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1 From the University of Jyväskylä, Jyväskylä, Finland (SC and AL); the University of Tennessee, Memphis (FT); the University of Kuopio, Kuopio, Finland (SC, HK, and AM); the University of Helsinki, Helsinki (MK and CL-A); the Central Hospital of Central Finland, Jyväskylä, Finland (AK); the Peurunka–Medical Rehabilitation Center, Laukaa, Finland (MA); and the University of Turku, Turku, Finland (JH and KV).
2 Supported by the Academy of Finland and the Finnish Ministry of Education. 3 Address reprint requests to S Cheng, Department of Health Sciences, University of Jyväskylä, PO Box 35, FIN-40014 Jyväskylä, Finland. E-mail: cheng{at}sport.jyu.fi.
ABSTRACT
Background: Very few studies have evaluated both parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D] and their effects on bone mass in children.
Objective: We studied the associations of serum 25(OH)D and intact PTH (iPTH) with bone mineral content (BMC) and bone mineral density (BMD) at different bone sites and the relation between serum 25(OH)D and iPTH in early pubertal and prepubertal Finnish girls.
Design: The subjects were 10–12-y-old girls (n = 193) at Tanner stage 1 or 2, who reported a mean (± SD) dietary calcium intake of 733 ± 288 mg/d. 25(OH)D, iPTH, tartrate-resistant acid phosphatase 5b (TRAP 5b), urinary calcium excretion, BMC, areal BMD, and volumetric BMD were assessed by using different methods.
Results: Thirty-two percent of the girls were vitamin D deficient [serum 25(OH)D 25 nmol/L], and 46% of the girls had an insufficient concentration (26–40 nmol/L). iPTH and TRAP 5b concentrations were significantly higher in the deficient group than in the insufficient and sufficient groups [iPTH: 43.9 ± 15.7 compared with 38.6 ± 11.2 pg/L (P = 0.049) and 32.7 ± 12.1 pg/L (P < 0.001), respectively; TRAP 5b: 12.2 ± 2.9 compared with 11.0 ± 2.8 U/L (P = 0.009) and 10.9 ± 1.9 U/L (P = 0.006), respectively]. The girls in the deficient group also had significantly lower cortical volumetric BMD of the distal radius (P < 0.001) and tibia shaft (P = 0.002). High iPTH concentrations were also associated with low total-body apparent mineral density and urinary calcium excretion (P < 0.007).
Conclusions: Vitamin D–deficient girls have low cortical BMD and high iPTH concentrations, which are consistent with secondary hyperparathyroidism. A low vitamin D concentration accompanied by high bone resorption (TRAP 5b) may limit the accretion of bone mass in young girls.
Key Words: 25-Hydroxyvitamin D • intact parathyroid hormone • nutrition • calcium intake • calcium excretion • bone mass • cortical bone density • biomarkers • prepubertal girls • secondary hyperparathyroidism
INTRODUCTION
Vitamin D, an essential nutrient with hormone-like activity, regulates calcium metabolism throughout the life cycle. In children, the regulation of calcium metabolism is imperative to ensure adequate growth and development of bone mass. In adults, and especially in the elderly, vitamin D appears to be required for the maintenance of bone mass and the prevention of osteoporosis and fractures through the intestinal absorption of calcium (1–4). Yet, little is known about the precise relation in humans between vitamin D and its effects on bone mass.
The production of 25-hydroxyvitamin D [25(OH)D] in the liver is dependent on vitamin D obtained from the diet and on exposure to ultraviolet light. Ultraviolet rays stimulate the conversion of provitamin D in skin to vitamin D, which is then available to the liver for hydroxylation to 25(OH)D. Thus, circulating 25(OH)D concentrations are considered to be reflective of total vitamin D content (5, 6). However, there is no consensus on the serum 25(OH)D concentration that would yield the most benefit for bone health (7, 8). Two studies suggest setting the threshold for vitamin D deficiency on the basis of the relation between serum 25(OH)D and serum intact parathyroid hormone (iPTH) according to the theory that the suppression of PTH is beneficial for bone (9, 10). Studies showed that mild vitamin D deficiency may lead to secondary hyperparathyroidism, with negative consequences for calcium metabolism and bone mineral density (BMD) (11–13). Very few studies evaluated both PTH and 25(OH)D in children, and the results of those studies were inconclusive (14, 15). The aims of the present cross-sectional study were to determine the association of serum 25(OH)D and iPTH concentrations with bone mineral content (BMC) and BMD at different bone sites and the relation between serum 25(OH)D and iPTH in prepubertal and early pubertal girls during winter.
SUBJECTS AND METHODS
Subjects
The subjects were 193 girls aged 10–12 y who resided in the city of Jyväskylä, Finland, which is located at 62° northern latitude. Their maturation status was characterized by Tanner stage 1 or 2, and they were participants in an intervention study to evaluate supplementation with calcium, vitamin D, and dairy products on the acquisition of bone mass during prepuberty. All the participants and their legal guardians provided written informed consent in accordance with the Ethical Committees of the University of Jyväskylä, the Central Hospital of Central Finland, and the Finnish National Agency of Medicines.
To be eligible for the study, the participants had to have no history of serious medical conditions or use of medication known to affect bone metabolism. For the determination of sexual development, the Tanner grading system was used (16). The Tanner system uses assessments of the pattern of development of pubic hair and breasts. If the Tanner stage assessment based on pubic hair differed from the assessment based on the breasts, the latter was used. A public health nurse assessed the Tanner stage of each subject.
Nutrition information
Dietary information was obtained from a food-intake diary kept for 3 successive days (2 weekdays, ie, ordinary school days, and 1 weekend day). We used a portion guidebook, which contains pictures of ordinary foodstuffs, dishes, and household measures. The participants were instructed by a study nurse to follow the guidebook and, with the assistance of their parents, to write down on a food-record sheet all the food items and dishes that they had eaten, including their quality. The food records were then coded into a nutrient-intake program (MICRO-NUTRICA), which was developed and is maintained by the Research Center of the Social Insurance Institution of Finland (Turku). The MICRO-NUTRICA database contains 62 dietary factors, 600 different food items, and 600 dishes commonly served in Finland. The program gives a reasonably good estimate of the intake of energy and of most nutrients (17).
Physical activity
Physical activity levels were assessed with the use of a self-reported questionnaire including items on the frequency, duration, and type of exercise performed during the subjects’ leisure time (18).
Laboratory assessments
After the subjects had fasted overnight, blood samples were taken between 0730 and 0900 during the periods 3–17 December 1999 (G1, n = 99) and 10 January–5 February 2000 (G2, n = 94). Serum samples were stored at -70 °C until analyzed. Serum 25(OH)D concentrations were measured by radioimmunoassay (Incstar Corporation, Stillwater, MN) (8). The intra- and interassay CVs were 10% and 15%, respectively. The reference range for 25(OH)D was 25–120 nmol/L. Serum iPTH concentrations were measured by using an immunoradiometric method (Nichols Institute, Juan San Capistrano, CA) (8), with 10–65 pg/L as the reference range. The intra- and interassay CVs were 4% and 3%, respectively.
Serum concentrations of tartrate-resistant acid phosphatase isoform 5b (TRAP 5b) (19) were measured as a marker of bone resorption by using a commercial immunoassay (BoneTRAP; SBA-Sciences, Oulu, Finland). The intra- and interassay CVs were 2.7% and 24.5%, respectively.
To estimate calcium excretion with the use of colorimetric assay photometry (iEMS reader MF; Thermo Labsystems Oy, Vantaa, Finland) (20), 12-h urine samples (1900–0700) were collected for 2 d for G1, and 24-h urine samples were collected for 1 d for G2. Calcium excretion was adjusted for creatinine, urine volume, hours of collection, and body weight. Because there were no significant differences in the ratio of calcium to creatinine or in calcium excretion between G1 and G2, the results were then pooled for the final statistical analysis. Urine samples were stored at -70 °C until analyzed.
Anthropometric measurements
Height and weight were determined with the subjects wearing only light clothing and no shoes. Height was determined by using a fixed wall scale. Weight was determined within 0.5 kg by using an electronic scale, which was calibrated before each measurement session. Body mass index (BMI) was calculated as weight (kg)/height2 (m).
Bone mass and density measurements
Dual-energy X-ray absorptiometry measurements
Bone area, BMC, and areal BMD of the whole body, femoral neck, total femur, and lumbar spine (L2–L4), as well as the lean soft tissue mass and fat mass of the whole body, were measured by using a Prodigy densitometer (GE Lunar Corp, Madison, WI). The precision of the repeated measurements was expressed as the percentage CV (CV%). CV% values at different bone sites ranged from 0.60 to 1.18 for BMC and from 0.86 to 1.31 for areal BMD. CV% values were 1.03 for lean soft tissue mass and 2.23 for fat mass. In addition, we applied the method developed by Brismar et al (21) for calculating the whole-body bone mineral apparent density (WBMAD):
RESULTS
We found that 32% of the girls had a serum 25(OH)D concentration below the reference range and were thus considered to be vitamin D deficient. Forty-six percent of the girls had a 25(OH)D concentration deemed to be insufficient. There were no significant differences between the three 25(OH)D concentration groups in body weight; height; BMI; fat mass; lean soft tissue mass; Tanner stage; physical activity; percentages of energy from dietary protein, fat, and carbohydrate; and vitamin D and energy intakes (Table 1). In our sample, 87% of the girls reported suboptimal vitamin D intake (< 5 µg/d). Compared with the girls in the sufficient group, those in the deficient group had significantly higher daily calcium intakes (Table 1). However, after adjustment for energy intake, no significant differences in calcium intake were observed between the groups.
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TABLE 1 . Comparison of physical characteristics between subjects in the vitamin D–deficient (25-hydroxyvitamin D [25(OH)D] 25 nmol/L), vitamin D–insufficient [25(OH)D = 26–40 nmol/L], and vitamin D–sufficient [25(OH)D >40 nmol/L] groups
A comparison of biomarkers between the subjects in the vitamin D–deficient, vitamin D–insufficient, and vitamin D–sufficient groups is shown in Table 2. The girls in the deficient and insufficient groups had significantly higher iPTH concentrations than did those in the sufficient group (P < 0.001 and P = 0.042, respectively). There were no significant differences in calcium excretion between the vitamin D status groups. However, concentrations of the bone resorption marker, TRAP 5b, were significantly higher in the deficient group than in the insufficient (P = 0.006) and sufficient (P = 0.015) groups.
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TABLE 2 . Comparison of biomarkers between subjects in the vitamin D–deficient (25-hydroxyvitamin D [25(OH)D] 25 nmol/L), vitamin D–insufficient [25(OH)D = 26–40 nmol/L], and vitamin D–sufficient [25(OH)D >40 nmol/L] groups1
There were no significant differences between the vitamin D status groups in the BMC and areal BMD of the total body, femur, or lumbar spine as measured by dual-energy X-ray absorptiometry (DXA). However, BMI and Tanner stage had significant influences on bone measurements. When Tanner stage and BMI were controlled for (Table 3), the BMC of the total femur was significantly higher in the deficient group than in the sufficient group (P = 0.04).
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TABLE 3 . Comparison of bone mass and density measured by dual-energy X-ray absorptiometry between subjects in the vitamin D–deficient (25-hydroxyvitamin D [25(OH)D] 25 nmol/L), vitamin D–insufficient [25(OH)D = 26–40 nmol/L], and vitamin D–sufficient [25(OH)D >40 nmol/L] groups1
Comparing bone measurements made with pQCT between the 3 vitamin D status groups (Table 4), we found that the girls in the deficient group had significantly lower whole-bone vBMD at the distal radius (P = 0.002) and tibia shaft (P = 0.02) than did those in the insufficient group; the girls in the deficient group also had significantly lower cortical vBMD at the distal radius than did those in the sufficient group (P < 0.001). The girls in the deficient group had significantly lower vBMD of the cortical bone in the distal radius than did those in the sufficient and insufficient groups (P < 0.001; Figure 1). Cortical vBMD of the tibia was significantly lower in the deficient group than in the insufficient group (P = 0.002). Serum vitamin D concentrations were apparently not related to the CSA or the vBMD of the subcortical bone of the tibia or of the trabecular bone of the radius. There was no influence of either BMI or Tanner stage on the subcortical vBMD of the tibia or on the trabecular vBMD of the radius.
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TABLE 4 . Comparison of bone mass and density measured by peripheral quantitative computed tomography between subjects in the vitamin D–deficient (25-hydroxyvitamin D [25(OH)D] 25 nmol/L), vitamin D–insufficient [25(OH)D = 26–40 nmol/L], and vitamin D–sufficient [25(OH)D >40 nmol/L] groups1
FIGURE 1. . Mean (± SD) cortical volumetric areal bone mineral density (vBMD) of the radius and tibia according to 25-hydroxyvitamin D [25(OH)D] concentration groups. *,**Significantly different from the vitamin D–deficient [25(OH)D concentration 25 nmol/L] group (ANOVA with Bonferroni adjustment): *P < 0.001, **P = 0.002.
In the linear regression model, we did not find any association between 25(OH)D and BMC or areal BMD at any of the skeletal sites measured by DXA. Nor was iPTH associated with BMC or areal BMD at any of the sites. However, there was a positive correlation between 25(OH)D and cortical vBMD of the distal radius (r = 0.294, P < 0.001), a negative correlation between iPTH and cortical vBMD of the distal radius (r = -0.168, P = 0.018), and a positive correlation between TRAP 5b and cortical vBMD of the distal radius (r = 0.261, P < 0.001). There was a curvilinear relation between 25(OH)D and iPTH (r = 0.311, P < 0.001; Figure 2) and between 25(OH)D and TRAP 5b (r = 0.286, P = 0.001). iPTH was associated with calcium excretion (r = 0.293, P = 0.001).
FIGURE 2. . Correlation between intact parathyroid hormone (iPTH) and 25-hydroxyvitamin D [25(OH)D] concentrations. Each circle represents an individual subject. The solid line is a cubic regression fit line.
The girls in the high-iPTH group (> 33 pg/L) had significantly larger bone areas measured by DXA and significantly larger CSAs measured by pQCT than did those in the low-iPTH group ( 33 pg/L) (data not shown), but there were no significant differences between the groups in BMC, areal BMD, and vBMD at the measured bone sites. However, the girls in the high-iPTH group had significantly lower WBMAD (P = 0.006; Figure 3) and calcium excretion (P = 0.004; Figure 3) than did those in the low-iPTH group both before and after adjustment for differences in BMI and Tanner stage. There was no significant interaction of 25(OH)D and iPTH on WBMAD or calcium excretion.
FIGURE 3. . Mean (± SD) whole-body bone mineral apparent density (WBMAD) and calcium excretion in groups of subjects having low ( 33 pg/L) or high (> 33 pg/L) intact parathyroid hormone (iPTH) concentrations. *,**Significantly different from the low-iPTH group (nonparametric ANOVA with Mann-Whitney U test): *P = 0.006, **P = 0.004.
When taking into account differences in blood-sample collection time (on average 37 d apart from December to January), we found that mean (± SD) 25(OH)D concentrations were significantly lower in G2 than in G1 (28.5 ± 10.9 compared with 36.4 ± 10.7 nmol/L; P < 0.001). These lower 25(OH)D concentrations in G2 were associated with elevated concentrations of iPTH (35.9 ± 12.4 and 41.7 ± 14.1 pg/L in G1 and G2, respectively; P = 0.003) and TRAP 5b (10.4 ± 1.2 and 12.2 ± 3.4 U/L in G1 and G2, respectively; P < 0.001). The time of blood-sample collection correlated significantly with 25(OH)D (r = -0.376, P < 0.001), iPTH (r = 0.227, P = 0.001), and TRAP 5b (r = 0.377, P < 0.001). There were no significant differences between G1 and G2 in the distribution of subjects in the 3 vitamin D–status groups.
DISCUSSION
In the present study, using a cutoff of 25 nmol/L for serum 25(OH)D concentration, we found that 32% of the girls, who had an average dietary calcium intake of 733 mg/d, were deficient in vitamin D. An additional 46% of the girls were considered to be vitamin D insufficient on the basis of a 25(OH)D concentration of 26–40 nmol/L. The percentages observed in the present study were higher than those found in our previous studies in adolescents and adults (8, 22). The girls in the vitamin D–deficient group had significantly higher iPTH and TRAP 5b concentrations than did those in the vitamin D–sufficient group. The girls in the deficient group also had significantly lower cortical vBMD of the distal radius and tibia shaft as measured by pQCT with and without adjustment for Tanner stage and BMI.
Researchers are in agreement that 25(OH)D is the best indicator for determining adequacy of vitamin D. However, the normal range (mean ± 2 SDs) for a group of healthy participants varies according to the geographic location of the study and the time of day or year that the samples are collected. Thus, the definition of vitamin D deficiency has varied between 10 and 50 nmol/L depending on the study (7). In Finland, the cutoff for vitamin D deficiency is < 25 nmol/L (22). This is based on the normal distribution of 25(OH)D concentrations found in earlier studies. However, many questions remain with regard to the 25(OH)D concentration required to maintain an adequate rate of calcium metabolism and to promote optimal skeletal growth.
Using an interval that encompasses the range of values between 25(OH)D deficiency and sufficiency (26–40 nmol/L, ie, vitamin D insufficiency) (8–10), we did not detect a negative influence on bone mass of insufficiency relative to sufficiency. However, the participants with insufficient 25(OH)D concentrations had higher iPTH and TRAP 5b concentrations, suggesting a subclinical influence that may not be evident by bone mass assessments until later in adolescence. This is in accordance with the results reported by Outila et al (8) in older adolescents. Using an ecologic study design, Oliveri et al (23) did not find a relation between limited winter sunlight and bone mass in prepubertal children. However, serum 25(OH)D concentrations were not quantified or examined in relation to the sufficiency of vitamin D stores. Unfortunately, at present, data are only available cross-sectionally, and the effects of fluctuations in vitamin D stores on the bones of growing children are unknown.
25(OH)D concentrations fluctuate according to dietary intakes and sunlight exposure. A Danish study indicated that the recommended daily intake of vitamin D is not sufficient to meet metabolic needs if exposure to sunlight is limited (24). In Finland, sunlight is limited during 9 mo of the year. We found that blood-sample collection times had a clear effect on 25(OH)D concentrations. The blood samples collected during January and February had significantly lower 25(OH)D concentrations (on average 22% lower) than did the blood samples collected during December, even though the distributions of subjects in the deficient, insufficient, and sufficient groups were similar between the 2 time periods. The lower 25(OH)D concentrations were also related to elevated iPTH and TRAP 5b concentrations. This finding indicates that limited exposure to sunlight is one of the reasons for vitamin D deficiency in late winter. Because all the samples were analyzed by the same person, who was blinded to the source of the samples, there was no bias regarding the assessments of 25(OH)D and iPTH. Dietary intakes may affect serum concentrations when sunlight is insufficient. Because dairy products were not fortified with vitamin D at the time the data were collected, the main dietary sources were fortified margarine (45% of intake) and fish (13% of intake), thus making it difficult for the subjects to achieve adequate vitamin D intake. In our sample, 87% of the girls reported suboptimal vitamin D intake, which suggests that vitamin D supplementation on an individual basis or through the food supply is indicated.
Whether or not to supplement persons with vitamin D has been a matter of some debate. The focus of this debate has been on the level of supplementation required to ensure target serum 25(OH)D concentrations during periods of limited sunlight (1, 10, 25). Guillemant et al (26) showed that supplementation with 2.5 mg (100 000 IU) vitamin D 3 times from September to January prevented the usual decrease in wintertime 25(OH)D concentrations in adolescent males. Had this level of supplementation been applied to our sample, the mean dietary intake would have been 20 µg/d. Currently, the Finnish recommended intake for girls aged 10–12 y is 5.0 µg/d (200 IU/d). The average vitamin D intake in the present study was 2.7 µg/d. This indicates that the vitamin D intake in our subjects was far below the level required to maintain 25(OH)D concentrations during the long, dark winter. However, there are no experimental data available on the concentration of vitamin D stores needed to meet the metabolic needs of prepubertal girls when exposure to sunlight is limited for extended periods of time.
Recent studies have suggested raising the threshold for vitamin D deficiency on the basis of the serum 25(OH)D concentration required to suppress serum iPTH concentrations. The 25(OH)D concentration that is needed to suppress PTH ranges from 30 nmol/L for elderly persons (27) to 40 nmol/L for adolescent females (8) and > 40–50 nmol/L for hospitalized patients (9). Other researchers reported a negative relation between serum 25(OH)D and iPTH but did not specify a cutoff concentration to mark vitamin D deficiency (14, 27–30).
Part of the difficulty in establishing what concentration of 25(OH)D can be considered deficient in relation to iPTH is the complex interrelation between vitamin D and iPTH in the maintenance of serum calcium. Although vitamin D stores are relatively stable from day to day, iPTH shows marked variation in response to 1,25-dihyroxyvitamin D and alterations in ionized serum calcium. This makes it difficult to obtain consistent results with the use of bone mass measurements or laboratory data. In our study, the low concentrations of 25(OH)D stores and high concentrations of iPTH and bone resorption marker (TRAP 5b) are consistent with secondary hyperparathyroidism as described in adults (11–13). The low WBMAD suggests that secondary hyperparathryoidism in children may compromise skeletal growth. The low urinary calcium excretion associated with high iPTH concentrations suggests that there is a compensatory mechanism to conserve calcium.
As far as we know, the present study is the first to provide results on the associations of vitamin D and iPTH with bone biomarkers and bone mass and density assessments at multiple bone sites and is the first such study to use pQCT. Our results clearly showed that, if the DXA results had been considered in isolation, we might have drawn a different conclusion, which may have led to misinterpretation of the role of 25(OH)D on bone. pQCT provides us with volumetric bone density, which is independent of bone size. We found significant differences only in volumetric bone density, including a significant difference between the vitamin D–deficient and vitamin D–sufficient groups in cortical vBMD measured with pQCT. This finding may indicate that in the deficient group, the secondary consolidation of mineral may have been limited. This finding is in accordance with the results reported by Khan and Bilezikian (31) that cortical vBMD decreases during hyperparathyroidism and that trabecular vBMD either increases or remains unchanged.
We did not find significant differences between the vitamin D–status groups in physical activity, maturation, weight, height, or vitamin D intake. The girls in the vitamin D–deficient group had significantly higher daily calcium and phosphorous intakes than did the girls in the vitamin D–sufficient group. However, after adjustment for energy intake, the differences between the groups disappeared. Dietary intake data were collected before the subjects and researchers knew the subjects’ vitamin D status; therefore, selective underreporting based on vitamin D status was unlikely.
Our study participants reported an average calcium intake of 733 mg/d. Most (92.4%) of the subjects had a calcium intake below the recommended intake (900 mg/d) set by the Finnish National Nutrition Council over a 3-d time period. The frequency of vitamin D deficiency may be higher in girls with low dietary calcium intake than in those with an adequate intake even when milk is fortified with vitamin D (32). A recent study provided some evidence that a very low dietary calcium intake could affect 25(OH)D concentrations (33). However, because the sample of girls who reported adequate dietary calcium intake was so small in the present study, we were unable to study how calcium intake influences serum 25(OH)D concentrations.
In summary, we showed in the present study that a substantial number of 10–12-y-old girls from central Finland who had a low calcium intake were either deficient in vitamin D (32%) or could be considered insufficient (46%) on the basis of their serum 25(OH)D concentrations. We showed that girls who are vitamin D deficient have lower cortical bone density and higher iPTH concentrations, results that are consistent with secondary hyperparathyroidism. A low vitamin D concentration accompanied by an elevated marker of bone resorption (TRAP 5b) may limit the accretion of bone mass. Limited exposure to sunlight and low dietary vitamin D intake are the main reasons for vitamin D deficiency during winter. Our results suggest that vitamin D supplementation on an individual basis or through the food supply is indicated for early pubertal and prepubertal girls. A larger sample size and longitudinal follow-up are necessary to provide consistent evidence for the effect of low vitamin D status on bone and calcium metabolism in growing children.
ACKNOWLEDGMENTS
We thank Erkki Helkala, Pia-Leena Salo, Miia Kemikangas, Leila Vilkki, and Marja Ylhäinen for their valuable work and technical assistance on this project.
SC was involved in the conception, management, design, and funding of the study, as well as in recruitment, data collection, analysis, and the writing of the paper. SC is the guarantor. FT was involved in the conception, management, and design of the study and in data analysis and paper preparation. HK was responsible for medical examinations and was involved in the management, design, and funding of the study, as well as in paper preparation. MK was involved in the vitamin D and iPTH analyses. AL was responsible for collecting the dietary information and for the nutrient analyses and was involved in paper preparation. AK was responsible for the medical examination and screening of the subjects and for the DXA assessments. AM was involved in the conception and design of the study and in biomarker analysis and paper preparation. MA was involved in funding and was responsible for medical examination of the subjects. JH was responsible for biomarker analyses and was involved in paper preparation. KV was involved in the design and funding of the study. CL-A was responsible for vitamin D and iPTH analysis and was involved in the design and funding of the study and in paper preparation. None of the authors had financial or personal-interest affiliations with the sponsors of this research.
REFERENCES