The serum vitamin D metabolome: What we know and what is still to discover

Abstract

Vitamin D, referring to the two forms, D2 from the diet and D3 primarily derived from phototransformation in the skin, is a prohormone important in human health. The most hormonally active form, 1α,25-dihydroxyvitamin D (1α,25(OH)₂D), formed from vitamin D via 25-hydroxyvitamin D (25(OH)D), is not only important for regulating calcium metabolism, but has many pleiotropic effects including regulation of the immune system and has anti-cancer properties. The major circulating form of vitamin D is 25(OH)D and both D2 and D3 forms are routinely measured by LC/MS/MS to assess vitamin D status, due to their relatively long half-lives and much higher concentrations compared to 1α,25(OH)₂D. Inactivation of both 25(OH)D and 1α,25(OH)₂D is catalyzed by CYP24A1 and 25-hydroxyvitamin D3 3-epimerase. Initial products from these enzymes acting on 25(OH)D3 are 24R,25(OH)₂D3 and 3-epi-25(OH)D3, respectively, and both of these can also be measured routinely in some clinical laboratories to further document vitamin D status. With advances in LC/MS/MS and its increased availability, and with the help of studies with recombinant vitamin D-metabolizing enzymes, many other vitamin D metabolites have now been detected and in some cases quantitated, in human serum. CYP11A1 which catalyzes the first step in steroidogenesis, has been found to also act on vitamins D3 and D2 hydroxylating both at C20, but with some secondary metabolites produced by subsequent hydroxylations at other positions on the side chain. The major vitamin D3 metabolite, 20S-hydroxyvitamin D3 (20S(OH)D3), shows biological activity, often similar to 1α,25(OH)₂D3 but without calcemic effects. Using standards produced enzymatically by purified CYP11A1 and characterized by NMR, many of these new metabolites have been detected in human serum, with semi-quantitative measurement of 20S(OH)D3 indicating it is present at comparable concentrations to 24R,25(OH)₂D3 and 3-epi-25(OH)D3. Recently, vitamin D-related hydroxylumisterols derived from lumisterol3, a previtamin D3 photoproduct, have also been measured in human serum and displayed biological activity in initial in vitro studies. With the current extensive knowledge on the reactions and pathways of metabolism of vitamin D, especially those catalyzed by CYP24A1, CYP27A1, CYP27B1, CYP3A4 and CYP11A1, it is likely that many other of the resulting hydroxyvitamin D metabolites will be measured in human serum in the future, some contributing to a more detailed understanding of vitamin D status in health and disease.

Keywords: 25-Hydroxyvitamin D3; Cytochrome P450; LC/MS/MS; Metabolome; Vitamin D.

Fig 1.

The structures of vitamins D3 and D2 with carbon numbering.

Fig. 2.

Major classical pathways for the synthesis, activation and inactivation of vitamin D3. Note that vitamin D2 is activated by CYP2R1 and CYP27B1 as shown for 25(OH)D3, can undergo a similar epimerization producing 3-epi-25(OH)D2 and can also be hydroxylated at C24 by CYP24A1.

Fig. 3.

The C23 and C24 oxidation pathways of 1α,25(OH)₂D3 metabolism by CYP24A1. A nonenzymatic product of the pathway, 1α,23-dihydroxy-24,25,26,27-tetranorvitamin D3, results from disproportionation of the C23 (oxo) aldehyde (2 × C23 aldehyde → 1 × C23 alcohol + 1 × C23 carboxylic acid [113]. 25(OH)D3 undergoes similar C23 and C24 oxidation pathways to those shown for 1α,25(OH)₂D3.

Fig. 4.

Pathways for the metabolism of vitamin D3 by CYP11A1. The bold arrow indicates the major pathway producing 20S(OH)D3. Further metabolism of the products of this pathway by other CYPs is shown in Table 3.