Abstract
Objective:
GC/DBP effects on response to vitamin D supplementation have not been well-studied. Thus we assessed free and total 25-OHD after vitamin D treatment across the 6 common GC haplotypes.
Design:
This double-blind, randomized study compared two vitamin D3 doses in healthy, urban-dwelling 6-month to 10-year-old children at-risk for vitamin D deficiency. Randomization was stratified by GC haplotype.
Methods:
Children were randomized to receive 2800 or 7000 International Units of vitamin D3 weekly. 25-OHD and 1,25(OH)2D were sampled at baseline and after 1 and 6 months of supplementation.
Results and Conclusions:
192 of 225 enrolled subjects completed the study. After one month, total 25-OHD increased with both doses, and were higher with 7000 IU/week (85.5 ± 22.8 nmol/L) compared to 2800 IU/week (76.8 ± 18.0 nmol/L), despite equivalent baseline levels. No further significant increase occurred at 6 months (89.8 ± 35.5 and 74.3 ± 18.3 nmol/L, respectively). Free 25-OHD similarly changed. 25-OHD differed among GC groups at baseline. Although no significant effects of individual GC haplotypes on incremental changes were evident, a trend towards an effect of combined “at risk” GC alleles on response was evident (P=0.06). Total 1,25(OH)2D showed modest increases, moreso with the larger dose. In urban-dwelling children at-risk for vitamin D deficiency, one month of vitamin D3 2800 IU/week increased 25-OHD across all GC haplotype groups, and somewhat enhanced with 7000 IU/week with no further significant increases after 6 months of supplementation. Free 25-OHD measures offer no monitoring advantage over total 25-OHD.
Keywords: Vitamin D Binding Protein; 25-hydroxyvitamin D; free 25-hydroxyvitamin D; 1,25 dihydroxyvitamin D; randomized clinical trial
INTRODUCTION
Although various vitamin D supplementation recommendations for children are available (1–4), few are targeted to children at increased risk for vitamin D deficiency, such as those living in extreme latitudes and for under-studied minorities where health care disparities exist. Even less information is available regarding the impact of genetic variants upon response to vitamin D supplementation. For safety reasons, routine vitamin D supplementation at or above 1000 IU daily has not been widely studied. The Institute of Medicine recommended Upper Tolerable Limit for daily vitamin D intake was revised in 2011 by age (1500 IU for 6–12 month-old children, 2500 IU for 1–3 year-olds, 3000 IU for 4–8 year-olds, and 4000 IU for 9–18 year-olds), leading to wider acceptance of higher vitamin D dosing for safe and ethical investigation (1).
In addition, the impact of genetic variants in vitamin D binding protein (DBP) and other vitamin D-related genes on vitamin D metabolism has become apparent (5–13). While circulating 25-OHD is in part determined by GC haplotype, limited information exists regarding effects on the response to vitamin D supplementation (14). In the current study we examine potential effects of variance in DBP/GC on response to vitamin D supplementation across the 6 common GC haplotypes. Furthermore, as free 25-OHD levels are considered the physiologically active fraction (15), we compare total and free 25-OHD responses as differences between these two measures would suggest that using total 25-OHD to monitor supplementation or therapy may be misleading.
Our previous work identified biochemical and nutritional determinants of 25-OHD levels in healthy children from a largely Hispanic, urban population of children living in Northeastern USA, with characteristics associated with high risk for vitamin D deficiency (16). Investigation of DBP/GC effects (5) confirmed a significant impact upon circulating 25-OHD and DBP concentrations, generating the hypothesis that the response to vitamin D supplementation may be dependent upon GC haplotype. We therefore undertook the current study to identify effect sizes of response to vitamin D and to identify design considerations for further studies within this understudied population.
METHODS
We aimed to identify differences between typical vitamin D supplementation (equivalent to the RDA of 400 IU daily at the time of performance of the study, 2010–2012) and a larger dose (equivalent to 1000 IU daily, the upper tolerable limit in this age group at that time, as recommended by the Institute of Medicine) (1), across the 6 common GC haplotype groups.
Subjects and Study design
Healthy, urban-dwelling children, aged 6 months to 10 years, and from a largely Hispanic community in New Haven, Connecticut were recruited to participate in a prospective, randomized, double-blinded vitamin D supplementation study. Families studied previously were contacted, and postings on www.clinicaltrials.gov (NCT01050387), in local medical offices and other community sites were employed. Exclusion criteria included any history of bone/mineral disorders or use of medications known to affect vitamin D metabolism (e.g., systemic glucocorticoids, pharmacologic vitamin D metabolites, or vitamin D supplements in excess of 400 IU/d). The primary outcome was change from baseline in total circulating 25-OHD between two dosing levels, and across the six commonly occurring GC haplotypes. Secondary outcomes included change from baseline of free 25-OHD levels, and in 1,25(OH)2D. Total serum calcium was measured as a safety measure. The study was approved by the Yale University Human Investigation Committee and written informed consent was obtained from the appropriate parent or guardian.
Initial visit and intervention
An initial visit to our research center included measurement of height, weight, and blood pressure. Erythematous and melanin-based skin tone were assessed at forehead and axillary sites using a Cortex DSM II reflectometer (CyberDERM, Broomall, PA) (17). A demographic questionnaire was completed, and a fasting blood sample was obtained for measurement of serum vitamin D metabolites, DBP, and biochemical variables detailed below. For subjects new to the cohort for whom genotype data was not available, DNA was collected for determination of GC haplotype. Speed of Sound (SOS) was determined at the radius using the Sunlight Pediatric Ultrasound device (BeamMed, Plantation, FL), as described previously (18). An extensive description of the baseline biochemical profile of this cohort is available elsewhere (19).
Subjects were randomized to receive either 2800 IU weekly (equivalent to the 400 IU daily, the RDA at the time of study) or 7000 IU of vitamin D3 weekly (equivalent to 1000 IU daily, the upper tolerable limit at that time) (1). Randomization was stratified by GC haplotype to achieve a balanced sample size across the 6 common haplotypes (1f/1f, 1f/1s, 1f/2, 1s/1s, 1s/2, and 2/2). Vitamin D3 was purchased as a 400 IU or 1000 IU per drop preparation (Carson Laboratories; Arlington Heights, Illinois), and repackaged by the Yale Investigational Pharmacy. Containers were relabeled without identification of vitamin D3 concentration to mask the dosing level to both subjects and investigators and distributed to caregivers with instructions to provide 7 drops weekly. Caregivers were telephoned weekly for reminders to provide timely dosing until visit two, 4 weeks later. Three random samples of each dose preparation were assayed for vitamin D3 ; all aliquots were within 12% of the expected concentration (mean of 109% for the 400 IU/drop preparation and 107% for the 1000 IU/drop preparation, Reinhold Vieth, University of Toronto).
Dietary food intake was obtained using a three-day food record and a multiple pass interview as described (20). Caregivers recorded intake during two weekdays and one weekend day, followed by a telephone interview using the multiple pass technique of related questions (amounts, preparation method, addition of condiments, etc). Data were analyzed using the Nutrition Data Software for Research (University of Minnesota).
Follow-up visits
The remaining 2 study visits occurred at 1 and 6 months following the initial visit. Serum 25-OHD and 1,25(OH)2D were repeated at the one-month visit, and again at the 6-month visit, together with serum DBP and directly measured free 25-OHD (dmf25-OHD). In addition, ultrasound speed-of-sound was repeated at the final visit, and caregivers were instructed to return vitamin D containers, which were weighed to assess overall compliance.
Analytical Methods
Biochemical assays
Serum calcium was performed using a Roche Diagnostics DPP modular autoanalyzer (Roche Diagnostics Corporation, Indianapolis, IN). Serum phosphorus, albumin and alkaline phosphatase activity were performed using standard colorimetric methods as described (19). Total serum 25-OHD (T25-OHD) and total serum 1,25(OH)2D (T1,25(OH)2D) were measured by radioimmunoassay kit methodology (DiaSorin, Stillwater, MN). Results of samples analyzed in our 25-OHD assay are consistently found to agree with the mid-range of outcomes for this assay in the international DEQAS standardization system (Vitamin D External Quality Assessment Scheme, London, UK) (21). The inter- and intra-assay coefficients of variation for the 25-OHD assay in our hands are 9.6% and 6.6%, respectively.
Serum DBP was determined by radial immunodiffusion methodology in the laboratory of one of the authors (22). Calculated free 25-OHD (cf25-OHD) and calculated free 1,25(OH)2D [cf1,25(OH)2D] were calculated using serum DBP and albumin concentrations, and their reported dissociation constants for these vitamin D metabolites respectively, as described (5). Direct measurement of free 25-OHD (dmf25-OHD) was performed using an ELISA-based kit (DIAsource ImmunoAssays; Louvain-la-Neuve, Belgium). We have previously shown cf25-OHD levels using this DBP methodology strongly correlate with dmf25-OHD (19).
Genotype analysis
The p.D432E (rs7041, occurring with either aspartate or glutamate at this residue) and p.T436K (rs4588, with tyrosine or lysine) SNPs of GC/DBP were genotyped as previously described (5). As there were no recombinants between these two loci, assignment of a diplotype for each subject, based on the three haplotype alleles – wild type (432D/436T, the electrophoretic variant 1f), polymorphic variant 432E (electrophoretic variant 1s), and polymorphic variant 436K (electrophoretic variant 2) – was unambiguous. Thus, each subject was assigned to one of the six commonly occurring haplotypes (1f/1f, 1f/1s, 1f/2, 1s/1s, 1s/2, and 2/2).
Statistical analysis
Descriptive statistics were used to summarize the data. Procedural means, frequencies for continuous and categorical variables. Multivariate analysis of covariance was performed as to evaluate effects of treatment, time and their related interaction on circulating 25-OHD and related outcomes (SAS v9.4). Parametric or non-parametric (ANOVA or Kruskal-Wallis) testing with multiple comparison testing was performed using GraphPad, PRISM® Software v8.00.
RESULTS
A total of 225 healthy children, aged 7 months to 10 years were enrolled from a largely Hispanic/LatinX background (74% of subjects). Phlebotomy and sample collection were completed in 203 (90%) children, who were randomized to vitamin D dose and continued in the study. Of these another 11 children dropped out or were lost to follow up, and 192 children completed the 6-month course of vitamin D supplementation (93% of those completing the initial visit). Characteristics of the recruited subjects and their baseline dietary mineral and vitamin D intake are shown in Table 1. The subjects were well-matched between treatment arms: serum 25-OHD (P=0.99), 1,25(OH)2D (P=0.40), calcium (P=0.51), and DBP (P=0.39) were no different between treatment arms at the baseline visit.
Table 1.
Characteristics of study subjects at baseline
| 2800 IU Weekly | 7000 IU Weekly | ||||
|---|---|---|---|---|---|
| Values | n | Values | n | ||
| Height (cm) | 111.4 ± 15.5 | 101 | 114.2 ± 16.9 | 100 | |
| Height (Z-Score) | 0.86 ± 1.2 | 100 | 0.77 ± 1.3 | 96 | |
| Weight (kg) | 23.1 ± 9.5 | 102 | 25.5 ± 9.5 | 100 | |
| BMI (kg/m2) | 18.0 ± 3.6 | 101 | 18.2 ± 4.8 | 102 | |
| Age (years) | 5.3 ± 2.3 | 102 | 5.9 ± 2.4 | 102 | |
| Gender | |||||
| Male | 54.9% | 56 | 45.1% | 46 | |
| Female | 45.1% | 46 | 54.9% | 56 | |
| Race/Ethnicity | |||||
| Hispanic/Latino | 75.5% | 77 | 77.2% | 78 | |
| African American | 23.5% | 24 | 15.8% | 16 | |
| Caucasian | 1% | 1 | 6.9% | 7 | |
| Other* | 1% | 1 | |||
| Axillary erythema score | 13.5 ± 2.6 | 101 | 13.7 ± 2.5 | 100 | |
| Axillary melanin score | 52.2 ± 12.6 | 101 | 51.7 ± 12.9 | 101 | |
| Forehead erythema score | 15.9 ± 8.3 | 101 | 15.7 ± 3.9 | 101 | |
| Forehead melanin score | 53.6 ± 12.2 | 101 | 54.2 ± 13.4 | 101 | |
| 25-OHD level (nmol/L) | 65.5 ± 16.5 | 102 | 58.3 ± 17.0 | 101 | |
| 1,25(OH)2D level (pmol/L) | 139.2 ± 38.4 | 98 | 136.8 ± 40.8 | 91 | |
| DBP level (nmol/L) | 4988 ± 550 | 101 | 5005 ± 516 | 101 | |
| Dietary calcium (mg/kg body weight) | 35.7 ± 15.7 | 87 | 36.2 ± 20.3 | 81 | |
| Protein (g/kg body weight) | 2.7 ± 0.9 | 87 | 2.7 ± 1.3 | 81 | |
| Energy (kcal/day) | 1377 ± 421 | 87 | 1419 ± 410 | 81 | |
| Vitamin D intake (IU/day) | 225 ± 87 | 87 | 214 ± 94 | 81 | |
Other refers to a combination of parentage and grand-parentage of any of the following ethnicities/heritage: African American and Hispanic, African American and Caucasian, African American and Native American, or Haitian.
Total 25-OHD
Baseline values (mean ± SD) for T25-OHD were 65.5 ± 16.5 nmol/L and 65.8 ± 17.0 nmol/L in the standard and higher dose groups, respectively. Fourteen children in each of the dosing groups had baseline T25-OHD less than 50 nmol/L; three children randomized to the lower dose group and none in the higher dose group had T25-OHD levels less than 37.5 nmol/L. There was a significant increase from baseline at both 1 and 6 months, with the higher dose resulting in an expected greater increment compared to the standard dose (Figure 1). Standard dosing resulted in a level of 76.8 ± 18.0 nmol/L (a mean incremental change of 11.8 nmol/L or 18%), whereas the higher dose resulted in a level of 85.5 ± 22.8 nmol/L (mean incremental change of 19.3 nmol/L or 29%) after 1 month of supplementation. At month 6, T25-OHD was 74.3 ± 18.3 nmol/L and 89.5 ± 35.5 nmol/L in the standard and higher dosing groups respectively (P<0.0001 for overall dose effect). The greater increment in T25-OHD occurred after one month of supplementation; differences between 1 and 6-month values were not significant (P=0.47). Two children in the lower dosing group, and one child in the higher dose group had T25-OHD less than 50 nmol/L at the end-of-study visit.
FIGURE 1:
Mean total circulating 25-OHD (T25-OHD) levels at baseline (left columns), after 1 month (middle columns), and after 6 months (right columns) in the standard dose group (dark bars) and higher dose groups (light bars). The overall dose effect indicated that the higher dose group achieved greater levels of 25-OHD after 1 and 6 months of supplementation (P < 0.0001). Most of the incremental change occurred after the first month). Error bars represent standard deviation. Sample size for each group is shown within each bar. To convert to ng/mL, divide the nmol/L value (shown) by 2.5.
There were few differences across the 6 GC haplotype groups in T25-OHD response to supplementation. There was a trend toward an overall haplotype effect (P=0.054), primarily due to the low increment in the 1f/1s (DETT) group, which was 11.5 ± 4.3 nmol/L lower at 1 month than that in the 1s/1s (EETT) group, which had the greatest response (P=0.008), and 19.3 ± 8.8 nmol/L less than the 1s/1s group after 6 months (P = 0.030), while other groups were no different from the 1s/1s group (Figure 2). There was no significant interaction between dosing level and haplotype.
FIGURE 2:
Mean total circulating 25-OHD (T25-OHD) levels for each of the GC haplotype groups are shown in separate panels. As in Figure 1, data at baseline (left columns), after 1 month (middle columns), and after 6 months (right columns) are shown for both the standard dose group (dark bars) and higher dose group (light bars). Incremental change to supplementation was generally comparable across the groups, with the 1f/1s (DETT) group showing the only significantly reduced increment, when compared to the group with the largest change (1s/1s, EETT; P= 0.008 at one month and P = 0.03 at 6 mos). Error bars represent standard deviation. Sample size for each group is shown within each bar. To convert to ng/mL, divide the nmol/L value (shown) by 2.5.
We then examined effects of the GC2 allele (having the variant lysine at residue 436 in DBP, “436K”, in contrast to the wild type tyrosine, “436T”) as we have previously shown the 436K variant to be associated with lower T25-OHD levels in a larger infant-toddler cohort. We confirmed that finding in the current study, as GC2 allele groups (1f/2, 1s/2, and 2/2) had lower T25-OHD at baseline than groups without a GC2 allele (1f/1f, 1s/1s, and 1f/1s) (P=0.0015). Specifically, T25-OHD was dependent upon the number of GC2 (436K) alleles present (Table 2). There was no effect of the GC2 allele on overall treatment response, however a significant interaction between treatment dose and GC2 allele was identified (P =0.043) after 6 months of supplementation; the increase in T25-OHD in the higher dose group was approximately twice as great for GC2 allele groups (35.5 ± 4.75 nmol/L, mean ± SD) than in its absence (19.3 ± 3.60 nmol/L). Only minimal differences in response between these groups were observed at the lower dosing level.
Table 2.
Gc1s and Gc2 allele effects on Total 25-OHD level (nmol/L). Values are Least Square Mean ± Standard Error. “K” refers to the lysine residue present at the 436 position in the Gc2 allele, and “E” refers to aspartate present at position 432 in the Gc1s allele.
| Baseline | 1 month | 6 months | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ALL | Low D | High D | P values | ALL | Low D | High D | P values | ALL | Low D | High D | P values | |||
| # of “K” (Gc2) alleles | ||||||||||||||
| 0 | 68.5 ± 1.4 (134) |
67.8 ± 2.0 (68) |
69.5 ± 2.0 (66) |
84.0 ± 1.8 (130) |
79.5 ± 2.5 (64) |
88.3 ± 2.5 (66) |
81.8 ± 2.6 (121) |
76.8 ± 3.7 (59) |
86.8 ± 3.7 (62) |
|||||
| 1 | 61.8 ± 2.2 (56) |
63.8 ± 3.2 (26) |
59.8 ± 3.0 (30) |
78.8 ± 2.7 (56) |
76.8± 4.0 (26) |
80.8 ± 3.7 (30) |
83.3 ± 3.9 (55) |
70.8 ± 5.8 (24) |
96.0 ± 5.2 (31) |
|||||
| 2 | 56.8 ± 4.7 (13) |
56.5± 5.8 (8) |
57.0 ± 7.4 (5) |
66.0 ± 5.8 (13) |
58.0 ± 7.1 (8) |
74.0 ± 9.0 (5) |
77.8 ± 8.1 (13) |
68.5 ± 10.1 (8) |
87.0 ± 12.7 (5) |
|||||
| P | ||||||||||||||
| K effect | 0.005 | 0.007 | 0.82 | |||||||||||
| Dose effect | 0.87 | 0.031 | 0.004 | |||||||||||
| K * dose | 0.53 | 0.59 | 0.26 | |||||||||||
| P (K effect on change from baseline) | 0.26 | 0.25 | ||||||||||||
| # of “E” (Gc1s) alleles | ||||||||||||||
| 0 | 62.5 ± 1.7 (90) |
62.0 ± 2.4 (47) |
62.8 ± 2.5 (43) |
78.8 ± 2.2 (84) |
74.8 ± 3.0 (43) |
82.5± 3.1 (41) |
84.3 ± 3.0 (86) |
74.0 ± 4.3 (43) |
94.5 ± 4.3 (43) |
|||||
| 1 | 65.0 ± 1.9 (73) |
64.5 ± 2.8 (34) |
65.3 ± 2.6 (39) |
77.8 ± 2.3 (74) |
73.8 ± 3.4 (34) |
80.8 ± 3.2 (40) |
75.5 ± 3.5 (67) |
71.0 ± 5.1 (30) |
80.0 ± 4.7 (37) |
|||||
| 2 | 75.0 ± 2.5 (40) |
75.8 ± 3.5 (21) |
74.0± 3.7 (19) |
92.8 ± 3.1 (41) |
87.0 ± 4.3 (21) |
98.3 ± 4.5 (20) |
89.8 ± 4.7 (36) |
81.3 ± 6.5 (18) |
98.0± 6.8 (18) |
|||||
| P | ||||||||||||||
| E effect | 0.0002 | 0.0002 | 0.0375 | |||||||||||
| Dose effect | 0.98 | 0.0025 | 0.0005 | |||||||||||
| E * dose | 0.91 | 0.89 | 0.46 | |||||||||||
| P (E effect on change from baseline) | 0.11 | 0.10 | ||||||||||||
| # of “risk alleles” | ||||||||||||||
| 0 | 75.0 ± 2.5 (40) |
75.8 ± 3.5 (21) |
74.0 ± 3.6 (19) |
92.81± 3.0 (41) |
87.0 ± 4.2 (21) |
98.3 ± 4.4 (20) |
89.8 ± 4.7 (36) |
81.3 ± 6.4 (18) |
98.0 ± 6.8 (18) |
|||||
| 1 | 65.3 ± 2.5 (44) |
62.0 ± 3.7 (19) |
68.8 ± 3.2 (25) |
76.8 ± 3.0 (44) |
70.3 ± 4.5 (19) |
83.0 ± 3.9 (25) |
72.3 ± 4.6 (38) |
67.8 ± 7.0 (16) |
77.0 ± 5.9 (22) |
|||||
| 2 | 64.8 ± 1.8 (79) |
66.0 ± 2.5 (43) |
63.3 ± 2.7 (36) |
81.0 ± 2.3 (75) |
79.3 ± 3.2 (39) |
82.8 ± 3.3 (36) |
82.3 ± 3.2 (76) |
77.5 ± 4.5 (39) |
87.0 ± 4.7 (37) |
|||||
| 3 | 59.3 ± 3.2 (27) |
58.0 ± 4.9 (11) |
60.3 ± 4.1 (16) |
78.3 ± 3.9 (26) |
74.8 ± 5.9 (11) |
82.0 ± 4.9 (15) |
85.5 ± 5.6 (26) |
65.3 ± 8.8 (10) |
105.5 ± 7.0 (16) |
|||||
| 4 | 56.8 ± 4.6 (13) |
56.5 ± 5.7 (8) |
57.0 ± 7.2 (5) |
66.0 ± 5.6 (13) |
58.0± 6.9 (8) |
74.0 ± 8.8 (5) |
77.8 ± 7.9 (13) |
68.5 ± 9.9 (8) |
87.0 ± 12.5 (5) |
|||||
| P | ||||||||||||||
| Risk allele effect | 0.0003 | 0.0001 | 0.10 | |||||||||||
| Dose effect | 0.71 | 0.0028 | 0.0001 | |||||||||||
| Allele* dose | 0.60 | 0.66 | 0.18 | |||||||||||
| P (allele effect on change from baseline) | 0.06 | 0.18 | ||||||||||||
Numbers in parentheses indicate n.
Furthermore, T25-OHD levels at baseline differed in the presence of the GC1s allele (glutamate at residue 432, “432E”, in contrast to aspartate, “432D”.) T25-OHD levels were greater as the number of GC1s alleles increased. This difference was evident after 1 and 6 months of supplementation (Table 2), but the response to vitamin D supplementation, after 1 or 6 months was no different across these groups.
Finally, in view of the effects of these alleles, we examined whether the total number of GC “risk” alleles impacted response to treatment. We combined the number of GC2 (436K) and GC1s (432D) alleles and classified each subject as having 0, 1, 2, 3, or 4 total risk alleles. Although T25-OHD decreased with increasing number of risk alleles (Table 2, P=0.0003), the response to treatment was not significantly different across the total risk allele groups.
DBP and free 25-OHD
Circulating DBP levels differed among GC haplotype groups at baseline (P<0.0001), with lower values in groups with the GC2 allele (1f/2, 1s/2, and 2/2) as expected (5), with no differences evident between treatment groups (P=0.39). After 6 months of supplementation DBP did not differ between treatment groups (P=0.40) and there was no change from baseline values (Figure 3A).
FIGURE 3:
A, Mean circulating levels of vitamin D binding protein (DBP) at baseline and after 6 months of supplementation in the standard dose group (dark bars) and higher dose groups (light bars).
Values were equivalent between dosing groups at baseline and did not change with supplementation. B, Mean calculated free 25-OHD (CF25-OHD) levels at baseline (left columns) and after 6 months (right columns) in the standard dose group (dark bars) and higher dose groups (light bars). Groups were comparable at baseline; a greater incremental change was seen with the higher dose (P = 0.0002), but there was no effect of haplotype group on the response to supplementation. C, Mean directly measured free 25-OHD (dmf25-OHD) levels at baseline (left columns) and after 6 months (right columns) in the standard dose group (dark bars) and higher dose groups (light bars). As with cf25-OHD, groups were comparable at baseline; a greater incremental change was seen with the higher dose (P < 0.0001), but there was no effect of haplotype group on the response to supplementation. For all panels, sample size for each group is shown within each bar. To convert to pg/mL, divide the pmol/L value (shown) by 2.4.
The response of circulating cf25-OHD and dmf25-OHD at the end-of-study 6-month time point were similar to that for T25-OHD. Baseline cf25-OHD (Figure 3B) was 17.2 ± 4.10 pmol/L in the standard dose group and 15.9 ± 4.00 pmol/L in the higher dose group with no differences between treatment (P=0.52) or GC haplotype groups (P=0.63). After 6 months of supplementation the standard dose group attained a value of 19.4 ± 5.20 pmol/L and the higher dose group increased to 23.5 ± 9.33 pmol/L; a significantly greater increment occurred with the higher dose (P=0.0002). There was no effect of haplotype on response to supplementation (P=0.24), and no interaction between haplotype and treatment level (P=0.50). Likewise, baseline dmf25-OHD levels (Figure 3C) were no different between treatment (P=0.79) or GC haplotype groups (P=0.14). Similarly, to cf25-OHD, the change from baseline was greater with the higher dose (P<0.0001), there was no effect of haplotype on response (P=0.22), and no interaction between haplotype and treatment level (P=0.58).
1,25(OH)2D
Finally, we examined the 1,25(OH)2D response to vitamin D supplementation (Figure 4). Baseline T1,25(OH)2D levels differed among haplotype groups as previously reported (15), but not between treatment groups (Table 1). Increases in both treatment groups occurred at both months 1 and 6 (P<0.0001); there was a modest treatment effect (P=0.013), explained by a 17.5 pmol/L greater increment in the higher dose group after one month of supplementation, however the difference between treatment arms was no longer apparent after 6 months. The T1,25(OH)2D response was no different across the GC haplotype groups (supplemental figure, P=0.59), and there were no significant changes in cf1,25(OH)2D with supplementation (data not shown).
FIGURE 4:
Mean total circulating 1,25(OH)2D [T1,25(OH)2D] levels at baseline (left columns), after 1 month (middle columns), and after 6 months (right columns) in the standard dose group (dark bars) and higher dose groups (light bars). The higher dose group achieved greater levels of T1,25(OH)2D after 1 month of supplementation (P = 0.013), but not after 6 months. There was no effect of GC haplotype group on T1,25(OH)2D levels. Error bars represent standard deviation. Sample size for each group is shown within each bar. To convert to pg/mL, divide the pmol/L value (shown) by 2.4.
There were no significant changes in serum calcium levels throughout the study, nor were there any significant changes in the skeletal ultrasound speed-of-sound parameter.
DISCUSSION
Various guidelines exist for vitamin D requirements in children. The long-standing IOM recommendation of 400 IU daily (200 IU daily in the first year of life) was modified in 2011 to an RDA of 600 IU daily for ages 1– 18 years and an AI of 400 IU daily for the first year of life was introduced (1). We therefore designed a comparison of doses using 2800 IU weekly (equivalent to 400 IU daily) and 7000 IU weekly (equivalent to 1000 IU daily, which at the time was the IOM designated upper tolerable limit for vitamin D intake) (1), in a highly pertinent cohort of children with multiple risk factors for vitamin D deficiency (including urban living, northern latitude, and minority status). To further investigate the well-established influences of GC haplotype on circulating 25-OHD (5), we aimed to determine if the response to vitamin D supplementation varied based on the 6 frequently occurring GC haplotypes. We confirmed effects of GC haplotype on baseline DBP levels, and on T25-OHD. We further demonstrated that increasing the number of “risk” alleles has an additive effect on reduction in T25-OHD (Table 2). We demonstrated a significant difference between the 2 vitamin D dosing regimens on circulating 25-OHD levels and found a slight trend for GC haplotype on the one month response, which was not evident at 6 months. Thus the “risk allele” level may have a small effect on response to supplementation, however the magnitude of this effect does not appear to be of clinical significance. There was a relatively robust response in the 2/2 group as suggested previously (10), however these changes were not statistically different from other haplotype groups, perhaps due to the smaller size of that group and lower baseline values. Finally, we confirmed that effects on total circulating 25-OHD levels were similar to that for free 25-OHD. The data suggest that there is no clinically meaningful difference in the total/free 25-OHD equilibrium across the GC haplotype groups in response to the doses of vitamin D supplementation provided in this study.
Both the standard and higher dose regimens increased T25-OHD in healthy children after one month of dosing, and the increase was equivalent across the 6 GC haplotype groups. These data demonstrate that liquid vitamin D3 drops providing 2800 IU or 7000 IU weekly are safe and effective means of attaining and maintaining appropriate vitamin D status in healthy children. Increases in T25-OHD after 1 month of supplementation persisted at similar levels after 6 months of supplementation. Overall, the increase in T25-OHD was 0.75 nmol/L and 0.5 nmol/L per ug of vitamin D3 in the low dose and high dose groups, respectively. This is consistent with the response in 25-OHD seen after vitamin D administration in postmenopausal women, with the expected lesser incremental change observed with increasing dose level (23). The difference in dose response became less significant by 6 months, suggesting that cumulative effects of long-term supplementation may diminish needs for routine high dose therapy. Thus, for long-term vitamin D supplementation, there does not seem to be clinically meaningful differences between the two dosages studied here, limiting the advantage to exceeding the RDA in children. Of the 14 children in each group with baseline T25-OHD less than 20 ng/mL, 2 in the lower dose group remained so, and one of the higher dose group at the end-of-study visit. Values were inconsistent between 1 and 6 month visits suggesting that poor adherence to therapy may have accounted for the limited response.
The increase in T25-OHD was comparable among the 6 common GC haplotypes, although the 1f/1s (DETT) group had a lesser rise than other haplotype groups at 1 month. This difference was not evident at 6 months, and there was no significant interaction between dose level and haplotype group, supporting the notion that vitamin D supplementation at typical levels does not require targeting doses to individuals of different haplotypes. Adjustment for dietary calcium intake, which was well-balanced between treatment arms, did not alter the interpretation of the findings.
Calculated free 25-OHD and directly measured free 25-OHD levels showed similar incremental changes as observed for total 25-OHD. Thus, it does not seem appropriate to recommend these refined measures beyond total 25-OHD levels for routine monitoring vitamin D supplementation, at least in this at-risk demographic group. Likewise, there is no need for specific attention to haplotype in this assessment.
Interestingly we identified a modest increase in 1,25(OH)2D levels after 1 month of vitamin D3 supplementation, suggesting a transient substrate effect contributing to circulating levels of this metabolite, which occurred across all genotypes. This effect may be more apparent in children as older individuals have not shown consistent changes in this regard.
Strengths of our study include a high retention rate of recruited subjects, with 84% of the enrolled cohort completing the study, and 93% of those who successfully completed the initial visit. Moreover, the cohort represents a group for which demographic issues represent a relatively high risk for vitamin D deficiency as compared to the overall US population, and target children in whom the skeletal consequences of deficiency are often the most severe. The study is limited in that subjects were screened to be healthy prior to enrollment, such that we did not test the efficacy of a therapeutic regimen for correction of vitamin D deficiency. We focused our attention on the risk alleles associated with the major GC haplotypes and did not explore other genetic influences on response. Finally, the study targeted a largely Hispanic, urban-dwelling community in the Northeastern USA, and thus the generalizability to other populations may be limited.
Overall these data support current IOM guidelines for vitamin D supplementation in healthy children and suggest that targeted dosing based on genetic variance in GC/Vitamin D Binding Protein is not indicated. This may relate to the lack of change in circulating DBP with supplementation across all haplotype groups. Finally, we provide further data that the measurement of free vitamin D in the setting of clinical monitoring for supplementation in children (with normal circulating DBP) offers little clinical advantage over measurement of total vitamin D. We are hopeful that the data from this study will be useful for future approaches to the study of vitamin D supplementation in children, as to inform refinements of related guidelines.
Supplementary Material
FUNDING SOURCES
This study was carried out with the support of the Thrasher Research Fund Award 02829-4 (TOC) and the National Institutes of Health (NICHD) Award 5RC1HD063562 to TOC. The study was made possible by the Yale Center for Clinical Investigation (supported by CTSA UL1 RR024139 from the NIH).
Footnotes
DECLARATION OF INTEREST
The authors have no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
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