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Background: High density lipoproteins (HDL) are complex lipoprotein particles involved in reverse cholesterol (C) transport and are negatively associated with the risk for coronary artery disease (CAD). We have described a disorder of familial HDL deficiency (FHD) due to abnormal cellular cholesterol efflux. In the present study, we investigated cellular cholesterol efflux on skin fibroblast from 15 probands with moderate to severe hypoalphalipoproteinemia, including one subject with Tangier disease (TD). We performed family studies on eight of these probands (269 individuals) with familial hypoalphalipoproteinemia (defined as a HDL-C <5th%, and with no known cause of HDL deficiency). We have previously shown that four of our FHD patients and patients with TD have mutations at the ABC1 gene, demonstrating that FHD is a heterozygous form of TD., Methods: On each subject, we carried out detailed biochemical analysis and determined apoA-I-mediated cellular cholesterol efflux using 3H-cholesterol labeled skin fibroblasts from study subjects compared with controls. TD has also been associated with abnormal cellular cholesterol efflux. Cell fusion experiments with polyethylene glycol (PEG) were carried out with fibroblasts from a subject with TD and one with FHD in order to determine whether the Tangier cells can complement the FHD defect. In all subjects with a reduced cellular cholesterol efflux, exons of the ABCA1 gene were sequenced., Results: Familial forms of HDL deficiency, defined as HDL-C levels <5th percentile, are a heterogeneous group of lipoprotein disorders. A reduced cellular cholesterol efflux has been identified in eight subjects from seven kindred (7/14 or 50% of probands tested), being reduced by a mean 59% of controls (range 49-63%). In four of these subjects, a mutation at the ABCA1 gene locus was identified. In three other subjects an efflux defect was idenfified but no critical mutation at the ABCA1 gene locus has been identified. In the remaining subjects, (7/14), no efflux defect was identified. Complementation studies reveal that the FHD defect is not corrected by Tangier cells, confirming that FHD and TD represent a spectrum of the same genetic defect., Conclusion: Familial hypoalphalipoproteinemia syndromes are phenotypically heterogeneous; one form is associated with abnormal cellular cholesterol efflux caused by heterozygous mutations at the ABCA1 gene, that defines familial HDL Deficiency while homozygous mutations or compound heterozygocity causes TD. Other forms are primary hypoalphalipoproteinemia of unknown cause, while the remaining cases are associated with hypertriglyceridemia with or without elevated apoB levels. We conclude that a cellular cholesterol defect is a relatively frequent cause of familial HDL deficiency and that a mutation at the ABCA1 gene can be identified in half of these patients.

Apo A-IMilano is a mutant form of apo A-I in which cysteine is substituted for arginine at amino acid 173. Subjects with apo A-IMilano are characterized by having low levels of plasma HDL cholesterol and apo A-I. To determine the kinetic etiology of the decreased plasma levels of the apo A-I in these individuals, normal and mutant apo A-I were isolated, radiolabeled with either 125I or 131I, and both types of apo A-I were simultaneously injected into two normal control subjects and two subjects heterozygous for apo A-IMilano. In the normal subjects, apo A-IMilano was catabolized more rapidly than the normal apo A-I (mean residence times of 5.11 d for normal apo A-I vs. 3.91 d for apo A-IMilano), clearly establishing that apo A-IMilano is kinetically abnormal and that it has a shortened residence time in plasma. In the two apo A-IMilano subjects, both types of apo A-I were catabolized more rapidly than normal (residence times ranging from 2.63 to 3.70 d) with normal total apo A-I production rates (mean of 10.3 vs. 10.4 mg/kg per d in the normal subjects). Therefore, in the subjects with apo A-IMilano, the decreased apo A-I levels are caused by rapid catabolism of apo A-I and not to a decreased production rate, and the abnormal apo A-IMilano leads to the rapid catabolism of both the normal and mutant forms of apo A-I in the affected subjects.

Tangier disease is a rare familial disorder characterized by extremely low levels of apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). In normal subjects, proapoA-I is secreted into plasma and converted to mature apoA-I by the cleavage of the amino-terminal six amino acids with the major isoprotein in plasma being mature apoA-I. In contrast, in Tangier disease there is a marked relative increase of proapoA-I as compared with mature apoA-I. ProapoA-I and mature apoA-I were isolated from normal and Tangier disease subjects, radio-labeled, and autologous apoA-I isoproteins injected into normal and Tangier subjects. The in vivo catabolism and conversion of proapoA-I and mature apoA-I in normal and Tangier disease subjects were quantitated. A comparison of the rate of catabolism of apoA-I isoproteins from plasma revealed a significantly faster rate of catabolism of both isoproteins of apoA-I in Tangier subjects when compared with normal subjects. The fractional conversion rate of proapoA-I to mature apoA-I was 3.9 d-1 in normal subjects and 3.6 d-1 in Tangier subjects. The results indicate that (a) apoA-I enters plasma as the pro isoprotein in both normal and Tangier subjects, (b) Tangier disease subjects have a normal fractional rate of conversion of proapoA-I to mature apoA-I, (c) proapoA-I is catabolized at the same rate as mature apoA-I in Tangier subjects, and (d) Tangier subjects catabolize both pro and mature apoA-I at a much greater rate than do normal subjects. Therefore, the relative increase in proapoA-I in Tangier disease is due to a marked decrease in mature apoA-I resulting from rapid catabolism of both pro- and mature apoA-I and not to defective conversion of proapoA-I to mature apoA-I.

The apolipoprotein A-I was isolated from the plasma of normal individuals and of three homozygous patients with Tangier disease by immunoprecipitation. The apoA-I isoforms were further fractionated by isofocusing on polyacrylamide gels. The physicochemical behavior of normal and Tangier apoA-I and of the isoproteins-2 and -4 was studied by monitoring the tryptophanyl fluorescence emission as a function of temperature, pH, and under exposure to guanidinium (guanidine) hydrochloride (GdmCl). Lipid-apoprotein complexes were generated by incubation with dimyristoylphosphatidylcholine and isolated by density gradient ultracentrifugation. Our results show that normal apoA-I and its isoprotein-4 associate with lipids to yield a complex containing 150-200 mol lecithin/mol apoA-I. The isoprotein-2 of normal apoA-I and the isoprotein-4 of Tangier apoA-I generate lipid-rich complexes with lecithin, while the isoprotein-2 of Tangier apoA-I shows only a limited association with lipids. ApoA-I normal and Tangier and their isoproteins-4 undergo a structural transition around 45 degrees C, which is not observed in the lecithin-apoA-I complexes. This transition is accompanied by an increased exposure of the tryptophanyl residues to the solvent. This transition was observed for the isoprotein-2 of apoA-I Tangier both in its lipid-free form and in the presence of lecithin. The pH denaturation of apoA-I and of the isoprotein-4 between pH 9 and 13 and between pH 7 and 2 is accompanied by a similar conformational transition. The transition occurs around pH 10.8 for the native apoproteins and is shifted towards respectively higher and lower pH's as result of the protective action of lipid binding on the protein conformation. Such an effect was not observed with the isoprotein-2 of apoA-I Tangier which is denatured at lower pH's both in its native form and in a lipid-protein mixture. Finally the denaturation of apoA-I by GdmCl indicates that apoA-I normal and Tangier undergo structural changes around 1 M GdmCl, whereas the apoA-I-Tangier-lecithin complex is more susceptible to denaturation than the complex with apoA-I normal. These data suggest that the apoA-I normal and Tangier and their isoproteins-4 are able to associate with lipids although the association between apoA-I Tangier with lecithin is weaker than that of apoA-I normal. The isoprotein-2 of normal apoA-I associates to a greater extent with lipids than the isoprotein-2 of Tangier apoA-I, whose structure differs from that of the isoprotein-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Corneal clouding is one of the manifestations of Tangier disease, an inherited disorder in which cholesterol-rich lipids are deposited in various tissues of the body. The cause of the corneal clouding is unknown. This study documents the clinical course and conjunctival biopsy findings of a 60-year-old man who was one of the earliest patients to be recognized with Tangier disease and in whom progressive corneal clouding developed in adult life. Noteworthy in the biopsy specimens were birefringent lipid particles that were predominantly present in degenerating pericytes of the conjunctival vessels.

Clinically significant hypolipidemia, although less common than hyperlipidemia, usually has important consequences that involve derangement of one or more of the major roles of lipoproteins. Deficiencies of lipoproteins are discussed under their classification as genetic disorders or as the presenting features of underlying disease.

Deficiencies of lipoproteins occur as genetic disorders or may be presenting features of underlying disease. Familial high density lipoprotein (HDL), or alpha-lipoprotein, deficiency so far described includes Tangier disease, Lecithin: cholesterol acyltransferase (LCAT) deficiency, A-I Variants syndrome and Fish-eye disease. In 1981 Vergani described a familial aggregation of low HDL-cholesterol (less than 33 mg/dl) and Apo A (about 50% of normal levels) in the presence of normal VLDL and LDL-cholesterol. LCAT and lipoprotein lipase activities, both extrahepatic and hepatic, were normal. By zonal ultracentrifugation HDL2 subclass was found to be reduced. HDL apoproteins, examined by isoelectric focusing in polyacrylamide gel, were qualitatively normal. No disorders to which low levels of HDL might be secondary (e.g., overweight, cigarette smoking, nephropathy, liver disease) are present in the affected members. The underlying biochemical defect is unknown but probably involves altered synthesis or catabolism of HDL. Familial hypo-alpha-lipoproteinemia is accompanied by a high prevalence of premature myocardial infarction and sudden death. The genetic analysis of the disorder is consistent with autosomal inheritance. The criteria for the definition of familial hypo-alpha-lipoproteinemia are, therefore, as follows: 1) low HDL-cholesterol level in the presence of normal VLDL and LDL-cholesterol levels; 2) absence of diseases or factors to which hypo-alpha-lipoproteinemia might be secondary; 3) presence of a similar lipoprotein pattern in a first degree relative.

Tangier disease is a rare autosomal recessive lipid transport disease characterized by the absence of the usual high density lipoproteins from plasma and cholesteryl ester storage in many organs. 25 cases of Tangier disease have been described so long. The predominant clinical symptoms include tonsilar hypertrophy, splenomegaly and peripheral neuropathy. The cholesteryl ester storage is limited to macrophages, Schwann's cells and intestinal smooth muscle cells. Hypocholest erolemia (less than 80mg/dl), hypertriglyceridemia (greater than 200 mg/dl), and the absence of high density lipoproteins in agarose electrophoresis are the major plasma abnormalities. The protein moiety of normal high density lipoprotein consists of apoprotein A-I and apoprotein A-II. In Tangier disease, serum concentrations of these apoproteins are reduced to less than 1% and 5-10%, respectively. Theories concerning the pathogenesis of Tangier disease are only incomplete and unproved up to now; however, a structural abnormality of apoprotein A-I causing an inability to bind to lipid or other proteins (apoprotein A-II) is consistent with several of the recent biochemical findings. The imbalance of cellular cholesterol metabolism caused by the absence of high density lipoproteins as well as the presumed role of these lipoproteins in cholesterol removal from cells are discussed in this article.

Platelet Activating Factor (PAF) (1-O-alkyl-2-acetyl sn-glycerol 3-phosphocholine) has been characterized by its ability to aggregate platelets at low concentrations and its profound hypotensive effects. There is evidence that the rate of catabolism of this compound in the plasma regulates its concentration. In humans, we and others have shown that a PAF acetylhydrolase is associated with low density lipoprotein (LDL). The LDL particle in the plasma of patients with Tangier disease is quite different from normal as its lipid core appears to be enriched with triacylglycerol. Thus, we have studied the potential of this abnormal lipoprotein to degrade PAF. The assay for PAF acetylhydrolase was based on the release of 3H from PAF that was labelled in the acetate moiety of the sn-2 position. Tangier disease plasma had approximately 3.3-fold higher PAF acetylhydrolase activity (208 +/- 9 nmol/min/mL) than controls (63 +/- 18 nmol/min/mL). This increase was brought about by an increase in the Vmax (400 +/- 40, Tangier disease; 54 +/- 5, controls) and Km for PAF (120 +/- 20 mumol/L, Tangier disease; 28 +/- 4 mumol/L, controls). The activity appears to be a specific acetylhydrolase rather than a phospholipase A2 as preincubation of the substrate with 0 to 100 mumol/L phosphatidylcholine did not affect the amount of [3H] acetate released. The role of PAF, and its degradation by LDL-bound PAF acetylhydrolase in the phenotypic expression of this patient with Tangier disease, is not known. However, this is the first patient so far described who has an increased ability to degrade PAF in the plasma.

A 65-year-old man with Tangier disease (analphalipoproteinemia) had had a progressive sensorimotor distal neuropathy with sensory ataxia for 1 year. Muscle biopsy demonstrated excess lipid vacuoles on histochemical and electron-microscopic techniques. Sural nerve biopsy showed a marked loss of large fibers and an increase in small myelinated fibers, with presence of remyelinating fibers and clusters of regeneration; a few aspects of active demyelination and some onion-like formations were also present. Lipid accumulation chiefly affected the Schwann cells of unmyelinated fibers and, to a lesser degree, of myelinated fibers, endoneurial fibroblast, and vasa nervorum. Teased fibers showed prevalent aspects of de-/remyelination and, often in association, marked myelin wrinkling suggesting axonal atrophy. This Tangier patient differs from known cases for the presence of a distal symmetrical sensorimotor polyneuropathy (not previously reported in Tangier disease) and because of the morphological findings of de-/remyelination coexisting with aspects of axonal atrophy and previous degeneration, and of lipid accumulation within striated muscle and vasa nervorum. This latter finding contrasts with the assumption that in Tangier disease vessel walls are not a site of lipid storage: probably the vasa nervorum are different, in this respect, from other vessels, because of the intense lipid metabolism of the nervous tissue. Thus we suggest that involvement of vasa nervorum in Tangier disease may be more important than previously suspected, possibly playing a role in the causation of neuropathy.

A 62-year-old man with clinical and biochemical findings consistent with homozygous Tangier disease is presented. Widespread atherosclerosis was present. Bile lipid analysis showed a low molar percentage of cholesterol with a low saturation index. The data suggest that high density lipoprotein cholesterol may act as a preferential precursor of biliary cholesterol. Coagulation and platelet studies indicated that the patient's platelets were hyper-responsive to aggregating agents and produced an increased amount of thromboxane B2. A platelet storage pool deficiency was also found.

The oxidative cross-linking effect exerted by OsO4 on lipids and the blackening of osmiophilic structures resulting from reduction of OsO4 constitute long-known phenomena. Nevertheless, osmic acid has rarely been used for lipid staining as various even non-lipid-containing tissue structures are more or less intensively blackened by OsO4. If one treats osmicated tissue sections with certain oxidants (e.g. ammonium persulfate) in aqueous solution, then all the black OsO4 reduction products which are exposed to the aqueous phase are oxidized and thereby bleached. This applies e. g. to all proteins and polar (hydrophilic) lipids. Only apolar lipids retain their more or less black color and can be stained subsequently with Sudan dyes. Based on this principles, a new procedure has been developed which is effective on paraffin sections obtained after previous block osmication. This OBS procedure can also be used for very precise localization of appropriate lipid structures as can be demonstrated in cases of pathologic lipid deposits in an-alpha- and in a-beta-lipoproteinaemia, in type III hyperlipoproteinaemia, and in diverse other dyslipoproteinaemias as well.

The interaction of human high density lipoproteins (HDL) with isolated human monocytes from control and Tangier patients was studied in tissue culture experiments. It was observed that normal monocytes, similar to mouse peritoneal macrophages, bind HDL to a cell surface receptor, internalize the bound HDL particles, transport the internalized HDL intracellularly through the cytoplasmic compartment without significant degradation, and ultimately resecrete intact HDL. The cellular interaction of Tangier monocytes with normal HDL was markedly different from control monocytes. HDL binding to Tangier monocytes was moderately increased and cell-associated HDL radioactivity was 6- to 10-fold enhanced in Tangier monocytes. The bulk of the internalized HDL, however, was detected in secondary lysosomes. Only minor amounts of the internalized HDL were resecreted from the Tangier monocytes, and most of this was degraded. These data suggest that the cellular metabolism of HDL is abnormal in Tangier monocytes. It is postulated that Tangier disease may be a disorder of intracellular membrane traffic in which HDL is diverted into the lysosomal compartment and degraded instead of being secreted through its regular transcellular route.

A 67-year-old woman, with the typical biochemical features of Tangier disease, had a syringomyelia-like syndrome which has now been observed in several patients with symptomatic onset in adult life. She developed progressive facial diplegia, bilateral wasting of hand muscles and loss of sensation over cranial, cervical and brachial dermatomes over 17 years. Nociception alone was first affected, then nociception and thermal discrimination and finally all modalities of sensation. Quantified tests of cutaneous sensation confirmed that sensation was normal in lower limbs but markedly abnormal in upper limbs. Biopsied fascicles of cutaneous nerves from clinically affected (forearm) and from clinically unaffected (leg) regions permitted a comparison of well-advanced and early pathologic lesions, respectively. The selective vulnerability of unmyelinated and small myelinated fibers in affected regions in this disorder has been confirmed. The earliest morphologic abnormalities of myelinated fibers, but seen infrequently, were mitochondrial enlargement and structural abnormality, aggregation of mitochondria and dense bodies and clusters of neurofilaments. Increased numbers of sudanophilic lipid droplets did not seem to form in Schwann cell cytoplasm prior to fiber degeneration. On the contrary, for myelinated fibers there appeared to be an altered process of axonal degeneration from that seen in Wallerian degeneration and in other axonal degenerations. Distinctive linear bands of closely-packed, minute, osmiophilic and clear lipid droplets formed and their further degradation appeared delayed. Although less clearly demonstrated, lipid droplets in Schwann cells of unmyelinated fibers also appeared to form following their degeneration. We would propose that in Tangier disease, the degradation of myelin ovoids to neutral lipid in Schwann cells does not appear to be delayed. However, further degradation of neutral lipid or its transport away from Schwann cells appears to be retarded.

To determine whether human small intestine synthesizes apoA-I, the major apoprotein of plasma high-density lipoproteins, we used immunofluorescence technics and monospecific antiserums to visualize apoA-I within intestinal epithelial cells from four normal subjects and one patient with Tangier disease. Biopsies from all subjects during fasting showed limited fluorescence. After lipid feeding intracellular apoA-I markedly increased in both normal subjects and the patient. During alimentary lipemia, mean plasma apoA-I levels (milligrams per deciliter) increased in four normal subjects from 161 +/- 12 (+/- S.E.M.) to 180 +/- 15 (P less than 0.05) and in the patient from 1.9 to 6.8. Normal plasma chylomicrons contained apoB, apoE and the C peptides but not apoA-I. The patient's chylomicrons contained ap0A-I. Normal and Tangier-disease intestinal-mucosa cells increase their content of apoA-I during chylomicron formation and subsequently contribute to plasma apoA-I levels. The low levels of apoA-I in Tangier disease are not due to a failure of intestinal synthesis but might be due to abnormal metabolism of chylomicron apoproteins.

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.

Tangier disease is a rare, autosomal recessive condition characterized by cholesterol ester deposition in reticuloendothelial cells, abnormal chylomicron remnants, decreased low-density lipoprotein levels, and a marked deficiency of high-density lipoproteins. Apolipoprotein A-I, a major protein constituent of chylomicrons and high-density lipoproteins, has been shown to be structurally and metabolically abnormal in this disease (apolipoprotein A-ITangier). A 63-year-old Tangier homozygous man is described, who underwent splenectomy because of thrombocytopenia and splenomegaly. Subsequently, a large orange mass developed at the base of the mesentery, with several smaller omental masses and thickening of the entire omentum due to infiltration with lipid-laden macrophages. Splenectomy appears to predispose to such deposition, since such masses have not been observed in other Tangier homozygotes. The spleen appears to play a significant role in the removal of abnormal lipoproteins in Tangier homozygotes; therefore, splenectomy may be contraindicated in Tangier disease.

To define the metabolic defect in Tangier disease, we studied the kinetics of [125I]-high-density lipoprotein apolipoproteins (apolipoproteins A-I and A-II) in 11 normal subjects, two obligate heterozygotes, and two homozygotes. Mean synthesis of apolipoproteins A-1 and A-11 was 8.24 mg per kilogram per day in the normal group, 7.94 in heterozygotes and 3.66 in homozygotes. The mean plasma-residence time for both apolipoproteins was 5.21 days in the normal subjects, 3.41 days in heterozygotes, and 0.52 days in homozygotes. In normal subjects and heterozygotes the apolipoproteins were catabolized at similar rates, whereas in homozygotes apolipoprotein A-I was catabolized at a much greater fractional rate than apolipoprotein A-II. These findings indicate that the deficiency of these apolipoproteins in Tangier disease is largely due to rapid and altered catabolism.

Normal high-density lipoproteins are absent from plasma in Tangier disease, and the disorder is characterized by accumulation of cholesteryl esters in several tissues, particularly those of the reticuloendothelial system. Electron microscopy of the abnormal high-density lipoproteins in the plasma of three patients with Tangier diseases revealed large (68-nm), flattened, translucent particles in all cases. These particles were most abundant in the plasma of the splenectomized patient. Restriction of dietary fat eliminated or drastically reduced the numbers of these particles among the Tangier high-density lipoproteins. Thus abnormal products of chylomicron metabolism that appear to occur in plasma in this disorder may be targets for phagocytosis and may be at least one source of the cholesteryl esters that accumulate in reticuloendothelial tissues in Tangier disease.