Armin Steinmetz

Apolipoprotein E phenotypes and hyperlipidemia

SummaryApolipoprotein E phenotypes were determined in 361 patients with hyperlipidemia and in controls. The E2 isoform was significantly more frequent in the group of hyperlipidemics (P<0.0005). This was not due to a higher frequency of E-2/2 homozygotes with type III hyperlipoproteinemia, but rather to a significantly higher frequency of E2 heterozygotes (P<0.0005). Subgrouping of hyperlipidemics into patients with a) hypertriglyceridemia, b) hypercholesterolemia and c) mixed hyperlipidemia revealed i) that isoform E2 was significantly more frequent in patients with hypertriglyceridemia (0.001>P>0.005), ii) that isoform E4 was significantly more frequent in patients with hypercholesterolemia (0.01>P>0.005) and iii) that isoforms E2 (P>0.005) and E4 (0.05>P>0.025) were both more frequent in patients with mixed hyperlipidemia. Roughly 20% of patients with mixed hyperlipidemia had one of the rare phenotypes E-4/4,-4/2 or-2/2. We conclude that alleles ε2 and ε4 both contribute to the susceptibility for, and/or phenotypic expression of hyperlipidemia. Whereas the gene ε2 seems to exert its influence on plasma lipoproteins by an abnormal gene product (E2) that has reduced binding activity to lipoprotein receptors, the mechanism underlying the association of the ε4 gene with hyperlipidemia is presently unclear.

Protection Against Atherogenesis in Mice Mediated by Human Apolipoprotein A-IV

Apolipoproteins are protein constituents of plasma lipid transport particles. Human apolipoprotein A-IV (apoA-IV) was expressed in the liver of C57BL/6 mice and mice deficient in apoE, both of which are prone to atherosclerosis, to investigate whether apoA-IV protects against this disease. In transgenic C57BL/6 mice on an atherogenic diet, the serum concentration of high density lipoprotein (HDL) cholesterol increased by 35 percent, whereas the concentration of endogenous apoA-I decreased by 29 percent, relative to those in transgenic mice on a normal diet. Expression of human apoA-IV in apoE-deficient mice on a normal diet resulted in an even more severe atherogenic lipoprotein profile, without affecting the concentration of HDL cholesterol, than that in nontransgenic apoE-deficient mice. However, transgenic mice of both backgrounds showed a substantial reduction in the size of atherosclerotic lesions. Thus, apoA-IV appears to protect against atherosclerosis by a mechanism that does not involve an increase in HDL cholesterol concentration.

Serum amyloid A (SAA): an acute phase protein and apolipoprotein.

Serum amyloid A (SAA) proteins comprise a family of apolipoproteins coded for by at least three genes with allelic variation and a high degree of homology between species. The synthesis of certain members of the family is greatly increased in inflammation. However, SAA is not often used as an acute-phase marker despite being at least as sensitive as C-reactive protein. SAA proteins can be considered as apolipoproteins since they associate with plasma lipoproteins mainly within the high density range, perhaps through amphipathic alpha-helical structure. It is not known why certain subjects expressing SAA develop secondary systemic amyloidosis. There is still no specific function attributed to SAA; however, a popular hypothesis suggests that SAA may modulate metabolism of high density lipoproteins (HDL). This may impede the protective function of HDL against the development of atherosclerosis. The potential significance of the association between SAA and lipoproteins needs further evaluation.

Genetic Control of Human Apolipoprotein E Polymorphism: Comparison of One- and Two-Dimensional Techniques of Isoprotein Analysis

SummaryGenetic polymorphism of human apolipoprotein E (apo E) has previously been demonstrated by one-dimensional isoelectric focusing (Utermann et al. 1977b) and by two-dimensional electrophoresis of apolipoproteins (Zannis et al. 1981), but the relationship between the results obtained by these methods remained unclear. We therefore performed comparative phenotyping by one-dimensional and two-dimensional electrophoresis. Apoproteins from very low-density lipoproteins (apo VLDL) prepared by ultracentrifugation or from an apo Erich lipoprotein fraction prepared by heparin/Mg++ precipitation, were used as a source of apo E. Six common phenotypes designated apo E-4/4, apo E-N/N, apo E-D/D, apo E-4/N, apo E-4/D, and apo E-N/D were differentiated irrespective of the technique used or the source of apolipoproteins, but the two-dimensional electrophoresis of apo VLDL and apo VLDL which had been treated with neuraminidase was the key for the correct genetic interpretation of those phenotypes exhibiting the E4 isoform of the protein. Each phenotype is characterized by the presence of either one or two of three major isoforms E2, E3, and E4 and by the presence of several minor sialylated forms of these proteins (apo Es) that have higher apparent molecular weights. The unsialylated major isoform apo E2 does not only differ in charge but also has a higher apparent mol.wt. (about 34,500) than the major isoforms apo E3 and apo E4 (mol. wt. about 33,000). Family studies including 90 matings with a total of 203 offspring confirmed the genetic one locus model of Zannis et al. (1981). Apo E phenotypes are controlled by three autosomal codominant alleles apo Ed, apo En, and apo E4 that specify for the E2, E3, and E4 isoforms respectively. Phenotypes apo E-D/D,-N/N, and-4/4 represent homozygotes and phenotypes apo E-4/N,-4/D, and-N/D heterozygotes for these alleles.The frequencies of apo E alleles in 1031 blood donors were apo E4=0.150, apo En=0.773, and apo Ed=0.077. Homozygosity for the allele apo Ed is associated with hyperlipoproteinemia type III. Hence a large number of the population (about 1%) are at risk for this specific lipoprotein disorder that is associated with premature atherosclerosis and xanthomatosis.

Influence of serum amyloid A on cholesterol esterification in human plasma

Lecithin-cholesterol acyltransferase (EC 2.3.1.43, LCAT) is the enzyme responsible for the formation of the bulk of cholesteryl ester in human plasma. The LCAT-reaction takes place mainly on high-density lipoproteins and requires an apolipoprotein as activator. Besides apolipoprotein (apo) A-I several other potent activator apolipoproteins (AIV, E and CI) were identified, furthermore apo A-II was shown to be a modulator of the enzymes reaction in the presence of apo A-I. Serum amyloid A, an apolipoprotein mainly associated with high-density lipoprotein, massively accumulates in plasma upon acute phase reactions. We therefore studied the possible influence of this acute phase reactant on cholesterol esterification in human plasma. There was a significant decrease of esterified cholesterol in plasma during acute phase reaction. We found a highly significant correlation between the unesterified part of plasma cholesterol and serum amyloid A levels (r = 0.694, P = 0.0001). Also, plasma LCAT activity was negatively correlated with serum amyloid A levels. Lipoproteins containing apo A-I and A-II (LpA-I: A-II) and lipoproteins containing apo A-I but no A-II (LpA-I) decreased significantly with the appearance in plasma of serum amyloid A. To study the influence of serum amyloid A on the LCAT reaction, artificial substrates were prepared either by a detergent dialysis procedure or by addition of apolipoprotein to a sonicated aqueous dispersion of lipid. In addition two different molar ratios of apolipoprotein/phospholipid (PC) (1:50 and 1:310) were chosen at a constant molar ratio of total cholesterol/PC of 1:20. The various substrates were incubated with purified LCAT enzyme. DMPC - or egg yolk phosphatidylcholine - cholesterol-[4-14C]cholesterol-serum amyloid A complexes per se did not stimulate LCAT activity significantly. However, apo serum amyloid A incorporated together with apo A-I by a detergent dialysis procedure lead at low concentrations of serum amyloid A to a marked increase in cholesteryl ester formation as compared to apo A-I alone but inhibited the cholesteryl ester formation at high concentrations. Thus, the low levels of esterified cholesterol in acute phase plasma are to some extent due to decreased plasma enzyme activity and in part may be due to interference of apo serum amyloid A with the natural substrate complexes of plasma HDL.

Lipoprotein(a) in Health and Disease

Lipoprotein(a) [Lp(a)] represents an LDL-like particle to which the Lp(a)-specific apolipoprotein(a) is linked via a disulfide bridge. It has gained considerable interest as a genetically determined risk factor for atherosclerotic vascular disease. Several studies have described a correlation between elevated Lp(a) plasma levels and coronary heart disease, stroke, and peripheral atherosclerosis. In healthy individuals, Lp(a) plasma concentrations are almost exclusively controlled by the apo(a) gene locus on chromosome 6q2.6-q2.7. More than 30 alleles at this highly polymorphic gene locus determine a size polymorphism of apo(a). There exists an inverse correlation between the size (molecular weight) of apo(a) isoforms and Lp(a) plasma concentrations. The standardization of Lp(a) quantification is still an unresolved task due to the large particle size of Lp(a), the presence of two different apoproteins [apoB and apo(a)], and the large size polymorphism of apo(a) and its homology with plasminogen. A working group sponsored by the IFCC is currently establishing a stable reference standard for Lp(a) as well as a reference method for quantitative analysis. Aside from genetic reasons, abnormal Lp(a) plasma concentrations are observed as secondary to various diseases. Lp(a) plasma levels are elevated over controls in patients with nephrotic syndrome and patients with end-stage renal disease. Following renal transplantation, Lp(a) concentrations decrease to values observed in controls matched for apo(a) type. Controversial data on Lp(a) in diabetes mellitus result mainly from insufficient sample sizes of numerous studies. Large studies and those including apo(a) phenotype analysis came to the conclusion that Lp(a) levels are not or only moderately elevated in insulin-dependent patients. In noninsulin-dependent diabetics, Lp(a) is not elevated. Conflicting data also exist from studies in patients with familial hypercholesterolemia. Several case-control studies reported elevated Lp(a) levels in those patients, suggesting a role of the LDL-receptor pathway for degradation of Lp(a). However, recent turnover studies rejected that concept. Moreover, family studies also revealed data arguing against an influence of the LDL receptor for Lp(a) concentrations. Several rare diseases or disorders, such as LCAT- and LPL-deficiency as well as liver diseases, are associated with low plasma levels or lack of Lp(a).

Differential distribution of apolipoprotein E isoforms in human plasma lipoproteins.

The polymorphism of apolipoprotein E (apo E) accounts for a substantial amount of the genetic variance of cholesterol levels In man. The e-2 allele lowers and the e-4 allele raises plasma and low density llpoproteln cholesterol levels as compared to the e-3 allele. Whereas the lower cholesterol levels in carriers of the e-2 allele can, at least In part, be attributed to the grossly deficient binding of apo E-2 to the apo B,E receptor, apo E-3 and E-4 bind to the same degree. We used gel filtration and uttracentrlfugatlon to separate lipoproteins and subsequent Immunoblottlng analysis to study the apo E Isoform distribution. We always found lipoproteins of lower density relatively enriched in apo E-4 and high density lipoproteins relatively depleted of apo E-4 as compared to apo E-3. This was also seen In plasma of heterozygous subjects that simultaneously express two apo E Isoforms. Also, the apo E-A-ll complex was directly shown by Immunoblottlng. Furthermore, when purified lodlnated apo E was incubated with plasma In vitro, apo E-4 also reassociated more with lipoproteins of lower density than apo E-3. We conclude that apo E-3 and apo E-4 have a different llpoprotein particle distribution in vivo. This differential llpoproteln distribution may account for differences In the metabolism between apo E-3 and E-4.

Multicenter Comparison of Micronized Fenofibrate and Simvastatin in Patients with Primary Type IIA or IIB Hyperlipoproteinemia

In 12 weeks of active treatment, we compared the efficacy and safety of a new (micronized) formulation of fenofibrate (F) (200 mg/day) with that of simvastatin (S) (20 mg/day), an inhibitor of hydroxy-methyl-glutaryl coenzyme A (HMG-CoA)-reductase. Men and women with primary hyperlipoproteinemia (HLP) with low-density lipoprotein (LDL) cholesterol level 180-300 mg/dl and triglyceride level < 500 mg/dl had dietary treatment for 8 weeks, and 133 (2 of 3 type IIa, 1 of 3 type IIb HLP) were randomized. The decrease in total cholesterol differed between type IIa patients (F - 17.9 vs. S - 25.8%), the decrease in triglyceride levels between the type II b groups (F - 52.8 vs. S - 14%), whereas the degree of decrease in LDL cholesterol (F - 20.9 vs. S - 34.9%) differed among all patients. Despite the difference in LDL cholesterol decrease, no difference was noted in total apolipoprotein (apo) B lowering (F - 20.8 and S - 26.5%). Increases in high-density lipoprotein (HDL) cholesterol (F + 18.5 vs. S + 15%) differed specifically in type IIb patients (F + 33.6 vs. S + 11.4%), accompanied by a more pronounced increase in apo AI with fenofibrate (F + 10.5% vs. S no change). Improvement in the ratios of total cholesterol/HDL cholesterol and apo AI/apo B occurred similarly with both drugs. Only fenofibrate, not simvastatin, decreased both fibrinogen (-10.3 vs. + 3.6%) and uric acid (-25% vs. no change) in type IIa and type IIb patients. Safety parameters reflected drug-specific known side effects, underscoring the safety of both drugs in addition to their efficacy in lipid lowering. Besides its advantages in type IIb hyperlipidemia, micronized fenofibrate proved a potent drug in decreasing total and LDL cholesterol and in very effectively decreasing apo B-containing lipoproteins, which is a recommendation for its use in primary hypercholesterolemia.

An angiotensin-converting enzyme gene variant is associated with acute myocardial infarction in women but not in men

We believe our data may speak to the issue of sexual dimorphism with respect to MI. Most studies have concentrated on men with this disease. In a recent study, Lindpaintner et al10 could find no relationship between the D/D genotype and AMI in subjects of the Physicians Health Study. However, this study consisted entirely of men. The D allele may provide an avenue to discern differences in the pathogenesis of MI in men and women.

Changes of apolipoprotein A-IV in the human neonate: evidence for different inductions of apolipoproteins A-IV and A-I in the postpartum period

The levels, isoforms and distribution of apolipoprotein A-IV (apo A-IV) were investigated in 127 term human umbilical cord sera. In addition, apo A-IV levels and isoforms were determined on the 3rd (n = 82) and 6th (n = 68) day following parturition and compared to apo A-I concentrations. Levels of apo A-IV were low in umbilical cord serum (5.7 +/- 1.9 mg/dl) as compared to adult serum (17.6 +/- 4.8 mg/dl). No difference was found between male and female neonates. The serum distribution of apo A-IV closely resembled that seen in the adult human. Apo A-IV concentrations dramatically increased during the first week of life reaching levels of 13.4 +/- 4.1 mg/dl on day 3 and 16.7 +/- 3.4 mg/dl on day 6 post-partum. During this time apo A-I levels did not change significantly (81.0 +/- 16.5 mg/dl in cord serum, 75.3 +/- 10.6 mg/dl and 84.2 +/- 14.5 mg/dl on day 3 and 6, respectively). Cord serum already exhibited the major serum apo A-IV isoforms seen in the adult. Isofocusing of apo A-IV also identified the known genetic polymorphism of apo A-IV. Among 127 cord sera studied we identified 109 homozygote normal patterns, apo A-IV (1-1), 16 heterozygotes, apo A-IV (1-2) and 2 individuals homozygote for the variant peptide, apo A-IV (2-2). We provide evidence that apo A-IV and apo A-I are differently induced in the human neonate during the beginning of the feeding period.(ABSTRACT TRUNCATED AT 250 WORDS)