Vitamin D the active compound
The active form of vitamin D, physiologically present in the human body, is 1,25-(OH)2-Vitamin D3 (calcitriol). Historically classified as a vitamin, calcitriol is actually a steroid hormone, classified as such due to its structure and synthesis pathway. Calcitriol forms a complex with the intracellular receptor protein VDR (vitamin D receptor) and acts as a transcriptional factor regulating the function of different hormone-sensitive genes of cells in various tissues. Calcitriol’s most prominent function is its control of parathyroid hormone (PTH), and calcium and phosphorus metabolism, thus it is directly involved in calcium absorption, bone metabolism and bone synthesis. More recent findings support the involvement of calcitriol in controlling the immune response, skin differentiation, the renin-angiotensin system, muscular function and neuronal function. Furthermore, calcitriol has been found to have inhibitory effects on cell proliferation in tumours, thus it has a role in cancer prevention and recovery from the disease.

Protohormone Vitamin D3
Vitamin D3 is produced from 7-dehydrocholesterol in the skin during a photochemical reaction with natural UVB light, resulting in Vitamin D3 protohormone. Vitamin D3 is hydroxylated into 25-hydroxyvitamin D3 in the liver. Further on in the metabolic pathway, 25-hydroxyvitamin D3 is metabolised in the kidneys by the enzyme 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1) to its active form, 1,25-dihydroxyvitamin D3 (calcitriol). The processing of 25-(OH)-Vitamin D3 into calcitriol is regulated according to tissue-specific demand and most of the vitamin D in the body is actually circulating as 25-(OH)-Vitamin D3 (25-(OH)-cholecalciferol). It is the most appropriate target for monitoring vitamin D levels in humans, since the presence of this molecule is the limiting factor for the production of calcitriol, and an immediate indicator of the individual irradiation level with UVB light.

Vitamin D2 not natural to humans
25-(OH)-Vitamin D2 (25-(OH)-ergocalciferol) has a structure closely related to 25-(OH)-vitamin D3 and differs from it because it possesses a double carbon bond and a methyl group. Unlike vitamin D3, which is naturally occuring in humans, vitamin D2 is derived from fungal and plant sources. In the human body 25-(OH)-Vitamin D2 is hydroxylated into 1,25-(OH)2-vitamin D2. It is believed that in this form, it plays an equivalent role to vitamin D3 as a steroid hormone. Vitamin D2 is easier and more economical to produce than vitamin D3 and therefore represents the majority of vitamin D used for supplements and prescribed drugs. However, 25-(OH)-vitamin D3 has been shown to be more effective in providing levels of vitamin D hormone in the human circulation, possibly due to a significantly more stable hormone-receptor complex compared to 25(OH)-vitamin D2. Since vitamin D2 is the major source of supplementation in several countries, such as the USA and Canada, monitoring vitamin D levels also requires the measurement of 25(OH)-vitamin D2.

Chromsystems analysis
25-OH-Vitamin D3 is thus a recognised clinical determinant for the diagnosis of bone mineralisation malfunction, whereas 25-OH-Vitamin D2 is measured for monitoring the therapy of vitamin D deficiency using vitamin D2.

The new Chromsystems HPLC reagent kit for the combined analysis of 25(OH)-vitamin D3 and 25(OH)-vitamin D2 has been  available since April 2007. This reagent kit allows the simultaneous and safe chromatographic determination of 25-OH-Vitamin D3 and 25-OH-Vitamin D2 on a simple isocratic HPLC system with UV detection. By means of efficient protein precipitation and selective solid phase extraction, any interfering components are removed. The analytes are quantified by the inclusion of a stable internal standard. This kit combines fast sample throughput with high cost efficiency. It is a refined version of the predecessor reagent kit, which had been evaluated for 25(OH)-vitamin D3 only. The new kit opens up new possibilities for vitamin D measurement in routine diagnostics, with an excellent separation capacity allowing reliable and reproducible determination of vitamin levels. Matching quality controls with two concentration levels and a calibrator are available. These products combine the accuracy and specificity of HPLC with the requirements of high throughput diagnostics for clinical settings.

CHROMSYSTEMS
Munich, Germany
Medica Stand 2/B46