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Current recommendations of the Adult Treatment Panel and the Children and Adolescents Treatment Panel of the National Cholesterol Education Program make the concentration of low-density lipoproteins cholesterol (LDL-C) in serum the basis for the classification and treatment of hypercholesterolemia. Numerous methodologies for the determination of serum LDL-C concentrations, in research and clinical laboratories, have been described. Here, we review the principles, performance, and limitations of major current methodologies for determining LDL-C concentrations. These methods include sequential and density-gradient ultracentrifugation, chromatographic and electrophoretic techniques, and precipitation methods. In addition, the advantages and disadvantages of estimating LDL-C concentration by the Friedewald equation, the most commonly used approach in clinical laboratories, are addressed.

We describe a gel isoelectric focusing procedure for resolving into at least five bands the arginine-rich protein of very-low-density lipoproteins, and use the method in diagnosis of hyperlipoproteinemia type 3. We find that deficiency of one band, designated E3, relative to E2, expressed as an E3/E2 ratio less than or equal to 0.3, is specifically associated with hyperlipoproteinemia type 3 as diagnosed by traditional criteria in a large family study. In addition, we used the procedure to test 47 referral samples with demonstrable beta-migrating very-low-density liproprotein grouped by very-low-density lipoprotein cholesterol/total triglyceride ratios of greater than or equal to 0.3, from 0.25 to 0.3, and less than 0.25. All 24 of the first group, three of six in the second, and only one of 17 in the third had E3/E2 ratios of less than or equal to 0.3. Also, two normolipidemic children, without detectable lipoprotein abnormalities but related to subjects with hyperlipoproteinemia type 3, had E3/E2 ratios of less than or equal to 0.3. Use of our procedure improves the specificity of diagnosis and allows sensitive detection of asymptomatic subjects who may be at risk of developing the disorder.

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We describe a one-day micro-scale procedure for determining the total lipid profile. Only 0.55 mL of plasma is needed for complete quantification of total cholesterol (TC), triglyceride (TG), and all lipoproteins. After precipitation with dextran sulfate and magnesium, the high-density lipoprotein (HDL) fraction was separated by centrifugation in an Eppendorf microcentrifuge. Very-low-density lipoprotein (VLDL) was separated from low-density lipoprotein (LDL) plus HDL in a Beckman TL 100 ultracentrifuge. TC, TG, and cholesterol in different lipoprotein fractions were measured enzymatically in a Baker "Encore II" automated analyzer. CVs, both within-day and day-to-day, were less than 3% for TG and TC, and less than 5% for HDL-C determinations. CVs for LDL-C and VLDL-C were less than 7.5% and 15%, respectively. Results by our micromethods (n = 66) agreed well with those by the conventional methods used at the Northwest Lipid Research Center, which are standardized against the Reference Methods of the Centers for Disease Control. Coefficients of correlation between the two methods were 0.98 for TC, 1.0 for TG, 0.98 for HDL-C, 0.94 for LDL-C, and 0.96 for VLDL-C. Results of electrophoresis on agarose gel and radioactivity-recovery studies indicate that our micro-centrifugation and slicing procedures result in clean separation of VLDL from other lipoproteins.