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Background: Chronic heart failure (CHF) seems to be associated with increased oxidative stress. However, the hypothesis that antioxidant nutrients may contribute to the clinical severity of the disease has never been investigated.Aims: To examine whether antioxidant nutrients influence the exercise capacity and left ventricular function in patients with CHF.Methods: Dietary intake and blood levels of major antioxidant nutrients were evaluated in 21 consecutive CHF patients and in healthy age- and sex-matched controls. Two indexes of the severity of CHF, peak exercise oxygen consumption (peak VO2) and left ventricular ejection fraction (LVEF), were measured and their relations with antioxidants were analysed.Results: Whereas plasma alpha-tocopherol and retinol were in the normal range, vitamin C (P=0.005) and beta-carotene (P=0.01) were lower in CHF. However, there was no significant association between vitamins and either peak VO2 or LVEF. Dietary intake (P<0.05) and blood levels of selenium (P<0.0005) were lower in CHF. Peak VO2 (but not LVEF) was strongly correlated with blood selenium: r=0.76 by univariate analysis (polynomial regression) and r=0.87 (P<0.0005) after adjustment for age, sex and LVEF.Conclusions: Antioxidant defences are altered in patients with CHF. Selenium may play a role in the clinical severity of the disease, rather than in the degree of left ventricular dysfunction. Further studies are warranted to confirm the data in a large sample size and to investigate the mechanisms by which selenium and other antioxidant nutrients are involved in CHF. [ABSTRACT FROM AUTHOR]

The isolated perfused rat kidney (IPK) model was used to assess initial renal function after 24 h preservation in 3 different cold storage solutions: EuroCollins (EC), a solution prepared according to the formulation of Belzer's solution (High-K UW) and a high Na-low K Belzer UW solution (High Na UW). GFR and FR were measured after 24 h cold storage in each of the solutions during 60 min, and were compared to values obtained in a control group in which renal function was measured immediately after the kidneys had been harvested. ATP and CP were measured in fresh renal tissue, in kidneys preserved for 24 h in each solution, in control IPK, and in reperfused IPK after they had been preserved for 24 h. Main results showed that preservation in either solution caused a dramatic decrease in GFR and in FR within the first 60 min following reperfusion of cold-stored kidneys. However FR was significantly higher in the High-Na UW group. ATP and CP content were decreased to ≈10% of basal values in all experimental groups after cold-storage. Normothermic reperfusion of IPK after cold-storage induced a restoration of ATP levels, but CP content decreased further. There was no significant difference in ATP and CP content between cold-storage solutions, nor any correlation between metabolic and functional parameters. [ABSTRACT FROM PUBLISHER]

NADPH oxidase (NOX) is a multimeric enzyme including a catalytic unit, gp91(phox), and several regulating subunits: p22(phox), p40(phox), p47(phox), p67(phox). This enzyme, also known as flavocytochrome b(588), is responsible for a deliberate production of superoxyde anion (O2*-). This enzyme, initially described in polynuclear neutrophils (NOX 2), belongs to a complex family of multimeric isoenzymes whose members are present in many cell types. NOXs are generally associated to cell signaling and they seem involved in physiological phenomena (vascular reactivity, proliferation and cellular migration...) as well as in many diseases. Lipids in general and poly unsaturated fatty acids (PUFA) in particular are able to modulate the activity of NOX in many models. With our fibroblastic model, we show that only arachidonic acid (AA) is able to activate the enzyme directly whereas many PUFA are able to induce a production of reactive oxygen species (ERO). Moreover the decrease of ERO production and NOX activity in fibroblasts triggered by PUFA does not depend on SOD activity but the time course of this decrease is associated with the expression of heme oxygenase 1 (HO-1). Besides a regulation by protein subunits, we propose, according to this model, a loop of regulation of NOX activity including a stimulation by lipids associated with an inhibition by HO-1. Thus, lipids, by interaction with phospholipase A2, release arachidonic acid which stimulates NOX, amplifying superoxyde anion production. This oxygen species may induce redox-sensitive gene transcription such as HO-1. Consequently this enzyme inhibits NOX activity and limits superoxyde anion production by heme degradation and CO production.

Oxidative stress decreases immune defences and is also suggested to participate in the activation of HIV virus replication. That is why we decided to explore some biomarkers of oxidative stress (reduced glutathione, lipoperoxides, true malondialdehyde and vitamin C) in 20 HIV positive patients whose HIV replication was determined by measurement of RNA viral load. Reduced glutathione is decreased in HIV positive patients, without correlation with the viral load. The patients mean content of lipoperoxides is twice that of controls but with such a large range that there is no statistical difference.

We describe a fast ED-HPLC method for ascorbic acid analysis, carried out on a new mixed mode chromatographic column. Due to its functional dodecylsilane group associating a quaternary ammonium, this column is simpler and easier to handle than previous ones because without any counter-ion in the mobile phase. As ascorbic acid and glutathione are very often measured on the same blood sample, it was efficient to validate a common pretreatment solution for ascorbic acid in plasma and glutathione in whole blood. This pretreatment is based on the use of a sulfosalycilic acid, N-ethylmaleimide, EDTA mix recently described. Plasma samples, stored after deproteinisation and without centrifugation, are stable for three weeks at -20 or -80 degrees C.

We conducted a cross-sectional pilot study on healthy pre-menopausal women (aged 45-50 years) living in Granada, in the south of Spain (n = 39) and Malmö, in the south of Sweden (n = 38) in order to compare their plasma carotenoid levels and to investigate the relationship between the differences in food consumption. Plasma concentrations of six carotenoids were measured using high performance liquid chromatography, habitual diet (at individual level) was estimated by food frequency questionnaires and 24-hour diet recalls were used for standardised measurement of diet at group-level. We found that women in Granada consumed more fruit and vegetables than women in Malmö. Plasma concentrations of beta-cryptoxanthin, lycopene, zeaxanthin, total carotenoids and alpha-tocopherol were higher in Granada than in Malmö, although plasma concentrations of alpha-carotene and retinol were higher in Malmö. Both within and between study centres, consumption of fruit and vegetables correlated positively with plasma concentrations of different carotenoids. The study showed that differences in consumption of fruit and vegetables between the two European centres were reflected in plasma carotenoid concentrations.

A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively., (Copyright 2000 Academic Press.)

The measurement of serum carotenoids by HPLC has been largely improved during the last 10 years. However these techniques still require much time and skills, and direct application of published methods is rarely satisfying. We report here the difficulties that we met to transfer some HPLC methods described in the literature to our laboratories. We propose some solution to overcome the problems that we have encountered, our experience will perhaps help out other biologists. We reported also some results obtained in healthy populations.

A bioluminescent D-luciferin-luciferase mixture is separated by gel filtration during the time course of the reaction. A simultaneous analysis with an UV-visible diode array detector and an on-line luminometer gives nonsuperimposable chromatograms. Luminescence recordings display three peaks, one associated with the enzyme (light-emitting species 1: LES(1)), and two other species free from the luciferase: LES(2), with a luciferyl-adenylate-like spectrum and LES(3). Production of these two species is nucleotide (ATP or 2'-dATP)- and pH-dependent. The chromatographic data presented here could lead to reconsideration of the generally assumed emission mechanism, which involves one emitter only. It could also suggest that each free emitting species is related to a colour of emission corresponding to the two defined wavelengths previously described ( approximately 575 and approximately 620 nm)., (Copyright 2000 Academic Press.)

By fluorescent results, it has been proposed that the product (excited oxyluciferin) bounded in the firefly luciferase emits the light. Also it is generally accepted that two forms (keto and enol) of the oxyluciferin emit red and yellow-green light, respectively. In order to demonstrate the existence of free emitting species using His-tagged luciferase (His-luc), N-terminal 6x His-tagged luciferase (Photinus pyralis) was prepared and expressed in E. coli. After immobilization of His-luc on the membrane by IDA-Ni(2+)-His complex, His-luc clearly showed spectral changes of the emission toward red light. The luciferase-free product obtained from enzymatic reaction mixture in the presence of ATP and dATP emits the light with maximal wavelengths of 575 and 620 nm, respectively. Based on these results, we suppose that two different emitting species or two different energy levels of the emitting species are responsible for two different color lights., (Copyright 1999 Academic Press.)

Background: Ischemia-reperfusion (IR) involves adhesion of leukocytes to the activated endothelium, leading to tissue damage. CD11/CD18 beta(2) integrins interact with their ligands on endothelial cells and may therefore represent a therapeutic target for the prevention of IR. We investigated the effects of three monoclonal antibodies (mAbs) that recognize epitopes of heavy or light chain of the beta(2) integrins on IR in kidneys., Methods: Uninephrectomized Fischer rats were subjected to 45 or 60 min of renal ischemia, treated with intravenously anti-beta(2) integrin monoclonal antibodies (anti-CD11a, anti-CD11b, and anti-CD18) 5 min prior to reperfusion, and compared to a nontreated group. Serum creatinine, blood urea nitrogen (BUN), and kidney histopathological damages were assessed at 1, 2, and 7 days after ischemia., Results: After 45 and 60 min of ischemia, serum creatinine and BUN were significantly higher in the control than in animals treated with anti-CD11a and anti-CD18 at 24 and 48 h. Administration of anti-CD11b had a beneficial effect on renal function after 45 min but not after 60 min of ischemia. Histologic and immunostaining studies demonstrated mild tubular necrosis and less leukocyte infiltration in the anti-CD11a- and anti-CD18-treated groups compared to the control group., Conclusion: These results indicate that selected antibodies to CD11a/CD18 may decrease kidney IR injury when administered prior to reperfusion., (Copyright 1999 Academic Press.)

Carotenoids are a family of pigments with at least 600 members. They derive from lycopene after steps of cyclisation, dehydrogenation and oxidation. It is their chemical structure that determines their physiochemical properties and, in part, their biological activities. About 50 carotenoids can be found in human diet and about 20 of them have been found in plasma and tissues. There is no RDA (Recommended Daily Allowance) for carotenoids. Quantities of carotenoids in diet are difficult to estimate, partly because methods used for the establishment of food composition tables were not specific and sensitive enough. Also, given values do not always take into account variations due to season and region of culture. Absorption of beta-carotene in humans has been the subject of numerous studies but only very little is known about other carotenoids. In general, absorption depends on bioavailability from the food matrix and solubility in micelles. After absorption through passive diffusion, carotenoids follow the chylomicrons metabolism. They are taken up by the liver and released in the blood stream in lipoproteins (VLDL). Carotenoids with no-substituted beta-ionone cycles (alpha and beta-carotene and beta-cryptoxanthin) have provitamin A activity. Highest activity has been found for all-trans beta-carotene. Not all steps of vitamin A biosynthesis and metabolism of other carotenoids have been clarified yet. Besides their provitamin A activity, carotenoids have numerous biological functions. They are efficient scavengers of free radicals, particularly of 1O2. In vitro they have been shown to protect LDL. However, results in vivo are inconsistent. Other functions include enhancement of gap junctions, immunomodulation and regulation of enzyme activity involved in carcinogenesis.

A laboratory-made spectroluminometer was used to analyse the light emitted by firefly (Photinus pyralis) luciferase reacting with several nucleotide derivatives. The analysis of the light emission in the presence of ATP or dATP provides some evidence that the enzyme has two nucleotide binding sites, each one leading to the formation of a complex emitting mainly at 575 nm (ATP) or 610 nm (dATP). AMP is able to displace dATP from the second site (610 nm) to the first one. Photoaffinity labelling of the second site by 8-azido-AMP gives similar results. The amplification effect of CoA and acetyl-CoA is also reconsidered according to this model.

A new photoaffinity probe with a benzophenone group, N-dibenzylphospho-N'-(4-benzoyl)-benzylguanidine (BzPG), has been synthesized on the basis on our previously described creatine kinase bisubstrate analog. BzPG is also a bisubstrate type analog whose photoinsertion is inhibited by the natural substrates of creatine kinase. When rabbit CK-MM is irradiated in the presence of BzPG then cleaved by CNBr, one labeled peptide can be purified by reverse phase HPLC and sequenced. This sequence of 31 amino acids (Ala30-Val60) contains a region which could be responsible for isoenzyme selectivity and another one just preceding the 11 amino acid peptide (Asp61-Thr70) very recently described as a putative creatine binding site. This second peptide was deduced from the comparison of 18 amino acid sequence alignments. We proposed the creatine binding site to be essentially a peptide from Lys39 to Val71.

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K+ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na+ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na+ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K+ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na+ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na+ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K+ UW solution. Liver preservation in the high-Na+ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution.

We used the isolated perfused rat liver model (IPRL) to assess parenchymal and nonparenchymal cell integrity after different conditions of storage and reperfusion. Two studies were performed. In study 1, the IPRL was applied to evaluate the effects of 30 min of normothermic reperfusion with Elohes solution, enriched William's medium (Wif), or Carolina rinse solution (CRS) following 24 h of cold preservation in high-K+ or high-Na+ UW solution. As indicated by creatine kinase-BB (CK-BB) release, reperfusion with CRS provided greater protection of endothelial cells after storage in high-K+ UW solution than after storage in high-Na+ UW solution. In study 2, livers were cold-preserved (24 h, 4 degrees C) in either high-K+ or high-Na+ UW solution, then flushed with either CRS or Wif solution at room temperature before reperfusion (120 min, 37 degrees C) with 5% albumin-William's medium E. There was no statistical difference between the rinse solutions for bile flow and transaminases release. However, CRS improved bile indocyanine green excretion, which is known to be a marker of parenchymal and nonparenchymal cell integrity. Therefore, we can assume that this rinse solution protects rat liver grafts from reperfusion-induced microvascular damage.

We describe a new compound, N-dibenzylphospho-N'-3-(2,6-dichlorophenyl)-propylguanidine (DPPG), and our study of its interaction with cytosolic CK. To our knowledge, it is the most potent inhibitor known for CK: the Ki value versus ADP was 330 nM and 110 nM for CK-MM and BB respectively. In view of the inhibition pattern, Ki(app) dependencies on the second substrate, and very low Ki values, we conclude that DPPG binds to the active site as a bisubstrate-type analog. This bisubstrate analog confirms different mechanisms for CK-MM and BB: in spite of a more than 80% similarity in amino-acid sequences, both isoenzymes are random at pH 8.6 but CK-BB has an ordered mechanism at pH 6.6.