Lung nitroxidative stress in mechanically-ventilated septic patients: A pilot study.

PURPOSE: During sepsis and mechanical ventilation oxidative stress is generated by endothelial and inflammatory lung cells. Our main objective was to study pulmonary NO (nitric oxide) production and nitroxidative stress in mechanically-ventilated septic patients. METHODS: We study 69 mechanically ventilated patients, 36 with sepsis and 33 without sepsis within the first 48 h of ICU admission compared with 33 mechanically ventilated patients without sepsis (MV) plus eight operating room patients without lung disease served as control healthy group (ORCG). Nitrite plus nitrate (NOx-), 3-nitrotyrosine and malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) were analyzed. RESULTS: BALF NOx-, BALF 3-nitrotyrosine, BALF MDA, and plasma NOx- were higher in the Sepsis than in MV patients (all p < .05). Both SG and MV patients had higher BALF NOx- than the healthy control group (p < .001). In the Sepsis patients, the ICU non-survivors had higher levels of BALF NOx- than ICU survivors 80(70-127) µM versus 31(15-47) µM, p < .001. CONCLUSIONS: We conclude that during early phases of sepsis there is an enhanced lung nitroxidative stress due to an increase of NO production leading to secondary NO-derived oxidants, which promote protein nitration and lipid peroxidation.

Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives.

Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with beta-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial beta-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

Formation of lipid-protein adducts in low-density lipoprotein by fluxes of peroxynitrite and its inhibition by nitric oxide.

Peroxynitrite (PN), the product of the diffusion-limited reaction between nitric oxide (*NO) and superoxide (O*-(2)), represents a relevant mediator of oxidative modifications in low-density lipoprotein (LDL). This work shows for the first time the simultaneous action of low-controlled fluxes of PN and *NO on LDL oxidation in terms of lipid and protein modifications as well as oxidized lipid-protein adduct formation. Fluxes of PN (e.g., 1 microM min(-1)) initiated lipid oxidation in LDL as measured by conjugated dienes and cholesteryl ester hydroperoxides formation. Oxidized-LDL exhibited a characteristic fluorescent emission spectra (lambda(exc) = 365 nm, lambda(max) = 417 nm) in parallel with changes in both the free amino groups content and the relative electrophoretic mobility of the particle. Physiologically relevant fluxes of *NO (80-300 nM min(-1)) potently inhibited these PN-dependent oxidative processes. These results are consistent with PN-induced adduct formation between lipid oxidation products and free amino groups of LDL in a process prevented by the simultaneous presence of *NO. The balance between rates of PN and *NO production in the vascular wall will critically determine the final extent of LDL oxidative modifications leading or not to scavenger receptor-mediated LDL uptake and foam cell formation.

Nitric oxide-oxygen radicals interactions in atherosclerosis.

Atherosclerosis is one of the most common diseases and the principal cause of death in western civilization. The pathogenesis of this disease can be explained on the basis of the 'oxidative-modification hypothesis,' which proposes that low-density lipoprotein (LDL) oxidation represents a key early event. Nitric oxide (*NO) regulates critical lipid membrane and lipoprotein oxidation events by a) contributing to the formation of more potent secondary oxidants from superoxide (i.e.: peroxynitrite), and b) its antioxidant properties through termination reactions with lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO). Relative rates of production and steady state concentrations of superoxide and *NO and cellular sites of production will profoundly influence the expression of differential oxidant injury-enhancing and protective effects of *NO. Full understanding of the physiological roles of *NO, coupled with detailed insight into *NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for intervention strategies directed toward oxidant-dependent atherogenic processes.

Direct evidence for apo B-100-mediated copper reduction: studies with purified apo B-100 and detection of tryptophanyl radicals.

Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.

Nitric oxide-oxygen radicals interactions in atherosclerosis

Atherosclerosis is one of the most common diseases and the principal cause of death in western civilization. The pathogenesis of this disease can be explained on the basis of the 'oxidative-modification hypothesis,' which proposes that low-density lipoprotein (LDL) oxidation represents a key early event. Nitric oxide (*NO) regulates critical lipid membrane and lipoprotein oxidation events by a) contributing to the formation of more potent secondary oxidants from superoxide (i.e.: peroxynitrite), and b) its antioxidant properties through termination reactions with lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO). Relative rates of production and steady state concentrations of superoxide and *NO and cellular sites of production will profoundly influence the expression of differential oxidant injury-enhancing and protective effects of *NO. Full understanding of the physiological roles of *NO, coupled with detailed insight into *NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for intervention strategies directed toward oxidant-dependent atherogenic processes.

Amino acid sequence and three-dimensional structure of the Tn-specific isolectin B4 from Vicia villosa.

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

Development of an immuno-lectin-enzymatic assay for the detection of serum cancer-associated glycoproteins bearing Tn determinant.

We report the development of an immuno-lectin-enzymatic assay (CA83.4) with the purpose of quantifying serum glycoproteins bearing Tn determinant (GalNAc alpha-O-Ser/Thr). An anti-Tn monoclonal antibody (83D4) is bound to the solid phase in order to capture glycoproteins. After the addition of a test sample, we used biotinylated isolectin B4 from Vicia villosa and avidin-peroxydase to act as a detection system. The linear relationship between CA83.4 determinations and the serum dilutions, the reproducibility of the dosage in intra- and inter-assay, and the specificity of the test for the N-acetylgalactosamine residue in a-glycosidic O-linkages, demonstrated the reliability of this trial. Self-recognition of Vicia villosa B4 molecules (K-D: 0.73x10(-6) M determined using biosensor technology) could determine an additional step of signal amplification in this assay. Using 0.25 units/ml of CA83.4 antigen as the cut-off level, higher values were found in 25/49 patients with breast cancer, 8/13 with colorectal carcinoma, 3/11 with lung cancer, but in none of the 49 patients with non-malignant diseases nor in 97 healthy controls. This first report on soluble Tn-glycoprotein detection assays suggests that Tn-glycoproteins are specific serological tumor markers and we believe that they could represent a valuable tool in the diagnosis of cancer.