age) were applied to pQCT and DXA scans.
The study received a favorable ethical opinion from the University of Surrey (UEC/2016/009/FHMS) and Federal University of Goiás Ethics Committees and from the Brazilian National Ethics Committee (CONEP) (CAAE 62149516.9.0000.5083, CEP-UFG nº2013222; CONPEP nº 1972029; respectively).
2.5. Statistical Analysis
Statistical analysis of the data was done using SPSS software for Windows (version 25.0; IBM Corp, Armonk, NY, USA). Data were tested for normal distribution using the Kolmogorov–Smirnov tests. Non-normally distributed variables were log transformed and reported on the original scale. Non-parametric tests were used when log transforming did not normalize the data. Descriptive statistics were determined for all variables. Continuous variables are presented as mean ± standard deviation (SD) for normally distributed variables or as median (25%, 75% percentiles) for not normally distributed. For categorical variables, the frequency and percentage are reported.
Baseline characteristics (age, weight, BMI, waist circumference, dietary intakes, and biomarkers) were compared between countries, by independent t-tests, or Mann–Whitney U tests if appropriate.
For each country separately, ANOVA, or the corresponding non-parametric Kruskal–Wallis test, were used to compare bone parameter measurements between age tertiles and between vitamin D status groups. Pearson’s correlation, or the corresponding non-parametric Spearman rho, were applied to investigate the correlation between bone parameter measurements and 25(OH)D or PTH concentrations as well as vitamin D and calcium intakes.
Regression models were used to investigate the association between bone parameters and age and anthropometric measurements.
Results are presented separately for each country as measurements derived from different methodologies.
A p value of <0.05 was considered significant.
4. Discussion
Vitamin D is essential for musculoskeletal health, and there is a consensus that serum concentrations should be at least 25 nmol/L to prevent detrimental effects on bone [
1,
2,
3]. The present cross-sectional analysis of healthy adult women showed that amongst Brazilian women living in England, all participants presented a normal radial z-score. Amongst women living in Brazil, only two participants (2.5%) presented with low bone mineral for age for the lumbar spine. Furthermore, bone parameters were significantly associated with weight and BMI in participants in both the England and Brazil cohorts, and with age in women living in Brazil only. This study also found that for women living in England, those with vitamin D deficiency (<25 nmol/L) had significantly lower total vBMD at the 66% site than those with greater concentrations, independent of age and BMI. This observation at the cortical site (66%) only is likely due to the fact that most bone loss is the result of intracortical and endocortical remodeling, which produces cortical porosity and cortical thinning. Therefore, any alteration of BMD would be first evidenced at the cortical (66%) in comparison to the trabecular (4%) site. There were no associations between 25(OH)D status and any of the measures from the DXA scan in participants from the Brazil cohort, although the range of vitamin D status was smaller and the generally better vitamin D status of this sub-group may have limited the observation of effects on the bone. Nevertheless, the finding of bone parameters being significantly associated with the 25(OH)D/PTH system in women living in England, but not in those living in Brazil, together with the finding that plasma PTH concentrations were negatively correlated with 25(OH)D concentrations in women living in the UK, but not in those living in Brazil, are relevant for speculating on the possible role of different lifestyles and climates in mediating the relationship between 25(OH)D, PTH, and bone.
The associations between bone parameters and weight and BMI suggest that the observed poorer bone health in this sample is likely to be due to lower weights. These findings are in agreement with several studies that have demonstrated positive associations between BMI and bone parameters, likely explained by a greater load on weight-bearing bones [
23,
24,
25]. In a cross-sectional study investigating a total of 412 Brazilian postmenopausal women, aged 40 to 75 years with BMD assessed by DXA at the lumbar spine, a higher BMI was associated with reduced osteoporosis risk [
26]. A study with 393 post-menopausal Brazilian women reported a lower prevalence of osteopenia and osteoporosis amongst obese women compared to those with eutrophic BMI (DXA-derived BMD assessment) [
27]. Another study in São Paulo, with 413 Brazilian women (52.5% <59 years and 47.5% >60 years) showed the BMI to be a positive predictor for DXA-derived BMD at the femoral neck [
28]. For women living in England, there was an inverse statistically significant correlation between vBMD at the 66% site (total and cortical) and weight. Some studies have reported similar findings of the inverse correlation in the cortical site of the radius observed in this study [
29,
30]. Some hypotheses to explain this include variances in bone geometry or bone damage, increased bone turnover, and a momentary reduction in vBMD due to increased mechanical loading. Further investigation is now required, especially in adult women under 50 years of age, to better understand these relationships [
29,
31,
32].
To date, there are still few longitudinal studies that have investigated the association of long-term lower calcium intake with bone health outcomes later in life, particularly from a younger starting age, i.e., <30–35 years. A longitudinal study of 5022 women (born between 1914 and 1948 and followed up for 19 years) with modest dietary calcium intake reported that only the lowest quintile of calcium intake (<751 mg) was associated with increased risk of fracture or osteoporosis [
33]. It has been suggested that the association between calcium and BMD might not be consistently linear, and a sufficient vitamin D status is likely to compensate for the negative effects of low calcium intake on bone [
34,
35,
36].
In the United Kingdom, the latest national survey reported mean dietary calcium intakes of 897 and 746 mg/day for men and women aged 19 to 64 years, respectively (NDNS years 7–8, 2015–2016). In Brazil, the latest national survey available to date reported that mean dietary calcium intakes were 546.4 and 476.4 mg/day for men and women aged 20 to 59 years, respectively (POF, 2008–2009) [
37]. In this study, 72.8% of participants had dietary calcium intakes below the RNI of 700 mg/day, while only 5.2% met the 1000 mg/day RDA reference. All participants had dietary vitamin D intakes below the 15 μg/day RDA recommended by the IOM [
21] and 99.2% had intakes below the RNI of 10 μg/day proposed by SACN [
20]. Despite this, there were no significant correlations between bone parameter measurements and habitual dietary vitamin D and calcium intakes, either for England or Brazil dwelling participants, perhaps again reflecting the narrow range of intakes and therefore lack of discriminatory power in this sample.
Elevated concentrations of serum PTH are associated with several adverse outcomes, particularly musculoskeletal outcomes [
38,
39,
40]. Secondary hyperparathyroidism may lead to bone loss due to increased bone turnover rates [
38,
41]. Several studies have shown 25(OH)D to be inversely correlated with PTH [
10,
38,
40,
42]. The present study confirms this with 25(OH)D concentrations being inversely correlated with PTH concentrations in healthy adult women. Additionally, 10.4% of participants had secondary hyperparathyroidism, with a higher prevalence amongst those with deficient and insufficient vitamin D status. The difference in the prevalence of secondary hyperparathyroidism was more evident between the two lower cut-off points for women living in England and between the two highest cut-off points for women living in Brazil, which might be explained by the significant difference in the mean and range of 25(OHD concentrations between the two groups. Due to the strong inverse correlation between 25(OH)D and PTH concentrations, individuals with deficient and insufficient vitamin D status would very likely benefit from vitamin D supplementation with a view to suppressing PTH secretion from the parathyroid gland.
Moreover, several studies have also shown an inverse correlation between serum PTH levels and BMD [
40,
41]. In this study, a negative correlation between PTH concentrations and total BMD at the 66% site was seen, although this was not significant after controlling for age and BMI. There were no associations with PTH levels and any of the measures from the DXA scan in participants from the Brazil cohort, which may be due to long-term detrimental consequences of higher PTH levels not yet being evident in the relatively young women in this study.
The associations between 25(OH)D, PTH, and bone mineral density (BMD) are still much debated. Evidence is more robust in specific subgroups, such as in those with low vitamin D levels [
39], in osteoporotic subjects [
9,
11], post-menopausal women [
9,
11,
43,
44], or in the elderly [
14,
38,
45]. The findings presented here are in accordance with previous reports of studies that included healthy younger adults as well as those that encompass the whole spectrum of vitamin D status (i.e., deficient, sufficient, and adequate), demonstrating the absence of any association between 25(OH)D or PTH and BMD [
13,
46,
47]. The reasons for no effects on bone parameters might be due to the lack of power within the deficient subgroup and the relatively young participants, who might not be currently affected by potential long-term detrimental outcomes from low 25(OH)D and high PTH concentrations. Further studies are now required to better understand the effects of inadequate concentrations of 25(OH)D or PTH on bone health specifically in younger otherwise healthy women and future health consequences.
The strengths of this study include the fact that both measurements used for bone density assessment, pQCT and DXA scan, are considered gold standard methods. The analysis controlled for possible confounders, such as age, weight, and BMI. A limitation of the study is that the DXA scan software reports using a combination of databases to calculate the Z-score, while for the pQCT data a specific published reference data was used. Ideally, the same method would have been used for both groups, and a DXA scan would be the preferred option, as it has been shown to be an accurate diagnostic clinical tool and would have provided a central measurement for all participants [
48]. It might be that there was insufficient statistical power in this sample to detect significant differences in bone parameters and serum 25(OH)D and PTH concentrations. This analysis would benefit from further measures of biochemical markers of bone turnover to further investigate the relationship of 25(OH)D, PTH, and bone health in these adult women.
In conclusion, weight and BMI were significantly correlated with bone parameters in both groups and age was significantly correlated with BMD at the femoral neck for women living in Brazil only. Although 25(OH)D concentrations were not correlated to bone parameters at any sites, in either country, PTH concentrations showed a significant correlation with total vBMD at the 66% site for women living in England. This analysis also showed that secondary hyperparathyroidism was more common amongst those with a deficient and insufficient vitamin D status. There were no significant correlations between bone parameters and the usual dietary intake of vitamin D and calcium, in either of the groups.