Dityrosine Research Articles

Flavonoid loaded nanoparticles as an effective measure to combat oxidative stress in Ribonuclease A

Flavonoids like quercetin and myricetin serve as naturally occurring antioxidants but their bioactivity is limited due to low aqueous solubility and oxidation under physiological conditions. In this current study, the antioxidant activity of quercetin and myricetin loaded chitosan nanoparticles during the induced oxidation of Ribonuclease A (RNase A) has been compared with the corresponding free flavonoids. Oxidation of RNase A leads to intermolecular dityrosine (DT) bond formation which shows a characteristic fluorescence emission around 405 nm. Although both quercetin and myricetin loaded nanoparticles initially exhibit lower antioxidant property compared to the free flavonoids, however, with increase in oxidant concentration over time the DT fluorescence showed greater increase for free flavonoids in comparison to the nanoparticles. The polyphenol loaded nanoparticles are also found to be effective in preventing bacterial cell damage in oxidizing medium. The slow release of flavonoids from the nanoparticles is responsible for their prolonged antioxidant effect in the oxidizing medium unlike the free flavonoids which are exhausted almost completely in the initial phase.

Processing milk causes the formation of protein oxidation products which impair spatial learning and memory in rats.

This study explored the effects of protein oxidation during milk processing on spatial learning and memory in rats. Increasing the heating time, fat content, and inlet air temperature during processing by boiling, microwave heating, spray-drying, or freeze-drying increases milk protein oxidation. Oxidative damage done to milk proteins by microwave heating is greater than that caused by boiling. Dityrosine (DT), as a kind of tyrosine oxidation product, is the most important marker of this process, especially during spray-drying. Rats received diets containing either SWM (spray-dried milk powder diet), FWM (freeze-dried milk powder diet), FWM + LDT (freeze-dried milk powder + low dityrosine diet, DT: 1.4 mg kg−1), or FWM + HDT (freeze-dried milk powder + high dityrosine diet, DT: 2.8 mg kg−1) for 6 weeks. We found that the SWM group, the FWM + LDT group, and the FWM + HDT group appeared to have various degrees of redox state imbalance and oxidative damage in plasma, liver, and brain tissues. Further, hippocampal inflammatory and apoptosis genes were significantly up-regulated in such groups, while learning and memory genes were significantly down-regulated. Eventually, varying degrees of spatial learning and memory impairment were demonstrated in those groups in the Morris water maze. This means that humans should control milk protein oxidation and improve the processing methods applied to food.

Comparison of oxidative stress on DNA, protein and lipids in patients with actinic keratosis, Bowen's disease and squamous cell carcinoma.

Detailed mechanisms on the effect of oxidative stress (OS), an etiological factor involved in photocarcinogenesis, remain to be fully elucidated. We used immunohistochemical methods to study OS in the DNA, protein and lipids of patients with actinic keratosis (AK), Bowen's disease (BD) and squamous cell carcinoma (SCC). Between January 2009 and December 2014, we treated 230 patients; 79 had AK, 61 had (BD) and 90 had cutaneous SCC; 28 healthy subjects served as the normal controls. OS on DNA, protein and lipids was assessed by the expression of 8-hydroxydeoxyguanosine (8-OHdG), dityrosine (DT) and malondialdehyde (MDA), respectively. 8-OHdG was significantly overexpressed in AK and BD lesions compared with surrounding non-lesional tissue, SCC lesions and the healthy controls. DT was more highly expressed in AK, BD and SCC than in the controls. There was no significant difference among AK, BD and SCC. The expression of MDA was higher in AK, BD and SCC lesions than the controls; SCC showed the highest expression. Our observations suggest that DNA oxidation plays an important role in the early stage of carcinogenesis, that protein oxidation is involved in all stages of carcinogenesis and that lipid oxidation is strongly implicated in the late stages of carcinogenesis.

Advanced glycation endproducts, dityrosine and arginine transporter dysfunction in autism - a source of biomarkers for clinical diagnosis

BackgroundClinical chemistry tests for autism spectrum disorder (ASD) are currently unavailable. The aim of this study was to explore the diagnostic utility of proteotoxic biomarkers in plasma and urine, plasma protein glycation, oxidation, and nitration adducts, and related glycated, oxidized, and nitrated amino acids (free adducts), for the clinical diagnosis of ASD.MethodsThirty-eight children with ASD (29 male, 9 female; age 7.6 ± 2.0 years) and 31 age-matched healthy controls (23 males, 8 females; 8.6 ± 2.0 years) were recruited for this study. Plasma protein glycation, oxidation, and nitration adducts and amino acid metabolome in plasma and urine were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Machine learning methods were then employed to explore and optimize combinations of analyte data for ASD diagnosis.ResultsWe found that children with ASD had increased advanced glycation endproducts (AGEs), Nε-carboxymethyl-lysine (CML) and Nω-carboxymethylarginine (CMA), and increased oxidation damage marker, dityrosine (DT), in plasma protein, with respect to healthy controls. We also found that children with ASD had increased CMA free adduct in plasma ultrafiltrate and increased urinary excretion of oxidation free adducts, alpha-aminoadipic semialdehyde and glutamic semialdehyde. From study of renal handling of amino acids, we found that children with ASD had decreased renal clearance of arginine and CMA with respect to healthy controls. Algorithms to discriminate between ASD and healthy controls gave strong diagnostic performance with features: plasma protein AGEs—CML, CMA—and 3-deoxyglucosone-derived hydroimidazolone, and oxidative damage marker, DT. The sensitivity, specificity, and receiver operating characteristic area-under-the-curve were 92%, 84%, and 0.94, respectively.ConclusionsChanges in plasma AGEs were likely indicative of dysfunctional metabolism of dicarbonyl metabolite precursors of AGEs, glyoxal and 3-deoxyglucosone. DT is formed enzymatically by dual oxidase (DUOX); selective increase of DT as an oxidative damage marker implicates increased DUOX activity in ASD possibly linked to impaired gut mucosal immunity. Decreased renal clearance of arginine and CMA in ASD is indicative of increased arginine transporter activity which may be a surrogate marker of disturbance of neuronal availability of amino acids. Data driven combination of these biomarkers perturbed by proteotoxic stress, plasma protein AGEs and DT, gave diagnostic algorithms of high sensitivity and specificity for ASD.

β-cyclodextrin encapsulated polyphenols as effective antioxidants.

Formation of dityrosine (DT) cross-linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein-polyphenol cross-linkages. To prevent the formation of such cross-linkages we have used β-cyclodextrin (β-CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/β-cyclodextrin (β-CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of β-CD and are no longer available for protein cross-linking.

Opposed Effects of Dityrosine Formation in Soluble and Aggregated α-Synuclein on Fibril Growth

Parkinson's disease is the second most common neurodegenerative disease. It is characterized by aggregation of the protein α-synuclein (α-syn) in Lewy bodies, mitochondrial dysfunction, and increased oxidative stress in the substantia nigra. Oxidative stress leads to several modifications of biomolecules including dityrosine (DiY) crosslinking in proteins, which has recently been detected in α-syn in Lewy bodies from Parkinson's disease patients. Here we report that α-syn is highly susceptible to ultraviolet-induced DiY formation. We investigated DiY formation of α-syn and nine tyrosine-to-alanine mutants and monitored its effect on α-syn fibril formation in vitro. Ultraviolet irradiation of intrinsically disordered α-syn generates DiY-modified monomers and dimers, which inhibit fibril formation of unmodified α-syn by interfering with fibril elongation. The inhibition depends on both the DiY group and its integration into α-syn. When preformed α-syn fibrils are crosslinked by DiY formation, they gain increased resistance to denaturation. DiY-stabilized α-syn fibrils retain their high seeding efficiency even after being exposed to denaturant concentrations that completely depolymerize non-crosslinked seeds. Oxidative stress-associated DiY crosslinking of α-syn therefore entails two opposing effects: (i) inhibition of aggregation by DiY-modified monomers and dimers, and (ii) stabilization of fibrillar aggregates against potential degradation mechanisms, which can lead to promotion of aggregation, especially in the presence of secondary nucleation.

The efficacy of donepezil administration on acetylcholinesterase activity and altered redox homeostasis in Alzheimer’s disease

Alzheimer’s disease (AD) is a serious multifactorial disorder with progressive neurodegenerative outcomes related with impaired redox homeostasis. Inhibition of the enzyme acetylcholinesterase (AChE), as one of the major therapeutic strategies, is considered to be offering only symptomatic relief and moderate disease modifying effect. We intended to investigate the effects of acetylcholinesterase inhibition via donepezil on protein carbonyl (PCO), advanced protein oxidation products (AOPP) and ischemia modified albumin (IMA) as protein oxidation markers and ferric reducing antioxidant power (FRAP), prooxidant–antioxidant balance (PAB), total thiol (T-SH), protein thiol (P-SH) as antioxidant status markers and also kynurenine (KYN), N-formyl kynurenine (N-FKYN) and protein bound dityrosine (DT) levels all in one demonstrating the redox homeostasis in Alzheimer patients also correlated with AChE activity. The AChE activity and PCO, KYN, N-FKYN and DT levels were found to be significantly higher in the AD group than the control group. The FRAP, T-SH and P-SH levels were significantly lower in the AD group than in the control group. The AChE activity was significantly higher both in donepezil treated and untreated groups when compared with the control group. PCO levels were significantly higher in Alzheimer’s untreated group than the healthy control and donepezil treated groups. AChE activity was positively correlated with PCO, IMA, PAB, KYN and N-FKYN levels and negatively correlated with FRAP, T-SH and P-SH levels in all participants. Our data showed that treatment with donepezil had ameliorating effects on redox homeostasis in Alzheimer patients. AChE inhibition seems to be exhibiting a potent antioxidant role and may inhibit protein oxidation by decreasing AChE activity in AD, thus medicinal natural substances exhibiting the similar mechanism of action with their antioxidant behaviours can be recommended for the emphasis on new drug new drug development. Further clinical and experimental studies are needed to support our current findings and conclusions.

The Aggregation Paths and Products of Aβ42 Dimers Are Distinct from Those of the Aβ42 Monomer.

Extracts of Alzheimer's disease (AD) brain that contain what appear to be sodium dodecyl sulfate-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated the Aβ monomer, consist primarily of Aβ42, and resist denaturation by chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in two ways: by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then used a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by the Aβ monomer. The results suggest that the cross-links disfavor fibril formation from Aβ dimers, thereby enhancing the concentration of soluble aggregates akin to those in aqueous extracts of AD brain. Thus, it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates.

DNA melting properties of the dityrosine cross-linked dimer of Ribonuclease A

Several DNA binding proteins exist in dimeric form when bound with DNA to be able to exhibit various biological processes such as DNA repair, DNA replication and gene expression. Various dimeric forms of Ribonuclease A (RNase A) and other members of the ribonuclease A superfamily are endowed with a multitude of biological activities such as antitumor and antiviral activity. In the present study, we have compared the DNA binding properties between the RNase A monomer and the dityrosine (DT) cross-linked RNase A dimer, and checked the inhibitory effect of DNA on the ribonucleolytic activity of the dimeric protein. An agarose gel based assay shows that like the monomer, the dimer also binds with DNA. The number of nucleotides bound per monomer unit of the dimer is higher than the number of nucleotides that bind with the each monomer. From fluorescence measurements, the association constant (Ka) values for complexation of the monomer and the dimer with ct-DNA are (4.95±0.45)×104M−1 and (1.29±0.05)×106M−1 respectively. Binding constant (Kb) values for the binding of the monomer and the dimer with ct-DNA were determined using UV–vis spectroscopy and were found to be (4.96±1.67)×104M−1 and (4.32±0.31)×105M−1 respectively. Circular dichroism studies shows that the dimer possesses significant effect on DNA conformation. The melting profile for the ct-DNA–dimer indicated that the melting temperature (Tm) for the ct-DNA–dimer complex is lower compared to the ct-DNA–monomer complex. The ribonucleolytic activity of the dimer, like the monomer, diminishes upon binding with DNA.

Lipid-lysine adducts and modified tyrosines as markers of oxidative stress in the second trimester of pregnancy and their association with infant characteristics.

Pregnancy is a physiological state accompanied by excessive levels of oxidative stress (OS), due to the increased demand and utilisation of oxygen. There is increasing evidence that maternally augmented OS exerts an adverse effect on pregnancy outcome. The aim of the present prospective study was to determine the association between the urinary concentration of relatively novel OS markers measured in the second trimester of pregnancy and the infant characteristics at birth. The maternal levels of urinary hexanoyl-lysine (HEL), propanoyl-lysine (PRL), dityrosine (DiY) and 3-nitrotyrosine (NY) were evaluated in generally healthy pregnant subjects to determine their association with birth weight, gestation at delivery and Apgar score. The observed levels of the markers were in agreement with those measured in healthy non-pregnant subjects in a previous study. A positive correlation was detected between HEL and PRL, as well as between HEL and DiY. Although the absence of a correlation between NY and the other markers has been previously noted in a non-pregnant population, a positive correlation in the pair PRL-NY (r=0.367; P<0.001) was observed in the present study. Maternal cigarette smoking was associated with increased urinary PRL levels (P=0.034). The most notable observation in the present study was that high levels of PRL and NY were associated with low Apgar scores at 1 and 5 min after birth (OR, 1.098 and 2.084 for PRL and NY, respectively; P<0.05). However, poor predictive accuracy was shown. For NY, the following results were obtained: Area under the curve (AUC), 0.818; sensitivity, 100%; specificity, 57%; positive predictive value (PPV), 11.54%; and negative predictive value (NPV), 100%. For PLR the values were as follows: AUC, 0.802; sensitivity, 100%; specificity, 62.6%; PPV, 13.05%; and NPV, 100%. DiY was negatively associated with preterm birth risk (OR=0.703; P=0.028). In conclusion, the results of the present study indicated the presence of OS in the second trimester of pregnancy, which was detected with damage to lipids and proteins and associated with an adverse Apgar score; however, the selected urinary markers exhibited poor positive predictive efficacy.

Abstract 14677: Oxidative Damage in Failed Clinical Bioprosthetic Heart Valve Explants

Introduction: Bioprosthetic heart valves (BHVs), fabricated from glutaraldehyde-fixed bovine pericardium or porcine aortic valves, have been widely used in valve replacement surgery. However, BHVs fail over time due to structural degeneration. Despite frequent observations of calcification-associated BHV failure, more than 25% of BHV structural failures occur without calcification. Previously, we reported that oxidative stress may have a pivotal role in noncalcific BHV failure. Moreover, oxidation-induced formation of dityrosine (DT) crosslinks via hydroxyl- and tyrosyl-radical mediated pathways was only detected in clinical BHV explants. Yet the mechanism of DT formation and its role in structural degeneration of clinical BHV is incompletely understood. Hypothesis: Oxidative damage to BHV is due to a specific spatial localization of DT crosslink formation mediated by myeloperoxidase (MPO) and/or iron (Fe) deposition in clinical BHV explants Method &amp; Results: BHVs from 7 human subjects were collected and subjected to mass spectrometry to measure DT in paraffin sections of BHV. 4 different regions of each BHV leaflet (free edge, sewing cushion, commissure, and middle region) were sampled as well as entire cross-sections of each cusp. DT was only detected in the mid–cusp (137.37±49.96 μmol/mol) and was not significantly different from DT quantitated in the cross-sections (191.29±65.98 μmol/mol). DT was not detected in other regions, implying a specific spatial localization of oxidative stress in BHV. MPO, a neutrophil derived enzyme, is responsible for reactive oxygen species (ROS) generation. Immunofluoresence microscopy demonstrated significant presence of MPO in the BHV, implying that MPO may be responsible for the DT formation in BHV. Fe deposition due to intracuspal hematomas and/or Fe-association with calcific deposits can also be responsible for ROS and DT formation. Fe was shown to be present in histology sections in failed BHV by Prussian Blue staining, indicating that Fe deposits may also in part be responsible for the observed DT formation. Conclusion: These results demonstrated a mid-leaflet localization of DT formation in clinically failed BHV explants. This may in part be mediated by MPO and/or Fe-deposits in clinical BHV.

Immunohistochemical localization of bilirubin oxidation in human placenta

Immunohistochemical localization of bilirubin oxidation in human placenta

Response of antioxidant compounds to selenium and salicylic acid in wheat cultivars under dry land conditions

In the present study, three dry land wheat cultivars, Azar 2, Sardary and Rasad, were tested for antioxidant enzyme activity, proline, malondialdehyde (MDA) and dityrosine (DT) content and grain yield after treatment with selenium and salicylic acid (SA). A factorial field experiment was carried out based on a completely randomized block design with three replicates. The experimental factors were three levels of salicylic acid (without SA; seed priming with 0.5 mM SA; seed priming + spraying with 1 mM SA) and two levels of selenium (0 and 20 g/ha). Significant increases in the activity of the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) enzymes and in the proline level were observed after treatment in the leaves of the three genotypes investigated, but this was associated with reduced MDA and DT content. The application of SA as seed priming and the foliar application of Se also increased the grain yield. These results suggest that cultivars exhibiting high antioxidant enzyme activity and proline content under dry land conditions may provide better drought tolerance in wheat.

Synthesis of a fluorescent and star-shaped 4-arm PEG with different functional groups at its ends

Dityrosine (DY), which is fluorescent and biocompatible, has multiple functional groups, including two amine and two carboxylic acid groups. By conjugating PEG arms to DY, we successfully synthesized a star-shaped 4-arm PEG with two carboxylic acid and two amine groups at its four ends. Two different kinds of molecules could be further linked to this 4-arm PEG.The chemical structures and molecular weights of the intermediate 2-arm PEG and 4-arm PEG were determined by 1H NMR spectroscopy and matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS).

Large-scale production of N, N′-diBoc-dityrosine and dityrosine by HRP-catalyzed N-Boc- l-tyrosine oxidation and one-step chromatographic purification

Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis. It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed by multistep chromatographic separations. In this study, we present an alternative method for the preparation of DY by HRP-catalyzed synthesis of N, N′-diBoc-dityrosine (DBDY) from N-Boc- l-tyrosine (BY). The presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products (e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions (for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To prepare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic acid.

Structure and conformational studies on dityrosine formation in the DNA binding domain of RFX5

The DNA binding protein RFX5 is a subunit of RFX complex involved in transcription regulation of MHCII molecules. The RFX complex binds to the X-box DNA through the DNA binding domain of RFX5. We have examined the formation of intramolecular tyrosine cross linking, dityrosine, in RFX5DBD under oxidative stress, through UV irradiation and enzymatic action of H 2O 2/peroxidase by fluorescence spectroscopic studies. Dityrosine (DT) was formed predominantly in alkaline condition showing its intense characteristic fluorescence emission. Homology modeling indicated Y 39 and Y 42 could be the potential tyrosine residues undergoing oxidative cross-linking. Conformational changes in RFX5DBD under oxidative stress were observed by CD measurements. The in vitro association of X-box DNA with RFX5DBD increased DT fluorescence significantly and protected RFX5DBD from UV irradiation as observed in SDS-PAGE followed by mass spectrometric analysis. Results indicate cross protection in both RFX5DBD and DNA under oxidative stress playing important role in protein modification.

Quantification of Modified Tyrosines in Healthy and Diabetic Human Urine using Liquid Chromatography/Tandem Mass Spectrometry

The quantification of urinary oxidized tyrosines, dityrosine (DiY), nitrotyrosine (NY), bromotyrosine (BrY), and dibromotyrosine (DiBrY), was accomplished by quadruple liquid chromatography-tandem mass spectrometry (LC/MS/MS). The sample was partially purified by solid phase extraction, and was then applied to the LC/MS/MS using multiple-reaction monitoring (MRM) methods. The analysis for the DiY quantification was done first. The residual samples were further butylated with n-butanol/HCl, and the other modified tyrosines were then quantified with isotopic dilution methods. MRM peaks of the modified tyrosines (DiY, NY, BrY, and DiBrY) from human urine were measured and the elution times coincided with the authentic and isotopic standards. The amounts of modified tyrosines in healthy human urine (n = 23) were 8.8 ± 0.6 (DiY), 1.4 ± 0.4 (NY), 3.8 ± 0.3 (BrY), and 0.7 ± 0.1 (DiBrY) µmol/mol of creatinine, respectively. A comparison of the modified tyrosines with urinary 8-oxo-deoxyguanosine, pentosidine, and Nε-(hexanoyl)lysine was also performed. Almost all products, except for NY, showed good correlations with each other. The amounts of the modified tyrosines (NY, BrY, and DiBrY) in the diabetic urine were higher than those in the urine from healthy people.

Immunohistochemical Detection of Oxidative Stress Biomarkers, Dityrosine and N&amp;sup.EPSILON.;-(hexanoyl)lysine, and C-Reactive Protein in Rabbit Atherosclerotic Lesions

Several lines of evidence have demonstrated that C-reactive protein (CRP) is associated with oxidative stress; however, the precise co-localization between CRP and oxidative stress markers in atherosclerotic lesions is not fully established. In this study, we focused on two oxidative stress markers, dityrosine (DY) and N(epsilon)-(hexanoyl)lysine (HEL), which had not previously been investigated in relation to CRP in atherosclerotic lesions. We investigated the production and localization of DY, HEL, and CRP in early-stage and moderately progressed fatty lesions of cholesterol-fed rabbits by immunohistochemistry using specific monoclonal antibodies to examine the co-localization between CRP and oxidative stress in atherosclerotic lesions. Rabbit atherosclerotic specimens were obtained from New Zealand White rabbits fed a diet containing 1.0% cholesterol for 12 weeks. All specimens were fixed in formalin for histological examinations. CRP-positive cells in rabbit early-stage and moderately progressed fatty lesions were detected mostly in the macrophage-derived foam cell-rich areas. Both DY and HEL were also detected in foam cell-rich areas in both lesions, where they were primarily co-localized with CRP-positive cells. Our results suggest that the generation of oxidative stress markers, DY and HEL, may be mediated by CRP in atherosclerotic lesions, and that CRP may be associated with oxidative stress in rabbit atherosclerotic lesions.

Metal catalyzed oxidation of tyrosine residues by different oxidation systems of copper/hydrogen peroxide

Metal-catalysed oxidation (MCO) reactions result in the formation of reactive oxygen species (ROS) in biological systems. These ROS cause oxidative stress that contributes to a number of pathological processes leading to a variety of diseases. Tyrosine is one residue that is very susceptible to oxidative modification and the formation of dityrosine (DT) and 3,4-dihydroxyphenylalanine (DOPA) have been widely reported in a number of diseases. However, the mechanisms of MCO of tyrosine in biological systems are poorly understood and require further investigation. In this study we investigated the mechanism of DT and DOPA formation by MCO using N-acetyl tyrosine ethyl ester as a model for tyrosine in proteins and peptides. The results showed that DT formation could be observed upon Cu2+/H2O2 oxidation at pH 7.4. Our results indicate that it is unlikely to be via Fenton chemistry since Cu+/H2O2 oxidative conditions did not lead to the formation of DT.

Increase in oxidative stress in kidneys of diabetic Akita mice.

New biomarkers for oxidative damage, were used to identify whether hyperglycemia caused oxidative stress in diabetic Akita mice. At 13 weeks of age, the tissues of these mice were obtained, and the levels of N(epsilon)-(hexanonyl)lysine (HEL) and dityrosine (DT) were measured, these being related to lipid peroxide-derived protein covalent modification and protein cross-linking. The levels of HEL and DT in the kidneys of Akita mice were significantly increased compared with the control mice without any accumulation of thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified protein. Immunopositive staining was clearly observed in the kidneys of the Akita mice when using the anti-HEL antibody or anti-DT antibody. These results suggest that hyperglycemia in Akita mice induced oxidative stress and increased these markers in the kidneys.