Separation of lipoproteins by capillary isotachophoresis combined with enzymatic derivatization of cholesterol and triglycerides.

Abstract

Combining specific enzymatic derivatization of cholesterol or triglycerides with capillary isotachophoresis (CITP), human serum lipoproteins are separated into 14 lipoprotein subfractions, monitored and quantitated by direct capillary UV detection. By comparing the separation patterns of human serum with the patterns of lipoprotein particles isolated by sequential ultracentrifugation it became evident that peaks 1-5 represent lipoproteins of the high density lipoprotein (HDL) fraction, peaks 6-8 embody the very low density lipoprotein (VLDL) fraction and chylomicrons, and peaks 7-14 represent the low density lipoprotein (LDL) fraction. Peaks 7 and 8 were found in the VLDL as well as in the LDL fraction. Using triglyceride-specific staining peaks 6-8 occurred prominently; and with cholesterol-specific staining, peaks 1-5 and 7-14 were prominent. The coefficient of variation, for the sum of the peak heights of a pooled serum, was 3.94 for triglyceride-specific staining and 2.32 for cholesterol-specific staining. A linearity range between 0.23 and 2.29 mM/L was found for triglyceride-specific staining and between 0.043 and 4.33 mM/L for cholesterol-specific staining. The practicability of the method was evaluated (i) using blood of humans before and 45 min after an oral fat load. Triglyceride-specific staining revealed a prominent increase in the VLDL fraction and chylomicrones containing peaks 6 and 7, and a minor increase in the HDL fraction containing peaks 3 and 4, and (ii) in patients with manifest hypothyroidism before and after thyroxine therapy. Cholesterol-specific staining demonstrated a massive decrease in the first peak of the HDL fraction and in peaks 9 and 11 of the LDL fraction regarding the hypo versus hyperthyroid state.