New micromethod for measuring cholesterol in plasma lipoprotein fractions

A method is described for the reliable, fast, and relatively inexpensive fractionation of plasma lipoproteins and quantitation of their cholesterol content. This procedure requires 350 microliter of plasma and can be completed within 3 h. Plasma lipoproteins (175 microliter of plasma) were prestained with Fat Red 7B and centrifuged (Beckman Airfuge) at plasma density (d = 1.006 kg/liter) and at a solvent density of 1.060 kg/liter, adjusted by adding solid KBr. Prestained centrifuged samples demonstrated the characteristic elevation of chylomicrons in phenotypes I and V, low-density lipoproteins of phenotype II, very-low-density lipoproteins in phenotype IV and V, and continuum of pink color throughout the centrifuge tube, diagnostic of the floating beta lipoprotein of type III. Centrifuged samples were separated into top and bottom fractions by aspiration. Cholesterol was quantitated with an enzymic oxygen-electrode analyzer (Beckman Cholesterol Analyzer). Correlation coefficients between cholesterol values for plasma from normal hyperlipidemic individuals obtained with the Beckman Analyzer vs. the Technicon AutoAnalyzer II and SMAC systems were 0.977 and 0.973, respectively.