Effect of ethyl esterification of phenolic acids on low-density lipoprotein oxidation.

Inhibition of copper-induced low-density lipoprotein (LDL) oxidation by phenolic acids and their ethyl esters was investigated. LDL oxidation was evaluated by the hydroperoxide concentration and the chromatographic pattern of apoprotein fractions after fast protein liquid chromatography (FPLC). Antiradical properties against 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) were also investigated, and lipophilicity determined by thin-layer chromatography. Caffeic acid at 5 microM and sinapic acid at 10 microM protected LDL against oxidation, inhibiting both hydroperoxide formation and the increase of apoprotein negative charge. Ferulic, gallic and p-hydroxy cinnamic acids were ineffective. Ethyl esterification increased the lipophilicity of the five acids, and enhanced the antioxidant properties of caffeic, sinapic and ferulic acids. Ethyl caffeate was protective at 1 microM. In contrast, gallic and p-hydroxy cinnamic ethyl esters were ineffective. Our results indicate that ethyl esterification of phenolic acids increases lipophilicity of their ethyl esters and may enable a better incorporation into the lipid layer of the LDL particle and the exertion of their antioxidant effect in the true site of lipoperoxidation. However, increasing lipophilicity is not the only mechanism able to potentiate preexisting antioxidant properties of molecules, and probably other mechanisms are implicated.

Implication of apolipoprotein E and the L-arginine-nitric oxide system in preeclampsia.

OBJECTIVE: During pregnancy, Apolipoprotein (Apo) E is synthesized in the placenta to facilitate the uptake of maternal lipoproteins. Preeclampsia is associated with an abnormal lipid profile. Apo E levels may affect the production of nitric oxide. We investigated whether Apo E variations could be related to the high lipid levels and nitric oxide secretion in preeclamptic women. METHODS: Blood samples from 15 normotensive women and 12 mild and 23 severe cases of preeclampsia were assayed for standard lipid profile, Apo E, and nitrate. Urine samples were analyzed for nitrate and cyclic GMP. RESULTS: In women with mild preeclampsia, the triglyceride concentration was significantly higher (p < 0.05) than in normotensive women (3.30 +/- 1.38 versus 2.31 +/- 0.92 g/L) and associated with a higher (p < 0.01) triglyceride/Apo E ratio (0.71; range = 0.40-1.70). In women with severe preeclampsia, the triglyceride/Apo E ratio was similar to normotensive women [0.39 (range = 0.18-1.19) versus 0.41 (range = 0.18-0.79)] associated with a normal triglyceride level and a twofold higher serum nitrate level [36 (range = 1-63 mumol/L) versus 14 (range = 1-37 mumol/L)]. CONCLUSION: The triglyceride/Apo E ratio is significantly higher in mild preeclampsia. In the severe form, this ratio is similar to that of normotensive pregnant women, probably due to a better uptake of triglyceride. Moreover, in the severe form, it is associated with a twofold normal serum nitrate level. Thus, Apo E and the nitric oxide status may be implicated in preeclampsia.

Oxidized-LDL induce apoptosis in HUVEC but not in the endothelial cell line EA.hy 926.

We studied the cytotoxic effect of copper-oxidized LDL in human primary human umbilical vein endothelial cells (HUVEC) and the immortalized EA.hy 926 cell line. Copper oxidized LDL (50-200 microg apoB/ml) induced concentration-dependent apoptotic cell death in HUVEC but did not induce apoptosis in EA.hy 926 cells. Only necrotic EA.hy 926 cells were evidenced at all copper oxidized LDL concentrations (25-200 microg apoB/ml), oxidation states (lightly, moderately and extensively copper-oxidized LDL) and incubation periods (4, 8 and 20 h). The different mechanisms of cell death induced by copper-oxidized LDL in EA.hy 926 cells and HUVEC may be related to various factors such as cytokines. In this study, we investigated whether interleukin-8 may be implicated in this process. The interleukin-8 production was increased in EA.hy 926 cells but not in HUVEC incubated with oxidized LDL. This increase in EA.hy 926 cells was associated with necrosis but not apoptosis. Nevertheless, the addition of interleukin-8 to HUVEC did not inhibit apoptosis induced by oxidized LDL. As the lower antioxidant capacity of EA.hy 926 cells results in higher sensitivity to oxidized LDL cytotoxicity (as we previously described), the redox status of cells may also control the form of endothelial cell death. In atherosclerotic lesions, the formation of apoptotic endothelial cells may result in part from the induction by oxidized LDL.

[New automatic test for the study of platelet response to variations in osmotic pressure: application in the evaluation of the viability of platelet concentrates]. / Nouveau test automatique d'étude de la réponse des plaquettes aux variations de pression osmotique: application à l'évaluation de la viabilité des concentrés plaquettaires.

Studying the osmotic resistance or swelling of platelets has often been suggested as a global test to assess the viability of those cells. A number of authors have also analysed the behaviour of platelets in hypotonic media by a variety of complementary methods (cell count, morphology, determinations of substances released, photometric measurement of aggregation induced by aggregating agents, etc). Most studies are currently based on the so-called "osmotic shock response" test, which measures according to time the light transmitted through platelet-rich plasma (PRP) after dilution in distilled water. In this study, the authors describe a new automated and reproducible test using slow dialysis to assess platelet osmotic resistance. The "Fragilimeter", a device initially described by the authors to characterise RBC fragility, has been adapted to the study of platelet osmotic behaviour. The variations in light transmission through a platelet suspension according to NaCl concentration are linked to the change in cellular volume and lysis and characterise the viability of the cells. The results obtained with normal platelets revealed the good reproducibility of the technique. The osmotic resistance is evaluated for two parameters: anticoagulant (citrate, EDTA) and cellular concentration. The test was applied to quality control of stored platelet concentrates for transfusion, prepared with different cell separators.

Urinary cGMP concentrations in severe primary pulmonary hypertension.

BACKGROUND: Prognostic evaluation of patients with primary pulmonary hypertension (PPH) requires right heart catheterisation. The development of accurate non-invasive methods for monitoring these patients remains an important task. Cyclic guanosine monophosphate (cGMP) is an indicator of the action of natriuretic peptides and nitric oxide on target cells. Plasma and urinary cGMP concentrations are raised in patients with congestive heart failure in whom they correlate closely with haemodynamic parameters and disease severity. The aim of the present study was to determine whether the urinary concentration of cGMP could be used as a non-invasive marker of haemodynamic impairment in patients with severe PPH. METHODS: Urinary cGMP concentrations were measured in 19 consecutive patients with PPH, seven with acute asthma, and 30 normal healthy controls. RESULTS: Patients with PPH had higher urinary cGMP concentrations than asthmatic patients or normal healthy controls (p = 0.001). Urinary cGMP concentrations were higher in patients with severe haemodynamic impairment--that is, those with a cardiac index (CI) of < or = 2 l/min/m2 (p = 0.002)--and urinary cGMP concentrations were inversely correlated with CI (r = -0.69, p = 0.002) and venous oxygen saturation (r = -0.65, p = 0.003). CONCLUSION: Urinary cGMP concentrations may represent a non-invasive indicator of the haemodynamic status of patients with severe PPH.

Protective effects of the lipophilic redox conjugate tocopheryl succinyl-ethyl ferulate on HIV replication.

Previously, we demonstrated that ferulate ethyl and tocopherol reduced HIV replication. In this study, we investigate whether the conjugation of both compounds (O-tocopheryl succinyl O-ethyl ferulate) can increase HIV inhibition. We show here for the first time that O-tocopheryl succinyl O-ethyl ferulate inhibits 80% of HIV replication (HIV-1 acute infection and HIV transmission), inhibits cell lipoperoxidation and prevents cellular glutathione consumption. Compared to ferulate ethyl and tocopheryl succinyl, O-tocopheryl succinyl O-ethyl ferulate inhibits more HIV replication. This may be due in part to the great increase in the lipophilicity of this compound.

Hemorheology and therapeutic hemapheresis.

Rheological parameters (plasma and blood viscosities, erythrocyte aggregation and deformability) are very relevant to a better understanding of the changes inflicted on the properties of blood in various hematological diseases (monoclonal gammopathies in particular). This study reports the measurement of rheological parameters (plasma and blood viscosity, RBC aggregation and deformability) in hematological disorders where abnormal blood properties have been described. All these diseases are treated by means of blood separators (therapeutic haemapheresis:plasma exchange, plasma processing, or erythrocyte exchange). We monitored plasma viscosity in 50 patients with monoclonal gammopathies: before and after each exchange (PE) or plasma processing (PP) sessions. Hyperviscosity was reduced in all cases after plasma exchange. Erythrocyte aggregation in gammopathies was also tested (15 multiple myelomas) treated by PE or PP, EA abnormalities and therapy-induced changes are described. EA is also modified by erythropheresis (seven sickle cell anemias). Measurement of rheological parameters are useful to the diagnosis of rheological abnormalities, as well as to assess the impact of therapeutic hemapheresis techniques like plasma exchange, plasma treatment, erythrocytapheresis.

Comparison of oxidized low-density lipoprotein toxicity on EA.hy 926 cells and human vein endothelial cells: influence of antioxidant systems.

We compared the susceptibility to oxidized LDL cytotoxicity of primary human umbilical vein endothelial cells (HUVEC) and EA.hy 926 cells. EA.hy 926 endothelial cells were more susceptible than HUVEC. To determine the basis of this difference, we evaluated the enzymatic antioxidant machinery in the two cell types. The antioxidant enzyme activities of superoxide dismutase, catalase and glutathione peroxidase were significantly lower in EA.hy cells than in HUVEC: 54%, 71% and 8% of the HUVEC enzyme activities respectively. Pre-incubation of the EA.hy 926 endothelial cells with glutathione peroxidase (100 IU/ml) inhibited the cytotoxic effect of oxidized LDL. Superoxide dismutase (300 or 600 IU/ml) and catalase (300 or 600 IU/ml) had no effect. Compared to HUVEC, the higher susceptibility of EA.hy 926 cells to oxidized LDL induced injury may be associated with lower antioxidant defences, in particular with lower glutathione peroxidase activity which is known to eliminate lipid hydroperoxides and thereby to prevent the formation of damaging peroxyl radical intermediates.

A method for simultaneous determination of plasma and erythrocyte antioxidant status. Evaluation of the antioxidant activity of vitamin E in healthy volunteers.

1. Since oxygen free radicals are directly involved in a variety of pathologies such as atherosclerosis, diabetes mellitus, inflammation and/or when a deficit of defences of the organism against radicals occurs, we developed a suitable and simple method to determine both the erythrocyte sensitivity to an oxidative stress and plasma antioxidant protective capacity. 2. This test is based on the introduction at 37 degrees C of a radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), within an erythrocyte suspension leading to a membrane alteration and ultimately to haemolysis. The latter can be quantified by determining the lacticodeshydrogenase activity released in the medium. The erythrocyte sensitivity to haemolysis and the volume of plasma inhibiting 50% of the haemolysis were determined. 3. Intra-assay CVs were 1.9% for erythrocyte sensitivity to oxidative stress and 3.4% for inhibitory 50% plasma volume. Inter-assay CVs for both erythrocyte sensitivity and inhibitory 50% plasma volume were 4%. 4. The reliability of this method was assessed and applied to test the protective effect of vitamin E, a well known antioxidant agent, in six healthy volunteers. Two weeks after daily administration of 500 mg of vitamin E, the mean plasma vitamin E concentration increased by 41% from 10.7 +/- 2.0 mg l-1 before treatment (P < 0.05). As the vitamin E concentration increased, the mean inhibitory 50% plasma volume and the percentage of haemolysed erythrocytes decreased respectively by 29% from 3.35 +/- 0.5 microliter (P < 0.05) and 18% from 71.5 +/- 3.8% (P < 0.05). No significative variation of these parameters was observed in six adult men without vitamin E supplementation. 5. Thus, this global and simple test permits an antioxidant status evaluation of a patient. It can be applied to various pathologies and allows the potency of new antioxidant molecules to be evaluated.

Oxidized low-density lipoprotein induces the production of interleukin-8 by endothelial cells.

The concentration of interleukin-8 (IL-8) and RANTES was measured in culture supernatants of human EA.hy 926 endothelial cells incubated with oxidized low-density lipoproteins (LDL). Oxidized LDL induced a 3-fold increase in IL-8 production (p < 0.01), whereas RANTES was not detected. Native LDL did not stimulate IL-8 production. IL-8 production in oxidized-LDL-treated cells was mediated by reactive oxygen species, as it was partially inhibited by catalase and completely inhibited by glutathione peroxidase and N-acetylcysteine (p < 0.01).

Immunocytochemical study of uptake of exogenous carrier-free copper-zinc superoxide dismutase by peripheral blood lymphocytes.

We recently demonstrated that exogenous copper-zinc superoxide dismutase (SOD) reduced HIV replication in tumor necrosis factor alpha activated chronically HIV-infected promonocytic U1 cell line and in peripheral blood mononuclear cells coculture. However, whether exogenous SOD penetrates the cellular membrane or acts extracellularly has been remained controversial. SOD has been considered as not to penetrate the cellular membrane because of its high molecular weight, thus the main site of action is presumed to be extracellular. In order to determine whether exogenous SOD penetrates inside the cell, we utilized a gentle immunocytochemical method to detect Mn and Cu,Zn SOD in peripheral blood lymphocytes incubated with various concentrations of exogenous carrier-free Cu,Zn SOD without prior permeabilization of cell membranes. After 24 hrs. the total SOD activity and immunocytochemical studies were performed. Here we demonstrate clearly that a large amount of carrier-free Cu,Zn SOD, added exogenously, penetrates the cellular membrane and increases total SOD activity.

Treatment of graft versus host disease (GVHD) by photopheresis?

Graft versus host disease (GVHD), whether acute or chronic, is a frightening complication of bone marrow allografts-indeed in many cases it can be life threatening. In chronic GVHD, the symptoms are less serious, but they can nevertheless be alarming. The mechanism of this reaction is very complex and the pathogenesis of chronic GVHD seems to be slightly different from that of acute GVHD. However, there is no doubt about the immune mechanisms. The production of numerous cytokines plays an important part and has also been des-cribed. Until the use of photopheresis, the only treatments that were effective to any degree have been immunosuppressive treatments. Extracorporeal photopheresis (ECP), a technique recently proposed in chronic GVHD, is promising and is believed to attack the actual cause of the disease (the role of cytotoxic T lymphocytes is now recognized). ECP is believed to have a complex mechanism of action, the explanation of an anti-T lymphocyte action of ECP seems too simple. We report the results of three patients suffering from chronic GVHD, refractory to the usual treatments. The schedule for ECP was a cycle of two treatments every 2 weeks. We recorded a complete remission for patient No. 1 (grade 1) with no relapse for now 3 years. In patient No. 2 (grade 2-3) a progressive improvement was observed in the various symptoms with, however, several episodes of aggravation. In patient No. 3 (grade 2-3), the skin symptoms improved and the lichen planus lesions healed after only 15 months of treatment (interrupted by two infectious episodes during which ECP was stopped). Although the study population was small, we may be justified in thinking that ECP can cause an improvement in chronic GVHD refractory to immunosuppressive treatment. These results should be confirmed by a rigorously designed multicentre study.

The storage quality of apheresis platelets--analysis of results from seven different cell separators.

Apheresis platelet concentrates (CAP) have been available for more than 20 years. Now, as a result of the use of, and progress in, automatic blood separation techniques, the quality of platelets is continually being improved so that we currently have access to purified concentrates which are not contaminated by red cells and contain only very few white cells. In view of the wide experience available to us, it was useful to monitor a number of qualitative parameters indicating platelet activation during storage. We monitored platelet aggregation, release of beta thromboglobulin, activation of complement and other basic parameters in seven platelet concentrates from seven cell separators (Cobe Spectra, MCS Haemonetics, CS3000+, Baxter, Excel Dideco, Autopheresis C Baxter, and V50 Haemonetics). We found that in the majority of cases aggregation was greatly reduced during storage. Release of beta thromboglobulin and activation of complement increased. pH remained within acceptable limits in some cases. However, in others (MCS, Excel), a decrease in pH < 6.0 was found. To conclude, although CAP, which are from a single donor, reduce the risks of transfusion, they are not protected from physical alteration and therefore from reduction in the clinical effects of transfusion. It is therefore necessary to improve the storage conditions of these cells in the future, for example by designing new plastics, or adding platelet storage media. Basic biological parameters should also be defined, within the framework of quality assurance, to evaluate platelet quality.

Antineutrophil cytoplasmic autoantibodies in autoimmune liver and inflammatory bowel diseases.

Perinuclear antineutrophil cytoplasmic autoantibodies have been described in inflammatory bowel diseases and in primary sclerosing cholangitis. Because the data concerning their occurrence are conflicting, we have used indirect immunofluorescence on ethanol-fixed neutrophils to test the sera from a large population of 382 patients with various liver and digestive diseases: in particular, from 27 patients with primary sclerosing cholangitis, 105 patients with autoimmune chronic active hepatitis, 30 patients with primary biliary cirrhosis and 124 patients with inflammatory bowel disease. The prevalence of the perinuclear antineutrophil cytoplasmic autoantibodies was 37% in ulcerative colitis and 15% in Crohn's disease. They would not be helpful in the differential diagnosis between these two inflammatory bowel diseases. Within the group of autoimmune liver diseases, perinuclear antineutrophil cytoplasmic autoantibodies were detected in 44% of sera from patients with primary sclerosing cholangitis and in 36% of sera from patients with type I autoimmune active hepatitis, but not in primary biliary cirrhosis. When primary sclerosing cholangitis was associated with an inflammatory bowel disease, the prevalence of these autoantibodies was 60%. They were 88% specific for primary sclerosing cholangitis and 86% specific for type I autoimmune active hepatitis. Despite their moderate sensitivity and specificity in primary sclerosing cholangitis, they remain the only serologic marker of this autoimmune liver disease. Moreover, they turned out to be a more sensitive marker for inflammatory bowel disease with associated primary sclerosing cholangitis.