RESUMO
Tryptophan (an essential amino acid) and its clinically important metabolite-kynurenine contribute to several fundamental biological processes and methods that allow their determination in biological samples are in demand. The novelty of the work was a demonstration of the utility of two polymers: 4-vinylpyridine crosslinked with trimethylolpropane trimethacrylate (poly(4VP-co-TRIM)) or 1,4-dimethacryloyloxybenzene (poly(4VP-co-14DMB))-in terms of human serum clean-up for simultaneous LC-MS determination of tryptophan and kynurenine. The goal was to achieve a reduction of the matrix effect, which is responsible for signal suppression, with minimal capture of analytes. The adsorption properties of the polymeric beads were studied by evaluating the adsorption kinetics and isotherms in model matrices. Therefore, the adsorption capacities of both molecules were not efficient, the tested 4-vinylpyridine-based copolymers have shown great promise (especially poly(4VP-co-TRIM)) as sorbents for serum clean-up. In the model human serum matrix, poly(4VP-co-TRIM) provided good recoveries of tryptophan and kynurenine (76% and 87%, respectively) and allowed for the reduction of the matrix effect. Performances of both copolymers were compared to those of commercially available sorbents (octadecylsilane, activated charcoal, and primary secondary amine).
Assuntos
Cinurenina , Espectrometria de Massa com Cromatografia Líquida , Polímeros , Piridinas , Triptofano , Humanos , Adsorção , Cinurenina/sangue , Cinurenina/análogos & derivados , Cinurenina/química , Espectrometria de Massa com Cromatografia Líquida/métodos , Polímeros/química , Piridinas/química , Piridinas/sangue , Triptofano/sangue , Triptofano/químicaRESUMO
Animals in urban areas often encounter novel and potentially stressful conditions. It is important to understand how wildlife cope with anthropogenic disturbance. To investigate this specific adaptation we live-trapped squirrels in two study sites in Warsaw: a forest reserve and an urban park and we estimated stress responses at three levels: long-term and medium-term stress (the level of stress hormones, i.e. cortisol and cortisone concentrations, in hair and feces) and acute reaction to human-induced stress (measured during handling with the aid of the three indices: breath rate, struggle rate, and vocalization). According to GLMM models no difference in the stress hormones level was found between the two populations. The only differences in cortisol concentrations clearly depended on the season, i.e. being higher in autumn and winter comparying to other seasons. There was no influence of sex, or reproductive status on stress hormones. Forest squirrels had significantly higher breath rates, suggesting they were more stressed by handling. There was no difference in the struggle rate between study areas, this index was mostly affected by season (i.e. being highest in winter). First-trapped squirrels vocalized less than during the subsequent trappings. Assumingly, during the first, and more stressful trapping, squirrels used 'freezing' and/or little vocalization, while during next captures they used alarm calls to warn conspecifics. Overall, we showed that the two squirrel populations differed only in terms of their breath rate. This suggests that they did not differ in medium-term and long-term stress in general, but they can differ in acute response to handling. This also suggests that both populations were similarly affected by environmental factors. The lack of clear effects may also be due to population heterogeneity. Thus, in order to assess the effects of anthropogenic stressors a broader range of indicators and diverse analytical methods, including behavioral analyses, should be employed.
Assuntos
Hidrocortisona , Sciuridae , Estresse Fisiológico , Animais , Sciuridae/fisiologia , Hidrocortisona/metabolismo , Hidrocortisona/análise , Estresse Fisiológico/fisiologia , Humanos , Masculino , Estações do Ano , Feminino , Cidades , Vocalização Animal/fisiologia , População UrbanaRESUMO
A new method for the determination of main glucocorticoids (cortisol, cortisone, and corticosterone) in hair by liquid chromatography-tandem mass spectrometry was developed. Glucocorticoids were extracted from hair shafts using methanol followed by solid-phase extraction. A validation test was performed using hair from three species of wild mammals with different body size (0.2-800 kg), lifestyle (terrestrial, burrowing and arboreal species), social organization (living in herds or solitary), and different predicted type of hair glucocorticoids: European bison (Bison bonasus), European hamster (Cricetus cricetus), and Eurasian red squirrel (Sciurus vulgaris). Regardless of the species evaluated, the method shows good linearity for all analytes accompanied by satisfactory accuracy (91-114%) and precision (RSD < 13%). Depending on the analyte and hair origin, the calculated limits of quantification were between 0.05 and 1.19 ng/mL, which corresponds to 1.28-31.51 pg/mg. Using cortisol and cortisone as examples, we have demonstrated that measuring multiple glucocorticoids simultaneously provides more comprehensive information than solely concentrating on one, thereby contributing to a more balanced and reliable interpretation of the acquired results. However, the utility of cortisol metabolites as markers of stress response in keratinized tissues should be substantiated by additional experimental studies on targeted animals. We posit that this paper could serve as a crucial catalyst to prompt such experiments.
Assuntos
Bison , Cortisona , Animais , Glucocorticoides/análise , Hidrocortisona/análise , Cromatografia Líquida/métodos , Cortisona/análise , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massa com Cromatografia Líquida , Cabelo/química , BiologiaRESUMO
Tryptophan metabolism plays an essential role in human health. In mammals, about 95% of dietary tryptophan is metabolized through the kynurenine pathway, which is associated with the development of several pathologies, including neurodegeneration. Some of the kynurenine pathway metabolites are agonists of the aryl hydrocarbon receptor involved in metabolic functions, inflammation, and carcinogenesis. Thus, their origins, fates, and roles are of widespread interest. Except for being produced endogenously, these metabolites can originate from exogenous sources (e.g., food) and undergo absorption in the digestive tract. Recently, a special focus on exogenous sources of tryptophan metabolites was observed. This overview summarizes current knowledge about the occurrence of the kynurenine pathway metabolites (kynurenines) in food and the analytical method utilized for their determination in different food matrices. Special attention was paid to sample preparation and chromatographic analysis, which has proven to be a core technique for the detection and quantification of kynurenines. A discussion of the fate and role of dietary kynurenines has also been addressed. This review will, hopefully, guide further studies on the impact of dietary kynurenines on human health.
Assuntos
Cinurenina , Triptofano , Animais , Humanos , Cinurenina/metabolismo , Triptofano/metabolismo , Inflamação , Dieta , Cromatografia , Mamíferos/metabolismoRESUMO
Fifty-seven samples of honey of different types and origins were screened for nicotine and nine mycotoxins (aflatoxin B1, aflatoxin B2, fusarenon X, ochratoxin A, penicillic acid, zearalenone, sterigmatocystin, gliotoxin, and patulin). The sample set consisted of monofloral, multifloral, nectar, honeydrew, cream, and artificial honey originating mainly from Poland. The physicochemical characterization of honey was performed by determining colour (by Pfund method), water content (by refractometry), total phenolics and flavonoids content (by spectrophotometry). For nicotine and mycotoxins determination a QuEChERS-based UHPLC-ESI-MS/MS method was developed and validated. Analyses were carried out in alkaline conditions to ensure patulin-methanol adduct formation and facilitate this mycotoxin detection. About 33% of tested honey samples were contaminated by nicotine or/and mycotoxins. However, the presence of mycotoxins was not related to herein evaluated physicochemical parameters of honey samples.
Assuntos
Mel , Patulina , Nicotina , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em TandemRESUMO
Catabolism of tryptophan (Trp) is modulated by physical activity and provides a pool of active compounds: Trp is considered a calmative agent, kynurenine (Kyn) and 3-hydroxykynurenine (3-HKyn) show neurotoxic effects, kynurenic acid (Kyna) and xanthurenic acid (XA) have neuroprotective properties like nicotinamide (NAm), while serotonin is the neurotransmitter. The study was conducted to investigate the dependence of exercise intensity, measured by plasma lactic acid (LA) concentration, on the level of Trp, its catabolites (serotonin, Kyn, 3-HKyn, Kyna and XA), and NAm in Thoroughbred horses. A total of 18 young race Thoroughbred horses were investigated during exercise tests. Blood samples for analysis were collected: at rest, 10 min after the end of the exercise, and 60 min after the end of the exercise. Plasma LA was determined by the enzymatic method, Trp, and other metabolites using liquid chromatography coupled with mass spectrometry. In horses performing intense exercise, the concentration of LA, Kyn, XA and NAm was increased, while Trp was decreased. Significant correlations were detected for exercise-induced increase in LA and 3-HKyn, XA, and NAm. Considering the scope of changes in analyzed data, there is an expected neutral effect on the health status of exercised horses.
RESUMO
The paper outlines a procedure based on ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry and QuEChERS pretreatment for the determination of patulin in selected berries and stone fruits. The mycotoxin was determined in the positive electrospray ionization mode utilizing the formation of its methanol adduct. The method was validated for 4 fruit matrices: strawberry (Rosaceae, Fragaria), raspberry (Rosaceae, Rubus), redcurrant (Rosaceae, Prunus) and sour cherry (Grossulariaceae, Ribes). The fruits were characterized by total phenolics, flavonoids, and anthocyanin contents and obtained results were within the ranges of 2.65-11.22 mg GAE/g, 1.06-4.67 mg CE/g, 0.05-1.44 mg cyd-3-glu/g, respectively. The limits of quantification ranged from 0.65 µg/kg to 3.01 µg/kg depending on the fruit type. The method was applied to 71 fruit samples collected in Poland. Analysis of moldy fruit samples has shown a high incidence of patulin contamination.
Assuntos
Fragaria , Patulina , Antocianinas/análise , Cromatografia Líquida de Alta Pressão/métodos , Frutas/química , Metanol/análise , Patulina/análise , Espectrometria de Massas em Tandem/métodosRESUMO
We examined the relationships of tryptophan (Trp) and the metabolites of the kynurenine pathway (KP) to the occurrence of type 2 diabetes (T2D) and metabolic risk factors in obese middle-aged women. The study included 128 obese women divided into two subgroups: a normoglycemic group (NG, n = 65) and a T2D group (n = 63). The concentrations of serum tryptophan (Trp), kynurenine (Kyn), 3-hydroxykynurenine (3HKyn), quinolinic acid (QA), and kynurenic acid (Kyna) were analyzed using ultra-high-performance liquid chromatography coupled with electrospray ionization/triple quadrupole mass spectrometry. Blood biochemical parameters and anthropometric parameters were measured. The women with T2D had significantly higher Trp, Kyna, Kyna/QA ratio, and Kyna/3HKyn ratio values than the NG women. Logistic regression analysis showed that the concentrations of Trp and Kyna and the values of the Kyna/3HKyn ratio were most strongly associated with T2D occurrence, even after controlling for confounding factors. The model with Trp level and Kyna/3HKyn ratio accounted for 20% of the variation in the presence of T2D. We also showed a different pattern of correlations between kynurenines and metabolic factors in the NG and T2D women, which was mostly reflected in the stronger relationship between BMI and KP metabolites in the NG obese women. An increase in Trp and Kyna levels with an accompanying increase in Kyna/3HKyn ratio value is associated with the occurrence of T2D in obese middle-aged women.
RESUMO
In this paper, we propose the first analytical procedure-using a screen-printed carbon electrode modified with carbon nanofibers (SPCE/CNFs)-for the detection and quantitative determination of an electroactive disubstituted fused triazinone, namely 4-Cl-PIMT, which is a promising anticancer drug candidate. The electrochemical performances of the sensor were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square-wave adsorptive stripping voltammetry (SWAdSV). The presence of carbon nanofibers on the sensor surface caused a decrease in charge-transfer resistance and an increase in the active surface compared to the bare SPCE. Under the optimised experimental conditions, the proposed voltammetric procedure possesses a good linear response for the determination of 4-Cl-PIMT in the two linear ranges of 0.5-10 nM and 10-100 nM. The low limits of detection and quantification were calculated at 0.099 and 0.33 nM, respectively. In addition, the sensor displays high reproducibility and repeatability, as well as good selectivity. The selectivity was improved through the use of a flow system and a short accumulation time. The SWAdSV procedure with SPCE/CNFs was applied to determine 4-Cl-PIMT in human serum samples. The SWAdSV results were compared to those obtained by the ultra-high-performance liquid chromatography coupled with electrospray ionization/single-quadrupole mass spectrometry (UHPLC-ESI-MS) method.
Assuntos
Antineoplásicos/análise , Carbono/química , Técnicas Eletroquímicas/instrumentação , Triazinas/análise , Espectroscopia Dielétrica , Eletrodos , Humanos , Limite de Detecção , Estrutura Molecular , Nanofibras/química , Propriedades de SuperfícieRESUMO
BACKGROUND: This study aimed to assess the importance of selected kynurenines measured in peritoneal fluid, lavage washings, and blood serum in patients with advanced gastric cancer (GC) based on the clinical and pathological staging of TNM for a more precise evaluation of the stage of the disease. METHODS: Data were collected from a prospectively maintained database of all patients operated on advanced GC between July 2018 and August 2020. In total, 98 patients were eligible for the analysis according to the REMARK guidelines. RESULTS: Among the various kynurenines analyzed in this study, we found that the median concentration of anthranilic acid (AA) in the peritoneal lavage washings was significantly higher in patients with positive nodes (pN1-3) compared to those with negative nodes (pN0) (P = 0.0100). Based on the ROC analysis, AA showed diagnostic utility in the differentiation of the pN staging (P = 0.0047). Furthermore, there was a positive correlation between AA in peritoneal fluid with stage pN (P = 0.0116) and a positive correlation between AA in peritoneal lavage washings with stage cT (P = 0.0101). We found that the median concentration of kynurenine (Kyn) in peritoneal lavage washings was significantly higher in patients with cM1 compared to cM0 patients (P = 0.0047). Based on the ROC analysis, Kyn showed diagnostic utility in cM staging differentiation (P < 0.0001). There was a positive correlation between peritoneal Kyn and stage of cM (P = 0.0079). CONCLUSIONS: AA and Kyn measured in peritoneal lavage indicate advanced GC and may be considered in the future as valuable adjunct tools in TNM staging of advanced GC.
RESUMO
In recent years, tryptophan metabolism via the kynurenine pathway has become one of the most active research areas thanks to its involvement in a variety of physiological processes, especially in conditions associated with immune dysfunction, central nervous system disorders, autoimmunity, infection, diabetes, and cancer. The kynurenine pathway generates several metabolites with immunosuppressive functions or neuroprotective, antioxidant, or toxic properties. An increasing body of work on this topic uncovers a need for reliable analytical methods to help identify and quantify tryptophan metabolites at physiological concentrations in biological samples of different origins. Recent methodological advances in the fabrication and application of electrochemical sensors promise a rise in the future generation of novel analytical systems. This work summarizes current knowledge and provides important suggestions with respect to direct electrochemical determinations of kynurenine pathway metabolites (kynurenines) in complex biological matrices. Measurement challenges, limitations, and future opportunities of electroanalytical methods to advance study of the implementation of kynurenines in disease conditions are discussed.
Assuntos
Cinurenina , TriptofanoRESUMO
Nowadays, development of analytical methods responding to a need for rapid and accurate determination of human metabolites is highly desirable. Herein, an electrochemical method employing a Nafion-coated glassy carbon electrode (Nafion/GCE) has been developed for reliable determination of kynurenine (a key tryptophan metabolite) using a differential pulse adsorptive stripping voltammetry. To our knowledge, this is the first analytical method to allow for kynurenine determination at the Nafion-coated electrode. The methodology involves kynurenine pre-concentration in 0.1 M H2SO4 in the Nafion film at the potential of +0.5 V and subsequent stripping from the electrode by differential pulse voltammetry. Under optimal conditions, the sensor can detect 5 nM kynurenine (for the accumulation time of 60 seconds), but the limit of detection can be easily lowered to 0.6 nM by prolonging the accumulation time to 600 seconds. The sensor shows sensitivity of 36.25 µAµM-1cm-2 and 185.50 µAµM-1cm-2 for the accumulation time of 60 and 600 seconds, respectively. The great advantage of the proposed method is easy sensor preparation, employing drop coating method, high sensitivity, short total analysis time, and no need for sample preparation. The method was validated for linearity, precision, accuracy (using a high-performance liquid chromatography), selectivity (towards tryptophan metabolites and different amino acids), and recovery. The comprehensive microscopic and electrochemical characterization of the Nafion/GCE was also conducted with different methods including atomic force microscopy (AFM), optical profilometry, time-of-flight secondary ion mass spectrometry (TOF-SIMS), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). The method has been applied with satisfactory results for determination of kynurenine concentration in a culture medium collected from the human ovarian carcinoma cells SK-OV-3 and to measure IDO enzyme activity in the cancer cell extracts.
RESUMO
Metabolites and enzymes involved in the kynurenine pathway (KP) are highly promising targets for cancer treatment, including gastrointestinal tract diseases. Thus, accurate quantification of these compounds in body fluids becomes increasingly important. The aim of this study was the development and validation of the UHPLC-ESI-MS/MS methods for targeted quantification of biologically important KP substrates (tryptophan and nicotinamide) and metabolites(kynurenines) in samples of serum and peritoneal fluid from gastric cancer patients. The serum samples were simply pretreated with trichloroacetic acid to precipitate proteins. The peritoneal fluid was purified by solid-phase extraction before analysis. Validation was carried out for both matrices independently. Analysis of the samples from gastric cancer patients showed different accumulations of tryptophan and its metabolites in different biofluids of the same patient. The protocols will be used for the evaluation of tryptophan and kynurenines in blood and peritoneal fluid to determine correlation with the clinicopathological status of gastric cancer or the disease's prognosis.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cinurenina/metabolismo , Neoplasias Gástricas/metabolismo , Espectrometria de Massas em Tandem/métodos , Líquido Ascítico/metabolismo , Humanos , Ácido Tricloroacético/metabolismo , Triptofano/metabolismoRESUMO
BACKGROUND: Immune modulatory factors like indoleamine 2,3-dioxygenase 1 (IDO1) generating kynurenine (Kyn) and receptor for advanced glycation end-products (RAGE) contribute to endometrial and cancer microenvironment. Using adequate experimental models is needed to learn about the significance of these molecular factors in endometrial biology. In this paper we study IDO1 activity and RAGE expression in the in vitro cultured primary human endometrial cells derived from cancerous and noncancerous tissue. METHODS: The generated primary cell cultures from cancer and noncancerous endometrial tissues were characterized using immunofluorescence and Western Blot for expression of endometrial and cancer markers. IDO1 activity was studied by Kyn quantification with High Performance Liquid Chromatography with Diode Array Detector. RESULTS: The primary cultures of endometrial cells were obtained with 80% success rate and no major genetic aberrations. The cells retained in vitro expression of markers (mucin MUC1 and HER2) or immunomodulatory factors (RAGE and IDO1). Increased Kyn secretion was associated with cancer endometrial cell culture in contrast to the control one. CONCLUSIONS: Primary endometrial cells express immune modulatory factors RAGE and IDO1 in vitro associated with cancer phenotype of endometrium.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias do Endométrio/patologia , Endométrio/patologia , Imunomodulação , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Microambiente Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/metabolismo , Endométrio/imunologia , Endométrio/metabolismo , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Cultura Primária de Células , Prognóstico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
Liquid chromatography-tandem mass spectrometry (LC-MS/MS), colorimetry, and bioassays were employed for the evaluation of the ability of endophytic bacterial strains to synthesize indole-related compounds (IRCs) and in particular indole-3-acetic acid (IAA). A total of 54 endophytic strains belonging to seven bacterial genera isolated from tissues of common and spelt wheat cultivars were studied. The endophytic bacteria isolated from different tissues of the tested wheat types were capable of IRCs production, including IAA, which constituted from 1.75% to 52.68% of all IRCs, in in vitro conditions via the tryptophan dependent pathway. The selected post-culture medium was also examined using a plant bioassay. Substantial growth of wheat coleoptile segments treated with the bacterial post-culture medium was observed in several cases. Our data suggest that the studied endophytic bacteria produce auxin-type compounds to support plant development. Summarizing, our approach to use three complementary methods for estimation of IRCs in different endophytic strains provides a comprehensive picture of their effect on wheat growth.
Assuntos
Colorimetria/métodos , Endófitos/metabolismo , Ácidos Indolacéticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Triticum/microbiologia , Bactérias/metabolismo , Bioensaio , Cromatografia Líquida , Endófitos/química , Endófitos/isolamento & purificação , Ácidos Indolacéticos/análise , Indóis/metabolismo , PolôniaRESUMO
The kynurenine pathway is the major tryptophan degradation routes generating bioactive compounds important in physiology and diseases. Depending on cell type it is initiated enzymatically by tryptophan-2,3-dioxygenase (TDO) or indoleamine-2,3-dioxygenase 1 and 2 (IDO1 and IDO2) to yield N-formylkynurenine as the precursor of further metabolites. Herein, we describe an accurate high-pressure liquid chromatography coupled with a diode array detector (HPLC-DAD) method to serve for IDO1 activity determination in human cancer cells cultured in vitro. Enzymatic activity was expressed as the rate of Ê-kynurenine generation by 1 mg of proteins obtained from cancer cells. Our approach shows the limit of detection and limit of quantification at 12.9 and 43.0 nM Kyn, respectively. Applicability of this method was demonstrated in different cells (ovarian and breast cancer)exposed to various conditions and has successfully passed the validation process. This approach presents a useful model to study the role of kynurenine pathway in cancer biology.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/análise , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Espectrometria de Massas em Tandem , Células Tumorais CultivadasRESUMO
A carbon nanofibers modified screen-printed carbon sensor (SPCE/CNFs) was applied for the determination of a novel promising anticancer agent candidate (ethyl 8-(4-methoxyphenyl)-4-oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3-carboxylate, EIMTC) using square-wave voltammetry (SWV). It is the first method for the quantitative determination of EIMTC. The modified screen-printed sensor exhibited excellent electrochemical activity in reducing EIMTC. The peak current of EIMTC was found to be linear in two concentration ranges of 2.0 × 10-9 - 2.0 × 10-8 mol L-1 and 2.0 × 10-8 - 2.0 × 10-7 mol L-1, with a detection limit of 5.0 × 10-10 mol L-1. The connection of flow-cell for the SPCE/CNFs with SWV detection allowed for the successful determination of EIMTC in human serum samples. Ultra-high-performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) acted as a comparative method in the serum samples analysis.
Assuntos
Antineoplásicos , Nanofibras , Carbono , Eletrodos , Humanos , Espectrometria de Massas em TandemRESUMO
The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines. Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode. This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method generates reliable results and can be further applied to other in vitro cellular models.
Assuntos
Cromatografia Líquida/métodos , Cinurenina/metabolismo , Espectrometria de Massas em Tandem/métodos , Técnicas de Cultura de Células , Meios de Cultura , HumanosRESUMO
The paper outlines the first report of application of a differential pulse voltammetry for simultaneous quantification of clinically important molecular markers - tryptophan and its metabolite - kynurenine. The analytes were determined in less than 60 s at the boron-doped diamond electrode modified in situ with bismuth film (BiF/BDDE). Proper adjustment of a supporting electrolyte composition allowed to obtain good separation of tryptophan and kynurenine oxidation peaks that appeared at potential of 0.88 and 1.05 V (vs. Ag/AgCl), respectively. Studies using an optical profilometer have confirmed an increase in electrode surface area after deposition of Bi film. At the optimized conditions, the obtained detection limits of tryptophan and kynurenine were at 30 nM concentrations. The method was validated for linearity, precision, accuracy, selectivity and recovery. We have investigated an impact of numerous relevant interfering organic compounds (including amino acids and different tryptophan metabolites of kynurenine pathway) on voltammetric signals of the measured analytes. Finally, for proof-of-technology, the sensor was used for tryptophan and kynurenine quantification in culture medium collected from human cancer cell lines (breast MDA-MB-231 and ovary SK-OV-3). The target molecules were analyzed directly, without any sample preparation step. The sensor showed good accuracy in presence of the sample matrix components that was confirmed by high performance liquid chromatography measurements. Our work emphasizes the advantages of application of the herein proposed, easy to fabricate voltammetric sensor, instead of popular chromatographic assays or previously proposed potentiometric immunosensor. The method might serve for rapid assessment of kynurenine pathway activity in cancer cells.
Assuntos
Meios de Cultura/metabolismo , Cinurenina/metabolismo , Neoplasias/metabolismo , Triptofano/metabolismo , Técnicas Biossensoriais , Boro/química , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Diamante/química , Técnicas Eletroquímicas , Eletrodos , Humanos , Cinurenina/análise , Neoplasias/química , Triptofano/análiseRESUMO
It is a common belief that reduction of disulfide bridges and alkylation of thiols in proteins are indispensable steps in proteomic sample preparation. Since this chemical procedure is often incomplete and prone to side reactions we reexamined its importance. We found that reduction and alkylation do not increase the depth of analysis and quality of proteomic quantification and therefore these steps are not essential in 'shotgun'-type investigations of proteomes. Moreover, we found that compared to a standard procedure using iodoacetamide for thiol-alkylation, sample preparation under conditions protecting thiols from oxidation improves quality of peptides and allows identifying of 10-20% more peptides and proteins. Excluding thiol-alkylation from proteomic sample preparation shortens the workflows and decreases the probability of biases resulting from occurrence of artificially modified peptides.