Colorimetric determination of cysteamine based on the aggregation of polyvinylpyrrolidone-stabilized silver nanoparticles.

A simple, colorimetric and visual method is described for the determination of cysteamine (CA) using polyvinylpyrrolidone-stabilized silver nanoparticles (PVP-AgNPs) as a colorimetric probe. The sensing method was based on the aggregation of PVP-AgNPs that led to the changes in the color and absorption profile of the probe. The aggregation of PVP-AgNPs in the presence of CA was evidenced by using transmission electron microscopy (TEM), zeta and dynamic light scattering (DLS) measurements. A distinct color transition could be observed with the naked eye from pale yellow color of PVP-AgNPs to purple. PVP-AgNPs probe showed an excellent selectivity towards CA versus other interfering biomolecules, cations and anions. Furthermore, the colorimetric probe had a linear response for CA from 0.1 to 1.0 µM concentration range with the limit of detection (LOD) of 4.9 nM. The prepared probe was successfully utilized for the determination of CA in blood serum as biological samples.

Cysteine-stabilized silver nanoparticles as a colorimetric probe for the selective detection of cysteamine.

The purpose of the present research was to design a method for the colorimetric determination of cysteamine. We have employed cysteine-stabilized silver nanoparticles (AgNPs) as a probe. The addition of cysteamine resulted in the quenching of the 400 nm surface plasmon resonance (SPR) band of the AgNPs. It was accompanied by the appearance of a new absorption band at 560 nm. The colour of the colloidal AgNPs changed from yellow to dark brown within a few seconds. The change in colour of the AgNPs was due to their aggregation induced by the addition of cysteamine. Significantly, other biomolecules such as arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutathione, glycine, methionine and 6-mercaptopurine did not cause any change in the colour of the AgNPs. The limit of detection (LOD) of the method was 0.37 µM. The mechanism of the aggregation of the AgNPs induced by cysteamine has also been described. The method has been applied for the detection of cysteamine in human blood serum.

Biothiols induced colour change of silver nanoparticles: A colorimetric sensing strategy.

A sensor for the detection and determination of bio-thiols (glutathione (GSH) and cysteamine (Cyste)) has been developed by integrating the distinguished distance related optical characteristics of silver nanoparticles with the simplicity of colorimetric technique. In presence of these analytes, shift in surface plasmon resonance (SPR) absorption of silver nanoparticles (AgNPs) with change in its colour was observed. Yellow coloured AgNPs solution becomes colourless in presence of GSH and changes to red in presence of Cyste. FTIR, TEM and DLS studies were used to confirm the mechanism. The difference in absorption of AgNPs in the absence and presence of GSH was found to vary linearly in the range 1.00×10-5M to 5.00×10-7M concentration range with limit of detection at 3.68×10-7M. The method can also be applied to quantify Cyste in the range 1.10×10-6M to 5.00×10-8M with limit of detection at 1.80×10-8M. The utility of the proposed colorimetric assay is validated by determination of GSH and Cyste in artificial blood serum.

MoS2/reduced graphene oxide nanocomposite for sensitive sensing of cysteamine in presence of uric acid in human plasma.

A hybrid nanocomposite of MoS2 nanosheets and reduced graphene oxide (rGO) was fabricated by a facile and effective method. The morphology and structure of the nanocomposite (MoS2-rGO) were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The MoS2 nanosheets were uniformly anchored on the rGO framework with strong adhesion. A glassy carbon electrode modified by drop-casting with MoS2-rGO was used for the electrochemical oxidation of cysteamine (CA) in the presence of uric acid (UA). Under optimum conditions, the anodic peak current of CA shows a linear relation with the CA concentration between 0.01 and 20µM with a detection limit of 7nM. The proposed electrochemical sensor was used for determination of CA in human plasma.

VNN1, a potential biomarker for pancreatic cancer-associated new-onset diabetes, aggravates paraneoplastic islet dysfunction by increasing oxidative stress.

In our previous clinical microarray analysis, we were the first to report on Vanin-1 (VNN1) as a novel clinically derived biomarker of pancreatic cancer-associated new-onset diabetes (PCAND). The functional mechanisms of VNN1 in the pathogenesis of PCAND, however, are not completely understood. In the present study, we further extend our previous clinical study to include laboratory research. The functions and mechanisms of neoplastic overexpressed VNN1 in PCAND have been explored using a co-culture model. Furthermore, the serum concentrations and discrimination power of downstream molecules of VNN1 were tested in a PCAND cohort. Pancreatic ductal adenocarcinoma (PDA) overexpressed VNN1 further aggravates paraneoplastic islet dysfunction; decreases in GSH/PPAR-γ concentrations and increases in ROS/cysteamine might be primary cause of this effect. Clinical serum analyses revealed that the expression profiles of these molecules were aberrant in the PCAND group. Our results further demonstrated that PCAND is a type of paraneoplastic diabetes. As the only clinically derived biomarker for PCAND screening available today, the biological role of VNN1 in triggering oxidative stress within the pancreatic microenvironment is important. The molecules downstream of VNN1 are also potential biomarkers for PCAND screening.

Pharmacokinetics of cysteamine bitartrate following intraduodenal delivery.

Cysteamine is approved for the treatment of cystinosis and is being evaluated for Huntington's disease and non-alcoholic fatty liver disease. Little is known about the bioavailability and biodistribution of the drug. The aim was to determine plasma, cerebrospinal fluid (CSF), and tissue (liver, kidney, muscle) cysteamine levels following intraduodenal delivery of the drug in rats pretreated and naïve to cysteamine and to estimate the hepatic first-pass effect on cysteamine. Healthy male rats (n = 66) underwent intraduodenal and portal (PV) or jugular (JVC) venous catheterization. Half were pretreated with cysteamine, and half were naïve. Following intraduodenal cysteamine (20 mg/kg), serial blood samples were collected from the PV or the JVC. Animals were sacrificed at specific time points, and CSF and tissue were collected. Cysteamine levels were determined in plasma, CSF, and tissue. The Cmax was achieved in 5-10 min from PV and 5-22.5 min from JVC. The PV-Cmax (P = 0.08), PV-AUC0-t (P = 0.16), JVC-Cmax (P = 0.02) and JVC-AUC0-t (P = 0.03) were higher in naive than in pretreated animals. Plasma cysteamine levels returned to baseline in ≤120 min. The hepatic first-pass effect was estimated at 40%. Peak tissue and CSF cysteamine levels occurred ≤22.5 min, but returned to baseline levels ≤180 min. There was no difference in CSF and tissue cysteamine levels between naïve and pretreated groups, although cysteamine was more rapidly cleared in the pretreated group. Cysteamine is rapidly absorbed from the small intestine, undergoes significant hepatic first-pass metabolism, crosses the blood brain barrier, and is almost undetectable in plasma, CSF, and body tissues 2 h after ingestion. Sustained-release cysteamine may provide prolonged tissue exposure.

Twice-daily cysteamine bitartrate therapy for children with cystinosis.

OBJECTIVE: Cystinosis causes renal and other organ failure. Regular 6-hourly cysteamine bitartrate (Cystagon; Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A formulation of cysteamine requiring less frequent dosing may improve compliance and possibly patient outcome. METHODS: Enteric-release cysteamine was prepared. For a period of 1 month, patients received their regular cysteamine dose every 6 hours (stage I). The patients then underwent pharmacokinetic and pharmacodynamic studies following washout periods using single-doses of cysteamine and enteric-release cysteamine (stage II). Finally, the patients commenced regular enteric-release cysteamine therapy (stage III). Weekly trough white blood cell (WBC) cystine levels were recorded. RESULTS: Seven children with cystinosis (mean age, 11.8 years; range, 8-17 years) who received cysteamine and enteric-release cysteamine (mean dose, 45 and 28.8 mg/kg body weight/day, respectively) had mean WBC cystine levels of 0.7+/-0.3 and 0.41+/-0.22 nmol half-cystine/mg protein in study stages I and III, respectively. Study stage II showed that the mean time (T(max)) to reach the maximum plasma cysteamine level (C(max)) was longer for enteric-release cysteamine than for cysteamine (176 minutes vs 60 minutes; P=.001), but the mean C(max) at the same dose was similar. Mean serum gastrin levels were similar after ingestion of cysteamine and enteric-release cysteamine. CONCLUSIONS: Twelve-hour enteric-release cysteamine, given at approximately 60% of the previous daily dose of cysteamine, was effective in maintaining trough WBC cystine levels within a satisfactory range.

A validated HPLC-fluorescence method with a semi-micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma.

A semi-micro column HPLC-fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) were isocratically separated within 12 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH(3)CN as a mobile phase. The purity and similarity of SBD-thiols by a multi-wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal-to-noise ratio of 3 were 0.16, 0.47 and 0.03 microm, respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572-1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and -0.35, respectively.

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.

Understanding intestinal cysteamine bitartrate absorption.

OBJECTIVES: To test the hypothesis that a controlled-release preparation of cysteamine, with fewer daily administrations, would improve the quality of life for patients with cystinosis. STUDY DESIGN: A specifically designed nasoenteric tube was used to administer cysteamine directly into the stomach, small intestine (SI) and colon and serial plasma cysteamine, serum gastrin and leukocyte cystine levels were measured. RESULTS: Eight control subjects (mean age 23.2 years) and 6 subjects with cystinosis (mean age 15.2 years) were studied. Cysteamine absorption (maximum concentration and area under the curve of the concentration-time gradient) was greater from the SI than stomach or cecum (P < .01). Leukocyte cystine depletion was greater after delivery of cysteamine into the SI than stomach or cecum; this effect was associated with the plasma cysteamine maximum concentration and area under the curve (P < .001 and < .02, respectively). Gastrin levels were not affected by site of drug delivery and were elevated only in patients with cystinosis with gastrointestinal symptoms. CONCLUSIONS: The absorption of cysteamine and the effect of this agent on leukocyte cystine depletion are more profound after SI administration. Enteric-coated cysteamine, targeted for SI release, may require fewer daily dosages. Not all patients with cystinosis require acid-suppression therapy.

Esomeprazole therapy for gastric acid hypersecretion in children with cystinosis.

Oral cysteamine therapy prevents natural disease progression in children with cystinosis, but it may cause severe gastrointestinal (GI) symptoms through gastric acid-hypersecretion. The purpose of this study was to assess the value of esomeprazole in controlling cysteamine-induced acid-hypersecretion and GI symptoms in children with cystinosis. Subjects underwent upper GI endoscopy and biopsy, serum gastrin and cysteamine measurements as well as acid secretion studies (basal, maximal and peak acid output, BAO, MAO, PAO) before and during esomeprazole therapy. A symptom score (maximum 14 points) was devised to monitor symptoms. Twelve children (mean age 5.8 years) were studied. Cysteamine ingestion resulted in mean MAO and PAO significantly higher than mean BAO, both before and during esomeprazole therapy. PAO was usually within 60 min of cysteamine ingestion. Esomeprazole therapy significantly reduced MAO (P<0.01) and PAO (P<0.01). The mean symptom score fell from 6.4 to 0.7 (P<0.0001) during esomeprazole therapy. The mean final dose of esomeprazole was 1.7 mg/kg per day (range 0.7 mg/kg per day to 2.75 mg/kg per day). Plasma cysteamine levels were not affected by acid-suppression therapy. One child had multi-nucleated parietal cells. Cysteamine-induced gastric acid-hypersecretion and GI symptoms are dramatically reduced with esomeprazole therapy. Esomeprazole does not alter cysteamine absorption and is very well tolerated in children.

Treatment of YAC128 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease.

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine --> cysteamine --> hypotaurine --> taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (< or = 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wild-type littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.

Determination of total cysteamine in human plasma in the form of its 2-S-quinolinium derivative by high performance liquid chromatography.

Cysteamine (mercaptamine) can be determined in plasma by liquid chromatography with ultraviolet detection after precolumn derivatization. The plasma is reduced with sodium borohydride in order to convert disulfides to thiols, and derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. The 2-S-quinolinium derivative of cysteamine is then separated from other thiols derivatives present in the plasma, and quantitated using high-performance liquid chromatography and then detection at 355 nm. Peaks from the main plasma thiols cysteine, cysteinylglycine, glutathione and homocysteine are also observed and can be measured as needed. The cystamine standards added to the plasma before the reduction step show that the response of the detector is linear within the range studied, from 0.1 to 40 micromol/L plasma. The imprecisions at the bottom and the top of the calibration range were 11.17 and 0.8% and the inaccuracies 8.64 and 1.50%, respectively, and the lower limit of quantitation was 0.1 nmol cysteamine in 1 ml of plasma.

Determination of total cysteamine in human serum by a high-performance liquid chromatography with fluorescence detection.

A convenient, reliable and rapid method for determination of total cysteamine in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves reduction of samples with dithiothreitol, derivatization of total cysteamine by addition of monobromobimane and protein precipitation by perchloric acid. The calibration curve was linear in the range 2-150 nmol ml-1 and the detection limit was 0.5 nmol ml-1. This method was successfully applied for a pharmacokinetic study of three cysteamine derivatives in healthy volunteers without any interference from coexisting substances.

Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine.

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 micromol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

Pathways of cysteine metabolism in MND/ALS.

Analysis of plasma from MND/ALS patients has shown no significant differences in metabolism of cysteine derivatives, although a sub-set of the population has raised glutamate values. Cysteine dioxygenase was found to have reduced activity in vitro, consistent with previous findings of a high plasma cysteine/sulphate ratio.

Determination of total cysteamine in urine and plasma samples by gas chromatography with flame photometric detection.

A sensitive and selective method for the determination of total cysteamine in urine and plasma samples by gas chromatography (GC) has been developed. After reduction of the sample with sodium borohydride, the liberated cysteamine was converted into its N,S-diisobutoxycarbonyl derivative and measured by GC with flame photometric detection using a DB-210 capillary column. The calibration curve was linear in the range 0.2-5.0 nmol, and the detection limit, at a signal-to-noise ratio of 3, was ca. 0.5 pmol injected. Using this method, total cysteamine in urine and plasma samples could be accurately and precisely determined without any interference from coexisting substances. Analytical results for the determination of total cysteamine in urine and plasma samples from normal subjects are presented.

A comparison of the effectiveness of cysteamine and phosphocysteamine in elevating plasma cysteamine concentration and decreasing leukocyte free cystine in nephropathic cystinosis.

Cysteamine (beta-mercaptoethylamine, MEA) is currently used to treat children with nephropathic cystinosis. In this study MEA was compared to phosphocysteamine (MEAP), a phosphorothioester that tastes and smells better than MEA, with respect to its ability to elevate plasma MEA and deplete leukocytes of cystine. Studies were performed in six children with nephropathic cystinosis ranging in age from 2 to 10 yr. After equimolar oral doses of either MEA or MEAP plasma cysteamine was determined at various times for 6 h. MEA was determined by sodium borohydride reduction followed by high-performance liquid chromatography separation and electrochemical detection. Leukocyte cystine was measured before and 1 and 6 h after drug administration. Peak plasma MEA was obtained 30 min to 1 h after a dose and was not significantly different when MEA (48.6 +/- 10.7, mean +/- SD) or MEAP (54.1 +/- 20.2) was given. Significant plasma MEA concentrations were seen as early as 15 min after an oral dose, indicating rapid absorption. Analysis of vomitus indicated that hydrolysis of the phosphate group of MEAP occurs in the stomach. The percent decrease in leukocyte cystine content obtained with MEA administration (61.9%) was not significantly different from the decrease observed when MEAP was administered (65.3%). MEA and MEAP appear to be equally effective in their cystine-depleting properties.

Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection.

Cysteamine is currently used to treat children with the inherited disorder nephropathic cystinosis. A method for the quantitative determination of this aminothiol in human plasma is presented. Whole plasma was reduced with sodium borohydride to convert disulfides to thiols. Cysteamine was then separated by high-performance liquid chromatography and detected electrochemically. The recovery of standard cysteamine added to plasma was 96.6 +/- 1.9%. In a patient with cystinosis, an oral dose of cysteamine was absorbed rapidly, with plasma cysteamine reaching a maximum of 56 microM 1 h after the dose. By 1.8 h the plasma cysteamine concentration had decreased to one-half the maximum value.

The determination of cysteamine in physiological fluids by HPLC with electrochemical detection.

Cysteamine, an amino thiol, was separated by rapid isocratic cation exchange chromatography and detected by electrochemical oxidation at a platinum electrode maintained at +0.45 V relative to an Ag/AgCl reference electrode. Eluent pH and electrode working potentials were optimized and the effects of alternative buffers and organic modifiers have been examined. On column sensitivity for cysteamine was 1.5 pmol at a signal-to-noise ratio of 5. Although the specificity was good, plasma samples required maximal sensitivity whereas urine samples required greater selectivity, which was achieved by use of lower working potentials. Cysteamine concentrations were determined in serial samples of plasma and urine from volunteers who had received a single oral dose of 200 mg of the drug. Cysteamine was rapidly oxidized in vivo, and detection required prior reduction with dithiothreitol before analysis.