Enzyme deficiency in cholesteryl ester storage idisease.

Cholesteryl ester storage disease has been shown to involve severe deficiency of acid cholesteryl ester hydrolase and triglyceride lipase activity in liver, spleen, and lymph node. The cholesteryl ester hydrolase was also deficient in aorta. Tissue storage of both cholesteryl esters and triglycerides is generalized. Both the lipid and enzymatic changes are very similar to those in Wolman's disease.

Properties of the plasma very low and low density lipoproteins in Tangier disease.

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 This secondary mechanism for C-apoprotein conservation is lost when dietary fat is withdrawn.LDL-2 (1.035 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

Familial hypercholesterolemia (one form of familial type II hyperlipoproteinemia). A study of its biochemical, genetic and clinical presentation in childhood.

Primary hyperbetalipoproteinemia (type II hyperlipoproteinemia) is a common disorder associated with premature vascular disease. It is frequently due to genetic abnormalities, some of which are expressed in childhood. We have examined the manner in which that form of hyperbetalipoproteinemia known as familial hypercholesterolemia may be expressed in 236 children aged 1-19 born of 90 matings in which one parent had hyperbetalipoproteinemia of this variety and one parent did not.Two Gaussian populations were fitted to the distribution of both low density lipoprotein cholesterol (C(LDL)) and plasma cholesterol (C) in these children and a likelihood ratio test strongly favored a two over a one population model for both C(LDL) (X(2) = 18.41, P < 0.0005) and C (X(2) = 7.81, P < 0.025). 45% of the children were in the population identified as affected; their mean C(LDL) was 229. The remaining 55% were in the normal population with a mean C(LDL) of 110 which was indistinguishable from that of an unrelated control population, aged 1-19. On the basis of an assumed frequency of hyperbetalipoproteinemia in the general population of 5%, the Edwards' test indicated that a polygenic model of inheritance was highly unlikely (expected, 22%; observed, 45%). The segregation ratio obtained from the derived intersection between the two population curves (C(LDL), 164 mg/100 ml; C, 235 mg/100 ml) was 45/55 (abnormal/normal). The percentage of abnormal children in the first decade (52%) significantly exceeded that in the second (39%) (P < 0.01). The ratios (II/N) were 50/47 and 55/84 in the offspring of affected female and male parents, respectively (X(2) = 3.819, 0.05 < P < 0.10). Only 10% of hyperbetalipoproteinemic children were considered to have hyperglyceridemia. These children, frequently, but not invariably, had a parent with hyperglyceridemia in addition to hyperbetalipoproteinemia (P < 0.05). None of the affected children who were examined had ischemic heart disease (IHD) and 7% had tendon xanthomas. Half of the parents (mean age, 37.4 yr) who were examined had IHD and three-quarters had xanthomas. The data agree well with the hypothesis that hyperbetalipoproteinemia is inherited as a monogenic trait with early expression in these children. More than one genetic defect within the group is not excluded, but retrospective analyses of the 345 first-degree adult relatives of the affected parents indicated that most of the abnormal parents probably represented familial hypercholesterolemia, rather than combined hyperlipidemia, the other most generally recognized form of familial hyperbetalipoproteinemia.

Selective measurement of two lipase activities in postheparin plasma from normal subjects and patients with hyperlipoproteinemia.

An assay has been developed for specific measurement of two different lipase activities in postheparin plasma. Lipoprotein lipase, derived from extrahepatic sources, is measured as protamine-inactivated lipase activity; hepatic lipase activity is protamine-resistant under the conditions of this assay. In 100 normal subjects, both enzyme activities were noted to be related to age and sex. Protamine-resistant lipase, which comprised 46-95% of the total activity, was highest in men over 18. Protamine-inactivated lipase activity was greatest in younger males and was age-correlated in women, doubling between the second and sixth decades. In 12 patients with hyperchylomicronemia, including five previously shown to have familial type I hyperlipoproteinemia, protamine-inactivated lipase activity was markedly reduced, whereas protamine-resistant lipase was below normal in only 1. The results were not due to lack of plasma activator, presence of plasma inhibitor, or diet, and the deficiency was not overcome by increasing the provoking dose of heparin from 10 U to 75 U/kg. Mean values for both lipase activities were not reduced in 32 other patients with hyperchylomicronemia, nine with "floating beta" lipoproteins (type III hyperlipoproteinemia), and 23 with hyperprebetalipoproteinemia (type IV). Mean protamine-resistant lipase activity was below normal in a group of four women with hypothyroidism, in whom protamine-inactivated lipase was not reduced. Both of the lipase activities were capable of hydrolyzing lipid in very low-density lipoproteins, but the relative rate of hydrolysis of chylomicrons by protamine-resistant lipase was markedly limited. These results indicate the importance of distinguishing between lipases of hepatic and extra-hepatic origin in the measurement of postheparin lipolytic activity.

A comparison of heritable abnormal lipoprotein patterns as defined by two different techniques.

Eight plasma lipoprotein patterns currently employed in attempts to identify different forms of familial dyslipoproteinemia have been compared by two methods. The first (NIH method) is based on paper electrophoretic patterns produced by the four lipoprotein classes obtained by paper electrophoresis in albuminated buffer, coupled with the measurement of the total cholesterol of three of these lipoprotein classes and ascertainment of abnormal flotation of beta-migrating lipoproteins. The second (Donner method) is based on lipoprotein patterns obtained in the analytical ultracentrifuge, adapted for computer analysis and complemented by chemical determination of the concentration of chylomicrons (lipoproteins of S(f) degrees > 400). Pooled samples representing patients with five types of familial hyperlipoproteinemia and three different forms of inherited lipoprotein dificiency were separately analyzed. For six of the eight pools, both methods provided a distinctive lipoprotein pattern in terms of changes in one or more variables. For the remaining two pools, type IV hyperlipoproteinemia and the heterozygote for Tangier disease, both methods provided identical but not unique patterns. The results indicate that lipoprotein analyses obtained by either method may be used interconvertibly in further genetic and other clinical studies.

On thelipoprotein abnormality in type 3 hyperlipoproteinemia.

Two lipoprotein species were isolated by starch block electrophoresis from the very low density lipoproteins (VLDL) (S(f) 20-400) of patients with type III hyperlipoproteinemia. One of these, alpha(2)-VLDL, had a content of lipid and protein and physical characteristics similar to VLDL from normal subjects or patients with other forms of hyperglyceridemia. The other species, beta-VLDL, contained more cholesterol and less triglyceride in relation to the protein, than normal VLDL. Only the apoprotein of low density lipoprotein was immunochemically detectable in the beta-VLDL; the proteins in the alpha(2)-VLDL reacted with antisera specific for low density lipoprotein and high density lipoprotein. The electrophoretic mobility of beta-VLDL was similar to that of low density lipoprotein and significantly less than that of alpha(2)-VLDL. Isolated beta-VLDL had a lesser mean flotation rate than alpha(2)-VLDL, but both alpha(2)- and beta-VLDL were found throughout the S(f) 20-400 flotation range.The relative quantities of alpha(2)- and beta-VLDL could be varied by changing the diet or by heparin administration. Most of the VLDL from type III patients on a high carbohydrate diet was in the alpha(2)-VLDL form. During fasting, alpha(2)-VLDL fell and beta-VLDL increased becoming the predominant species of VLDL. Heparin-induced acceleration of triglyceride clearance also increased beta-VLDL and decreased alpha(2)-VLDL. These findings suggest a precursor-product relationship between the alpha(2)- and beta-forms of VLDL.

Isolation and characterization of an abnormal high density lipoprotein in Tangier Diesase.

The nature of the high density lipoproteins has been investigated in five patients homozygous for Tangier disease (familial high density lipoprotein deficiency). It has been established that Tangier high density lipoproteins, as isolated by ultracentrifugation, are morphologically heterogenous and contain several proteins (Apo B, albumin, and Apo A-II). An abnormal lipoprotein has been isolated from the d = 1.063-1.21 g/ml ultracentrifugal fraction by agarose-column chromatography which contains apoprotein A-II as the sole protein constituent. In negative-stain electron microscopy, these lipoproteins appeared as spherical particles 55-75 A in diameter. By a variety of criteria (immunochemical, polyacrylamide electrophoresis, amino acid composition, and fluorescence measurements), apoprotein A-I the major apoprotein of normal high density lipoproteins and the C apoproteins were absent from this lipoprotein. As demonstrated by (125)I very low density lipoprotein incubation experiments with Tangier plasma, C apoproteins did not associate with lipoproteins of d = 1.063-1.21 g/ml. Tangier apoprotein A-II, isolated to homogeneity by delipidation of the apoprotein A-II-containing lipoprotein or Sephadex G-200 guanidine-HCl chromatography of the d = 1.063-1.21 g/ml fraction, was indistinguishable from control apoprotein A-II with respect to amino acid composition and migration of tryptic peptides in urea-polyacrylamide electrophoresis. The ability of Tangier apoprotein A-II to bind phospholipid was demonstrated by in vitro reconstitution experiments and morphological and chemical analysis of lipid-protein complexes. It is concluded that normal high density lipoproteins, as defined by polypeptide composition and morphological appearance, are absent from Tangier plasma and that as a consequence, the impairment of C apoprotein metabolism contributes to the hypertriglyceridemia and fasting chylomicronemia observed in these patients.

Studies on the protein defect in Tangier disease. Isolation and characterization of an abnormal high density lipoprotein.

High density lipoproteins (d 1.063-1.210 g/ml) were isolated from the plasma of normal individuals (HDL) and seven homozygous patients with Tangier disease (HDLt). In Tangier patients, the concentration of protein in the high density region (HDLt) was only 0.5-4.5% of normal. Immunochemical studies, including mixing experiments conducted in vivo and in vitro, indicated that HDLt was different from HDL. HDLt was the only high density lipoprotein detectable in the plasma of Tangier homozygotes. In heterozygotes both HDL and HDLt were present. HDLt was not detected in the plasma of over 300 normal persons and 10 patients with secondary high density lipoprotein deficiency and appeared to be a unique marker for Tangier disease.ApoHDL contained two major apoproteins designated apoLp-Gln-I and apoLp-Gln-II; together they comprised 85-90% of the total protein content. Both of the major HDL apoproteins were present in apoHDLt; but apoLp-Gln-I was disproportionately decreased with respect to apoLp-Gln-II, the ratio of their concentrations being 1: 12 in apoHDLt as compared with 3: 1 in apoHDL. Several minor apoprotein components which together comprise 5-15% of apoHDL were present in approximately normal proportions in apoHDLt. In the HDL of Tangier patients it was estimated that, compared with normal individuals, the concentration of apoLp-Gln-I was decreased about 600-fold and the concentration of apoLp-Gln-II about 17-fold. The decrease in these apoproteins was not due to preferential segregation with the lipoprotein fractions of d < 1.063 g/ml or with the plasma proteins of d > 1.21 g/ml. Tangier apoLp-Gln-I and apoLp-Gln-II appeared to be immunochemically identical with their normal counterparts, and no differences between the two sets of apoproteins were detected on polyacrylamide gel electrophoresis at pH 9.4 or 2.9. These results are most compatible with the hypothesis that the hereditary defect in Tangier disease is a mutation in an allele-regulating synthesis of apoLp-Gln-I.

Evidence for the identity of the major apoprotein in low density and very low density lipoproteins in normal subjects and patients with familial hyperlipoproteinemia.

The major apoprotein(s) from human plasma low density lipoproteins was isolated and compared with a major protein fraction (fraction I) from very low density lipoproteins (VLDL). Fraction I had been previously found to comprise approximately 40% of the total protein of VLDL. Fraction I from VLDL and apoLDL from normal subjects were indistinguishable in amino acid compositions and circular dichroic spectra. They yielded indistinguishable displacement curves of LDL-(125)I by radioimmunoassay and formed immunoprecipitin lines of complete identity. Fraction I from VLDL of normal subjects was compared with the fraction isolated from patients with familial types II, III, IV, and V hyperlipoproteinemia. There were no detectable differences between any of these fractions in amino acid compositions, circular dichroic spectra, and immunochemical properties. It was, therefore, concluded that short of peptide mapping or determination of amino acid sequence, fraction I from VLDL of each subject with familial hyperlipoproteinemia appears to be identical with fraction I and apoLDL from normal individuals.A new convenient method of preparation of soluble apoLDL, modified from a procedure previously described from this laboratory, is presented.

Biochemical studies in a patient with a Tangier syndrome.

The chemical composition of the major classes of lipids were evaluated in the plasma and in various other tissues of a 68-year-old woman with a syringomyelia-like syndrome affecting cranial, cervical and brachial regions. No tonsillar abnormalities were apparent on visual examination of the oropharynx but the absence of alpha-lipoproteins on serum lipoprotein electrophoresis prompted the tentative diagnosis of Tangier disease. The diagnosis was confirmed by lipid, lipoprotein and apolipoprotein analyses of the plasma. The plasma cholesterol was low (93-113 mg/dl) and the triglyceride concentration normal (133-160 mg/dl). The very low density lipoproteins had normal chemical composition and morphology, but migrated with beta rather than pre-beta mobility on paper electrophoresis. Low density lipoproteins were deficient in cholesteryl esters and enriched in triglycerides; their electrophoretic mobility and morphology were normal. A small amount of high density lipoprotein (approximately 1.4 mg/dl) was recovered from the plasma. This contained few particles of the size of normal high density lipoprotein and polyacrylamide gel electrophoresis of the lipid-free protein demonstrated a disproportionate increase in the A-II apolipoprotein. All of these abnormalities are consistent with Tangier disease. The serum concentration of glycosphingolipids was approximately 40% lower than normal, with the most marked reductions in the glucosylceramide (GL-1a) and trihexosylceramide (GL-3a) fractions. The relative quantity of long chain fatty acids (23 or more carbons) in serum sphingomyelin was reduced about 38% of that in control sera. Serum lecithin:cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity was 25% of normal and the reduced activity was shown not to be related to a change of enzyme specificity or to a lack of appropriate substrate. These findings are likely related to the HDL deficiency which characterizes Tangier disease. A biopsy sample of apparently normal tonsil contained three to four times the normal amount of cholesterol, and the increase was due entirely to abnormal quantities of cholesteryl esters. Of great interest was the chemical documentation of increased cholesteryl esters in a nerve biopsy specimen. These findings indicate that the neurologic as well as the reticuloendothelial manifestations of Tangier disease may be related to cholesteryl ester accumulation. Lipoprotein profiles, their triglyceride and cholesterol concentration, and LCAT activity were obtained on the plasma of 7 closely related members of the kinship. None of these relatives were found to have the biochemical derangement of Tangier disease.

The heart in Tangier disease. Severe coronary atherosclerosis with near absence of high-density lipoprotein cholesterol.

Cardiac necropsy findings are described in a 72-year-old man with Tangier disease whose plasma total cholesterol levels averaged 70 mg/dL, low-density lipoprotein cholesterol level was 45 mg/dL, and high-density lipoprotein cholesterol level was 1.4 mg/dL, and who had coronary artery bypass grafting for severe atherosclerotic coronary artery disease. At necropsy, 24 of the 72 (33%) 5-mm segments of the 4 major (right, left main, left anterior descending, and left circumflex) native coronary arteries and 4 of the 27 (15%) 5-mm segments of the saphenous vein aortocoronary bypass conduits were narrowed by more than 75% in cross-sectional area by atherosclerotic plaques. The plaques were composed primarily (91% to 97%) of fibrous tissue. Oil red O staining, polarized light microscopy, and electron microscopy revealed cholesterol deposits in the plaques and in the walls of coronary arteries, saphenous vein grafts, and aorta. Such deposits also were found in foam cells of histiocytic origin, fibroblasts in all four cardiac valves, and in Schwann cells of cardiac nerves.

Abnormal concentration and anomalous distribution of apolipoprotein A-I in Tangier disease.

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

Preparation and properties of an apoprotein derivative of human serum beta-lipoprotein.

The aim of this study was to develop a convenient method for the preparation and study of a soluble delipidated form of human serum beta-lipoprotein. This was achieved by succinylation and delipidation with ether-ethanol (3ratio1). The succinylated apoprotein was soluble in either 0.13 M Tris-HCl buffer, pH 8.2 (for beta-lipoprotein prepared by ultracentrifugation) or in the same Tris buffer to which 5 mM sodium decyl sulfate was added (for heparin-Mn precipitated beta-lipoprotein). The immunological activity of beta-lipoprotein or its apoprotein were markedly altered by succinylation. Whereas the succinylated beta-lipoprotein appeared as one peak in the analytical ultracentrifuge, the succinylated apoprotein appeared as two. Under the electron microscope beta-lipoprotein and succinylated beta-lipoprotein were indistinguishable, appearing as uniform preparations of spherical particles 215 to 220 A in diamater.