Fish eye syndrome: a molecular defect in the lecithin-cholesterol acyltransferase (LCAT) gene associated with normal alpha-LCAT-specific activity. Implications for classification and prognosis.

We have identified the molecular defect in two siblings presenting with classical clinical and biochemical features of Fish Eye disease (FED), including corneal opacities, HDL cholesterol < 10 mg/dl, normal plasma cholesteryl esters, and elevated triglycerides. In contrast to previously reported patients with FED who are unable to esterify HDL-associated cholesterol, our patients' plasma lecithin-cholesterol acetyltransferase (alpha-LCAT)-specific activities assayed using an HDL-like proteoliposome substrate were 12.7-25.7 nmol/micrograms (19.5 +/- 1.8 in controls). In addition, significant residual cholesterol esterification was present in VLDL/LDL-depleted plasma, confirming the presence of HDL-associated alpha-LCAT activity. DNA sequence analysis of the proband's LCAT gene identified deletion of the triplet coding for leu300, which resulted in the loss of a restriction site for MlnI. Digestion of PCR-amplified DNA using MlnI established that both siblings are homozygous for this defect. Expression of LCAT300-del. in human embryonic kidney-293 cells revealed normal mRNA and intracellular LCAT concentrations. However, reduced amounts of LCAT300-del., which had a normal specific alpha-LCAT activity, were present in the media. In summary, we report the first case of FED associated with a mutant enzyme that has a normal alpha-LCAT-specific activity. The functional significance of this LCAT gene defect has been established in an in vitro expression system, which demonstrates that very small amounts of this functional LCAT mutant enzyme accumulate in the media. Characterization of LCAT300-del. established that selective alpha-LCAT deficiency is not a prerequisite for the development of FED. On the basis of our combined results, we propose that the residual amounts of total plasma LCAT activity and not its distribution on lipoproteins primarily determines the heterogeneity in phenotypic expression observed in familial LCAT deficiency syndromes.

Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin: cholesterol acyltransferase deficiency and fish-eye disease.

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.

Immunochemical study of the plasma low and high density lipoproteins in Tangier disease.

Major disturbances of the lipoproteins in Tangier serum have been investigated using electrophoretic and immunochemical techniques. Previously described anomalies concerning the striking deficiency in HDL and the very low levels of apo A-I and apo A-II in Tangier patients are illustrated and explained. Anomalies concerning the fast LDL of Tangier serum are attributed to different forms of apo B not previously described. These data are strengthened by the features of a 2-dimensional electrophoresis method elaborated in the laboratory which allows apoproteins to separate in the second dimension. These apoproteins are obtained by the delipidation of the lipoproteins fractionated in a first polyacrylamide discontinuous gel. This method clearly shows the distribution of apoproteins in the first lipoprotein track and is in perfect accordance with the new concept of lipoprotein particles.

Isoelectric focusing (IEF) and immunofixation for determination of disialotransferrin.

OBJECTIVES: A new simplified method for detection and quantitation of disialontransferrin in serum is described. DESIGN AND METHODS: The method is based on polyacrylamide gel isoelectric focusing, direct immunofixation with a specific antibody, and measurement by computerized scanning densitometry. Disialotransferrin levels were determined in 24 teetotallers and 34 alcoholics at 3 moments during detoxification. Three groups of drinkers were arranged: group 1 (without), group 2 (with light), and group 3 (with severe hepatitis). RESULTS: The metho showed very good reproducibility and accuracy with a coefficient of variation between 5 to 8%. Alcoholic patients could be clearly separated from teetotallers, with a specificity of 100% and a sensitivity of 94%. After 12 days of alcohol withdrawal, disialotransferrin values declined in alcoholics but remained slightly high. They were not influenced by the severity of liver disease. No significant difference was found between the 3 groups. CONCLUSIONS: An easy-to-perform, sensitive, and inexpensive method has been developed to quantify disialotransferrin that can be used by laboratories almost everywhere.

Heterogeneity of HDL in crossed immunoelectrophoresis with continuous gradient polyacrylamide gel in the first dimension and one or two antibody layers in the second dimension.

We describe a new method of immunoelectrophoresis with a continuous gradient polyacrylamide gel in the first dimension and an agarose-dextran gel in the second dimension with one or two layers of antibody. The use of a polyacrylamide gel in the first dimension allows better resolution of lipoproteins than with crossed immunoelectrophoresis using agarose gel in both dimensions. The use of two layers of antibody in the second dimension also enhances the specificity of characterization and the resolution of the separation. Thus, using a layer of anti-apo A-I combined with a layer of anti-apo A-II, three particles containing only apo A-I and three containing both apo A-I and A-II could be separated.

Absence of "A"-esterase activity in the serum of a patient with Tangier disease.

The levels of apolipoprotein A-I, A-II and B in subjects who are homozygous or heterozygous for Tangier disease are reported and compared with the amount of "A"-esterase in the serum. The "A"-esterases hydrolyse toxic organophosphate pesticides and are currently classified by the nomenclature committee of the International Union of Biochemistry as arylesterases (EC 3.1.1.2) although recent evidence has cast doubt on this classification. The apolipoprotein data are consistent with previous data reported for a number of Tangier patients. The homozygote has a marked reduction in apo A-I and A-II levels and a 30% reduction in apo B. The heterozygotes have about a 50% reduction of apo A-I, a slight reduction in apo A-II and no change in apo B. These apolipoprotein values correspond to a marked reduction in HDL cholesterol for the homozygote and substantial reductions in the heterozygotes. The "A"-esterase activity is zero in one homozygote while heterozygotes have about 5% of the levels in control subjects. Arylesterase activity appears to be essentially normal. The data thus support previous observations that the HDL "A"-esterase activity is greatly reduced in those conditions where HDL apo A-I is markedly reduced, e.g., in "Fish-eye" Disease.

Relationship between red blood cell antioxidant enzymatic system status and lipoperoxidation during the acute phase of malaria.

OBJECTIVE: To investigate the relationship between oxidative stress and aggrevation of the disease in patients with malaria. METHODS AND RESULTS: In the present study lipoperoxidation was demonstrated during the acute phase of malaria by a significant decrease in polyunsaturated fatty acids (PUFA). The lowest values of PUFA were obtained for C20:4 and C22:6, which were the main targets of reactive oxygen species (ROS) when parasitemia was higher than 1%. Similarly, plasma vitamins E and A were significantly reduced during the acute phase of malaria owing to their consumption in part as antioxidants. However, evaluation of the antioxidant enzymatic system in red blood cells of malaria patients indicated no significant difference from controls. Only superoxide dismutase activity tended to decrease when parasitemia increased. CONCLUSION: The results suggest that superoxide radicals are the main ROS produced during the acute phase of malaria, and that rejuvenation of RBC during hemolysis involving increased enzyme activities interacts to protect RBC from excessive superoxide radical production.

Preliminary report on a case of apolipoproteins CI and CII deficiency.

A combined deficiency of Apo C-I and C-II assessed by mono and bidimensional electrophoresis as well as immunoelectrophoresis is described. It was discovered after a 'check up' in a 70-yr-old woman consulting for a vertebral pain. Lipoprotein disorders correspond to a particular form of Fredrickson's type V. They consisted of types I and IV, with decreased HDL of low electrophoretic mobility, increased VLDL of high electrophoretic mobility, and without LDL. A decrease of Apo A-I, A-II, B and C-III was observed. Data correspond for the most part with all those actually known to characterize Apo C-II deficiency. HDL3 predominance in decreased HDL fraction and strongly decreased CE/TC ratio could be dependent of Apo C-I deficiency. The association of these two apolipoprotein deficiencies, the genes of which are located on chromosome 19, suggest a common defect on the pathway of their biosynthesis possibly located at the gene level. In spite of these numerous anomalies, the affection appears well tolerated.

Modified lipid-protein interactions in Tangier beta lipoprotein (LDL2) demonstrated by fluorescence quenching.

Fluorescence quenching by iodide ions has been found to be higher in isolated Tangier low density lipoprotein (LDL2) than in isolated normal LDL2. Apolipoprotein (apo) B-100 is the main protein component of these lipoproteins and its tryptophanyl residues (Trp) are known to be the most hydrophobic and to be responsible for protein fluorescence. Trp exposure can thus be calculated; it was 0.50 in Tangier and 0.42 and 0.41 in insulin-dependent diabetics (IDD) and normal controls, respectively. The greater fluorescence quenching of Tangier LDL2 reveals a shallower embedding of Trp which is principally due to a lowered free cholesterol (FC) level in the shell and a smaller lipid core, itself dependent on a drop in cholesterol esters (CE). This is in accordance with the electrophoretic properties of Tangier LDL2 and suggests that Tangier LDL2 may be considered to be modified.

Reduction of dietary saturated fatty acids correlates with increased plasma lecithin cholesterol acyltransferase activity in humans.

OBJECTIVE: Increased HDL-cholesterol (HDL-C) concentrations have been associated with lower coronary heart disease risk. On the other hand, dietary fats are known to influence the fatty acid profile of plasma lipids, including phospholipids that are substrates of lecithin cholesterol acyltransferase (LCAT), an important enzyme in HDL metabolism. The purpose of this study was to examine the association between the saturated fatty acid (SFA) intake and LCAT activity. DESIGN: An interventional study was performed in a monk community of 25 men. SETTING: A French monk community, South West of France. SUBJECTS AND INTERVENTIONS: The basal diet of the study cohort contained SFA in a proportion of 13.5% of their total energy intake (TEI). They were submitted to two experimental isocaloric diets containing either 8.4% of the TEI in SFA (diet A) or 11% (diet B), each lasting 5 weeks. RESULTS: The elevation of SFA in diet B was mainly obtained by decreasing carbohydrates. The only significant difference among total fats between diets A and B was the myristic acid content (0.6 and 1.2% of TEI, respectively). The elevation in SFA in diet B resulted in a significant increase of HDL-C (P<0.04), while plasma apo A-I concentration and LCAT activity both decreased (P<0.02). CONCLUSION: Altogether, these results are consistent with a negative effect of SFA on reverse cholesterol transport.

A fish-eye disease-like familial condition with massive corneal clouding and dyslipoproteinemia. Report of clinical, histologic, electron microscopic, and biochemical features.

Fish-eye disease (FED) is a rare familial condition characterized by progressive bilateral corneal clouding and dyslipoproteinemia previously described in one family and an unrelated woman of Swedish descent. Biochemical studies have clearly demonstrated the existence of this entity as a unique dyslipoproteinemia. We present a non-Swedish family of Mediterranean ancestry afflicted with bilateral corneal clouding and lipoprotein analysis consistent with FED-like state. This family's biochemical profile, corneal button histology, and electron microscopy of one member are reviewed. Other dyslipoproteinemias causing corneal changes are considered. Corneal tissue and familial biochemical analyses differed significantly from previous descriptions. On the basis of these findings, explanation of pathologic deposition and disease mechanism is proposed.

[M monoclonal macroglobulinemia induced in BALB/C mice. Effect on blood cholesterol and its significance]. / Macroglobulinémie M monoclonale provoquée chez la souris BALB/C. Incidence sur la cholestérolémie et intérêt.

BALB/C mice have been inoculated with a monoclonal IgM secreting hybridoma and have developed an hypocholesterolemia strongly dependent of the hypermacroglobulinemia M obtained (P less than 0.001). Cholesterolegram shows cholesterol is carried on the monoclonal IgM fraction. This result has been established in comparison with a lot of mice treated with the same no secreting hybridoma and a lot of untreated-mice, in these cases cholesterolemia is not modified. The whole aminoacids sequence of this IgM being nearly achieved, it is thinked of check the hypocholesterolemic activity of several parts of this immunoglobulin.

[Demonstration of the anti-lipid peroxidation effect of 3',5,7-trihydroxy-4'-methoxy flavone rutinoside: in vitro study]. / Mise en évidence de l'effet antilipoperoxydant du 7 rutinoside de la 3',5,7-trihydroxy-4'-methoxy flavone: étude in vitro.

Diosmin (DI) 3'5,7-trihydroxy-4'-methoxy flavone rutinoside is a member of the flavonoid family, some of whom have antioxidant or free radical scavenger properties. Paw oedema induced by doxorubicin in rats is reduced by DI as observed by SAUVAIRE et al (1989). This suggests a free radical scavenger activity for DI. In this work we demonstrate that such activity is able to protect isolated human LDL from oxidation in vitro. The study of the electrophoretic mobility of oxidized LDL and of total-MDA values as lipoperoxidation-marker indicates an oxidation inhibiting effect higher than 70% for 0.16 mM DI in LDL mixtures containing 50 mM Cu2+ or 4.3 mU/ml xanthine-oxidase and incubated during 20 and 6 hours respectively. Owing to the high level of the oxidizing conditions and the vitamin E (48 mM), vitamin A (1.4 mM) and beta-carotene (2.4 mM) content of the LDL mixtures, it is concluded that DI is clearly able to complement the antioxidant effect of isoprenoids which are naturally present in LDL mixtures.

[In vivo study of the antilipoperoxidant effect of 3',5,7-trihydroxy-4'-methoxy flavone 7 rutinoside]. / Etude in vivo de l'effet antilipoperoxydant du 7 rutinoside de la 3',5,7-trihydroxy-4'-méthoxy flavone.

In 3-month-old Wistar rats carrageenan and CCl4 injected intraperitoneally induce an acute phase reaction which is characterized by a marked increase in alpha 1, alpha 2, beta serum globulins. This reaction corresponds to a large increase in these globulins in the first case and a smaller one in the second. A lipoperoxidant effect is demonstrated by the serum lipoprotein mobility as the lipoperoxidation index (in MDA units) or the decrease in serum vitamin A and E concentrations. This effect is also greater in the first case than in the second one. In the same way the lipoperoxidant effect is shown in liver microsomes but with a lower amplitude in the first case than in the second one. The treatment of rats by intraperitoneal injection of diosmine (150 mg/kg per week) during the 8 weeks which precede the injection of carrageenan or CCl4 results in: i) a marked decrease in the acute-phase reaction and a lower one in the lipoperoxidant effect, in serum; ii) a decrease in the CCl4 induced lipoperoxidant effect in liver microsomes. It may be concluded that diosmine, not injected at the same time as carrageenan or CCl4, but during the previous 8 weeks is sufficiently well distributed in the whole body to produce a marked inhibition of the acute phase reaction and a perceptible effect on lipoperoxidation. It may be considered an effective complement to the natural antioxidant defences of the organism (vitamins A and E).

[First observations on the main plasma parameters of oxidative stress in homozygous sickle cell disease]. / Premières observations sur les principaux paramètres plasmatiques du stress oxydatif chez le drépanocytaire homozygote.

The present study report 7 cases of sickle homozygous disease which have been analysed using markers of the oxidative-stress, 26 african male subjects were studied: 7 Hb SS subjects (age: m = 20) and 19 control subjects (Hb AA, age: m = 40). Plasma concentrations of F-MDA, T-MDA, TBARS, alpha tocopherol, retinol and beta carotene were measured. Plasma MDA and TBARS mean levels increased in sickle homozygous patients more than in controls. However, only TBARS mean concentrations were significantly increased between patients and controls: TBARS: 4.14 +/- 1.49 nMol/ml for Hb SS versus 2.10 +/- 1.21 nMol/ml for Hb AA (P less than 0.005). Vitamin A and vitamin E concentrations were significantly lower in Hb SS than in Hb AA. Beta carotene was significantly increased in patients vs controls. The significant increase of TBARS explains the great importance of the oxidative damage, whereas the significant decrease of vitamins A and E, may contribute, at least for a part, to maintain the autoxidation process or reveals its intensity in these patients.

[Interferon and blood tumor necrosis factor in 95 patients with HIV infection]. / Interféron et facteur de nécrose tumorale sériques chez 95 patients infectés par le VIH.

We have measured TNF-alpha and interferon alpha in 95 HIV positive patients and 20 healthy subjects. TNF-alpha was higher in the HIV+ patients (P = 0.0001) and was correlated to the CD4 cell count (P = 0.02) and cholesterol (negatively) (P = 0.04). Interferon-alpha was correlated to the wasting syndrome (P = 0.002), hypertriglyceridemia (P = 0.004) and haematocrit (P = 0.04).

[Membrane fatty acids and blood antioxidants in 77 patients with HIV infection]. / Acides gras membranaires et anti-oxydants sériques chez 77 patients infectés par le VIH.

We have measured the fatty acid (FA) composition of erythrocyte membranes and plasma anti-oxidants in HIV+ patients. Saturated FA are higher and poly-unsaturated FA lower than in controls (P = 0.02). Selenium (Se) is lower in patients less than 400 CD4 cells/mm3 (P = 0.002). Vitamin A is lower in the HIV+ regardless of the CD4 cell count. Se and vitamin A are correlated to nutritional markers (body mass index and albumin).

[Lp(a) and weight loss in obese patients]. / Lp(a) et perte de poids chez l'obèse.

High concentrations of lipoprotein (a) (Lp(a)) are associated with an increased risk of atherosclerotic vascular disease. Lp(a) synthesis is mainly under genetic control but many endocrine disturbances may modulate Lp(a) plasmatic concentrations. There is no agreement upon Lp(a) variations in patients under a hypocaloric diet. This study was undertaken to assess this point in obese females subjected to a 1100 kcal/d diet. Ninety-two obese patients (42.4 +/- 10.4 yr old, BMI 33.9 +/- 5.6 kg/m2) came once a week as out patients during 9 weeks. Lp(a) concentrations distribution was highly skewed. The threshold Lp(a) concentration for a significant cardiovascular risk is estimated at 0.3 g/l. Concentrations above 0.3 g/l were found in 29/92 patients (31%). If the patients were distributed in 2 groups according to their Lp(a) values (< or = ou > 0.3 g/l), the BMI, total cholesterol or triglycerides were not different. There were no significant correlation between Lp(a) and age, total cholesterol or triglycerides. After 9 weeks BMI and total cholesterol values decreased (-1.6 +/- 3.4 kg/m2 and -0.17 +/- 0.68 mmol/l, respectively). Lp(a) concentrations were unchanged (0.3 +/- 0.3 vs 0.3 +/- 0.3 g/l). There were no significant correlation between Lp(a) variations and age, BMI or initial Lp(a) concentrations. No significant decrease of Lp(a) could be detected even in the sub-group of patients with initial concentrations of Lp(a) > 0.3 g/l or even in a sub-group with Lp(a) > 0.7 g/l (n = 7). Under our conditions, weight loss is not associated with a decrease of Lp(a) concentrations suggesting that in a given obese a single determination is enough to assess his Lp(a)-related atherosclerotic risk.