We found differences in the total, genotype-corrected free, and bioavailable 25(OH)D concentrations and the DBP concentrations among GC genotypes, indicating that the biological response could possibly be modified by the availability of 25(OH)D. Moreover, some genotypes with low 25(OH)D concentrations also had low PTH concentrations, which is contrary to the usual finding for this relationship. Interestingly, the free and bioavailable 25(OH)D concentrations were high in these groups, which could explain the low PTH concentration.
Previous studies have shown the critical role of GC genotypes and serum GC protein concentration for vitamin D metabolism [11, 12, 17, 18]. The 25(OH)D concentrations have been suggested to differ among DBP phenotypes and genotypes. In this study, we wanted to extend the observations to diplotypes and haplotypes. We discovered a significant difference in 25(OH)D concentrations among the SNP rs4588 genotypes. A similar difference was also found among the diplotypes when two SNPs, rs4588 and rs7041, were combined. Among the SNP rs4588, the lowest 25(OH)D concentration was seen in individuals with the GC2/2 genotype and the highest in individuals with GC1/1. Within diplotypes, the difference was significant between genotypes 1S/2 and 1 F/2, the 25(OH)D concentration being lowest in genotype 1S/1 F and highest in 1 F/2. Our results are in line with earlier studies [18–21]. Most of the studies have focused on the two common SNPs rs4588 and rs7041, but they are seldom used in combination to form diplotypes.
In this study, the DBP concentrations varied significantly among the GC genotypes. The lowest DBP concentration was found in GC2/2 and the highest in GC1/1. In diplotypes, the highest concentration was found in GC1F/1 F and the lowest in GC2/2. A significant difference in DBP concentration emerged between GC1F/2 and GC2/2. Also haplotype-corrected DBP values differed; the highest value was observed in GC1S and the lowest in GC2. Variation in DBP concentrations based on DBP genotypes has been reported also in previous studies, but the results have not been consistent [11, 18, 21]. Powe et al. [11] reported that the T-allele at rs7041 was associated with decreased concentrations of DBP, and allele A at rs4588 was associated with higher DBP, after accounting for rs7041. In a study conducted with African American and Caucasian American subjects, GC1S homozygotes had the highest DBP concentration, GC2 had an intermediate concentration, and GC1F had the lowest concentration [18]. Similar to our results, a Danish study observed that GC2 homozygotes generally had lower DBP concentrations than the other genotypes [21].
However, criticism has been directed at some of the measurement protocols used by earlier studies for determining the DBP concentration. It has been suggested that studies based on monoclonal antibodies [11, 18] may be inaccurate because the assay might not have equal affinity for all genotypes. The use of polyclonal antibodies instead has been proposed [22].
Based on the free hormone hypothesis, the free fractions could correlate better with biological actions of vitamin D than the total 25(OH)D [9]. In order to test the free and the bioavailable hypotheses, we calculated the concentrations of unbound “free” 25(OH)D and bioavailable 25(OH)D. A Norwegian study found that adjusting for DBP phenotype-specific (SNPs rs4588 and rs7041 combined) binding coefficients affected the calculated free and bioavailable concentrations by up to 37.5 % [10]. Thus, additional genotype adjustment was made for diplotype- and haplotype-specific binding coefficients. In our study, serum free or bioavailable 25(OH)D concentrations did not differ significantly between the genotypes. However, after adjustment for diplotype-specific binding coefficients a significant difference emerged in free and bioavailable values among the haplotypes and diplotypes. The highest concentrations of free and bioavailable 25(OH)D were found in GC 2/2 and the lowest in haplotype GC1F and diplotypes GC1S/2, GC1S/2, and 1S/1 F. Powe et al. [11] stated that genetic polymorphism explained almost 10 % of the variation in 25(OH)D levels. In their study, the T-allele at rs7041 was associated with decreased levels of vitamin D-binding protein in both black and white Americans. In white individuals, the A-allele at rs4588 was associated with decreased levels of total 25(OH)D.
An inverse association between 25(OH)D and PTH has been found in numerous studies. PTH has therefore been suggested to be used as a health outcome reference for optimal vitamin D status. However, the cut-off values for PTH and 25(OH)D differ considerably among studies, making this very difficult. Moreover, large variation exists in PTH at specific 25(OH)D concentrations [13]. In our previous study in Finnish children and adolescents, we observed that GC2 phenotype was associated with the lowest 25(OH)D concentrations and that there was an inverse association between 25(OH)D and PTH within the genotypes [23]. Interestingly, the GC2/2 genotype, which had the lowest 25(OH)D, also had the lowest PTH concentration, which is contrary to the association in general. Here, we found differences in PTH concentrations among the diplotypes and a similar linear trend in haplotypes. GC2/2, with the second lowest 25(OH)D among diplotypes, also had the lowest PTH.
In accord with our finding, a Danish study observed that rs4588-AA carriers had the lowest prevalence of 25(OH)D > 50 nmol/L in a vitamin D fortification group, but in the control group the SNP rs4588 carriers had the lowest prevalence of low vitamin D status. They state that this may suggest that rs4588 carriers have low, yet stable 25(OH)D concentrations. The rs4588-AA genotype had also lower PTH levels and 25(OH)D concentrations relative to rs4588-CC or rs4588-CA carriers [20]. In comparison, the genetic variance in GC significantly contributed to circulating DBP as well as to 25(OH)D in a study in North American children [24]. However, in that study both total and free 25(OH)D correlated inversely with PTH, and this correlation was independent of DBP genotype. Interestingly, we also noted a difference in PTH concentrations among SNP rs705124, which has not been reported in earlier studies. Thus, our results emphasize the importance of the genetic variation in DBP in vitamin D and mineral metabolism.
In addition to renal production of 1,25(OH)2D3, there is local production in non-renal tissues. Based on our previous findings and the results in this paper, we hypothesize that free 25(OH)D may enter the parathyroid glands and might be converted locally to 1,25(OH)2D3 with the help of cytochrome p450 27B1 enzyme (CYP27B1) in the cell. The higher amount of 1,25(OH)2D3 could suppress the production of PTH and explain the lower concentration of PTH with the GC2/2 genotype. Our results support those of Ritter et al. [25], who demonstrated the presence of 25(OH)D 1-alpha-hydroxylase (1alpha-OHase) in cultured bovine parathyroid cells and showed that the enzyme was functionally active, converting 25(OH)D to 1-hydroxylated metabolites and inducing the major enzyme involved in the degradation of calcitriol, 25(OH)D 24-hydroxylase (24-OHase, CYP24A1). This together with the fact that vitamin D receptor (VDR) was expressed in the cells suggests an autocrine/paracrine function for locally produced 1,25(OH)2D3. They showed that 25(OH)D suppressed the production of PTH without the involvement of conversion to 1,25(OH)2D3, but possibly by interaction with VDR. The experiments were done in serum-free medium, indicating that no DBP was needed for the internalization and that the effects could be due to free 25(OH)D. Therefore, local production of 1alpha-OHase suggests an autocrine/paracrine role in regulating parathyroid function and may mediate, in part, the suppression of PTH by calcium and FGF-23 [25].
A Canadian study concluded that the biological effect of vitamin D on PTH concentration is mainly independent of DBP concentrations [12]. Another North-American study reported that genetic variance in GC significantly contributed to circulating DBP as well as to 25(OH)D [24]. However, both total and free 25(OH)D were correlated inversely with PTH, and this correlation was independent of DBP genotype. This reinforces our hypothesis that free and bioavailable 25(OH)D may be good biomarkers of vitamin D status and action in cells than serum 25(OH)D per se.
One of the limitations of this study is that it was not designed to demonstrate a difference in the concentration of 25(OH)D, DPB, or PTH in the different genotypes. The study derives from a larger study focusing on phosphorus intake and bone outcomes. The sample size was calculated to find a 4 % difference (standard deviation, SD-0.050 g/cm2) in distal radius bone mineral density between the highest and lowest phosphorus intake tertiles.
Other limitation of this study is that the affinity constants of DBP by Arnauld et al. [6] are derived by experimental evidence from one individual’s human serum sample. It has not been investigated whether post-translational modification of Gc may influence affinity for vitamin D metabolites. In addition, the Gc affinity constant for 25(OH)D3 in published literature ranges by several orders of magnitude from 1.9 × 10-10 to 1.5 to 10–8 [1]. It is also known that Gc protein circulates several fold above 25(OH)D concentrations, with only approximately 5 % of circulating Gc protein occupied by vitamin D metabolites.
As we do not have the full protein sequence, we cannot be certain whether we have some of the other Gc-isoforms. According to Arnaud’s analysis, binding affinity of these more rare isoforms may vary as much as 12 fold. Nevertheless, the other isoforms are quite rare. Arnauld’s affinity constants are estimated for 25(OH)D3 and not for 25(OH)D2 but this is not a problem for the interpretation of the results as vitamin D2 is not found in foods and only in a few supplements in Finland as food is fortified with vitamin D3 and most of the supplements are vitamin D3.