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Since the development of coronary heart disease (CAD) is affected by a specific pattern of plasma high density lipoprotein (HDL) effects it may be useful to know whether this occurs already in childhood. In this study we evaluated particle size distribution of HDL by gradient gel electrophoresis and the determination of cholesterol esterification rate (FER(HDL)) in plasma depleted of apo B lipoproteins in 221 children (108 boys and 113 girls) aged 4 months to 20 years. Total plasma- (TC), low-density lipoprotein- (LDL-C) and HDL- (HDL-C) cholesterol, HDL unesterified cholesterol (HDL-UC) and plasma triglycerides (TG) were also measured. There were no significant gender and age differences with respect to the plasma TC, LDL-TC and TG but concentration of HDL-TC increased with age. Post-pubertal girls had significantly higher relative concentrations of HDL2b compared to boys (30.4% vs 17.2%), while HDL3b,c was lower in post-pubertal girls (8.7% vs. 16.5%). FER(HDL) correlated inversely with HDL2b and positively with HDL3b,c particles and was significantly higher in boys of the post-pubertal group compared to girls (16.9%/h vs 12.5%/h). While in girls there was a positive correlation between age and HDL-C, HDL-UC and the relative concentration of HDL2b no significant correlation were observed in boys. In girls the increase in TC showed a significant correlation with a simultaneous increase in HDL-C, HDL-UC and HDL2b. In boys TC correlated significantly with changes in TG only. When HDL2b and HDL3b,c cholesterol levels are calculated from HDL-C concentration and per cent distribution the differences between males and females are further emphasized. These data indicate that HDL particle size distribution is age- and gender-dependent.

We studied the particle size distribution of plasma high-density lipoproteins (HDL) by gradient gel electrophoresis and by assay of cholesterol esterification rate (FERHDL) in plasma depleted of very low (VLDL) and low-density (LDL) lipoproteins in 32 hypertensive women (53 +/- 10 y old) and in an age-matched group of 21 apparently healthy women. There were no significant differences between the groups with respect to their plasma total, HDL- and LDL-cholesterol. The plasma triglyceride (TG) concentration was significantly higher in the group of hypertensive women, and HDL-free cholesterol was significantly lower in the hypertensive group. There were highly significantly differences in the relative proportions of HDL subclasses between the hypertensive and control women: HDL2b was significantly lower and HDL3b,c was significantly higher in hypertensive patients compared to controls. FERHDL was 12 +/- 4%/h in the control group and 18 +/- 6%/h in patients with hypertension (p < 0.001). There was a strong positive correlation between FERHDL and the relative content of HDL3b,c, and a strong negative correlation between FERHDL and HDL2b in both groups. Comparison of subgroups of healthy and hypertension pre- and postmenopausal women revealed, apart from age, no significant differences between the healthy subjects in any of the above parameters.

A decreased level of HDL cholesterol (HDL-C) is the most common lipoprotein abnormality seen in people with premature coronary artery disease (CAD). In many cases, HDL-C reduction in patients with CAD may be the result of increased apo B-containing lipoprotein production by the liver with secondary hypoalphalipoproteinemia. Primary hypoalphalipoproteinemia is seen in approximately 4% of people with CAD. We report findings in four subjects with severe familial HDL deficiency (HDL-C << 5th percentile for age and sex; 0.08 to 0.38 mmol/L) in three French-Canadian kindreds with autosomal codominant inheritance. By inclusion criteria, all four subjects had normal fasting triglycerides and none were diabetic. HDL particle size by gradient gel electrophoresis revealed small HDL particles (estimated Stokes' diameter, 8.14 to 8.30 nm). Apo AI analysis by polyacrylamide gel electrophoresis and use of isoelectrofocusing gels in affected subjects revealed normal molecular weight (28.3 kD) and normal isoelectrofocusing point but a relative increase in proapoliprotein AI, with near-normal levels of proapolipoprotein AI in plasma, suggesting normal secretion of apo AI. Quantitative Southern blot analysis of the apo AI-CIII-AIV gene cluster reveals no gene rearrangements or allele deletion. Haplotypes of the apo AI gene, determined by use of the restriction enzymes Pst I, Xmn I, and Sst I and of the apo AII gene by use of the enzyme Msp I, did not reveal segregation of the low HDL-C trait with either the apo AI or the AII gene. Sequence analysis of the promoter region of the apo AI gene reveals heterozygosity for guanine-to-adenine substitution at position 76 in two kindreds with no evidence of segregation with the low HDL trait. None of the patients had mutations of the lipoprotein lipase gene common in subjects of French-Canadian descent. Haplotype analysis of the lipoprotein lipase gene did not show segregation with the low HDL trait. Plasma lecithin: cholesterol acyltransferase (LCAT) activity was found to be within normal levels in affected subjects and in nonaffected first-degree relatives. None of the affected subjects had clinical manifestations of Tangier disease. Two of the four cases examined, both men, had severe CAD and had undergone revascularization procedures. The third is a younger brother of one of these probands and the fourth is a 30-year-old woman, and both were free of clinical CAD. However, in none of the families did the low HDL trait unequivocally cosegregate with CAD.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied the heterogeneity of high-density lipoproteins (HDL) in plasma of 110 subjects, using three different methods: (a) gradient gel electrophoresis (GGE); (b) electroimmunoassay, to measure the concentration of lipoprotein particles containing apoprotein (apo) AI but no apo-AII (LP AI); and (c) cholesterol esterification rate (FERHDL) in very-low- and low-density lipoprotein-depleted plasma. There were two study groups: patients with hypertension, whose plasma lipid profile was similar to their respective controls, and patients with hypoalphalipoproteinemia (hypoalpha), whose family members served as controls. Values for FERHDL were significantly higher in both risk groups than in their respective controls. LP AI was significantly decreased only in the hypoalpha subjects. Generally, LP AI and FERHDL were inversely related. LP AI correlated strongly with plasma HDL-cholesterol, apo AI, and LP AI/AII; FERHDL correlated with those values inversely. LP AI, but not FERHDL, correlated with HDL free cholesterol. On the other hand, FERHDL correlated strongly with plasma concentrations of triglycerides and with the plasma ratio of total/HDL-cholesterol while LP AI did not. GGE determination of the composition of HDL subspecies showed that both FERHDL and LP AI were significantly related to the content of HDL2b particles: FERHDL inversely, LP AI directly; the relative amount of HDL3b,c particles correlated only with FERHDL. We conclude that GGE and FERHDL can be used to quantify both the apparently protective (HDL2b) and risk-associated (HDL3b,c) particles, whereas the concentration of LP AI in plasma mainly reflects the concentration of the HDL2 subpopulation.

Classic (complete) lecithin:cholesterol acyltransferase (LCAT) deficiency and Fish-eye disease (partial LCAT deficiency) are genetic syndromes associated with markedly decreased plasma levels of high density lipoprotein (HDL) cholesterol but not with an increased risk of atherosclerotic cardiovascular disease. We investigated the metabolism of the HDL apolipoproteins (apo) apoA-I and apoA-II in a total of five patients with LCAT deficiency, one with classic LCAT deficiency and four with Fish-eye disease. Plasma levels of apoA-II were decreased to a proportionately greater extent (23% of normal) than apoA-I (30% of normal). In addition, plasma concentrations of HDL particles containing both apoA-I and apoA-II (LpA-I:A-II) were much lower (18% of normal) than those of particles containing only apoA-I (LpA-I) (51% of normal). The metabolic basis for the low levels of apoA-II and LpA-I:A-II was investigated in all five patients using both exogenous radiotracer and endogenous stable isotope labeling techniques. The mean plasma residence time of apoA-I was decreased at 2.08 +/- 0.27 d (controls 4.74 +/- 0.65 days); however, the residence time of apoA-II was even shorter at 1.66 +/- 0.24 d (controls 5.25 +/- 0.61 d). In addition, the catabolism of apoA-I in LpA-I:A-II was substantially faster than that of apoA-I in LpA-I. In summary, genetic syndromes of either complete or partial LCAT deficiency result in low levels of HDL through preferential hypercatabolism of apoA-II and HDL particles containing apoA-II. Because LpA-I has been proposed to be more protective than LpA-I:A-II against atherosclerosis, this selective effect on the metabolism of LpA-I:A-II may provide a potential explanation why patients with classic LCAT deficiency and Fish-eye disease are not at increased risk for premature atherosclerosis despite markedly decreased levels of HDL cholesterol and apoA-I.

The relationship between the fractional rate of cholesterol esterification (FERHDL) in very low density lipoprotein (VLDL)- and low density lipoprotein (LDL)-depleted plasma and the particle size distribution of high density lipoproteins (HDL) were studied in: a) a control group of 9 apparently healthy men (42 +/- 11 years); b) 15 septuagenarians (76 +/- 6 years) who had no clinical signs of coronary artery disease; and c) 32 outpatients with essential hypertension of different stages of severity (51 +/- 10 years). There were small differences between the groups with respect to their plasma total and HDL-cholesterol and plasma triglyceride levels. However, there was a highly significant increase in FERHDL in patients with hypertension compared to control and older men. The HDL of hypertensive patients had a markedly increased relative content of HDL3b, while their HDL2b fraction was reduced by over 50% compared to the other groups. Overall, there was a strong positive correlation between FERHDL and HDL3b (r = 0.89; P less than 0.001) and a negative correlation between FERHDL and HDL2b (r = -0.61; P less than 0.001) and HDL3a (r = -0.77; P less than 0.001). These findings confirm our previous conclusions that FERHDL reflects the relative HDL subclass distribution. In addition, we demonstrate that FERHDL is increased in hypertensive male subjects regardless of the stage of hypertension, i.e., whether or not organic lesions have already become manifest (stage III and stages I plus II, respectively).

Familial hypoalphalipoproteinemias (HA) are a heterogenous group of disorders characterized by various degrees of HDL deficiency. Differential diagnosis involves clinical and biochemical evaluation after intervention designed to correct known secondary causes of low HDL. Two specific HAs are discussed in this report: 1. primary isolated HA (PIHA) is a poorly characterized entity with an apparent autosomal dominant transmission and distinct abnormalities in the structure and function of HDL. 2. Lecithin: cholesterol acyltransferase (LCAT) deficiency syndromes are caused by a number of different genetic defects that lead to at least two distinct clinical presentations i.e. familial LCAT deficiency and fish eye disease. PIHA is an example of a genetic disorder whose diagnosis would greatly be improved by the availability of molecular diagnostic tests. Conversely, the effect of the genetic heterogeneity of LCAT deficiency syndromes on diagnosis is best overcome by utilizing existing biochemical measurement of LCAT activity and the plasma cholesterol esterification rate.

Fish eye disease (FED) is an extremely rare familial disorder characterized by severe HDL deficiency and extensive corneal opacities. This disorder appears to be a variant of familial lecithin: cholesterol acyltransferase (LCAT) deficiency in which the enzyme remains partly active yet the ability of the enzyme to esterify cholesterol in high-density lipoprotein (HDL) has been lost. The rarity of this disorder has limited advances in our understanding of the pathophysiology of the HDL deficiency. However, we here describe the clinical and biochemical presentation of a family with FED who are of Dutch descent. The proposition presented with HDL deficiency and corneal opacity. Subsequently, they were diagnosed as having FED by the absence of LCAT activity against a small proteoliposome substrate despite the presence of half-normal LCAT mass and a near-normal ratio of unesterified to total cholesterol in plasma. Heterozygotes presented with half-normal LCAT activity, but not with decreased HDL. With the identification of this three-generation family, renewed investigation of this intriguing disorder of HDL is now possible.

The familial hypoalphalipoproteinemias are a heterogeneous group of rare lipoprotein disorders characterized by extremely low levels of plasma high density lipoproteins (HDL) and, in most cases, autosomal recessive inheritance. Most of these conditions present distinctive and diagnostic clinical and laboratory abnormalities. In spite of the marked reductions in HDL, however, many of these conditions are not associated with premature atherosclerosis. This is true of Tangier disease, Fish Eye disease, lecithin: cholesterol acyltransferase deficiency, and of some variants of apo Al. Another condition, defined as a primary and familial decrease in HDL-cholesterol levels in the absence of other lipoprotein abnormalities. that is associated with premature atherosclerosis was originally called Familial Hypoalphalipoproteinemia but is better referred as to Familial Isolated Hypoalphalipoproteinemia. At present, the prevalence, inheritance, and the underlying defect(s) in this disorder are unknown. Decreased or absent synthesis of apo A-I due to a gene defect is the cause of apo A-I/C-III and apo A-I/C-III/A-IV deficiency. However, the etiology of the low levels of HDL is unclear for most of the remaining familial hypoalphalipoproteinemias. Increased catabolism, decreased synthesis and altered equilibration of HDL between intra- and extravascular spaces have all been suggested as underlying causes of low plasma HDL. Whatever their causes, these disorders are associated with altered HDL composition and altered equilibration of cholesterol amongst the various lipoprotein classes. The absence of consistent correlation with premature atherosclerosis in many of these conditions suggests that the protective effect of HDL may reside in a quantitatively small, but metabolically active subfraction of HDL particles.

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.

Cholesteryl ester transfer from solid-phase bound HDL to endogenous plasma HDL or VLDL/LDL was determined in 50 patients with primary disorders of lipid metabolism and 27 normolipidemic subjects. Transfer to the plasma HDL pool was significantly reduced in familial hypercholesterolemia, familial combined hyperlipidemia, hypoalphalipoproteinemia and dysbetalipoproteinemia. Subfractionation of HDL revealed that the lipid transfer to HDL3 was significantly reduced in all patient groups while transfer to HDL2 was increased in those with dysbetalipoproteinemia and familial hypertriglyceridemia. Transfer to LDL and VLDL was increased only in patients with dysbetalipoproteinemia and hypoalphalipoproteinemia. Reduced transfer to HDL occurred in samples with altered HDL composition; particularly where HDL-triglyceride was significantly increased and HDL-cholesteryl esters were reduced. Transfer of cholesteryl ester to HDL3 was significantly decreased in patients with vascular disease. These findings indicate that impaired interaction of cholesteryl ester transfer protein with the HDL3 pool may contribute to the risk of coronary heart disease in patients with specific plasma lipid abnormalities.

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.

Erythrocyte membrane (EM) abnormalities in a 16-yr-old boy with hypoalphalipoproteinemia resembling fish eye disease (FED-LS) were investigated. The proband's erythrocytes had markedly decreased osmotic fragility with target cells observed in the peripheral film. Analysis of his EM lipids revealed normal cholesterol and phospholipid content but a marked increase in phosphatidylcholine with concomitant decreases in phosphatidylethanolamine and sphingomyelin. Of the EM enzymes examined, acetylcholinesterase and superoxide dismutase activities were decreased while those of Na+-K+ ATPase, catalase and glutathione reductase were normal. 51Cr erythrocyte survival in the patient was slightly decreased. The observed changes in a number of structural and functional properties of erythrocytes in this disorder are indistinguishable from those previously described in homozygotes for familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Thus, it is possible that in both of these disorders an abnormality of plasma LCAT activity causes, either directly or indirectly, functional and structural changes in the erythrocyte membrane.

A 16-year-old boy presented with bilateral arcus cornealis and markedly decreased plasma high density lipoprotein cholesterol. The plasma lipoprotein abnormalities, as well as decreased mass and activity of lecithin:cholesterol acyltransferase (LCAT), were similar to those described in patients with fish eye disease. Increased number of target cells and decreased osmotic fragility of the proband's erythrocytes were noted. The proband's father and one of his brothers showed intermediate plasma lipoprotein and LCAT alterations. The father's erythrocytes also showed abnormal osmotic fragility. The mother of the propositus had normal plasma lipoproteins and erythrocyte osmotic fragility, but her LCAT activity was also low. Many of these features suggest a disorder similar to fish eye disease which is clinically and biochemically distinct from other hypoalphalipoproteinemias.

We have investigated the possibility that the mild proteinuria in a patient with Tangier disease is directly related to the plasma HDL-deficiency through excretion of apolipoprotein A-I (Mr 28,300). An increased urinary excretion of a 29,000 polypeptide was observed in this patient. However, western blot analysis of the urine showed that this protein was not apolipoprotein A-1 or its precursor form. Subsequent investigations identified the urinary protein as immunoglobulin light chains. The elevated excretion may be a consequence of the patient's plasma polyclonal gammopathy which had resulted from a chronic infection.

A 56-year-old man, the offspring of a consanguineous first cousin marriage, presented with clinical, morphological, and biochemical features of familial deficiency of high-density lipoproteins (Tangier disease). Of 8 first- and second-degree relatives examined, 4 had either plasma apo A-I or HDL cholesterol concentrations 2 standard deviations below normal population mean on at least 1 occasion. The patient and a majority of his relatives also had high plasma apo B concentrations relative to their levels of cholesterol. Adipose tissue biopsy was undertaken to study HDL interaction with the patients' cells. Specific uptake of HDL3 was demonstrated in adipocytes of this patient, but was decreased relative to a control of similar fat cell size. However, no marked difference in fat cell cholesterol content was observed between the Tangier patient and the control. Thus it appears unlikely that adipocytes play a role in the etiology of Tangier disease.

We studied the ability of lipid-transfer factors in plasma to promote transfer, to endogenous lipoproteins, of [3H]cholesteryl ester from high-density lipoprotein (HDL) covalently bound to Sepharose 4B beads. After incubation for 2 h at 37 degrees C, 12 to 14% of the [3H]cholesteryl ester had been transferred to the lipoproteins of the plasma, in the proportions 57% to HDL and 43% to low- and very-low-density lipoproteins. This process was a function of the amount of plasma present and was stimulated by addition of partly purified lipid-transfer protein. Transfer also depended on the concentration of donor HDL but was independent of the amount of acceptor lipoprotein. This simple evaluation of cholesteryl ester transfer does not require removal of lipoproteins from the plasma before incubation.

Serum high density lipoproteins (HDL) are a protective factor against atherosclerosis. Many epidemiological studies show a strong inverse relationship between serum HDL-cholesterol (HDL-C) and the likelihood of developing coronary artery disease (CAD). HDL-C levels appear to be a stronger predictive factor for CAD than serum total or low density lipoprotein cholesterol (LDL-C) levels. In the presence of high HDL-C levels, the development of CAD is unlikely even in persons with increased total or LDL-C. Conversely, subjects with low serum levels of HDL-C are at increased risk even if their total and LDL-C is within the "desirable" range. A number of studies have also shown that increasing HDL-C levels is associated with both primary and secondary prevention of atherosclerosis. HDL-C levels should be estimated in individuals with family and/or personal history of premature atherosclerosis, even if they have normal total cholesterol. Only the current problems with the accuracy and precision of the serum HDL-C assay prevent it from being the single most important test for assessment of the lipid risk factors for CAD. The most frequent causes of low HDL-C are smoking, obesity, and hypertriglyceridemia. Treatment of low HDL-C includes removal of these factors, and if this is ineffective, use of drugs. Gemfibrozil and niacin are most effective in raising serum HDL-C, although a number of other medications can markedly improve the total:HDL-C ratio.