An Improved,Single Step Standardized Method of Lipid Extraction from Human Skeletal Muscle Tissue

摘要

A new method of lipid extraction from human skeletal muscle is presented.The quantitative extraction of lipid metabolites such as phospholipids,triglycerides and cholesterol,simultaneously,in a single experiment is evaluated by ~1H-NMR spectroscopy.Thirty-seven normal cryopreserved muscle samples were pulverized at liquid nitrogen temperature and treated with methanol and chloroform at different ratios (i.e.,1:2,1:3,and 1:4,by vol).The methanol-chloroform ratio,at which maximum extraction occurred (1:3,by vol),was further used to study the efficiency of lipid extraction by simultaneous and sequential treatment of the solvents;phospho-lipids and cholesterol were extracted better when the tissue was treated first with methanol and subsequently,with chloroform.Further,the quantities of the lipid extracts obtained from muscle tissue by treatment of methanol and chloroform sequentially (1:3 ratio,by vol) with and without addition of a known quantity of standard lipids,were compared with those obtained from commonly used methods (Folch's and Bligh and Dyer's).The present method of sequential use of methanol and chloroform in 1:3 ratio provided 44.9%,41.9%,and 22.8% higher yields of triglyceride,phospholipids,and cholesterol,respectively,compared to Folch's method and 73.5%,54.1%,and 46.9% higher yields compared to Bligh and Dyer's method in spite of adding yield of re-extraction in the latter two methods.In the recovery experiment after addition of 1 and 2mg of these individual lipid components,average recovery was 84.5% with the present method compared to 70.7% with Folch's method and 62.2% with the Bligh and Dyer's method.The results show that the present method is superior in terms of extraction of lipid components such as phospholipids,triglycerides,and cholesterol compared to commonly used methods.The major advantage of this method is that the total lipids are extracted in a single step.