Phenol-soluble modulin α induces G2/M phase transition delay in eukaryotic HeLa cells.

Staphylococcus aureus is a gram-positive bacterium responsible for a wide range of infections. Host cell cycle alteration is a sophisticated mechanism used by pathogens to hijack the defense functions of host cells. We previously demonstrated that S. aureus MW2 (USA400) bacteria induced a G2/M phase transition delay in HeLa cells. We demonstrate here that this activity is triggered by culture supernatant compounds. Using size exclusion chromatography of the MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin α (PSMα) peptides as the likely candidates for this effect. Indeed, synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus Los Angeles County clone (LAC) wild-type with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1-4. PSMα-induced G2/M transition delay correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. By testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production. We show that PSMs secreted by S. aureus alter the host cell cycle, revealing a newly identified mechanism for fostering an infection.

Lipid peroxidation in all-in-one admixtures for preterm neonates: impact of amount of lipid, type of lipid emulsion and delivery condition.

AIM: To evaluate the effect of lipid emulsion composition and delivery condition on lipid peroxidation in typical all-in-one parenteral admixtures for preterm neonates. METHODS: Malonedialdehyde (MDA) concentrations were assessed in different all-in-one admixtures. We evaluated the effects of fat blend (three lipid emulsions) and the amount of lipids, as well as the effects of protecting bags and/or tubing from ambient light and storage for 72 h. MDA was measured by liquid chromatography/mass spectrometry. RESULTS: Three hundred and sixty samples were collected from 114 admixtures. Neither the type of lipid (p = 0.43) nor the interaction between light and type of lipid (p = 0.49) had any influence on final MDA concentrations, but the increase in MDA concentration at 24 h (T(24)) was related to light exposure (p < 0.001). The increase in MDA concentration was related to the increase in lipid amount in the admixture at T(0) (r = 0.77) and T(24) (r = 0.86). MDA concentrations in solutions stored for 72 h showed no significant increase, with no difference between the three lipid emulsions (p = 0.69). CONCLUSION: All-in-one admixtures may be interesting for the parenteral nutrition of preterm neonates. Protection from light and restricting the amount of lipid to what is required for appropriate energy provision are essential to limit lipid peroxidation.

Preserved residual renal function is associated with lower oxidative stress in peritoneal dialysis patients.

BACKGROUND: Residual renal function (RRF) correlates with survival in peritoneal dialysis (PD). We investigated the association between oxidative stress and RRF in PD. METHODS: Adequacy of dialysis, total and free malondialdehydes (MDA), and lipid hydroperoxides (LHP) were obtained from 23 stable PD patients. RESULTS: Free MDA level decreased with total weekly Kt/V urea (r = -0.51, P = 0.013) and urinary Kt/V (KRU) (r = -0.53, P = 0.009), but not with peritoneal Kt/V. Similar results were found with LHP level. In multivariate analysis, total weekly Kt/V urea and KRU remained associated with free MDA and LHP, independently of gender, nutritional or inflammatory status, and peritoneal permeability. CONCLUSION: A preserved RRF is associated with lower serum levels of lipid peroxidation products among PD patients.

Structural equations to model relationships between pulmonary function, fatty acids and oxidation in cystic fibrosis.

OBJECTIVE: To illustrate the advantages of structural equation models in biomedical research using the complex example of cystic fibrosis. MATERIAL AND METHODS: 595 blood samples from 312 patients were tested. The model studied the effects of age, BMI and clinical condition on seven major latent variables: pulmonary function, lipid oxidation status, vitamins A and E, glutathione, carotenoids, two essential fatty acids and arachidonic acid. RESULTS: The model confirmed previous associations: positive (fatty acids, arachidonic acid, carotenoids and vitamins with pulmonary function and with lipid oxidation) and negative (glutathione with pulmonary function). It also verified the decrease in fatty acids during bronchial exacerbation and the increase in fatty acids and lipid oxidation after antibiotic treatment. Above all, the model revealed new positive associations between lipid oxidation and carotenoid levels and between lipid oxidation and vitamin A and E levels. CONCLUSIONS: Structural equations dealt easily with the great number of outcome variables of the example. They deserve a central place in biomedical issues involving too many correlated factors to help physicians and statisticians conceive biological models that best represent reality.

Sensitization by docosahexaenoic acid (DHA) of breast cancer cells to anthracyclines through loss of glutathione peroxidase (GPx1) response.

Docosahexaenoic acid (DHA, a lipid of marine origin) has been found to enhance the activity of several anticancer drugs through an oxidative mechanism. To examine the relation between chemosensitization by DHA and tumor cells antioxidant status, we used two breast cancer cell lines: MDA-MB-231, in which DHA increases sensitivity to doxorubicin, and MCF-7, which does not respond to DHA. Under these conditions, reactive oxygen species (ROS) level increased on anthracycline treatment only in MDA-MB-231. This was concomitant with a decreased cytosolic glutathione peroxidase (GPx1) activity, a crucial enzyme for protection against hydrogen and lipid peroxides, while major antioxidant enzyme activities increased in both cell lines in response to ROS. GPx-decreased activity was accompanied by an accumulation of glutathione, the GPx cosubstrate, and resulted from a decreased amount of GPx protein. In rat mammary tumors, when a DHA dietary supplementation led to an increased tumor sensitivity to anthracyclines, GPx1 activity was similarly decreased. Furthermore, vitamin E abolished both DHA effects on chemotherapy efficacy enhancement and on GPx1 inhibition. Thus, loss of GPx response to an oxidative stress in transformed cells may account for the ability of peroxidizable targets such as DHA to enhance tumor sensitivity to ROS-generating anticancer drugs.

Polyunsaturated fatty acids modulate NOX 4 anion superoxide production in human fibroblasts.

The strong ROS (reactive oxygen species) production, part of an antioxidant response of human fibroblasts triggered by DHA (docosahexaenoic acid; C(22:6,n-3), served as a model for deciphering the relative contribution of NOX (NADPH oxidase) to ROS production, as the role of this enzymatic system remains controversial. Using hydroxyethidium fluorescence for fibroblast ROS production, RT (reverse transcriptase)-PCR for NOX 4 mRNA quantification and mRNA silencing, we show that ROS production evolves in parallel with the catalytic activity of NOX and is suppressed by siNOX 4 (small interference oligonucleotide RNA directed against NOX 4) silencing. Apocynin and plumbagin, specific inhibitors of NOX, prevent ROS production in this cellular model and confirm the role of NOX 4 for this production. Furthermore, we show that, in cell lysates, NOX 4 activity can be modulated by PUFAs (polyunsaturated fatty acids) at the micromolar level in the presence of calcium: NOX 4 activity is increased by arachidonic acid (C20:4,n-6) (approximately 175% of the control), and conjugated linoleic acid (C18:2 [9Z,11E]) is a potent inhibitor (50% of the control). Unexpectedly, intracellular superoxide dismutase does not participate in the modulation of this ROS production and the opposite effects of some PUFAs, described in our experiments, could suggest another way of regulating NOX activity.

Fatty acids platelets and oxidative markers following intravenous n-3 fatty acids administration in cystic fibrosis: An open pilot observational study.

BACKGROUND: An imbalance in the ratio of arachidonic acid and docosahexaenoic acid (DHA) was found in cystic fibrosis (CF) affected tissues and was suggested to promote inflammation. Several studies have shown that the long chain n-3 fatty acids reduced inflammatory activity while others have highlighted prooxidant activity of DHA at high concentrations. The aim of our study was to evaluate the effects of an intravenous fish-oil emulsion enriched with n-3 FA in patients with CF on plasma and platelet FA composition and peroxidation markers. METHODS: 13 patients with CF received one IV emulsion per week of 2 mL/kg fish-oil n-3 emulsion for 12 weeks. RESULTS: There was a significant increase in 20:5 n-3 and 22:6 n-3 platelet FA composition, no variation in 20:4 n-6, a decrease in n-9. There was no variation in plasma FA composition. Specific urinary markers of lipid peroxidation derived from n-3 and n-6 showed a very high level before infusion compared with usual values in healthy subjects which was not affected by treatment. A significant weight loss and a decrease in reduced glutathione were observed in adult patients. CONCLUSIONS: The intravenous administration of n-3 FA in CF patients induced a significant modification in platelet FA composition but no modification of oxidative markers. However, the weight loss and the decreased level in reduced glutathione observed in adult patients may suggest a potential deleterious activity for some patients. Further studies are necessary to determine the optimal dose and route for long chain FA administration required to reach a potential beneficial effect.

Evaluation of IGL-1 preservation solution using an orthotopic liver transplantation model.

AIM: To compare, in a pig liver transplantation model, the protective effect of UW with that of IGL-1, a high-sodium preservation solution containing polyethylene glycol (PEG) as an oncotic supply. METHODS: All livers were harvested and grafted orthotopically according to standard techniques. The livers were washed out and preserved for 7 h in IGL-1 (n = 6) or in UW solution (n = 7) at 4 degree centigrade. In a sham group (n = 4), the livers underwent a 60-min warm ischemia at 37 degree centigrade. The hepatocellular injury was assessed in organ preservation solution washed out from the graft at the end of ischemic storage (before revascularization), and in serum 2 h after reperfusion and daily for up to 6 d. RESULTS: Livers preserved in IGL-1 solution released markedly less AST than that preserved in the UW solution before and after revascularization (P < 0.05). Besides, the activity of creatine kinase-BB, a marker of sinusoidal lining cells injury, was higher in the UW group than in the IGL-1 group (P < 0.05). Histological results showed less necrotic regions in livers preserved in IGL-1 solution; however, no difference was observed for inflammation. CONCLUSION: IGL-1 liquid effectively protects parenchymal and non-parenchymal cells against preservation-reperfusion injuries.

Systemic markers of the redox balance and apolipoprotein E polymorphism in atherosclerosis: the relevance for an integrated study.

Prospective studies have demonstrated that an imbalance between oxidative damage and antioxidative protection can play a role in the development and progression of atherosclerosis. Also, genotypes with the apolipoprotein E epsilon4 allele have been associated with an increase risk for this pathology. Based on this knowledge, the aim of this study was to evaluate indicators of the redox balance, trace elements, and apolipoprotein E allelic profile in subjects from the Lisbon population with clinically stable atherosclerosis, at risk for atherosclerotic events, and in healthy subjects for comparison. The activities of superoxide dismutase in erythrocytes and glutathione peroxidase in whole blood, plasma total thiols, and serum ceruloplasmin were kept unchanged among the three groups. Serum alpha- tocopherol was increased in atherosclerotic patients. Total malondialdehyde in serum and protein carbonyls in plasma, which are indicators of lipid and protein oxidative damage, respectively, reached their highest values in risk subjects. The concentrations of potassium and calcium, in plasma and in blood cells, were slightly elevated in patients and might reflect an electrolytic imbalance. Regarding the apolipoprotein E polymorphism, atherosclerotic patients had an increased incidence of the high-risk genotypes for atherogenesis (epsilon3/epsilon4 and epsilon4/epsilon4). A multivariate model applied to the general population using most of the parameters clearly separated the three groups at study (i.e., the healthy group from the steady-state group of risk disease and from the atherosclerotic one). As shown by us, the usefulness of biochemical and complementary genetic markers is warranted for a better knowledge on atherosclerosis molecular basis.

Delta Hemolysin and Phenol-Soluble Modulins, but Not Alpha Hemolysin or Panton-Valentine Leukocidin, Induce Mast Cell Activation.

Mast cells are located at host interfaces, such as the skin, and contribute to the first-line defense against pathogens by releasing soluble mediators, including those that induce itching and scratching behavior. Here, we show that delta-hemolysin (Hld) and phenol soluble modulins (PSMs) PSMα1 and PSMα3, but not alpha-hemolysin (Hla) or Panton-Valentine leukocidin (PVL), induce dose-dependent tryptase, and lactate dehydrogenase (LDH) release by the HMC-1 human mast cell line. Using supernatants from isogenic strains, we verified that tryptase and LDH release was Hld- and PSMα-dependent. PSMα1 and Hld production was detected in 65 and 17% of human Staphylococcus aureus-infected skin abscess specimens, respectively, but they were produced in vitro by all clinical isolates. The results suggest that Hld and PSM-α1 produced in vivo during S. aureus skin infections induce the release of mast cell mediators responsible for itching and scratching behavior, which may enhance skin to skin transmission of S. aureus via the hands. As Hld and PSMs are upregulated by accessory gene regulator (agr), their association may contribute to the elective transmission of S. aureus strains with a functional agr system.

Differential sensitization of cancer cells to doxorubicin by DHA: a role for lipoperoxidation.

Polyunsaturated fatty acids have been reported to enhance the cytotoxic activity of several anticancer drugs. In the present study, we observed that doxorubicin chemosensitization of breast cancer cell lines by docosahexaenoic acid (DHA, a long-chain omega-3 polyunsaturated fatty acid) was cell-line selective, affecting MDA-MB-231 and MCF-7 dox (a doxorubicin-resistant cell line) but not the parental MCF-7 cell line. DHA supplementation led to an increase in membrane phospholipid DHA level, but did not induce changes in intracellular [(14)C]doxorubicin accumulation. In MDA-MB-231, doxorubicin efficacy enhancement by DHA was linked to an increase in malondialdehyde level, a final product of lipid peroxidation. DHA elicited by itself a 3.7-fold malondialdehyde level increase, additive to that induced by doxorubicin. Addition of doxorubicin to DHA further increased the glutathione level, indicative of the generation of an oxidative stress. In contrast to MDA-MB-231, doxorubicin did not increase the malondialdehyde level in MCF-7, although DHA induced lipid peroxidation. Therefore in MCF-7, lipid peroxidation induced by DHA itself was not sufficient to trigger an oxidative stress and to subsequently increase sensitivity to doxorubicin. These data indicate that the differential effect of DHA among cells on drug toxicity results from a differential oxidative response to doxorubicin. Chemosensitization through fatty acids appears as a new promising adjuvant therapeutic paradigm, since omega-3 fatty acids are physiological molecules found in food and are nontoxic in vivo.

Circulating markers of oxidative stress and liver fibrosis in Sudanese subjects at risk of schistosomiasis and hepatitis.

Epidemiological studies in the developing world are frequently biased by the simultaneous presence of several infectious pathogens. In the present study, we examined the usefulness of circulating markers of oxidative stress and liver fibrosis to investigate the distinct forms of chronic liver inflammations associated with schistosomiasis and viral hepatitis, respectively. The study was performed in a Sudanese population exposed to Schistosoma. Circulating hyaluronic acid (HA) was used as a marker of liver fibrosis; the severity of schistosomiasis was determined by ultrasonic examination; viral hepatitis infection was ascertained by circulating anti-hepatitis antibodies. Serum markers were examined also in Sudanese subjects not exposed to Schistosoma infection and in French control subjects. We found a drastic decrease of lycopene levels in the subjects exposed to schistosomiasis in comparison with non-exposed Sudanese and French control subjects. Retinol, alpha-tocopherol and five carotenoids were unchanged. Lycopene depletion was unlikely to be due to variations of nutritional origin, since the lycopene/beta-carotene ratio was five-fold lower in the population at risk of schistosomiasis than in the other groups. We found that high HA serum levels were associated with severe periportal fibrosis but not with viral infection. Conversely, levels of the oxidized lipid malondialdehyde (MDA) were associated with viral infection but not with the severity of schistosomiasis, even though the two infections had additive effects. We concluded that serum markers are valuable tools for investigating the complex effects of co-existing factors of chronic liver inflammation.

Kinetic measurement by LC/MS of gamma-glutamylcysteine ligase activity.

gamma-Glutamylcysteine ligase (GCL) combines cysteine and glutamate through its gamma carboxyl moiety as the first step for glutathione (GSH) synthesis and is considered to be the rate-limiting enzyme in this pathway. The enzyme is a heterodimer, with a heavy catalytic and a light regulatory subunit, which plays a critical role in the anti-oxidant response. Besides the original method of Seelig designed for the measurement of a purified enzyme, few endpoint methods, often unrefined, are available for measuring it in complex biological samples. We describe a new, fast and reliable kinetic LC/MS method which enabled us to optimize its detection. l-2-Aminobutyrate is used instead of cysteine (to avoid glutathione synthetase interference) as triggering substrate with saturating concentrations of glutamate and ATP; the gamma glutamylaminobutyrate formed is measured at m/z=233 at regular time intervals. Reaction rate is maximum because ATP is held constant by enzymatic recycling of ADP by pyruvate kinase and phosphoenolpyruvate. The repeatability of the method is good, with CV% of 6.5 and 4% for catalytic activities at, respectively 0.9 and 34 U/l. The affinities of rat and human enzymes for glutamate and aminobutyrate are in good agreement with previous published data. However, unlike the rat enzyme, human GCL is not sensitive to reduced glutathione and displays a more basic optimum pH.

Dihydroxynonene mercapturic acid, a urinary metabolite of 4-hydroxynonenal, as a biomarker of lipid peroxidation.

The objective of our study was to compare the information obtained through the use of three different urinary biomarkers of lipoperoxidation during the time course of a bromotrichloromethane (BrCCl3) induced oxidative stress in rats. These biomarkers were malondialdehyde (MDA) measured by LC/MS after derivatization, the isoprostane 8-iso-PGF2alpha measured by enzyme immunoassay and 1,4-dihydroxynonene mercapturic acid (DHN-MA), the major 4-hydroxynonenal urinary metabolite [1], measured by LC-MS. Male Wistar rats received a single dose of 100 microL/kg BrCCl3 per os and lipid peroxidation was estimated every day for a 4-day-period after treatment. MDA, 8-iso-PGF2alpha and DHN-MA significantly increased in response to BrCCl3 treatment for this period of time, and DHN-MA showed the main increase during the 24-48 h period after treatment.

[Evaluation of IGL-1, a new organ preservation solution: preclinical results in renal transplantation]. / Evaluation de IGL-1, une nouvelle solution de conservation d'organes: résultats pré-cliniques en transplantation rénale.

INTRODUCTION: Renal ischaemia and reperfusion lesions partly determine short-term and long-term graft survival. Organ preservation conditions appear to play a decisive role. This article presents the preclinical experimental results obtained in renal transplantation with an extracellular organ preservation solution, in which polyethylene glycol (PEG) is used as colloid. METHODS AND RESULTS: The effects of inversion of Na+ and K+ gradients in the IGL-1 preservation solution compared to UW and replacement of hydroxylethyl starch (HES) by PEG were evaluated in an ex vivo isolated, perfused rat kidney model and then in a pig renal autotransplantation model. In these experimental models, after 24 hours of static storage, the sodium reabsorption fraction correlated with the quality of tubular function of the kidney and the glomerular filtration rate were constantly better in the IGL-1 group than in the UW group. In vivo, in the pig, resumption of renal function was significantly better in the IGL-1 group and histological examination demonstrated a significant reduction of expression of Major Histocompatibility Complex (MHC) type II, an indirect marker of inflammation, but also a reduction of markers of apoptosis and fibrosis for kidneys preserved in IGL-1. CONCLUSION: In animal renal transplantation, IGL-1 ensures better resumption of renal function than UW, which currently remains the "gold standard"preservation solution. Further studies must be conducted to determine whether this new generation solution can replace UW as the reference solution.

[Influence of hemodialysis on total and free malondialdehyde measured by a new HPLC method]. / Influence de l'hémodialyse sur les concentrations de malonedialdéhyde total et libre, mesurées par une nouvelle technique HPLC spécifique.

Accurate evaluation of oxidative stress is needed for patients on chronic hemodialysis (HD), as cardiovascular risk level seems related to it. Oxidative stress is often evaluated by measuring an end product of lipoperoxidation named malondialdehyde (MDA). However, the most common technique for measuring MDA, the Thio Barbituric Acid Reactive Substances method (TBARS), is known to be sensitive but poorly specific. We measured true total and free plasma MDA in fifty-four unselected patients on long-term HD, before and after HD sessions, by a new, highly specific HPLC method. Total and free MDA were higher before than after dialysis. Essentially, free MDA was decreased by HD but its fractional decrease was lower than that of urea or creatinine. This confirms that, in fact, free MDA is more or less bound to low molecular weight compounds and/or suggests that MDA may be produced mainly during HD sessions. We propose this new tool to further explore the relationship between oxidative stress, HD and true MDA.

Dietary polyunsaturated fatty acids and heme iron induce oxidative stress biomarkers and a cancer promoting environment in the colon of rats.

The end products of polyunsaturated fatty acid (PUFA) peroxidation, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and isoprostanes (8-iso-PGF2α), are widely used as systemic lipid oxidation/oxidative stress biomarkers. However, some of these compounds have also a dietary origin. Thus, replacing dietary saturated fat by PUFAs would improve health but could also increase the formation of such compounds, especially in the case of a pro-oxidant/antioxidant imbalanced diet. Hence, the possible impact of dietary fatty acids and pro-oxidant compounds was studied in rats given diets allowing comparison of the effects of heme iron vs. ferric citrate and of ω-6- vs. ω-3-rich oil on the level of lipid peroxidation/oxidative stress biomarkers. Rats given a heme iron-rich diet without PUFA were used as controls. The results obtained have shown that MDA and the major urinary metabolite of HNE (the mercapturic acid of dihydroxynonane, DHN-MA) were highly dependent on the dietary factors tested, while 8-iso-PGF2α was modestly but significantly affected. Intestinal inflammation and tissue fatty acid composition were checked in parallel and could only explain the differences we observed to a limited extent. Thus, the differences in biomarkers were attributed to the formation of lipid oxidation compounds in food or during digestion, their intestinal absorption, and their excretion into urine. Moreover, fecal extracts from the rats fed the heme iron or fish oil diets were highly toxic for immortalized mouse colon cells. Such toxicity can eventually lead to promotion of colorectal carcinogenesis, supporting the epidemiological findings between red meat intake and colorectal cancer risk. Therefore, the analysis of these biomarkers of lipid peroxidation/oxidative stress in urine should be used with caution when dietary factors are not well controlled, while control of their possible dietary intake is needed also because of their pro-inflammatory, toxic, and even cocarcinogenic effects.

Longitudinal study of oxidative status in 312 cystic fibrosis patients in stable state and during bronchial exacerbation.

In cystic fibrosis (CF), there is an imbalance in the oxidant/antioxidant system, leading to oxidative damage. The aim of this study was to assess antioxidant-scavenger deficiencies and lipid peroxide variations in three clinical situations (stable status, acute exacerbation, and after intravenous antibiotic treatment). The objective was also to correlate oxidative stress with age, nutritional status, and respiratory function. The study included prospectively 312 consecutive patients and 53 controls. Antioxidants (vitamin A, vitamin E, carotenoids, and glutathione) and oxidative markers (malondialdehyde and lipid peroxides) were measured in plasma. Regression analyses were performed. Antioxidant levels were lower in CF patients than in controls. These levels decreased during acute exacerbation and increased after antibiotic treatment. Carotenoid levels were not modified by infection or age. Only vitamin A and carotenoid levels were positively correlated to body mass index. Antioxidant levels were correlated to forced expiratory volume at 1 sec. Lipid peroxidation markers were lower in patients than in controls. Their levels decreased during infection, and increased after antibiotic treatment. Impaired lung function was correlated with elevated malondialdehyde levels. In conclusion, this study demonstrates antioxidant deficiency in a very large cohort of CF patients. Carotenoid and vitamin E deficiencies occur early in the course of the disease. Antioxidants decrease with bronchial infection. By contrast, nutritional disorders did not modify antioxidant levels during acute exacerbations. Thus, pulmonary disorders rather than nutritional disorders seem to be essential in the imbalance of the oxidant/antioxidant system. Results concerning glutathione and oxidative-marker levels highlighted the fact that their plasma values do not reflect oxidative stress in the respiratory tract.

Fast liquid chromatography-mass spectrometry glutathione measurement in whole blood: micromolar GSSG is a sample preparation artifact.

We describe a new, fast (6 min) and reliable method to measure reduced or oxidized glutathione (GSH) or (GSSG) in whole blood. The method is based on a LC/MS measurement in positive electrospray ionization mode after a chromatographic separation on a specific column which does not need any counter-ion in the mobile phase, improving the sensitivity of detection. A 50 microl sample of whole blood is sufficient for analysis. We demonstrate that the lack of an alkylating agent during the sample preparation brings out an underestimation of GSH and an artefactual production of GSSG, corresponding to 2-3% of GSH. The simultaneous use of N-ethyl-maleimide and a strong deproteinising acid prevents these two drawbacks. This efficient and new method of preparation and analysis lets us show that, unexpectedly, GSH is stable in whole blood for some hours and that deproteinised samples can be stored without GSH loss for at least three weeks at -20 or -80 degrees C. The reference interval, measured on 22 volunteers, on blood samples collected either with heparin or with EDTA, is 1310 +/- 118 microM for GSH and 0.62 microM for GSSG. The within-run precision of this method, with gamma glutamyl-glutamic acid as an internal standard, evaluated in three successive series (n = 30), lies between 2.1 and 4.8% for a GSH level at 580 or 1150 microM. The one step sample preparation we propose seems well suited for GSH routine measurements in hospital laboratories and avoids any underestimation of GSH, a now well accepted biomarker of oxidative stress.

Malondialdehyde adduct to hemoglobin: a new marker of oxidative stress suitable for full-term and preterm neonates.

Oxidative stress may play a central role in the onset of many diseases during the neonatal period. Malondialdehyde (MDA) is a marker of lipid peroxidation. The aim of this study was to evaluate a new marker, the malondialdehyde adduct to hemoglobin (MDA-Hb), which is measured in red blood cells (RBCs) and thus does not require that an additional blood sample be drawn. In this prospective study, we first adapted the measurement method previously described to Hb solutions obtained from washed RBCs and then evaluated the suitability of the method for use in neonates. MDA-Hb concentrations were measured by liquid chromatography-mass spectrometry. We compared the concentrations of MDA-Hb between preterm and term neonates. Erythrocyte samples were collected at birth from 60 healthy neonates (29 full-term and 31 preterm), as well as from 50 preterm neonates with uncomplicated postnatal evolution during the first months of life. We found a significantly higher MDA-Hb concentration at birth in preterm neonates (P = 0.002). During the first months of life, MDA-Hb concentrations were 9.4 nanomol/g Hb in hospitalized preterm neonates. MDA-Hb could be used to assess oxidative stress in preterm neonates. Together with clinical variables, it could be a useful marker for oxidative stress exposition in these higher risk patients.