Daniel Pometta

CHARACTERIZATION OF SUBPOPULATIONS OF LIPOPROTEIN PARTICLES ISOLATED FROM HUMAN CEREBROSPINAL FLUID

The aim of the present study was to define lipoprotein complexes within cerebrospinal fluid (CSF) in terms of their apolipoprotein composition, using fractionation procedures considered optimal for maintaining lipoprotein structural integrity. Five apolipoproteins were identified, namely apolipoproteins A-I, A-IV, D, E and J. These were differentially distributed amongst lipoprotein particles of which three major subpopulations were identified. CSF-LpAI (20.1 +/- 3.8 nm) was enriched in apolipoprotein A-I and contained the major proportion (> 50%) of apolipoproteins D, E and J. CSF-LpE, of similar size to CSF-LpAI (20.2 +/- 3.1 nm), was composed principally of apolipoprotein E, with minor quantities of apolipoproteins A-I, A-IV, D and J. Elimination of these particles from cerebrospinal fluid by immunoabsorption revealed a third subpopulation of significantly greater diameter (32.0 +/- 6.8 nm). The majority (62%) of apolipoprotein A-IV was also present in this fraction. The study demonstrates the structural and size heterogeneity of lipoproteins in cerebrospinal fluid. This may reflect the lipid transport processes within the central nervous system.

Differences in Lipoprotein Subfraction Composition and Distribution Between Type I Diabetic Men and Control Subjects

Subfractions of very-low-density and low-density lipoproteins were examined in 10 male normolipidemic type I (insulin-dependent) diabetic patients before and after improvement of metabolic control and were compared with subfractions from male control subjects matched to the diabetic patients at entry for age, body mass index, plasma cholesterol, and plasma triglycerides. Two consistent differences in subfraction composition were noted between the diabetic patients at entry and the control subjects. First, subfractions from diabetic patients tended to be cholesterol-ester poor and triglyceride rich; this was particularly marked for the low-density lipoprotein subfractions. Second, the subfractions from pretreatment diabetic patients contained higher proportions of non-apolipoprotein B apolipoproteins. This compositional anomaly, but not the lipid modifications, responded to but was not completely normalized by improved glycemic control, which was also accompanied by reductions in the plasma concentrations of all subfractions. Treatment modified subfraction distribution so that the lipoprotein profile of posttreatment diabetic patients more closely resembled the profile observed in the control subjects. These changes were achieved without significant modification of daily insulin dose. In the context of blood lipid risk factors, the results argue for the need to maintain optimal insulinization even in apparently normolipidemic diabetic patients to avoid modifications of the lipoprotein pattern toward a potentially more atherogenic profile.

Underexpression of the apolipoprotein E4 isoform in an Italian population.

Apolipoprotein (apo) E polymorphism was examined in a population of Italian blood donors. A significantly reduced frequency of the epsilon 4 allele was observed in comparison to a combined Caucasian population. Apo E polymorphism was also associated with significant differences in plasma lipid and lipoprotein levels. Notably, total and low-density lipoprotein cholesterol as well as triglycerides were increased, whereas high-density lipoprotein cholesterol was decreased in carriers of the E4 isoform. This is the first report of a significantly lower frequency of the apo E4 isoform in a European population. The reduced occurrence of an apo E isoform, which is associated with a more atherogenic lipid/lipoprotein profile, may be a contributory factor to the relatively lower incidence of cardiovascular disease in the Italian population.

Characterization of a human high density lipoprotein-associated protein, NA1/NA2. Identity with SP-40,40, an inhibitor of complement-mediated cytolysis.

Two peptides, NA1 and NA2, which we previously suggested to be associated with high density lipoproteins (HDLs), have been purified. Polyclonal antibodies against each peptide and a monoclonal antibody against NA2 have been used to further characterize them and their association with HDL. Immunoblotting studies revealed that the peptides form a complex of molecular mass of approximately 80 kd. Agarose gel filtration showed coelution of NA1/NA2 and apolipoprotein (apo) A-I, the structural protein of HDL. This was confirmed by fast protein liquid chromatography, which further indicated that up to 60% of NA1/NA2 was located within the lower density range of the HDL spectrum. Complementary studies with anti-apo A-I immunoaffinity columns provided evidence that at least 40% of NA1/NA2 was associated with HDL, an association easily disrupted by ultracentrifugal manipulation. Finally, partial amino acid sequences showed virtually complete homology with a recently identified protein, SP-40,40, or cytolysis inhibitor. The protein is suggested to have a powerful inhibitory effect on complement-mediated cell lysis. Our results could thus furnish an explanation for the previously observed modulating influence of HDL on complement activity.

Immunoaffinity fractionation of high-density lipoprotein subclasses 2 and using anti-apolipoprotein A-I and A-II immunosorbent gels

High-density lipoprotein (HDL) subclasses 2 and 3 prepared by density gradient ultracentrifugation have been further fractionated by immunoaffinity chromatography using antibody affinity gels targetting the major HDL apolipoproteins, A-I and A-II. Fractions containing A-I without A-II (AI w/o AII) and A-I with A-II (AI w AII) were isolated from both density ranges. Whereas there were similar concentrations of the major subfraction (HDL3(AI w AII] in both males and females, the remaining subfractions were present in higher concentrations in females as compared to males, in the order HDL3 (AI w/o AII) less than HDL2(AI w AII) less than HDL2(AI w/o AII). The difference was most marked for HDL2 (AI w/o AII), where plasma concentrations in females were almost 3-fold greater than in males. Compositional analyses indicated that the plasma concentrations of the fractions, rather than their compositions, were the major determinants of male-female differences in HDL levels. In contrast, fractions defined by similar apolipoprotein criteria and isolated from different density subclasses (i.e., HDL2(AI w/o AII) vs. HDL3(AI w/o AII) and HDL2(AI w AII) vs. HDL3(AI w AII] showed major compositional differences. This is suggestive of distinct lipoprotein particles.

Decreased HDL cholesterol in prepubertal and pubertal children of CHD patients

Prepubertal boys and pubertal girls and boys selected because of the occurrence of acute myocardial infarction in their father or mother were characterized by a low HDL cholesterol when compared to healthy controls. This observation extends our previous observations of low HDL cholesterol in adult relatives of CHD patients to the children of the same patients. The occurrence of an HDL abnormality in young children demonstrates that the low HDL cholesterol precedes the occurrence of cardiovascular disease. Low HDL cholesterol was associated with increased VLDL cholesterol in prepubertal children and pubertal boys without significant increase in VLDL triglycerides. Smoking and drinking habits, and physical activity which are environmental factors known to affect HDL did not differ between these children and the controls and cannot account for the observed differences. The results show that the low HDL cholesterol is detectable early in life in close relatives of CHD patients, persists throughout the lifespan and is possibly genetically determined.

High density lipoprotein cholesterol in male relatives of patients with coronary heart disease

To study factors that play a role in the familial occurrence of coronary heart disease, very low density lipoprotein (VLDL) triglycerides, low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol were measured after preparative ultracentrifugation in first degree male relatives of coronary patients and in control subjects. The HDL cholesterol concentration was significantly lower in relatives of 20--71 years old than in controls. No increase of serum and LDL cholesterol was found. A low level of HDL cholesterol was observed even in the younger relatives who are less likely to have cardiovascualr disease. In older relatives low HDL cholesterol was found in the presence or absence of clinical evidence of coronary artery disease. The HDL-cholesterol concentration was inversely related to the VLDL triglycerides both in relatives and controls, but the regression lines were different ((P less than 0.001) for the relative (y = --0.166x + 0.43) and for the controls (y = 0.191x + 0.49). A low HDL cholesterol level appears to be a marker of relatives of coronary patients.

Distribution of apolipoprotein E between free and A-II complexed forms in very-low- and high-density lipoproteins: functional implications

The stability of apolipoprotein E/lipoprotein associations has been examined as a function of apolipoprotein E phenotype. Visualisation by immunoblotting showed plasma apolipoprotein E to be present in two forms; the free form and, as previously described, an E-A-II complex. In very low density lipoproteins isolated by gel filtration from subjects with E3/3 and E4/3 phenotypes, apolipoprotein E was present essentially in the free form (ratio free: complex of 12.2 and 37.5, respectively). Exploiting ultracentrifugation as the disruptive agent, very-low-density lipoproteins thus isolated were shown to have substantially lower ratios (5.6 and 5.4, respectively) reflecting preferential loss of free apolipoprotein E. In high-density lipoproteins isolated by gel filtration from E3/3 phenotypes, apolipoprotein E was largely present as an E-A-II complex (80.3%). In contrast, the majority of apolipoprotein E in high-density lipoproteins from E4/3 phenotypes was present in the free form (58.7%). In both phenotypes, the content of free apolipoprotein E was markedly reduced by ultracentrifugation. The results confirm the notion that the formation of the E-A-II complex is a major determinant of the stability of apolipoprotein E-high-density lipoprotein associations. Moreover, that the predominant, ancestral isoform, apolipoprotein E3, exists largely as an E-A-II complex in higher density lipoproteins has important functional implications for this plasma source of apolipoprotein E.

Immunofractionation of high density lipoprotein subclasses 2 and 3: Similarities and differences of fractions isolated from male and female populations

High density lipoprotein (HDL) subclasses 2 and 3 isolated from male and female populations were further subfractionated by immunoaffinity techniques. Each subclass gave rise to 2 fractions: one contained apolipoprotein (apo) A-I but no apo A-II (LpAI); the other contained apo A-I and apo A-II (LpAI,AII). The bulk fraction (HDL-3(LpAI,AII)) comprised over 70% of total HDL and was present in similar concentrations in both populations. There were, however, significant male-female differences in plasma levels of the minor HDL-3 fraction i.e. HDL-3(LpAI). Females had significantly higher plasma concentrations of both fractions within HDL-2. These fractions also exhibited strong, positive correlations with total HDL cholesterol concentrations, both in males as well as females. It suggests that metabolic activities giving rise to both HDL-2(LpAI) and HDL-2(LpAI,AII) determine plasma HDL cholesterol concentrations. Several similarities were noted between the male and female populations. Triglyceridaemia was negatively correlated with HDL-2 derived fractions and positively correlated with the bulk fraction HDL-3(LpAI,AII). Compositional data showed that the fraction (LpAI) had a lower esterified cholesterol to total cholesterol ratio than the fraction (LpAI,AII), the differences being more apparent at the HDL-3 level. Additionally, analysis of the surface components of HDL-3 fractions suggested that (LpAI,AII) had a greater potential than (LpAI) for absorbing lipoprotein surface material. Finally, the relative concentrations of the individual components of fractions within the same population and defined by the same apolipoprotein criterion showed highly significantly, positive correlations. Such correlations were not apparent for apolipoprotein dissimilar fractions. These observations could reflect a metabolic link between apolipoprotein similar fractions.

HDL lipids in close relatives of coronary heart disease patients. Environmental and genetic influences.

Female first-degree relatives of CHD patients differed, after the age of forty, from the normal control population by their low HDL cholesterol. Between the ages of 20 and 40 years a slight but significant increase in HDL triglycerides was observed. Except for hormonal contraception which induces significant lowering of HDL cholesterol in the first degree relatives, the observed differences in HDL lipids did not seem related to environmental factors but rather to be genetically determined. In contrast, the low HDL cholesterol observed in the wives of the CHD patients appeared to be related to differences in alcohol intake.