Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C > T and c.1286A > C variants affect fibrin clot properties in Polish venous thromboembolic patients.

BACKGROUND AND AIMS: Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients. METHODS: In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness. RESULTS: There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 µM (n = 71, 18.3%), compared to patients with tHcy ≤15 µM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 µM compared to tHcy ≤15 µM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 µM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 µM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05). CONCLUSIONS: Our study indicates that patients with MTHFR variants and tHcy >15 µM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine-Chromatographic Studies.

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography-mass spectrometry (GC-MS) based method was elaborated to identify and quantify TCA in human urine. The GC-MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1-50 µmol L-1 range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC-MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.

Organic Acids Secreted by Lactobacillus spp. Isolated from Urine and Their Antimicrobial Activity against Uropathogenic Proteus mirabilis.

The natural microbiota of the urinary tract includes Lactobacillus spp., which secrete molecules with antimicrobial properties and have antagonistic activity against many pathogens. This paper focuses on the antibacterial effect of Lactobacillus strains isolated from urine against clinical strains of Proteus mirabilis isolated from kidney stones and from urine with coexisting urolithiasis. The study involved analyzing the main antimicrobial molecules secreted by Lactobacillus. In order to indicate which agent had the strongest antimicrobial effect, the supernatants were made alkaline and treated with catalase and high temperature. Both treated and untreated supernatants were analyzed for their activity. Exposing uropathogens to all untreated cell-free supernatants of Lactobacillus significantly reduced their growth, and it was established that these properties were related to organic acid secretion by these strains. Using LC-MS/MS and spectrophotometric techniques, lactic, citric, and succinic acids were determined qualitatively and quantitatively. The influence of these acids on the P. mirabilis growth and biofilm formation and their influence on membrane permeability were also investigated. The results indicate that organic acids secreted by Lactobacillus strains have a high antibacterial potential and could be used as novel agents in the treatment of urinary tract infections caused by P. mirabilis.

The Influence of UV Varnishes on the Content of Cysteine and Methionine in Women Nail Plates-Chromatographic Studies.

The main purpose of this work was to determine if the use of hybrid nail polishes causes changes in concentration of the most important sulfur amino acids that build nail plate structures, cysteine and methionine. We found that the average contents of cysteine and methionine in studied samples before the use of hybrid manicure were 1275.3 ± 145.9 nmol mg-1 and 111.7 ± 23.8 nmol mg-1, respectively. After six months of hybrid manicure use, the average amount of these sulfur amino acids in studied samples were 22.1% and 36.5% lower in the case of cysteine and methionine, respectively. The average amounts of cysteine and methionine in nail plate samples after the use of hybrid manicures were 992.4 ± 96.2 nmol mg-1 and 70.9 ± 14.8 nmol mg-1, respectively. We also confirmed that in studied women the application of UV light varnishes reduced the thickness of the nail plate, from 0.50 ± 0.12 mm before to 0.46 ± 0.12 mm after the use of the hybrid manicure.

Simultaneous Determination of Ciprofloxacin and Ofloxacin in Animal Tissues with the Use of Capillary Electrophoresis with Transient Pseudo-Isotachophoresis.

We have developed a precise and accurate method for the determination of ciprofloxacin and ofloxacin in meat tissues. Our method utilizes capillary electrophoresis with a transient pseudo-isotachophoresis mechanism and liquid-liquid extraction during sample preparation. For our experiment, a meat tissue sample was homogenized in pH 7.00 phosphate buffer at a ratio of 1:10 (tissue mass: buffer volume; g/mL). The extraction of each sample was carried out twice for 15 min with 600 µL of a mixture of dichloromethane and acetonitrile at a 2:1 volume ratio. We then conducted the electrophoretic separation at a voltage of 16 kV and a temperature of 25 °C using a background electrolyte of 0.1 mol/L phosphate-borate (pH 8.40). We used the UV detection at 288 nm. The experimentally determined LOQs for ciprofloxacin and ofloxacin were 0.27 ppm (0.8 nmol/g tissue) and 0.11 ppm (0.3 nmol/g tissue), respectively. The calibration curves exhibited linearity over the tested concentration range of 2 to 10 nmol/g tissue for both analytes. The relative standard deviation of the determination did not exceed 15%, and the recovery was in the range of 85-115%. We used the method to analyze various meat tissues for their ciprofloxacin and ofloxacin contents.

Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane.

Biothiols are extremely powerful antioxidants that protect cells against the effects of oxidative stress. They are also considered relevant disease biomarkers, specifically risk factors for cardiovascular disease. In this paper, a new procedure for the simultaneous determination of human serum albumin and low-molecular-weight thiols in plasma is described. The method is based on the pre-column derivatization of analytes with a thiol-specific fluorescence labeling reagent, monobromobimane, followed by separation and quantification through reversed-phase high-performance liquid chromatography with fluorescence detection (excitation, 378 nm; emission, 492 nm). Prior to the derivatization step, the oxidized thiols are converted to their reduced forms by reductive cleavage with sodium borohydride. Linearity in the detector response for total thiols was observed in the following ranges: 1.76-30.0 mg mL-1 for human serum albumin, 0.29-5.0 nmol mL-1 for α-lipoic acid, 1.16-35 nmol mL-1 for glutathione, 9.83-450.0 nmol mL-1 for cysteine, 0.55-40.0 nmol mL-1 for homocysteine, 0.34-50.0 nmol mL-1 for N-acetyl-L-cysteine, and 1.45-45.0 nmol mL-1 for cysteinylglycine. Recovery values of 85.16-119.48% were recorded for all the analytes. The developed method is sensitive, repeatable, and linear within the expected ranges of total thiols. The devised procedure can be applied to plasma samples to monitor biochemical processes in various pathophysiological states.

Determination of homocysteine thiolactone in human urine by capillary zone electrophoresis and single drop microextraction.

A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of homocysteine thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The background electrolyte was 0.1 M pH 4.75 phosphate buffer. Effective separation was achieved within 6.04 min under the separation voltage of 24 kV (~110 µA). The LOQ and LOD for HTL were 50 and 25 nM urine, respectively. The calibration curve in urine showed linearity in the range of 50-200 nM, with R2 0.9995. The intra- and inter-day precision and recovery were 4.0-14.5% (average 8.7% and 9.3%) and 92.7-115.5% (average 103.6% and 104.8%), respectively. The procedure was successfully applied to analysis of urine samples.

Gas Chromatography-Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva.

Gas chromatography-mass spectrometry technique (GC-MS) is mainly recognized as a tool of first choice when volatile compounds are determined. Here, we provide the credible evidence that its application in analysis can be extended to non-volatile sulfur-containing compounds, to which methionine (Met), homocysteine (Hcy), homocysteine thiolactone (HTL), and cysteine (Cys) belong. To prove this point, the first method, based on GC-MS, for the identification and quantification of Met-related compounds in human saliva, has been elaborated. The assay involves simultaneous disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and acetonitrile (MeCN) deproteinization, followed by preconcentration by drying under vacuum and treatment of the residue with a derivatizing mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS). The validity of the method was demonstrated based upon US FDA recommendations. The assay linearity was observed over the range of 0.5-20 µmol L-1 for Met, Hcy, Cys, and 1-20 µmol L-1 for HTL in saliva. The limit of quantification (LOQ) equals 0.1 µmol L-1 for Met, Hcy, Cys, while its value for HTL was 0.05 µmol L-1. The method was successfully applied to saliva samples donated by apparently healthy volunteers (n = 10).

2-(3-Hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic Acid, Novel Metabolite of Pyridoxal 5'-Phosphate and Cysteine Is Present in Human Plasma-Chromatographic Investigations.

It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5'-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L-1, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.

A Simplified Method for Simultaneous Determination of α-Lipoic Acid and Low-Molecular-Mass Thiols in Human Plasma.

α-Lipoic acid, glutathione, cysteine, and cysteinylglycine can be applied as therapeutic agents in civilization diseases such as diabetes mellitus, cardiovascular diseases, and cancers. On the other hand, a higher concentration of homocysteine can result in health problems and has been indicated as an independent risk factor for cardiovascular disease and accelerated atherosclerosis. Here, the first simplified HPLC-UV assay that enables simultaneous determination of α-lipoic acid and low-molecular-mass thiols in plasma, reduces the number of steps, shortens the total time of sample preparation, and limits the amount of single-use polypropylene laboratory materials is described. The assay is based on reversed-phase high performance liquid chromatography with UV detection and simultaneous reduction of disulfide bound with tris(2-carboxyethyl)phosphine and the selective pre-column derivatization of the thiol group with 1-benzyl-2-chloropyridinium bromide. Linearity in the detector responses for plasma samples were observed in ranges: 0.12-5.0 nmol mL-1 for α-lipoic acid; 2.0-20.0 nmol mL-1 for glutathione, cysteinylglycine, and homocysteine; and 40.0-400.0 for cysteine. The LODs for α-lipoic acid and low-molecular-mass thiols were 0.08 and 0.12 nmol mL-1, respectively, while LOQs were 0.12 and 0.16 nmol mL-1, respectively. The usefulness of the proposed method has been proven by its application to real samples.

Higher Levels of Low Molecular Weight Sulfur Compounds and Homocysteine Thiolactone in the Urine of Autistic Children.

In this study, the levels of concentration of homocysteine thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10-8), Cys (p = 1.49 × 10-10) and CysGly (p = 1.06 × 10-8) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.

Microvascular circulatory dysregulation driven in part by cystathionine gamma-lyase: A new paradigm for cardiovascular compromise in the preterm newborn.

OBJECTIVE: H2 S may explain the dysregulation of microvascular tone associated with poor outcome following preterm birth. In adult vasculature, H2 S is predominantly produced by CSE. We hypothesized that vascular CSE activity contributes to microvascular tone regulation during circulatory transition. METHODS: Preterm (GA62) and full-term (GA69) guinea pig fetuses and neonates were studied. Microvascular blood flow was assessed by laser Doppler flowmetry. Thiosulfate, primary urinary metabolite of H2 S, was determined by high-performance liquid chromatography. Real-time H2 S production was assessed using a microrespiration system in fetal and postnatal (10, 24 hours) skin and heart samples. CSE contribution was investigated by inhibition via propargylglycine. RESULTS: In preterm animals, postnatal H2 S production capacity in peripheral vasculature increased significantly and was significantly reduced by the inhibition of CSE. Urinary thiosulfate correlated with both microvascular blood flow and capacity of the vasculature to produce H2 S. H2 S produced via CSE did not correlate directly with microvascular blood flow. CONCLUSIONS: In preterm neonates, H2 S production increases during fetal-to-neonatal transition and CSE contribution to total H2 S increases postnatally. CSE-dependent mechanisms may therefore underpin the increase in H2 S production over the first 72 hours of life in preterm human neonates, associated with both central and peripheral cardiovascular instability.

Paraoxonase 1 Q192R genotype and activity affect homocysteine thiolactone levels in humans.

Genetic or nutritional deficiencies in 1 carbon and homocysteine (Hcy) metabolism elevate Hcy-thiolactone levels and are associated with cardiovascular and neurologic diseases. Hcy-thiolactone causes protein damage, cellular toxicity, and proatherogenic changes in gene expression in human cells and tissues. A polymorphic cardio-protective enzyme, paraoxonase 1 (PON1), hydrolyzes Hcy-thiolactone in vitro. However, whether Hcy-thiolactone hydrolysis is a physiologic function of the PON1 protein and whether polymorphisms in the PON1 gene affect Hcy-thiolactone levels in humans was unknown. Here we show that the PON1-192 genotype, which affects the enzymatic activity of the PON1 protein, also affected urinary Hcy-thiolactone levels, normalized to creatinine. Carriers of the PON1-192R allele had significantly lower Hcy-thiolactone/creatinine levels than individuals carrying the PON1-192Q allele. Individuals with low serum PON1 paraoxonase activity had significantly higher Hcy-thiolactone/creatinine levels compared with individuals with high paraoxonase activity. In contrast, Hcy-thiolactone/creatinine levels were unaffected by serum PON1 arylesterase activity or by PON1 protein levels. Taken together, these findings suggest that PON1 hydrolyzes Hcy-thiolactone in humans and that the interindividual variations in PON1 genotype/activity can modulate the pathology of hyperhomocysteinemia.-Perla-Kaján, J., Borowczyk, K., Glowacki, R., Nygård, O., Jakubowski, H. Paraoxonase 1 Q192r genotype and activity affect homocysteine thiolactone levels in humans.

Determination of nikethamide by micellar electrokinetic chromatography.

A simple, fast, sensitive and reproducible micellar electrokinetic chromatography (MEKC)-UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000g), separation by MEKC and ultraviolet-absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L sodium dodecyl sulfate and 6.5% ACN. Effective separation was achieved within 5.5 min under a voltage of 21 kV (~90 µA) using a standard fused-silica capillary (effective length 51 cm, 75 µm i.d.). The determined limit of detection for NKD in urine was 1 µmol/L (0.18 µg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4-280 µmol/L (0.71-49.90 µg/mL), with R2 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.

Application of Butylamine as a Conjugative Reagent to On-Column Derivatization for the Determination of Antioxidant Amino Acids in Brain Tissue, Plasma, and Urine Samples.

(1) Antioxidants are involved in body protection mechanisms against reactive oxygen species. Amino acids such as glutathione (GSH) and N-acetylcysteine (NAC) are known to be involved in providing protection against oxidative lethality. A quick and simple method for the determination of NAC and GSH in various biological matrices such as urine, plasma, and homogenates of brain tissues has been developed and described in this work. (2) The assay is based on reversed phase high performance liquid chromatography with spectrofluorimetric detection and on-column derivatization. Butylamine and o-phthaldialdehyde have been used as derivatization reagents. Since o-phthaldialdehyde constitutes a part of the mobile phase, the derivatization reaction and chromatographic separation occur simultaneously. (3) Linearity in the detector response for NAC in human urine was observed in the range of 5-200 nmol mL-1, and NAC and GSH in the brain tissue homogenates were observed in the range of 0.5-5 nmol mL-1 and 0.5-15 nmol mL-1, respectively. Human plasma linearity ranges covered 0.25-5.00 nmol mL-1 and 0.5-15 nmol mL-1 for NAC and GSH, respectively. The LODs for NAC and GSH were 0.01 and 0.02 nmol mL-1 while the LOQs were 0.02 and 0.05 nmol mL-1, respectively. The usefulness of the proposed method was proven through its application to real samples.

Simultaneous determination of total homocysteine, cysteine, glutathione, and N-acetylcysteine in brain homogenates by HPLC.

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10 min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20 µmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2 µmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.

Determination of lipoic acid in human urine by capillary zone electrophoresis.

Fast, simple, and accurate CE method enabling determination of lipoic acid (LA) in human urine has been developed and validated. LA is a disulfide-containing natural compound absorbed from the organism's diet. Due to powerful antioxidant activity, LA has been used for prevention and treatment of various diseases and disorders, e.g. cardiovascular diseases, neurodegenerative disorders, and cancer. The proposed analytical procedure consists of liquid-liquid sample extraction, reduction of LA with tris(2-carboxyethyl)phosphine, derivatization with 1-benzyl-2-chloropyridinium bromide (BCPB) followed by field amplified sample injection stacking, capillary zone electrophoresis separation, and ultraviolet-absorbance detection of LA-BCPB derivative at 322 nm. Effective baseline electrophoretic separation was achieved within 6 min under the separation voltage of 20 kV (∼80 µA) using a standard fused-silica capillary (effective length 51.5 cm, 75 µm id) and BGE consisted of 0.05 mol/L borate buffer adjusted to pH 9. The experimentally determined limit of detection for LA in urine was 1.2 µmol/L. The calibration curve obtained for LA in urine showed linearity in the range 2.5-80 µmol/L, with R2 0.9998. The relative standard deviation of the points of the calibration curve was lower than 10%. The analytical procedure was successfully applied to analysis of real urine samples from seven healthy volunteers who received single 100 mg dose of LA.

Fast and simple MEKC sweeping method for determination of thiosulfate in urine.

A new method for determination of thiosulfate in human urine has been developed and validated. Analytical procedure is very simple and consists of only few steps: derivatization of thiosulfate with 2-chloro-1-methylquinolinium tetrafluoroborate, centrifugation of a mixture, separation of so-formed derivative by micellar electrokinetic chromatography with sweeping and UV detection at 375 nm. A fused-silica capillary with an inlet to detector length of 51.5 cm and a total length of 60 cm (75 µm id) was served as a separation column. The separation voltage of 20.5 kV (∼160 mA) and buffer solution consisting of 0.055 mol/L sodium phosphate (pH 8), 25% acetonitrile, and 0.035 mol/L sodium dodecyl sulfate were found to be the most suitable conditions for the effective separation. The limit of quantification for thiosulfate was 4 µmol/L urine. The method was validated and calibrated for thiosulfate in the range of 4-64 µmol/L (R(2) = 0.9997). The relative standard deviation of the points of the calibration curve varied from 1.2 to 4.8% RSD.

Quantification of homocysteine and cysteine by derivatization with pyridoxal 5'-phosphate and hydrophilic interaction liquid chromatography.

A simple and rapid assay using pyridoxal 5'-phosphate (PLP) as a derivatizing reagent was developed for the simultaneous determination of homocysteine (Hcy) and cysteine (Cys) in human plasma. Derivatization with PLP affords UV-absorbing tetrahydrothiazine and thiazolidine derivatives of Hcy and Cys, respectively. Separation of these derivatives was achieved in 5 min using a hydrophilic interaction liquid chromatography, followed by UV detection at 330 nm. Linearity in detector response was observed over the range of 0.25-20 µM for Hcy and 10-300 µM for Cys. The limit of quantification (LOQ) values for Hcy and Cys were 0.25 and 2.5 µM, respectively. The method was successfully applied to plasma samples donated by apparently healthy volunteers.

Antioxidant efficacy of Kalanchoe daigremontiana bufadienolide-rich fraction in blood plasma in vitro.

CONTEXT: The main source of bufadienolides is toad venom; however, plants such as members of Kalanchoe Adans. (Crassulaceae) genus may also synthesize these bioactive substances. OBJECTIVE: This is the first study on antioxidant effects and cytotoxicity of bufadienolide-rich fraction isolated from Kalanchoe daigremontiana Raym.-Hamet & H. Perrier. MATERIALS AND METHODS: The methanolic fraction was extracted from the plant roots and contained 0.48 mg bufadienolides/mg of dry mass (11α,19-dihydroksytelocinobufagin, bersaldegenin-1-acetate, bersaldegenin-1,3,5-orthoacetate, 19-(acetyloxy)-3ß,5ß,11α,14-tetrahydroxyl-12-oxo-bufa-20,22-dienolide and 19-(acetyloxy)-1ß,3ß,5ß,14-tetrahydroxyl-bufa-20,22-dienolide, mainly). The cytotoxicity of K. daigremontiana fraction was evaluated in an in vitro experimental model of blood platelets. The viability of blood platelets was determined on the basis of a release of lactate dehydrogenase. RESULTS: The fraction scavenged DPPH• radicals, with EC50 of 21.80 µg/mL. Studies on an experimental model of blood plasma under peroxynitrite-induced oxidative stress revealed that the plant preparation had moderate antioxidant properties. Levels of 3-nitrotyrosine and thiol groups indicated that the protective effect of K. daigremontiana was significant mainly for its concentration of 50 µg/mL. No effect was found in prevention of oxidation of low-molecular plasma thiols (glutathione, cysteine and cysteinylglycine). Simultaneously, measurements of lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) indicated that the examined fraction might be effective antioxidant at broader concentration range, that is 1-5 and 25-50 µg/mL for hydroperoxides and TBARS generation, respectively. No cytotoxicity was observed at the concentration range of 1-50 µg/mL. CONCLUSIONS: Based on the obtained results, we suggest that antioxidant activity may additionally contribute to beneficial properties of K. daigremontiana-derived extracts.