Familial apolipoprotein CII deficiency: plasma lipoproteins and apolipoproteins in heterozygous and homozygous subjects and the effects of plasma infusion

Abstract. Plasma lipoproteins and apolipoproteins have been studied in a kindred with familial apolipoprotein CII (apo CII) deficiency. As in two other recently documented pedigrees, apo CII deficiency appeared to be transmitted as an autosomal recessive trait. The homozygous state was characterized by gross fasting hypertriglyceridaemia, complete absence of apo CII from plasma and failure of plasma to activate lipoprotein lipase. Post-heparin plasma hepatic triglyceride lipase activity was normal. Hypertriglyceridaemia reflected chylomicronaemia and elevated Sf 100–400 and Sf 20–100 lipoprotein concentrations; lipoproteins of Sf 12–20 (LDL₁), Sf 0–12 (LDL₂), F_1.23.5–9 (HDL₂) and F_1.20–3.5 (HDL₃) were greatly reduced in concentration. Low density lipoproteins (1.006–1.063 g/ml), isolated by preparative ultracentrifugation, and high density lipoproteins, isolated by heparin/Mn⁺⁺, were triglyceride-enriched. Electroimmunoassays revealed additionally low plasma concentrations of apolipoproteins AI, AII and B and very high concentrations of apolipoproteins CIII and E in the homozygote. The parents of the proband (heterozygotes) were normotriglyceridaemic, and had normal lipoprotein lipid concentrations and normal apolipoprotein AI, AII, B, CIII and E concentrations, in spite of having low apo CII concentrations. Activation of lipoprotein lipase in the homozygote by intravenous infusion of 200 ml fresh-frozen plasma rapidly reduced the plasma concentrations of chylomicrons and very low density lipoproteins (VLDL). Within VLDL, the decrease in concentration occurred sequentially in the Sf 100–400 and Sf 20–100 subclasses. These changes were associated during a 4-day study period with reciprocal increases in LDL₁, LDL₂, HDL₂ and HDL₃. The plasma concentrations of apo AI and apo B also increased, associated with a less marked fall in that of apo CIII; the apo AII and apo E concentrations were unchanged. These observations support other evidence that apo CII is a cofactor for the catabolism of chylomicrons and both major subfractions of VLDL by lipoprotein lipase in man, and that human LDL₁ and LDL₂ are derived, at least in part, from triglyceride-rich lipoprotein catabolism. They also suggest that both major subfractions of HDL acquire additional components during triglyceride-rich lipoprotein catabolism. In normal subjects the plasma apo CII concentration appears to be greatly in excess of that required for adequate activation of lipoprotein lipase.     

Apolipoprotein B metabolism in subjects with deficiency of apolipoproteins CIII and AI. Evidence that apolipoprotein CIII inhibits catabolism of triglyceride-rich lipoproteins by lipoprotein lipase in vivo.

Previous data suggest that apolipoprotein (apo) CIII may inhibit both triglyceride hydrolysis by lipoprotein lipase (LPL) and apo E-mediated uptake of triglyceride-rich lipoproteins by the liver. We studied apo B metabolism in very low density (VLDL), intermediate density (IDL), and low density lipoproteins (LDL) in two sisters with apo CIII-apo AI deficiency. The subjects had reduced levels of VLDL triglyceride, normal LDL cholesterol, and near absence of high density lipoprotein (HDL) cholesterol. Compartmental analysis of the kinetics of apo B metabolism after injection of 125I-VLDL and 131I-LDL revealed fractional catabolic rates (FCR) for VLDL apo B that were six to seven times faster than normal. Simultaneous injection of [3H]glycerol demonstrated rapid catabolism of VLDL triglyceride. VLDL apo B was rapidly and efficiently converted to IDL and LDL. The FCR for LDL apo B was normal. In vitro experiments indicated that, although sera from the apo CIII-apo-AI deficient patients were able to normally activate purified LPL, increasing volumes of these sera did not result in the progressive inhibition of LPL activity demonstrable with normal sera. Addition of purified apo CIII to the deficient sera resulted in 20-50% reductions in maximal LPL activity compared with levels of activity attained with the same volumes of the native, deficient sera. These in vitro studies, together with the in vivo results, indicate that in normal subjects apo CIII can inhibit the catabolism of triglyceride-rich lipoproteins by lipoprotein lipase.     

Effects of apolipoproteins on lipoprotein lipase activity of human adipose tissue.

The effect of apolipoproteins isolated from HDL and VLDL on the activity of lipoprotein lipase (LPL) of adipose tissue was studied. The CII apoprotein was found to activate LPL. This activation was strongly inhibited by CI, AI (apo-Lp-Gln I), and the arginine-rich apoprotein, whereas AII and CIII exhibited a considerably lower inhibitor effect.     

Catabolism of very low density lipoproteins in experimental nephrosis

The effects of experimental nephrosis in rats, produced by puromycin aminonucleoside, include an elevation of plasma levels of all lipoprotein density classes and the appearance of high density lipoprotein (HDL) rich in apoprotein (apo) A-I and deficient in apo A-IV and apo E. The hyperlipoproteinemia is associated with an increase in hepatic synthesis of lipoproteins. The possible role of decreased very low density lipoprotein (VLDL were obtained from nonfasting animals by ultracentrifugation at d 1.006 and included chylomicrons) catabolism and its relationship to the apolipoprotein composition of nephrotic high density lipoproteins (1.063 less than d less than 1.210, or 1.072 less than d less than 1.210 [HDL]) was explored. When 125I-VLDL was injected, the faster plasma clearance of lower molecular weight apolipoprotein B (apo BL) compared with that of higher molecular weight apo BH which is seen in normal rats was not observed in nephrotic rats. Less labeled phospholipid, apo C, and apo E were transferred from VLDL to higher lipoprotein density classes. Heparin-releasable plasma lipoprotein lipase and hepatic lipase activities were decreased by 50% in nephrotic rats compared with pair-fed controls. Perfusion of livers with medium that contained heparin released 50% less lipase activity in nephrotic rats than in controls. When heparin was injected intravenously, significant decreases in plasma levels of triglycerides and significant increases in levels of free fatty acids were observed in both groups of animals. In the nephrotic rats, 86% of the free fatty acids were in the lipoprotein fractions, as compared with 16% in the controls. Heparin treatment did not restore to normal the decreased apo BL clearance in nephrotic rats but it produced an increased amount of apo A-IV and apo E in the plasma HDL. In vitro addition of partially pure lipoprotein lipase to whole serum from nephrotic rats significantly increased the content of apo E in HDL. We conclude that the abnormal apoprotein composition of HDL in experimental nephrosis is the result of altered entry of apolipoproteins from triglyceride-rich lipoproteins, probably because of decreased lipolysis.     

Composition of very low density lipoproteins and in vitro effect of lipoprotein lipase.

In order to clarify the relationship between composition and lipolytic responses to lipoprotein lipase (LPL), very low density lipoproteins (VLDL) from rats or humans were incubated with a commercially available LPL or with a partially purified LPL from postheparin human plasma and fatty acids released from VLDL were determined in vitro. VLDL from rats fed a diet containing 0.25% cholesterol for 6 months were rich in cholesterol and poor in triglycerides, and released less fatty acids from incubation with LPL than those from control rats. VLDL from normo-and hypertriglyceridemic human subjects were incubated with LPL. The fatty acid release poorly correlated with the apoprotein ratios of VLDL, apo C-III/C-II, B/E, and C/E with the exception of apo B/C, but it correlated well with the ratio of triglyceride/either one of the surface components including total apoproteins, free cholesterol and phospholipids in VLDL or the ratio of the triglyceride/total sum of the surface components. The correlation coefficients between fatty acid release and a ratio of triglyceride/total surface components were 0.774 (using the commercially available LPL) and 0.786 (using the partially purified human LPL). The fatty acid release increased after pretreatment of VLDL with phospholipase A2. The phospholipid content of VLDL was reduced without significant changes in other VLDL components. Thus, the responses of VLDL to LPL treatment may depend mainly upon the surface: core relationship of VLDL rather than its apoprotein composition except in rare clinical cases such as apo C-II deficiency.     

Mechanism of action of gemfibrozil on lipoprotein metabolism.

Gemfibrozil is a potent lipid regulating drug whose major effects are to increase plasma high density lipoproteins (HDL) and to decrease plasma triglycerides (TG) in a wide variety of primary and secondary dyslipoproteinemias. Its mechanism of action is not clear. Six patients with primary familial endogenous hypertriglyceridemia with fasting chylomicronemia (type V lipoprotein phenotype) with concurrent subnormal HDL cholesterol levels (HDL deficiency) were treated initially by diet and once stabilized, were given gemfibrozil (1,200 mg/d). Each patient was admitted to the Clinical Research Center with metabolic kitchen facilities, for investigation of HDL and TG metabolism immediately before and after 8 wk of gemfibrozil treatment. Gemfibrozil significantly increased plasma HDL cholesterol, apolipoprotein (apo) AI, and apo AII by 36%, 29%, and 38% from base line, respectively. Plasma TG decreased by 54%. Kinetics of apo AI and apo AII metabolism were assessed by analysis of the specific radioactivity decay curves after injection of autologous HDL labeled with 125I. Gemfibrozil increased synthetic rates of apo AI and apo AII by 27% and 34%, respectively, without changing the fractional catabolic rates. Stimulation of apo AI and apo AII synthesis by gemfibrozil was associated with the appearance in plasma of smaller (and heavier) HDL particles as assessed by gradient gel electrophoresis and HDL composition. Postheparin extra-hepatic lipoprotein lipase activity increased significantly by 25% after gemfibrozil, and was associated with the appearance in plasma of smaller very low density lipoprotein particles whose apo CIII:CII ratio was decreased. These data suggest that gemfibrozil increases plasma HDL levels by stimulating their synthesis. Increased transport (turnover) of HDL induced by gemfibrozil may be significant in increasing tissue cholesterol removal in these patients.     

Fat feeding in humans induces lipoproteins of density less than 1.006 that are enriched in apolipoprotein [a] and that cause lipid accumulation in macrophages.

Formula diets containing lard or lard and egg yolks were fed to six normolipidemic volunteers to investigate subsequent changes in the composition of lipoproteins of d less than 1.006 g/ml and in their ability to bind and be taken up by receptors on mouse macrophages. Both formulas induced the formation of d less than 1.006 lipoproteins that were approximately 3.5-fold more active than fasting very low density lipoproteins (VLDL) in binding to the receptor for beta-VLDL on macrophages. Subfractionation of postprandial d less than 1.006 lipoproteins by agarose chromatography yielded two subfractions, fraction I (chylomicron remnants) and fraction II (hepatic VLDL remnants), which bound to receptors on macrophages. However, fraction I lipoproteins induced a 4.6-fold greater increase in macrophage triglyceride content than fraction II lipoproteins or fasting VLDL. Fraction I lipoproteins were enriched in apolipoproteins (apo) B48, E, and [a]. Fraction II lipoproteins lacked apo[a] but possessed apo B100 and apo E. The apo[a] was absent in normal fasting VLDL, but was present in the d less than 1.006 lipoproteins (beta-VLDL) of fasting individuals with type III hyperlipoproteinemia. The apo[a] from postprandial d less than 1.006 lipoproteins was larger than either of two apo[a] subspecies obtained from lipoprotein (a) [Lp(a)] isolated at d = 1.05-1.09. However, all three apo[a] subspecies were immunochemically identical and had similar amino acid compositions: all were enriched in proline and contained relatively little lysine, phenylalanine, isoleucine, or leucine. The association of apo[a] with dietary fat-induced fraction I lipoproteins suggests that the previously observed correlation between plasma Lp(a) concentrations and premature atherosclerosis may be mediated, in part, by the effect of apo[a] on chylomicron remnant metabolism.     

Relationships between the metabolism of high-density and very-low-density lipoproteins in man: studies of apolipoprotein kinetics and adipose tissue lipoprotein lipase activity

Abstract. In order to gain further insight into the relationship between high-density lipoprotein (HDL) metabolism and plasma triglyceride transport, measurements were made of HDL cholesterol concentration, apoprotein (apo) AI and AII metabolism, very-low-density lipoprotein (VLDL) apo B metabolism, and heparin-elutable adipose tissue lipoprotein lipase (LPL) activity in seventeen subjects with a wide range of plasma triglyceride concentrations (0.8–25 mmol/l).
The fractional catabolic rate (FCR) of VLDL apo B was directly related to LPL activity ( r =+ 0.80), providing evidence that the activity of the enzyme in adipose tissue is a determinant of the rate of lipolysis of VLDL in man. HDL cholesterol concentration was a positive function of both VLDL apo B FCR ( r =+ 0.74) and LPL activity, a finding consistent with previous evidence for the origin of a proportion of HDL cholesterol from 'surface remnants' liberated during VLDL catabolism. The FCRs of both apo AI and apo AII were inversely related to VLDL apo B FCR (AI, r = - 0.52; AII, r = - 0.69) and to LPL activity. The synthetic rate of apo AII, but not that of apo AI, was positively correlated with VLDL apo B synthesis ( r =+ 0.71). Thus, the metabolism of the major proteins of HDL in man appears to be closely associated with VLDL metabolism.     

Evaluation of the roles of lipoprotein lipase and hepatic lipase in lipoprotein metabolism: in vivo and in vitro studies in man

Abstract. The roles of lipoprotein lipase (LPL) and hepatic lipase in very low density lipoprotein (VLDL) and VLDL remnant metabolism were investigated by (1) in vivo studies where the kinetics of VLDL-apo B removal were measured in patients with non-functioning lipoprotein lipase systems, and (2) in vitro studies where the relative capacities of hepatic lipase and LPL to hydrolyse the triglyceride (TG) of different lipoprotein substrates was measured. The results indicated that VLDL-apo B removal was not impaired in patients with non-functional LPL, nor was there any apparent abnormality in the conversion of VLDL-apo B to intermediate- (IDL) and low (LDL) density lipoprotein-apo B. Post-heparin plasma hepatic lipase activity against VLDL was normal in these subjects. Purified normal hepatic lipase had a similar K_m for VLDL-TG hydrolysis (1.57 mmol/l) to that of LPL (1.49 mmol/l). However, at equal lipoprotein TG concentration, hepatic lipase had increasing activity with lipoproteins of decreasing particle size, in the order chylomicrons ≪ VLDL of Sf 100–400 < VLDL of Sf 60–100 < VLDL of Sf 20–60 < IDL. The mean contribution of hepatic lipase to VLDL-TG hydrolysis by post-heparin plasma was 35% in normal controls, but the contribution to IDL-TG hydrolysis was significantly higher (mean = 58%). It is concluded that hepatic lipase plays a significant role in VLDL and, especially, IDL metabolism, at least in patients with non-functioning lipoprotein lipase.     

Distribution of apolipoprotein(a) in the plasma from patients with lipoprotein lipase deficiency and with type III hyperlipoproteinemia. No evidence for a triglyceride-rich precursor of lipoprotein(a).

Lipoprotein(a) consists of a low-density lipoprotein containing apolipoprotein (apo) B-100 and of the genetically polymorphic apo(a). It is not known where and how lipoprotein(a) is assembled and whether there exists a precursor for lipoprotein(a). We have determined the phenotype, concentration, and distribution of apo(a) in plasma from patients with lipoprotein lipase (LPL) deficiency (type I hyperlipoproteinemia, n = 14), in apo E 2/2 homozygotes with type III hyperlipoproteinemia (n = 12) and in controls (n = 16). In the two genetic conditions, there is grossly impaired catabolic conversion of apo B-100-containing precursor lipoproteins to low-density lipoproteins. Considering apo(a) type, the plasma concentration of apo(a) was normal in type III patients but significantly reduced in LPL deficiency. Despite the defects in the catabolism of other apo B-containing lipoproteins, the distribution of apo(a) was only moderately affected in both metabolic disorders, with 66.7% (type I) and 74.7% (type III) being present as the characteristic lipoprotein(a) in the density range of 1.05-1.125 g/ml (controls 81.6%). The remainder was distributed between the triglyceride-rich lipoproteins (type I 12.4%, type III 8.5%, controls 4.7%) and the lipid-poor bottom fraction (type I 19.3%, type III 15.3%, controls 12.6%). In all conditions most apo(a) (57-88%) dissociated from the triglyceride-rich lipoproteins upon recentrifugation and was recovered as lipoprotein(a). These data suggest that lipoprotein(a) is not generated from a triglyceride-rich precursor. Lipoprotein(a) may be secreted directly into plasma or may be formed by preferential binding of secreted apo(a) to existing low-density lipoprotein.     

The role of hepatic lipase in lipoprotein metabolism.

Hepatic lipase (HL) is one of two major lipases released from the vascular bed by intravenous injection of heparin. HL hydrolyzes phospholipids and triglycerides of plasma lipoproteins and is a member of a lipase superfamily that includes lipoprotein lipase and pancreatic lipase. The enzyme can be divided into an NH2-terminal domain containing the catalytic site joined by a short spanning region to a smaller COOH-terminal domain. The NH2-terminal portion contains an active site serine in a pentapeptide consensus sequence, Gly-Xaa-Ser-Xaa-Gly, as part of a classic Ser-Asp-His catalytic triad, and a putative hinged loop structure covering the active site. The COOH-terminal domain contains a putative lipoprotein-binding site. The heparin-binding sites may be distributed throughout the molecule, with the characteristic elution pattern from heparin-sepharose determined by the COOH-terminal domain. Of the three N-linked glycosylation sites, Asn-56 is required for efficient secretion and enzymatic activity. HL is hypothesized to directly couple HDL lipid metabolism to tissue/cellular lipid metabolism. The potential significance of the HL pathway is that it provides the hepatocyte with a mechanism for the uptake of a subset of phospholipids enriched in unsaturated fatty acids and may allow the uptake of cholesteryl ester, free cholesterol and phospholipid without catabolism of HDL apolipoproteins. HL can hydrolyze triglyceride and phospholipid in all lipoproteins, but is predominant in the conversion of intermediate density lipoproteins to LDL and the conversion of post-prandial triglyceride-rich HDL into the post-absorptive triglyceride-poor HDL. It has been suggested that enzymatically inactive HL can play a role in hepatic lipoprotein uptake forming a 'bridge' by binding to the lipoprotein and to the cell surface. This raises the interesting possibility that production and secretion of mutant inactive HL could promote clearance of VLDL remnants. We have described a rare family with HL deficiency. Affected patients are compound heterozygotes for a mutation of Ser267Phe that causes an inactive enzyme and a mutation of Thr383Met that results in impaired secretion of HL and reduced specific activity. Human HL deficiency in the context of a second factor causing hyperlipidemia is strongly associated with premature coronary artery disease.     

Bovine milk lipoprotein lipase transfers tocopherol to human fibroblasts during triglyceride hydrolysis in vitro.

Lipoprotein lipase appears to function as the mechanism by which dietary vitamin E (tocopherol) is transferred from chylomicrons to tissues. In patients with lipoprotein lipase deficiency, more than 85% of both the circulating triglyceride and tocopherol is contained in the chylomicron fraction. The studies presented here show that the in vitro addition of bovine milk lipoprotein lipase (lipase) to chylomicrons in the presence of human erythrocytes or fibroblasts (and bovine serum albumin [BSA]) resulted in the hydrolysis of the triglyceride and the transfer of both fatty acids and tocopherol to the cells; in the absence of lipase, no increase in cellular tocopherol was detectable. The incubation system was simplified to include only fibroblasts, BSA, and Intralipid (an artificial lipid emulsion containing 10% soybean oil, which has gamma but not alpha tocopherol). The addition of lipase to this system also resulted in the transfer of tocopherol (gamma) to the fibroblasts. Addition of both lipase and its activator, apolipoprotein CII, resulted in a further increase in the cellular tocopherol content, but apolipoprotein CII alone had no effect. Heparin, which is known to prevent the binding of lipoprotein lipase to the cell surface membrane, abrogated the transfer of tocopherol to fibroblasts without altering the rate of triglyceride hydrolysis. Thus, in vitro tocopherol is transferred to cells during hydrolysis of triglyceride by the action of lipase, and for this transfer of tocopherol to occur, the lipase itself must bind to the cell membrane.     

Hydrolysis of phospholipids by purified milk lipoprotein lipase. Effect of apoprotein CII, CIII, A and E, and synthetic fragments

Different pyrene-labeled phospholipid monolayer vesicles were used as substrates for the bovine milk lipoprotein lipase activity. The effects of synthetic fragments of apoprotein C II were measured on the hydrolysis of 1-myristoyl-2[9(1pyrenyl)-nonanoyl] phosphatidylcholine in vesicles: The activating capacity of fragments 30-78 and 43-78, 50-78 and 55-78, compared to entire apo CII, were similar to that obtained with hydrolysable triglycerides. Our study shows that the longer the carboxy terminal fragment is, the higher is the activation. The phospholipid hydrolysis activity represents in the presence of apo C II, 36% of the triglycerides hydrolysis activity. Phospholipid hydrolysis is less dependent on activator than triglycerides hydrolysis (100% and 300% of increase with apo CII for phosphatidyl-choline and triglycerides respectively). The ratio hydrolysis without apo C II/hydrolysis with apo CII was different when other phospholipids than myrystoyl-phospatidylcholine were assayed: phosphatidyl-serine, ethanolamine, -choline, -glycerol, or diglycerides and butanoylglycerols. Fragment CIII(1) (1-40) which did not bind to lipids, had no inhibitory effect. The entire sugar moiety and the first 40 amino acids are not required for the total inhibition of LPL. Inhibition was also obtained with Apo A I, A II,C I and fragments of apo E.     

Combined hyperlipidemia in transgenic mice overexpressing human apolipoprotein Cl.

We have generated transgenic mice over-expressing human apolipoprotein CI (apo CI) using the native gene joined to the downstream 154-bp liver-specific enhancer that we defined for apo E. Human apo CI (HuCI)-transgenic mice showed elevation of plasma triglycerides (mg/dl) compared to controls in both the fasted (211 +/- 81 vs 123 +/- 52, P = 0.0001) and fed (265 +/- 105 vs 146 +/- 68, P < 0.0001) states. Unlike the human apo CII (HuCII)- and apo CIII (HuCIII)-transgenic mouse models of hypertriglyceridemia, plasma cholesterol was disproportionately elevated (95 +/- 23 vs 73 +/- 23, P = 0.002, fasted and 90 +/- 24 vs 61 +/- 14, P < 0.0001, fed). Lipoprotein fractionation showed increased VLDL and IDL + LDL with an increased cholesterol/triglyceride ratio (0.114 vs 0.065, P = 0.02, in VLDL). The VLDL apo E/apo B ratio was decreased 3.4-fold (P = 0.05) and apo CII and apo CIII decreased in proportion to apo E. Triglyceride and apo B production rates were normal, but clearance rates of VLDL triglycerides and postlipolysis lipoprotein "remnants" were significantly slowed. Plasma apo B was significantly elevated. Unlike HuCII- and HuCIII-transgenic mice, VLDL from HuCI transgenic mice bound heparin-Sepharose, a model for cell-surface glycosaminoglycans, normally. In summary, apo CI overexpression is associated with decreased particulate uptake of apo B-containing lipoproteins, leading to increased levels of several potentially atherogenic species, including cholesterol-enriched VLDL, IDL, and LDL.     

Apoprotein profile of plasma and chylous ascites lipoproteins.

Plasma and chylous ascites lipoproteins were compared in a rare case of exudative enteropathy associated with stenosis of the thoracic duct. All ascites lipoproteins separated by ultracentrifugation showed a much higher triglyceride/protein ratio and a lower cholesterol ester content than their plasma counterparts. Polyarcylamide gel electrophoresis of apoproteins before and after fractionation on Sephadex G-200 essentially showed, compared to normal plasma, a reduction of apoprotein C in VLDL and HDL particularly obvious for apoCIII1 and CIII2. Ascites lipoproteins were characterized by an increased apoA content in chylomicrons, VLDL and LDL and a reduced percentage of apo CII and CIII1, mostly in chylomicrons, VLDL and HDL. The differences in composition between plasma and ascites might be explained by different origins for the various apoprotein subfractions. The transsudation of chyle into the abdominal cavity might divert some peptides of intestinal origin from their normal entry into the circulation.     

Role of lipoprotein lipase in the regulation of high density lipoprotein apolipoprotein metabolism. Studies in normal and lipoprotein lipase-inhibited monkeys.

Mechanisms that might be responsible for the low levels of high density lipoprotein (HDL) associated with hypertriglyceridemia were studied in an animal model. Specific monoclonal antibodies were infused into female cynomolgus monkeys to inhibit lipoprotein lipase (LPL), the rate-limiting enzyme for triglyceride catabolism. LPL inhibition produced marked and sustained hypertriglyceridemia, with plasma triglyceride levels of 633-1240 mg/dl. HDL protein and cholesterol and plasma apolipoprotein (apo) AI levels decreased; HDL triglyceride (TG) levels increased. The fractional catabolic rate of homologous monkey HDL apolipoproteins injected into LPL-inhibited animals (n = 7) was more than double that of normal animals (0.094 +/- 0.010 vs. 0.037 +/- 0.001 pools of HDL protein removed per hour, average +/- SEM). The fractional catabolic rate of low density lipoprotein apolipoprotein did not differ between the two groups of animals. Using HDL apolipoproteins labeled with tyramine-cellobiose, the tissues responsible for this increased HDL apolipoprotein catabolism were explored. A greater proportion of HDL apolipoprotein degradation occurred in the kidneys of hypertriglyceridemic than normal animals; the proportions in liver were the same in normal and LPL-inhibited monkeys. Hypertriglyceridemia due to LPL deficiency is associated with low levels of circulating HDL cholesterol and apo AI. This is due, in part, to increased fractional catabolism of apo AI. Our studies suggest that variations in the rate of LPL-mediated lipolysis of TG-rich lipoproteins may lead to differences in HDL apolipoprotein fractional catabolic rate.     

Familial apolipoprotein AI and apolipoprotein CIII deficiency. Subclass distribution,composition, and morphology of lipoproteins in a disorder associated with premature atherosclerosis.

Lipoprotein classes isolated from the plasma of two patients with apolipoprotein AI (apo AI) and apolipoprotein CIII (apo CIII) deficiency were characterized and compared with those of healthy, age- and sex-matched controls. The plasma triglyceride values for patients 1 and 2 were 31 and 51 mg/dl, respectively, and their cholesterol values were 130 and 122 mg/dl, respectively; the patients, however, had no measurable high density lipoprotein (HDL)-cholesterol. Analytic ultracentrifugation showed that patients'' S degrees f 0-20 lipoproteins possess a single peak with S degrees f rates of 7.4 and 7.6 for patients 1 and 2, respectively, which is similar to that of the controls. The concentration of low density lipoprotein (LDL) (S degrees f 0-12) particles, although within normal range (331 and 343 mg/dl for patients 1 and 2, respectively), was 35% greater than that of controls. Intermediate density lipoproteins (IDL) and very low density lipoproteins (VLDL) (S degrees f 20-400) were extremely low in the patients. HDL in the patients had a calculated mass of 15.4 and 11.8 mg/dl for patients 1 and 2, respectively. No HDL could be detected by analytic ultracentrifugation, but polyacrylamide gradient gel electrophoresis (gge) revealed that patients possessed two major HDL subclasses: (HDL2b)gge at 11.0 nm and (HDL3b)gge at 7.8 nm. The major peak in the controls, (HDL3a)gge, was lacking in the patients. Gradient gel analysis of LDL indicated that patients'' LDL possessed two peaks: a major one at 27 nm and a minor one at 26 nm. The electron microscopic structure of patients'' lipoprotein fractions was indistinguishable from controls. Patients'' HDL were spherical and contained a cholesteryl ester core, which suggests that lecithin/cholesterol acyltransferase was functional in the absence of apo AI. The effects of postprandial lipemia (100-g fat meal) were studied in patient 1. The major changes were the appearance of a 33-nm particle in the LDL density region of 1.036-1.041 g/ml and the presence of discoidal particles (12% of total particles) in the HDL region. The latter suggests that transformation of discs to spheres may be delayed in the patient. The simultaneous deficiency of apo AI and apo CIII suggests a dual defect in lipoprotein metabolism: one in triglyceride-rich lipoproteins and the other in HDL. The absence of apo CIII may result in accelerated catabolism of triglyceride-rich particles and an increased rate of LDL formation. Additionally, absence of apo CIII would favor rapid uptake of apo E-containing remnants by liver and peripheral cells. Excess cellular cholesterol would not be removed by the reverse cholesterol transport mechanism since HDL levels are exceedingly low and thus premature atherosclerosis occurs.     

Concentrations of apoprotein CII, CIII, and E in total serum and in the apoprotein B-containing lipoproteins, determined by a new enzyme-linked immunosorbent assay

We describe a new enzyme-linked immunosorbent assay (ELISA) that permits direct determination of apoprotein (apo) CII, CIII, and E in total serum as well as in apo B-containing lipoprotein particles. To validate this ELISA technique, we studied several aspects of the assay: its specificity, the influence of the conditions of conservation of plasma and of lipoprotein fractions, the effect of delipidation, and its reproducibility. We measured the concentrations of apo CII, CIII, and E in total serum and in apo B-containing lipoproteins from a pool of normal sera and in sera from 75 healthy subjects. After sequential ultracentrifugation, the content of apo CII, CIII, and E in the major lipoprotein fractions was also determined. Total serum or plasma could be stored at -20 or -50 degrees C for at least six weeks and the isolated lipoprotein fractions for as long as four weeks, which suggests a protective effect of total serum on lipoprotein particle structure. Advantages of this ELISA include (a) its specificity, sensitivity, and reliability; (b) better discrimination than determination of total serum apoprotein; (c) easier application and greater rapidity; and (d) the possibility of application to population screening.     

Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles.

Hypertriglyceridemia is common in the general population, but its mechanism is largely unknown. In previous work human apo CIII transgenic (HuCIIITg) mice were found to have elevated triglyceride levels. In this report, the mechanism for the hypertriglyceridemia was studied. Two different HuCIIITg mouse lines were used: a low expressor line with serum triglycerides of approximately 280 mg/dl, and a high expressor line with serum triglycerides of approximately 1,000 mg/dl. Elevated triglycerides were mainly in VLDL. VLDL particles were 1.5 times more triglyceride-rich in high expressor mice than in controls. The total amount of apo CIII (human and mouse) per VLDL particle was 2 and 2.5 times the normal amount in low and high expressors, respectively. Mouse apo E was decreased by 35 and 77% in low and high expressor mice, respectively. Under electron microscopy, VLDL particles from low and high expressor mice were found to have a larger mean diameter, 55.2 +/- 16.6 and 58.2 +/- 17.8 nm, respectively, compared with 51.0 +/- 13.4 nm from control mice. In in vivo studies, radiolabeled VLDL fractional catabolic rate (FCR) was reduced in low and high expressor mice to 2.58 and 0.77 pools/h, respectively, compared with 7.67 pools/h in controls, with no significant differences in the VLDL production rates. In an attempt to explain the reduced VLDL FCR in transgenic mice, tissue lipoprotein lipase (LPL) activity was determined in control and high expressor mice and no differences were observed. Also, VLDLs obtained from control and high expressor mice were found to be equally good substrates for purified LPL. Thus excess apo CIII in HuCIIITg mice does not cause reduced VLDL FCR by suppressing the amount of extractable LPL in tissues or making HuCIIITg VLDL a bad substrate for LPL. Tissue uptake of VLDL was studied in hepatoma cell cultures, and VLDL from transgenic mice was found to be taken up much more slowly than control VLDL (P < 0.0001), indicating that HuCIIITg VLDL is not well recognized by lipoprotein receptors. Additional in vivo studies with Triton-treated mice showed increased VLDL triglyceride, but not apo B, production in the HuCIIITg mice compared with controls. Tissue culture studies with primary hepatocytes showed a modest increase in triglyceride, but not apo B or total protein, secretion in high expressor mice compared with controls. In summary, hypertriglyceridemia in HuCIIITg mice appears to result primarily from decreased tissue uptake of triglyceride-rich particles from the circulation, which is most likely due to increased apo CIII and decreased apo E on VLDL particles. the HuCIIITg mouse appears to be a suitable animal model of primary familial hypertriglyceridemia, and these studies suggest a possible mechanism for this common lipoprotein disorder.     

Abnormal concentrations of CII, CIII, and E apolipoproteins among apolipoprotein B-containing, B-free, and A-I-containing lipoprotein particles in hemodialysis patients

Serum concentrations of total cholesterol, triglycerides, and apolipoproteins (apo) A-I, B, CII, CIII, and E in 36 hemodialysis patients and nine anephric patients were compared with the concentrations in 34 normolipidemic subjects. The dialysis patients displayed a moderate hypertriglyceridemia (1.94 +/- 0.12 vs 1.09 +/- 0.11 mmol/L in controls, mean +/- SEM; P less than 0.001), apo CIII concentrations were also increased (130.2 +/- 2.1 vs 108.4 +/- 0.7 mg/L; P less than 0.001), whereas apo CII (34.5 +/- 0.5 vs 36 +/- 0.5 mg/L; P less than 0.05), apo E (22.7 +/- 0.3 vs 27.9 +/- 0.2 mg/L; P less than 0.001), and apo A-I (1.18 +/- 0.05 vs 1.31 +/- 0.04 g/L; P less than 0.05) were decreased. Concentrations of serum apo B were normal (0.86 +/- 0.03 vs 0.97 +/- 0.07 g/L). In the hemodialysis patients, apo CIII concentrations were increased in apo B-containing lipoproteins (30.1 +/- 0.5 vs 25.0 +/- 0.1 mg/L; P less than 0.001), whereas CII and E were decreased below control values (14.4 +/- 0.2 vs 16.8 +/- 0.1, and 8.2 +/- 0.2 vs 11.4 +/- 0.1 mg/L, respectively; P less than 0.001 each). By calculation, non-B-containing lipoproteins in the hemodialysis group had increased concentrations of apo CIII (100.1 +/- 2.1 vs 83.3 +/- 0.7 mg/L; P less than 0.001) and decreased amounts of apo E (14.5 +/- 0.4 vs 16.4 +/- 0.3 mg/L; P less than 0.001); apo CII content was unchanged (20.1 +/- 0.5 vs 19.3 +/- 0.5 mg/L). Results for apo CII, CIII, and E among apo A-I-containing lipoproteins in both normolipidemic and hemodialysis groups were similar to those in non-B-containing lipoproteins. Finally, the sole significant (P less than 0.01) difference between the anephric and hemodialysis groups was the lower apo E concentrations in the former group. Accumulation of triglyceride-rich lipoproteins in hemodialysis patients may thus be related to the enrichment of apo CIII in apo B-containing lipoproteins and to a marked decrease in the apo CII and E contents.