Detection of Vitamin D and Its Major Metabolites

Abstract

In the interest of full disclosure, the author wishes to inform the readers that he has been a paid consultant to the DiaSorin Company. INTRODUCTIONVitamin D is a 9,10-secosteroid and is treated as such in the numbering of its carbon skeleton ( Fig. 47.1). Vitamin D occurs in two distinct forms: vitamin D 2 and vitamin D 3 . As shown in Figure 47.1, vitamin D 3 is a 27-carbon derivative of cholesterol; vitamin D 2 is a 28-carbon molecule derived from the plant sterol ergosterol. Besides containing an extra methyl group, vitamin D 2 differs from vitamin D 3 in that it contains a double bond between carbons 22 and 23. The most important aspects of vitamin D chemistry center on its cis-triene structure. This unique cis-triene structure makes vitamin D and related metabolites susceptible to oxidation, ultraviolet (UV)-light-induced conformational changes, heat-induced conformational changes, and attack by free radicals. As a rule, the majority of these transformation products have lower biological activity than vitamin D. It is important to note that, in humans, vitamins D 2 and D 3 provide similar potency (although some controversy exists as discussed in Chapter 61), and in this chapter the term vitamin D refers to both compounds.Metabolic activation of vitamin D is achieved through hydroxylation reactions at both carbon 25 of the side chain and, subsequently, carbon 1 of the A ring. Metabolic inactivation of vitamin D takes place primarily through a series of oxidative reactions at carbons 23, 24, and 26 of the side chain of the molecule. These metabolic activations and inactivations are well characterized and result in a plethora of vitamin D metabolites ( Fig. 47.2). Of the compounds shown in Figure 47.2, only four, vitamin D, 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH) 2 D), and 1,25-dihydroxyvitamin D (1,25(OH) 2 D) have been extensively quantitated, and to date only two of those, namely, 25(OH)D and 1,25(OH) 2 D, provide any clinically relevant information. However, the quantitation of vitamin D and 24,25(OH) 2 D can provide important information in a research environment. Thus, this chapter addresses the quantitation of these four important vitamin D compounds. Further, it is not the intent of this chapter to address the detailed history of vitamin D metabolite analysis, as this can be obtained from CH 3 previous reviews [1e3]. Rather, the intent of this chapter is to describe how we currently measure vitamin D and its major metabolites in our laboratory, as well as to discuss the appropriate clinical judgments in the selection of a given compound for analysis.The first semiquantitative assay for vitamin D was a bioassay based on the rat-line test [4]. This assay was cumbersome, expensive, and relatively inaccurate. Real progress in vitamin D analysis was not achieved until the advent of high-specific-activity 3 H-labeled vitamin D 3 compounds [5]. The introduction of these tracers led to the development of competitive protein-binding assays (CPBA) for vitamin D and 25(OH)D [6,7]. A sho...

Genetic ancestry, skin reflectance and pigmentation genotypes in association with serum vitamin D metabolite balance

“…[55-57] If this model is applicable to racial/ethnic differences in vitamin D metabolism, then differences in the relative levels of 25(OH)D 3 and 24,25(OH) 2 D 3 , which we and others have observed, might be consistent with a higher average production rate of 1,25(OH) 2 D 3 among African Americans. In general, there is not conclusive evidence regarding racial/ethnic differences in calcitriol, in part due to inaccuracy with earlier assays [58], and also because of the fact that studies do not account for diurnal variation [59]. …”

Can vitamin D metabolite measurements facilitate a “treat-to-target” paradigm to guide vitamin D supplementation?

“…It is logical that such differences are due, at least in part, to between-individual variation in vitamin D absorption, degradation, and distribution. However, vitamin D absorption has not been widely studied, as assays for cholecalciferol have historically been challenging [4]. Nonetheless, existing, albeit limited, data do document substantial between-individual variation in vitamin D absorption [5–8], which could contribute to variability in 25(OH)D observed following supplementation.…”

Optimal bone fracture repair requires 24R,25-dihydroxyvitamin D3 and its effector molecule FAM57B2

“…For rescue experiments, mice were subcutaneously injected for 15 days, starting 72 hours after surgery, with either 24R,25(OH) 2 D 3 (6.7 μg/kg daily in 50 μl propylene glycol with trace ethanol for vitamin D metabolite solubility); 1,25(OH) 2 D 3 (67 ng/kg); or propylene glycol (with trace ethanol) vehicle. The difference in the rescue doses of the 2 metabolites is based on the fact that systemic levels of 1,25(OH) 2 D 3 are 10-to 100-fold lower than circulating levels of 24R,25(OH) 2 D 3 under normal physiological conditions (58) in-depth understanding of FAM57B2 structure and function could yield a novel therapeutic target for fracture repair as well as OA. Alternatively, treatment with 24R,25(OH) 2 D 3 [or 24R,25(OH) 2 D 3 analogs] or with LacCer could represent a novel approach for the therapeutic management of fractures.…”