640 MICROMETHOD FOR THE RAPID ANALYSIS OF PLASMA LIPIDS

Abstract

The quantitation of plasma triglycerides (TG), cholesterol (C), cholesteryl esters (CE), and fatty acids (FFA) presently requires expensive, time-consuming multiple analyses; sample volumes are too large to permit repeated assays in the newborn. To overcome these problems, we have developed a thin-layer chromatographic (TLC) technique which simultaneously estimates these lipids. Blood is drawn into a 44.7μl heparinized capillary tube. One to ten μl of plasma is applied directly to the aquaphilic, preadsorbent area of a silica gel TLC plate. After thorough drying, the plate is developed to 10cm (20 minutes) with petroleum ether:ethyl ether: acetic acid-90:10:1 which quantitatively extracts the four lipids from the plasma and readily separates them. Color is developed with a cupric acetate stain, and the lipid content of each spot is estimated by visual comparison with standards or quantified by photodensitometry. This technique provides linear color responses over these ranges: TG, .5-10μg; C, .3-5μg; CE, .5-5μg; FFA, .5-5μg, quantities present in 10μl of normal or hyperlipemic plasma. The method's accuracy is ± 5% and its precision is ± 3%. Results are available within ninety minutes and the estimated cost is approximately one dollar per test. With TLC we have: determined the C, CE, and TG content of normal and hyperlipemic plasma; monitored on a daily, and even hourly basis, lipid levels of small premature infants receiving IV fat; and screened for hyperlipidemia. This microtechnique should facilitate the study of fat metabolism in infants.

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