Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

OBJECTIVES: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. DESIGN AND METHODS: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated haemoglobin (HbA1c) was measured by HPLC. RESULTS: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. CONCLUSIONS: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin.     

Allantoin as a marker of oxidative stress in human erythrocytes

Abstract Background: Uric acid is the final product of purine metabolism in humans. It was determined that this compound has important antioxidative properties and it may be oxidized to allantoin by various reactive oxygen species. Therefore, the measurement of allantoin may be useful for the determination of oxidative stress in humans. Methods: We measured allantoin and uric acid in human plasma and erythrocytes obtained from patients with chronic renal failure before hemodialysis (n=30) and blood donors (n=30). We used a method based on selective isolation of allantoin from deproteinized plasma and erythrocyte lysate samples on AG 1-X8 resin and its derivatization to glyoxylate-2, 4-dinitrophenylhydrazone. Separation of glyoxylate-2, 4-dinitrophenylhydrazone from interfering substances was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. Uric acid was determined by reversed phase HPLC with UV/VIS detection at 292 nm. Results: We found significant differences in allantoin and uric acid concentration between the patients with chronic renal failure and the control group both in plasma (20.5+/-6.5 mumol/L and 323.9+/-62.9 mumol/L vs. 2.1+/-1.1 mumol/L and 270.1+/-62.3 mumol/L, p<0.05) and erythrocytes [82.8+/-39.1 nmol/g hemoglobin (Hb) and 110.7+/-28.8 nmol/g Hb vs. 20.1+/-6.1 nmol/g Hb and 82.1+/-23.7 nmol/g Hb, p<0.05]. Conclusions: Significant higher levels of allantoin in both plasma and erythrocytes of patients with chronic renal failure indicate that allantoin may be used as a good marker of oxidative stress. Clin Chem Lab Med 2008;46:1270-4.     

How urine analysis reflects oxidative stress--nitrotyrosine as a potential marker

Enhanced oxidant stress involved in the pathogenesis of cardiovascular (heart failure, atherosclerosis, ischemia-reperfusion injury), neurodegenerative (M. Alzheimer), metabolic (hypercholesterolemia, diabetes) and inflammatory disorders is mimicked by non-intermittent therapy with nitrovasodilators. We used this latter therapy model to study urinary 3-nitrotyrosine (n-tyr) excretion as a potential biomarker that may reflect the enhanced generation of reactive oxygen species. Namely, free or protein-bound n-tyr is formed in the organism by nitration of tyrosine (residues) via peroxynitrite (reaction product of NO&z.ccirf; and O(2)(-)&z. ccirf;). Free n-tyr content was analyzed by gas chromatography in urine obtained from healthy human subjects under a nitrite-limited diet during a two-day non-intermittent transdermal administration of glyceroltrinitrate (GTN; 0.4 mg/h) with or without vitamin C (Vit-C; 55 microg/kg/min) as antioxidant. Concomitant with the development of complete vascular tolerance (loss of dilator action), a progressive increase in urinary n-tyr excretion (up to 186+/-9 microg/day) was demonstrated in volunteers given GTN only. In contrast, when Vit-C was added, the GTN-induced increases in urinary n-tyr content were significantly suppressed (up to 130.20+/-6.91 microg/day), whereas Vit-C alone even decreased urinary n-tyr content (down to 34.00+/-5.66 microg/day), which was below control values (56.0+/-3.4 microg/day). Thus, urinary n-tyr may serve as a biomarker to detect changes in oxidant stress and thereby to evaluate the efficacy of therapeutic interventions aimed at reducing oxidant stress under various pathophysiological conditions.     

Nitrotyrosine: new findings as a marker of postprandial oxidative stress

Oxidative stress plays an important role in diabetic vascular complications. It has been shown that an imbalance in the ratio of nitric oxide to superoxide anion due to a prevalence of the superoxide anion leads to an alteration in vascular reactivity. Under these conditions an increase in peroxynitrite (ONOO-) production, resulting from the reaction between nitric oxide (NO) and superoxide (O2-), may be hypothesised. ONOO- is responsible for nitration of tyrosine residues in proteins; therefore the presence of nitrotyrosine (NT) in plasma proteins is considered indirect evidence of ONOO- production. NT has been found in the plasma of patients with diabetes, but it is not detectable in the plasma of healthy controls. NT plasma values are correlated with plasma glucose concentrations, and further studies exploring the effects of acute hyperglycaemia on NT formation confirmed that NT is produced both in normal subjects during hyperglycaemic clamp and in working hearts from rats during hyperglycaemic perfusion. Postprandial hypertriglyceridemia and hyperglycaemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridaemia and hyperglycaemia induce an endothelial dysfunction through an oxidative stress; however, the specific roles of these two factors are matters for debate. In a clinical study, high-fat load and glucose alone each produced a decrease in endothelial function and an increase in NT in normal subjects and patients with diabetes. These effects were more pronounced when high-fat load and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters, but reduced the effects of high-fat load, glucose alone, and both high-fat load and glucose on endothelial function and NT Long-term simvastatin treatment was accompanied by a smaller increase in postprandial triglycerides, which was followed by smaller variations in endothelial function and NT. This study showed an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycaemia on endothelial function, suggesting oxidative stress as a common mediator of these effects. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug. These studies indicate that ONOO- is generated in diabetes, suggesting the possible involvement of ONOO- in the development of diabetic complications.     

The potential use of urine cell free DNA as a marker for cancer

Introduction: Although the role of circulating cell free DNA in cancer has been widely demonstrated, less is known about the role of urine cell free DNA (UcfDNA). UcfDNA can serve as a ‘liquid biopsy’ for urological and non-urological tumors, as it carries information on DNA from cells exfoliated in urine and from circulation.

Areas covered: We review the studies on UcfDNA as a source of biomarkers for cancer, focusing on the new techniques and the differences between urological and non-urological tumors. We searched Pubmed for articles published between 1998 and 2016 with the following key words and phrases: ‘urine’ and ‘cell free DNA’ or ‘liquid biopsy’ or ‘cancer’.

Expert commentary: Despite the few papers published on this topic, UcfDNA is an important component of ‘liquid biopsy’, a useful and non-invasive tool for cancer diagnosis, prognosis and treatment monitoring, containing a wide range of genetic information.     

Evaluation of a novel colorimetric assay for free oxygen radicals as marker of oxidative stress

BackgroundFree oxygen radicals play an important role in the pathogenesis of many diseases including cardiovascular disease, diabetes, hypertension, cancer and aging. Several methods were developed for the direct or indirect measurement of oxygen free radical and its byproducts. The free oxygen radicals monitor (FORM) is a novel point-of-care system for the rapid measurement of free oxygen radicals (FORT) in blood. We have carefully evaluated the use of this assay for batch analysis of plasma samples in a research environment with respect to factors affecting its performance, including storage temperature and duration.MethodsWe determined the effect of storage, hemolysis, variability and reproducibility of the FORT in blood and plasma.ResultsPlasma FORT correlated significantly with hsCRP (P < 0.0001) and CHOL/HDL ratio (P < 0.02). While hsCRP results have shown agreater range of assay variability (27.82%–53.92%), FORT measurements in the same samples have less assay variability (5.63%–9.61%). Collected whole blood can be kept on ice for up to 7 h prior to plasma isolation without affecting FORT values. Storage of plasma has no effect on FORT when stored at 4 °C for up to 3 weeks (R² = 0.685). Comparable values can be obtained in samples stored for up to 3 months at ? 80 °C (R² = 0.5888) but not at ? 20 °C.ConclusionsThe day to day variability of FORT, as assessed by multiple measures in a group of controls over time, is minimal. FORT assay isstable when stored at ? 80 °C for a couple of months or at 4 °C for a few weeks. FORT correlation with hsCRP and other lipid markers provides an interesting insight and a novel link between oxidative stress, inflammation and lipid metabolism.     

Using redox potential as a feasible marker for banked blood quality and the state of oxidative stress in stored red blood cells

BackgroundStored red blood cells (RBCs) may undergo oxidative stress over time, with functional changes affecting oxygen delivery. Central to these changes are oxidation‐reduction (redox) reactions and redox potential (RP) that must be maintained for cell function. RP imbalance can lead to oxidative stress that may contribute to storage lesions. This study''s purpose was to identify changes in RP over time in banked RBCs, and among RBCs of similar age.MethodsMultiple random RBC segments from RBC units were tested (n = 32), ranging in age from 5 to 40 days, at 5‐day intervals. RP was recorded by measuring open circuit potential of RBCs using nanoporous gold electrodes with Ag/AgCl reference. RP measures were also performed on peripheral venous blood from 10 healthy volunteers. RP measures were compared between RBC groups, and with volunteer blood.ResultsStored RBCs show time‐dependent RP increases. There were significant differences in Day 5 RP compared to all other groups (p ≤ 0.005), Day 10–15 vs. ages ≥ Day 20 (p ≤ 0.025), Day 20–25 vs. Day 40 (p = 0.039), and all groups compared to healthy volunteers. RP became more positive over time suggesting ongoing oxidation as RBCs age; however, storage time alone was not predictive of RP measured in a particular unit/segment.ConclusionsThere are significant differences in RP between freshly stored RBCs and all others, with RP becoming more positive over time. However, storage time alone does not predict RP, indicating RP screening may be an important measure of RBC oxidative stress and serve as an RBC quality marker.     

Urinary hydrogen peroxide: a probable marker of oxidative stress in malignancy

BACKGROUND: Urinary hydrogen peroxide was postulated to be a biomarker of oxidative stress. We estimated urinary hydrogen peroxide along with other established parameters of oxidative stress in malignancies where oxidative stress is well documented. METHODS: The oxidative stress markers tested were concentrations of erythrocyte glutathione, erythrocyte malonaldehyde (MDA) and plasma hydroperoxide, and activities of plasma glutathione-S-transferase (GST) and erythrocyte catalase. Urinary hydrogen peroxide was measured by a modified ferrous ion oxidation xylenol orange version-2 (FOX-2) method on a spot random sample of urine. RESULTS: In healthy controls (n=10), erythrocyte glutathione concentration was 4.41+/-0.057mg/g of hemoglobin, plasma hydroperoxide was 2.5+/-0.07 micromol/l, erythrocyte MDA was 0.9+/-0.15 nmol/ml of packed cell suspension and erythrocyte catalase and plasma GST were 74.66+/-9.2/s/ml of packed cell suspension and 6.12+/-0.84 IU/l, respectively. In cancer patients (n=25), erythrocyte glutathione, plasma hydroperoxide and erythrocyte MDA were 9.32+/-0.42 mg/g of hemoglobin, 6.2+/-0.13 micromol/l and 2.3+/-0.27 nmol/ml of packed cell suspension, respectively; and activities of erythrocyte catalase and plasma GST were 151.04+/-6.5/s/ml of packed cell suspension and 10.9+/-0.36 IU/l, respectively. Urinary hydrogen peroxide concentration was 15+/-9.8 micromol/l in the healthy controls and 56.3+/-3.9 micromol/l in cancer patients. CONCLUSION: Urinary hydrogen peroxide may be a marker of oxidative stress in malignancies.     

The use of stroma-free hemoglobin solutions as blood substitute

Solutions of stroma free human hemoglobin and of polymerized hemoglobin were used for perfusion of isolated organs of the experimental animal (Wistar rats). Both preparations proved to be suited for maintainance of sufficient oxygen transport. Following intravenous infusion of greater amounts of hemoglobin or polymerized hemoglobin in the experimental animals (3.0 g/kg body weight), the renal losses amounted 30% and less than 10% respectively. Half live was only two hours in the case of hemoglobin and 13 hours with the polymerized hemoglobin, despite molecular weight was only doubled in the latter preparation. Since the minor amount of the different hemoglobin preparation was excreted by the kidneys, the greater amount was stored in the organism or metabolized respectively. The intravenous infusion did not cause chemically demonstrable signs of liver toxicity. Neither bilirubin concentration nor enzyme activity showed significant alterations. Additionally, all animals survived the high dosed intravenous infusions. However, histological evaluations showed distinct alterations caused by the hemoglobin preparations. Hemoglobin was found inside the liver parenchymal cells. The liver cells and kidney cells showed signs of toxic effects. The polymerized hemoglobin was not found in liver parenchymal cells. In contrast to hemoglobin this preparation was stored inside the Kupffer's cells. In contrast to hemoglobin the polymerized form behaves like erythrocytes. The rapid elimination of hemoglobin (half live only two hours) renders this substance unsuited for blood substitution. However, half life of polymerized hemoglobin is 13 hours, and from this point of view the polymerized form is suited for blood substitution. Considering the histologically demonstrable alterations additional experiments are required before hemoglobin solutions are used in human subjects.     

Obesity and the clinical use of serum GGT activity as a marker of heavy drinking

OBJECTIVE: Gamma-glutamyl transferase (GGT) is a widely used clinical marker of alcohol abuse. However, although obesity may also elevate serum GGT activities, the effects of overweight on the interpretation of GGT testing have remained poorly defined. MATERIAL AND METHODS: GGT activities from 1147 moderate drinkers and 449 abstainers who were classified according to body mass index (BMI) were compared with those of 208 heavy drinkers admitted for detoxification. RESULTS: GGT upper normal limits, defined based on normal weight abstainers (men 53 U/L; women 45 U/L) were lower than those based on moderate drinkers (men 68 U/L; women 50 U/L). The relative increases in GGT activities in male moderate drinkers with overweight (54%) or obesity (125%) exceeded the corresponding changes found in women (25% and 75%, respectively). The BMI-dependent variation on the sensitivity of GGT for correctly classifying heavy drinkers ranged from 29% to 67%. The rates of false-positive values in the subgroups from low to high BMI varied from 0% to 27%, respectively. CONCLUSIONS: The data indicate that the diagnostic value of serum GGT testing could be improved by using reference data derived from databases of abstainers with normal weight or BMI-based categorization of reference ranges.     

Obesity and the clinical use of serum GGT activity as a marker of heavy drinking

Objective. Gamma‐glutamyl transferase (GGT) is a widely used clinical marker of alcohol abuse. However, although obesity may also elevate serum GGT activities, the effects of overweight on the interpretation of GGT testing have remained poorly defined. Material and methods. GGT activities from 1147 moderate drinkers and 449 abstainers who were classified according to body mass index (BMI) were compared with those of 208 heavy drinkers admitted for detoxification. Results. GGT upper normal limits, defined based on normal weight abstainers (men 53?U/L; women 45?U/L) were lower than those based on moderate drinkers (men 68?U/L; women 50?U/L). The relative increases in GGT activities in male moderate drinkers with overweight (54%) or obesity (125%) exceeded the corresponding changes found in women (25% and 75%, respectively). The BMI‐dependent variation on the sensitivity of GGT for correctly classifying heavy drinkers ranged from 29% to 67%. The rates of false‐positive values in the subgroups from low to high BMI varied from 0% to 27%, respectively. Conclusions. The data indicate that the diagnostic value of serum GGT testing could be improved by using reference data derived from databases of abstainers with normal weight or BMI‐based categorization of reference ranges.     

Comparative analysis of plasma and erythrocyte 7-ketocholesterol as a marker for oxidative stress in patients with diabetes mellitus

OBJECTIVES: To reveal increased lipid peroxidation in diabetics by quantification of cholesterol oxidation products (COPs) not only in plasma, but also in erythrocytes. DESIGN AND METHODS: We quantified 7-ketocholesterol (7-kCho) by gas chromatography-mass spectrometry as a surrogate measure for COPs. These assays were performed on both plasma and erythrocytes in 20 control subjects and 20 treated patients with relatively poorly controlled Type 2 diabetes. RESULTS: Both plasma and erythrocyte 7-kCho levels in diabetics were significantly higher than those in control subjects. Although neither plasma nor erythrocyte 7-kCho levels were associated with markers for glucose tolerance in diabetics, a negative correlation of serum HDL-cholesterol levels with erythrocyte, but not plasma, 7-kCho levels was found. CONCLUSION: Increased oxidative stress in diabetics affects oxidation of cholesterol. Assays of COPs not only in plasma, but also in erythrocytes, may yield complementary information in lipid peroxidation.     

Hexosaminidase as a new potential marker for larynx cancer

ObjectivesLarynx squamous cell carcinoma is one of the most common forms of cancer in the area of the neck. The aim of our study was to investigate the activities of N-acetyl-β-d-hexosaminidase (HEX) in larynx cancer compared with the specimens from the healthy space of the tumor that served as controls.Design and methodsLarynx cancer (n = 15) and normal healthy tissue around the tumor (n = 15) were collected from the patients during total laryngectomy. Specimens were immediately frozen in ? 80 °C. To assess hexosaminidase activity, release of p-nitrophenol from p-nitrophenol derivatives was used.ResultsWe observed a significantly higher activity of the investigated enzyme in all laryngeal cancer specimens compared with that in healthy tissue homogenates. The differences were statistically significant.ConclusionsIt could be assumed that HEX may release particular sugars from the ends of oligosaccharide chains of glycocalyx proteins, changing adhesive forces binding together cells, and the communication between cells and elements of extracellular matrix.     

Effects of an oxidative stress on human hemoglobin: a multiwavelength visible spectrometry study.

This study was carried out to investigate hemoglobin behavior and the role of cell membrane during oxidative stress of human red blood cells induced by a water-soluble radical initiator, 2,2'-azobis(amidino-propane) hydrochloride (AAPH) and compare the observed data to the one obtained with purified human haemoglobin solution. The different forms of hemoglobin were identified and quantified by multiwavelength visible spectrometry using multiple linear regression analysis. Hemolysis was quantified by the Drabkin method. Oxidative stress on purified hemoglobin solutions induced an early formation of Hb(+). In intact erythrocytes, no modified form of haemoglobin was found. Only the hemoglobin released by hemolysis in the extracellular medium was notified in the same way as purified haemoglobin. Thus, the cell membrane appears to protect intraerythrocytic hemoglobin toward an extracellular oxidative stress. Oxidative stress-induced by hemolysis does not seem to be due to changes in intraerythrocytic hemoglobin forms.     

Hexosaminidase as a new potential marker for middle ear cholesteatoma

OBJECTIVES: The study aim was to investigate the activities of hexosaminidase (HEX) in cholesteatoma tissue compared with that in normal skin. DESIGN AND METHODS: The enzyme activities were determined using the Chatterjee et al. method in the modification of Zwierz et al. in cholesteatoma and skin. RESULTS: Significantly higher activity of hexosaminidase was observed in cholesteatoma tissue compared with the skin. CONCLUSIONS: Hex may be considered as a new pathogenetic factor in that destructive lesion.     

Ubiquinol: a potential biomarker for tissue energy requirements and oxidative stress

BACKGROUND: Coenzyme Q (CoQ) has been suggested as a biomarker for tissue redox status. The aims are (1) to compare ubiquinol-9, ubiquinol-10, ubiquinone-9, ubiquinone-10, total CoQ content and CoQ redox ratio in quadriceps muscle, heart, brain and liver tissues of mdx mice with wild-type controls; and (2) to determine if ubiquinol content and CoQ redox ratio changes are associated with pathological findings in mdx mouse. METHODS: CoQ contents were determined in homogenized quadriceps muscle, heart, liver and brain of age-matched mdx and wild-type control mice by HPLC-EC. Light and electron microscopy studies were conducted using standard pathology methods. RESULTS: Ubiquinol-9 and ubiquinol-10 concentrations are significantly increased in quadriceps and heart muscle of mdx mouse. Increased redox ratios of coenzyme Q(9) and coenzyme Q(10) are also evident in quadriceps, heart and liver tissues in mdx mouse, but not brain. Pathological examination shows marked myofiber regeneration and evidence of mitochondrial proliferation for mdx muscle. CONCLUSIONS: Evidence that changes in ubiquinol content and CoQ redox ratio are related to pathological features in mdx skeletal and heart myofibers suggests that tissue ubiquinol content and CoQ redox ratio may be useful biomarkers for evaluating muscle disorders associated with mitochondrial proliferation and defects in oxidative phosphorylation.