Effect of Vitamin D Supplementation on Free and Total Vitamin D

A Comparison of Asians and Caucasians

Jaya Sujatha Gopal-Kothandapani; Lucy Faith Evans; Jennifer S. Walsh; Fatma Gossiel; Alan S. Rigby; Richard Eastell; Nick J. Bishop


Clin Endocrinol. 2019;90(1):222-231. 

In This Article


Baseline Clinical Characteristics

The baseline clinical characteristics of the study subjects are shown in Table 1. The White Caucasian and Asian men did not differ with respect to age, weight, height, BMI and waist to hip ratio. The mean (SD) calcium intake (grams/d) was adequate and equal between the Asians [1031.23(382.6)] and White Caucasians [1065.87(266.88)]. The median (range) vitamin D intake (IU/d) in comparison with the UK recommended daily intake was low and similar between the groups, 140(74 276) and 120(20 524) in Asians and White Caucasians, respectively. White Caucasians had a Fitzpatrick skin type of 1-3 and Asians 3-5. White Caucasians predominantly had Gc1s-2 haplotype and did not have Gc1f-1f and Gc1f-2. South Asians predominantly had Gc1s-1s haplotype and East Asians Gc1f-1f haplotype. Gc2-2 and Gc1s-2 were not found in East Asians (Table 2). Table 2 shows the relationship between haplotype frequency and ethnicity. There was a significant association between the two (P = 0.052, Fisher's exact test). There were more Asians with Gc1f-1f haplotype than expected [5 vs 2.5]. Similarly, more Caucasians with Gc2-1s haplotype than expected [11 vs.8.5].

Table 2 shows the differences in baseline total 25OHD status between the different haplotypes and ethnicity. However, the numbers were too small for statistical comparison.

Influence of Ethnicity on Measured Parameters at Baseline and Following Intervention

Twenty-nine participants of the 60 recruited had a serum total 25OHD level of <30 mnmol/L at baseline, of which 60% (18/30) were Asian and 36% (11/30) were White Caucasian (Table 1). Asians had significantly lower serum total 25OHD and DBP levels, but similar measured and calculated free 25OHD levels compared to White Caucasians at baseline (Table 3). At 4 weeks, the changes in serum total 25OHD, DBP and calculated free 25OHD levels were similar between Asians and White Caucasians; however, the increase in directly measured serum free 25OHD level was significantly greater in Asians 18.1(9.4) vs 12.2 (13.3) pmol/L in Caucasians (P = 0.0464) (Table 3) (Figure 2A, B and C). Although the observed significance was marginal, we considered it to be acceptable for a preliminary study.

Figure 2.

A, B, C shows the change in total, free 25OHD and VDBP following vitamin D dosing. Three-part box plot showing ethnicity in x-axis and change in total 25OHD, VDBP and free 25OHD in y-axis. D, shows the change in serum free 25OHD according to mean D binding protein (DBP) with shaded circles representing Caucasians and clear circles for Asians [Colour figure can be viewed at wileyonlinelibrary.com]

There was a significant interaction (P = <0.01) between ethnicity and mean DBP in relation to change in free 25OHD following dosing. In Caucasians, a lower mean DBP was associated with a larger in an increase in measured free 25OHD; this was not true for Asians, in whom the relationship of free 25OHD with mean DBP was positive, that is as mean DBP increased, the change in free 25OHD also increased (Figure 2D).

We found no clear effect of DBP haplotype within the ethnic group on change in either total or free 25OHD, either alone or in combination with other factors.

Baseline PTH concentration was higher in Asians than in White Caucasians (P = 0.0019) (Table 3). Following intervention, no significant changes in PTH levels were noted in either of the groups (Table 3). No increase in urine calcium: creatinine ratio was noted at either 1 or 4 weeks postintervention.

Bland-Altman plot at baseline shows clear evidence of bias and one outlying observation (Figure 3A). Passing-Bablok plot at baseline shows small and nonsignificant systematic bias (intercept = 0.54, 95% CI -3.54, 3.05 pmol/L). There is a significant proportional bias (slope = 0.43, 95% CI 0.24, 0.71 pmol/L) (Figure 3C).

Figure 3.

A and B, shows the Bland-Altman plots. Bold line indicates mean difference with 95% limits of agreement on either side. A, demonstrates systematic bias between calculated (using fixed affinity constants) and directly measured serum free 25OHD at baseline and B, which is postsupplementation, does not. Mean serum free 25OHD (directly measured) is represented in x-axis, and the difference between the directly measured and calculated free 25OHD is represented on the y-axis. Bland-Altman plots the mean of X and Y vs the difference between X and Y. C and D, shows the Passing and Bablok plots. Bold line indicates the calculated regression. With 95% CI either side. The line of no difference is drawn at 45 degrees. C, demonstrates no systematic bias but a significant proportional bias at baseline. D, demonstrates neither systematic nor proportional bias postsupplementation. Serum free 25OHD (directly measured) is represented in x-axis and the calculated free 25OHD (using fixed affinity constants) represented on the y-axis. Passing and Bablok is a nonparametric regression for method comparison. It fits a straight line between two variables (X and Y) where both are measured with error. The intercept of the straight line represents the systematic bias between X and Y. The null hypothesis is zero intercept. If the 95% confidence (CI) crosses zero, the systematic bias is not statistically significant. The slope measures the amount of proportional bias. The null hypothesis is a slope of one. If the 95% CI cuts across on the proportional bias, it is not statistically significant. If the two methods are identical, then they will share a common intercept (zero) and a common slope (one) [Colour figure can be viewed at wileyonlinelibrary.com]

Method comparison between directly measured and calculated serum free 25OHD concentration using fixed affinity constant for the DBP genotype (Gc1f-1f)

Bland-Altman plot postsupplementation shows no evidence of bias (Figure 3B).

Passing-Bablok plot postsupplementation shows a large negative systematic bias (−13.29 pmol/L). This is not statistically significant (95% CI -33.82, 2.99 pmol/L). There is no significant proportional bias (slope = 1.04, 95% CI 0.54, 1.70 pmol/L) (Figure 3D).

Relationship of PTH With Total and Free 25OHD

There was a statistically significant negative correlation between PTH and total 25OHD at baseline (r = −0.442; P = 0.0006) and postintervention (r = −0.452; P = 0.0004). However, the relationship of PTH at baseline with measured free 25OHD did not reach significance at baseline (r = −0.245; P = 0.0634) or following intervention (r = −0.061; P = 0.6446).

Relationship of Skin Type With Total and Free 25OHD

There was no statistically significant difference between skin types and baseline total 25OHD concentrations by one-way ANOVA (F (4,54) = 2.08, P = 0.0956). Postdosing total 25OHD also did not differ between skin types.

Relationship of DBP Genotypes on Total and Free 25OHD

There were no significant differences by DBP genotype for baseline total 25OHD (F ratio = 1.0075, P = 0.4225) or serum free 25OHD (F ratio = 0.4838, P = 0.7868). Following intervention, subjects with Gc1f-1s haplotype (high affinity for 25OHD) showed the greatest increment in serum total 25OHD (increment 60.65 (17.3) nmol/L; baseline 32.9 (14.0) nmol/L). Subjects with the lowest affinity haplotype Gc2-2 had the smallest increment in serum total 25OHD (increment 48.9 (6.9) nmol/L, baseline 18.4 (3.6) nmol/L). The increment in serum direct free 25OHD levels was greatest in subjects with the Gc2-1s (16.6 (10.2) pmol/L; low-affinity haplotype), and lowest in subjects with Gc1f-1f (10.1 (11.9) pmol/L highest affinity haplotype). None of these differences reached statistical significance, however.

Relationship of Dietary Calcium and Vitamin D Intake With PTH, DBP, Total and Free 25OHD

There was no relationship of dietary calcium intake at baseline with baseline PTH, DBP, total or free 25OHD, or change in any of those parameters, either for the whole group or by ethnicity.