Page 342 - Vitamin D and Cancer

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15 Assessment of Vitamin D Status in the 21 Century 329
products have less biological activity than vitamin D. Research has now demonstrated
that vitamin D is much less bioactive than vitamin D in humans [3, 4] although a
2 3
recent study disputes this finding [5]. The parent compounds, vitamins D and D
2 3
are sometimes referred to as calciferol.
Hydroxylation reactions at both carbon 25 of the side chain and, subse-
quently, carbon 1 of the A ring result in metabolic activation of vitamin D.
Metabolic inactivation of vitamin D takes place primarily through a series of
oxidative reactions at carbons 23, 24, and 26 of the molecule’s side chain. This
metabolic activation and inactivation are well characterized and result in a
plethora of vitamin D metabolites [6]. Of these metabolites, only 25(OH)D and
1,25-dihydroxyvitamin D provide any clinically relevant information. 25(OH)D
2
and 25(OH)D are commonly known as calcifediol and the 1,25(OH) D metabo-
3 2
lites as calcitriol. The assay of these vitamin D metabolites will be discussed in
this chapter.

15.3 Methods of 25(OH)D Quantitation

The assessment of circulating 25(OH)D started its journey approximately 4 decades
ago with the advent of the competitive protein-binding assay (CPBA) [1]. From that
early time to the present we have progressed to radioimmunoassay (RIA), high-
performance liquid chromatography (HPLC) and liquid chromatography coupled
with mass spectrometry (LC/MS). A detailed procedural description of these meth-
ods can be reviewed in a recent publication [7]. I will provide a brief description of
each technique in this text.

15.3.1 Competitive Protein-Binding Assay

A major factor responsible for the explosion of information on vitamin D
metabolism and its relation to clinical disease was the introduction of a CPBA
for 25(OH)D. John Haddad, Jr., introduced this CPBA almost 4 decades ago
[1]. The assay assessed circulating 25(OH)D concentrations using the vitamin
D-binding protein (DBP) as a primary binding agent and H-25(OH)D as a
3
3
reporter. Although this CPBA was valid, it was also relatively cumbersome.
Technicians had to extract the sample with organic solvent, dry it under nitro-
gen, and purify it using column chromatography. This assay was suitable for the
research laboratory but did not meet the requirements of a high-throughput
clinical laboratory.
The major difficulty in measuring 25(OH)D is attributable to the molecule itself.
25(OH)D is probably the most hydrophobic compound measured by protein-binding
assay (PBA), which constitutes either CPBA or radioimmunoassay (RIA). The fact
that the molecule exists in two forms, 25(OH)D and 25(OH)D , compounds the
2 3
difficulties with its quantitation by PBA. 25(OH)D’s lipophilic nature renders it