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The metabolism of apolipoproteins A-I and A-II, as well as other high density lipoprotein (HDL) constituents, was studied in patients with homozygous familial HDL deficiency (Tangier disease) prior to and after plasma exchange or HDL infusion. Mean plasma apoA-I, apoA-II, and HDL cholesterol values in homozygotes (n = 2) were 2.0 mg/dl, 2.7 mg/dl, and 1.5 mg/dl, respectively, and in a normal control subject were 125.1 mg/dl, 23.0 mg/dl, and 53.0 mg/dl, respectively. Based on radioiodinated apoA-I and apoA-II kinetic studies in the baseline state, synthesis rates for apoA-I and apoA-II in mg/kg/day were 3.81 and 1.61, respectively, in one homozygote (patient B) and 11.82 and 1.99, respectively, in the normal subject. ApoA-I and apoA-II plasma residence times in days were 0.22 and 0.81, respectively, in the homozygote, and 4.04 and 4.44, respectively, in the normal subject. These data indicate that this homozygote had both a moderate decrease in the synthetic rates of apoA-I and apoA-II, as well as a marked decrease in the plasma residence times of these two apolipoproteins. In one homozygote (patient A) following a complete plasma exchange during cardiopulmonary bypass, plasma HDL cholesterol, apoA-I, and apoA-II levels were very similar to pre-exchange values within 64 hr after exchange. A second homozygote (patient B) received HDL intravenously as well as 125I-labeled apoA-I and 131I-labeled apoA-II. Following infusion, the residence time in days for HDL subfractions, HDL2b, HDL2a, and HDL3 were 0.1, 0.8, and 2.7, respectively. HDL protein and phospholipid both had a monoexponential decay, with residence times of 0.7 days, while HDL triglyceride disappeared monoexponentially with a residence time of 0.5 days. HDL cholesterol had a biexponential decay, with the residence time of the slow component being 0.7 days. Plasma and HDL apoA-I decayed down to baseline values significantly faster than did plasma and HDL apoA-II. ApoA-II specific radioactivity decreased throughout the course of the infusion study in both plasma and HDL, while apoA-I specific radioactivity decreased slightly, then rose, and subsequently declined in both plasma and HDL. The data indicate that the rapid and altered catabolism of apoA-I and apoA-II in Tangier homozygotes persists despite major increases in the plasma pool size of these proteins. In addition, following HDL infusion, HDL2b and HDL2a disappeared at a faster rate than HDL3, HDL cholesterol and triglyceride were catabolized at a faster rate than HDL protein and phospholipid, and apoA-I disappeared more rapidly than apoA-II. These observations may have important implications with regard to the catabolism of HDL subfractions and constituents in normal man.

The action of a bacterial acyltransferase similar in overall reaction mechanism to the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been studied using normal plasma and lipoproteins and plasma from LCAT-deficient patients. The microbial enzyme (GCAT) catalyzed acyl transfer using phosphatidylcholine and cholesterol in all of the lipoprotein fractions, presumably because it has no apolipoprotein cofactor. In addition, the enzyme was capable of hydrolyzing cholesteryl ester in lipoproteins but not in small unilamellar vesicles nor in micellar dispersions containing low amounts of Triton X-100. This suggests that cholesteryl ester is exposed on the surface of lipoprotein particles or that it may be transferred there quickly from the interior. Although considerable interconversion of radiolabeled cholesterol and cholesteryl ester could be demonstrated upon treatment of normal plasma or lipoproteins with the enzyme, there was little change in the actual amount of either steroid. This indicates that the rate of cholesteryl ester formation is very similar to the rate of hydrolysis. The relative proportions of cholesterol and cholesteryl ester in normal plasma are therefore near the equilibrium ratio for the reaction carried out by GCAT, or the ratio is controlled by the properties of the lipoproteins themselves. During reaction with the microbial acyltransferase, the ratio of cholesterol to cholesteryl ester in plasma from LCAT-deficient patients was reduced substantially, suggesting that the enzyme may have some practical applications.

This study compared lovastatin and gemfibrozil therapy for effects on lipid and lipoprotein levels in 22 normolipidemic patients with reduced high-density lipoprotein cholesterol levels. Most patients had coronary heart disease. A randomized, crossover design consisted of two drug phases (lovastatin and gemfibrozil) alternating with placebo. Lovastatin reduced total and low-density lipoprotein cholesterol and apolipoprotein B levels by 28%, 34%, and 24%, respectively. These were unaffected by gemfibrozil. Both drugs reduced very low-density lipoprotein and intermediate-density lipoprotein cholesterol levels by 30% to 40%. Both caused small but significant increases in high-density lipoprotein cholesterol, but not in apolipoproteins A-I or A-II. Both significantly lowered ratios of total (and low-density lipoprotein) cholesterol-to-high-density lipoprotein cholesterol, but lovastatin more than gemfibrozil. Thus, for normolipidemic patients with low levels of high-density lipoprotein cholesterol, neither drug markedly raised high-density lipoprotein levels, but lovastatin produced the better overall change in lipoprotein cholesterol and apolipoprotein B levels.

Hypocholesterolemia (less than 120 mg/dl) was found in 200 out of 3,700 patients admitted consecutively to a University Department of Internal Medicine. In 91, hypocholesterolemia as defined above could be confirmed in a second serum sample on the following day. Mortality during hospitalization in this group was 32%. When diagnoses were grouped into 5 categories, prognosis was particularly poor in heart (36%) and liver disease (31%) and in malignancies (33%), while patients with malabsorption syndromes had a considerably better prognosis (10% mortality). Hypocholesterolemia was associated with a low cholesterol/phospholipid ratio and may be among the first signs of further deterioration of disease.

Reverse correlation between content of cholesterol in lipoproteins of high density (LPHD) and severity of clinical manifestations was observed in patients with obliterating atherosclerosis of legs. Concentrations of cholesterol, triglycerides and LPHD cholesterol were normal in the majority of patients but dyslipoproteinemia of various types including hypo-alpha-lipoproteinemia, was noted in some patients. The molar ratio "cholesterol/phospholipids" was increased in erythrocytes of the patients examined, demonstrating the disbalance in cholesterol turnover between the cell membranes and blood lipoproteins.

Erythrocyte membrane abnormalities in 3 members of a family with a hereditary haemolytic syndrome (HHS) were compared to those previously described in a family with lecithin:cholesterol acyltransferase (LCAT) deficiency. Despite similarities including an increase in membrane phosphatidylcholine, a decrease in phosphatidylethanolamine, stomatocytosis, and a marked decrease in erythrocyte osmotic fragility a number of differences were observed. These included membrane cholesterol content (increased in homozygotes with LCAT deficiency), changes in sodium and potassium content and Na+,k+-ATPase activity (the latter being increased in HHS), changes in acetylcholinesterase and sulfhydryl group latency (present in LCAT deficiency, but not in HHS) and 2,3 DPG content (decreased in HHS, normal in LCAT deficiency. Full compensation of the erythrocyte defect occurred in HHS but the homozygotes for LCAT deficiency were slightly anaemic. It is concluded that, although similar abnormalities in phospholipid composition, osmotic fragility, and erythrocyte morphology exist in these two disorders, the molecular nature of the erythrocyte membrane structural and functional changes in HHS and LCAT deficiency is clearly different.

This review assesses current knowledge of the clinical, genetic, and biochemical features of familial high density lipoprotein (HDL) deficiency syndromes. The focus is on HDL deficiency states occurring in the absence of severe hypertriglyceridemia or lecithin/cholesterol acyltransferase deficiency. Specific entities falling within this category include Tangier disease, familial HDL deficiency with planar xanthomas, familial apolipoprotein A-I and C-III deficiency (formerly known as apolipoprotein A-I absence), familial deficiency of apolipoprotein A-I and C-III, fish-eye disease, familial hypoalphalipoproteinemia, and apolipoprotein A-I variants (apo A-I Milano, apo A-I Marburg, apo A-I Giessen, and apo A-I Munster 1-3). Diffuse corneal opacification and premature coronary artery disease are common features in many of these kindreds. No striking clinical abnormalities have been noted in patients with currently known apolipoprotein A-I variants, possibly because these subjects are heterozygotes for their respective defects. The HDL deficiency in many of these disorders has been associated with abnormalities or deficiencies of apolipoprotein A-I. Further research will undoubtedly define the defects in all the disorders that have been described, uncover new mutations, as well as provide additional insights into the precise relationship between HDL deficiency and atherosclerosis.

Restriction enzyme analysis of the apolipoprotein A-I (apo A-I) gene was performed in two patients with fish eye disease and one with Tangier disease. Despite the marked deficiency of high density lipoprotein and concomitantly of apo A-I in these two conditions, no evidence was found for major deletions or insertions in the apo A-I gene.

Mechanisms responsible for hypertriglyceridemia in Tangier disease were elucidated by an analysis of the plasma post-heparin lipolytic activities and the structural and metabolic properties of very low (VLDL) and low (LDL) density lipoproteins. The levels of lipoprotein lipase activity in six Tangier patients were significantly lower (P less than 0.001) than in 40 control subjects (8.1 +/- 3.3 (+/- S.D.) vs. 14.1 +/- 3.7 units/ml). In contrast, the levels of hepatic triacylglycerol lipase were higher (P less than 0.01) than in normal controls (14.4 +/- 3.9 vs. 9.3 +/- 4.0 units/ml). Because kinetic parameters such as Km or Vmax cannot be obtained with naturally occurring triacylglycerol-rich lipoproteins, the pseudo-first-order rate constant (k1) of triacylglycerol hydrolysis was used to assess the effectiveness of triacylglycerol-rich lipoproteins as substrates for lipoprotein lipase. The k1 values for Tangier VLDL (k1 = 0.017 +/- 0.002 min-1) were significantly lower (P less than 0.001) than the k1 values (0.036 +/- 0.008 min-1) for control VLDL. Both the Tangier and control LDL2 are similar in their resistance to the action of lipoprotein lipase, as shown by their low k1 values (0.002 +/- 0.001 and 0.001 +/- 0.001 min-1, respectively). The major compositional difference between the lipoproteins of Tangier disease and normal subjects was a significant increase in the percent content of apolipoprotein A-II in all lipoprotein particles with d less than 1.063 g/ml, with the greatest increase occurring in VLDL and the lowest in LDL2. These results were interpreted as indicating that, in Tangier disease, there is a lower reactivity of VLDL with lipoprotein lipase which may in part be attributed to the abnormal apolipoprotein composition. This finding, in conjunction with the reduced levels of lipoprotein lipase activity, may explain the hypertriglyceridemia in Tangier disease.

Tangier disease is a disorder characterized by low levels of apo-A-I and high density lipoproteins. The defect in Tangier disease is an abnormal A-I apolipo protein, designated apo-A- ITangier . In normal subjects, apo-A-I is secreted as proapo -A-I with subsequent extracellular conversion to mature apo-A-I. The major form in normal plasma is mature apo-A-I with small amounts of proapo -A-I. In Tangier disease, proapo -A- ITangier is present in roughly equivalent concentrations compared to mature apo-A- ITangier . It has been proposed that the defect in Tangier disease is in the conversion of pro- to mature apo-A- ITangier . To test this, proapo -A-I was isolated from normal and Tangier subjects, and the conversion to the mature form by plasma from normal and Tangier subjects was analyzed. Incubation of radiolabeled normal proapo -A-I in normal plasma anticoagulated with heparin was associated with progressive conversion to mature apo-A-I over 24 h (initially 85% of the radioactivity was in the proapo -A-I isoform; at 24 h 33% radioactivity remained in the pro-isoform). Proapo -A- ITangier was also converted to the mature isoform during 24 h of incubation in normal plasma. Initially, 84% of radioactivity was in proapo -A- ITangier , and by 24 h the radioactivity in this isoprotein had decreased to 36%. A similar pattern of conversion was also observed when proapo -A- ITangier was incubated in Tangier plasma. The proteolytic conversion of both normal proapo -A-I and proapo -A- ITangier was unaffected by the serine protease inhibitors phenylmethylsulfonyl fluoride (1 mM) or aprotinin (200 Kallikrein-inactivating units/ml), but was inhibited by EDTA (0.1%). These results indicate that proapo -A- ITangier can be converted to mature apo-A- ITangier by the converting enzyme in normal plasma. In addition, plasma from a Tangier subject can convert both normal and Tangier proapo -A-I to the mature form. These results establish that proapo -A- ITangier can be rapidly converted to mature apo-A- ITangier , and there is no deficiency of the converting enzyme activity in Tangier disease.

As the alpha-cholesterol concentration decreased in blood plasma of healthy men, relative content of oleic and linoleic acids was also decreased in phospholipids of HDL2 subfraction without any alterations in HDL3 fraction. In the patients with ischemic heart disease content of linoleic acid was lower in the phospholipids and the ratio free cholesterol (HDL2 phospholipids was higher as compared with the group of healthy persons exhibiting the similar low content of cholesterol in HDL. The molar ratio free cholesterol) phospholipids was higher in the HDL3 fraction of the patients as compared with healthy persons, however composition of fatty acids was unaltered in the phospholipids Activity of lecithine cholesterol acyltransferase was lower in the patients as compared with healthy persons exhibiting the same content of cholesterol in HDL. Administration of heparin into the patients was responsible for an increase in the level of unsaturated phospholipids in HDL2 fraction (while they decreased in VLDL/as well as for a decrease in the ratio cholesterol/phospholipids. The data obtained suggest that alterations in the physico-chemical properties of the fraction studied might affect the antiatherogenic characteristics of HDL.

The incorporation of [3H] thymidine and the synthesis of glycosaminoglycans (GAG) by cultured human aortic smooth muscle cells were studied in the presence of human sera with high HDL cholesterol concentration, high LDL cholesterol concentration or with normal serum lipoprotein concentrations. The sera were prepared either conventionally (CPS, platelet factor present) or from platelet poor plasma by recalcification (PPPS). As compared to normolipidaemic sera, HDL-aemic CPS decreased the incorporation of thymidine but LDL-aemic sera had no effect. HDL-aemic sera decreased markedly the synthesis of sulphated GAG but had no effect on the synthesis of hyaluronic acid (HA). Therefore, the sulphated GAG/HA ratio was decreased. The decrease in sulphated GAG was observed only in the presence of CPS, not in the presence of PPPS. LDL-aemic sera decreased the synthesis of hyaluronic acid causing an increase in the sulphated GAG/HA ratio. This effect was observed in the presence of both CPS and PPPS. The results suggest that determination of the sulphated GAG/HA ratio in aortic smooth muscle cell cultures provides a useful method for estimating the atherogeneity of various sera. The anti-atherogenic effect of HDL-aemic sera seems to be dependent on the platelet factor while the atherogenic effect of LDL-aemic sera may be independent of the platelet factor.

This study describes a variant of hypo-alpha-lipoproteinemia in a 57-year-old male patient. The total plasma cholesterol level was 258 mg/dl with 64% in esterified form. The concentration of triglycerides was 205 mg/dl. The lipoprotein electrophoretic pattern revealed the absence of alpha-lipoproteins, whereas the other lipoproteins showed an intermediate electrophoretic mobility. The concentration of HDL cholesterol (heparin: MgCl2 precipitation) was extremely low (3 mg/dl). The activity of lecithin; cholesterol acyltransferase (LCAT) and the postheparinlipolytic activity were within the normal range. Determination of apolipoproteins revealed a marked deficiency of both apoprotein A-I (17 mg/dl) and apolipoprotein A-II (11 mg/dl). The concentration of apolipoprotein-B was elevated (186 mg/dl). Unlike the clinical manifestations of Tangier disease, our patient did not show skin lesion, abnormal tonsils, hepatosplenomegaly, peripheral neurologic abnormalities or corneal deposits. Also in contrast to Tangier disease our patient had coronary artery disease with myocardial infarction accompanied with severe occlusive peripheral arterial disease.

Blood plasma contains lipoproteins of which the structure and the composition are not constant during the time and that depend on their metabolic evolution from their synthesis rate in intestine or liver to their utilization in the liver or the peripheral tissues. The composition and the pathway of the different lipoprotein classes (chylomicrons, VLDL, LDL, HDL) are reviewed, as also the main pathological perturbations that attack them.

The absence of normal high density lipoproteins (HDL) in Tangier disease is well established, but the properties of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in this disorder have not been well defined. The profiles obtained by analytic ultracentrifugation and the chemical composition, morphology, and electrophoretic mobility of Tangier and normal VLDL and LDL were compared. Apolipoproteins were fractionated by gel chromatography and characterized by amino acid analysis, polyacrylamide-gel electrophoresis, and immunochemical reactivity. Concentrations of low density lipoproteins of S(f) (o) 0-12 were reduced in three of six Tangier plasmas studied by analytic ultracentrifugation. Accumulation of intermediate density lipoproteins (S(f) (o) 12-20) was not observed. Two subjects with hypertriglyceridemia had normal VLDL (S(f) (o) 20-400) levels, suggesting that abnormalities of chylomicron metabolism probably account for the hypertriglyceridemia frequently observed in this disorder. Tangier VLDL migrate more slowly than normal VLDL on paper electrophoresis, yet their morphology, gross chemical composition, and qualitative apolipoprotein content are similar. Quantitative abnormalities in C-apolipoproteins, however, were observed in Tangier VLDL. When patients were consuming unrestricted diets, C apoproteins accounted for 19-49% of the protein in lipoproteins of d < 1.006 g/ml. Ingestion of low-fat, high-carbohydrate diets reduced the VLDL-C-apoprotein content in all Tangier patients (mean = 17% of VLDL protein vs. 43% in controls). These findings suggested that a major proportion of the C apoproteins in Tangier plasma is associated with chylomicrons or their remnants, perhaps because the C-apoprotein reservoir normally provided by HDL is absent. This secondary mechanism for C-apoprotein conservation is lost when dietary fat is withdrawn.LDL-2 (1.035 < d < 1.063) from Tangier and control plasma had identical electrophoretic mobilities. Tangier LDL-2 had slightly smaller median diameters (210-225 A vs. 230-240 A in controls) and a quite different composition than normal LDL-2. Triglyceride accounted for a mean of 29% of Tangier LDL-2 mass (control = 6%) and the cholesteryl ester content was reduced by about 50%. Thus, HDL may be required for the generation of chemically normal LDL. Alternatively, the fundamental defect in Tangier disease may involve all lipoprotein classes.

Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.

Erythrocyte morphology, kinematic viscosity, and membrane lipid composition have not been well documented in heterozygous hypobetalipoproteinemia (HBL). In our study of nine subjects with HBL from two kindreds, three presented with schistocytes (3-6%) while two of them also showed acanthocytosis (25-35%) on their peripheral blood films. The other affected family members had a normal RBC morphology despite a decreased kinematic viscosity of RBC suspension at 37 degrees C (log eta = 0.20 +/- 0.02 vs. log eta = 0.26 +/- 0.02) in all nine subjects; erythrocyte osmotic fragility however was normal. Although some subjects showed acanthocytosis total cholesterol (0.63 +/- 0.05 mol x 10(-6)/mg membrane protein) and sphingomyelin/lecithin ratio (0.905 +/- 0.042) of RBC membranes were normal while plasma from the subject with schistocytosis had a significantly decreased sphingomyelin/lecithin ratio in plasma (17.2 +/- 2.4 vs. 29.1 +/- 3.3) suggesting that sphingomyelin and lecithin are not freely exchangeable between plasma and the outer surface of RBC membranes.

The siblings presented here are the third family found in Japan with familial LCAT deficiency. Their post-heparin plasma lipoprotein lipase and hepatic triglyceride lipase activities were measured selectively by an immunochemical method. Plasma triglyceride levels were elevated, and post-heparin plasma lipoprotein lipase was decreased only in a patient with nephropathy, while hepatic triglyceride lipase activities were within reference limits in both patients. The plasma concentrations of apo A-I, apo A-II, and apo B were reduced in both patients. On the other hand, the plasma concentration of apo E was markedly increased. Enzyme replacement therapy by plasma transfusion in the propositus resulted in marked improvement of deranged compositions of triglyceride-rich lipoproteins. Also, improvement of the plasma apo E concentration was demonstrated, while the improvement of post-heparin lipase did not occur. These results suggest that LCAT may play an important physiological role in triglyceride metabolism as well as in cholesterol metabolism.

High density lipoprotein (HDL) infusion and partial plasma exchange were undertaken in two patients homozygous for Tangier disease. Serum samples and ultracentrifugally isolated serum fractions were analysed over a period of 7 days post infusion by agarose electrophoresis, two-dimensional immunoelectrophoresis (employing antibodies to HDL, HDL3, Apoprotein A-I, and Apoprotein A-II), Apoprotein A radioimmunoassay, and analytical polyacrylamide electrophoresis. The following observations were made: (a) immediately after HDL substitution the broad-beta band, normally visible upon agarose electrophoresis of Tangier plasma, resolved into a distinct beta and pre-beta band; (b)as HDL was catabolized, an abnormal alpha-migrating lipoprotein was generated which contained Apoprotein A-II as protein constituent; and (c) there was a proferential loss of Apoprotein A-I from HDL and the plasma compartment in the course of HDL catabolism. The results suggest that the defect in Tangier disease resides with enhanced catabolism or defective synthesis of Apoprotein A-I.

The molecular defect in Tangier disease is unknown. We have compared the electrophoretic and immunoreactive properties of Tangier disease and normal apolipoprotein A-I using four monoclonal antibodies. We verified that the molecular weight, pI and CNBr-cleaved fragments of Tangier disease and normal apolipoprotein A-I were not different, excluding the possibility that dimers, aggregates or fragments of apolipoprotein A-I could be responsible for its rapid catabolism in this disease.

We compared the plasma protein patterns of the two living patients suffering from fish-eye disease with those of appropriate controls, using high-resolution two-dimensional electrophoresis. Quantitative abnormalities were detected in plasma polypeptides corresponding to the isoforms of apolipoproteins A-I and A-II. The disease was characterized by normal concentrations of proapo A-I but dramatically subnormal concentrations of the other apo A-I isoforms and apo A-II. No significant difference was detected in the concentrations of other plasma proteins. These findings are discussed in relation to other apolipoprotein disorders.

A double-antibody radioimmunoassay was used to determine the concentration and distribution of apolipoprotein A-I (apoA-I) in the plasma of patients with Tangier disease. Obligate heterozygotes for Tangier disease had apoA-I levels that were 50% or less of controls, even when estimates of high-density lipoprotein cholesterol concentration were normal. Plasma apoA-I levels in Tangier homozygotes were at most 3% of normal. Most of the apoA-I in the plasma of homozygotes sedimented in the ultracentrifuge at density 1.21 g/ml, and no more than 20% was recovered in the plasma lipoprotein fraction. Radioimmunoassay demonstrated no immunochemical differences between Tangier and control apoA-I.

The plasma decay of three groups of iodinated apoproteins on human very low density lipoproteins were evauluated in two normals, two subjects with endogenous hypertriglyceridemia and another two with dysbetalipoproteinemia. The apo beta decay was more rapid than that of the C apoproteins in all patients. The apo beta decay was more rapid for the normals than for either the subjects with hypertriglyceridemia or dysbetalipoprotenemia. The apo C protein had an irregular decay in the normals but decayed less irregularly for the hypertriglyceridemics. The arginine rich apoprotein had a decay somewhat similar to apo C protein in the normals. The apo beta protein of the alpha2 very low density lipoprotein of a dysbetalipoproteinemic was consistent with a precursor relationship to the apo beta of beta very low density lipoprotein of this subject, but the arginine rich apoprotein was not.

Isoforms of apolipoprotein A-I (apo A-I) from subjects with Tangier disease were characterized, and their ability to recombine with normal high density lipoproteins (HDL) was studied. In contrast to normal serum, in which isoprotein 4 is the dominant species [79 +/- 1.8% (mean +/- SD)], the Tangier serum contained much less total apo A-I (approximately equal to 1% of that in normal serum), and isoproteins 2 and 4 were present in roughly equivalent amounts (35.3 +/- 2.5% and 42.7 +/- 3.6%, respectively). The Tangier isoprotein 2 was shown to correspond to pro-apo A-I, having a six-amino acid amino-terminal extension with the sequence: Arg-His-Phe-Trp-Gln-Gln-. The Tangier isoprotein 4 had the same amino-terminal sequence as normal circulating plasma apo A-I. Its association with normal HDL (70%) was similar to the association of normal apo A-I with HDL (80-90%) in recombination experiments. In marked contrast to this behavior, very little (less than 10%) of Tangier isoprotein 2 (pro-apo A-I) associated with HDL in recombination experiments. These results suggest that the underlying defect in Tangier disease may be a faulty conversion of pro-apo A-I to mature apo A-I, either due to a defect in the converting enzyme activity or to a further specific structural defect in Tangier apo A-I. The failure of Tangier pro-apo A-I to associate with HDL may be at least partially responsible for the HDL deficiency in Tangier subjects.

Quantitative alterations were found in content and the ratio of apoproteins "C" and "E" from lipoproteins of very low density (LPVLD) and of high density (LPHD). In lipoproteinlipolysis of LPVLD the ratio of total apoprotein-inhibitors to protein-activators was increased in men with hyperalphalipoproteinemia; the lipoproteinlipolytic activity appears to depend on content and composition of specific LPVLD apoproteins. Dissimilar alterations in content of "C" and "E" apoproteins, studied in LPVLD and LPHD simultaneously, occurred when high and low concentrations of cholesterol-LPHD (CS-LPHD) were used. At the same time, content of apoE and cholesterol esters, occurring both in LPVLD and LPHD, was uniformly altered under conditions of CS-LPHD content variation. The data obtained suggest that content of blood plasma CS-LPHD is related to quantitative composition of apoproteins involved in regulation of lipoprotein metabolism in blood plasma.

Isolated high density lipoprotein (HDL) from the two living fish eye disease patients have been incubated in vitro with autologous lipoprotein depleted plasma or with lipoprotein depleted plasma from domestic pig (Sus domesticus), with and without the presence of LCAT inhibitor for 24 hours at 0 and 37 degrees C. The lecithin:cholesterol acyltransferase (LCAT) activity in lipoprotein depleted pig plasma increased the abnormally low cholesteryl ester content of the fish eye disease HDL particles from about 20 to 100% and increased their exceptionally small mean particle size, probably by particle fusion, to a range which is representative of normal HDL3. Both esterification and particle enlargement were totally blocked by the LCAT inhibitor. Incubation of concentrated fish eye disease HDL with autologous lipoprotein depleted plasma for 24 hours at 37 degrees C resulted in a small increase in its cholesteryl ester percentage to 37%, without affecting the apparent HDL particle size. This finding confirms a deficiency of HDL lecithin:cholesterol acyltransferase activity (alpha-LCAT) in fish eye disease. The observed normalization of both HDL cholesteryl ester percentage and particle size by lipoprotein depleted pig plasma which contains virtually no cholesteryl ester transfer activity indicates that the latter is not a requisite for esterification of the free cholesterol of fish eye disease HDL.

Cholesterol efflux was studied from cholesteryl esterladen mouse peritoneal macrophages in the presence of Tangier lipoproteins derived from fasting and postprandial sera of three patients homozygous for Tangier disease (analphalipoproteinemia). The d greater than 1.063 g/ml fractions isolated from fasting patients and 3 hr and 18 hr after an oral fat load were all effective in cellular cholesterol removal. By contrast, the d greater than 1.063 g/ml fractions isolated 6 hr and 12 hr after fat ingestion did not affect net removal of cellular cholesterol. The d greater than 1.21 g/ml protein fractions derived from fasting as well as postprandial sera were all effective in removing cholesterol. D 1.063-1.21 g/ml fractions from fasting Tangier patients contained HDLT. In the corresponding postprandial fractions, in addition to HDLT, apoB-100- and apoB-48-containing lipoproteins were present. Furthermore, the 6 hr and 12 hr postprandial Tangier HDL fractions contained apoB-immunoreactive proteins of lower molecular weight. The abnormal activity of the elastase/alpha 1-antitrypsin proteolytic system and the abnormal fibronectin concentration we found in Tangier plasma suggests a possible relationship to the in vivo degradation of apoB. The peculiar type of membrane-bound lipid droplets in Tangier splenic macrophages points to a lipoprotein source of lipid accumulation which possibly originates from the uptake of chylomicrons or chylomicron-derived particles. It is concluded that cholesteryl ester storage in Tangier macrophages results from an imbalance of cholesterol influx and efflux. In the absence of HDL, the net increase of cholesterol caused by abnormal lipoproteins in certain postprandial states cannot be fully compensated by effective efflux and ultimately leads to macrophage cholesteryl ester accumulation.

The results of lipoprotein studies performed in 67 members of 10 kindreds with familial hypoalphalipoproteinemia are presented. Probands were ten patients referred to the Lipid Clinic of the National Institute of Cardiology for evaluation of their lipid profile, all of whom had history of a definite myocardial infarction occurring before they had reached age 60. Their only plasma lipid abnormality was a reduction of the cholesterol fraction associated with high-density lipoproteins (C-HDL) below the corresponding age-sex specific fifth percentile. We studied 57 other individuals including probands spouses and available first-degree relatives. All participants were clinically examined and their complete coronary risk factor profile was assessed. After a 12 hour fast, plasma samples were obtained for lipid analysis, total cholesterol and trïglycerides were measured by enzymatic methods, C-HDL was determined in the supernatant after plasma precipitation with magnesium chloride: phosphotungstic acid. Low-density lipoprotein cholesterol (C-LDL) was estimated with the formula proposed by DeLong. For every family a pedigree was constructed and statistically analyzed to assess the within-family clustering of low C-HDL levels and the pattern of transmission of the abnormal phenotypes. Mean C-HDL level for propositii was 24.3 mg/dl, the corresponding value for the C-LDL/C-HDL ratio (atherogenic index) was 4.9 and it was elevated above 3.5 (average coronary risk) in 7/10 (70%) of these patients. Obesity, defined by a Quetelet index above 28 in men and 26 in women was present in 4/10 (40%) of the probands, 3/10 (30%) had stable non insulin-dependent diabetes mellitus, prevalence figures for the other coronary risk factors was very low. In addition to probands, the hypoalphalipoproteinemic phenotype was found in 30/57 (52.6%) subjects including spouses (two cases) and first-degree relatives. For these 30 cases the mean C-HDL value was 32 mg/dl. In all ten reported kindred, we clearly observed either horizontal, vertical or both types of within-family transmission of the aberrant phenotype. This finding was considered to be most compatible with the presence of a primary genetic abnormality affecting C-HDL metabolism in all these kindreds in spite of the fact that some of the probands had some conditions like diabetes or obesity that are sometimes associated with secondary reductions of C-HDL. Due to the lack of a reliable biochemical marker, the distinction between primary and secondary hypoalphalipoproteinemia in individual cases is frequently impossible, for this reason the diagnostic assessment depends on family studies.(ABSTRACT TRUNCATED AT 400 WORDS)

The content of A1, A2, and E apolipoproteins in blood plasma of males (40-59 years of age) with normo-, hyper-, and hypoalphalipoproteinemia was determined by rocket immunoelectrophoresis. Considerable changes were revealed in the alphalipoprotein cholesterol/A1-apo ratio which were evidence of increased capacity of HDLP for transporting cholesterol in hyperalphalipoproteinemia and a decrease in this HDLP function in hypoalphalipoproteinemia irrespective of their concentration in blood plasma. A reverse correlative connection was revealed between the A1-apo content and the E-apo content in dysalphalipoproteinemia. It is suggested that the changes in the content of A1-apo and E-apo in blood plasma are significant in lipoprotein metabolism in dysalphalipoproteinemia.

We have isolated and charachterized a subfraction of high density lipoproteins, rich in apolipoprotein E, from the plasma of patients afflicted with familial lecithin:cholesterol acyltransferase deficiency. Prepared by successive ultracentrifugal flotation, affinity chromatography on heparin-agarose, and affinity chromatography on conconavalin A-agarose, the subfraction contained disc-shaped lipoproteins that measured 14--40 nm in diameter and 4.4--4.5 nm in thickness. The major components were apolipoprotein E, phosphatidylcholine, and unesterified cholesterol, though other apolipoproteins and lipids were present in small amounts. A second subfraction of high density lipoproteins, isolated during the chromatography, contained apolipoproteins A-I and A-II, but no apolipoprotein E. This subfraction included disc-shaped lipoproteins, 13--24 nm in diameter, as well as small round particles, 5.7 nm in diameter. Both subfractions contained similar proportions of total protein relative to lipid, similar amounts of unesterified cholesterol relative to phosphatidylcholine, and a similar distribution of phosphatidylcholine fatty acid.

Native fish-eye disease plasma, which is deficient of both high density lipoproteins (HDL) and lecithin-cholesterol acyltransferase activity (alpha-LCAT), processing the free cholesterol of these lipoproteins, has been supplemented with normal isolated HDL2 or HDL3 and incubated in vitro at 37 C. After incubation for 0, 7.5 and 24 hr the very low density (VLDL) and low density (LDL) lipoproteins as well as HDL were isolated, and their contents of triglycerides, phospholipids and free, esterified and total cholesterol were quantified. The resulting net mass transfer of the different lipids revealed a functioning transfer of cholesteryl esters and all other analyzed lipids between the lipoproteins, although no de novo esterification of the HDL cholesterol by LCAT in this plasma occurred. In accordance with previous findings there was a functioning esterification process of the free cholesterol of the combined VLDL and LDL of fish-eye disease plasma. The present reports make it reasonable to conclude that the lack of HDL cholesterol esterification in this disease is not a result of a deficiency of cholesteryl ester transfer or lipid transfer activities.

Measurement of plasma lecithin:cholesterol acyltransferase (LCAT) activity was used to segregate unaffected family members (n = 8) from heterozygotes (n = 8) and homozygotes (n = 2) in a large LCAT-deficient kindred. The activity was absent in the homozygotes and was decreased to 50% of normal in the heterozygotes. Endogenous cholesterol esterification rate measurements did not differentiate the heterozygotes from the unaffected family members or normal subjects. The heterozygotes had significantly higher fasting plasma triglycerides, apo B, and lower HDL-cholesterol and apo AI than the unaffected family members. The HDL of the heterozygotes had the same mass of free cholesterol and triglyceride, but the mass of cholesteryl ester was reduced by 47%. The differences were not related to abnormal postheparin lipolytic activities. However, cholesteryl ester transfer activity in the lipoprotein-free (d greater than 1.21 bottom) fraction of plasma was significantly (P less than .05) decreased in the heterozygotes when compared to unaffected members. We conclude that the low LCAT activity is the likely cause of the qualitative and quantitative differences in the plasma lipoproteins of the heterozygotes in this family with LCAT deficiency. However, the low HDL and apo A-I levels are not associated with either a family or personal history of premature atherosclerosis.

The analysis of UV spectra of human platelet particulates showed the existence of an evident protein component ordered in alpha-helical conformation in normal subjects. This ordered component was enhanced in type IIa hyperbetalipoproteinemia and, on the contrary, very low in a case of heterozygous familial hypobetalipoproteinemia. In the latter condition almost all proteins were indeed disordered as random coils. These data thus suggest that cholesterol significantly contributes to the control of molecular arrangement of platelet membrane proteins.

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.

Structural, compositional, and functional abnormalities were found in the erythrocyte membranes of homozygotes for LCAT deficiency. Similar but less pronounced abnormalities were also present in the heterozygotes for this disorder. Some of the membrane alterations, which included decreased osmotic fragility, changes in phospholipid composition, and membrane sulfydryl group latency, as well as changes in the activity of membrane p-nitrophenylphosphatases and acetylcholinesterase, may be secondary to the changes in plasma lipids. However, since plasma lipids (and LCAT activity) were normal in the hereozygotes, the existence in both the homo- and the heterozygotes of erythrocyte membrane abnormalities unrelated to plasma LCAT activity seems likely.

We determined cholesteryl ester transfer activity in whole plasma and in lipoprotein-depleted plasma of normolipidemic subjects and of patients with severe high-density lipoprotein (HDL) deficiency: Tangier disease, lecithin:cholesterol acyltransferase (LCAT) deficiency, and "fish-eye" disease. Transfer rates in plasma were positively correlated (r = 0.950) with rates measured in the absence of the endogenous lipoproteins. This suggests that lipoprotein composition and content may not affect total cholesteryl ester transfer activity in normolipidemic and the HDL-deficient subjects. Cholesteryl ester transfer from solid-phase-bound HDL to plasma lipoproteins was decreased by 39% in fish-eye disease and 33% in LCAT deficiency but increased by 57% in Tangier disease, as compared with normal values. Changes were similar for lipoprotein-depleted plasma from the same individuals. Transfer to plasma HDL was significantly decreased in all HDL-deficient patients, whereas transfer to very-low- and low-density lipoproteins was increased only in Tangier disease. Differences in transfer rates between the patients studied appeared to reflect the LCAT activity and the need to transport cholesteryl ester rather than the HDL cholesterol concentration. Thus, the concentration of HDL in plasma does not directly affect total cholesteryl ester transfer activity in HDL deficiency.

Tangier disease is characterized by a deficiency of high-density lipoproteins and of their major protein constituent, apolipoprotein (apo) A-I. We used high-resolution two-dimensional electrophoresis to examine the principal plasma apolipoproteins (A-I, A-II, A-IV, E, C-II, and C-III) of three persons with Tangier disease, one homozygous patient and his two heterozygous children, comparing the patterns with those for healthy subjects. Characteristic abnormalities were found in the distribution of the isoproteins of apo A-I, there being a normal concentration of pro apo A-I but dramatically decreased concentrations of the other apo A-I isoproteins. We also found hitherto-undescribed polypeptide abnormalities in apo C-III: sialylated and nonsialylated forms of apo C-III appear as double spots having the same isoelectric points but different molecular masses. No other substantial difference was detected in the polypeptide distribution of the other plasma apolipoproteins.

Low levels of high-density lipoprotein cholesterol (HDL-C) are associated with increased risk of coronary heart disease (CHD). Therefore, assessment of the mode of inheritance of HDL-C is of importance. HDL-C concentrations in 3,074 nuclear families in the multiethnic Jerusalem Lipid Research Center study population were analyzed for possible involvement of major genes in determination of low levels of this trait. Complex segregation analysis under the mixed model of inheritance (major gene and multifactorial components) was performed on transformed HDL-C concentrations after adjustment for age, sex, and environmental measures. Evidence for segregation of a recessive major gene for depressed HDL-C, with an allele frequency of q = 0.06, in addition to multifactorial transmission (H = 0.45) was found in these families. Estimates from the mixed model were homogeneous across the different ethnic groups. When the substantial multifactorial background was excluded from the model, we found evidence for an additive (codominant) Mendelian gene (d = 0.48), which demonstrates the necessity of using the mixed model. Our previous results were inconclusive with respect to the involvement of a major gene in determination of high levels of HDL-C. However, we tentatively postulate an uncommon recessive gene for low levels of HDL-C in the Israeli population in addition to polygenic and environmental determinants.

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.