Cysteine In Human Plasma Research Articles

A ratiometric fluorescent BODIPY-based probe for rapid and highly sensitive detection of cysteine in human plasma.

Biological thiols, especially low molecular weight thiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a pivotal role in physiological and pathological systems. Thus, the detection of biothiols is highly important for early diagnosis of diseases and evaluation of disease progression. Herein, we developed a highly selective and sensitive ratiometric fluorescent 8-Cl BODIPY-based probe with high fluorescence quantum yields. The probe displayed a sensitive response to Cys and Hcy over other biothiols, which can be visualized colorimetrically and/or fluorescently. The probe was successfully applied to detect Cys in human plasma, demonstrating its great value for practical application in biological sample analysis.

Highly selective and sensitive detection of cysteine with a graphene quantum dots-gold nanoparticles based core-shell nanosensor

Reduced graphene quantum dots (r-GQDs) coated gold nanoparticles (AuNPs) core-shell structure is developed as a nanosensor for highly selective and sensitive colorimetric detection of cysteine. The reduction of nitrogen doped GQDs (N-GQDs) by NaBH4 produces r-GQDs, which serve as reductant for facilitating the conversion of HAuCl4 to AuNPs. The abundant hydroxyl groups and amino groups of r-GQDs not only promote the reduction of Au (III) into Au (0), but also facilitate the effective coating of r-GQDs onto AuNPs. The obtained core-shell structured AuNPs@r-GQDs are well dispersed and exhibit an intensive surface plasma band at 525nm. After the adsorption of Ag+ on the surface of AuNPs@r-GQDs, cysteine as cross-linking agent induces the aggregation of AuNPs@r-GQDs and leads to their color change, which allows the sensitive colorimetric detection of cysteine and offers a detection limit low to 5.6nM. More importantly, AuNPs@r-GQDs nanosensor exhibits very high detection selectivity towards cysteine against glutathione (GSH), even at a GSH concentration of 10000-fold higher than that of cysteine. The practicability of this nanosensor is demonstrated by the discriminative determination of cysteine in human plasma.

Fabrication and characterization of Cu(OH)2/CuO nanowires as a novel sensitivity enhancer of the luminol–H2O2chemiluminescence system: determination of cysteine in human plasma

Cu(OH)2/CuO nanowires as a sensitivity enhancer in luminol–H2O2chemiluminescence system and its application for the determination of cysteine.

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.

Determination of Thiols by Fluorescence using Au@Ag Nanoclusters as Probes

A simple and label-free fluorescent assay for the sensitive determination of biological thiols was developed using Au@Ag nanoclusters. The sensing approach was based on the strong affinity of thiols to silver on the surface of the nanoclusters. In the presence of thiol-containing amino acids, the fluorescence of the Au@Ag nanoclusters was quenched due to the formation of a non-fluorescent coordination complex via the robust Ag-S bond, which allowed the determination of thiol-containing amino acids in a very simple and rapid way. Under the optimal conditions, an excellent linear relationship was present due to quenching of the Au@Ag nanoclusters over cysteine concentrations between 20 nM and 80 µM with a low detection limit of 5.87 nM. Glutathione was determined between 2 µM and 70 µM with a detection limit of 1.01 µM. In addition, the results reveal that the fluorescent assay has excellent selectivity toward thiol-containing amino acids compared to non-thiol containing amino acids. Moreover, the assay was successfully used to determine cysteine in human plasma, and thus Au@Ag nanoclusters are a suitable fluorescent probe for biological applications.

Simultaneous determination of total homocysteine and cysteine in human plasma by capillary zone electrophoresis with pH-mediated sample stacking

An accurate, simple and sensitive method for simultaneous determination of total homocysteine (Hcy) and total cysteine (Cys) in human plasma has been developed and validated. Hcy and Cys play a major role in metabolism and cellular homeostasis. An elevated level of plasma Hcy is considered an important risk factor or marker for several diseases, in particular cardiovascular disease, while Cys is involved in a variety of important cellular functions such as protein synthesis, detoxification and metabolism. The proposed analytical procedure consists of reduction of title thiol dimers and unsymmetrical disulfides with tris(2-carboxyethyl)phosphine, and derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by pH-mediated stacking capillary electrophoresis separation and ultraviolet-absorbance detection of Hcy-CMQT and Cys-CMQT derivatives at 355 nm. Effective baseline electrophoretic separation was achieved using a standard fused-silica capillary (effective length 91.5 cm, 75 μm id) and 0.1 mol L−1, lithium acetate buffer adjusted to pH 4.75. The limit of quantification (signal to noise ratio of 9) for derivatized Hcy and Cys in plasma was 2 μmol L−1. The calibration curves obtained for Hcy and Cys in plasma showed linearity in the range 2–20 μmol L−1, with R2 = 0.9986 and 20–300 μmol L−1, with R2 = 0.9998, respectively. The relative standard deviation of the points of the calibration curve was lower than 7%. The method can be used for a routine monitoring of total Hcy and total Cys levels in plasma of human subjects.

Validation and clinical application of an UHPLC method for simultaneous analysis of total homocysteine and cysteine in human plasma

Several studies indicate that high levels of homocysteine (Hcy) and L-cysteine (L-Cys) are independent risk factors for cardiovascular disease. The validation and clinical application of an ultra HPLC method for analysis of Hcy and L-Cys is described. The reported method is simple, sensitive, rapid, precise, and less aggressive than other previously reported methods. The effect of the derivatization reaction time, pH, and organic solvent contents in the mobile phase are described and discussed. Optimized conditions resulted in excellent peak shapes. Results of method validation showed a good linearity (r(2) ≥ 0.993) over the investigated concentration ranges and were observed for both compounds. The LOD and LOQ were 0.05 μM and 0.15 μM for Hcy and 0.24 μM and 0.80 μM for L-Cys, respectively. Validation results proved that the method precision was good and the accuracy was satisfactory. This validated method was successfully applied in an epidemiological study to measure and compare the prevalence of Hcy and L-Cys high levels in plasma of Portuguese type 2 diabetic patients with and without angiopathy. The study results showed that prevalence of hyperhomocysteinemia and hypercysteinemia were at least two times higher in diabetic patients with angiopathy compared to diabetics without angiopathy.

Interaction of N-acetylcysteine and Cysteine in Human Plasma

N-acetyl-L-cysteine (NAC), a well-known antioxidant, has been successfully used as adjuvant therapy for late-stage childhood cerebral adrenoleukodystrophy (c-ALD); however, the mechanisms of NAC action are poorly understood. Previous research indicates that NAC serves as a precursor to L-cysteine (Cys), the rate-limiting substrate in the biosynthesis of glutathione (GSH), a potent, endogenous antioxidant. We hypothesized that NAC acts by liberating protein-bound Cys in plasma in an NAC concentration-dependent manner, which increases unbound Cys available for GSH biosynthesis. Human plasma was incubated for 1 h with varying, clinically relevant concentrations of NAC (0-1000 µg/mL). The effect of this interaction over time was evaluated by incubating plasma for 5-90 min with 100 µg/mL NAC. Unbound and bound Cys and NAC were separated by ultrafiltration, and concentrations were measured using high-performance liquid chromatography-mass spectrometry. Significant increases in unbound Cys were observed with increasing NAC concentrations. Also, Cys plasma protein binding decreased from 85% (10 µg/mL NAC) to approximately 0% (1000 µg/mL). Total endogenous Cys was 66% unbound at 5 min after incubation. These results demonstrate that NAC liberates endogenous, protein-bound Cys in human plasma at clinically relevant NAC concentrations. A greater understanding of NAC actions will aid in the optimization of NAC therapy including its use in c-ALD.

High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography–electrospray tandem mass spectrometry

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography–electrospray tandem mass spectrometry (LC–MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC–MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40μmol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60μmol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43μmol/mmol creatinine, respectively. Bland–Altman difference plots of method comparison between LC–MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.

Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years ( n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.

Determination of cysteine in human plasma by high-performance liquid chromatography and ultraviolet detection after pre-column derivatization with 2-chloro-1-methylpyridinium iodide

This paper describes the development and validation of a reversed-phase HPLC method with isocratic conditions for the analysis of total cysteine in human plasma. The essential steps in the assay include derivatization of cysteine via thiol group with 2-chloro-1-methylpyridinium iodide (CMPI), separation of the formed cysteine S-pyridinium derivative from endogenous thiol derivatives, usually present in biological samples, by ion-pair reversed-phase HPLC, and ultraviolet detection. Free cystine and protein-bound cysteine are converted to their reduced counterpart by the use of 2-mercaptoethanol. The standard curve is linear over the concentration range from 20 to 300 nmol ml −1. The recoveries are from 93.9 to 105.9% and imprecision was at most 2.5%. Optimal conditions are determined for all steps of the analytical procedure.

Determination of cysteine by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode without deoxygenation

Capillary zone electrophoresis was employed for the determination of cysteine using an end-column amperometric detection with a gold/mercury amalgam microelectrode, at a constant potential of 0.02–0.06 V vs. saturated calomel electrode. In this procedure deoxygenation is not necessary. The electrochemical characteristics at the microelectrode, the effect of the concentration of the buffer and the separation voltage across the capillary on the migration time and separation efficiency, and the dependence of the injection voltage and time on the detection signal, the separation efficiency and coulometric efficiency has been investigated. The calibration plot was found to be linear over four orders of magnitude and the limit of detection was 5.8·10 −8 mol/l (or 14. 5 amol). The method was applied to the determination of cysteine in human plasma, blood and urine.

Quantification of S-carboxymethyl-(R)-cysteine in human plasma by high-performance ion-exchange liquid chromatography/atmospheric pressure ionization mass spectrometry.

The determination of S-carboxymethyl-(R)-cysteine (SCMC) in human plasma during extended bioequivalence studies demands a rapid, accurate and selective assay technique. A liquid chromatographic/mass spectrometric method was developed which involves rough protein precipitation followed by high-performance liquid chromatographic separation with an ion-exchange column and atmospheric pressure ionization (API) mass spectrometric detection, with the instrument operating with electrospray ionization (ESI) and in the selected-ion monitoring mode. The drug and the internal standard S-[(R)-1-carboxyethyl]-(R)-cysteine (SCEC) are detected by focusing the first quadrupole of the triple stage system on MH+ ions, thus permitting elimination of endogenous interfering substances and allowing a detection limit of 0.05 microgram ml-1. The chromatographic run time is 16 min and the method has sufficient sensitivity, precision, accuracy and selectivity for routine analyses of clinical plasma samples containing SCMC at concentrations in the range 0.2-20 micrograms ml-1. In summary, this LC/MS-based assay of SCMC demonstrates advantages of easy sample preparation, low limit of quantification (200 ng per ml of human plasma) without any derivatization step, high specificity and rapid sample analysis with an overall throughput of more than 60 analyses per day.

Determination of total homocysteine in human plasma by isocratic high-performance liquid chromatography.

A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.