Extraction of omega- and omega-1-hydroxylauric acids with ethyl acetate results in formation of acetoxy products.
In studies of the regiospecific metabolism of lauric acid by monooxygenase enzymes, incubation medium from the reaction of [14C] lauric acid and microsomes is routinely acidified and extracted with ethyl ether. Separation of products by HPLC shows the formation of omega(12)- and omega-1(11)-hydroxylauric acids by rat liver microsomes. In a comparison of several extraction solvents, we observed that the extraction of incubation media with ethyl acetate alone was associated with the appearance of a major new metabolite peak concomitant with loss of the 12- and 11-hydroxylaurate peaks by 62 and 23%, respectively. No difference was observed between extraction procedures in the per cent recovery of radiolabel. Similar results were observed with extraction of lauric acid metabolites formed by fish liver microsomes. Acidification of the incubation media prior to ethyl acetate extraction prevented formation of the new metabolite peak. In experiments to identify the new metabolite(s), 11- and 12-hydroxylaurate products formed by rat liver microsomes were acetylated and found to show an HPLC retention time similar to that of the unknown metabolite peak formed with ethyl acetate extraction. Mass spectrometric analysis further showed that the methyl esters of 11- and 12-acetoxylauric acids were very similar to that of the methyl ester derivative of the unknown metabolite fraction. Thus, data indicate that the acetoxy derivatives of 11- and 12-hydroxylauric acids were formed by direct reaction of ethyl acetate with the laurate products formed by microsomal monooxygenase enzymes. This extraction procedure can produce a major shift in the amount and ratio of omega- and omega-1-hydroxylaurates measured in the incubation media.
D000085:Acetates / Q000378:metabolism*; D000107:Acetylation; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005399:Fishes; D006900:Hydroxylation; D007850:Lauric Acids / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000032:analysis* / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains
D000085:Acetates; D007850:Lauric Acids; C007650:ethyl acetate