Extract from <Emphasis Type="Italic">Conyza canadensis</Emphasis> as a modulator of plasma protein oxidation induced by peroxynitrite <Emphasis Type="Italic">in vitro</Emphasis>

The antioxidative activity of the extract from Conyza canadensis in plasma treated with peroxynitrite (ONOO⁻) (0.1 mM) was studied. C. canadensis is known to possess a broad set of pharmacological effects because of content of various antioxidants, antiplatelet and anticoagulant compounds. The aim of our study was to assess if this extract protects plasma proteins against oxidative/nitrative damages induced by ONOO⁻. The plasma components are continuously exposed to reactive oxygen/nitrogen species action. Peroxynitrite evokes oxidative stress and induces undesirable effects in biological systems and causes damage to biomolecules. The extract from Conyza (50–2500 mg/ml) caused a dose-dependent reduction of protein nitration by 90%. The oxidation of plasma proteins was diminished by about 75%. ONOO⁻ oxidized the plasma thiol groups and this process was inhibited by tested extract. The level of reduced protein thiols was increased thrice at the lowest concentration of extract (50 mg/ml). The highest concentration of extract decreased twice the level of protein thiols in reduced forms and increased the homocysteine level about 4.5 times. The obtained results demonstrated that the extract from Conyza possesses antioxidative properties in vitro, protects plasma proteins against toxicity induced by peroxynitrite and has modulating effects on thiol/disulfide redox status.     

Mechanisms for the formation of protein-bound homocysteine in human plasma

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Greater than 70% of homocysteine in circulation is protein-bound. An in vitro model system using human plasma has been developed to study mechanisms of protein-bound homocysteine formation and establish the equilibrium binding capacities of plasma for homocysteine. Addition of homocysteine to plasma caused an initial rapid displacement of cysteine and a subsequent increase in protein-bound homocysteine. This rapid reaction was followed by a slower oxygen-dependent reaction forming additional protein-bound homocysteine. To determine the equilibrium binding capacity of plasma proteins for homocysteine, plasma was treated with 0.5-10 mM dl-homocysteine for 4 h at 37 degrees C under aerobic conditions. Under these conditions the equilibrium binding capacity was 4.88 +/- 0.51 and 4.74 +/- 0.68 micromol/g protein for male (n = 10) and female (n = 10) donors, respectively. The mechanism of protein-bound homocysteine formation involves both thiol-disulfide exchange and thiol oxidation reactions. We conclude that plasma proteins have a high capacity for binding homocysteine in vitro.     

Plasma total homocysteine level and methylenetetrahydrofolate reductase 677C>T genetic polymorphism in Japanese patients with rheumatoid arthritis

Hyperhomocysteinemia is a known risk factor of cardiovascular disease. Homocysteine has been also linked to inflammation in rheumatoid arthritis (RA). In this study, we investigated the relationship between plasma homocysteine levels and single nucleotide polymorphism (SNP) of the gene coding for methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the biosynthesis of homocysteine, and the correlation between the plasma homocysteine levels and generally used inflammatory markers (C-reactive protein, erythrocyte sedimentation rate and matrix metalloproteinase-3) in 96 Japanese patients with RA. Plasma homocysteine levels in patients with the MTHFR 677TT genotype were significantly higher than in those with the 677CC genotype (p < 0.05). In addition, plasma homocysteine levels were increased along with the elevation of general inflammatory markers. Therefore, we conclude that homocysteine might affect the inflammatory status of patients, and the MTHFR 677C>T SNP could be a predictive factor of hyperhomocysteinemia in patients with RA.     

Determination of reduced and oxidized homocysteine and related thiols in plasma by thiol-specific pre-column derivatization and capillary electrophoresis with laser-induced fluorescence detection

A new sensitive and rapid capillary electrophoresis (CE) assay for measuring reduced and oxidized thiols in human plasma has been developed. To prevent oxidation of the thiols, whole blood was immediately centrifuged after collection and the plasma proteins were precipitated with perchloric acid. The reduced thiols in the supernatant were derivatized quantitatively at 25°C, pH 7.5 with a fluorescent reagent, fluorescein-5-maleimide (FM). The total plasma concentration of thiols, including the fraction coupled to proteins, was assayed after an initial reduction of the disulfide linkage in plasma with dithiothreitol. The separation of FM-thiols was performed in an acetonitrile/10 mM sodium phosphate–50 mM SDS buffer [25:75 (v/v); pH 7.0] using a fused-silica capillary (57 cm×75 μm I.D.) at 45°C. A 3-mW argon-ion laser (λ_ex 488 nm/λ_em 520 nm) was employed for FM-thiol detection. With the electric field of 530 V/cm, the time needed for the separation of FM-homocysteine, FM-glutathione and FM-N-acetylcysteine was less than 8 min. The lower limit of detection was 3 μM for the total thiols and 10 nM for the reduced thiols. The method was applied to the determination of homocysteine levels in plasma from patients with end-stage renal disease.     

Correlation between maternal plasma homocysteine and zinc levels in preeclamptic women

Women with preeclampsia have been shown to have elevated blood levels of the metabolite homocysteine, and alterations in blood levels of zinc and copper have also been reported. This study measured plasma levels of zinc, copper, and homocysteine in women with preeclampsia and in women with healthy, normotensive pregnancies. For the patients with preeclampsia compared with controls, significantly higher mean plasma levels were found of homocysteine (16.39 vs 9.45 nmol/mL; p≤0.001), zinc (15.53 vs 11.93 μg/g protein; p < 0.05), and copper (47.90 vs 31.60 μg/g protein; p=0.001). The ratio of plasma Cu/Zn levels tended to be higher in preeclamptic women and could be taken as an index of inflammatory reaction, but the difference was not significant. Homocysteine concentrations correlated positively with plasma zinc concentrations in women with preeclampsia (r=0.588, p=0.003) but not in women with healthy pregnancies. No correlations were observed between plasma levels of homocysteine and copper. Thus, the present study found evidence that preeclampsia might be associated with hyperhomocysteinemia and elevated blood levels of zinc and copper. Furthermore, elevated blood levels of zinc were significantly associated with hyperhomocysteinemia in preeclampsia. More studies are warranted to investigate further any relationship between altered homocysteine metabolism and levels of zinc and copper in preeclampsia.     

A method for measurement of free and bound plasma cyst(e)ine

A method for the measurement of free and bound cyst(e)ine and the sum thereof has been developed. In adult human blood, cyst(e)ine is distributed equally between that which is bound to plasma proteins and that which is free. Cyst(e)ine is bound predominantly to albumin, and this binding is not an in vitro artifact. Cysteine bound to plasma proteins may be displaced by homocysteine which competes for the available sulfhydryl groups of plasma proteins. Rats starved for 8 days had a significant decrease in both plasma free cyst(e)ine and bound cysteine. These data suggest that present methods for the determination of plasma cyst(e)ine under-estimate the quantity of cyst(e)ine in the plasma available for cellular metabolism.     

Gas chromatographic–mass spectrometric determination of total homocysteine in human plasma by stable isotope dilution: method and clinical applications

The detection and quantitation of slight increases of plasma homocysteine levels is of growing interest. This has prompted us to develop a highly sensitive and accurate capillary gas chromatography–mass spectrometry (GC–MS) method. The method proved to be highly sensitive (DL=0.17 μmol/l) with between- and within-run precision less than 6% and 7%, respectively. Reference values of plasma total homocysteine have been determined for men (n=39) and women (n=36), showing a significant difference (P=0.003) between gender. Preliminary results in cerebrovascular accidents and in venous thrombosis are presented.     

Simultaneous detection of N-acetyl-L-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis

N-acetyl-l-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.     

The Oxidative Stress May be Induced by the Elevated Homocysteine in Schizophrenic Patients

The mechanisms of oxidative stress in schizophrenic patients are not fully understood. In the present study, we investigated the effect of elevated level of homocysteine (Hcys) on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in plasma, the level of carbonyl groups in plasma proteins, as well as the amount of 3-nitrotyrosine in plasma proteins isolated from schizophrenic patients. Patients hospitalised in I and II Psychiatric Department of Medical University in Lodz, Poland were interviewed with special questionnaire (treatment, course of diseases, dyskinesis and other EPS). According to DSM-IV criteria all patients had diagnosis of paranoid type. They were treated with antipsychotic drugs (clozapine, risperidone, olanzapine). Mean time of schizophrenia duration was about 5 years. High-performance liquid chromatography was used to analyse the total level of homocysteine in plasma. Levels of carbonyl groups and 3-nitrotyrosine residues in plasma proteins were measured by ELISA and a competition ELISA, respectively. The lipid peroxidation in plasma was measured by the level of TBARS. Our results showed that in schizophrenic patients the amount of homocysteine in plasma was higher in comparison with the control group. We also observed a statistically increased level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine in plasma proteins from schizophrenic patients. Moreover, our experiments indicate that the correlation between the increased amount of homocysteine and the oxidative stress exists. Considering the data presented in this study, we suggest that the elevated Hcys in schizophrenic patients may stimulate the oxidative stress.     

Maternal antioxidant concentrations after uncomplicated pregnancies

Background: To analyse the post-partum concentrations of intra- and extra-cellular blood antioxidants in women with uncomplicated pregnancies.

Methods: Whole blood and plasma thiols, plasma vitamin E and C, serum cholesterol and triglyceride, ferric reducing ability of plasma (FRAP) concentrations were compared between women delivered by caesarean section (n=17) or spontaneous delivery (n=10). A repeated mixed model was used for statistical analysis.

Results: The majority of whole blood thiols increased significantly in both groups the first days post-partum. However, within the caesarean group free cysteine, oxidised cysteine, homocysteine and glutathione and plasma cysteine and homocysteine levels dropped significantly after 24?h, while FRAP levels peaked significantly in this group. Plasma vitamin E levels decreased significantly in both groups within 24 to 48?h after delivery. Independent of the way of delivery whole blood and plasma thiols were significantly increased and vitamin E levels were significantly decreased 3?months post-partum while plasma vitamin C levels and FRAP were unchanged compared to ante-partum levels.

Discussion: Decreased plasma vitamin E levels shortly post-partum are associated with decreased lipid peroxidation. The 24?h post-partum drop of some plasma and whole blood thiols in the caesarean group may be due to prolonged fasting.     

Oxidized modification of fragments <Emphasis Type="Italic">D</Emphasis> and <Emphasis Type="Italic">E</Emphasis> from fibrinogen induced by ozone

Ozone-induced free-radical oxidation of fragments D and E from fibrinogen has been studied. The methods of elastic and dynamic light scattering in combination with electrophoresis of unreduced samples have shown the acceleration of enzymatic covalent crosslinking of molecules of oxidation-modified fragment D under the action of factor XIIIa. UV and IR spectroscopy shows that free-radical oxidation of amino acid residues of polypeptide chains catalyzed by ozone affects the cyclic and amino groups, giving rise to generation of mainly oxygen-containing products. Comparison of the IR spectra obtained for the oxidation-modified D and E fragments revealed more significant transformation of functional groups for the D fragment. EPR spectroscopy showed that the rotational correlation time of spin labels bound to the ozonized proteins decreased in comparison with the non-ozonized proteins. The rotation correlation time of the radicals covalently bound to the ozonized D and E fragments suggests that D fragment of fibrinogen is more sensitive to free-radical oxidation followed by local structural changes. Possible causes of different degrees of oxidation for fragments D and E are discussed.     

Predicting protein homocysteinylation targets based on dihedral strain energy and pKa of cysteines

A multitude of complex diseases have been linked to elevated homocysteine levels; however, till date there is no plausible explanation for a single amino acid's involvement in so many diseases. Since homocysteine is a reactive thiol amino acid and the majority of plasma homocysteine is protein thiol bound, we hypothesized that homocysteine might bind to accessible cysteine residues in target proteins, thereby modulating its structure or function or both. The parameters that dictate homocysteine-protein interaction are not well understood, and the few known homocysteine binding proteins were identified by a candidate protein approach. In this study, we identified potential homocysteine interacting proteins based on cysteine content, solvent accessibility of cysteine residues, and dihedral strain energies and pKa of these cysteines. Pathway mapping of the cysteine-rich proteins revealed that proteins in the coagulation cascade, notch receptor-mediated signaling, LDL endocytosis, programmed cell death, and extracellular matrix proteins were significantly over-represented with cysteine-rich proteins, and we believe that homocysteine has a high probability to bind to proteins in these pathways. In fact, several clinical studies have implicated high homocysteine levels to be associated with diseases like thrombosis, neural tube defects, and so forth, which result from dysfunction of one or more of the proteins identified in our study. Further, we successfully validated our prediction parameters on the proteins that have already been experimentally shown to bind homocysteine, and our structural analysis argues a plausible explanation for these prior reported protein interactions with homocysteine that could not be previously explained.     

Daily Variations of Homocysteine Concentration May Influence Methylation of DNA in Normal Healthy Individuals

The regulation of genetic expression is tightly controlled and well balanced in the organism by different epigenetic mechanisms such as DNA methylation and histone modifications. DNA methylation occurring after embryogenesis is seen mainly as an irreversible event. Even small changes in genomic DNA methylation might be of biological relevance, and several factors influencing DNA methylation have been identified so far, one being homocysteine. In this study, genomic DNA methylation was analyzed and homocysteine plasma levels were measured over a 24 h period in 30 healthy students (15 males and 15 females) exposed to a standard 24 h regime of daytime activity alternating with nighttime sleep. Plasma homocysteine concentrations were measured using HPLC detection. DNA was extracted from whole EDTA blood, and genomic DNA methylation was assessed by fluorescently labeled cytosine extension assay. Both homocysteine and DNA methylation showed 24 h variation. Homocysteine showed a significant daily rhythm with an evening peak and nocturnal nadir in all subjects (p<0.001). Males showed higher overall homocysteine levels compared to females (p=0.002). Genomic DNA methylation showed a significant rhythm with increased levels at night (p=0.021), which was inverse to plasma homocysteine levels.     

Relation of Plasma Homocysteine to Plasma Amyloid Beta Levels

Background Elevated plasma homocysteine and amyloid β (Aβ) have been associated with Alzheimer’s disease (AD). We investigated the cross-sectional association between these biomarkers. Methods We used linear regression to relate plasma homocysteine and Aβ adjusting for age, gender, creatinine, APOE-ε4, and ethnic group in 327 persons aged 78 ± 6.6 years. Results Plasma homocysteine correlated with age, serum creatinine, plasma Aβ40 and Aβ42, and was inversely correlated with serum vitamin B12, and folate. Aβ42, but not Aβ40, was related to later development of dementia. Homocysteine was related to higher Aβ40 levels (coefficient = 2.0; P < 0.0001) and this association was attenuated after adjustment for creatinine (coefficient = 1.0; P < 0.0001). The crude association between homocysteine and Aβ42 was weaker (coefficient = 0.5; P = 0.01) and became non-significant after adjustment for creatinine (coefficient = 0.4; P = 0.06). These associations were unrelated to ethnicity, the presence of APOE-ε4 or dementia. Analyses by quartiles of homocysteine showed that these association were driven primarily by the fourth quartile. Conclusions Plasma homocysteine is directly related to Aβ40. The association with Aβ42 is not significant. These results seem to indicate that homocysteine is related to aging but not specifically to AD. Special issue dedicated to John P. Blass.     

γ-Glutamyl semialdehyde and 2-amino-adipic semialdehyde: biomarkers of oxidative damage to proteins

Reactive oxygen species are formed in the body by several natural processes and by induced oxidative stress. The reactive oxygen species may react with the various biomolecules of the body, including proteins. In order to assess the impact of oxidative damage to proteins, we have tried to identify oxidized amino acids in blood proteins which might serve as biomarkers of oxidative damage. When oxidative damage is induced into bovine serum albumin by metal-catalysed oxidation systems, the aldehyde groups formed can be derivatized by fluoresceinamine (FINH₂). Following acid hydrolysis of FINH₂-derivatized protein, two major oxidation products, γ-glutamyl semialdehyde (GGS) and 2-amino-adipic semialdehyde (AAS), were found and identified by HPLC and MS. Isolation and identification of oxidized amino acids from homopolymers (poly-Arg,-Pro,-Lys,-Trp or -Leu) confirmed that GGS can originate from Arg or Pro, while AAS is an oxidation product of Lys. When oxidative stress was induced in rats by treatments with t-butyl hydroperoxide or acrolein, rat plasma protein levels of GGS and AAS were found to be significantly higher compared with control rats. The AAS-content in serum albumin or in total plasma proteins collected from eight different mammalian species was found to be inversely proportional to their maximum lifespan potential. The content of AAS in plasma proteins of untreated adult rats showed a positive correlation with the age of the rat. In young rats a negative correlation with age was found for both GGS and AAS. We conclude that GGS or AAS may be useful novel biomarkers of oxidative damage to proteins in vivo.     

Synthesis and characterization of fluorinated homocysteine derivatives as potential molecular probes for ¹⁹F magnetic resonance spectroscopy and imaging

A N-trifluoroacetyl-protected amino acid containing a thioester function, 2,2,2-trifluoro-N-(2-oxo-tetrahydrothiophen-3-yl)acetamide (TFA-tHcy), has been synthesized and characterized. It was then used to prepare a fluorine-labeled N-homocysteinylated protein, ¹⁹F-Hcy-εN-Lys-albumin, that was characterized by SDS-PAGE, MALDI-TOF-MS, UV-vis and ¹⁹F NMR spectroscopy. On average, four N-trifluoroacetylhomocysteine residues were covalently conjugated to human serum albumin through the N-substituted homocysteine thiolactone. The in situ homocysteinylation of human plasma proteins with TFA-tHcy has also been performed and has led to the formation of N-homocysteinylated proteins, with albumin modification accounting for ca. 75% of all fluorine-labeled human plasma proteins. The synthesized fluorinated molecular probes can be potentially used as informative molecular probes for in vivo ¹⁹F magnetic resonance spectroscopy and imaging.     

New iodo‐acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido‐redox status

Two new iodoacetamide‐substituted cyanines, C3NIASO3 and C5NIASO3, were synthesized starting from hemicyanine and were utilized for labeling plasma proteins. Specificity, sensitivity and feasibility for SH residues was tested utilizing an equimolar mixture of standard proteins and with normal plasma. Oxidized plasma proteins following H₂O₂ exposure and plasma from patients with focal glomerulosclerosis were analyzed as models of altered protein oxido‐redox status. Following optimization of the assay (dye/protein ratio, pH), C3NIASO3 and C5NIASO3 gave a sensitivity slightly better than N‐hydroxysuccinimidyl dyes for plasma proteins and were successfully employed for differential display electrophoresis (DIGE). Twenty‐nine proteins were detected in normal plasma after 2‐DE while less proteins were detected in plasma of patients with glomerulosclerosis. Following massive ‘in vitro' oxidation with H₂O₂, C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis of two new iodoacetamide cyanines that can be utilized for the analysis of plasma proteins with 2‐DE and DIGE. They are also indicated for the definition of the oxido‐redox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.     

S-homocysteinylation of transthyretin is detected in plasma and serum of humans with different types of hyperhomocysteinemia

While the association of homocystinuria with disease is known for more than four decades, mild hyperhomocysteinemia has been detected more recently as a risk factor for a number of diseases. However, the mechanism which apparently renders (even mild) hyperhomocystenemia harmful is not known. Following reports on N-homocysteinylation of proteins by the homocysteine derivative homocysteine thiolactone, it has been suggested that homocysteinylation of proteins may contribute to the induction of biological effects by homocysteine. This has prompted us to study by electrospray ionization mass spectrometry homocysteinylation of transthyretin (TTR) in plasma and serum of humans with different types of hyperhomocysteinemia. We did not detect any N-homocysteinylation, but found pronounced S-homocysteinylation of TTR, if the concentration of total homocysteine was high. Our findings support a possible role of S-homocysteinylation of proteins in the mediation of detrimental effects of hyperhomocysteinemia. Careful study of posttranslational modifications of individual proteins may contribute to a better understanding of diseases associated with hyperhomocysteinemia.     

Plasma levels of total, free and protein bound thiols as well as sulfane sulfur in different age groups of rats

The redox status of plasma thiols can be a diagnostic indicator of different pathological states. The aim of this study was to identify the age dependent changes in the plasma levels of total, free and protein bound glutathione, cysteine and homocysteine. The determination was conducted in plasma of three groups of rats: 1) young (3-month-old), 2) middle aged (19-month-old), and 3) old (31-month-old). Total levels of glutathione, cysteine and homocysteine and their respective free and protein-bound fractions decreased with age. The only exception was a rise in free homocysteine concentration in the middle group, which indicates a different pattern of transformations of this thiol in plasma. The drop in the level of protein-bound thiols suggests that the antioxidant capacity of plasma diminishes with age, which, consequently, leads to impaired protection of -SH groups through irreversible oxidation. The plasma sulfane sulfur level also declines with age, which means that aging is accompanied by inhibition of anaerobic sulfur metabolism.