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

Combining specific enzymatic derivatization of cholesterol or triglycerides with capillary isotachophoresis (CITP), human serum lipoproteins are separ ated 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 ultracen trifugation it became evident that peaks 1-5 represent lipoproteins of the high density lipoprotein (HDL) fraction, peaks 6-8 embody the very low dens ity 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, pea ks 1-5 and 7-14 were prominent. The coefficient of variation, for the sum o f the peak heights of a pooled serum, was 3.94 for triglyceride-specific st aining and 2.32 for cholesterol-specific staining. A linearity range betwee n 0.23 and 2.29 mM/L was found for triglyceride-specific staining and betwe en 0.043 and 4.33 mM/L for cholesterol-specific staining. The practicabilit y of the method was evaluated (i) using blood of humans before and 45 min a fter an oral fat load. Triglyceride-specific staining revealed a prominent increase in the VLDL fraction and chylomicrones containing peaks 6 and 7, a nd 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 firs t peak of the HDL fraction and in peaks 9 and 11 of the LDL fraction regard ing the hypo versus hyperthyroid state.