Radioimmunoassay studies of human apolipoprotein E.

This report describes the development and first applications of a sensitive and specific double antibody radioimmunoassay for human apoplipoprotein E (apoE). ApoE was purified from the very low density lipoproteins of hypertriglyceridemic patients by heparin-agarose affinity chromatography, DEAE-cellulose chromatography, and preparative polyacrylamide gel electrophoresis. The purified apoprotein had an amino acid composition characteristic of apoE and resulted in the production of monospecific antisera when injected into rabbits. The radioimmunoassay, which was carried out in the presence of 5 mM sodium decyl sulfate, had a working range of 0.8-12 ng. The withinassay coefficient of variation was 9% and the coefficient of variation for systematic between-assay variability was 3%. Prior delipidation of samples with organic solvents did not alter their immunoreactivity. In 26 normal volunteers, the mean plasma apoE concentration was 36 +/- 13 microgram/ml. Hyperlipidemic patients (n = 68) had higher mean apoE levels. A single patient with type III hyperlipoproteinemia had a plasma apoE level of 664 microgram/ml. The plasma apoE level was independently related to plasma cholesterol and triglyceride levels in a population of 108 normal and nonchylomicronemic hyperlipidemic patients. The multiple correlation coefficient for this relationship was 0.73. Thus, variation in plasma cholesterol and triglyceride concentrations described 53% of the variation in apoE concentrations in this population. The lipoprotein distribution of apoE was investigated by agarose column chromatography and ultracentrifugation of plasma. Agarose column chromatography demonstrated that all or nearly all plasma apoE is associated with lipoproteins. In plasma from normal volunteers and hypercholesterolemic patients, apoE was found in two discrete lipoprotein classes: very low density lipoproteins and a set of lipoprotein particles with size and density characteristics similar to HDL2. In hypertriglyceridemic patients, nearly all apoE was associated with the triglyceride-rich lipoproteins.

Association of plasma lipoproteins with postheparin lipase activities.

Studies were designed to explore the association of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities with lipoproteins in human postheparin plasma (PHP). The major peak of LPL activity after gel filtration of PHP eluted after the triglyceride-rich lipoproteins and just before the peak of low density lipoprotein (LDL) cholesterol. When PHP contained chylomicrons, an additional peak of LPL activity eluted in the void volume of the column. Most HTGL activity eluted after the LDL and preceded the elution of high density lipoprotein cholesterol. LPL activity in preheparin plasma eluted in the same position, relative to lipoproteins, as did LPL in PHP. Gel filtration of purified human milk LPL mixed with plasma or isolated LDL produced a peak of activity eluting before LDL. During gel filtration of PHP in high salt buffer (1 M NaCl) or after isolation of lipoproteins by ultracentrifugation in high salt density solutions, most of the lipase activity was not associated with lipoproteins. LPL activity was removed from PHP by elution through immunoaffinity columns containing antibodies to apolipoprotein (apo) B and apo E. Since lipoproteins in PHP have undergone prior in vivo lipolysis, LPL activity in PHP may be bound to remnants of chylomicrons and very low density lipoproteins.

Identification of a new structural variant of human apolipoprotein E, E2(Lys146 leads to Gln), in a type III hyperlipoproteinemic subject with the E3/2 phenotype.

A type III hyperlipoproteinemic subject having the apolipoprotein E (apo E) phenotype E3/2 was identified. From isoelectric focusing experiments in conjunction with cysteamine treatment (a method that measures cysteine content in apo E), the E2 isoform of this subject was determined to have only one cysteine residue, in contrast to all previously studied E2 apoproteins, which had two cysteines. This single cysteine was shown to be at residue 112, the same site at which it occurs in apo E3. From amino acid and sequence analyses, it was determined that this apo E2 differed from apo E3 by the occurrence of glutamine rather than lysine at residue 146. When phospholipid X protein recombinants of the subject's isolated E3 and E2 isoforms were tested for their ability to bind to the human fibroblast apo-B,E receptor, it was found that the E3 bound normally (compared with an apo E3 control) but that the E2 had defective binding (approximately 40% of normal). Although they contained E3 as well as E2, the beta-very low density lipoproteins (beta-VLDL) from this subject were very similar in character to the beta-VLDL from an E2/2 type III hyperlipoproteinemic subject; similar subfractions could be obtained from each subject and were shown to have a similar ability to stimulate cholesteryl ester accumulation in mouse peritoneal macrophages. The new apo E2 variant has also been detected in a second type III hyperlipoproteinemic subject.

Human intestinal lipoproteins. Studies in chyluric subjects.

To explore the role of the human intestine as a source of apolipoproteins, we have studied intestinal lipoproteins and apoprotein secretion in two subjects with chyluria (mesenteric lymphatic-urinary fistulae). After oral corn oil, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) output in urine increased in parallel to urinary triglyceride. One subject, on two occasions, after 40 g of corn oil, excreted 8.4 and 8.6 g of triglyceride together with 196 and 199 mg apoA-I and on one occasion, 56 mg apoA-II. The other subject, after 40 g corn oil, excreted 0.3 g triglyceride and 17.5 mg apoA-I, and, after 100 g of corn oil, excreted 44.8 mg apoA-I and 5.8 mg apoA-II. 14.5+/-2.1% of apoA-I and 17.7+/-4.3% of apoA-II in chylous urine was in the d < 1.006 fraction (chylomicrons and very low density lipoprotein). Calculations based on the amount of apoA-I and apoA-II excreted on triglyceride-rich lipoproteins revealed that for these lipid loads, intestinal secretion could account for 50 and 33% of the calculated daily synthetic rate of apoA-I and apoA-II, respectively. Similarly, subject 2 excreted 48-70% and 14% of the calculated daily synthetic rate of apoA-I and apoA-II, respectively. Chylous urine contained chylomicrons, very low density lipoproteins and high density lipoproteins, all of which contained apoA-I. Chylomicrons and very low density lipoproteins contained a previously unreported human apoprotein of 46,000 mol wt. We have called this apoprotein apoA-IV because of the similarity of its molecular weight and amino acid composition to rat apoA-IV. In sodium dodecyl sulfate gels, chylomicron apoproteins consisted of apoB 3.4+/-0.7%, apoA-IV 10.0+/-3.3%, apoE 4.4+/-0.3%, apoA-I 15.0+/-1.8%, and apoC and apoA-II 43.3+/-11.3%. Very low density lipoprotein contained more apoB and apoA-IV and less apoC than chylomicrons. Ouchterlony immunodiffusion of chylomicron apoproteins revealed the presence of apoC-I, apoC-II, and apoC-III. In contrast, plasma chylomicrons isolated during a nonchyluric phase revealed a markedly altered chylomicron apoprotein pattern when compared with urinary chylomicrons. The major apoproteins in plasma chylomicrons were apoB, apoE, and the C peptides: no apoA-I or apoA-IV were present in sodium dodecyl sulfate gels indicating that major changes in chylomicron apoproteins occur during chylomicron metabolism. When incubated in vitro with plasma, urinary chylomicrons lost apoA-I and apoA-IV and gained apoE and apoC. Loss of apoA-I and apoA-IV was dependent upon the concentration of high density lipoproteins in the incubation mixture. These studies demonstrate that the human intestine secretes significant amounts of apoA-I and apoA-II during lipid absorption. Subsequent transfer of apoproteins from triglyceride-rich lipoproteins to other plasma lipoproteins may represent a mechanism whereby the intestine contributes to plasma apoprotein levels.

Role of apolipoprotein E-containing lipoproteins in abetalipoproteinemia.

Detailed studies of apolipoprotein E (apoE)-containing lipoproteins in abetalipoproteinemia have been performed in an attempt to resolve the apparent paradox of a suppressed low density lipoprotein (LDL) receptor pathway in the absence of apoB-containing lipoproteins. It was hypothesized that apoE-containing high density lipoproteins (HDL) in abetalipoproteinemia might functionally substitute for LDL in regulation of cholesterol metabolism in these patients. The mean (+/-standard deviation) plasma concentration of apoE in nine patients with abetalipoproteinemia was 44.8+/-8.2 mug/ml, slightly higher than the corresponding value for a group of 50 normal volunteers, 36.3+/-11 mug/ml. Fractionation of plasma lipoproteins by agarose column chromatography or by ultracentrifugation indicated that in abetalipoproteinemia, plasma apoE was restricted to a subfraction of HDL. This was in contrast to the results obtained with plasma from 30 normal volunteers, in whom apoE was distributed between very low density lipoproteins (VLDL) and HDL. Consequently, the mean apoE content of HDL in abetalipoproteinemia (44.8 mug/ml) was more than twice that found in the normal volunteers (20.3 mug/ml).ApoE-rich and apoE-poor subfractions of HDL(2) were isolated by heparin-agarose affinity chromatography. ApoE comprised a mean of 81% of the protein mass of the apoE-rich subfraction. Compared with the apoE-poor subfraction, the apoE-rich HDL(2) was of larger mean particle diameter (141+/-7 vs. 115+/-15 A) and had a higher ratio of total cholesterol/protein (1.01+/-0.11 vs. 0.63+/-0.14). Plasma and HDL fractions from three patients were studied with respect to their ability to compete with (125)I-LDL in specific binding to receptors on cultured human fibroblasts. The binding activity of plasma from patients (per milligram of protein) was about half that of plasma from normal volunteers. All binding activity in the patients' plasma was found to reside in the HDL fraction. The binding activity of the patients' HDL (on a total protein basis) was intermediate between that of normal HDL and normal LDL. However, the large differences in binding between patients' HDL and normal HDL entirely disappeared when data were expressed in terms of the apoE content of these lipoproteins. This suggested that the binding activity was restricted to that subfraction of HDL particles that contain apoE. These apoE-rich HDL particles had calculated binding potencies per milligram of protein 10-25 times that of normal LDL. Direct binding studies using (125)I-apoE-rich HDL(2) and (125)I-apoE-poor HDL(2), confirmed the suggestion that binding is restricted to the subfraction of HDL particles containing apoE. The apoE-rich HDL(2) were found to be very potent inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in cultured fibroblasts, providing direct evidence of the ability of these lipoproteins to regulate cholesterol metabolism. On the basis of binding potencies of apoE-rich HDL, apoE concentrations, and the composition of apoE-rich HDL, it could be calculated that apoE-rich HDL in abetalipoproteinemia have a capacity to deliver cholesterol to tissues via the LDL receptor pathway equivalent to an LDL concentration of 50-150 mg/dl of cholesterol. Thus, these apoE-rich lipoproteins are capable of producing the suppression of cholesterol synthesis and LDL receptor activity previously observed in abetalipoproteinemia.

High density lipoprotein metabolism in man.

The turnover of (125)I-high density lipoprotein (HDL) was examined in a total of 14 studies in eight normal volunteers in an attempt to determine the metabolic relationship between apolipoproteins A-I (apoA-I) and A-II (apoA-II) of HDL and to define further some of the determinants of HDL metabolism. All subjects were first studied under conditions of an isocaloric balanced diet (40% fat, 40% carbohydrate). Four were then studied with an 80% carbohydrate diet, and two were studied while receiving nicotinic acid (1 g three times daily) and ingesting the same isocaloric balanced diet. The decay of autologous (125)I-HDL and the appearance of urinary radioactivity were followed for at least 2 wk in each study. ApoA-I and apoA-II were isolated by Sephadex G-200 chromatography from serial plasma samples in each study. The specific activities of these peptides were then measured directly. It was found that the decay of specific activity of apoA-I and apoA-II were parallel to one another in all studies. The mean half-life of the terminal portion of decay was 5.8 days during the studies with a balanced diet.Mathematical modeling of the decay of plasma radioactivity and appearance of urinary radioactivity was most consistent with a two-compartment model. One compartment is within the plasma and exchanges with a nonplasma component. Catabolism occurs from both of these compartments. With a balanced isocaloric diet, the mean synthetic rate for HDL protein was 8.51 mg/kg per day. HDL synthesis was not altered by the high carbohydrate diet and was only slightly decreased by nicotinic acid treatment. These perturbations had effects on HDL catabolic pathways that were reciprocal in many respects. With an 80% carbohydrate diet, the rate of catabolism from the plasma compartment rose by a mean of 39.1%; with nicotinic acid treatment, it fell by 42.2%. Changes in the rate of catabolism from the second compartment were generally opposite those in the rate of catabolism from the plasma compartment, suggesting that these two catabolic pathways may be reciprocally regulated.

Apolipoprotein and size heterogeneity in human umbilical cord blood low density lipoproteins.

Neonatal umbilical cord blood plasma low density lipoproteins (LDL, d = 1.019-1.063 g/ml) were subfractionated by density gradient ultracentrifugation into seven fractions (from 1.024 to 1.062 g/ml); the bulk of the LDL mass was in a density region of 1.034-1.042 g/ml. Apolipoprotein B by 10% SDS-polyacrylamide gel electrophoresis varied inversely with density, with only trace amounts present in the most dense fraction. The distribution of apolipoprotein B molecular weight forms was assessed by both 3% SDS-polyacrylamide gel electrophoresis and relative aminoacyl mass determination. Lower molecular weight forms of apolipoprotein B (B74 and B26) increased relative to apolipoprotein B100 with increasing density, ranging from undetectable in fraction 1 to apolipoproteins B26 and B74 comprising 30% of the total mass of apolipoprotein B in fraction 6. No apolipoprotein B48 was detectable in the LDL. Apolipoprotein E as determined by both SDS-polyacrylamide gel electrophoresis and radioimmunoassay increased with density with a maximum (14% of the protein) in the most dense fraction, fraction 7. Apolipoprotein A-I by SDS-polyacrylamide gel electrophoresis increased with increasing density and was the major apolipoprotein in fraction 7. Electron microscopic analysis revealed spherical particles whose diameters decreased with increasing density, ranging from 28.6 nm in the top fraction (fraction 1) to 15.6 nm in the bottom fraction (fraction 7). Gradient gel electrophoresis revealed that most of the fractions contained several different sized particles. The bottom fraction (fraction 7), enriched in apolipoproteins E and A-I, had a unique, poorly defined peak at 14.6 nm on gradient gel electrophoresis and showed a tendency to pack hexagonally upon electron microscopy. The unusual composition and apolipoprotein distribution in neonatal LDL fractions suggests that the LDL in the neonate are metabolically very diverse.

Comparison of properties of four inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Four inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase have been approved for treatment of hypercholesterolemia. Three of these are fungal metabolites or derivatives thereof: lovastatin, simvastatin, and pravastatin. The fourth, fluvastatin, is totally synthetic. Its structure, containing a fluorophenyl-substituted indole ring, is distinct from that of the fungal metabolites. Lovastatin and simvastatin are administered as prodrugs, which undergo in vivo transformation to active inhibitory forms; fluvastatin and pravastatin are administered as active agents. The HMG-CoA reductase inhibitors are all effective in reducing plasma concentrations of low density lipoprotein. They have differing pharmacokinetic properties, which may be of importance in some patients. All of these drugs are very well tolerated, and there do not appear to be major differences in toxicity or adverse effects. When LDL reductions > 30% are needed, simvastatin is the most cost-effective HMG-CoA reductase inhibitor. However, these drugs are most commonly used in dosages that reduce LDL-C by 20-30%. For this degree of LDL reduction, fluvastatin is the most cost-effective HMG-CoA reductase inhibitor.

Metabolism of meso-2,3-dimercaptosuccinic acid in lead-poisoned children and normal adults.

Meso-2,3-dimercaptosuccinic acid (DMSA, or succimer) is an oral chelating agent for heavy-metal poisoning. While studying the urinary elimination of unaltered DMSA, altered DMSA (i.e., its mixed disulfides), and lead in children with lead poisoning, we observed a pattern of urinary drug elimination after meals suggestive of enterohepatic circulation. The excretion of lead in urine patterned the elimination of altered DMSA rather than the parent molecule. In addition, the half-life of elimination of DMSA via the kidney was positively associated with blood lead concentration. Two additional crossover studies of DMSA kinetics were conducted in normal adults to confirm the presence of enterohepatic circulation of DMSA after meals. In one, increases in plasma total DMSA concentration were observed after meals in all six subjects; these increases were prevented by cholestyramine administration 4, 8, and 12 hr after DMSA. In the second, the administration of neomycin also prevented increases in DMSA after meals. These studies indicate that 1) a metabolite(s) of DMSA undergoes enterohepatic circulation and that microflora are required for DMSA reentry; 2) in children, moderate lead exposure impairs renal tubular drug elimination; and 3) a metabolite of DMSA appears to be an active chelator.

A human in vivo model for the determination of lead bioavailability using stable isotope dilution.

Beverages stored in lead-crystal glass accumulate extraordinary concentrations of lead. We obtained a lead-crystal decanter manufactured with lead from Australia, where the ratio of 206Pb/207Pb is distinctly different from that in the United States. We sought to determine the bioavailability of crystal-derived lead, using the technique of stable isotope dilution in blood. We conducted a single-dose, nonrandomized cross-over study in which participants were admitted to the Clinical Research Center twice, 1 week apart. During the first admission, subjects ingested sherry obtained from the original bottle. During the second admission, they ingested sherry that had been stored in the crystal decanter and that had achieved a lead concentration of 14.2 mu mol/l. After ingesting decanter-stored sherry, mean blood lead rose significantly (p = 0.0003) from 0.10 to 0.18 mu mol/l, while mean 206Pb/207Pb fell from 1.202 to 1.137 (p = 0.0001). On average, 70% of the ingested dose of lead was absorbed. We conclude that lead derived from crystal glass is highly bioavailable; repeated ingestions could cause elevated blood lead concentration. The technique of stable isotope dilution lends itself to the study of the bioavailability of lead in other matrices, including soil.

Dynamics of apolipoprotein E metabolism in humans.

The dynamics of human apoE metabolism were explored by examining the effects of alimentary lipemia and postheparin lipolysis on the plasma level and lipoprotein distribution of apoE. In the studies of alimentary lipemia, fasting and postprandial plasma samples were obtained from five normal adult males, each of whom drank 100 g of corn oil. Although no change in the plasma concentration of apoE accompanied alimentary lipemia, a major redistribution of apoE among lipoproteins occurred. The portion of apoE associated with triglyceride-rich lipoproteins as assessed by agarose column chromatography increased by a mean of 44%. Furthermore, in the two subjects in whom multiple postprandial samples were taken, there were striking linear correlations between plasma triglyceride concentrations and the fraction of apoE in triglyceride-rich lipoproteins (r = 0.96 and 0.73). In contrast, the plasma concentration of apoE fell in each of the seven studies of postheparin lipolysis. The fall averaged 17% of the control plasma apoE level. In hypertriglyceridemic patients, the decline in plasma triglyceride concentration preceded the decline in apoE concentration, suggesting that the decline in apoE was due to removal of remnants of triglyceride-rich lipoproteins. Lipoprotein fractionation demonstrated substantial loss of apoE from triglyceride-rich lipoproteins; the data suggested that this loss of apoE from triglyceride-rich lipoproteins was due both to removal of apoE from plasma and to transfer of apoE to an HDL fraction. During the recovery phase, as plasma triglyceride levels rose, opposite changes occurred: the plasma apoE level rose, apoE in triglyceride-rich lipoproteins increased in concentration, and apoE in HDL decreased in concentration. Furthermore, it became apparent during the recovery phase that apoE in triglyceride-rich lipoproteins was composed of two discrete subfractions. The first subfraction consisted of apoE on larger, probably recently synthesized lipoproteins; the second consisted of apoE on much smaller lipoproteins. These studies provide evidence in intact humans for a dynamic traffic of apoE between triglyceride-rich lipoproteins and high density lipoprotein. This traffic is a prominent phenomenon of normal alimentary lipemia and of lipolysis. By modulating the lipoprotein distribution of apoE, it probably plays a key functional role in lipoprotein metabolism.-Blum, C. B. Dynamics of apolipoprotein E metabolism in humans.

Role of dietary intervention in the primary prevention of coronary heart disease. Individuals with high-normal or elevated serum cholesterol levels should be placed on cholesterol-lowering diets.

A large and convincing body of evidence links increased coronary risk with elevated plasma levels of low-density lipoprotein (LDL) cholesterol. Cholesterol in atherosclerotic lesions originates from that circulating in the blood bound to LDL. Even mild degrees of hypercholesterolemia (cholesterol greater than 180 mg/dl) when due to increased levels of LDL are associated with increased risk. Lowering plasma levels of LDL has been clearly shown to reduce coronary risk. We are able to modify plasma levels of LDL by restricting the dietary content of cholesterol and saturated fats. Such diets are safe and can be adhered to by large populations. Available information, reviewed here in detail, supports vigorous efforts to lower cholesterol levels by dietary means, even in the patient with so-called mild hypercholesterolemia. The evidence is overwhelming, the risk is nil, and the potential benefits are substantial.

Incorporation of radioactive phospholipid into subclasses of high-density lipoproteins.

The incorporation of orally administered phospholipid into plasma high-density lipoproteins (HDL) was studied in three subjects. Plasma was analyzed by equilibrium density gradient ultracentrifugation, 5, 6, and 8 h after ingestion of 1.1 g [3H-choline, 14C-dilinoleoyl]phosphatidylcholine. At all time points in all subjects, there was a peak of phosphatidylcholine specific activity in fractions of density approximately 1.10-1.13 g/ml, corresponding to the subclass previously designated HDL2a. There was also a more variable, smaller peak of specific activity of phospholipids in HDL2b (1.063-1.100 g/ml) and in fractions of density approximately 1.19 g/ml. In the 1.10-1.13 fraction, 97 and 71%, respectively, of the 3H and 14C radioactivity were in phospholipids. The 3H/14C ratio was similar in phospholipids of HDL subfractions, the d less than 1.07 fraction, and in the administered phospholipid. The results show preferential transfer or exchange or absorbed phosphatidylcholine into specific subclasses of HDL.

Cholestyramine: an effective, twice-daily dosage regimen.

Nine patients with familial hypercholesterolemia (type II hyperlipoproteinemia) and two normal volunteers were studied to ascertain the effectiveness of cholestyramine in lowering plasma cholesterol when a twice-a-day dosage regimen was compared with the same total dosage administered four times a day. The study showed that in these subjects the two regimens were equally effective. The ability to administer cholestyramine twice daily in working adults and school children should greatly enhance adherence to this agent.

Elevated levels of apolipoprotein E in the high density lipoproteins of human cord blood plasma.

The concentrations and lipoprotein distributions of apolipoprotein E (apoE) in normal human umbilical cord blood plasma were determined. The mean plasma apoE level of 95 neonates was considerably higher than that of 49 normal adults (58.1 vs 35.8 micrograms/ml). This elevation of apoE levels was in striking contrast to the lower than adult levels of cholesterol (72 mg/dl vs 185 mg/dl), triglyceride (37.8 mg/dl vs 97.6 mg/dl), and LDL cholesterol (25 mg/dl vs 110 mg/dl) in neonatal plasma. For the group of 95 neonates, the plasma apoE concentration correlated significantly with total plasma cholesterol concentration (r = 0.60), with LDL cholesterol concentration (r = 0.27) and with HDL cholesterol concentration (r = 0.50). Among the neonates, 87% of plasma apoE was associated with a less dense subfraction of high density lipoprotein compared to a mean of 58% for 30 normal adults. Thus, for neonates, despite hypolipidemia, the absolute concentration of apoE in HDL (50 micrograms/ml) was 2.5 times that of adults (20 micrograms/ml). We speculate that the very low level of neonatal VLDL, providing limited substrate for lipolysis, may result in retarded removal of apoE from plasma and the observed high level of apoE in neonatal HDL. We hypothesize that in the fetus and neonate, as has been demonstrated in abetalipoproteinemia, apoE-rich HDL may functionally substitute for LDL in delivering cholesterol to cells.

Independent effects of dietary saturated fat and cholesterol on plasma lipids, lipoproteins, and apolipoprotein E.

Nine normolipidemic males (18-37 years) were fed formula diets containing (as % of calories) egg white protein (15%), glucose polymer:sucrose, 3:1 (54%), and fats (31%) as one of the following: corn oil (corn), corn oil plus 1 gram/day cholesterol (corn+), coconut oil (coco), coconut oil plus 1 gram/day cholesterol (coco+). Two dietary periods of 18 days each were separated by 1 month during which plasma lipid levels returned to prestudy values. A given dietary period consisted of 9 days of either corn or coco feeding allowed by 9 days of corn+ or coco+, respectively. Fasting plasma samples were taken the last 3 days of each 9-day interval. Lipids were determined by standard procedures and the apoE levels in lipoprotein fractions isolated by discontinuous density gradient ultracentrifugation were determined by radioimmunoassay. The biochemical variables measured were: total plasma, VLDL, IDL + LDL, and HDL, cholesterol, triglyceride, and apoE levels, as well as the apoE of plasma d greater than 1.17 g/ml. The effects of apoE phenotype, the type of dietary oil (corn versus coco), the presence or absence of dietary cholesterol, and the day of sampling within triplicates on the above variables were assessed statistically. The type of oil had the only significant effect on any variable. At P less than 0.01, the coconut oil diets were associated with significant elevations (as compared to corn oil) of the following nine variables: total, VLDL, IDL + LDL, and HDL cholesterol; total, VLDL, and IDL + LDL apoE; total and VLDL triglycerides.

Apolipoprotein E synthesis in human kidney, adrenal gland, and liver.

Human tissues were incubated in vitro with radiolabeled amino acids to determine whether plasma apolipoproteins are synthesized in human kidney. Subsequently, tissue extracts were screened with antisera directed against apolipoprotein E (apo E), apolipoprotein B (apo B), apolipoprotein AI (apo AI), and bulk apolipoproteins of high density lipoprotein (HDL). Newly synthesized apo E, but not apo AI or apo B, was identified in kidney and adrenal cortex. Estimates of relative rates of apo E synthesis in vitro suggest that a substantial portion of adrenal and kidney protein synthesis is committed to apo E synthesis. The relative rate of apo E synthesis was 4-6 times greater in kidney cortex than in kidney medulla. Analysis of immunoreactive apo E showed that kidney and adrenal apo E species have the same electrophoretic mobility in NaDodSO4/polyacrylamide gels as does plasma apo E. Further characterization by high resolution two-dimensional gel analysis indicated that the isoforms of newly synthesized kidney and adrenal apo E correspond to specific isoforms of plasma apo E. These findings suggest that apolipoproteins arising from peripheral tissues may play an important role in lipid transport and metabolism.