Gaetano Corso

Oxidative structural modifications of low density lipoprotein in homozygous familial hypercholesterolemia

Patients with homozygous familial hypercholesterolemia (FH), as a result of the increased levels and prolonged residence time of low density lipoprotein (LDL) in plasma, have a strong tendency toward accumulation of LDL-cholesterol in the arterial wall, causing premature atherosclerosis. This phenomenon may enhance per se the physiological degradation of both protein and lipid component of LDL, which be more susceptible to oxidative damage induced by oxygen radicals. It is well known that LDL may undergo oxidative modification before being taken up by macrophages which are then transformed into foam cells. It has been suggested that platelet-activating factor (PAF) may play an important role in atherogenesis and PAF catabolism is known to be mediated by serum acetylhydrolase, an enzyme that is normally associated with LDL. Thus, the present study was designed to investigate the structural properties of LDL, including acetylhydrolase activity, in homozygous FH as compared to normolipidemic subjects before and after xanthine/xanthine oxidase-mediated oxidation. We studied 8 homozygous FH patients matched with 8 normolipidemic volunteers. Lipids of LDL fraction were extracted and verified by thin layer chromatography (TLC) analysis. Fatty acids were methylated and injected into a gas chromatograph/mass spectrometer. Vitamin E in LDL was determined by high performance liquid chromatography (HPLC). As an index of susceptibility of LDL to oxidative modifications, the formation of lipid-conjugated dienes was continuously monitored at 234 nm. Lipid peroxidation was also evaluated from the amount of both lipid peroxides (LPO) and malonyldialdehyde (MDA) content. Apolipoprotein (apo) B-100 on LDL was carried on polyacrylamide and agarose gel electrophoresis. In the homozygous FH patients, the relative content of cholesteryl ester was slightly increased. Interestingly, the relative amount of arachidonic acid (20:4) was constantly increased in each lipid fraction in homozygous FH patients. The amount of vitamin E was not significantly different in the patient group from that in the control group. However, LDL from patients carried lower levels of vitamin E (nmol/mg LDL) than controls (2.7 +/- 0.4 vs. 2.9 +/- 0.3 P = NS). The results shows that lag time (min) was decreased (82 +/- 19 vs. 111 +/- 21; P < 0.05) and the maximal rate of diene production and total diene production was increased in homozygous FH patients. Mean levels of MDA were similar in both groups before oxidation, but levels after initiation of oxidation were significantly higher in the patient group. In contrast, mean levels of LPO were already higher in patients before oxidation (58 vs. 27 nmol/mg of protein; P < 0.05), and after initiation of oxidation were also significantly higher at each time points. When oxidized LDL was run on a polyacrylamide gel, an extensive apo B-100 fragmentation replaced by lower molecular mass fragments ranging from 45,000 to 205,000 m.wt., was observed only in LDL from homozygotes. Relative LDL agarose gel mobility shows that LDL from patients migrated higher than LDL of controls. Finally acetylhydrolase activity associated with LDL in patients was significantly reduced as compared to controls. Thus, in homozygous FH patients, LDL appeared more susceptible to oxidation in vitro; the indices for LDL oxidizability were all significantly different from those of controls. This phenomenon might be due to prolonged residence time of LDL in these patients, as suggested from high basal LPO levels and lower vitamin E levels carried by LDL. This hypothesis may explain together with the high content of arachidonic acid, the enhanced susceptibility of LDL from homozygous FH patients to oxidative damage.

Vitamin D status in patients affected by Smith-Lemli-Opitz syndrome

SummarySmith-Lemli-Opitz syndrome (SLOS) is an inborn error of cholesterol biosynthesis characterized by developmental delay and multiple malformations.Some of the patients have skin photosensitivity and therefore tend to avoid direct exposure to sunlight.SLOS patients typically have low concentrations of cholesterol and abnormally high concentrations of its precursor 7-dehydrocholesterol (7-DHC) in biological fluids and tissues. 7-DHC is also a precursor in the cutaneous synthesis of vitamin D. Sunlight exposure plays a major role in this pathway and reactions transforming 7-DHC into vitamin D and then into 25-hydroxyvitamin D are known not to be specifically regulated. The aim of this study was to evaluate vitamin D status in SLOS patients. We measured 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D serum concentrations and markers of calcium metabolism in five SLOS patients.Despite abnormally high concentrations of 7-DHC, circulating concentrations of vitamin D metabolites were not significantly different from appropriate controls matched for sex, age and season of blood collection. The analysis of historical serum samples stored in our laboratory from the same cases plus 10 other SLOS patients further supported these findings. Our data suggest that SLOS patients have a peculiar vitamin D metabolism that protects them from vitamin D intoxication.This appears to be due in most cases to decreased transformation of 7-DHC into 25-hydroxyvitamin D, perhaps depending on reduced sunlight exposure as a consequence of photosensitivity. Possible alternative mechanisms are discussed.

Sterol profiling in red blood cell membranes and plasma of newborns receiving total parenteral nutrition.

Background and Objectives: Total parenteral nutrition (TPN) is a lifesaving therapy in children with intestinal failure, frequently complicated by liver dysfunction. Plant sterols (phytosterols) of lipid emulsions have been supposed to contribute to cholestasis in TPN-treated children. The present study aimed to evaluate the plasma and red blood cell membrane (RBCM) phytosterol levels in newborns after a short period of TPN. Patients and Methods: Phytosterols, cholesterol, and other sterol levels were quantified by gas chromatography-mass spectrometry in 15 healthy control infants, 22 patients after TPN, and 11 patients before TPN. Sterols of lipid emulsions were quantified. Results: Plasma and RBCM phytosterol levels were, respectively, on average 56 μmol/L and 83 μmol/g per protein in patients after TPN, 13 μmol/L and 15 μmol/g per protein in patients before TPN, and 9 μmol/L and 13 μmoL/g per protein in control infants (P < 0.05 for differences). The days of TPN and the total amount of infused lipids correlated significantly with RBCM phytosterol (P < 0.05); correlations for plasma were positive but not significant. No correlation was observed with plasma bilirubin, γ-glutamyltransferase, or alanine transaminase. Conclusions: Plasma and RBCM phytosterols increase significantly in newborns after a short period of TPN. Higher phytosterol levels were observed in some patients that could have been due to their individual variability in phytosterol metabolism and/or clearance. A greater accumulation of phytosterols in membranes may induce TPN-related cholestasis.

Increased low-density lipoprotein peroxidation in elderly men.

BACKGROUND Oxidative modification of low-density lipoprotein (LDL) appears to play a pivotal role in atherogenesis. The specific role played by LDL peroxidation in aging is not known. Since estrogens may protect LDL from peroxidation in vitro and in vivo, we chose to investigate only men of various ages. OBJECTIVE To determine whether LDL from healthy elderly men was differently susceptible to peroxidation than LDL of young and adult men. SUBJECTS AND METHODS LDL was isolated from 15 normolipidemic young (aged 19-23 years), 17 adult (aged 35-55 years), and 16 elderly (aged 77-90 years) healthy men. None of the men included in the study was a smoker or a hypertensive. LDL peroxidation was achieved by exposure to 5 mumol/l copper sulfate for 18 h at 37 degrees C, and some markers of lipid peroxidation (estimating various levels of peroxidation) were evaluated. RESULTS The levels of lipid peroxides in LDL from our elderly men were already higher under basal conditions than were those both of adult and of young men. LDL from elderly men was more susceptible to peroxidation than was that of adult and young men. Furthermore, the lag time correlated inversely to age (r = -0.68, P < 0.01), whereas lipid peroxide and malonyldialdehyde levels correlated highly to age (r = 0.79 and r = 0.77, P < 0.0002 and P < 0.0012, respectively). With aging the vitamin E content in LDL decreased whereas the arachidonic fatty acid content increased. More importantly, the relationship between the vitamin E content and the lag time made evident the parallel increase in lag time and in vitamin E level with aging. The vitamin E concentration also correlated inversely to levels of thiobarbituric acid-reactive substances in LDL from elderly patients (r = -0.61, P < 0.05). CONCLUSIONS The present study shows that LDL peroxidation increases with age. This phenomenon may favor the progression of atherosclerosis in elderly men.

Serum lipids and apolipoproteins in children with the Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome (SLOS, McKusick 270400) is an autosomal recessive multiple congenital malformation/mental retardation syndrome caused by a deficiency of 3β-hydroxysteroid Δ 7 -reductase (7-dehydrocholesterol Δ 7 -reductase). As a result, markedly reduced cholesterol (C) concentration and accumulation of the precursor 7-dehydrocholesterol (7-DHC) and derivatives 8-dehydrocholesterol (8-DHC) and 19-nor-5,7,9(10)-cholestatrienol are found in serum and tissues (Salen et al 1996; Tint et al 1994). Although the essential metabolic defect was well defined in the cells, the lipoprotein system responsible for cholesterol transport in the blood has not been evaluated in detail until now. Here we report findings of serum lipids and apolipoproteins determined in SLOS children not treated with cholesterol.

Smith–Lemli–Opitz syndrome with extremely low plasma cholesterol

V. Bzdüch1*, D. Behülovä2, L. Kozäk3, J. Sï kodovä2, E. Vëghovä4, A. Dello Russo5, G. Corso5 and F. Bauer6 First Department of 1Pediatrics and 2Clinical Biochemistry, University Childrens Hospital, Bratislava, Slovakia; 3 Research Institute of Child Health, Brno, Czech Republic; 4 Department of Clinical Genetics, Faculty Hospital, Bratislava, Slovakia; 5 Department of Biochemistry and Medical Biotechnology, Federico II University, Naples, Italy; 6 Department of Neonatology, Novë Zämky, Slovakia * Correspondence: First Department of Pediatrics, University Childrens Hospital, Limbova 1, 833 40 Bratislava, Slovakia

Occurrence of the same peroxidative compounds in low density lipoprotein and in atherosclerotic lesions from a homozygous familial hypercholesterolemic patient: a case report

Oxidative modification of low density lipoprotein (LDL) and its byproducts may play a fundamental role in atherosclerosis. We report an in vitro analysis of LDL peroxidative compounds in an homozygous familial hypercholesterolemic (HFH) patient who subsequently died. During the autopsy, we analyzed lipids extracted directly from different atherosclerotic plaques, and we also provided an immunocytochemical analysis using the specific monoclonal antibody MDA2 (directed against malondialdeyde-lysine epitopes of oxidized LDL). The results showed that the same species of peroxidative compounds were present both in LDL in vitro and in lipids extracted directly from atherosclerotic lesions. Moreover, the immunocytochemistry analysis revealed a positive staining of atherosclerotic plaques, confirming the presence of LDL oxidation-specific epitopes. Although observation of a single case is necessarily limited, our findings are consistent with the hypothesis that oxidative modification of LDL is involved in human atherogenesis.

Automated enzymatic determination of urinary nitrates in humans

Abstract Urinary nitrate determinations are used to evaluate the in vivo synthesis of nitric oxide, the major endothelial-derived relaxing factor. In the present study, the enzymatic method using the Aspergillus nitrate reductase was applied to an autoanalyzer to determine urinary nitrate in 17 healthy men. This method was fast and required a small amount of biologic sample (7 μL). Linearity of the assay was between 0.062 μmol/mL and 1.00 μmol/mL; accuracy, measured as percentage of nitrate recovered, ranged from 96% to 118%; between-day imprecision, assessed as coefficient of variation, ranged from 3.5% to 4.5%. The mean urinary nitrate concentration was 0.73 ± 0.29 μmol/mL (range, 0.26 to 1.26 μmol/mL). To carefully quantify nitrate production, urinary nitrate excretion rates were also measured. Rates averaged 0.71 ± 0.18 μmol/min (range, 0.44 to 0.98 μmol/min). Nitrate excretion rates were much less variable than nitrate concentrations. Nitrate excretion rates, but not concentrations, differed significantly in smokers (n = 7, 0.54 ± 0.08 μmol/min) and nonsmokers (n = 10, 0.85,± 0.11 μmol/min). In conclusion, (1) the nitrate reductase assay applied to an autoanalyzer is an accurate, fast, and convenient method for the determination of urinary nitrates in humans; (2) urinary nitrate excretion rate is less variable than nitrate concentration; and (3) cigarette smoking is associated with a diminished nitrate excretion rate. Further studies are needed to assess the nitric oxide generation rate in these subjects.