Abstract
The level of free fatty acids in intact tissues has been found to be low but is known to rise in proportion to the extent of autolysis. Therefore, the high levels of free fatty acid reported in the cardiac lipids of rats fed rapeseed oil were reinvestigated using two different procedures for homogenization. Quick freezing and pulverization at dry ice temperature followed by lipid extraction was found to give lower values of free fatty acids (200 microgram/g of wet heart tissue) than the more commonly used technique of employing rotating blade-type homogenizers (greater than 1700 microgram/g of wet heart tissue). The amount of diglycerides was found to be 3 times greater when the latter method was used. The high levels of free fatty acid and diglyceride suggest that extensive autolysis occurs during homogenization with a rotating blade-type homogenizer. Freezing and pulverization at dry ice temperature is therefore recommended for determining intact lipid classes in rat heart.
Highlights
After the addition of methyl heptadecanoate as internal standard, each of the lipid classes was transesterified; TG, DG, free fatty acid (FFA), and PL were reacted with 5% HCl in anhydrous methanol [19] and cholesteryl ester (CE) was reacted with NaOCH,CH,OH [20]
The resultant methyl esters were purified by thinlayer chromatography (TLC) using hexanediethyl ether 9O:lO as developing solvent, and analyzed by gas-liquid chromatography (GLC) using a column packed with 5% butanediol succinate on Chromosorb G 80/100 mesh operated at 190°C
Preparation of the heart homogenate by method B resulted in significantly higher levels of FFA and DGand similar levels of TG and CE, as compared to the results obtained by method A
Summary
Thirty Sprague-Dawley male rats, 3 weeks of age, were randomly placed into three groups of 10 each, and for 0, 3, and 7 days were fed a diet containing rapeseed oil. Method B involved cutting the frozen heart into small pieces, placing these pieces into CHC13CH30H 2:l kept at O"C, and homogenizing them with a Virtis 45 homogenizer. This homogenate was allowed to warm up to room temperature (approximately 1 hr). To prevent partitioning of lipid material into the CH30H-H20 phase, the lipids were not washed [8,9]; a quantitative recovery was obtained [18].
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