Abstract
We have devised a high performance thin-layer chromatography (HPTLC) densitometry method to resolve the major lipid classes of brain tissue. We used DEAE-Sephadex column chromatography to separate the total lipid into neutral and acidic lipid fractions. The lipid fractions were then spotted on separate HPTLC plates and chromatographed in one dimension using two solvent systems. Quantitation was by in situ densitometry with absolute amounts of the lipid classes determined from co-chromatographed standards. An internal standard was also used to improve the precision. The individual lipid classes of rat whole brain, human brain gray and white matter, rat and bovine myelin, and bovine oligodendroglia were quantitated. Human brain phosphatidylethanolamine plasmalogen was also quantitated. Sensitivity was increased by using the cupric acetate charring reagent, which we found to be more sensitive than the conventional sulfuric acid-dichromate reagent. Total lipid (less than 400 micrograms) was quantitated from 5 mg of tissue wet weight. The limit of detection, on HPTLC, for the individual lipid classes was below 20 ng.
Highlights
We havedevised a high performance thin-layer chromatography (HPTLC) densitometry method to resolve the major lipidclassesof brain tissue
Standard curves for each non-acidic lipid class (Fig. 3) and each acidic lipid class (Fig. 4) indicate that each lipid classhad a different densitometric response, which was nonlinear over the range of 1.O- 10.0 pg
Data for each lipid class, expressed as a percent of total lipid, and total lipid, expressed on an absolute basis, are shown in comparison to data reported by other investigators (Table 1 and Table 2)
Summary
We havedevised a high performance thin-layer chromatography (HPTLC) densitometry method to resolve the major lipidclassesof brain tissue. This procedure affords a Abbreviations: TLC, thin-layer chromatography; HPTLC, high performance thin-layer chromatography; LPC, lysophosphatidylcholine; SPM, sphingomyelin; PC, choline phospholipids; PE, ethanolamine phospholipids; PS, serine phospholipids; C, cholesterol; TG, triacylglycerols; CE, cholesteryl esters; FA, fatty acids; CB, cerebrosides; SULF, sulfatides; IS, internal standard; PI, inositol phospholipids. The columns were further eluted with 10 ml of chloroform-methanol-water 30:60:8 and the non-acidic lipids were collected in this fraction.
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