Apolipoprotein C-ll deficiency syndrome due to apo C-IIHamburg: clinical and biochemical features and Hphl restriction enzyme polymorphism

We have characterized the clinical and biochemical features of three siblings of a kindred with severe hypertriglyceridaemia due to apolipoprotein C-II (apo C-II) deficiency caused by the mutation described as apo C-IIHamburg. The clinical syndrome is characterized by recurrent pancreatitis in two of three affected individuals, with discrete hepatosplenomegaly in all three patients and cholelithiasis in one. Eruptive xanthomas and lipemia retinalis were absent. Plasma lipoproteins were characterized by fasting chylomicronaemia, reduced low density lipoproteins (LDL) and low high density lipoproteins (HDL). The marked hypertriglyceridaemia could be corrected promptly by infusion of normal plasma. Apolipoprotein C-II (apo C-II) levels in homozygotes were very low (0.01 mg dl-1), and mean apo C-II levels in heterozygotes were lower (2.08 +/- 0.11 mg dl-1) than in normal family members (3.38 +/- 0.75 mg dl-1). Lipoprotein lipase and hepatic triglyceride lipase activities in post-heparin plasma were normal. Zonal ultracentrifugation revealed a marked increase in triglyceride-rich lipoproteins and reduced LDL and HDL. LDL consisted of two fractions with higher hydrated density of the main fraction compared with normals with a trend to normalization on a fat-free diet. The molecular defect in the apo C-II Hamburg gene has been previously identified as a donor splice site mutation in the second intron. This leads to abnormal splicing of the apo C-II Hamburg mRNA and apo C-II deficiency in plasma. The mutation causes the loss of an HphI restriction enzyme site present in the normal apo C-II gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein C-II deficiency syndrome. Clinical features, lipoprotein characterization, lipase activity, and correction of hypertriglyceridemia after apolipoprotein C-II administration in two affected patients.

Two patients (brother and sister, 41 and 39 yr of age, respectively) have been shown to have marked elevation of plasma triglycerides and chylomicrons, decreased low density lipoproteins (LDL) and high density lipoproteins (HDL), a type I lipoprotein phenotype, and a deficiency of plasma apolipoprotein C-II (apo C-II). The male patient had a history of recurrent bouts of abdominal pain often accompanied by eruptive xanthomas. The female subject, identified by family screening, was asymptomatic. Hepatosplenomegaly was present in both subjects. Analytical and zonal ultracentrifugation revealed a marked increase in triglyceride-rich lipoproteins including chylomicrons and very low density lipoproteins, a reduction in LDL, and the presence of virtually only the HDL3 subfraction. LDL were heterogeneous with the major subfraction of a higher hydrated density than that observed in plasma lipoproteins of normal subjects. Apo C-II levels, quantitated by radioimmunoassay, were 0.13 mg/dl and 0.12 mg/dl, in the male and female proband, respectively. A variant of apo C-II (apo C-IIPadova) with lower apparent molecular weight and more acidic isoelectric point was identified in both probands by two-dimensional gel electrophoresis. The marked hypertriglyceridemia and elevation of triglyceride-rich lipoproteins were corrected by the infusion of normal plasma or the injection of a biologically active synthesized 44-79 amino acid residue peptide fragment of apo C-II. The reduction in plasma triglycerides after the injection of the synthetic apo C-II peptide persisted for 13-20 d. These results definitively established that the dyslipoproteinemia in this syndrome is due to a deficiency of normal apo C-II. A possible therapeutic role for replacement therapy of apo C-II by synthetic or recombinant apo C-II in those patients with severe hypertriglyceridemia and recurrent pancreatitis may be possible in the future.     

Plasma lipoproteins in familial lecithin:cholesterol acyltransferase deficiency: lipid composition and reactivity in vitro

Plasma lipoproteins from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been fractioned by preparative ultra-centrifugation and gel filtration and their lipid content and reactivity studied. All of the lipoproteins are abnormal with respect to lipid concentration or relative lipid content. The low density lipoproteins (LDL) and high density lipoproteins (HDL) appear to react normally with partially purified LCAT from normal plasma. Also, the lipids of the very low density lipoproteins (VLDL) and LDL, like those of the corresponding lipoproteins of normal plasma, are indirectly altered by the action of LCAT on normal HDL. Thus, during incubation in vitro VLDL cholesteryl ester is increased and VLDL triglyceride is decreased, as described by others for VLDL from hyperlipemic plasma, and both the unesterified cholesterol and lecithin of the VLDL and LDL are decreased. The patients' VLDL and LDL are abnormal, however, in that they lose unesterified cholesterol and lecithin to normal HDL in the absence of LCAT. Also, the patients' HDL lose these lipids to erythrocyte membranes in the absence of the enzyme.Our results provide further evidence that the abnormal cholesterol and phospholipid composition of the patients' lipoproteins is caused by the LCAT deficiency. They support the postulate that an excess of unesterified cholesterol and lecithin develops as VLDL are converted to LDL and HDL and suggest that in the absence of LCAT this excess lipid distributes among plasma lipoproteins and plasma membranes. They further suggest that LCAT normally reduces this excess lipid through a combination of direct and indirect effects.     

Fish eye disease: a new familial condition with massive corneal opacities and dyslipoproteinaemia Clinical and laboratory studies in two afflicted families

Abstract. Fish eye disease (FED) is characterized by severe corneal opacities, causing impaired vision, and dyslipoproteinaemia: hypertriglyceridaemia, raised levels of very low density lipoproteins (VLDL), triglyceride enrichment of low density liproteins (LDL) and reduction of high density lipoproteins (HDL). The disease is described in two unrelated families. In both there was a high proportion of low HDL in relatives without eye disease.
VLDL, LDL and HDL had normal electrophoretic mobilities. The concentrations of VLDL cholesterol and triglycerides were increased fivefold. LDL cholesterol levels were normal but LDL triglycerides markedly increased. HDL cholesterol was reduced by 90% as were the levels of HDL apolipoproteins. The major part of HDL cholesterol was in the HDL₃ fraction. FED HDL were smaller than normal with molecular weights of 115,000 daltons.
Lecithin: cholesterol acyltransferase activity and amount of cholesterol esters in serum were normal. Postheparin lipoprotein and hepatic lipases showed normal or subnormal values.
Clinically FED differs from other familial conditions with deficiency of HDL such as Tangier disease, LCAT-deficiency and Milano-AI-apoprotein disease. In spite of the extremely low HDL cholesterol FED is not characterized by premature atherosclerosis. Mechanisms for the dyslipoproteinaemia are discussed.     

The plasma lipoproteins in familial chylomicronemia. Analysis by zonal ultracentrifugation

Familial chylomicronemia is a rare genetic disorder attributable to the absence of lipoprotein lipase activity or the absence of apo-CII, i.e., the cofactor for the same enzyme. Plasma lipoproteins were analyzed by zonal ultracentrifugation under rate flotation conditions in four patients with lipoprotein lipase deficiency and two patients with apo-CII deficiency. Lipoproteins of density less than 1.006 gm/ml, and particularly lipoproteins with Sf greater than 100, were present in very high concentrations. Low levels of density greater than 1.006 gm/ml lipoproteins were observed. This fraction was composed of some different and discrete lipoprotein populations: intermediate-density lipoproteins (in three of six patients, density = 1.006 to 1.019 gm/ml); low-density lipoprotein LDL2 (in all patients, density = 1.019 to 1.045 gm/ml); low-density lipoprotein LDL3 (in all patients, density = 1.045 to 1.063 gm/ml); high-density lipoprotein HDL2 (in four of six patients); and high-density lipoproteins HDL3 (in all patients). LDL3 was never observed in normal participants by means of zonal ultracentrifugation; this subclass of low-density lipoproteins seems to correspond to LDL particles of very low Sf (2 to 5) previously identified by analytical ultracentrifugation in patients with severe hypertriglyceridemia. LDL3 was isolated by means of zonal ultracentrifugation as a single and discrete peak in all patients. Lipoproteins of density greater than 1.006 gm/ml were rich in triglycerides and poor in cholesterol in comparison with normal lipoproteins. The heterogeneity of low-density lipoproteins (particularly the appearance of LDL3), low levels of total high-density lipoproteins, and lower HDL3 flotation rate than normal are typical aspects of serum lipoproteins in these patients. No significant differences in the lipoprotein profiles of the patients with lipoprotein lipase deficiency in comparison with patients with apo-CII deficiency were found. In both groups of patients, the plasma lipoproteins profile and the altered lipoprotein composition could be related to the impaired catabolism of triglyceride-rich lipoproteins caused by the absence of lipoprotein lipase activity.     

Concentrations of apolipoproteins B, C-I, C-II, C-III, E and lipids in serum and serum lipoproteins of normal subjects during alimentary lipaemia

The effect of alimentary lipaemia, induced by ingestion of 100 g of fat (cream), on lipids and apolipoprotein B, C-I, C-II, C-III and E levels was evaluated in 16 normotriglyceridaemic subjects. Apolipoprotein concentrations were determined by enzyme immunoassay in whole serum and, in a subsample of seven subjects, in serum lipoprotein fractions of d less than 1.006 kg/l (supernatant) containing very low density lipoproteins (VLDL) and chylomicrons and of d greater than 1.006 kg/l (infranatant) containing low density lipoproteins (LDL) and high density lipoproteins (HDL). Triglyceride concentrations in the supernatant were increased by 164% 3 h after the fat ingestion. The level of alimentary lipaemia was positively related to the fasting serum concentrations of triglycerides and apolipoproteins C-II and C-III and inversely related to the fasting concentration of apolipoprotein E in the infranatant lipoprotein fraction with d greater than 1.006 kg/l. No significant changes in the serum levels of apolipoproteins B, C-I and E were found during the postprandial lipaemia. By contrast, serum concentrations of apolipoproteins C-II and C-III were decreased (compared to the fasting levels) 6 h after the fatty meal. In the 3-h samples all apolipoproteins were increased in the supernatant (VLDL + chylomicrons) and a corresponding decrease was observed in the infranatant (LDL + HDL). In conclusion, this study shows that in normotriglyceridaemic subjects a high serum level of apo C-II is not associated with a more rapid clearance of alimentary lipaemia than a low level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of apolipoproteins B, C-I, C-II, C-III and E in sera from normal men and their relation to serum lipoprotein levels

The concentrations of apolipoproteins B, C-I, C-II, C-III and E (by enzyme immunoassay), and cholesterol, triglycerides and phospholipids both in while serum and in serum very low (VLDL), low (LDL) and high (HDL) density lipoproteins, HDL2 and HDL3, were determined in sera from 29 randomly selected normolipidemic men, age 40-60 years, in Stockholm, Mean values, +/- SD, were for, apolipoprotein B, 720 +/- 162; C-I, 63 +/- 14; C-II, 27 +/- 11; C-III, 125 +/- 57; and for E, 25 +/- 6 mg/l. A skewness to the right of the distributions was found for apolipoproteins B and C-II and for serum triglycerides and VLDL lipids. The relations between the different variables were studied by linear correlation analysis. Several significant correlations existed between the lipoprotein levels. Apolipoprotein C-I, C-II and C-III were significantly correlated with each other, whereas neither apolipoprotein B nor apolipoprotein E was correlated with any other apolipoprotein. The following significant, positive correlations existed between the apolipoproteins and total serum lipids and/or lipids of lipoprotein density classes: apolipoprotein B with serum cholesterol and LDL lipids, apolipoprotein C-I with HDL3 cholesterol, apolipoprotein C-II with serum triglycerides and VLDL lipids, apolipoprotein C-III with serum cholesterol and phospholipids. Apolipoprotein E showed no correlation with either serum lipids or lipoproteins.     

Gemfibrozil treatment potentiates oxidative resistance of high-density lipoprotein in hypertriglyceridemic patients

BACKGROUND: Studies suggest that both oxidized low and high density lipoprotein (LDL and HDL) play a role in the pathogenesis of atherosclerosis. Gemfibrozil is widely used and is reported to increase cholesterol of LDL and HDL in hypertriglyceridemic patients. The aim of this study was to investigate the effect of gemfibrozil treatment on the oxidative status of lipoprotein particles in Fredrickson phenotype IV hypertriglyceridemic patients. METHODS: Twenty-two patients, aged 38-64 years, with fasting plasma triglyceride concentrations between 2.90 and 8.97 mmol L(-1), were recruited and were given gemfibrozil 300 mg three times daily for 12 weeks. Venous blood samples were collected before gemfibrozil treatment, after 4, 8, or 12 weeks of treatment, and 4 weeks after termination of treatment, and used to analyse the plasma lipid profile, isolate lipoproteins, and analyse the chemical composition and in vitro oxidation of lipoprotein particles. RESULTS: Gemfibrozil treatment resulted in a decrease in plasma total triglyceride levels and the triglyceride content of all lipoproteins. Plasma total cholesterol levels were decreased as a result of a decrease in very low density lipoprotein (VLDL) cholesterol levels. A slight increase in LDL cholesterol levels was observed, whereas the thiobarbituric acid-reactive substances (TBARS) of LDL were decreased and the lag and peak time of LDL to oxidation were unchanged and maximal diene production was decreased. Plasma HDL cholesterol levels, the surface-to-core ratio of HDL particles, and the resistance of HDL to oxidation were increased. CONCLUSION: The decreased TBARS and diene production of LDL, increased HDL cholesterol levels, and increased resistance of HDL to oxidation may, in part, explain why gemfibrozil treatment was found to be generally beneficial in terms of protection against coronary heart disease.     

Effect of heparin-induced lipolysis on the distribution of apolipoprotein e among lipoprotein subclasses. Studies with patients deficient in hepatic triglyceride lipase and lipoprotein lipase

In normal subjects, apolipoprotein E (apo E) is present on very low density lipoproteins (VLDL) (fraction I) and on particles of a size intermediate between VLDL and low density lipoproteins (LDL) (fraction II). The major portion of apo E is, however, on particles smaller than LDL but larger than the average high density lipoproteins (HDL) (fraction III). To investigate the possible role of the vascular lipases in determining this distribution of apo E among the plasma lipoproteins, we studied subjects with primary deficiency of either hepatic lipase or of lipoprotein lipase and compared them with normal subjects. Subjects with familial hepatic triglyceride lipase deficiency (n = 2) differ markedly from normal in that fraction II is the dominant apo E-containing group of lipoproteins. When lipolysis of VLDL was enhanced in these subjects upon release of lipoprotein lipase by intravenous heparin, a shift of the apo E from VLDL into fractions II and III was observed. In contrast, apolipoproteins CII and CIII (apo CII and CIII, respectively) did not accumulate in intermediate-sized particles but were shifted markedly from triglyceride rich lipoproteins to HDL after treatment with heparin. In subjects with primary lipoprotein lipase deficiency (n = 4), apo E was confined to fractions I and III. Release of hepatic triglyceride lipase by heparin injection in these subjects produced a shift of apo E from fraction I to III with no significant increase in fraction II. This movement of apo E from large VLDL and chylomicron-sized particles occurred with little hydrolysis of triglyceride and no significant shift of apo CII or CIII into HDL from triglyceride rich lipoproteins. When both lipoprotein lipase and hepatic triglyceride lipase were released by intravenous heparin injection into normal subjects (n = 3), fraction I declined and the apo E content of fraction III increased by an equivalent amount. Either moderate or no change was noted in the intermediate sized particles (fraction II). These data strongly support the hypothesis that fraction II is the product of the action of lipoprotein lipase upon triglyceride rich lipoproteins and is highly dependent on hepatic triglyceride lipase for its further catabolism. In addition, the hydrolysis by hepatic triglyceride lipase of triglyceride rich lipoproteins in general results in a preferential loss of apo E and its transfer to a specific group of large HDL.     

Lipoprotein spectrum analysis of uremic patients maintained on chronic hemodialysis.

It is known that patients maintained on chronic hemodialysis have elevated plasma lipids. In order to establish whether the type of kidney pathology is related to a specific lipoprotein abnormality, we measured plasma lipoprotein in eight patients with glomerulonephritis, two patients with polycystic kidney disease and nine patients that had been surgically nephrectomized. The concentration and composition of plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) in patients were compared to plasma lipoproteins in a control group. In all patient groups, the lipoprotein alterations appeared identical. VLDL were elevated 3 to 4 fold, LDL were decreased 15--35% and HDL were decreased 30--45% when compared to values of our control group. Since no differences in the lipoprotein spectrum were found among the patient groups, it appears that the hypertriglyceridemia of chronic uremia is due to the uremic state per se and is not related to a specific pathology of the kidney.     

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.     

Abnormalities in very low, low and high density lipoproteins in hypertriglyceridemia. Reversal toward normal with bezafibrate treatment.

The effects of triglyceridemia on plasma lipoproteins were investigated in 16 hypertriglyceridemic (HTG) subjects (222-2,500 mg/dl) before and after the initiation of bezafibrate therapy. Bezafibrate caused a mean reduction of 56% in plasma triglyceride and increased the levels of lipoprotein and hepatic triglyceride lipases by 260 and 213%, respectively. The natures of very low density lipoprotein (VLDL), isolated at plasma density and of low and high density lipoprotein (LDL and HDL), separated by zonal ultracentrifugation, were determined. HTG-LDL appears as multiple fractions whereas HTG-HDL is seen predominantly as HDL3. HTG-VLDL is relatively poor in apoproteins and triglycerides but enriched in free and esterified cholesterol. HTG-LDL (main fraction) is depleted of free and esterified cholesterol but enriched in apoprotein and triglyceride. It is also denser and smaller than normal. HTG-HDL3 is denser than N-HDL3 and demonstrates compositional abnormalities similar to those of HTG-LDL. With the reduction of the VLDL mass, all abnormalities revert towards normal. This is accompanied by an increase in LDL-apoprotein B and cholesterol levels, which indicates an increased conversion of VLDL to LDL. Significant correlations between plasma triglyceride and the degree of all abnormalities are shown. The data obtained during treatment corroborate these relationships. The observations support the concept that most abnormalities reflect the degree of triglyceridemia. We suggest that plasma core-lipid transfer protein(s) is an effector of the abnormal cholesteryl ester distribution. Its prolonged action on increasingly large and slowly metabolized VLDL populations would entail a correspondingly excessive transfer of cholesteryl ester to VLDL and of triglyceride to LDL and HDL. It is calculated that, in moderate HTG, LDL and HDL contain only 50% of the normal cholesterol load. It is suggested that cholesteryl ester redistribution in HTG might be important in regulating metabolic events.     

Metabolic Studies in an Unusual Case of Asymptomatic Familial Hypobetalipoproteinemia with Hypoalphalipoproteinemia and Fasting Chylomicronemia

A new kindred with asymptomatic hypobetalipoproteinemia is reported. The proband, age 67, differs from previously described cases in several respects: (a) unusually low levels of low density lipoprotein (LDL) cholesterol (4-8 mg/dl); (b) normal triglyceride levels; (c) low levels of high density lipoprotein; (d) mild fat malabsorption; and (e) a defect in chylomicron clearance. On a high-carbohydrate diet his plasma triglyceride levels, instead of rising, actually fell. Turnover of triglycerides in very low density lipoproteins (VLDL) was low (2.8 mg/kg per h). Fractional catabolic rate of LDL protein was just above the normal range (0.655/d) but net turnover was <10% of normal (0.65 mg/kg per d). The half-life of his chylomicrons was 29 min, five times the normal value. Postheparin lipoprotein lipase activity was normal and apolipoprotein C-II, the activator protein for lipoprotein lipase, was present and functional. Apolipoprotein C-III(1), however, was not detected in the VLDL fraction, a finding previously reported in patients with abetalipoproteinemia. Fecal excretion of cholesterol was almost twice normal; total sterol balance was increased by congruent with40%. The unusual features in the proband that distinguish him from previously described cases and from his affected first-degree relatives suggested that, in addition to the basic gene defect affecting LDL metabolism, he might have a second abnormality affecting clearance of chylomicrons and VLDL. The ratio of apolipoprotein E(3) to E(2) in his VLDL fraction was 0.93, just below the lower limit of normal, suggesting heterozygosity for E(3) deficiency. Whether or not this contributes to his hypertriglyceridemia remains to be established.     

Chronic hepatitis C beta-interferon-induced severe hypertriglyceridaemia with apolipoprotein E phenotype E3/2

The mechanisms of hypertriglyceridaemia and changes in plasma lipoprotein subfractions by beta-interferon treatment were studied in a hepatitis C patient with apo E phenotype E3/2. Plasma levels of triglyceride (TG) were increased by treatment with 6 x 10(6) beta-interferon and reached 8.06 mmol/l at 4 weeks of treatment. Low energy and low fat diet reduced them to half the maximal level. Plasma levels of LDL1 (1.019 < d < 1.045)-C, LDL2 (1.045 < d < 1.063)-C, HDL2-C and HDL3-C were 0.39, 0.31, 0.21 and 0.28 mmol/l, respectively, which are low, but the plasma levels of IDL, which is a remnant of TG-rich lipoproteins, was normal at 7 weeks of treatment. The distribution of plasma lipoprotein subfractions returned to normal after interferon treatment was discontinued. The mass and activity of lipoprotein lipase (LPL) were reduced to half the baseline level by interferon treatment. The activity of hepatic triglyceride lipase (HTGL) which transforms IDL to LDL was normal. The patient's apo E phenotype was E3/2; with that phenotype the removal of TG-rich lipoproteins and IDL through the receptors of the remnant and LDL is impaired. But the IDL plasma level was normal, probably because of normal HTGL activity and high LDL-receptor activity. Lymphocyte LDL-receptor activity was double that of the control. We conclude that interferon caused the low mass and activity of LPL which in turn caused the hypertriglyceridaemia. And no retention of the remnant of TG-rich lipoproteins in this patient with apo E3/2 and low levels of LDL subfractions was due to the active removal of them through LDL-receptors as well as the impaired production of them by suppression of LPL by interferon.     

Lipoprotein fractionation by a precipitation method. A simple quantitative procedure.

A method is described for quantitation of the three major classes of serum lipoproteins. After precipitation of very low density lipoprotein (VLDL) using sodium dodecyl sulphate, the cholesterol and triglyceride content of this lipoprotein class is directly measured. In a second aliquot serum high density lipoprotein (HDL) lipids are measured after precipitation of VLDL and low density lipoprotein (LDL). LDL cholesterol and triglyceride contents are calculated by difference. The procedure requires 2 ml serum, and sensitivity is adequate to permit lipoprotein analyses on umbilical cord serum. Close agreement is observed between this precipitation method and preparative ultracentrifugation.     

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.     

Apolipoprotein B-100 deficiency. Intestinal steatosis despite apolipoprotein B-48 synthesis

We describe a child, the issue of phenotypically normal parents, who had fat malabsorption, both intestinal and hepatic steatosis, and serum cholesterol and triglyceride concentrations of 38 and 63 mg/dl, respectively. Lipoprotein electrophoresis, Ouchterlony double diffusion, and electron microscopy demonstrated that normal low density lipoproteins (LDL: 1.006 less than rho less than 1.063 g/ml) were absent. Lipoprotein particles in the rho less than 1.006-g/ml fraction were triglyceride rich, very large (93.2 +/- 35.1 nm), and contained the B-48 but not the B-100 apoprotein; both species of apolipoprotein (apo) B were found in the parents' lipoproteins. These chylomicrons and chylomicron remnants were present even in the patient's fasting plasma, which suggested prolonged dietary fat absorption. Plasma levels of high density lipoprotein lipids and proteins were low, and the phosphatidylcholine/sphingomyelin ratio was reduced as in typical abetalipoproteinemia. The monosialylated form of apo C-III was not identified on polyacrylamide gel electrophoresis, which suggested that this protein was elaborated only with very low density lipoproteins (VLDL). A radioimmunoassay for apo B employing a polyclonal antisera to plasma LDL gave apparent plasma apo B levels of 0.6, 66, and 57 mg/dl in the patient and his father and mother, respectively. The displacement curve generated by the parents' VLDL and LDL did not did not differ from control lipoproteins. The patient's chylomicron-chylomicron remnant fraction displaced normal LDL over the entire radioimmunoassay range, but the efficiency of displacement was strikingly less than with B-100 containing lipoproteins. If the patient's B-48 protein is not qualitatively abnormal, these results confirm very limited immunochemical cross-reactivity between at least one major epitope on B-100 and the epitopes expressed on B-48. The apo B defect in this patient appears to be recessive. It abolishes B-100 production and may additionally limit the formation of B-48.     

Lipoprotein profiles in plasma and interstitial fluid analyzed with an automated gel-filtration system

BACKGROUND: High-quality methods for lipoprotein characterization are warranted in studies on various metabolic diseases. MATERIALS AND METHODS: An automated system for size-exclusion chromatography (SEC) of lipoproteins using commercially available components is described. Cholesterol or triglyceride content in separated lipoproteins from plasma and interstitial fluid (IF) was continuously determined on-line using microlitre sample volumes. RESULTS: The lipoprotein assay showed a good concordance with the classic ultra-centrifugation/precipitation technique using fresh or frozen samples. Determination of lipoproteins in IF obtained from vacuum-induced skin blisters from 18 healthy subjects revealed that very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol levels were 18%, 19% and 25%, respectively, of concomitant plasma concentrations. The size-exclusion chromatography (SEC) system also allows for triglyceride determination on-line and it could be shown that the system is advantageous for an accurate determination of triglycerides in conditions when there are high levels of glycerol, e.g. in mice and in patients with hyperglycerolaemia (pseudo-hypertriglyceridaemia). CONCLUSIONS: The described system should be of value in studies where detailed lipoprotein analysis is warranted and particularly when significant sample series with small volumes are available. Our data also suggest that there is a 4-5.5-fold concentration gradient between plasma and IF for the three major plasma lipoproteins.     

Hepatic and extrahepatic triglyceride lipase activity in uraemic patients on chronic haemodialysis.

In eleven patients with chronic renal insufficiency treated by intermittent haemodialysis and in ten normal subjects, hepatic and extrahepatic triglyceride lipase activity of post heparin plasma was selectively measured, utilizing the different sensitivity of both enzymes to inhibition by protamine sulphate. In uraemic patients, hepatic triglyceride lipase activity was significantly decreased and extrahepatic triglyceride lipase activity was normal when compared with the control group. The uraemic subjects showed a moderate hypetriglyceridaemia; their serum cholesterol level, however, was normal. The high triglyceride concentration was due to an increase of very low density lipoproteins and low density lipoproteins of the density between 1.006 and 1.019 g/ml (LDL1). The concentration of low density lipoproteins of the density between 1.019 and 1.063 g/ml (LDL2) was decreased. LDL2 were relatively rich in triglycerides when compared with LDL2 from the control group.     

Lipoprotein metabolism during acute inhibition of lipoprotein lipase in the cynomolgus monkey.

To clarify the role of lipoprotein lipase (LPL) in the catabolism of nascent and circulating very low density lipoproteins (VLDL) and in the conversion of VLDL to low density lipoproteins (LDL), studies were performed in which LPL activity was inhibited in the cynomolgus monkey by intravenous infusion of inhibitory polyclonal or monoclonal antibodies. Inhibition of LPL activity resulted in a three- to fivefold increase in plasma triglyceride levels within 3 h. Analytical ultracentrifugation and gradient gel electrophoresis demonstrated an increase predominantly in more buoyant, larger VLDL (Sf 400-60). LDL and high density lipoprotein (HDL) cholesterol levels fell during this same time period, whereas triglyceride in LDL and HDL increased. Kinetic studies, utilizing radiolabeled human VLDL, demonstrated that LPL inhibition resulted in a marked decrease in the catabolism of large (Sf 400-100) VLDL apolipoprotein B (apoB). The catabolism of more dense VLDL (Sf 60-20) was also inhibited, although to a lesser extent. However, there was a complete block in the conversion of tracer in both Sf 400-100 and 60-20 VLDL apoB into LDL during LPL inhibition. Similarly, endogenous labeling of VLDL using [3H]leucine demonstrated that in the absence of LPL, no radiolabeled apoB appeared in LDL. We conclude that although catabolism of dense VLDL continues in the absence of LPL, this enzyme is required for the generation of LDL.