The Diagnostic and Prognostic Roles Played by Homocysteine and Other Aminothiols in Patients with Chronic Kidney Disease.

We examined standard clinical and laboratory biochemical parameters, as well as the levels of aminothiols in the blood and urine (homocysteine (Hcy), cysteine (Cys), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH)) via capillary electrophoresis in patients with CKD at stages II-V. Patient outcomes were assessed after five years. To complete forecasting, correlation and ROC analysis were performed. It was found that the levels of Cys and Hcy in blood plasma were earlier markers of CKD starting from stage II, while the levels of SAM and SAM/SAH in urine made it possible to differentiate between CKD at stages II and III. Blood plasma Hcy and urinary SAM and SAM/SAH correlated with mortality, but plasma Hcy concentrations were more significant. Thus, plasma Hcy, urine SAM, and SAM/SAH can be considered to be potential diagnostic and prognostic markers in patients with CKD.

Neuroprotective Effect of Platinum Nanoparticles Is Not Associated with Their Accumulation in the Brain of Rats.

Platinum nanoparticles (nPts) have neuroprotective/antioxidant properties, but the mechanisms of their action in cerebrovascular disease remain unclear. We investigated the brain bioavailability of nPts and their effects on brain damage, cerebral blood flow (CBF), and development of brain and systemic oxidative stress (OS) in a model of cerebral ischemia (hemorrhage + temporary bilateral common carotid artery occlusion, tBCAO) in rats. The nPts (0.04 g/L, 3 ± 1 nm diameter) were administered to rats (N = 19) intraperitoneally at the start of blood reperfusion. Measurement of CBF via laser Doppler flowmetry revealed that the nPts caused a rapid attenuation of postischemic hypoperfusion. The nPts attenuated the apoptosis of hippocampal neurons, the decrease in reduced aminothiols level in plasma, and the glutathione redox status in the brain, which were induced by tBCAO. The content of Pt in the brain was extremely low (≤1 ng/g). Thus, nPts, despite the extremely low brain bioavailability, can attenuate the development of brain OS, CBF dysregulation, and neuronal apoptosis. This may indicate that the neuroprotective effects of nPts are due to indirect mechanisms rather than direct activity in the brain tissue. Research on such mechanisms may offer a promising trend in the treatment of acute disorders of CBF.

Impact of glutathione on acute ischemic stroke severity and outcome: possible role of aminothiols redox status.

OBJECTIVE: Acute brain ischemia is accompanied by a disruption of low-molecular-weight aminothiols (LMWTs) homeostasis, such as homocysteine (Hcy), cysteine (Cys), and glutathione (GSH). We investigated the redox balance of LMWTs in blood plasma and its influence on ischemic stroke severity and the functional outcome in patients with an acute period. PATIENTS AND METHODS: A total of 177 patients were examined. Total and reduced forms of LMWTs were determined in the first 10-24 h. Stroke severity and functional state were estimated using the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRs) at admission and after 21 days. RESULTS: Patients with high levels of total Hcy (> 19 µM) showed significantly reduced redox statuses of all LMWTs. Patients with low total GSH levels (≤ 1.07 µM) were at an increased risk of higher stroke severity (NIHSS > 10) compared to patients with a total GSH level > 2.64 µM (age/gender-adjusted odds ratio: 4.69, 95% CI: 1.43-15.4). DISCUSSION: (1) low total GSH level can be considered as a novel risk marker for the severity of acute stroke in conditions of low redox status of LMWTs and (2) high Hcy levels associated with low redox status of LMWTs.

Urine S-Adenosylmethionine are Related to Degree of Renal Insufficiency in Patients with Chronic Kidney Disease.

OBJECTIVE: To determine whether urine S-adenosylmethionine (SAM) might be an indicator of chronic kidney disease (CKD). METHODS: We investigated urine levels of SAM and related metabolites (S-adenosylhomocysteine and homocysteine cysteine) in 62 patients (average age, 65.9 years) with CKD (stages II-V). RESULTS: Patients with stages III-V CKD stages have significantly decreased urine levels and SAM/S-adenosylhomocysteine ratio and also cysteine/homocysteine ratio in blood plasma (P <.05), compared with patients with stage II CKD. Urine SAM levels allowed us to distinguish patients with mildly decreased kidney function from those with moderate to severe renal impairment (AUC, 0.791; sensitivity, 85%; specificity, 78.6%). CONCLUSIONS: Our study results demonstrate that urine SAM is a potent biomarker for monitoring renal function decline at early CKD stages. Urine SAM testing confers an additional advantage to healthcare professionals in that it is noninvasive.

Low S-adenosylmethionine/ S-adenosylhomocysteine Ratio in Urine is Associated with Chronic Kidney Disease.

OBJECTIVE: To evaluate the association of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in urine with chronic kidney disease (CKD). METHODS: Case-control study including 50 patients with CKD and 20 healthy volunteers. RESULTS: SAM level and SAM/SAH ratio in urine were significantly lower in patients than in control individuals (P <.001 and P = .01, respectively). The estimated glomerular filtration rate was associated with the SAM level (P = .04) and the SAM/SAH ratio in urine (P = .01). CONCLUSION: CKD is associated not only with the decline in the SAM level but also with the decrease in the SAM/SAH ratio in urine. Thus, use of the urinary SAM/SAH ratio as a noninvasive diagnostic indicator of renal function seems promising.

Determination of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine in urine using solvent-modified micellar electrokinetic chromatography.

A new approach for direct determination of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methylthioadenosine (MTA) in urine was developed based on MEKC by using SDS modified with isobutanol in the presence of PEG-300. Analytes were first extracted with grafted phenylborononic acid. Using a 50 µm internal diameter silica capillary of 32 cm total length filled with 0.05 M SDS, 0.05 M H3 PO4 , 5% (v/v) isobutanol, and 10% (v/v) PEG-300, LOQ of 0.15 µM for SAM and SAH, and 0.2 µM for MTA was reached. Accuracy was 92% for MTA, 109% for SAH, and 105% for SAM, intra- and interday imprecision were <2.5 and ≤3%, respectively. The total time of analysis for one sample was 10 min. Analysis of 30 urine samples from healthy volunteers showed that the median SAM and SAH levels were 12.1 and 0.73 µM, respectively. MTA levels, which were determined in urine for the first time (according to our data), were 0.43 µM, and these values correlated well with the SAM level (r = 0.748, p < 0.01).

Determination of S-adenosylmethionine and S-adenosylhomocysteine in blood plasma by UPLC with fluorescence detection.

A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100 × 2.1 mm × 1.8 µm C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2 mL/min) from 2 to 20%. The eluent was initially composed of 10 mM KH2PO4 with 10 mM acetic acid and 25 µM heptafluorobutyric acid. The total analysis time was 11 min. Validation of the method included detection of the limit of detection (2 nM), limit of quantification (5 nM), accuracy (97.2-101%), and intra- and interday precision (2.2-9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8 nM, respectively.

Disturbance of thiol/disulfide aminothiols homeostasis in patients with acute ischemic stroke stroke: Preliminary findings.

OBJECTIVES: To determine the disruption of low-molecular-weight aminothiols (LMWTs: cysteine, cysteinylglycine, homocysteine, and glutathione) homeostasis in blood plasma during the acute and early subacute stages after ischemic stroke. PATIENTS AND METHODS: We admitted 41 patients with primary large-artery atherosclerosis and cardioembolic stroke in the carotid arteries within the first 6-24 h from the moment of neurologic symptoms development. We included 31 patients with chronic cerebral ischemia in the control group. Total LMWT levels and their reduced forms were measured in blood plasma on the 1st, 3rd, 7th, and 15th days after stroke. RESULTS: Our study demonstrated a decrease of cysteine and cysteinylglycine reduced forms and an increase of total glutathione and cysteine levels. There were no differences in LMWT levels among stroke subtypes (large-artery atherosclerosis and cardioembolic stroke). The decrease (or increase) in GSH and Hcy redox status on the 3rd day after stroke was associated with severe neurological deficit. Total Hcy (1st day), Cys (3rd day) and CG(7th day) levels were associated with the size of cerebral infarction area. Logistic regression analysis indicated that reduced homocysteine, total cysteinylglycine levels, and cysteine redox status at admission were predictive factors for ischemic stroke occurrence with a probability of 86.2% (p < 0.001). CONCLUSIONS: LMWTs may indicate the severity of neurological deficit and the size of the cerebral infarct, and their complex determination can be of diagnostic importance both at an early stage of ischemic stroke development and during its monitoring.

Metoprolol and Nebivolol Prevent the Decline of the Redox Status of Low-Molecular-Weight Aminothiols in Blood Plasma of Rats During Acute Cerebral Ischemia.

Cerebral ischemia has previously been shown to cause a systemic decrease in levels of the reduced forms of low-molecular-weight aminothiols [cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)] in blood plasma. In this study, we examined the effect of beta-adrenergic receptor (ß-AR) antagonists metoprolol (Met) and nebivolol (Neb) on the redox status of these aminothiols during acute cerebral ischemia in rats. We used a model of global cerebral ischemia (bilateral occlusion of common carotid arteries with hypotension lasting for 10 minutes). The antagonists were injected 1 hour before surgery. Total and reduced Cys, Hcy, and GSH levels were measured 40 minutes after the start of reperfusion. Neb (0.4 and 4 mg/kg) and Met (8 and 40 mg/kg) treatment increased the levels of reduced aminothiols and the global methylation index in the hippocampus. The treatments also prevented any decrease in reduced aminothiol levels in blood plasma during ischemia. Although both of these drugs eliminated delayed postischemic hypoperfusion, only Neb reduced neuronal damage in the hippocampus. The results indicate an essential role of ß1-AR blockage in the maintenance of redox homeostasis of aminothiols in the plasma and brain during acute cerebral ischemia.

Capillary electrophoresis coupled with chloroform-acetonitrile extraction for rapid and highly selective determination of cysteine and homocysteine levels in human blood plasma and urine.

A rapid and selective method has been developed for highly sensitive determination of total cysteine and homocysteine levels in human blood plasma and urine by capillary electrophoresis (CE) coupled with liquid-liquid extraction. Analytes were first derivatized with 1,1'-thiocarbonyldiimidazole and then samples were purified by chloroform-ACN extraction. Electrophoretic separation was performed using 0.1 M phosphate with 30 mM triethanolamine, pH 2, containing 25 µM CTAB, 2.5 µM SDS, and 2.5% polyethylene glycol 600. Samples were injected into the capillary (with total length 32 cm and 50 µm id) at 2250 mbar*s and subsequent injection was performed for 30 s with 0.5 M KОН. The total analysis time was less than 9 min, accuracy was 98%, and precision was <2.6%. The LOD was 0.2 µM for homocysteine and 0.5 µM for cysteine. The use of liquid-liquid extraction allowed the precision and sensitivity of the CE method to be significantly increased. The validated method was applied to determine total cysteine and homocysteine content in human blood plasma and urine samples obtained from healthy volunteers and patients with kidney disorders.

Plasma low-molecular-weight thiol/disulphide homeostasis as an early indicator of global and focal cerebral ischaemia.

OBJECTIVE: Recent studies have shown that cerebral ischaemia causes not only local, but also systemic oxidative stress. This leads to oxidation of thiol-containing compounds, including low-molecular-weight thiols (cysteine, glutathione, homocysteine and others). Therefore, the aim of this work was to verify the hypothesis that the thiol/disulphide homeostasis of low-molecular-weight thiols is disturbed in the early stages of cerebral ischaemia. METHODS: Two experimental rat models of ischaemia were used: a global model of vascular ischaemia (clamping the common carotid arteries + haemorrhage) and focal ischaemia (middle cerebral artery occlusion). The total levels of thiols and their reduced forms were measured before surgery and after 40 minutes of reperfusion (global) or 3 hours (focal) ischaemia. RESULTS: The global ischaemia model caused a marked (2.5-4 times, P < 0.01) decrease in the plasma thiol/disulphide redox state, and focal ischaemia caused an even larger decrease (30-80 times, P < 0.001). DISCUSSION: These results suggest that plasma low-molecular-weight thiols are actively involved in oxidation reactions at early stages of cerebral ischaemia; therefore, their reduced forms or redox state may serve as a sensitive indicator of acute cerebrovascular insufficiency.

Capillary electrophoresis and phenylboronic acid solid phase extraction for the determination of S-adenosylmethionine/S-adenosylhomocysteine ratio in human urine.

An approach that allows direct analysis of the ratio of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) by using CE is presented. The analytes were extracted on phenylboronic acid phase and eluted with 100 mmol/L HCl. CE separation of the analytes took place in the transient isotachophoresis mode with addition of NaCl and meglumine to the samples. The sensitivity (S/N = 3) and quantification limit (S/N = 10) of the method were 0.07 and 0.2 µmol/L, respectively, using a silica capillary with 50 µm internal diameter and 30.5 cm total length. The BGE was 0.02 mol/L Tris with 1 mol/L HCOOH (pH 2.2), and the separation voltage was 15-17 kV. Accuracy of SAM and SAH analysis in urine was 96 and 105%, respectively; interday precision for the SAM/SAH ratio was within 6%. The theoretical plate number exceeded a million. Total analysis time was 8.5 min.

Capillary electrophoresis coupled with 1,1'-thiocarbonyldiimidazole derivatization for the rapid detection of total homocysteine and cysteine in human plasma.

A simple and rapid approach is described for the determination of total plasma cysteine and homocysteine using capillary electrophoresis. Human plasma samples were reduced with dithiothreitol and then processed with 1,1'-thiocarbonyldiimidazole in acetonitrile. After centrifugation, the sample supernatant was injected directly into a capillary by applying negative voltage and analytes were stacked after alkaline post-injection. Using a 50µm i.d. silica capillary of 35cm total length, filled with 0.1M triethanolamine, 0.15M formic acid, and 50µM hexadecyltrimethylammonium bromide (pH 3.9), we reached a limit of quantification of 2.5µM for homocysteine. Accuracy was 94.7-105.1%, intra- and inter-day imprecisions were <2.5 and <3%, respectively. The total analysis time was 6min. Furthermore, liquid-phase extraction with isopropanol led to a fourfold increase in sensitivity.