Improved staining characteristics of serum lipids after halogenation and esterification of thin-layer chromatograms

We describe a procedure for treatment of thin-layer chromatographic serum lipid patterns so that they may be stained by dyes for evaluation by densitometry. After development of the chromatogram [Clin. Chem. 18, 384 (1972)] the plates are dried and sprayed with butyryl chloride. This esterifies the free cholesterol. After drying, the plate is treated with iodine monobromide, to add iodline to the double bonds. The triacylglycerols (triglycerides), cholesterol esters, free cholesterol, and phospholipids are now all in the form of esters with no unsaturated double bonds. The free fatty acids are also now saturated. The chromatogram is now stained with basic fuchsine in acetate buffer (0.1 mol/liter, pH 5.0). Excess stain is removed with a buffered solution of guanidine hydrochloride. Erythrosine B may also be used. With basic fuchsine the background will be a uniform pink. With erythrosine B the background is white, but the stain tends to be washed out of the free fatty acids. The chromatograms are evaluated by densitometry, with use of a 540-nm filter for basic fuchsine and a 520-nm filter for erythrosine B. The stained chromatograms and densitometric scans accurately represent the relative concentration of the various lipid fractions as compared to that of an internal standard, and correlate with the nature of the disease being explored.