Suzanne Salmon

Sensitivity of tryptophan and related compounds to oxidation induced by lipid autoperoxidation. Application to human serum low- and high-density lipoproteins

Tryptamine, serotonin and tryptophan are readily oxidized during the Cu2+-catalyzed peroxidation of arachidonic acid (AA) at neutral pH and under certain experimental conditions which determine their relative susceptibility to oxidation. Thus, in AA micelles, fluorescence spectroscopy demonstrates that positively-charged indoles interact with negatively-charged micelles while Trp remains in the aqueous phase. As a result, serotonin and tryptamine are preferentially oxidized. In egg phosphatidylcholine liposomes loaded with AA, the three substrates interact with vesicles and undergo lipid-induced oxidation. EDTA inhibits the formation of thiobarbituric-reactive substances (TBARS) and prevents the indoles from oxidation. Owing to the intricate contact between the lipidic core and the apolipoproteins, the Trp residues of human serum LDL and HDL3 are very rapidly oxidized, i.e., at least one order of magnitude faster than Tyr HDL and Lys LDL, which are believed to be involved in the binding of these lipoproteins to their cell receptors. Cupric ions are rather specific for the lipid-induced autoxidation of Trp residues of lipoproteins whereas in micelles and liposomes, Mn2+ and Fe2+ can lead to TBARS production and to oxidation of indoles. This specificity is surprising considering the known ability of Fe2+ to catalyze LDL modification (measured by TBARS production) during their incubation with various cells. Biological consequences of the easy lipid-induced oxidation of biologically important indoles are discussed.

Immunological detection of low-density lipoproteins modified by malondialdehyde in vitro or in vivo

We describe an ELISA technique able to recognize malondialdehyde-modified low-density lipoproteins (LDL). For this purpose we produced antibodies to malondialdehyde-LDL, specific for the malondialdehyde modification of LDL; these antibodies recognized essentially malondialdehyde-LDL. Coating ELISA plates with the antibodies to malondialdehyde-LDL and using peroxidase-labelled antibodies to LDL, which reveal only apolipoprotein B, we obtained an accurate method of detecting malondialdehyde-modified apolipoprotein B. Preliminary studies demonstrated that this method allows the detection of lipoproteins containing malondialdehyde-modified apolipoprotein B in the serum of patients with cardiovascular diseases.

Modified apolipoprotein pattern after irradiation of human high-density lipoproteins by ultraviolet B

The ultraviolet B-induced destruction of tryptophan residues and lipid peroxidation of high-density lipoproteins is accompanied by the immediate and marked structural modification of the apolipoproteins, as revealed by SDS-polyacrylamide gel electrophoresis and immunoblot with specific monoclonal antibodies. Formation of several polymers of apolipoprotein A-I, apolipoprotein A-II or both apolipoproteins occurred, although apolipoprotein A-II did not contain any Trp residue. These results suggest that initial photochemical damage can be transferred via intramacromolecular processes to other sites within the same apolipoprotein and by intermacromolecular reactions from apolipoprotein A-I to other apolipoproteins. In both cases, lipid peroxidation enhances the propagation of the initial photochemical damage. The physiological significance of this work is discussed with respect to the low-light doses required for the alterations of the high-density lipoproteins.

Monoclonal antibodies to low density lipoprotein used for the study of low- and very-low-density lipoproteins, in “ELISA” and immunoprecipitation technics

Seven monoclonal antibodies to low-density lipoprotein were studied by the ELISA for their reactivity with LDL or VLDL. Cotitration experiments showed that five of them are addressed to different antigenic epitopes. Two of the monoclonal antibodies were temperature independent whereas the others had a decreased binding activity at 37 degrees C compared to that obtained at 25 degrees C or 4 degrees C, suggesting the presence of antibodies directed to sequence or conformation epitopes, respectively. All antibodies reacted with both LDL and VLDL; four of them had a higher affinity for LDL and two others for VLDL. Immunoprecipitation of LDL and/or VLDL was observed upon immunodiffusion with certain pairs of antibodies. This may allow the use of pairs of monoclonal antibodies to LDL for the quantitative determination of apolipoprotein B in serum LDL and VLDL.

Carbon disulfide modification and impaired catabolism of low density lipoprotein.

Carbon disulfide interacts in vitro with low density lipoprotein (LDL), resulting in an increased electrophoretic mobility of the particle, due to a decrease in free amino groups of apolipoprotein B-100. The processing of carbon disulfide-modified LDL through the apo B/E receptor pathway of cultured human fibroblasts is decreased as compared to that of native LDL, depending on the level of modification. Carbon disulfide-modified LDL is recognized and degraded by the scavenger pathway of macrophages, but to a lesser extent than acetylated LDL. Carbon disulfide modification decreases the ability of the LDL to down-regulate sterol synthesis and to stimulate cholesterol esterification in fibroblasts. Carbon disulfide-modified LDL markedly stimulates cholesteryl ester formation in macrophages, albeit to a lesser extent than acetylated LDL. These results indicate that after carbon disulfide modification the LDL catabolism is shifted to the scavenger pathway, and are consistent with the fact that carbon disulfide intoxication accelerates the appearance of atherosclerotic lesions.

Cyclic AMP decseases LDL catabolism and cholesterol synthesis in the human hepatoma cell line HepG2

Summary A 24h pretreatment of the human hepatoma cell line HepG2 with dibutyryl cyclic AMP in the presence of theophylline induced a dose dependent decrease in low density lipoprotein binding, uptake and degradation. This effect is most likely due to a reduction of the LDL receptor number. Sterol synthesis from sodium acetate is markedly inhibited, either in the presence or absence of LDL, whereas synthesis from mevalonic acid is unchanged. Cyclic AMP also induced a decrease in hydroxy methyl glutaryl coenzyme A reductase activity. These effects of cyclic AMP might be involved in some hormonal regulation of the LDL pathway and cholesterol metabolism in the liver.

The affinity of type I collagen for lipid in vitro

Abstract Acid-soluble type I collagen was extracted from rat dermis and subcutaneous tissue, purified by three different precipitation procedures and the collagen-bound lipids were then compared. Pure delipidated type l collagen from rat skin was also incubated with pure human low-density lipoproteins. The lipids which bound to this collagen were analyzed as a function of collagen and LDL concentrations in the medium. The nature of the collagen-bound lipids depended on the lipid donor and the amount of lipids bound seemed to vary with the structural organization of the collagen (among other factors).

The antidiabetic drug Metformin decreases cholesterol metabolism in cultured human fibroblasts

The effect of the hypoglycemic biguanide drug Metformin was investigated after a 72 h pretreatment of human cultured fibroblasts. Metformin induced a moderate increase in low density lipoprotein binding, uptake and internalization (25% increase after treatment with 5 X 10(-4) M of drug). A decrease in sterol, fatty acid and triacyglycerol synthesis from sodium acetate was observed after pretreatment with the drug, with a dose-dependent effect in the range of 5 X 10(-5) to 5 X 10(-4) M (50% reduction of sterol synthesis after treatment with Metformin 5 X 10(-4) M). This effect was also observed in fibroblasts from a patient with homozygous familial hypercholesterolemia. Cholesterol esterification studied by incorporation of radiolabeled oleic acid was reduced by Metformin (40% of control after treatment with Metformin 5 X 10(-4) M) whereas incorporation into triacylglycerols was less impaired. These effects of Metformin on cholesterol metabolism were observed either in the presence or in the absence of low density lipoproteins. Moreover, Metformin also reduced cholesterol esterification in J774 monocyte-macrophage cells. Metformin also induced a decrease of hydroxymethylglutaryl coenzyme A reductase activity in cultured fibroblasts and a reduction of acyl-coenzyme A: cholesterol-O-acyltransferase activity in cultured fibroblasts and J774 cells.

Lipoproteines associeés aux lipoproteines B dans les lipoproteines de basse densite de serum humain

Abstract Small amounts of lipoprotein C and lipoprotein D could be observed in low density lipoproteins (1.030–1.055 g/ml), using electroimmunomigration and polyacrylamide gel electrophoresis. Lipoprotein structures containing several apolipoproteins such as lipoprotein (B + C) or (B + D) were not detected in these low density lipoproteins. Lipoproteins C and D could not be separated from lipoprotein B by using gel filtration and affinity chromatography on heparin-Sepharose. Apolipoproteins C-III and D measured by electroimmunoassay are 3.2 ± 1.2% and 1.15 ± 0.06%, respectively, of the proteins found in the density range 1.030–1.055 g/ml, so there is, therefore, about 1 mol of apolipoprotein C-III and 0.1 mol of apolipoprotein D per mol of apolipoprotein B.

Étude par immunoélectrophorèse bidimensionnelle de la dissociation spontanée des lipoprotéines de basse densité

Summary Plasma-lipoproteins isolated between d 1.020 and d 1.055 g/ml were partially delipidated with ethyl ether at 4°C. This treatment induces a transformation of the lipoproteins which is evolutive during several days. Bidimensional immunoelectrophoresis reveals the lipoproteins dissociation and the appearance of 4 immunologically different fractions. The time dependent formation of these subunits is slowed down by EDTA and less efficiently by antioxydants. Once started, the dissociation can be accelerated by heating at 37°C or by UV exposition. Another lipopeptide is more easily revealed by anti VLDL antiserum. It can be shown in the native and in the partially or completely delipidated LDL. Its presence does not depend on lipoprotein dissociation.