Physicians as Gatekeepers in the Use of Medical Marijuana

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A new chromatographic system for vitamin D, and its metabolites: resolution of a new vitamin D, metabolite M. F. HOLICK and H. F. DELUCA* Department of Biochemistry, University of Wisconsin, Nadison, Wisconsin 53706 ABSTRACT A simple yet powerful new chromatographic procedure for vitamin D3 and its metabolites is described. Liquid-gel partition chromatography on Sephadex LH-20 using a solvent of various percentages of CHC13 in Skellysolve B (petroleum ether, bp 6'749OC) permits excellent resolution of vitamin Da, 25-hydroxyvitamin D3, and their more polar metabolites. Of special importance is the resolution of the metabolites of vitamin D3 more polar than 25-hydroxychole- calciferol. Because of this resolution, a new metabolite of vita- min D3 has been demonstrated in the plasma of rats and in the intestines of chicks given 100 IU of vitamin DI-~,~-~H. limited to separating metabolites of vitamin Da that are similar in polarity. Silicic acid chromatography of vitamin DI and its metabolites was first described by Norman and DeLuca (9) and later modified (1-3). Suda et al. (7) and Suda, DeLuca, Schnoes, Tanaka, and Holick (8) used a multi- bore silicic acid system (10) to resolve three metabolites from hog plasma that are more polar than 25-HCC. Although silicic acid chromatography is widely used for metabolism studies of vitamin D3 and 25-HCC, this method has many disadvantages. The silicic acid column requires a gradient generating system, and after the chromatography is completed, the silicic acid bed must be discarded. The results reported in this paper also demonstrate that a number of metabolites more polar than 25-HCC cannot be resolved on silicic acid columns. Sephadex gel chromatography of lipophilic compounds in organic solvents has been studied by many investi- gators using methyl ether, hydroxypropyl ether, and acetylated derivatives of various Sephadex products for gel permeation, liquid-liquid partition, and adsorption chromatography (11-1 3). Nystrom and Sjovall (11) observed that the behavior of bile acids and steroids with mixed solvents on methylated Sephadex could not be explained by either liquid-liquid partition, adsorption, or gel filtration alone. Van Baelen, Heyns, and De Moor (14) investigated the potential of Sephadex LH-20 for separating various urinary estrogen metabolites and noted that a use of different solvents or mixtures of solvents may render the procedure more suitable for practical application. Re- cently, Ellingboe, Nystrom, and Sjovall (15) synthesized hydrophobic long-chain alkyl ethers of Sephadex and reported straight- and reversed-phase chromatography for various steroids, bile acids, and triglycerides. SUPPLEMENTARY KEY WORDS Sephadex LH-20 * cholecalciferol . intestine * 25-hydroxy cholecalciferol plasma . chick . rat ]&OGRESS IN liquid chromatographic methods for vitamin D and its metabolites has been limited to various procedures with Celite, silicic acid, and alumina (1-4). Recently, countercurrent distribution was employed for separating the more polar metabolites of vitamin D3 (5). Celite liquid-liquid partition chromatography has been used effectively for the purification of polar vitamin D3 metabolites (6-8) and serves as a powerful tool for sepa- rating various metabolites of the vitamin. This type of chromatography, however, is time-consuming, with re- coveries which vary between 60 and SO%, and its use is Abbreviations: 25-HCC, 25-hydroxycholecalciferol; 21,25- DHCC, 21,25-dihydroxycholecalciferol;25,26-DHCC, 25,26-di- hydroxycholecalciferol ; RVD, retention volume of vitamin Da metabolites relative to vitamin DI. * Address requests for reprints to H. F. DeLuca, Department of Biochemistry, University of Wisconsin, Madison, Wis. 53706. 460 JOURNAOLF LIPIDRESEARCHVOLUME12, 1971 Downloaded from by guest, on June 7, 2018

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