DIS1RIBUTION OF CHOLESTEROL WITHIN HIGH DENSITY LIPOPROTEINS FRACTIONATED BY IMMUNOAFFINITY CHROMATOGRAPHY.

DIS1RIBUTION OF CHOLESTEROL WITHIN HIGH DENSITY LIPOPROTEINS FRACTIONATED BY IMMUNOAFFINITY CHROMATOGRAPHY

INTRODUCTION

Cholesterol enjoys notoriety as a pro or anti-atherogenic lipid by virtue of the lipoprotein with which it is associated. This simplistic interpretation is most evident in the positive and negative correlations with the atherosclerotic process manifested by cholesterol associated with the most abundant lipoprotein species, respectively low density (LDL) and high density (HDL) lipoproteins 1,2. The statement requires qualification, however, when lipoprotein sub-populations are contemplated. Thus, within the HDL density spectrum, the protective influence is largely believed to reside within the ultracentrifugally­ defined lower density HDL-2 subclass 3. HDL-3 enjoys much less support as an anti-atherogenic lipoprotein particle, a somewhat unsatisfactory state of affairs when considering mechanistic explanations of the function of HDL. Notably, particles of the size of HDL-3 are the principal acceptors of cellular cholesterol, the initiating step in reverse cholesterol transport 4. In addition, they can act as a repository for lipids shed from triglyceride-rich lipoproteins, greatly facilitating the catabolic removal of these potentially atherogenic lipoprotein particles 5. Further subfractionation of lipoproteins into, hopefully, metabolically homogenous sub-populations is one approach which should yield more satisfying explanations. This is the rationale behind the studies described in the present report. HDL subClasses 2 and 3, as defined by the physico-chemical criteria of ultracentrifugation, have been further fractionated using an immunoaffinity approach 6,7 targetting the predominant HDL apolipoprotein (apo) components, apos A-I and A-II. We have examined the cholesterol distribution of such immunoaffinity-defined fractions within HDL-2 and 3 isolated from healthy male and female populations. Furthermore, the distribution has been analysed in sub-groups corresponding to the lowest and highest total HDL-cholesterol quartiles of the same populations.

MATERIALS AND METHODS

Study populations

Healthy male (n=35) and female (n=33) subjects were recruited from the university hospital and medical research centre in Geneva.

Basic clinical characteristics of these groups have been described previously 8. Average HDL-cholesterol values were 1.22±0.19mm/l for men and 1.55±0.33mm/l for women.

The populations were also segregated into quartiles based on values of total HDL-cholesterol. For the male population, quartiles 1 and IV averaged HDL-cholesterol levels of 0.99±0.09mm/l (range 0.80- 1.07mm/l) and 1.49±0.13mm/l (range 1.29-1.60mm/l). Corresponding values for female quartiles I and IV were 1.19±0.1Omm/1 (range 0.98- 1.28mm/l) and 2.03±0.26mm/l (range 1.76-2.46mm/l).

Lipoprotein fractionation

Serial fractionation of high density lipoproteins from fasting plasma by ultracentrifugation and immunoaffinity chromatography was achieved as described 8. The immunoaffinity procedure gave rise to two types of lipoprotein particle, described according to the presence of apos A-1 and A-11. Thus LpAI,AII contains both apos, whereas LpAI contains apo A-1 but no apo A-11. The particles are further defined by their subclass source ie HDL-2 or HDL-3.

Other analyses

Lipid and protein measurements and statistical analyses were performed as described previously 8,9,10

RESULTS

Fig. 1 shows the distribution of cholesterol within the subfractions in both the male and female populations. Cholesterol was principally associated with HDL3-LpAI,AII and concentrations were not significantly different between the males and females. In contrast,

highly significant differences (p<0.0001 for HDL-2 derived subfractions ; p=0.0001 for HDL3-LpAI) between populations were observed for the remaining subfractions . These differences were particularly marked for HDL2-LpAI and HDL2-LpAI,AII, being respectively 100% and 60%

higher in the female group. Of the cholesterol associated with (AI,AII), 31% was within the HDL-2 density range in men, compared to 39% for women. For (AI), the male group had 54% of associated cholesterol within HDL-2, whereas the female group had 65% within the lower density subclass. Total HDL-cholesterol (measured after phosphotungstate precipitation) correlated strongly with HDL2-LpAI and HDL2-LpAI,AII cholesterol, with coefficients of +0.66 and +0 .67 (men) and +0.83 and +0.82 (women) respectively. These contrast with

the coefficients observed for the quantitatively major fraction, HDL3- LpAI,AII cholesterol, with values of +0.53 (men) and +0.46 (women).

Cholesterol of both HDL-2 derived fractions showed negative correlations with plasma triglyceride levels. For HDL2-LpAI these were -0.53 for men and -0.36 for women: corresponding correlations for HDL2-LpAI,AII were -0.39 and -0.30 respectively.

The lipoprotein association of cholesterol was also examined in the first and fourth quartiles of both populations. For the female population, significantly lower cholesterol levels were found in quartile I in both HDL-2 and 3 density ranges (25.9±5.0 v 58.1±9.4mg/dl (p<O.OOOI) and 41.5±4.9 v 50.7±6.5mg/dl (p=0.015)). Further subfractionation (Fig. 2) showed that differences at the HDL-3 level were due to the (AI) lipoproteins (p=0.0005), there being similar concentrations of HDL3-LpAI,AII (p=0.09). Both immunoaffinity defined fractions from HDL-2 were highly significantly increased in quartile IV (LpAI, p=0.0002; LpAI,AII, p=0.0001).

When cholesterol associated with the subfractions was correlated with plasma triglyceride levels (Table 1) some interesting differences between the two quartiles emerged. Thus, for HDL-2, negative correlations were observed for LpAI for quartile I, but with LpAI,AII for quartile IV. Contrasting results w·ere also evident for HDL-3 derived immunoaffinity-derived particles (Table 1): triglycerides were correlated in a positive manner with HDL3-LpAI,AII from quartile I, but negatively with the same fraction from quartile IV.

With respect to quartiles established for the male population, qualitatively similar conclusions could be drawn from an analysis of the lipoprotein association of cholesterol. Namely, quartile I had significantly lower cholesterol concentrations within subclasses HDL-2 (14.8±3.1 v 29.4±5.7mg/dl; p=0.0007) and HDL-3 (37.7 ±6.0 v 48.1±2.6mg/dl; p=0.005). Likewise, HDL2-LpAI (p=0.002) and HDL2- LpAI,AII (p=0.0004) were also significantly lower in quartile 1 (Fig. 2). Within HDL-3, LpAI,AII cholesterol was significantly higher (p=0.004) in quartile IV, but there were no significant differences (p=0.12) in cholesterol concentrations of LpAI (Fig. 2).

As with the female population, the male quartiles differed somewhat when examined in terms of triglyceridaemia. Whereas negative correlations were observed with fractions originating from HDL-2 of both quartiles, divergent correlations were found for HDL-3 derived fractions (Table 1). Both fractions of quartile I were positively correlated with triglyceride levels, in contrast to negative correlations exhibited for the same fractions from quartile IV.

The dual fractionation procedure adopted herein provides a more precise definition of the cholesterol distribution within the high density lipoprotein spectrum. The results demonstrate the importance of both HDL2-LpAI and HDL2-LpAI,AII in determining total plasma levels of HDL-cholesterol. This is evident from the strength of the correlation coefficients when comparing total HDL-cholesterol with cholesterol in each subfraction. Moreover, male-female differences in HDL levels largely reside in the concentrations of HDL2-LpAI and HDL2-LpAI,AII (Fig. 1). Finally, within the same population, it is these two subfractions that essentially differentiate subjects in the first and fourth quartiles of HDL plasma concentrations. It suggests that physiological events giving rise to both fractions are important in determining HDL-cholesterol levels.

Although the major proportion of HDL-2 cholesterol is present in LpAI,AII in both males and females, it would appear that HDL2-LpAI is the more sensitive indicator of HDL-cholesterol levels. Thus, concentrations of HDL2-LpAI cholesterol are 2.5 to 3 fold higher in quartile IV as compared to quartile I. In contrast, levels of HDL2- LpAI,AII cholesterol in quartile IV are less than double those in quartile I. Further, in quartile I of both populations, only HDL2-LpAI cholesterol shows a strong correlation with total HDL-cholesterol. Interestingly, subjects in quartile IV also showed strong correlations between total HDL-cholesterol and HDL2-LpAI,AII cholesterol, again in both sexes. The latter is one observation that differentiates the two quartiles. Another is the correlation between triglyceridaemia and cholesterol levels. Notably, HDL3-LpAI,AII cholesterol showed a positive correlation with triglycerides in quartile I, but a negative correlation in quartile IV. Other differences were also evident (Table 2), although there was less of a parallel in the response of the male and female quartiles.

Overall, the results suggest that the combination of distinct fractionation procedures can be helpful in further defining the association of cholesterol with high density lipoproteins. It should provide information useful in determining the relative importance of the different subfractions to the anti-atherogenic effect of HDL.

ACKNOWLEDGEMENTS

The work reported herein was supported by grants 3.999-0.86 and 32.9484-88 from the Swiss National Research Fund.

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