DNPH Research Articles

Pesticidal Activities of Some Schiff Bases Derived from Benzoin, Salicylaldehyde, Aminophenol and 2,4 Dinitrophenyl Hydrazine

The pesticidal activities of three Schiff bases namely PDH [N- (1 phenyl 2 hydroxy 2 phenyl ethylidine) 2¢4¢ dinitrophenyl hydrazine], PHP [N(1-phenyl 2 hydroxy -2 phenyl ethylidine) 2¢ hydroxy phenyl imine] and HHP [N (2-hydroxy benzylidine) 2¢ hydroxy phenyl imine] derived from benzoin, salicylaldehyde, 2 aminophenol and 2,4 dinitrophenyl hydrazine were evaluated against Tribolium castaneum. Probit mortality were studied by surface film treatment method at different doses of the compounds. The results showed that the LD50 values as recorded after 24 hours of treatment were 15.1268, 3.0922 and 3.0922 mg /cm2 for PDH, PHP and HHP, respectively as compared with 0.2416 mg/cm2 for a standard pesticide l-cyhalothrin. With Schiff base complexes of cobalt(II), copper(II) and zinc(II), the LD50 values were 1.5206, 14.8576 and 3.2829 mg/cm2 respectively. Better results were obtained when recorded with longer treatment time. These three compounds may, therefore, be considered as potent pesticides. Keywords: Pesticidal activities; Schiff bases; Surface film treatment method. © 2009 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245(Online). All rights reserved. DOI: 10.3329/jsr.v1i3.2585 J. Sci. Res. 1 (3), 641-646 (2009)

Ratiometric Raman spectroscopy for quantification of protein oxidative damage

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement because it requires only one conjugation reaction, uses a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is approximately 0.33 pmol carbonyl per measurement. Fluorescence and/or immunoassay-based techniques only detect a signal from the labeling molecule and, thus, yield no structural or quantitative information for the modified protein, whereas the RMRS technique allows protein identification and protein carbonyl quantification in a single experiment.

The effect of Hibiscus anthocyanins on 2, 4-dinitrophenylhydrazine-induced hepatotoxicity in rabbits

This study examines the effects of anthocyanin extract of the dried calyces ofHibiscus sabdariffa Linn on the 2, 4-dinitrophenylhydrazine (2, 4-DNPH)-induced toxic side effects in rabbits liver. The side effects of DNPH which include tissue lipid peroxidation and depletion of antioxidant defenses were induced in rabbits with a single intraperitoneal administration of the toxicant (28 mg kg-1 body weight) at the end of a 14-day treatment with Hibiscus anthocyanin extract (100 mg kg-1 body weight twice daily) and after the animals have been fasted overnight. Four hours after the toxicant administration, liver and blood of the experimental animals were analyzed for changes in the activities of hepatic enzyme markers (alanine and aspartate aminotransferases; ALT and AST respectively). The hepatic levels of malondialdehyde (MDA), soluble protein as well as antioxidant molecules, superoxide dismutase (SOD) and reduced glutathione (GSH) were also determined. The anthocyanin extract significantly (p < 0.05) lowered the serum levels of the hepatic enzymes ALT and AST and reduced oxidative liver damage. The extract also significantly counteracted the depleting action of DNPH on the liver protein, SOD and GSH levels. Relative to the DNPH treated group, the extract significantly inhibited the formation of MDA in the liver. The data generated in this investigation show that the anthocyanin extract of H. sabdariffa dried calyces has potent protective properties against 2, 4-dinitrophenylhydrazine-induced oxidative hepatic toxicity in rabbits. Key words: Anthocyanin, antioxidant defenses, 2, 4-dinitrophenylhydrazine, hepatic enzyme markers, Hibiscus sabdariffa, lipid peroxidation.

Analysis of Protein Oxidation Markers α-Aminoadipic and γ-Glutamic Semialdehydes in Food Proteins Using Liquid Chromatography (LC)−Electrospray Ionization (ESI)−Multistage Tandem Mass Spectrometry (MS)

To elucidate the formation of protein oxidation biomarkers alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) in food proteins was the main purpose of the present study. Food proteins, namely, myofibrillar proteins, alpha-lactalbumin, and soy proteins, as well as bovine serum albumin (BSA), were suspended in a piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer and oxidized by Fe(3+) and H(2)O(2) while kept in an oven for 14 days at 37 degrees C. For the analysis of semialdehydes, a derivatization procedure with p-aminobenzoic acid (ABA) and NaCNBH(3) followed by liquid chromatography (LC)-electrospray ionization (ESI)-multistage tandem mass spectrometry (MS) was performed. For comparative purposes, the dinitrophenylhydrazine (DNPH) method was also employed as a routine method to assess carbonyl gain. Both semialdehydes were specifically and accurately detected by LC-MS in all oxidized proteins proving that GGS and AAS are formed as a consequence of the oxidation of lysine, proline, and arginine amino acid residues from BSA and other food proteins. Proteins from an animal source and, particularly, BSA were more susceptible to undergo oxidative reactions than soy proteins. The results from the present paper highlight the significance of using both semialdehydes as protein oxidation indicators in meat and dairy products. The analysis of GGS and AAS in real food systems would contribute to the understanding of the precise mechanisms involved in food protein oxidation and shed light on the fate of oxidizing amino acids during food processing and storage.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by beta-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-beta (PDGFR-beta), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-beta. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-beta was diminished by approximately 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-beta-dependent signal transduction pathway, including PDGFR-beta, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-beta.

Antimicrobial Activity of Some Schiff Bases Derived from Benzoin, Salicylaldehyde, Aminophenol and 2,4 Dinitrophenyl Hydrazine

The antibacterial and antifungal activities of three schiff bases were evaluated against some pathogenic bacteria and fungi. Parallel experiments were also carried out with standard drugs (Kanamycin for bacteria and Nystatin for fungi). Two compounds [N-(1-phenyl-2-hydroxy-2phenylethylidine)-2‘,4’ dinitrophenyl hydrazine, abbreviated as PDH and N-(2-hydroxy benzylidine)-2’-hydroxy imine, abbreviated as HHP] showed significant antimicrobial activities. The rest one [N-(1-phenyl 2-hydroxy-2 phenyl ethylidine) 2’-hydroxy phenyl imine, abbreviated as PHP] showed moderate activity. All these three compounds were found to possess pronounced cytotoxic effect. These compounds can be considered as potent antimicrobial agents.

Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis.

Protein carbonylation, the non-enzymatic addition of aldehydes or ketones to specific amino acid residues, has been implicated in the pathophysiology of multiple sclerosis. In this study, we investigated whether protein carbonyls also accumulate in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE). Western blots analysis after derivatization with dinitrophenyl hydrazine (oxyblot) showed elevated protein carbonylation at the time of maximal clinical disability. During the same period glutathione levels were substantially reduced, suggesting a causal relationship between these two markers. In contrast, lipid peroxidation products accumulated in EAE spinal cord well before the appearance of neurological symptoms. Carbonyl staining was not restricted to inflammatory lesions but present throughout the spinal cord particularly in neuronal cell bodies and axons. By 2-dimensional-oxyblot, we identified several cytoskeletal proteins, including beta-actin, glial acidic fibrillary protein, and the neurofilament proteins as the major targets of carbonylation. These findings were confirmed by pull-down experiments, which also showed an increase in the number of carbonylated beta-actin molecules and a decrease in that of oxidized neurofilament proteins in EAE. These data suggest the possibility that oxidation targets neurofilament proteins for degradation, which may contribute to axonal pathology observed in multiple sclerosis and EAE.

Laboratory Evaluation of an Aldehyde Scrubber System Specifically for the Detection of Acrolein

We demonstrate the use of an aldehyde scrubber system to resolve isobaric aldehyde/alkene interferences in a proton transfer reaction mass spectrometer (PTR-MS) by selectively removing the aldehydes from the gas mixture without loss of quantitative information for the alkene components. The aldehyde scrubber system uses a bisulfite solution, which scrubs carbonyl compounds from the gas stream by forming water-soluble carbonyl bisulfite addition products, and has been evaluated using a synthetic mixture of acrolein and isoprene. Trapping efficiencies of acrolein exceeded 97%, whereas the transmission efficiency of isoprene was better than 92%. Quantification of the PTR-MS response to acrolein was validated through an intercomparison study that included two derivatization methods, dinitrophenylhydrazine (DNPH) and O-(4-cyano-2-ethoxybenzyl)hydroxylamine (CNET), and a spectroscopic method using a quantum cascade laser infrared absorption spectroscopy (QCL) instrument. Finally, using cigarette smoke as a complex matrix, the acrolein content was assessed using the scrubber and compared with direct QCL-based detection.

Vitamin A supplementation induces a prooxidative state in the striatum and impairs locomotory and exploratory activity of adult rats

Although vitamin A has been reported to be essential to brain homeostasis, some central nervous system (CNS)-associated deleterious effects may be induced by vitamin A or by its metabolites. In this work, we investigated the effects of acute and chronic vitamin A supplementation at therapeutic (1000 or 2500 IU/kg/day) or excessive (4500 or 9000 IU/kg/day) doses on the redox state of the rat striatum. We found a 1.8- to 2.7-fold increase of lipid peroxidation in the striatum after acute or chronic supplementation (TBARS method). Therapeutic doses induced a 1.6- to 2.2-fold increase of protein carbonylation (dinitrophenylhydrazine (DNPH) derivatization). Vitamin A supplementation induced a 1.2- to 1.4-fold decrease of protein thiol content acutely and chronically. Superoxide dismutase (SOD) activity, assessed through the inhibition of epinephrine's autoxidation, was increased in a dose-dependent manner chronically. Acutely, both therapeutic and excessive vitamin A doses induced a 1.8- to 2.2-fold decrease of catalase (CAT) activity, as determined through the rate of decrease of hydrogen peroxide (H 2O 2). Glutathione peroxidase (GPx) activity did not change in this experimental model. Some vitamin A doses decreased the non-protein thiol content only chronically. Vitamin A supplementation decreased the striatal non-enzymatic antioxidant defenses (TRAP assay). Furthermore, our results show that vitamin A supplementation impaired the SOD/CAT ratio. Moreover, we observed a 1.6- to 2.0-fold decrease of locomotion and exploration in an open field after vitamin A supplementation. Therefore, our results suggest that vitamin A supplementation induces oxidative stress in the rat striatum and that it may be related to a metabolic impairment in such brain area.

Determination of Carbonyl Compounds in Exhaled Cigarette Smoke

Abstract This paper presents the findings on a quantitative evaluation of carbonyl levels in exhaled cigarette smoke from human subjects. The cigarettes evaluated include products with 5.0 mg ‘tar’, 10.6 mg ‘tar’ and 16.2 mg ‘tar’, where ‘tar’ is defined as the weight of total wet particulate matter (TPM) minus the weight of nicotine and water, and the cigarettes are smoked following U.S. Federal Trade Commission (FTC) recommendations. The measured levels of carbonyls in the exhaled smoke were compared with calculated yields of carbonyls in the inhaled smoke and a retention efficiency was obtained. The number of human subjects included a total of ten smokers for the 10.6 mg ‘tar’, five for the 16.2 mg ‘tar’, and five for the 5.0 mg ‘tar’ product, each subject smoking three cigarettes. The analyzed carbonyl compounds included several aldehydes (formaldehyde, acetaldehyde, acrolein, propionaldehyde, crotonaldehyde and n-butyraldehyde), and two ketones (acetone and 2-butanone). The smoke collection from the human subjects was vacuum assisted. Exhaled smoke was collected on Cambridge pads pretreated with a solution of dinitrophenylhydrazine (DNPH) followed by high performance liquid chromatography (HPLC) analysis of the dinitrophenylhydrazones of the carbonyl compounds. The cigarette butts from the smokers were collected and analyzed for nicotine. The nicotine levels for the cigarette butts from the smokers were used to calculate the level of carbonyls in the inhaled smoke, based on calibration curves. These were generated separately by analyzing the carbonyls in smoke and the nicotine in the cigarette butts obtained by machine smoking under different puffing regimes. The comparison of the level of carbonyl compounds in exhaled smoke with that from the inhaled smoke showed high retention of all the carbonyls. The retention of aldehydes was above 95% for all three different ‘tar’ levels cigarettes. The ketones were retained with a slightly lower efficiency. Acetone was retained in the range of 90% to 95%. The retention for 2-butanone showed a larger scatter compared to other results but it also appeared to be slightly less absorbed than the aldehydes, with an average retention around 95%. The retention of acetaldehyde and acetone by human smokers was previously reported in literature and the findings from this study are in very good agreement with these result.

Commuter Exposures to Vocs in Various Transportation Modes

MS7-05 Abstract: Because it can be challenging to measure personal exposure on large population scales, modeling exposure using stochastic methods to characterize microenvironmental exposures can be a more efficient way of estimating population exposure. However, there is little data on exposures to contaminants such as volatile organic compounds (VOCs) in different microenvironments away from the home. As part of a study to quantify exposure to volatile organic compounds (VOCs) in different nonhome microenvironments, multiple modes of transportation were sampled simultaneously during morning and evening rush hours in the summer of 2003 (once each time of day, with n = 10 for VOCs and n = 10 for aldehydes) and the winter of 2004 (twice for each time of day, with n = 14 for VOCs and n = 15 for aldehydes). The VOCs include hydrocarbons and chlorinated compounds sampled using thermal desorption tubes and aldehydes sampled using dinitrophenylhydrazine (DNPH) -coated silica cartridges. Modes sampled were walking, bus, train/subway, urban and highway car. The walking, bus, and urban modes followed a similar route, whereas the train and highway car traveled between a major U.S. urban metropolitan area and an outlying suburban town. The walking route was not sampled in the winter. Summary statistics were calculated and time of day, seasons, and mode were compared. Overall coefficient of variations (CVs) for all compounds, except for 1,3-butadiene (CV = 2.0), were below 1. Each mode was compared with the train mode as a reference on a day-by-day basis. Relative to the train route, the bus and car routes had higher concentrations in the summer, but in the winter, there were less consistent patterns. Summer had higher concentrations except for benzene, in which winter mean concentrations were higher (3.1 μg/m3 in winter vs 2.3 μg/m3 in summer). Formaldehyde was significantly higher in the summer, with a mean of 12.8 μg/m3 versus 8.3 μg/m3 in winter. Overall, exposures to VOCs in transportation modes tend to be lower than that of indoor environments in homes, workplaces, or shops and restaurants. Given the small sample size of this study, it is difficult to describe the full temporal or modal variability; however, results suggest that when developing stochastic models for personal exposure, it may be reasonable to assume a single distribution for transportation as a microenvironment rather than mode- or time-specific distribution.

Synthesis and antimicrobial evaluation of some pyrazolo-thiazolyl alkoxy-1H-isoindole-1, 3(2H)-dione derivatives

1,3-Thiazolidine-2,4-dione 2 has been synthesized by the cyclisation reaction of thiourea and chloroacetic acid in the presence of ethanol. The reaction of compound 2 with substituted aromatic aldehyde afforded the corresponding derivatives of substituted 5-benzylidene-1,3-thiazolidinone-2,4-dione 3a–d, which upon reflux with ω-bromoalkoxyphthalimide gave 2-{[-5-(substituted benzylidine)-2,4-dioxo-1,3-thiazolidine-3-yl]alkoxy} -1H-isoindole-1,3(2H)-dione 4a–i. Further, compounds 4a–i were treated with phenyl hydrazine and 2,4 dinitro phenyl hydrazine in the DMF to yield the title compound 2-[5-oxo-2,3-substituted diphenyl-2H-pyrazolo[3,4-d][1,3]thiazol-6(5H)-yl)alkoxy]-1H-isoindole-1,3(2H)-dione 5a–r. Structures of newly synthesized compounds were established based on elemental analysis, IR, 1H NMR and mass spectral data. Synthesized compounds have been assayed for their antibacterial activities against B. subtilis, K. pneumoniae, P. aeruginosa and S. aureus and antifungal activities against A. fumigatus and C. albicans.

Ascorbic Acid Levels in Hepatitis and Non-Hepatitis Subjects in University of Calabar Teaching Hospital (UCTH), Calabar

The ascorbic acid levels of thirty (30) hepatitis (sero-positive) subjects who presented themselves as donors at the serology unit of the Department of Hematology and blood transfusion, University of Calabar Teaching Hospital (UCTH), Calabar, Nigeria were assessed alongside thirty (30) non-hepatitis (Sero- negative) subjects as controls. Using the 2, 4 dinitrophenyl hydrazine (spectrophotometric method), the mean ascorbic acid level for the control subjects was 1.05±0.45mg/dl while that of the hepatitis subjects was 1.06±0.42mg/dl. There was no significant difference between the mean ascorbic acid levels of hepatiti s subjects, when compared with that of the controls (P>0.05). On separation of the subjects, based on their sex, there was also found to be no significant differences (P>0.05).

Simple Sensitive Spectrophotometric Determination of Vanadium in Biological and Environmental Samples

Novel, rapid, highly sensitive and selective spectrophotometric method for the determination of traces of vanadium (V) in environmental and biological samples, pharmaceutical and steel samples was studied. The method is based on oxidation of 2,4‐ dinitro phenyl hydrazine(2,4‐DNPH) by vanadium (V) followed by coupling reaction with N‐(1‐naphthalene‐1‐yl)ethane‐1,2‐diamine‐dihydrochloride (NEDA) in acidic medium to give red colored derivative or on oxidation of 4‐Amino Pyridine by vanadium (V) followed by coupling reaction with NEDA in basic medium to give pink colored derivative. The red colored derivative having an λmax 495 nm which is stable for 8 days and the pink colored derivative with 525 nm is stable for more than 7 days at 350C. Beer′s law is obeyed for vanadium (V) in the concentration range of 0.02 ‐ 3.5 μg mL–1 (red derivative) and 0.03 – 4.5 μg mL–1 (pink derivative) at the wave length of maximum absorption. The optimum reaction conditions and other analytical parameters were investigated to enhance the sensitivity of the present method. The detailed study of various interferences made the method more selective. The proposed method was successfully applied to the analysis of vanadium in natural water samples, plant material, soil samples, synthetic mixtures, pharmaceutical samples and biological samples. The results obtained were agreed with the reported methods at the 95 % confidence level. The performance of proposed method was evaluated in terms of Student′s t‐test and Variance ratio f‐test which indicates the significance of proposed method over reported method.

Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins

This study was aimed at establishing whether oxidative stress induced by acute depletion of brain glutathione (GSH) is sufficient to generate protein carbonyls (PCOs). To this end, rat brain slices were incubated separately with the GSH depletors 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU) and diethyl maleate (DEM), and protein carbonylation was assessed on Western blots after derivatization with dinitrophenyl hydrazine. Incubation with 1 mM BCNU or 10 mM DEM for 2 hr decreased GSH levels by > 70%. Under these conditions the carbonylation of several proteins (40-120 kDa) increased by 2-3 fold. Isolation of carbonylated proteins showed that augmented PCOs represents a rise in the amount of oxidized protein. The iron chelator deferoxamine, the superoxide scavenger rutin and the H2O2 quencher dimethylthiourea all prevented DEM-induced protein carbonylation and lipid peroxidation (TBARS), indicating that the underlying mechanism involves the iron-catalyzed generation of hydroxyl radicals from H(2)O(2) (Fenton reaction). Inhibition of catalase activity with sodium azide and aminotriazole, and glutathione peroxidase activity with mercaptosuccinic acid did not increase PCOs or TBARS, suggesting that increased production of reactive oxygen species (ROS) rather than compromised cellular antioxidant defenses is the cause for the accumulation of H2O2 after GSH depletion. PCO formation was not affected by the xanthine oxidase inhibitor oxypurinol but it was reduced by SKF-525A and carbonyl cyanide 3-chlorophenylhydrazone, indicating that the microsomal monooxygenase system and the mitochondrial electron transport system are the major sources of ROS. Consistent with these findings, subcellular fractionation studies showed that mitochondria and synaptosomes are the major PCO-containing organelles. These results were also supported by the anatomic distribution of PCOs in brain. Our observations may be important in the context of multiple sclerosis where decreased GSH, mitochondrial dysfunction, excessive production of ROS, and increased protein carbonylation have all been reported.

Determination of the GABA analogue succinic semialdehyde in urine and cerebrospinal fluid by dinitrophenylhydrazine derivatization and liquid chromatography–tandem mass spectrometry: Application to SSADH deficiency

Succinic semialdehyde (SSA) accumulates in the inborn error of meta- bolism succinic semialdehyde dehydrogenase deficiency owing to impaired enzymatic conversion to succinic acid. We developed a stable-isotope dilution liquid chromato- graphy-tandem mass spectrometry method for the determination of SSA in urine and cerebrospinal fluid samples. Stable-isotope-labelled [13C4]SSA, serving as internal standard, was prepared by reaction of ninhydrin with L-[13C5]glutamic acid. SSA in body fluids was converted to its dinitrophenylhydrazine (DNPH) derivative, without sample purification prior to the derivatization procedure. The DNPH derivative of SSA was injected onto a C18 analytical column and chromatography was performed by isocratic elution. Detection was accomplished by tandem mass spectrometry operating in the negative multiple-reaction monitoring mode. The limit of detection was 10 nmol/L and the calibration curves over the range 0-500 pmol of SSA showed good linearity (r2 > 0.99). The intra-day coefficient of variation (n = 10) for urine was 2.7% and inter-day coefficient of variation (n = 5) for urine was 8.5%. The average recoveries performed on two levels by enriching urine and cerebrospinal fluid samples ranged between 85 and 115%, with coefficients of variation < 8%. The method enabled the first determination of normal values for SSA in urine and pathological values of SSA in urine and cerebrospinal fluid samples derived from patients with succinic semialdehyde dehydrogenase deficiency.

High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein

The oxidative modification of proteins has been shown to play a major role in a number of pathological processes. One such modification is the addition of the carbonyl groups to the amino acid residue in proteins. For the measurement of the carbonyl groups in low concentration protein samples, we have modified the ELISA (enzyme-linked immunosorbent assay) method that was developed by Buss et al. [Buss, I. H; Chan, T. P.; Sluis, K. B.; Domigan, N. M.; Winterbourn, C. C. Protein carbonyl measurement by a sensitive ELISA method. Free Radic. Biol. Med. 23:361–366; 1997 [14]]. In the modified method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH). The protein-conjugated DNPH was probed by a commercial anti-DNPH antibody, and then a second antibody conjugated with horseradish peroxidase was added for quantification. The method was calibrated using oxidized albumin, and required only 5 μg protein. This obviated the need to concentrate protein in experimental and clinical samples with low amounts of protein. In addition the effect of TCA on carbonyl measurement is eliminated. The standard curve was linear in the range of 0–3.36 nmol carbonyls/mg protein, which is the range within which clinical samples fell. The results correlated well with the colorimetric carbonyl assay. The method was used to analyze the amount of protein carbonyl in aqueous humor and diluted plasma samples.

Supplementation With Mixed Fruit And Vegetable Juice Concentrates Attenuates Oxidative Stress Markers In Trained Athletes

Intensive exercise enhances the formation of free radicals and other reactive oxygen and nitrogen species (RONS) in animals and humans resulting in the generation of compounds like malondialdehyde (MDA) and carbonyl-proteins (CP). Reduced formation of these oxidative stress parameters during physical exercise by supplementation with antioxidants has not been reported. Purpose To investigate the influence of a) supplementation with mixed fruit and vegetable juice concentrates and b) of exercise duration with a defined intensity on the concentrations of MDA and CP after intense mountain biking for two hours. Methods 5 healthy trained men (male; 37 ± 5yrs; VO2max 55 ± 5mL ± kg−1 ± min−1) supplemented daily with 4 capsules of a phytonutrient preparation from fruits and vegetables. Subjects performed a two hours lasting mountain bike exercise to the top of a mountain (altitude 1500m, distance 30km) at 75% of the individual VO2max, followed by a moderate downhill performance (same route) at 45% of the individual VO2max. Capillary blood was collected from the finger tip before the exercise, on the top of the mountain immediately after intense exercise (=two hours) and the third collection at the end of exercise (=three hours). After collection blood plasma samples were separated by centrifugation at 4 ± C and plasma levels of MDA were measured by a HPLC method, measurement of CP was performed after derivatization with dinitrophenylhydrazine (DNPH) by a chemiluminescence technique. A two-sided Student's t-test was performed for testing the significance of differences with p <0.05 as the criterion for significance. Results Both parameters were significantly decreased before exercise (MDA: 2.43 ± 0.39 vs. 1.64 ± 0.52 μM; CP: 0.46 ± 0.05 nmol/mg protein vs. 0.23 ± 0.02 nmol/mg protein), after 2 hours of intense exercise (MDA: 2.68 ± 0.45 vs. 1.85 ± 0.50 μM; CP: 0.55 ± 0.18nmol/mg protein vs. 0.32 ± 0.019nmol/mg protein) and after prolonged exercise of 3 hours (MDA: 2.47 ± 0.73 to 1.64 ± 0.47 μM; CP: 0.5 ± 0.08nmol/mg protein to 0.33 ± 0.09nmol/mg protein) by supplementation compared to the non supplemented phase. No significant difference in the concentrations of both parameters throughout the time course of exercise was estimated except the CP values between the first and second blood collection in the supplemented phase (CP: 0.29 ± 0.02 nmol/mg vs. 0.315 ± 0.02 nmol/mg). Conclusions Thus we demonstrate a benefit of the supplementation with the phytonutrient preparation concerning oxidative protein damage and lipid peroxidation products in human plasma. The chosen exercise duration at a certain intensity had modest influence to the concentrations of oxidative stress markers.

High-Density Lipoproteins Prevent the Oxidized Low-Density Lipoprotein–Induced Endothelial Growth Factor Receptor Activation and Subsequent Matrix Metalloproteinase-2 Upregulation

The atherogenic oxidized low-density lipoprotein (oxLDL) induces the formation of carbonyl-protein adducts and activates the epidermal [corrected] growth factor receptor (EGFR) signaling pathway, which is now regarded as a central element for signal transduction. We aimed to investigate whether and by which mechanism the anti-atherogenic high-density lipoprotein (HDL) prevents these effects of oxLDL. In vascular cultured cells, HDL and apolipoprotein A-I inhibit oxLDL-induced EGFR activation and subsequent signaling by acting through 2 separate mechanisms. First, HDL, like the aldehyde scavenger dinitrophenyl hydrazine, prevented the formation of oxLDL-induced carbonyl-protein adducts and 4-hydroxynonenal (HNE)-EGFR adducts. Secondly, HDL enhanced the cellular antioxidant defenses by preventing (through a scavenger receptor class B-1 (SR-BI)-dependent mechanism) the increase of intracellular reactive oxygen species (ROS) and subsequent EGFR activation triggered by oxLDL or H2O2. A pharmacological approach suggests that this protective effect of HDL is independent of cellular glutathione level and glutathione peroxidase activity, but it requires catalase activity. Finally, we report that oxLDL upregulates both membrane type 1 (MT1)-matrix metalloproteinase-1 (MT1-MMP) and MMP-2 through an EGFR-dependent mechanism and that HDL inhibits these events. HDLs block in vitro oxLDL-induced EGFR signaling and subsequent MMP-2 activation by inhibiting carbonyl adducts formation and cellular oxidative stress. These effects of HDL may participate to reduce cell activation, excessive remodeling, and alteration of the vascular wall.

Induction of protein oxidation by cobalt and chromium ions in human U937 macrophages

Metal particles and ions from hip prostheses have the potential to induce the production of reactive oxygen species (ROS), making them prime suspects for disturbing the cellular balance of oxidants/antioxidants (redox state of the cell). To better understand the cellular effect of metal ions from metal-on-metal prostheses, the aim of this study was to examine the effect of cobalt (Co 2+) and chromium (Cr 3+) ions on protein oxidation in human U937 macrophages. Protein oxidation was measured by Western blot using antibodies directed against dinitrophenylhydrazine (DNP)-derivatized protein carbonyls, the most commonly measured products of protein oxidation in biological samples. Three DNP-derived proteins were detected. The first has a molecular weight of 16 kDa and is expressed at a very low level. The second has a molecular weight of 48 kDa and its level is not regulated by metal ions. The third is a 69 kDa protein and its level is regulated by Co 2+ and Cr 3+ ions. Therefore, the last band served as a marker of protein oxidation in the present study. Results showed that Co 2+ and Cr 3+ ions induced a time- and dose-dependent protein oxidation reaching 6.5 and 2.9 times the control after 72 h, respectively, which were inhibited by the antioxidant glutathione monoethyl-ester. Finally, results showed that the oxidized proteins are mainly found in the cytoplasmic fraction of the cells and are absent from the nucleus. In conclusion, our results suggest that metal ions from metal-on-metal prostheses have the potential to modify the redox state of cells both locally (periprosthetic environment) or systematically (circulating cells). The long term effect of these ions on protein oxidation in vivo remains to be investigated.