Physicians as Gatekeepers in the Use of Medical Marijuana

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Physicians as Gatekeepers in the Use of Medical Marijuana ( physicians-as-gatekeepers-the-use-medical-marijuana )

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The present investigation was undertaken to evaluate gel chromatography as a means of resolving vitamin D metabolites that exhibit similar rates of migration by silicic acid chromatography. The use of Sephadex LH-20 with various mixed solvents as a rapid and efficient method for investigating vitamin D metabolism is de- scribed. A new metabolite of vitamin D3 in intestinal lipid extracts from chicks and in the plasma of rats that received 100 IU of tritiated vitamin D3 is also reported. EXPERIMENTAL METHODS All solvents were of analytical grade unless otherwise stipulated. SkellysolveB (predominantly n-hexane) was redistilled (67-69 "C) before use. Reference compounds 21,25-dihydrox ycholecalciferol (21,25-DHCC), 25,26- dihydroxycholecalciferol (25,26-DHCC), and hog plasma peak Vb were isolated as described previously (7, 8). Sample Preparation The plasma extracts were prepared according to the modified extraction procedure reported by Lund and DeLuca (16). Groups of four rats (weighing 400 g) fed a low vitamin D diet (17) were injected intraperitoneally with 100 IU of radiochemically pure vitamin D I - ~ , ~ (specific activity 9.0 X lo4dpm/IU) in 0.02 ml of EtOH. 24 hr later, blood was collected either by cardiac punc- ture or decapitation, giving 36 m l of plasma after centri- fugation. This was extracted with methanol-chloroform 2 :1 (v/v) and prepared as previously described (7). In other experiments chicks were maintained on a vitamin D-deficient diet for 4 wk. Under light ether anesthesia, groups of three chicks were injected in the wing vein with either 10 I U or 100 I U of vitamin Dz- l,2-3H (specific activity 10,750 dpm/IU) in 0.05 ml of EtOH and sacrificed 24 hr later. The entire small in- testine (each weighed ca. 3 g) from each group was pooled, washed with saline, and frozen at -20°C. The frozen tissue was ground in a meat grinder which had been previously cooled with dry ice. The tissue was homogenized in a Waring Blendor for 1 min in 150 ml of MeOH-CHC13 2 : l (v/v). 100 ml of distilled HzO and 50 ml CHCl, were added 2 hr later; this resulted in the separation of the phases. The chloroform phase was collected and washed with an equal volume of tap water and stored at 4°C overnight. The chloroform phase was then collected and evaporated to dryness with a flash evaporator. Each of the dried samples was redissolved in 0.2-1.0 mi of CHClx-Skellysolve B 65:35 (v/v). C'hromatography Columns 20 g of Sephadex LH-20, a hydroxypropyl ether deriva- Piscataway, N.J.), was slurried in 70 ml of the appro- priate solvent (CHCl3-Skellysolve B, either 3:7, 1:1, or 65:35 [./VI). After 24 hr of equilibration, the slurry was poured into a 60 X 1.1 cm glass column containing 15 ml of solvent. The stopcock was opened at the same time the slurry was poured and it was allowed to settle by gravity with free solvent flow. The column was washed with at least 50 ml of solvent before the sample was ap- plied. Air pressure of 1-2 lb was applied to the CHCl3- Skellysolve B 65:35 columns in order to obtain a flow rate of 0.66 ml/min; the other two systems were run under gravity. Silicic acid chromatography of the lipid extracts was carried out according to the procedure of Ponchon and DeLuca (1). Columns 60 X 1.1 cm were packed with 25 g of silicic acid (18) in SkellysolveB. The column was eluted with consecutive additions of 400 ml of 100% diethyl ether, 300 ml of 5% (v/v) methanol in diethyl ether, and 200 ml of 50% (v/v) methanol in diethyl ether to a holding chamber of an exponential gradient generating apparatus. The column was stripped with 200 ml of MeOH that was applied directly to the column. The constant volume mixing chamber contained 230 ml of Skellysolve B. - ~ H tive of Sephadex G-25 (Pharmacia Fine Chemicals, Inc., HOLICKANDD~Luc.4Chromatographyof VitaminD3andItsMetabolites 461 Measurement of Radioactivity Radioactivity was determined by liquid scintillation counting in a Packard Tri-Carb model 3375 equipped with an automatic external standard system. Samples were evaporated to dryness with a stream of air, dis- solved in toluene-counting solution (2 g of 2,5-diphenyl- oxazole and 100 mg of 1,4-bis-2-[4-methyl-5-phenyl- oxazolyl]-benzene per liter of toluene), and counted. RESULTS Chromatographic Properties To avoid the problem of the Sephadex LH-20 floating in CHC13,a mixed solvent system was used. Although it has been reported that alkanes are too nonpolar for Sephadex LH-20 (15), when Skellysolve B was mixed with CHCla in varying amounts it complemented the CHC13 by making the gel bed suitable for the chromatography. This lipophilic solvent also improved the chromatog- raphy of vitamin D3 and its metabolites to the extent that resolution was generally superior to silicic acid chromatography . In most of the experiments, vitamin D3-1,2-3Hwas used as a reference and the elution volumes of the other metabolites were expressed relative to it. The relative values for each of the metabolites on different batches and different quantities of Sephadex LH-20 were re- producible. Downloaded from www.jlr.org by guest, on June 7, 2018

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