Calciferol

VITAMIN D

The Nobel Nobel prize for chemistry for 1928 was awarded to Adolf Windaus "for his studies on the constitution of the sterols and their connection with vitamins". The vitamin in question was vitamin D and Windaus was the first scientist to receive an award mentioning vitamins.
A comprehensive review of the vitamin D history viewed through the contribution of Windaus may be found in a paper by Wolf G (J Nutr 2004, 134, 1299).

Vitamin D2 (calciferol) originates from irradiation at about 260nm of ergosterol while vitamin D3 is formed by irradiation of a provitamin molecule (7-dehydrocholesterol) present in the skin and gut lining. From a physiological point of view, Steenbock H et al. discovered the formation of an anti-rachitic factor in rats under UV irradiation (J Biol Chem 1924, 61, 405). Vitamin D3 was isolated around 1936 (Brockmann) from the very potent liver oil of the tuna fish. The unsaponifiable fraction was partitioned between 90% methanol and petroleum ether to separate vitamins A and D. The residue from the petroleum phase contained vitamin D3 to be purified by multiple chromatography. The total synthesis was done around 1952 (Woodward, J Amer Chem Soc, 74, 4223). When tested on rats, the two vitamins are equally potent (vitamin D3 is more active than D2 in the chick test). The international unit has been so defined as 40 i.u./µg. Natural fish liver oils vary from about 100 i.u./g in cod to 200,000 i.u./g in tuna fish.
DeLuca HF (Holick M F et al., Biochemistry 1971, 10, 2799), Norman AW (Biochem Biophys Res Commun 1971, 42, 1082), and Kodicek E (Fraser DR et al., Nature 1970, Nature 228, 764) independently reported the existence of an active metabolite, 1,25-dihydroxyvitamin D3, which was produced in the kidneys. From these observations it was surmised that vitamin D3 was hydroxylated in the liver to become 25-hydroxyvitamin D3, the major circulating form of the vitamin, and then converted to 1,25-dihydroxyvitamin D3 in the kidneys. Thus, this final metabolic product is now considered as the metabolically active form of vitamin D3, which carried out its functions in initiating intestinal calcium transport. As the generation of vitamin D3 by UV-mediated photosynthesis is the main source of this secosteroid compound, it can be considered that vitamin D3 is not a true vitamin but a true hormone. 

It is now well known that vitamin D is a central player in calcium and bone metabolism, but more recently, important immunomodulatory effects have been attributed to this vitamin (Baeke F et al., Mol Aspects Med 2008, 29, 376).  Vitamin D deficiency/insufficiency has been associated with muscle weakness and a high incidence of various chronic diseases such as cardiovascular disease, cancer, multiple sclerosis, and type 1 and 2 diabetes (Zittermann A et al., Nutrients 2010, 2, 408). Most importantly, low vitamin D status has been found to be an independent predictor of all-cause mortality (Zittermann A et al., Curr Opin Clin Nutr Metab Care 2009, 12, 634). A review of the new trends in the physiological actions of vitamin D has been released in 2009 (Dixon KM et al., Int J Biochem Cell Biol 2009, 41, 982) and may be found in my book (Lipids – Nutrition and health, Claude Leray, CRC Press).

Initially, the determination of vitamin D was based on a reaction between antimony trichloride giving a yellow color with l max 500nm.Removal of vitamin A and E was necessary. Partition chromatography is actually used with success.

	vitamin D2	Vitamin D3
Molecular weight	396.6	384.6
l max	265	265
Molar extinction coefficient	18,200	18,200
m.p.	116°C	83°C

For humans, the requirements are about 100 i.u./day for a man but 400 i.u./day for a child or a pregnant woman.

The analysis of the vitamin D metabolites is dominated by immunoassays and receptor binding assays but an efficient profiling of major vitamin D metabolites has been described using a Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry (Aronov PA et al., Anal Bioanal Chem 2008, 391, 1917). A review of that approach has been reviewed (Higashi T et al., J Chromatogr B 2010,878, 1654).