H B Brewer

Abnormal in vivo metabolism of apolipoprotein E4 in humans.

Apolipoprotein E (apoE) is important in modulating the catabolism of remnants of triglyceride-rich lipoprotein particles. It is a polymorphic protein with the three common alleles coding for apoE2, apoE3, and apoE4. ApoE3 is considered the normal isoform, while apoE4 is associated both with hypercholesterolemia and type V hyperlipoproteinemia. We quantitated the kinetics of metabolism of apoE4 in 19 normolipidemic apoE3 homozygotes and 1 normolipidemic apoE4 homozygote, and compared this with the metabolism of apoE3 in 12 normolipidemic apoE3 homozygotes. In the apoE3 homozygous subjects, apoE4 was catabolized twice as fast as apoE3, with a mean plasma residence time of 0.37 +/- 0.01 d (+/- SEM) and 0.73 +/- 0.05 (P less than 0.001), respectively. When plasma was fractionated into the lipoprotein subclasses, the greatest amount of labeled apoE4 was present on very low density lipoproteins, while the largest fraction of labeled apoE3 was associated with high density lipoproteins. The plasma apoE concentration was decreased in an apoE4 homozygote compared with the apoE3 homozygotes (3.11 mg/dl vs. 4.83 +/- 0.35 mg/dl). The reduced apoE4 concentration was entirely due to a decreased apoE4 residence time in the apoE4 homozygote (0.36 d vs. 0.73 +/- 0.05 d for apoE3 in apoE3 homozygotes). These results indicate that apoE4 is kinetically different than apoE3, and suggest that the presence of apoE4 in hypercholesterolemic and type V hyperlipoproteinemic individuals may play an important pathophysiological role in the development of these dyslipoproteinemias.

Familial apolipoprotein E deficiency.

A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.

Plasma apolipoprotein A-1 absence associated with a marked reduction of high density lipoproteins and premature coronary artery disease.

A 45-year-old woman with corneal opacification and severe coronary artery disease was noted to have the following plasma lipid levels (mg/dl, +/- SD): total cholesterol 111 +/- 13, triglyceride 62 +/- 6, very low density lipoprotein cholesterol 4 +/- 1, low density lipoprotein cholesterol 106 +/- 14, and high density lipoprotein (HDL) cholesterol 1 +/- 1 (normal, 50 +/- 14). Her two offspring and one brother were found to have HDL cholesterol values (mg/dl) of 23, 20, and 20, respectively. The percentage of cholesterol in the esterified form in the patients plasma was normal at 70%. Lipoprotein electrophoresis showed no alpha lipoprotein band, and no HDL was detectable when plasma was subjected to analytic ultracentrifugation. Only trace amounts of lipids were noted within the HDL density region following preparative ultracentrifugation. Mean plasma apolipoprotein (apo) A-ll, apo B, and apo C-ll plasma levels were 13.8%, 130.6% and 26.6% of normal, respectively. The ratio of apo B to cholesterol within LDL was elevated. Apo A-l, the major HDL protein constituent, was immunologically undetectable in this patients plasma. A decreased HDL cholesterol concentration has been associated with premature coronary artery disease. These data indicate that plasma apo A-l absence results in a striking reduction in HDL, is associated with premature coronary artery disease, and represents a new distinct disease entity.

Coronary heart disease prevalence and other clinical features in familial high-density lipoprotein deficiency (Tangier disease).

Abstract High-density (HD) lipoprotein cholesterol levels have been inversely associated with the incidence of coronary heart disease. The clinical features were reviewed and the prevalence of clin...

Quantitation of plasma apolipoproteins in the primary and secondary prevention of coronary artery disease.

Lipids are transported in the circulation by lipoproteins, which consist of lipids (cholesterol, triglycerides, and phospholipids) and proteins (called apolipoproteins). Apolipoproteins have many physiologic functions in lipoprotein metabolism, acting as structural proteins for lipoprotein particles, cofactors for enzymes, and ligands for cell-surface receptors. Table 1 summarizes the major apolipoproteins and their known functions. Table 1. Major Apolipoproteins and Their Functions* Traditionally, lipoproteins have been separated on the basis of their hydrated densities; the major density classes of lipoprotein particles include chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) [1]. Figure 1 depicts the metabolism of these lipoproteins. Chylomicrons are intestinal lipoproteins that transport dietary lipids to peripheral tissues and the liver. They are triglyceride rich and contain one form of apolipoprotein B (apo B), apo B-48. The triglycerides in chylomicrons are hydrolyzed by the endothelial enzyme lipoprotein lipase, which requires apolipoprotein C-II (apo C-II) as a cofactor [2]. The resulting chylomicron remnants are removed from the circulation by the liver through a process that involves the binding of apolipoprotein E (apo E) on the chylomicron remnants to a putative hepatic remnant receptor (or apo E receptor) [3]. Very low-density lipoproteins are triglyceride-rich lipoproteins secreted by the liver and contain another form of apo B, apo B-100. These triglycerides are also hydrolyzed by lipoprotein lipase, with conversion to the more dense VLDL remnants, or intermediate-density lipoprotein. Some VLDL remnants are removed from the circulation by the liver through an apo E-mediated process, but others are further hydrolyzed by the endothelial enzyme hepatic lipase, ultimately resulting in conversion to LDL. Low-density lipoprotein transports cholesterol ester to various peripheral tissues, but a substantial amount of LDL is eventually removed from the circulation by the liver when apo B-100 is bound to the hepatic LDL receptor [4]. Low-density lipoprotein can undergo oxidative modification, producing a form of oxidized LDL that can cause cholesterol loading in cells [5]. Figure 1. Schematic diagram of lipoprotein metabolism. High-density lipoproteins are synthesized and secreted by the intestine and the liver and also are generated by hydrolysis of triglyceride-rich lipoproteins [6, 7]. The major apolipoproteins in HDL are apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) (Table 1). High-density lipoprotein stimulates the efflux from cells of unesterified cholesterol, which is then converted to the esterified form by lecithin-cholesterol acyltransferase Figure 1, a plasma enzyme activated primarily by apo A-I [8]. As small, dense HDL3 accumulates cholesteryl ester, it is transformed into larger, less dense HDL2. High-density lipoprotein cholesteryl ester can be transferred to apo B-containing lipoproteins by the cholesteryl ester transfer protein [9]. This may be one important route of human reverse cholesterol transport [10]. High-density lipoprotein is a substrate for hepatic lipase, which hydrolyzes HDL phospholipids and triglycerides, creating smaller HDL particles [7] (Figure 1). Plasma Lipoproteins and Coronary Artery Disease The risk for premature atherosclerotic coronary artery disease is directly correlated with plasma concentrations of LDL cholesterol [11, 12] and inversely correlated with levels of HDL cholesterol (reviewed in reference 13). The independent association of plasma triglycerides with coronary artery disease risk is less certain [14, 15]. Interventions designed to decrease plasma LDL concentrations are effective in the primary prevention of coronary artery disease [16-18]. Lowering plasma LDL is also very effective in secondary prevention of coronary artery disease (reviewed in [19]), decreasing overall mortality rate [20], decreasing cardiovascular events [21-25], and producing an objective regression of atherosclerotic disease [22-26]. Although one primary prevention trial suggested an independent benefit of increasing plasma HDL cholesterol concentrations [18], no clinical trials have been done specifically to evaluate the effect of selectively increasing HDL in primary or secondary prevention of coronary artery disease. Despite the association of plasma lipid levels with coronary artery disease risk, many patients with premature coronary artery disease do not have very high levels of LDL cholesterol or very depressed HDL cholesterol concentrations. Therefore, investigators continue to search for other clinical markers that will allow better prediction of coronary artery disease risk and can be used to guide therapeutic decisions to prevent or treat coronary artery disease. Quantitation of plasma apolipoproteins was proposed as one such clinical tool. In this review, we assess evidence regarding the clinical utility of apolipoprotein quantitation and review the use of plasma apolipoprotein concentrations in the primary and secondary prevention of coronary artery disease. We focus primarily on the apolipoproteins for which the most data and the most clinical evidence exist that are relevant to coronary artery disease: apo A-I, apo B, and lipoprotein(a) (Lp[a]). For each of these apolipoproteins, we address the question of whether quantitation of the apolipoprotein enhances the ability to predict coronary artery disease risk in healthy persons or recurrent events in patients with established coronary artery disease, and we suggest how knowledge of the plasma apolipoprotein concentration might influence clinical management. We retrieved 82 articles from the English-language literature for the years 1975 to 1993 using MEDLINE (key words: apolipoproteins, quantitation, and coronary artery disease) and review of article bibliographies. We examined all retrospective and prospective studies of apolipoprotein quantitation that used some measure of coronary artery disease as a criterion for patient selection, including acute myocardial infarction, classic angina pectoris, and angiographic evidence of severe coronary artery disease [22, 23]. Many of the studies were designed to address the predictive value of the test for the development of coronary artery disease; relatively few studies assessed the predictive value of the test for recurrent events in patients with established coronary artery disease. We found 71 retrospective cross-sectional studies, including 7 in children and adolescents, and 11 prospective studies. For each apolipoprotein, we discuss the retrospective studies as a group and specific studies where appropriate; each of the studies in children and each prospective study are discussed individually. More than 90% of studies were done in men, and therefore we cannot generalize results to women. In addition, because assays for apolipoprotein quantitation have not been standardized, we included a section addressing some of the methodologic issues in apolipoprotein quantitation. Issues regarding Assay Methods and Standardization The lack of standardization and reference methods for apolipoprotein assays is a limitation to the general application of apolipoprotein quantitation in clinical practice [27-31]. Variation in apolipoprotein measurements among laboratories can be substantial. A collaborative study initiated by the International Federation of Clinical Chemistry evaluated differences in apo A-I and apo B quantitation among 28 laboratories, 25 of which were company laboratories [29]. The overall interlaboratory coefficient of variation was 7% for apo A-I and 19% for apo B. After uniform calibration of assay standards, the coefficients of variation decreased to 5% and 6%, respectively. Among the factors resulting in this variation are preanalytical factors such as differences in sampling and storage conditions [32]. In addition, matrix effects (additives, stabilization processes) on the immunoreactivity of standards also can be a source of bias in apolipoprotein quantitation [33]. Assays for apo A-I and apo B are widely available and are frequently done by commercial laboratories. These laboratories use methods that often result in good intralaboratory reproducibility, with coefficients of variation within laboratories that are generally less than 4% [29]. However, some of these assays may be subject to interference by high plasma triglyceride levels [27]. Therefore, apo A-I and apo B measurements in patients with very high hypertriglyceride levels (>4.5 mmol/L [400 mg/dL]) should be interpreted with caution. Several commercially available Lp(a) assay kits are used by research laboratories for Lp(a) quantitation. However, an Lp(a) assay has yet to be approved by the Food and Drug Administration for clinical use. As in the case of apo A-I and apo B, there has been no standardization of Lp(a) assays [34, 35]. Lipoprotein(a) is an acute-phase reactant [36] and should not be quantitated within several weeks after an acute illness or surgical procedure. Apolipoprotein A-I Background Apolipoprotein A-I is the major apolipoprotein in HDL and serves various structural and functional roles in HDL metabolism (reviewed in reference 6). It is probably important in protecting against premature atherosclerosis. Genetic defects that cause the inability to synthesize apo A-I cause very low plasma concentrations of HDL cholesterol and premature coronary artery disease in the fourth and fifth decades [37-40]. Conversely, an increased rate of apo A-I production causes high plasma levels of HDL cholesterol and may be associated with protection from premature coronary artery disease based on familial longevity [41]. Furthermore, overexpression of human apo A-I in transgenic mice inhibits the development of atherosclerosis [42]. Cross-Sectional Studies Many retrospective

Human Apolipoprotein A-I and A-II Metabolism

The kinetics of the major apolipoproteins (apo) of plasma high density lipoproteins (HDL), apoA-I and apoA-II, were examined in a total of 44 individual tracer studies in 22 normal male and female subjects. Following the intravenous injection of radioiodinated HDL, the specific radioactivity decay of apoA-I within HDL (residence time, 5.07 +/- 1.53 days), as determined by column chromatography, was significantly (P < 0.01) faster than that of apoA-II (residence time, 5.96 +/- 1.84 days). The specific radioactivity decay of apoA-I within HDL when labeled on HDL or as apoA-I was found to be almost identical. Similar results were obtained for apoA-II. Analysis of simultaneous paired radiolabeled apoA-I and apoA-II studies revealed that the mean apoA-I plasma residence time (4.46 +/- 1.04 days) was significantly (P < 0.01) shorter than that for apoA-II (4.97 +/- 1.06 days). Females had significantly (P < 0.01) higher apoA-I plasma concentrations (124 +/- 24 mg/dl) and apoA-I synthesis rates (13.58 +/- 2.23 mg/kg. day) than did males (108 +/- 16 mg/dl, and 11.12 +/- 1.92 mg/kg. day, respectively). Plasma apoA-I levels were correlated with plasma apoA-I residence times, but not synthesis rates; and apoA-II concentrations were correlated only with apoA-II whole body residence times. ApoA-I and apoA-II plasma residence times were inversely correlated with plasma triglyceride levels. These data are consistent with the following concepts: 1) labeling of apoA-I and apoA-II as apolipoproteins or on HDL does not affect their specific radioactivity decay within HDL; 2) the mean residence time of apoA-I both in plasma and in HDL is significantly shorter than that of apoA-II; 3) the increased apoA-I levels seen in female subjects are due to increased apoA-I synthesis; and 4) the plasma apoA-I residence time, which is inversely correlated with plasma triglyceride levels, is an important determinant of apoA-I concentration in both males and females.-Schaefer, E. J., L. A. Zech, L. L. Jenkins, T. J. Bronzert, E. A. Rubalcaba, F. T. Lindgren, R. L. Aamodt, and H. B. Brewer, Jr. Human apolipoprotein A-I and A-II metabolism.

The inverse association of plasma lipoprotein(a) concentrations with apolipoprotein(a) isoform size is not due to differences in Lp(a) catabolism but to differences in production rate.

Lipoprotein(a) (Lp[a]) is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL) but contains an additional protein called apolipoprotein(a) (apo[a]). Apo(a) is highly polymorphic in size, and there is a strong inverse association between the size of the apo(a) isoform and the plasma concentration of Lp(a). We directly compared the in vivo catabolism of Lp(a) particles containing different size apo(a) isoforms to establish whether there is an effect of apo(a) isoform size on the catabolic rate of Lp(a). In the first series of studies, four normal subjects were injected with radio-labeled S1-Lp(a) and S2-Lp(a) and another four subjects were injected with radiolabeled S2-Lp(a) and S4-Lp(a). No significant differences in fractional catabolic rate were found between Lp(a) particles containing different apo(a) isoforms. To confirm that apo(a) isoform size does not influence the rate of Lp(a) catabolism, three subjects heterozygous for apo(a) were selected for preparative isolation of both Lp(a) particles. The first was a B/S3-apo(a) subject, the second a S4/S6-apo(a) subject, and the third an F/S3-apo(a) subject. From each subject, both Lp(a) particles were preparatively isolated, radiolabeled, and injected into donor subjects and normal volunteers. In all cases, the catabolic rates of the two forms of Lp(a) were not significantly different. In contrast, the allele-specific apo(a) production rates were more than twice as great for the smaller apo(a) isoforms than for the larger apo(a) isoforms. In a total of 17 studies directly comparing Lp(a) particles of different apo(a) isoform size, the mean fractional catabolic rate of the Lp(a) with smaller size apo(a) was 0.329 +/- 0.090 day-1 and of the Lp(a) with the larger size apo(a) 0.306 +/- 0.079 day-1, not significantly different. In summary, the inverse association of plasma Lp(a) concentrations with apo(a) isoform size is not due to differences in the catabolic rates of Lp(a) but rather to differences in Lp(a) production rates.

Variation in lipoprotein(a) concentrations among individuals with the same apolipoprotein (a) isoform is determined by the rate of lipoprotein(a) production.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to, but metabolically distinct from, LDL. Factors regulating plasma concentrations of Lp(a) are poorly understood. Apo(a), the protein that distinguishes Lp(a) from LDL, is highly polymorphic, and apo(a) size is inversely correlated with plasma Lp(a) level. Even within the same apo(a) isoform class, however, plasma Lp(a) concentrations vary widely. A series of in vivo kinetic studies were performed using purified radiolabeled Lp(a) in individuals with the same apo(a) isoform but different Lp(a) levels. In a group of seven subjects with a single S4-apo(a) isoform and Lp(a) levels ranging from 1 to 13.2 mg/dl, the fractional catabolic rate (FCR) of 131I-labeled S2-Lp(a) (mean 0.328 day-1) was not correlated with the plasma Lp(a) level (r = -0.346, P = 0.45). In two S4-apo(a) subjects with a 10-fold difference in Lp(a) level, the FCRs of 125I-labeled S4-Lp(a) were very similar in both subjects and not substantially different from the FCRs of 131I-S2-Lp(a) in the same subjects. In four subjects with a single S2-apo(a) isoform and Lp(a) levels ranging from 9.4 to 91 mg/dl, Lp(a) concentration was highly correlated with Lp(a) production rate (r = 0.993, P = 0.007), but poorly correlated with Lp(a) FCR (mean 0.304 day-1). Analysis of Lp(a) kinetic parameters in all 11 subjects revealed no significant correlation of Lp(a) level with Lp(a) FCR (r = -0.53, P = 0.09) and a strong correlation with Lp(a) production rate (r = 0.99, P < 0.0001). We conclude that the substantial variation in Lp(a) levels among individuals with the same apo(a) phenotype is caused primarily by differences in Lp(a) production rate.

Elevated plasma lipoprotein(a) in patients with the nephrotic syndrome.

Lipoprotein(a) (Lp[a]) is a plasma lipoprotein composed of lipids and two major protein components, apolipoprotein B ([apo]B) and apo(a) [1, 2]. Plasma concentrations of Lp(a) have been correlated with risk for atherosclerotic vascular disease [3, 4]. The distribution of plasma Lp(a) levels is highly skewed toward lower concentrations, with more than two thirds of the population having levels lower than 20 mg/dL. Apolipoprotein(a) exhibits a striking size polymorphism [1], with the apo(a) isoproteins ranging in approximate size from 420 kd to 840 kd [5]. The apo(a) isoforms are heritable in an autosomal codominant fashion and Lp(a) levels are under strong genetic control [6]. One important factor in determining plasma Lp(a) concentration is the apo(a) isoprotein phenotype itself, with an inverse correlation between the size of the apo(a) isoprotein and the plasma Lp(a) concentration [1]. Lipoprotein(a) concentrations also vary substantially within each apo(a) isoform class, due largely to differences in the rate of production [7]. However, the genetic and metabolic factors that modulate Lp(a) concentrations remain poorly understood. End-stage renal disease has been associated with elevations in plasma Lp(a) levels. Analysis of patients treated with long-term hemodialysis revealed that Lp(a) levels were approximately three times higher than those of matched healthy controls [8, 9]. However, studies of Lp(a) concentration in patients with end-stage renal disease that control for apo(a) isoform have not been reported. Patients with proteinuria have also been shown to have increased Lp(a) levels [10, 11]; however, the proteinuria was relatively low grade and the patients were not classically nephrotic. Therefore, we undertook a comprehensive evaluation of Lp(a) concentrations in a series of 62 patients with classic nephrotic syndrome, controlling for apo(a) isoform, and compared the results with data obtained in a healthy control group. Methods A total of 62 patients (26 women, 36 men; mean age, 47.2 2.2; age range, 18 to 76 years) with the nephrotic syndrome were studied. The patients were consecutively entered at one clinic (University Clinic, Freiburg, Germany) from 1990 to 1992. All patients had severe proteinuria, low serum albumin, hyperlipoproteinemia, and edema. Patients were divided into two subgroups: those with primary kidney disease (n = 47) and those with diabetic nephropathy (n = 15). The histologic findings of patients with primary kidney disease as determined by renal biopsy were membranous glomerulonephritis (n = 22), focal and segmental glomerulosclerosis (n = 13), minimal change disease (n = 7), mesangiocapillary glomerulonephritis (n = 1), and immunotactoid glomerulonephritis (n = 1). In three patients with primary glomerulonephritis tissue, a diagnosis could not be obtained. Patients with diabetic glomerulopathy were diagnosed as having diabetic nephropathy according to the duration of underlying type I (n = 4) or type II (n = 11) diabetes mellitus and the presence of retinopathy. Patients with diabetic nephropathy were all being treated with pharmacologic therapy with an angiotensin-converting inhibitor at the time they were sampled. Ninety-one healthy members of the staff of the National Institutes of Health acted as controls and were studied in 1991. Thirteen patients with primary kidney disease were resampled 6.7 0.7 months after remission of the nephrotic syndrome was induced by immunosuppressive therapy. Four patients had normal Lp(a) levels, and nine exhibited high levels (> 30 mg/dL) before remission. A second follow-up was performed in six of the nine patients with high Lp(a) 13.1 0.5 months after they achieved remission. Drugs used for induction of remission were cyclosporine/prednisone (n = 4), chlorambucil/prednisone (n = 6), prednisone monotherapy (n = 1), and acetylsalicylic acid/prednisone (n = 1). One patient had a spontaneous remission. Blood sampling was done when patients were free of steroids and chlorambucil, but four patients still were being treated with cyclosporine monotherapy. Diabetic patients were followed for a total period of 19 2.3 months. Ten patients developed end-stage renal disease. Because two patients died of myocardial infarction and one had no detectable Lp(a) levels, serum was analyzed in seven patients 9.8 0.9 months after initiation of hemodialysis (n = 5) or peritoneal dialysis (n = 2). The remaining five patients are still nephrotic. Blood samples were collected after an overnight fast of at least 12 hours. All chemistry, lipid, and apolipoprotein measurements were performed on fresh samples. At least two 24-hour urine specimens were collected and subjected to gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% to 25%) (Phast System, Pharmacia; Uppsala, Sweden) to determine selectivity of protein excretion. Lipid and Apolipoprotein Analysis Very-low-density lipoprotein was isolated by ultracentrifugation [12]. Cholesterol and triglycerides were determined enzymatically (Boehringer-Mannheim, Mannheim, Germany). Apolipoprotein B (apoB) concentrations were determined by rate nephelometry on the Array nephelometer (Beckman; Palo Alto, California). Lyophilized control serum was used as a calibrator as previously described [13]. Lipoprotein(a) concentrations in the nephrotic patients were determined by two independent methods. A commercial two-site immunoradiometric assay (Pharmacia) was performed on fresh samples from the nephrotic patients. Lipoprotein(a) was remeasured in the same samples stored at 20C for less than 1 year by a differential enzyme-linked immunosorbent assay (ELISA) based on the method of Fless and colleagues [14]. A monoclonal antibody against apo(a) (2-D1, Cappel; Durham, North Carolina) was used to coat microtiter plates at a concentration of 10 g/mL. This antibody recognizes all sizes of apo(a) (on immunoblotting) and does not cross-react with plasminogen. After blocking with 2% bovine serum albumin, plasma samples at a 1:5000 dilution were added to wells and incubated for 60 minutes at 37 C. A sheep, polyclonal anti-apoB (Biodesign; Kennebunkport, Maine) labeled with horseradish peroxidase was added to the wells at a 1:2000 dilution and incubated for 60 minutes. Substrate was then added and absorbance read at 450 nm. The standard was a secondary plasma standard calibrated against two commercial standards (Terumo; Elkton, Maryland; Immuno; Vienna, Austria). Two controls were run with each assay. Intra-assay and interassay coefficents of variation were less than 3% and less than 10%, respectively. Between the two assays (ELISA and radioimmunoassay), a good correlation (r = 0.884) was found. Lipoprotein(a) concentrations in control subjects were measured using only the differential ELISA. The values reported here are those from the ELISA. Apoprotein(a) Isotyping Apoprotein(a) isoform determination was done on plasma using a modification of an immunoblotting technique, as previously described [15]. Briefly, plasma samples were delipidated twice in chloroform-methanol 8:5 (vol/vol) and washed twice with phosphate-buffered saline. The samples were reduced with 100 mmol/L dithiothreitol in 8 mol/L urea and incubated at 37 C for 30 minutes. The samples were solubilized in a solution containing 75% glycerol, 0.5% bromophenol blue, and 10% sodium dodecyl sulfate and applied to 7.5% polyacrylamide gel electrophoresis with 0.1% crosslinker. Gels were run for approximately 2.5 hours at 60 mA. Subsequent immunoblotting was carried out as described previously [16]. After transfer of the proteins to Immobilon polyvinylidene difluoride transfer membrane (Millipore; Bedford, Massachusetts), incubation of the membranes with antibodies was performed using an anti-apo(a) monoclonal antibody (2-D1, 1:2000; Cappel) as first antibody and the Vectastain ABC anti-mouse test kit (Vector Laboratories; Burlingame, California) for detection. Several plasma samples of a defined apo(a) isoform were used as standards on each gel. One nephrotic patient had no detectable apo(a) band, 30 patients had only one apo(a) band, and 29 patients had two apo(a) bands. In both patients and controls with two apo(a) bands, one band was usually of substantially greater intensity. For purposes of statistical analysis, these patients and controls were assigned a single apo(a) isotype based on the apo(a) band with the greater immunostaining. This dominant band accounts for the majority of Lp(a) in plasma. Statistical Analysis Data are given as mean SE. Data analysis was performed using the Statistical Analysis System software package (SAS Institute; Cary, North Carolina) using the Wilcoxon rank-sum tests or the Student t-test for group comparisons of continuous variables with non-normal or normal population distributions, respectively. Point estimates and confidence interval estimates were calculated by nonparametric procedures [17, 18]. P values less than 0.05 were considered to be statistically significant. Results The clinical and laboratory characteristics of the nephrotic patients are shown in Table 1. Patients with primary kidney disease were younger, had lower body mass indices, and had lower serum creatinine and serum albumin values than did patients with diabetic nephropathy (P < 0.05 for all values). The lipid and apolipoprotein data for the nephrotic patients are shown in Table 2 and compared with those of the control sample (n = 91). Total cholesterol, low-density lipoprotein cholesterol, triglyceride, and apoB concentrations were elevated in both groups of patients compared with healthy controls (P < 0.05). Lipoprotein(a) levels were also elevated in the nephrotic patients compared with the control group (P < 0.01). Sixty percent of the nephrotic patients had Lp(a) levels greater than 30 mg/dL, whereas only 18% of the controls had levels in this range. The percentage of elevated Lp(a) levels were identical: >30 mg/dL among patients with primary kidney

PLASMA-TRIGLYCERIDES IN REGULATION OF H.D.L.-CHOLESTEROL LEVELS

Plasma-high-density-lipoprotein (H.D.L.) cholesterol concentrations are lower in patients with coronary-artery disease than in control subjects. In an investigation of the relationship of H.D.L. cholesterol to other lipid and lipoprotein parameters in normal and hyperlipoproteinaemic subjects inverse correlations were found between H.D.L. cholesterol and very-low-density-lipoprotein (V.L.D.L.) cholesterol, and between H.D.L. cholesterol and plasma-triglyceride levels. Mean H.D.L.-cholesterol concentrations in normal subjects were 50 mg/dl, and in hyperlipoproteinaemic patients they were: type I, 17 mg/dl; type II, 44 mg/dl; type III, 38 mg/dl; type IV, 37 mg/dl; and type V, 27 mg/dl. H.D.L.-cholesterol levels were lowest in patients with fasting chylomicronaemia and were diminished in hypertriglyceridaemic subjects, suggesting a relationship between the metabolism of triglyceride-rich lipoproteins and H.D.L.