Total lipoproteins

Ultracentrifugation for the preparation of the standard lipoproteins and the total lipoprotein fraction from the serum was carried out using an RP55 rotor in an Hitachi 55 P-2 ultracentrifuge (Hitachi Koki Co., Tokyo). 

The standard lipoproteins or the total lipoprotein fraction containing 20-100 pg of protein are applied onto the HPLC apparatus for monitoring by A gg. The loaded volume of normolipidemic human serum for monitoring of total cholesterol (TC), choline-containing phospholipids (PL) and triglycerides (TG) in a post column effluent are 10 pi, 20 pi and 50 pi, respectively, according to detection limit of each lipid in this HPLC technique as described in Section 6. In the case of the hyperlipidemic subjects, the loaded volume of serum decreases with an increase in serum lipid concentration. 

Serum lipoproteins are detected by Aggg in the case of the standard lipoproteins or the total lipoprotein fraction isolated by the ultracentrifugation. 

Fig. 3. Elution patterns of the total lipoprotein fraction by a single TSK GEL column. Column (a), G3000SW (b), G4000SW (c), G5000PW (d), G6000PW. Eluent 0.1 M Tris-HCl buffer (pH 7.4). Flow rate 1.0 ml/min. Loaded volume 20 pi. Peaks 1, chylomicrons 2, VLDL 3, LOL 4, HDL 5, albumin.

The total lipoprotein fraction (d<1.210) prepared from serum by the ultracentrifugation was analyzed in the same HPLC conditions as in Fig. 7. As presented in Fig. 8, well separated peaks corresponding to chylomicrons+VLDL, LDL, HDL2 and HDLj are observed by monitoring at A2gQ. The elution volume of each lipoprotein peak in Fig. 8 is consistent with that of the reference standards in Fig. 7. The concentration of total cholesterol, triglycerides and phospholipids in each ml of eluate are plotted in the same figure. For all lipoprotein classes, the peak position of protein is consistent with those of the three lipid components. 

Recovery of the standard lipoproteins and the total lipoprotein fraction was examined on the basis of total cholesterol concentration, and was found to be satisfactory as follows 85.3 6.0 % for chylomicrons+VLDL, 94 5 % for LDL,... 

Fig. 8. Elution pattern of the total lipoprotein fraction (d<1.210) from human serum. Peaks 1, chylomicrons+VLDL 2, LDL 3, HDL2 4, HDL3. HPLC conditions as in Fig. 7.

As described in Section 3.4, the elution patterns of serum lipoproteins can be obtained by monitoring A gg with use of the total lipoprotein fraction prepared from serum by ultracentrifugation. A few examples obtained for individual human subjects are shown in Fig. 9. The level of each lipoprotein is found to vary with individual subjects. Especially in the case of pathological subjects, there are distinct patterns with respect to each disease. 

Although the separation profile of the total lipoprotein fraction can be given by monitoring of protein moiety at A gg, the concentration of each lipoprotein class could not be calculated from peak area because of the difficulties in estimating lipoprotein levels caused by the following factors evaluation of turbidity, different molar coefficient of A2gg due to each lipoprotein, coelution of serum proteins with HDL fractions. At this point of view, a selective detection method of lipid components by enzymatic reaction using whole serum is useful for lipoprotein analysis as described in the next section. 

In the case of the total lipoprotein fraction, serum lipoproteins can be monitored both by A20q and Aggg. On the other hand, serum lipoproteins can be... 

Fig. 10. Elution patterns of protein (A280) and total cholesterol (A550) for human serum. Column G5000PW+G3000SW+G3000SW+G3000SW. Sample (A), 20 pi of whole serum (B), 20 pi of the total lipoprotein fraction (d<1.210). Flow rate 1.0 ml/min for eluent 0.15 M NaCl), 0.35 ml/min for enzyme solution (Determiner TC"555"). Reaction temperature 40°C. Peaks 1, LDL 2, HDLg 3, HDL3 4, human serum albumin.

Age Number of subjects Total cholesterol (mg./lOO cc.) Total lipoproteins (mg./lOO cc.) a-Lipo- proteins (mg./lOO cc.) -Lipo- protein (mg./lOO cc.) Ratio jSlot lipoproteins... 

The surface area is an important determinant of lipoprotein function. When compared on the basis of the number of lipoprotein particles ml, about 90% of the total lipoprotein particles in the plasma are HDL (Table 3). By contrast, in males, HDL have only about half of the total lipoprotein surface area. In females, HDL contributes about 75% of the total lipoprotein surface area because of the relative abundance of HDL. In terms of the core volume of lipoproteins, LDL contains about half the lipoprotein core volume in both males and females. 

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