A method for the determination of total and reduced methimazole in various biological samples.

A simple, rapid, reproducible and sensitive method based on HPLC with ultraviolet detection was developed for the determination of methimazole (MMI) in animal tissues and plasma samples. Under the optimum experimental conditions, the calibration curves for MMI were linear in the tested range 0.5-20 mg kg(-1) tissue sample (mg l(-1) plasma) with correlation coefficients better than 0.99. The performance of the proposed method was tested for the determination of MMI levels in brain, liver, thyroid gland and plasma of MMI-treated hens, as well as in their eggs and embryos. The proposed method reduces time and simplifies the sample preparation procedure.

Maternal transfer of methimazole and effects on thyroid hormone availability in embryonic tissues.

Methimazole (MMI) is an anti-thyroid drug used in the treatment of chronic hyperthyroidism. There is, however, some debate about its use during pregnancy as MMI is known to cross the mammalian placenta and reach the developing foetus. A similar problem occurs in birds, where MMI is deposited in the egg and taken up by the developing embryo. To investigate whether maternally derived MMI can have detrimental effects on embryonic development, we treated laying hens with MMI (0.03% in drinking water) and measured total and reduced MMI contents in the tissues of hens and embryos at different stages of development. In hens, MMI was selectively increased in the thyroid gland, while its levels in the liver and especially brain remained relatively low. Long-term MMI treatment induced a pronounced goitre with a decrease in thyroxine (T4) content but an increase in thyroidal 3,5,3'-triiodothyronine (T3) content. This resulted in normal T3 levels in tissues except in the brain. In chicken embryos, MMI levels were similar in the liver and brain. They gradually decreased during development but always remained above those in the corresponding maternal tissues. Contrary to the situation in hens, T4 availability was only moderately affected in embryos. Peripheral T3 levels were reduced in 14-day-old embryos but normal in 18-day-old embryos, while brain T3 content was decreased at all embryonic stages tested. We conclude that all embryonic tissues are exposed to relatively high doses of MMI and its oxidised metabolites. The effect of maternal MMI treatment on embryonic thyroid hormone availability is most pronounced for brain T3 content, which is reduced throughout the embryonic development period.

Treatment of chronic hemodialysis patients with low-dose fenofibrate effectively reduces plasma lipids and affects plasma redox status.

Dyslipidemia is common in chronic hemodialysis patients and its underlying mechanism is complex. Hemodialysis causes an imbalance between antioxidants and production of reactive oxygen species, which induces the oxidative stress and thereby may lead to accelerated atherosclerosis. Statins have been found to be little effective in end-stage kidney disease and other lipid-lowering therapies have been only scarcely studied. The study aimed to assess the effect of low-dose fenofibrate therapy on plasma lipids and redox status in long-term hemodialysis patients with mild hypertriglyceridemia.Twenty seven chronic hemodialysis patients without any lipid-lowering therapy were included in a double-blind crossover, placebo-controlled study. The patients were randomized into two groups and were given a sequence of either 100 mg of fenofibrate per each hemodialysis day for 4 weeks or placebo with a week-long wash-out period between treatment periods. Plasma lipids, high sensitive C-reactive protein (CRP), urea, creatinine, electrolytes, phosphocreatine kinase (CK), GOT, GPT and plasma thiols (total and free glutathione, homocysteine, cysteine and cysteinylglycine) were measured at baseline and after each of the study periods. Plasma aminothiols were measured by reversed phase HPLC with thiol derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate.Fenofibrate therapy caused a significant decrease of total serum cholesterol, LDL cholesterol and triglycerides and an increase of HDL cholesterol. The treatment was well tolerated with no side-effects but there was a small but significant increase of CK not exceeding the upper limit of normal range. There were no changes of serum CRP, potassium, urea, and creatinine and liver enzymes during the treatment. Neither total nor total free cysteinylglycine and cysteine changed during the study but both total and free glutathione increased during the therapy with fenofibrate and the same was observed in case of plasma homocysteine.The study shows that a treatment with reduced fenofibrate dose is safe and effective in reducing serum triglycerides and cholesterol in chronic dialysis patients and may shift plasma aminothiol balance towards a more antioxidative pattern.

Fast analysis of wine for total homocysteine content by high-performance liquid chromatography.

Alimentary methionine is believed to be the main source for plasma homocysteine. Recent literature supplies information about homocysteine content in daily food components, but not in wine, an attractive complement of the evening meal in some western countries. In this communication, a simple and fast high-performance liquid chromatography method for determination of total homocysteine in wine is described. The two steps procedure relies on reduction of the disulfide forms of homocysteine with tris-(2-carboxyethyl)phosphine and on-column derivatization with o-phthaldialdehyde followed by separation and fluorescence detection. The entire analysis time, including sample work-up, amounts 14 min. The calibration performed with wine matrix, spiked with homocystine within the practical concentration range, proved linear response of the detector. The proposed method was applied for the analysis of 32 different types of wines for total homocysteine. The average concentration of the analyte was 10.31 (±4.25) µM and 6.11 (±3.44) µM for red (n = 23) and white (n = 9) wines, respectively.

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.

Effects of the commercial extract of aronia on oxidative stress in blood platelets isolated from breast cancer patients after the surgery and various phases of the chemotherapy.

Since the extract from berries of Aronia melanocarpa presents antioxidative properties in plasma and in blood platelets, not only from healthy group, but also from patients with benign breast diseases and in patients with invasive breast cancer before surgery, the aim of our present study was to evaluate the oxidative stress by measuring the level of various biomarkers of this process such as the generation of superoxide anion radicals (O(2)(-·)), the amount of carbonyl groups and 3-nitrotyrosine in proteins or the amount of glutathione in blood platelets isolated from breast cancer patients after the surgery and after various phases of the chemotherapy in the presence of A. melanocarpa extract (Aronox) in vitro. We demonstrated in platelet proteins from patients with invasive breast cancer (after the surgery and after various phases of the chemotherapy) higher level of carbonyl groups than in control healthy group. The level of 3-nitrotyrosine in platelet proteins from patients with invasive breast cancer was also significantly higher than in healthy subject group. We observed an increase of other biomarkers of oxidative stress such as O(2)(-·) and a decrease of GSH in platelets from patients with breast cancer (after the surgery and after various phases of the chemotherapy) compared to the healthy group. In model system in vitro our results showed that the commercial extract from berries of A. melanocarpa due to antioxidant action, significantly reduced the oxidative/nitrative stress in platelets from patients with invasive breast cancer caused by the surgery and various phases of the chemotherapy.

The elevated homocysteine stimulates changes of haemostatic function of plasma isolated from breast cancer patients.

The aim of our study was to explain the effect of elevated homocysteine (measured by HPLC) on haemostatic activity of plasma from breast cancer patients (fibrin polymerization and lysis; the thrombin and prothrombin time), because homocysteine (Hcys) induces changes in haemostasis, as well blood clotting as fibrinolysis. Patients were hospitalized in Department of Oncological Surgery, Medical University of Lodz, Poland. All patients have not had preadjuvant therapy, and samples from patients were taken before surgery. We observed that changes of selected parameters of haemostatic properties of plasma, e.g., the prothrombin time and thrombin time were prolonged in plasma from invasive breast cancer when compared with the control group (healthy subjects) and patients with benign breast diseases. Our results showed also that the correlation between the increased amount of Hcys and changes of selected parameters of haemostasis in invasive breast cancer patients exists. Considering the data presented in this study, we suggest that the elevated Hcys in invasive breast cancer patients may induce the changes of haemostatic properties of plasma isolated from these patients.

An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine.

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with ε-amino group of a protein lysine residue generating N-Hcy-protein. This reaction impairs and alters protein's function and has been implicated in atherothrombotic disease. Here, we describe new high-performance liquid chromatography assays for the determination of Hcy-thiolactone, protein N-linked Hcy, and Hcy based on an on-column derivatization with o-phthaldialdehyde and fluorescence detection. The on-column derivatization generates narrow peaks, which allows fast run times (3-5 min) and facilitates determination of N-linked Hcy directly from acid hydrolysates of plasma protein. Utility of these assays was demonstrated with human urine and plasma samples.

Identification and origin of Nε-homocysteinyl-lysine isopeptide in humans and mice.

Homocysteine (Hcy) metabolites, Hcy-thiolactone and N-Hcy-proteins, have been linked to the pathology of human cardiovascular and neurodegenerative diseases. Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. N-Hcy-protein, in which Hcy is linked via isopeptide bond to ε-amino group of a protein lysine residue, forms in a post-translational reaction of Hcy-thiolactone with proteins. Here, we identify a novel metabolite, Nε-Hcy-Lys, in human and mouse plasma, and show that this metabolite is elevated in genetic (cystathionine ß-synthase deficiency in humans and mice, methylenetetrahydrofolate reductase deficiency in mice) or dietary (high Met diet in mice) deficiencies in Hcy metabolism. We also show that Nε-Hcy-Lys is generated by proteolytic degradation of N-Hcy-protein in mouse liver extracts. Our data indicate that free Nε-Hcy-Lys is an important pathology-related component of Hcy metabolism in humans and mice.

On-column derivatization with o-phthaldialdehyde for fast determination of homocysteine in human urine.

A new assay for urinary homocysteine is described. The assay relies on an on-column derivatization with o-phthaldialdehyde, using a reversed-phase HPLC column and detection/quantification by fluorescence. The analysis time for reduced and total homocysteine, including sample work-up, was 5 and 13 min, respectively. Quantification limit was 25 nM.

L-carnitine modulates blood platelet oxidative stress.

The oxidative stress induced by acute exertion may interfere with blood platelet activation. The beneficial effect of L-carnitine (gamma-trimethylamino-beta-hydroxybutyric acid) on oxidative stress in blood platelets has not been fully investigated; however, different studies indicate that this compound modulates platelet functions. The aim of our study was to assess the effects of L-carnitine on platelet activation and oxidative/nitrative protein damage (determined by the levels of protein carbonyl groups, thiol groups, and 3-nitrotyrosine residues) in resting blood platelets or platelets treated with peroxynitrite (ONOO(-), a strong physiological oxidant) in vitro. We also investigated the effects of L-carnitine on the level of platelet glutathione and on the formation of superoxide anion radicals O2(-*), lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS) in blood platelets stimulated by thrombin (a strong physiological agonist), and platelet aggregation induced by adenosine diphosphate (a strong physiological stimulator). We have observed that carnitine decreases platelet activation (measured by platelet aggregation, the generation of O2(-*), and TBARS production). Moreover, our results in vitro demonstrate that carnitine may protect against oxidation of thiol groups induced by ONOO(-). Thus, carnitine may have some protectory effects against oxidative changes induced in blood platelets.

Analysis of orange juice for total cysteine and glutathione content by CZE with UV-absorption detection.

For total content of cysteine (Cys) and glutathione (GSH) a sample (500 microL) of neat orange juice or commercially available soft drinks is treated according to the procedure consisted of four essential steps: reduction of disulfide forms of the analytes to their thiol counterparts with Tris 2-carboxyethylphosphine, derivatization of thiols to their S-quinolinium derivatives with 2-chloro-1-methylquinolinium tetrafluroborate, separation of so-formed derivatives by capillary zone electrophoresis with ACN stacking, and detection and quantitation with the use of ultraviolet detector at 355 nm. Commercially available bare fused-silica capillary served as the CE column. The method is linear in a wide range of concentrations covering practical application. The coefficient of correlation between analyte concentrations within the range from 2.5 to 30.0 micromol/L and the detector response was for Cys and GSH 0.998 and 0.997, respectively. The imprecision for above-mentioned range was between 6.7 and 3.8, and 3.6 and 1.1% RSD for Cys and GSH, respectively. The method was applied to several samples of fresh juices and commercial orange beverages.

[Homocysteine and glutathione metabolism in steroid-treated relapse of idiopathic nephrotic syndrome]. / Metabolizm homocysteiny i glutationu w zaostrzeniu idiopatycznego zespolu nerczycowego leczonym glikokortykosteroidami.

UNLABELLED: Changes in metabolism of aminothiols may have an influence on endothelial function or change the red-ox balance. The aim of study was designed to assess changes in plasma aminothiols': proatherogenic (homocysteine-HCY) and antiatherogenic (glutathione-GSH) metabolism in nephrotic syndrome in children. MATERIAL AND METHODS: The study group included 77 nephrotic children (aged 2-18 years) divided into four groups, i.e. in acute phase of the disease (24), during steroid-induced (24), steroid-free (12) and in long-term remission (17). Twenty five healthy children served as controls. GSH and HCY in plasma were assessed by high-performance liquid chromatography. Fraction of protein-bound and free aminothiols was assessed and albumin saturation was calculated. RESULTS: GSH and its fractions' concentrations were comparable to healthy subject, however in early relapse free fraction was significantly higher than in late remission. The albumin saturation with GSH was significantly higher in early than in late relapse. Total HCY concentration was decreased in early relapse, elevated after 2 week and comparable to controls after 8 week of treatment. HCY free fraction and albumin saturation were elevated within first 2 weeks. Children in long-term remission showed elevated total concentration of HCY and GSH and their protein bound fractions when compared to controls. Albumin saturation with these aminoacids was higher as well. CONCLUSION: The study showed aminothiol imbalance in children in first weeks of relapse of nephrotic syndrome.

Fully automated method for simultaneous determination of total cysteine, cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection.

A fully automated HPLC method for the simultaneous determination of total thiols in plasma samples has been developed. The method involves reductive conversion of disulfides to their reduced counterparts with the use of tris(2-carboxyethyl)phosphine. After reduction the newly formed sulfhydryl groups are reacted with 2-chloro-1-methylquinolinium tetrafluoroborate to form 2-S-quinolinium derivatives followed by deproteinization by dialysis. The reaction products are separated by reversed-phase HPLC, detected and quantified by UV absorbance detection at 355nm. The recommended HPLC procedure enables measurement of four main plasma aminothiols cysteine, cysteinylglycine, glutathione, and homocysteine with low imprecision (mean relative standard deviations within calibration range, 3.47%, 5.34%, 4.25% and 3.26%, respectively) and good sensitivity. Accuracy, expressed as the mean measured amount as percentage of added amount, was within 97.5-103.0%, 98.3-102.5%, 96.3-99.5% and 97.1-99.1%, respectively. The lower limit of quantification for all thiols was 0.5microM. The whole unattended instrument acquisition time amounts 13min.

Determination of thiosulfate in human urine by high performance liquid chromatography.

Thiosulfate is a sulfate analogue with a thiosulfur substituent and is found in human samples. Its concentration in urine is increased in some diseases and after exposure to hydrogen sulfide gas. We have developed a sensitive, simple and cheap method for thiosulfate determination in urine. The method is based on precolumn derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate followed by reversed-phase liquid chromatography separation and ultraviolet detection of 1-methyl-2-thioquinolone at 375 nm. The calibration curve for thiosulfate was linear in the tested range 0.5-50 micromol L(-1) with correlation coefficient better than 0.999. The analytical recovery and relative standard deviation values for precision within the calibration range were from 90.1% to 104.2% and from 2.39% to 5.59%, respectively. The lower limit of detection and quantitation were 0.3 and 0.5 micromol L(-1), respectively. The mean (range) concentration of thiosulfate normalized against creatinine for apparently healthy seven women and six men was 2.21 (1.45-2.77) and 2.51 (1.36-4.89)mmol mol(-1) creatinine, respectively. We monitored thiosulfate in urine samples from one volunteer for 24 h. The urinary excretion of thiosulfate was 21.4 micromol per 24 h. This method can be used for routine clinical monitoring thiosulfate in urine. Cysteine and cysteinylglycine can be measured concurrently, if needed.

Determination of endogenous thiols and thiol drugs in urine by HPLC with ultraviolet detection.

Analysis of urine for endogenous thiols and thiol drugs content by HPLC with ultraviolet detection is addressed. Other methodologies for detection and determination of thiols in urine are only mentioned. Outline of metabolism, role of main biological thiols in physiological and pathological processes and their reference concentrations in urine are presented. In particular, urine sample preparation procedures, including reduction of thiol disulfides, chemical derivatization and reversed-phase HPLC separation steps are discussed. Some experimental details of analytical procedures for determination of endogenous thiols cysteine, cysteinylglycine, homocysteine, N-acetylcysteine, thioglycolic acid; and thiol drugs cysteamine, tiopronin, D-penicillamine, captopril, mesna, methimazole, propylthiouracil and thioguanine are reviewed.

Reversed-phase liquid chromatography method for the determination of total plasma thiols after derivatization with 1-benzyl-2-chloropyridinium bromide.

A high-performance liquid chromatography method was developed for simultaneous detection and quantitation of total cysteine, glutathione, homocysteine and cysteinylglycine in human plasma. The two key steps in the analysis are reduction of disulfides and treatment with 1-benzyl-2-chloropyridinium bromide, which rapidly and quantitatively reacts with thiol groups to form stable S-pyridinium derivatives with intense UV absorption. The derivatives are well separated on a Zorbax SB C(18) column using reversed-phase high-performance liquid chromatography and monitored at 315 nm. The calibration graphs were linear over concentration ranges covering most experimental and clinical cases with a regression coefficients better than 0.999. The detection and quantitation limits for all analytes were 0.2 and 0.5 micromol/L, respectively. The recoveries were 99.25-101.68%. The intra- and interassay imprecisions were 0.88-4.24 and 1.68-5.14%, respectively. The method was applied for plasma samples donated by apparently healthy volunteers.

Oxidative/nitrative modifications of plasma proteins and thiols from patients with schizophrenia.

OBJECTIVE: The level of various specific biomarkers of oxidative stress in plasma from schizophrenic patients, as well as biomarkers (the level of isoprostanes) in urine in schizophrenic patients was described. The aim of our present study was to evaluate biomarkers of oxidative stress by oxidative/nitrative modifications of plasma proteins (by measuring the level of carbonyl groups and 3-nitrotyrosine in proteins) from patients with schizophrenic disorders and from control group. We also investigated the level of low-molecular-weight thiols [glutathione (GSH), cysteine (CSH), cysteinylglycine (CGSH) and homocysteine] in plasma obtained from schizophrenic patients and from healthy volunteers. Patients hospitalized in the 1st and 2nd Psychiatric Department of the Medical University in Lodz, Poland were interviewed with a special questionnaire (treatment, course of diseases, dyskinesis and other extrapyramidal syndromes). According to DSM-IV criteria, all patients had a diagnosis of paranoid type. They were treated with antipsychotic drugs (clozapine, risperidone, olanzapine). The mean duration of schizophrenia was about 5 years. METHODS: Levels of carbonyl groups and 3-nitrotyrosine residues in plasma proteins were measured by ELISA and a competition ELISA, respectively. High-performance liquid chromatography was used to analyze free thiols in plasma. RESULTS: We observed a statistically increased level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine in plasma proteins from schizophrenic patients. In schizophrenic patients the amount of homocysteine in plasma was higher compared with the control group; the level of GSH, CSH and CGSH was decreased. This indicates that reactive oxygen species and reactive nitrogen species may stimulate oxidative/nitrative modifications of plasma proteins in schizophrenic patients. CONCLUSION: Considering the data presented in this study, we suggest that the amount of carbonyl groups and 3-nitrotyrosine in plasma proteins may be important indicators of protein damage in vivo in schizophrenia.

PAMAM G4 dendrimers lower high glucose but do not improve reduced survival in diabetic rats.

For nearly a decade poly(amidoamine) (PAMAM) dendrimers G4 were claimed unnegligible cytotoxic agents. Here we monitored whether in vivo cytotoxic effect of PAMAM G4 (0.5 micromol kg(-1) day(-1)) may be compromised by its ameliorating effect on severe hyperglycaemia in chronic streptozotocin-diabetic Wistar rats. PAMAM G4 significantly reduced the 60-day overall survival in long-term experimental diabetes: treated animals were 6.7 times more likely to die than control animals (p<0.025). PAMAM G4 significantly reduced numerous biochemical parameters in blood, including glucose, glycated haemoglobin or protein oxidation, cholesterol and triglycerides, but apparently unchanged plasma insulin peptide C. Terminal blood glucose in PAMAM-treated animals was significantly higher in survivors, pointing to the possible preventive role of glycation in reducing of PAMAM G4 cytotoxicity. Our results provide the first in vivo evidence that PAMAM G4 is able to lower plasma glucose and suppress long-term markers of diabetic hyperglycaemia. Nevertheless, this beneficial influence cannot override PAMAM G4 cytotoxic effects in the increased mortality of streptozotocin-diabetic rats.

Method for determination of total cysteamine in human plasma by high performance capillary electrophoresis with acetonitrile stacking.

An analytical procedure enabling routine analysis of human plasma for total cysteamine (CASH) has been developed and validated. The method includes reduction of CASH disulfides to thiol with tri-n-butylphosphine, derivatization of the thiol with 2-chloro-1-methylquinolinium tetrafluoroborate, separation of CASH 2-S-quinolinium derivate from those of plasma endo- and exogenous thiol derivatives by capillary zone electrophoresis based on acetonitrile stacking and quantitation with the use of ultraviolet detection. A large volume of sample is injected to achieve analyte concentration directly on the capillary, according to the transient pseudo-isotachophoresis principle, before the separation step takes place. Method performance characteristics, for example recovery, calibration, precision, limit of detection and limit of quantitation are presented. The procedure was applied to analysis of plasma samples donated by apparently healthy volunteers spiked with known amounts of cystamine standard solution.