Perturbation of human coronary artery endothelial cell redox state and NADPH generation by methylglyoxal.

Diabetes is associated with elevated plasma glucose, increased reactive aldehyde formation, oxidative damage, and glycation/glycoxidation of biomolecules. Cellular detoxification of, or protection against, such modifications commonly requires NADPH-dependent reducing equivalents (e.g. GSH). We hypothesised that reactive aldehydes may modulate cellular redox status via the inhibition of NADPH-generating enzymes, resulting in decreased thiol and NADPH levels. Primary human coronary artery endothelial cells (HCAEC) were incubated with high glucose (25 mM, 24 h, 37°C), or methylglyoxal (MGO), glyoxal, or glycolaldehyde (100-500 µM, 1 h, 37°C), before quantification of intracellular thiols and NADPH-generating enzyme activities. Exposure to MGO, but not the other species examined, significantly (P<0.05) decreased total thiols (∼35%), further experiments with MGO showed significant losses of GSH (∼40%) and NADPH (∼10%); these changes did not result in an immediate loss of cell viability. Significantly decreased (∼10%) NADPH-producing enzyme activity was observed for HCAEC when glucose-6-phosphate or 2-deoxyglucose-6-phosphate were used as substrates. Cell lysate experiments showed significant MGO-dose dependent inhibition of glucose-6-phosphate-dependent enzymes and isocitrate dehydrogenase, but not malic enzyme. Analysis of intact cell or lysate proteins showed that arginine-derived hydroimidazolones were the predominant advanced glycation end-product (AGE) formed; lower levels of N(ε)-(carboxyethyl)lysine (CEL) and N(ε)-(carboxymethyl)lysine (CML) were also detected. These data support a novel mechanism by which MGO exposure results in changes in redox status in human coronary artery endothelial cells, via inhibition of NADPH-generating enzymes, with resultant changes in reduced protein thiol and GSH levels. These changes may contribute to the endothelial cell dysfunction observed in diabetes-associated atherosclerosis.

Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes.

Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP(+) in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0-2.5mM, 2-3h, 37°C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500µM. Incubation with radiolabeled MGO (0-500µM, 0-3h, 37°C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP(+) binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.

Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged overweight men: a randomized, placebo-controlled, crossover trial.

BACKGROUND: Olive plant leaves (Olea europaea L.) have been used for centuries in folk medicine to treat diabetes, but there are very limited data examining the effects of olive polyphenols on glucose homeostasis in humans. OBJECTIVE: To assess the effects of supplementation with olive leaf polyphenols (51.1 mg oleuropein, 9.7 mg hydroxytyrosol per day) on insulin action and cardiovascular risk factors in middle-aged overweight men. DESIGN: Randomized, double-blinded, placebo-controlled, crossover trial in New Zealand. 46 participants (aged 46.4 ± 5.5 years and BMI 28.0 ± 2.0 kg/m(2)) were randomized to receive capsules with olive leaf extract (OLE) or placebo for 12 weeks, crossing over to other treatment after a 6-week washout. Primary outcome was insulin sensitivity (Matsuda method). Secondary outcomes included glucose and insulin profiles, cytokines, lipid profile, body composition, 24-hour ambulatory blood pressure, and carotid intima-media thickness. RESULTS: Treatment evaluations were based on the intention-to-treat principle. All participants took >96% of prescribed capsules. OLE supplementation was associated with a 15% improvement in insulin sensitivity (p = 0.024) compared to placebo. There was also a 28% improvement in pancreatic ß-cell responsiveness (p = 0.013). OLE supplementation also led to increased fasting interleukin-6 (p = 0.014), IGFBP-1 (p = 0.024), and IGFBP-2 (p = 0.015) concentrations. There were however, no effects on interleukin-8, TNF-α, ultra-sensitive CRP, lipid profile, ambulatory blood pressure, body composition, carotid intima-media thickness, or liver function. CONCLUSIONS: Supplementation with olive leaf polyphenols for 12 weeks significantly improved insulin sensitivity and pancreatic ß-cell secretory capacity in overweight middle-aged men at risk of developing the metabolic syndrome.

Targeting the AGE-RAGE axis improves renal function in the context of a healthy diet low in advanced glycation end-product content.

AIM: Mouse chow is commonly high in advanced glycation end-products, known contributors to diabetic nephropathy. The aim of this study was to evaluate if targeting of the AGE-RAGE axis was still effective in the context of a diet low in AGE content, which is more comparable to diets consumed by individuals with type 1 diabetes. METHODS: C57BL/6J wild-type and mice deficient in the receptor for AGEs (RAGE-KO) consumed a diet low in AGE content. Groups of mice were given (i) vehicle; (ii) streptozotocin; or (iii) streptozotocin + AGE lowering therapy (alagebrium chloride) and followed for 24 weeks. RESULTS: Diabetic mice had high urinary albumin excretion rates, hyperfiltration and release of urinary Kim-1, not seen in diabetic RAGE-KO mice. Diabetic mice also had renal fibrosis, measured by glomerulosclerosis, tubulointerstitial expansion, TGF-ß1 and glomerular collagen-IV deposition which almost all improved by RAGE-KO or alagebium. Diabetic mice had a greater renal burden of AGEs and increased expression of renal specific PKC-α phosphorylation, which was improved in RAGE-KO mice, or those treated with alagebrium. CONCLUSION: Diabetic mice given a low-AGE diet still developed renal disease, which could be attenuated by targeting of the AGE-RAGE axis.

Quantification of hydroxyl radical-derived oxidation products in peptides containing glycine, alanine, valine, and proline.

Proteins are a major target for oxidation due to their abundance and high reactivity. Despite extensive investigation over many years, only limited quantitative data exist on the contributions of different pathways to the oxidation of peptides and proteins. This study was designed to obtain quantitative data on the nature and yields of oxidation products (alcohols, carbonyls, hydroperoxides, fragment species) formed by a prototypic oxidant system (HO(•)/O(2)) on small peptides of limited, but known, amino acid composition. Peptides composed of Gly, Ala, Val, and Pro were examined with particular emphasis on the peptide Val-Gly-Val-Ala-Pro-Gly, a repeat motif in elastin with chemotactic activity and metalloproteinase regulation properties. The data obtained indicate that hydroperoxide formation occurs nonrandomly (Pro > Val > Ala > Gly) with this inversely related to carbonyl yields (both peptide-bound and released). Multiple alcohols are generated at both side-chain and backbone sites. Backbone fragmentation has been characterized at multiple positions, with sites adjacent to Pro residues being of major importance. Summation of the product concentrations provides clear evidence for the occurrence of chain reactions in peptides exposed to HO(•)/O(2), with the overall product yields exceeding that of the initial HO(•) generated.

Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage.

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.

High plasma thiocyanate levels in smokers are a key determinant of thiol oxidation induced by myeloperoxidase.

Smokers have an elevated risk of atherosclerosis but the origins of this elevated risk are incompletely defined, though evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall. We hypothesized that smokers would have a high level of thiocyanate (SCN(-)), a preferred substrate for MPO, which in turn would predispose to thiol oxidation, an established independent risk factor for atherosclerosis. In this study it is shown that on exposure to MPO/H(2)O(2), thiols on plasma proteins from nonsmokers were increasingly oxidized with increasing added SCN(-) concentrations. Plasma from smokers contained significantly higher endogenous levels of SCN(-) than that from nonsmokers (131±31 vs 40±24 µM, P<0.0001). When plasma from smokers and nonsmokers was exposed to MPO/H(2)O(2)-stimulated oxidation, a strong positive correlation (r=0.8139, P<0.0001) between the extent of thiol oxidation and the plasma SCN(-) concentrations was observed. Computational calculations indicate a changeover from HOCl to HOSCN as the major MPO-generated oxidant in plasma, with increasing SCN(-) levels. These data indicate that plasma SCN(-) levels are a key determinant of the extent of thiol oxidation on plasma proteins induced by MPO, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage to proteins in smokers.

Amino acid, peptide, and protein hydroperoxides and their decomposition products modify the activity of the 26S proteasome.

Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal-lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH(4), which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ≥1µM for oxidized amino acids and peptides and ≥10µM for oxidized proteins, compared with ca. 100µM for H(2)O(2), indicating that H(2)O(2) is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials.

Removal of amino acid, peptide and protein hydroperoxides by reaction with peroxiredoxins 2 and 3.

Prxs (peroxiredoxins) are a ubiquitous family of cysteine-dependent peroxidases that react rapidly with H2O2 and alkyl hydroperoxides and provide defence against these reactive oxidants. Hydroperoxides are also formed on amino acids and proteins during oxidative stress, and they too are a potential cause of biological damage. We have investigated whether Prxs react with amino acid, peptide and protein hydroperoxides, and whether the reactions are sufficiently rapid for these enzymes to provide antioxidant protection against these oxidants. Isolated Prx2, which is a cytosolic protein, and Prx3, which resides within mitochondria, were reacted with a selection of hydroperoxides generated by γ-radiolysis or singlet oxygen, on free amino acids, peptides and proteins. Reactions were followed by measuring the accumulation of disulfide-linked Prx dimers, via non-reducing SDS/PAGE, or the loss of the corresponding hydroperoxide, using quench-flow and LC (liquid chromatography)/MS. All the hydroperoxides induced rapid oxidation, with little difference in reactivity between Prx2 and Prx3. N-acetyl leucine hydroperoxides reacted with Prx2 with a rate constant of 4 × 10(4) M-1 · s-1. Hydroperoxides present on leucine, isoleucine or tyrosine reacted at a comparable rate, whereas histidine hydroperoxides were ~10-fold less reactive. Hydroperoxides present on lysozyme and BSA reacted with rate constants of ~100 M-1 · s-1. Addition of an uncharged derivative of leucine hydroperoxide to intact erythrocytes caused Prx2 oxidation with no concomitant loss in GSH, as did BSA hydroperoxide when added to concentrated erythrocyte lysate. Prxs are therefore favoured intracellular targets for peptide/protein hydroperoxides and have the potential to detoxify these species in vivo.

Cellular effects of photogenerated oxidants and long-lived, reactive, hydroperoxide photoproducts.

Reaction of radicals and singlet oxygen ((1)O(2)) with proteins results in both direct damage and the formation of long-lived reactive hydroperoxides. Elevated levels of protein hydroperoxide-derived products have been detected in multiple human pathologies, suggesting that these secondary oxidants contribute to tissue damage. Previous studies have provided evidence for protein hydroperoxide-mediated inhibition of thiol-dependent enzymes and modulation of signaling processes in isolated systems. In this study (1)O(2) and hydroperoxides have been generated in J774A.1 macrophage-like cells using visible light and the photosensitizer rose bengal, with the consequences of oxidant formation examined both immediately and after subsequent (dark-phase) incubation. Significant losses of GSH (≤50%), total thiols (≤20%), and activity of thiol-dependent proteins (GAPDH, thioredoxin, protein tyrosine phosphatases, creatine kinase, and cathepsins B and L; 10-50% inhibition) were detected after 1 or 2 min photo-oxidation. Non-thiol-dependent enzymes were not affected. In contrast, NADPH levels increased, together with the activity of glutathione reductase, glutathione peroxidase, and thioredoxin reductase; these increases may be components of a rapid global cytoprotective cellular response to stress. Neither oxidized thioredoxin nor radical-mediated protein oxidation products were detected at significant levels. Further decreases in thiol levels and enzyme activity occurred during dark-phase incubation, with this accompanied by decreased cell viability. These secondary events are ascribed to the reactions of long-lived hydroperoxides, generated by (1)O(2)-mediated reactions. Overall, this study provides novel insights into early cellular responses to photo-oxidative damage and indicates that long-lived hydroperoxides can play a significant role in cellular damage.

Deleterious effects of reactive aldehydes and glycated proteins on macrophage proteasomal function: possible links between diabetes and atherosclerosis.

People with diabetes experience chronic hyperglycemia and are at a high risk of developing atherosclerosis and microvascular disease. Reactions of glucose, or aldehydes derived from glucose (e.g. methylglyoxal, glyoxal, or glycolaldehyde), with proteins result in glycation that ultimately yield advanced glycation end products (AGE). AGE are present at elevated levels in plasma and atherosclerotic lesions from people with diabetes, and previous in vitro studies have postulated that the presence of these materials is deleterious to cell function. This accumulation of AGE and glycated proteins within cells may arise from either increased formation and/or ineffective removal by cellular proteolytic systems, such as the proteasomes, the major multi-enzyme complex that removes proteins within cells. In this study it is shown that whilst high glucose concentrations fail to modify proteasome enzyme activities in J774A.1 macrophage-like cell extracts, reactive aldehydes enhanced proteasomal enzyme activities. In contrast BSA, pre-treated with high glucose for 8 weeks, inhibited both the chymotrypsin-like and caspase-like activities. BSA glycated using methylglyoxal or glycolaldehyde, also inhibited proteasomal activity though to differing extents. This suppression of proteasome activity by glycated proteins may result in further intracellular accumulation of glycated proteins with subsequent deleterious effects on cellular function.

Cellular effects of peptide and protein hydroperoxides.

Hydroperoxides are major products of the reactions of radicals and singlet oxygen with amino acids, peptides, and proteins. These species can generate radicals in the presence of metal ions and oxidize thiols via nonradical reactions, but the effects of these materials on cells are poorly understood. In this study the exposure of murine macrophage-like cells to preformed peptide or protein hydroperoxides is shown to result in hydroperoxide consumption and cellular thiol oxidation; these effects precede loss of cell viability. N-acetyltryptophan methyl ester hydroperoxides, but not the decomposed species, decreased total cellular thiols and GSH, with the latter occurring more rapidly. Time-dependent inhibition of lysosomal cathepsins B and L was also observed, together with diminished caspase 3/7 activity. A number of other cytosolic thiol- and non-thiol-dependent enzymes were not affected significantly. Hydroperoxides formed on BSA did not deplete total thiols or GSH within cells, although such reactions are rapid in model systems. In contrast, selective inhibition of cathepsins B and L (but not cathepsin D or arylsulfatase) of the endosomal-lysosomal system was detected, consistent with localization within these compartments. Decomposed BSA hydroperoxides did not induce these effects, indicating a requirement for the hydroperoxide group. The differences between these hydroperoxides are ascribed to their mechanisms of penetration into cells. Overall these studies provide valuable data on the initial cellular events arising from exposure to exogenous protein and amino acid peroxides and indicate that cellular thiols are a major target. This selective oxidation may modulate cellular redox balance and subsequent cell behavior.

Modulation of the cellular expression of circulating advanced glycation end-product receptors in type 2 diabetic nephropathy.

BACKGROUND: Advanced glycation end-products (AGEs) and their receptors are prominent contributors to diabetic kidney disease. METHODS: Flow cytometry was used to measure the predictive capacity for kidney impairment of the AGE receptors RAGE, AGE-R1, and AGE-R3 on peripheral blood mononuclear cells (PBMCs) in experimental models of type 2 diabetes (T2DM) fed varied AGE containing diets and in obese type 2 diabetic and control human subjects. RESULTS: Diets high in AGE content fed to diabetic mice decreased cell surface RAGE on PBMCs and in type 2 diabetic patients with renal impairment (RI). All diabetic mice had elevated Albumin excretion rates (AERs), and high AGE fed dbdb mice had declining Glomerular filtration rate (GFR). Cell surface AGE-R1 expression was also decreased by high AGE diets and with diabetes in dbdb mice and in humans with RI. PBMC expression of AGE R3 was decreased in diabetic dbdb mice or with a low AGE diet. CONCLUSIONS: The most predictive PBMC profile for renal disease associated with T2DM was an increase in the cell surface expression of AGE-R1, in the context of a decrease in membranous RAGE expression in humans, which warrants further investigation as a biomarker for progressive DN in larger patient cohorts.

Quantification of protein modification by oxidants.

Proteins are major targets for oxidative damage due to their abundance and rapid rates of reaction with a wide range of radicals and excited state species, such as singlet oxygen. Exposure of proteins to these oxidants results in loss of the parent amino acid residue, formation of unstable intermediates, and the generation of stable products. Each of these events can be used, to a greater or lesser extent, to quantify damage to proteins. In this review the advantages and disadvantages of a number of these approaches are discussed, with an emphasis on methods that yield absolute quantitative data on the extent of protein modification. Detailed methods sheets are provided for many of these techniques.

Evidence for chronically elevated serum protein oxidation in systemic lupus erythematosus patients.

Serum protein oxidation levels in people with the autoimmune disease systemic lupus erythematosus (SLE) have previously been shown to (a) be elevated at a single time point and (b) correlate with disease activity. This study investigates whether this elevation is a chronic phenomenon, by analysis of multiple serum samples collected from 21 SLE patients and nine controls over a period of up to 38 months. Protein thiols were chronically decreased in SLE patients with stable or variable disease activity compared to controls, whilst protein-bound carbonyls and glycine were chronically increased. 2D-gel analysis of carbonyl distribution showed albumin and immunoglobulins to be particularly affected. In SLE patients with stable disease activity, higher long-term protein oxidation correlated with higher long-term disease activity. SLE patients with variable disease activity exhibited varying correlations between protein oxidation and disease activity markers. These results further support a role for oxidative stress in the pathogenesis of SLE.

Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins.

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.

Identification of plasma proteins that are susceptible to thiol oxidation by hypochlorous acid and N-chloramines.

Hypochlorous acid (HOCl), the major strong oxidant produced by myeloperoxidase, reacts readily with free amino groups to form N-chloramines. Although HOCl and N-chloramines play an important role in the human immune system by killing bacteria and invading pathogens, they have also been shown to cause damage to tissues, which is believed to contribute to a number of diseases. It has been shown previously that N-chloramines react more readily with protein thiols than with other targets in plasma, but the nature of the plasma thiol-containing proteins oxidized is unknown. In this study, the ability of N-chloramines to selectively oxidize thiol-containing plasma proteins was determined using the thiol-specific probe, 5-iodoacetamidofluorescein, combined with two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments were performed with N-chloramines formed on Nalpha-acetyl-lysine, Nalpha-acetyl-histidine (HisCA), glycine, taurine, and ammonia. With the exception of HisCA, the N-chloramines were more efficient than HOCl at oxidizing plasma thiols. The thiol-containing plasma proteins alpha1-antitrypsin and transthyretin were found to be oxidized in addition to albumin, with this treatment resulting in the inactivation of alpha1-antitrypsin. A similar selectivity of reaction and extent of thiol oxidation were also observed with myeloperoxidase in the presence of hydrogen peroxide and chloride ions.

Serum protein oxidation and apolipoprotein CIII levels in people with systemic lupus erythematosus with and without nephritis.

Increased oxidative stress is a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). This study compares serum protein oxidation levels in SLE patients without and with renal involvement (lupus nephritis); the latter have a significantly poorer prognosis. Similar increases in protein carbonyls and decreases in protein thiols were observed in both SLE groups compared to controls. Protein carbonyl distribution, determined by Western blotting of 2D gels, was similar in both SLE groups, suggesting factors other than oxidation also play a role in SLE complications. 2D electrophoresis examined the serum proteome further. Six proteins were significantly decreases in non-renal SLE patients compared to controls; five were identified by mass spectrometry, including one isoform of pro-atherogenic apoCIII. Total apoCIII levels (assessed by ELISA) in lupus nephritis patients were significantly elevated compared to controls or non-renal SLE patients. Thus, levels of oxidized proteins and apoCIII may be useful biomarkers in SLE studies.

Increased levels of serum protein oxidation and correlation with disease activity in systemic lupus erythematosus.

OBJECTIVE: To examine protein oxidation in systemic lupus erythematosus (SLE) and to correlate levels of protein oxidation products with disease activity. METHODS: Serum was collected from SLE patients and healthy control subjects. Protein-bound carbonyls and the pro-oxidant enzyme myeloperoxidase (MPO) were quantified by enzyme-linked immunosorbent assay. Protein thiols were quantified using 5,5'-dithionitrobenzoic acid. Protein-bound amino acids and methionine, tyrosine, and phenylalanine oxidation products were quantified by acid hydrolysis and high-performance liquid chromatography. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Levels of anti-double-stranded DNA (anti-dsDNA) antibodies were measured by radioimmunoassay. RESULTS: Compared with control subjects, SLE patients exhibited elevated levels of protein carbonyls (0.108 +/- 0.078 versus 0.064 +/- 0.028 nmoles/mg of protein; P = 0.046), decreased levels of protein thiols (3.9 +/- 1.1 versus 4.9 +/- 0.7 nmoles/mg of protein; P = 0.003), decreased levels of protein-bound methionine (P = 0.0007), and increased levels of protein-bound methionine sulfoxide (P = 0.0043) and 3-nitrotyrosine (P = 0.0477). SLE patients with high SLEDAI scores or elevated anti-dsDNA antibody levels exhibited increased oxidation compared with patients with low SLEDAI scores or low antibody levels. Serum MPO levels were decreased in SLE patients (P = 0.03), suggesting that this enzyme is not responsible for the enhanced protein oxidation. CONCLUSION: We found elevated levels of multiple markers of protein oxidation in sera from SLE patients compared with controls, and these levels correlated with disease activity. The findings suggest that protein oxidation may play a role in the pathogenesis of chronic organ damage in SLE.