Physicians as Gatekeepers in the Use of Medical Marijuana

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2.o I I,'1"' 0 20 40 60 80 IO 1.0ml FRACTIONS FIG. 1. Separation of vitamin Dr and 25-HCC on Sephadex LH-20 in various solvent mixtures. Column: 1.1 X 60 cm con- taining 20 g of Sephadex LH-20. Solvent CHClt-Skellysolvr B; (A)65:35, ( B ) l : l ,(C)3:7;flowrate,0.66ml/min. Fig. 1 illustrates the effects of different solvent mix- tures (ix., CHCl3 in Skellysolve B) on the Sephadex LH-20 separation of 25-HCC from vitamin D3. The retention volume for 25-HCC relative to vitamin D3 increased as the percentage of CHC13 in Skellysolve B decreased from 65% to 30%. It was noted, however, that as the amount of CHC13 decreased so did the swelling of the gel. This decreased the void volume of the column and elution volume for vitamin D3.The CHC13-Skelly- solve B 3:7 and 1:1 methods appeared to be the best systems for routine separations of vitamin D3 and 25- HCC. Because the Sephadex LH-20 did not swell very well in CHCl3-Skellysolve B 3 : 7 this system was not investigated further. CHC13-Skellysolve B 1 : 1 was a suitable solvent for Sephadex LH-20 and is the method of choice for purifying vitamin D3and 25-HCC. The capability of the CHCl~-Skellysolve B 65 :35 system in resolving the various vitamin Ds metabolites in rat plasma as compared with that obtained with a silicic acid column is clearly demonstrated in Fig. 2. The peak V region, which appears to be homogeneous on silicic acid, sepa- rates into four distinct peaks, Fl,, L'u, Vb, and V,, on Sephadex LH-20. It should be noted that when the peak V fraction from silicic acid columns is chromatographed 462 JOURNOAF LIPIDRESEARCHVOLUME12. 1971 25-HCC alone on LH-20, V,, Vu,V,, and V,all appear in the chromatogram, illustrating that they are found in the I' region of silicic acid. Peak IZZ(vitamin D3) and peak ZV (25-HCC) appeared to be homogeneous in both systems. The compound(s) corresponding to the peak VI region of the silicic acid chromatogram was stripped off the Sephadex column with 100 ml of CHC13-Skellysolve B 7 : 3 . Although the column swelled as the percent of CHC13 increased, the flowrate was kept constant by slightly increasing the air pressure. After the chroma- tography was completed, the column was regenerated by passing 100 nil of CHCl3-Skellysolve B 65 : 35 through the column. The various peak V assignments for the Sephadex LH-20 chromatograms were determined by cochromatography of each of the Sephadex LH-20 peaks with 21,25-DHCC, 25,26-DHCC, and hog plasma peak Vb;they are summarized in Table 1. A new metabolite(s) V,,which is more polar than 25-HCC, was detected in rat plasma. This metabolite also appeared in the intestine from chicks given 100 I U of tritiated vitamin D3,but could not be detected in the case of a 10-IU dose (see Fig. 3). Another metabolite that cochromatographed with 21,25-DHCC on three different chromatographic systems (Le., Celite liquid- liquid partition (7),silicicacid (l),and Sephadex LH-20 with CHC13-Skellysolve B 65:35) also became evident in the intestinal extracts from chicks given 100 IU of tritiated vitamin D3.Blood present in the intestine prob- ably does not account for the appearance of these metabo- lites in that tissue extract. On silicic acid, however, all of the peak T' metabolites seemed to comigrate as a single peak in the peak I' region of the Chromatogram (see Figs. 2 and 3). Applications The Sephadex LH-20 chromatographic systems de- scribed in this report offer several advantages in column TABLE 1 ELUTIONVOLUMEOSF VITAMIDNt METABOLITES RELATIVTEO VITAMIDNOON SEPHADELXH-20 (CHCII-SKELLYSOLBVE6 5 :35) V,* RVDt ml Peak VI, Peak V, (25,26-DHCC)ยง Peak V I I 25-IHCC, Peak I 25 0.56 Peak 111 (vitamin D3) 44 1.oo Peak IV (25-HCC) 65 1.47 PeakV,, 93 2.11 Peak V , (21,25-DHCC)$ * Elution volume. tRetention volumes of vitamin Dr metabolites relative to vitamin Da. $ 21,25-dihydroxycholecaIciferol. $! 25,26-dihydroxycholecalciferol. 152 3.45 183 4.16 248 5.63 254 5.77 Downloaded from www.jlr.org by guest, on June 7, 2018

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