Cysteine In Human Urine Research Articles

Development of a Novel Benzimidazole-Based Probe and Portable Fluorimeter for the Detection of Cysteine in Human Urine.

The measurement of cysteine in human urine and live cells is crucial for evaluating biological metabolism, monitoring and maintaining the immune system, preventing tissue/DNA damage caused by free radicals, preventing autoimmune diseases, and diagnosing disorders such as cystinuria and cancer. A method that uses a fluorescence turn-on probe and a portable fluorescence spectrometer device are crucial for highly sensitive, simple, rapid, and inexpensive cysteine detection. Herein, we present the synthesis and application of a benzimidazole-based fluorescent probe (ABIA) along with the design and development of a portable fluorescence spectrometer device (CysDDev) for detecting cysteine in simulated human urine. ABIA showed excellent selectivity and sensitivity in detecting cysteine over homocysteine, glutathione, and other amino acids with the response time of 1 min and demonstrated a detection limit of 16.3 nM using the developed CysDDev. Further, ABIA also demonstrated its utility in detecting intracellular cysteine, making it an excellent probe for bio-imaging assay.

LC–MS/MS procedure for the simultaneous determination of N-acetyl-S-(1-naphthyl)cysteine and N-acetyl-S-(2-napthyl)cysteine in human urine

A two-dimensional liquid chromatographic electrospray-ionization tandem mass spectrometric (LC/LC–ESI–MS/MS) procedure for the simultaneous determination of the expected mercapturic acids of naphthalene (1- and 2-naphthylmercapturic acids; 1- and 2-NpMA) and of the well-established parameter for benzene biomonitoring (S-phenylmercapturic acid; PhMA) in human urine was developed, validated and applied to human urine samples. Apart from sample acidification, the enrichment of analytes and sample clean-up as well as the separation of all analytes were completely automated using both a restricted access material column (RAM C18) and a core-shell biphenyl material. Sensitive, specific and reliable detection of all target substances, with limits of detection ranging from 0.03 to 0.04 μg/L, was achieved using structurally well matching isotope-labelled internal standard substances for each analyte. Intraday and interday precision were determined, ranging from 2.2 to 4.3%, and mean accuracy from 98.4 to 100.8%. Due to the low limits of detection, the good precision and accuracy of the developed procedure, it is well suited for application in biomonitoring studies to evaluate the validity of mercapturic acids as biomarkers after naphthalene exposure.

Colorimetric sensor for cysteine in human urine based on novel gold nanoparticles

Herein, a novel, high sensitive, and specific colorimetric sensor for cysteine (Cys) based on pectinase protected gold nanoparticles (P@AuNPs) has been demonstrated for the first time. The P@AuNPs were synthesized by “MW-assisted heat method” and were characterized by UV–vis, TEM, FT-IR and zeta potential techniques. Cys could cause the aggregation of P@AuNPs due to formation of the strong covalent Au-S bond and electrostatic binding. As the Cys concentration increased, the color of the solutions gradually changed from wine-red to blue as well as the large absorption band shifted from 523 to 650nm upon P@AuNPs aggregation. In addition, various experimental parameters such as pH, the amount of P@AuNPs and incubation time were investigated for the optimum sensing conditions. The concentration of Cys could be determined by monitoring with the naked eye or a UV–vis spectrometer. The proposed colorimetric sensor showed an extreme selectivity toward the determination of Cys in the presence of 20-fold all other different interferents. Under optimum conditions, this method exhibited two good linear ranges from 4.85×10−9 to 3.02×10−4M (R2=0.996) and 3.25×10−3 to 1.03×10−2M (R2=0.999), with a low detection limit of 4.6×10−9M. Moreover, this colorimetric sensor was successfully applied to the detection of Cys in human urine samples, demonstrating its great value for practical application in biological systems.

Colorimetric detection of cysteine using noncrosslinking aggregation of fluorosurfactant-capped silver nanoparticles

In this work, it was found that the rapid aggregation of the nonionic fluorosurfactant-capped silver nanoparticles via noncrosslinking mechanism could be induced by cysteine, but not by other amino acids and biomolecules. The high specificity toward cysteine was due to that the nonionic fluorosurfactant ligands allowed for thiol–silver interaction while prohibited the binding of other functional groups on the surface of the silver nanoparticles. Based on these interesting results, we developed a novel, selective and rapid colorimetric method for the determination of cysteine by using the nonionic fluorosurfactant-capped silver nanoparticles as a probe. Under optimum experimental conditions, the calibration curve was found to be linear over the range of 1.5–6.0μM for cysteine with a detection limit of 0.05μM. The applicability of the proposed method has been validated by determining cysteine in human urine and plasma samples with satisfactory results. Recoveries from spiked urine and plasma samples were in the range of 94.8–111.3%.

Simultaneous Measurement of N-Acetyl-S-(2-cyanoethyl)-cysteine and N-Acetyl-S-(2-hydroxyethyl)-cysteine in Human Urine by Liquid Chromatography-Tandem Mass Spectrometry

Acrylonitrile, possibly carcinogenic to humans, is mainly present in tobacco smoke and undergoes metabolism to form N-acetyl-S-(2-cyanoethyl)-cysteine (CEMA) and N-acetyl-S-(2-hydroxyethyl)-cysteine (HEMA). A method based on the direct dilution to simultaneously identify and quantify CEMA and HEMA in human urine by rapid resolution liquid chromatography-electrospray ionization tandem mass spectrometry (RRLC-MS-MS) was validated for assessing smoking-related acrylonitrile exposure. The recovery rates of the whole analytical procedure were 98.2-106.0% and 97.1-112.7% for HEMA and CEMA, respectively. The linear range of standard solutions was 0.5-100.0 ng/mL for CEMA and was 0.2-40.0 ng/mL for HEMA. RRLC using a small particle size column was combined with a tandem mass spectrometry system, which lowered the detection limit of analytes, reduced the ion suppression of mass and shortened the analysis time. The proposed method was successfully applied for the analysis of 126 urine samples from smokers and nonsmokers.

Styryl quinolinium/G-quadruplex complex for dual-channel fluorescent sensing of Ag+ and cysteine

Based on the disruption and recovery of fluorescence of styryl quinolinium (SQ) dye/G-quadruplex complex, a label-free method for dual-channel detection of Ag + and cysteine is developed. The above sensing system exhibits high sensitivity and good selectivity for Ag + , even in the presence of other metal ions (such as Na + , Mg 2+ , Mn 2+ , Ba 2+ , Ca 2+ , Pb 2+ , Zn 2+ , Cd 2+ , Hg 2+ and Cu 2+ ). Due to cysteine (Cys) is a strong Ag + -binder and competes with quadruplex-forming G-rich oligonucleotides for Ag + binding, and results in the reformation of G-quadruplex structure and the recovery of fluorescence. Thus SQ dye/G-quadruplex complexes are not only a potential primary sensor toward Ag + , but also a secondary sensor toward Cys. Neither the Ag + -sensing nor the Cys-sensing systems required labeled oligonucleotides. Under the optimal conditions, this method is capable of detection of Ag + in the range of 0.1–15.0 μM with a detection limit of 26.0 nM. In addition, the proposed method allows a good response to cysteine with a linear range of 0.008–10 μM, and the limit of detection is found to be 2.0 nM. Moreover, this method has been applied successfully to the sensing of cysteine in human urine with satisfactory recovery.

Specific determination of cysteine in human urine by capillary micellar electrokinetic chromatography

AbstractThis paper describes a method for the analysis of cysteine in human urine using capillary micellar electrokinetic chromatography and on‐column reaction with 2,2′‐dipyridyl disulfide. In this reaction cysteine is quantitatively transformed into a mixed disulfide concomitantly with formation of an equimolar amount of 2‐thiopyridone that is further separated by capillary micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of cysteine is thus estimated indirectly from the result of 2‐thiopyridone determination. The linear detection range for concentration versus peak area for the assay is from 0.05 to 5 mM (correlation coefficient 0.989) with a detection limit of 2.5 μM and a limit of quantitation of 8.5 μM. The inter‐day reproducibility of the peak area was 2.18% and the inter‐day reproducibility of the migration time 0.51%. The method is relatively rapid, simple, and can be easily automated. Moreover, its detection limit covers the concentration range at which cysteine is present in biological samples such as human urine.

Determination by liquid chromatography of free and total cysteine in human urine in the form of its S-quinolinium derivative

A reversed-phase high-performance liquid chromatographic method for the determination of free and total cysteine in urine is described. The method involves reductive conversion of cysteine dimer and cysteine mixed disulphides to their reduced counterpart with the use of tri- n-butylphosphine, ultraviolet-labeling with 2-chloro-1-methylquinolinium tetrafluoroborate, and liquid chromatographic separation with isocratic conditions. In developing this method the following parameters were investigated and optimized: the time, pH and reagent excess in the derivatization step, and mobile phase buffer concentration, pH, organic modifier and column temperature in the separation step. The method provides quantitative information on free and total cysteine based on assays with derivatization before and after reduction with tri- n-butylphosphine. The calibration graph, obtained with the use of normal urine spiked with growing amounts of cystine, was linear over the concentration range covering most experimental and clinical cases. The assay has a low pmol detection and quantitation limits, low imprecision and high recovery. The method was validated for urine samples received from several donors. Cystine was chosen as a primary calibrator for these assays.

Electrochemical response of new carbon electrodes bulk modified with cobalt phthalocyanine to some thiols in the presence of heptane or human urine

The method for preparation of solid working electrodes from carbon powder proposed earlier has been simplified and the conditions of bulk modification of the new electrodes with Co(II) phthalocyanine (CoPC) has been investigated using thiol oxidation as an example. The dependence of operational potential and of the analytical signal on the concentration of the modifier CoPC has been studied. The maximum sensitivity is at 12–15% (w/w) CoPC. The second-order rate constants K 1 o and electron transfer constants K el value for some thiols have been evaluated. It was shown that K 1 o value are large (∼10 4 l mol −1 s −1), i.e. CPrEM shows a high catalytic effect and the K el value is relatively low (∼10 −4 cm s −1). The sensitivity for mercaptoethanol determination in a petroleum fraction (heptane) diluted 1:200 in the range 0.25–3.25 μM is 6.37 nA μM −1. That for cysteine in human urine diluted 1:100 in the range 1–12 μM is 8.89 nA μM −1. The detection limit for mercaptoethanol in the diluted petroleum is 0.8×10 −7 M and that of cysteine in diluted human urine is 2×10 −7 M.

Determination of thiocompounds by liquid chromatography with amperometric detection at a Nafion/indium hexacyanoferrate film modified electrode

Polynuclear indium(III) hexacyanoferrate(II, III) thin films are electrodeposited on a glassy carbon (GC) electrode through potential cycling between 1.1 and 0.0 V vs. SCE in a fresh modification solution containing potassium, indium(III) and hexacyanoferrate(III) ions. During the negative scans, the cationic In 3+ species interact with the simultaneously generated anionic Fe(CN) 4− 6 to yield a sparingly soluble mixed-valent coating on the electrode. Coated with Nafion film, the chemically modified electrode (CME) shows a better stability. The CME shows a single set of reversible, well-defined peaksc significantly separated from the solution responses. The apparent surface coverage is found to be 6.3×10 −9 mol/cm 2. This CME exhibits an efficient catalytic activity towards the rapid oxidation of thiocompounds in cyclic voltammetry. Cysteine (Cys), glutathione (GSH), 6-thiopurine (6-TP) and methimazole (MMI) have good and stable current responses at the CME in liquid chromatography with electrochemical detection (LC–ECD). The linear ranges are over three orders of magnitude and the detection limits are 1.6 pmol for Cys, 20 pmol for GSH, 20 pmol for 6-TP and 1.6 pmol for MMI. The method has been successfully applied to assess the purity of methimazole, the contents of cysteine in human urine and other thiocompounds in artificial urines.

Determination of N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine in human urine after experimental exposure to styrene

An assay for determination of one of the styrene mercapturic acids [(N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine] was developed. Styrene mercapturic acid in urine samples was determined using high performance liquid chromatography (HPLC) with electrochemical detection. Urine samples from 6 individuals who had been experimentally exposed to styrene (205–220 mg/m 3, 2 hrs, 50 W) were analysed using our assay. The absorption of styrene during the exposure was 3.6 mmol (range 3.2–4.1) or 64% of the amount inhaled. N-acetyl-S-(2-phenyl-2-hydroxyethyl)cysteine could not be identified in the urine collected during the exposure and up to 5 hrs from the end of the exposure. If present, the metabolic yield from absorbed styrene was less than 1%. Our results, in contrast to what have been reported from animal experiments, indicate that, in man, gluthatione conjugation of styrene to mercapturic acids is a metabolic pathway of less importance and/or that styrene-glutathione conjugates in man undergo a metabolism different from that which has been reported in rats.

Gas chromatographic method for the determination of N-acetyl-S-(N-methylcarbamoyl)cysteine, a metabolite of N,N-dimethylformamide and N-methylformamide, in human urine

A simple method has been developed for the determination of N-acetyl-S-(N-methylcarbamoyl)cysteine in human urine. Treatment of a urine sample (1 ml) with ethanol (2 ml) and potassium carbonate (1.5 g) produces ethyl N-methylcarbamate, which is extracted into ethanol and measured by packed column gas chromatography with nitrogen-sensitive detection. The limit of quantitation in human urine is 1 μg/ml and the between-sample coefficient of variation is 5–11%. Simultaneously, N,N-dimethylformamide, N-methylformamide and formamide can also be determined.

3-(S-cysteinyl)glutaric acid, S-(1-methyl-2-carboxyethyl-cysteine and S-(1,2-dimethyl-2-carboxyethyl)cysteine in human urine

3-(S-Cysteinyl)glutaric acid, S-(1-methyl-2-carboxyethyl)cysteine, and S-(1,2-dimethyl-2-carboxyethyl)cysteine have been found in normal human urine as minor components.

S-(2-hydroxy-2-carboxyethylthio)cysteine and S-(carboxymethyltio)cysteine in human urine

S-(2-hydroxy-2-carboxyethylthio)cysteine and S-(carboxymethyltio)cysteine in human urine