The effect of hemolysis on plasma oxidation and nitration in patients with sickle cell disease

This study aimed to determine the effect of haemolysis on plasma oxidation and nitration in sickle cell disease (SCD) patients. Blood was collected from haemoglobin (Hb)A volunteers and homozygous HbSS patients who had not received blood transfusions in the last 3 months. Haemolysis was characterised by low levels of haemoglobin and haptoglobin and high levels of reticulocyte, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), plasma cell-free haemoglobin, bilirubin, total lactate dehydrogenase (LDH) and dominance of LDH-1 isoenzyme. Plasma 8-isoprostane, protein carbonyl and nitrotyrosine levels were measured to evaluate oxidised lipids, oxidised and nitrated proteins, respectively. Plasma nitrite-nitrate levels were also determined to assess nitric oxide (NO) production in both SCD patients and controls. Markers of haemolysis were significantly evident in SCD patients compared to controls. Plasma 8-isoprostane, protein carbonyl and nitrotyrosine levels were markedly elevated in SCD patients compared to controls. Linear regression analysis revealed a significant inverse correlation between haemoglobin and reticulocyte counts and a significant positive correlation of plasma cell-free haemoglobin with protein carbonyl and nitrotyrosine levels. The obtained data shows that increased haemolysis in SCD increases plasma protein oxidation and nitration.     

Reduction in extracellular superoxide dismutase activity in African-American patients with hypertension

Superoxide anions react with nitric oxide to form peroxynitrite and hence reduce the bioavailability of nitric oxide in the arteries. Extracellular superoxide dismutase (EC-SOD) is a major superoxide scavenger in human plasma and vascular tissues. The objective of this study is to assess whether essential hypertension is associated with an alteration in EC-SOD activity. In this report, blood samples were obtained from hypertensive (n=39) and normotensive (n=37) African-Americans. Plasma EC-SOD activity was measured using in-gel activity staining and spectrophotometric assays, EC-SOD protein level was measured using Western blotting, nitrotyrosine was measured using slot blotting, 8-isoprostane was measured with an enzyme immunoassay, and plasma copper and zinc concentrations were measured using an atomic absorption assay. Our data demonstrate that the copper, zinc, and plasma EC-SOD protein concentrations in the hypertensive and normotensive subjects are indistinguishable. Compared to normotensive controls, hypertensive patients have significantly reduced plasma EC-SOD activity. Plasma nitrotyrosine and 8-isoprostane levels are significantly higher in the hypertensive patients than in normotensive controls. Results from this study suggest that a reduction in EC-SOD activity in hypertensive patients is not due to a down-regulation of the SOD3 gene (encoding EC-SOD) or deficiency in mineral cofactors. Furthermore, the reduced EC-SOD activity might be at least partially responsible for the increased oxidative stress, as reflected by increased plasma nitrotyrosine and 8-isoprostane, in hypertensive subjects.     

Free radical reaction products and antioxidant capacity in beating heart coronary artery surgery compared to conventional bypass

Oxygen-derived free radicals are important agents of tissue injury during ischemia and reperfusion. The aim of this study was to investigate changes in protein and lipid oxidation and antioxidant status in beating heart coronary artery surgery and conventional bypass and to compare oxidative stress parameters between the two bypass methods. Serum lipid hydroperoxide, nitric oxide, protein carbonyl, nitrotyrosine, vitamin E, and β-carotene levels and total antioxidant capacity were measured in blood of 30 patients undergoing beating heart coronary artery surgery (OPCAB, off-pump coronary artery bypass grafting) and 12 patients undergoing conventional bypass (CABG, on-pump coronary artery bypass grafting). In the OPCAB group, nitric oxide and nitrotyrosine levels decreased after reperfusion. Similarly, β-carotene level and total antioxidant capacity also decreased after anesthesia and reperfusion. In the CABG group, nitric oxide and nitrotyrosine levels decreased after ischemia and reperfusion. However, protein carbonyl levels elevated after ischemia and reperfusion. Vitamin E, β-carotene, and total antioxidant capacity decreased after ischemia and reperfusion. Significantly decreased nitration and impaired antioxidant status were seen after reperfusion in both groups. Moreover, elevated protein carbonyls were found in the CABG group. The off-pump procedure is associated with lower degree of oxidative stress than on-pump coronary surgery.     

Homocysteine from endothelial cells promotes LDL nitration and scavenger receptor uptake

We recently reported that methionine-loaded human umbilical vein endothelial cells (HUVECs) exported homocysteine (Hcy) and were associated with hydroxyl radical generation and oxidation of lipids in LDL. Herein we have analysed the Hcy-induced posttranslational modifications (PTMs) of LDL protein. PTMs have been characterised using electrophoretic mobility shift, protein carbonyl ELISA, HPLC with electrochemical detection and Western blotting of 3-nitrotyrosine, and LDL uptake by scavenger receptors on monocyte/macrophages. We have also analysed PTMs in LDL isolated from rheumatoid (RA) and osteo-(OA) arthritis patients with cardiovascular disease (CVD). While reagent Hcy (< 50 microM) promoted copper-catalysed LDL protein oxidation, Hcy released from methionine-loaded HUVECs promoted LDL protein nitration. In addition, LDL nitration was associated with enhanced monocyte/macrophage uptake when compared with LDL oxidation. LDL protein nitration and uptake by monocytes, but not carbonyl formation, was elevated in both RA and OA patients with CVD compared with disease-matched patients that had no evidence of CVD. Moreover, a direct correlation between plasma total Hcy (tHcy) and LDL uptake was observed. The present studies suggest that elevated plasma tHcy may promote LDL nitration and increased scavenger receptor uptake, providing a molecular mechanism that may contribute to the clinical link between CVD and elevated plasma tHcy.     

Considerations of blood parameters of largemouth bass, Micropterus salmoides

To evaluate physiological stress resulting from environmental influences, a haematological study of a natural population of largemouth bass, Micropterus salmoides , was undertaken to establish a 'normal' blood profile. Several parameters including haemoglobin concentration, haematocrit, erythrocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, plasma protein concentration and glucose levels were investigated. Haematocrit, haemoglobin, and total plasma protein were positively correlated with fish length. Haemoglobin and haematocrit were positively correlated with fish age while mean corpuscular haemoglobin, concentration was negatively correlated with fish age. Both haemoglobin and haematocrit values were related to erythrocyte counts.     

Haemolysis during sample preparation alters microRNA content of plasma

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.     

In vivo rates of erythrocyte glutathione synthesis in adults with sickle cell disease

Despite reports of lower GSH concentration in sickle cell disease (SCD), the in vivo kinetic mechanism(s) responsible for GSH deficiency is unknown. To determine whether suppressed synthesis was responsible for the lower erythrocyte GSH concentration, we used a primed intermittent infusion of [(2)H(2)]glycine to measure erythrocyte GSH synthesis in vivo in 23 individuals with homozygous beta(s) SCD and 8 healthy controls. Erythrocyte cysteine concentration, the rate-limiting precursor for GSH synthesis, plasma markers of oxidant damage, and dietary intakes of energy and protein were also measured. Compared with values of controls, SCD subjects had significantly lower erythrocyte GSH (P < 0.04) and cysteine concentrations (P < 0.004) but significantly faster fractional rates of GSH synthesis (P < 0.02). The absolute rates of GSH synthesis in SCD subjects compared with control subjects was greater by approximately 57% (P = 0.062). However, the concentrations of markers of oxidative damage, plasma derivatives of reactive oxygen metabolites, plasma nitrotyrosine, urinary isoprostane-to-creatinine ratio, and GSH-to-GSSG ratio, as well as dietary intakes of energy, protein, and GSH precursor amino acids, were not different between SCD subjects and controls. The findings of this study suggest that the lower erythrocyte GSH of SCD patients is not due to suppressed synthesis or impaired regeneration but rather to increased consumption. In addition, the lower erythrocyte cysteine concentration plus the faster rate of GSH synthesis strongly suggest that the endogenous cysteine supply is not sufficient to meet all anabolic demands; hence, cysteine may be a conditionally essential amino acid in individuals with SCD.     

Hypoxia-induced acute lung injury in murine models of sickle cell disease

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human alpha/sickle beta-globin (halphabetaS) transgene (SCD mice) or are heterozygous for the normal murine beta-globin gene and express the halphabetaS transgene (mbeta+/-, halphabetaS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing *NO generation and predisposing the lung to vaso-occlusion.     

Muscle and blood redox status after exercise training in severe COPD patients

Beneficial effects of exercise training in patients with chronic obstructive pulmonary disease (COPD) are acknowledged. However, high-intensity exercise may enhance muscle oxidative stress in severe COPD patients. We hypothesized that high-intensity exercise training of long duration does not deteriorate muscle redox status. In the vastus lateralis and blood of 18 severe COPD patients and 12 controls, before and after an 8-week training program, protein oxidation and nitration, antioxidant systems, and inflammatory cytokines were examined. At baseline, COPD patients showed greater muscle oxidative stress and superoxide dismutase activity and circulating inflammatory cytokines than controls. Among COPD patients, muscle and blood protein carbonylation levels were correlated. Both groups showed training-induced increase in VO(2) peak and decreased blood lactate levels. After training, among the COPD patients, blood protein nitration levels were significantly reduced and muscle protein oxidation and nitration levels did not cause impairment. Muscle and blood levels of inflammatory cytokines were not modified by training in either patients or controls. We conclude that in severe COPD patients, high-intensity exercise training of long duration improves exercise capacity while preventing the enhancement of systemic and muscle oxidative stress. In addition, in these patients, resting protein oxidation levels correlate between skeletal muscle and blood compartments.     

Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite

The protective effects of resveratrol (3, 4', 5-trihydroxystilbene; present naturally in different plants) against the oxidative/nitrative damage of human plasma proteins induced by peroxynitrite (ONOO-) were studied and compared with those of deferoxamine (DFO; a natural siderophore isolated from Streptomyces pilosus), which is a typical and well-known antioxidant. We also studied the effect of ONOO- on plasma lipid peroxidation and the role of tested antioxidants in this process. ONOO- at the used concentrations (0.01-1 mM) showed toxicity to human plasma components. Exposure of plasma to ONOO- (0.1 mM) resulted in an increase of the level of carbonyl groups and nitrotyrosine residues in plasma proteins (approximately 4-fold and 76-fold, respectively) and in a distinct augmentation of lipid peroxidation (approximately 2-fold). In the presence of 0.1-mM resveratrol, a distinct decrease of carbonyl group formation and tyrosine nitration in plasma proteins caused by 0.1-mM ONOO- was observed (by approximately 70% and 65%, respectively). Addition of 0.1-mM DFO to plasma also distinctly reduced the level of carbonyl groups and nitrotyrosines caused by 0.1-mM ONOO- (by approximately 50% and 60%, respectively). Moreover, these antioxidants also inhibited plasma lipid peroxidation induced by ONOO- (0.1 mM). The obtained results indicate that in vitro resveratrol, like well-known antioxidant DFO, has inhibitory effects on ONOO- -mediated oxidation of proteins and lipids in human plasma.     

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol. 49:1993–2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized ～1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P < 0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.     

Eosinophil peroxidase nitrates protein tyrosyl residues. Implications for oxidative damage by nitrating intermediates in eosinophilic inflammatory disorders.

Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase have primarily focused on its unusual substrate preference for bromide (Br(-)) and the pseudohalide thiocyanate (SCN(-)) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO(2)(-)), a major end-product of nitric oxide ((.)NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically relevant concentrations of NO(2)(-). Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO(2)(-) with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and NO(2)(-). EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN(-) concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o'-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory conditions characterized by recruitment and activation of eosinophils.     

Haematological effects of prolonged sublethal hypoxia on channel catfish*Ictalurus punctatus (Rafinesque)

Sub-adult channel catfish, Ictalurus punctatus, were exposed to sublethal hypoxia for 24, 48 or 72 h. Haematological parameters were used to assess the physiological state of the catfish subjected to prolonged, sublethal hypoxia. Haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentreation, plasma lactic acid and plasma glucose deviated significantly from control values in channel catfish exposed to hypoxia for 24, 48 or 72 h. Haematocrit, total plasma protein, total erythrocyte counts, total leukocyte counts mean corpuscular volume and differential leukocyte counts were not sensitive indicators to the catfish's physiological status. After five days, of reacclimation to oxygen levels saturation, channel catfish continued to exhibit haematological responses to prolonged hypoxia.     

Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anaemia

Temporal changes in the levels of denatured haemoglobin (Heinz bodies) and fluorescent lipid peroxidation products in the red cells of rabbits administered phenylhydrazine have been followed. Heinz bodies were maximal just before the period when most of the cell destruction occurred, whereas lipid peroxidation products were maximum when reticulocyte levels were highest. This implies that lipid peroxidation occurs mainly in immature cells and that haemoglobin denaturation is more likely than lipid peroxidation to be a major contributor to haemolysis.     

Increased formation of S-nitrothiols and nitrotyrosine in cirrhotic rats during endotoxemia

Plasma S-nitrosothiols are believed to function as a circulating form of nitric oxide that affects both vascular function and platelet aggregation. However, the formation of circulating S-nitrosothiols in relation to acute and chronic disease is largely unknown. Plasma S-nitrosothiols were measured by chemiluminescence in rats with biliary cirrhosis or controls, and the effect of lipopolysaccharide (LPS) on their formation was determined. Plasma S-nitrosothiols were increased in rats with cirrhosis (206 +/- 59 nM) compared to controls (51 +/- 6 nM, p <.001). Two hours following injection of LPS (0.5 mg/kg) plasma S-nitrosothiols increased to 108 +/- 23 nM in controls (p <.01) and to 1335 +/- 423 nM in cirrhotic rats (p <.001). The plasma clearance and half-life of S-nitrosoalbumin, the predominant circulating S-nitrosothiol, were similar in control and cirrhotic rats, confirming that the increased plasma concentrations were due to increased synthesis. Because reactive nitrogen species, such as peroxynitrite, may cause the formation of S-nitrosothiols in vivo, we determined the levels of nitrotyrosine by gas chromatography/mass spectrometry as an index for these nitrating and nitrosating radicals. Hepatic nitrotyrosine levels were increased at 7.0 +/- 1.2 ng/mg in cirrhotic rats compared to controls (2.0 +/- 0.2 ng/mg, p <.01). Hepatic nitrotyrosine levels increased by 2.3-fold and 1.5-fold in control and cirrhotic rats, respectively, at 2 h following injection of LPS (p <.01). Strong positive staining for nitrotyrosine was shown by immunohistochemistry in all the livers of the rats with cirrhosis. We conclude that there is increased formation of S-nitrosothiols and nitrotyrosine in biliary cirrhosis, and this is markedly upregulated during endotoxemia.     

Protection of erythrocytes by the macrophage synthesized antioxidant 7,8 dihydroneopterin

Neopterin and the reduced form, 7,8-dihydroneopterin (78NP), are pteridines released from macrophages when stimulated with γ-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations of 78NP can inhibit or reduce red blood cell haemolysis induced by 2,2′-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred μM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 μmole HOCl/10⁷ RBC. Fifty μM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 μM 78NP reduced dityrosine formation in H₂O₂/Fe⁺⁺ treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.     

Plasma protein carbonyl levels and breast cancer risk

To study the role of oxidative stress in breast cancer risk, we analysed plasma levels of protein carbonyls in 1050 cases and 1107 controls. We found a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels (OR = 1.2, 95% CI = 0.9-1.5; OR = 1.5, 95% CI = 1.2-2.0; OR = 1.6, 95% CI = 1.2-2.1, for the 2(nd), 3(rd) and 4(th) quartile relative to the lowest quartile, respectively, P for trend = 0.0001). The increase in risk was similar for younger (<50 years) and older women, more pronounced among women with higher physical activity levels (0.7 hrs/week for 4(th) quartile versus lowest quartile OR = 2.0, 95% CI = 1.4-3.0), higher alcohol consumption (> or = 15 grams/day for 4(th) quartile versus lowest quartile OR = 2.3, 95% CI = 1.1-4.7), and hormone replacement therapy use (HRT, OR = 2.6, 95% CI = 1.6-4.4 for 4(th) quartile versus lowest quartile). The multiplicative interaction terms were statistically significant only for physical activity and HRT. The positive association between plasma protein carbonyl levels and breast cancer risk was also observed when the analysis was restricted to women who had not received chemotherapy or radiation therapy prior to blood collection. Among controls, oxidized protein levels significantly increased with cigarette smoking and higher fruit and vegetable consumption, and decreased with alcohol consumption >30 grams per day. Women with higher levels of plasma protein carbonyl and urinary 15F(2t)-isoprostane had an 80% increase in breast cancer risk (OR = 1.8, 95% CI = 1.2-2.6) compared to women with levels below the median for both markers of oxidative stress. In summary, our results suggest that increased plasma protein carbonyl levels may be associated with breast cancer risk.     

Protein carbonyl content in erythrocyte membranes in type 2 diabetic patients.

Protein carbonyl groups result from free radical-induced protein oxidation; their level in tissues and plasma is a relatively stable marker of oxidative damage. Protein carbonyl contents in erythrocyte membranes were investigated in the type 2 diabetic patients with good (n = 16) and poor (n = 30) glycemic control. Diabetic patients were classified as patients with (n = 20) and without (n = 26) angiopathy. Protein carbonyl content was evaluated using the 2,4-dinitro-phenyl-hydrazine method. Protein carbonyl content and GHb levels were significantly higher in both patients with poor and good glycemic control than in control subjects (p < 0.001 in each case). There was a significant difference in protein carbonyl content between patients with poor and good glycemic control (p < 0.001). Diabetic patients with angiopathy had significantly higher protein carbonyl content and GHb levels than the diabetic patients without angiopathy (p < 0.001). These results suggest that impaired glycemic control is connected to protein oxidation, and protein oxidation may be related to underlying metabolic abnormalities and complications of diabetes.     

Plasma protein carbonyls in nonpregnant, healthy pregnant and preeclamptic women

Increased reactive oxygen species (ROS) and lipid peroxidation may be implicated in the pathogenesis of preeclampsia by causing cell (membrane) damage and impaired endothelial function. Carbonyl derivatives of proteins, or protein carbonyls, may be sensitive biomarkers of ROS-mediated damage. The aim of the study was to compare levels of protein carbonyls in plasma of preeclamptic, healthy pregnant and healthy nonpregnant women. Plasma protein carbonyls were measured in 47 preeclamptic, 45 healthy pregnant and 22 healthy nonpregnant women by using a sensitive ELISA-method. ANOVA, the unpaired t-test and Pearson's correlation were used for statistical analysis. Preeclamptic women had significantly higher plasma protein carbonyl levels than healthy pregnant women (P < 0.0001). Healthy pregnant women showed significantly higher protein carbonyl levels (P < 0.001) as compared to nonpregnant controls. The higher levels of protein carbonyls as compared to nonpregnant controls suggest that increased oxygen free radical damage occurs in normal pregnancy and to a much higher extent in preeclampsia.     

Identification of oxidized plasma proteins in Alzheimer's disease

The modification of proteins by reactive oxygen species is central to the pathology of Alzheimer's disease (AD). Previously, we have observed specific oxidized proteins in blood plasma of AD subjects [Biochem. Biophys. Res. Commun. 275 (2000) 678]. Plasma from AD subjects and age-matched controls was subjected to two-dimensional gel electrophoresis (2-DE). Oxidized proteins with new carbonyl groups were detected by reaction with 2,4-dinitrophenylhydrazine, followed by Western blotting with anti-DNP antibody. Seven principal oxidized protein spots (isoelectric point=4.7-5.5; molecular mass=45-65 kDa) were observed, with varying levels of oxidation in plasma samples from both AD and non-AD subjects. Matrix-assisted laser desorption mass spectroscopy (MALDI-TOF/MS) revealed that these oxidized proteins were isoforms of fibrinogen gamma-chain precursor protein and of alpha-1-antitrypsin precursor. These proteins exhibited a two- to sixfold greater specific oxidation index in plasma from AD subjects when compared to controls. Both these proteins have been previously implicated in the pathology of the disease. It is possible that oxidized isoforms of these proteins may serve as biomarkers for AD.