BIOM-13. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS ASSAY TO MEASURE GLUTATHIONE, GLUTATHIONE DISULFIDE, CYSTEINE, AND CYSTINE IN HUMAN BRAIN AND HUMAN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi12-vi13
Author(s):  
Miao Liu ◽  
Tigran Margaryan ◽  
Nader Sanai ◽  
Artak Tovmasyan
Keyword(s):  
Oxidative Stress ◽  
Human Brain ◽  
Aminolevulinic Acid ◽  
Brain Homogenate ◽  
Gradient Elution ◽  
Glutathione Disulfide ◽  
Oxidative Stress Biomarkers ◽  
Human Glioblastoma ◽  
Independent Calibration ◽  
Development And Validation

Abstract BACKGROUND Oxidative stress is implicated in many pathological conditions. Herein, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of oxidative stress biomarkers glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys) and cystine (CySS) in human brain and glioblastoma tissue. METHODS Freshly-acquired human glioblastoma tissue was homogenized with N-ethylmaleimide solution to prevent thiol oxidation. Analytes were then extracted from homogenate samples by protein precipitation with 2% sulfosalicylic acid (SSA). Stable isotope-labeled analytes were used as internal standards. Independent calibration curves for thiols and disulfides were prepared in analytical solutions. Three levels of quality controls were prepared in human brain homogenate. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS Linear regression model was used to cover a concentration ranging 0.4-100 µmol/L for GSSG/CySS and 1-400 µmol/L for GSH/Cys. Chromatographic separation was optimized on Intrada Amino Acid column with total run time of 5 min using gradient elution. For all analytes the maximum coefficient of variation for intra- and inter-day precision was 11.4% and the accuracy was within 80.9-113.7% in analytical solution and matrix. The analytes were stable in brain homogenate for 1 hour and 3 hours at room temperature (RT) and 4 °C, respectively. Stability at -80°C was demonstrated for at least 35 days in human brain homogenate. Stability of stock and working solutions was demonstrated for at least 4 hours (RT) and 25 days (-20°C). CONCLUSIONS A bioanalytical method to quantify GSH, GSSG, Cys, and CySS is successfully developed and validated. The method is currently applied to measure thiols and related disulfides in human glioblastoma tissue undergoing 5-aminolevulinic acid sonodynamic therapy (NCT04559685).

DDRE-08. A BIOANALYTICAL LC-MS/MS ASSAY TO QUANTIFY TOTAL AND UNBOUND LY3214996, ABEMACICLIB AND ITS ACTIVE METABOLITES IN HUMAN PLASMA, CEREBROSPINAL FLUID AND HUMAN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi75-vi76
Author(s):  
Tigran Margaryan ◽  
Mackenna Elliott ◽  
Garry Hook ◽  
Nader Sanai ◽  
Artak Tovmasyan
Keyword(s):  
Cerebrospinal Fluid ◽  
Human Plasma ◽  
Equilibrium Dialysis ◽  
Brain Homogenate ◽  
Gradient Elution ◽  
Recurrent Glioblastoma ◽  
Active Metabolites ◽  
Unbound Fraction ◽  
Human Glioblastoma ◽  
Four Levels

Abstract BACKGROUND Here, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of total and unbound concentrations of ERK inhibitor LY3214996, CDK4/6 inhibitor abemaciclib and its M2 and M20 active metabolites in human plasma, cerebrospinal fluid and human glioblastoma tissue. METHODS Analytes were extracted from patient plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Four levels of quality controls were used during validation. The method was validated according to FDA guidelines and CAP/CLIA regulations. Equilibrium dialysis was performed to determine unbound fraction of four analytes in plasma and brain tissue. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS The method was validated over a concentration range from 0.2-500 nmol/L for all four analytes. The analytical separation was optimized on Phenomenex Kinetex™ F5 column with total run time of 3.8 min using gradient elution. For all the analytes the maximum coefficient of variation for intra- and inter-day precision was 12.0% and the accuracy was within 90.2-119.7% in all matrixes. The analytes are stable in plasma and brain homogenate for at least 19 hours and 6 hours at room temperature (RT), respectively. Stability of stock and working solutions was demonstrated for at least 15 hours (RT) and 28 days (2-8°C). The unbound fraction of the analytes in pooled human plasma and brain were determined to be 0.371 and 0.065 for LY3214996, 0.026 and 0.013 for abemaciclib, 0.052 and 0.008 for M2, 0.024 and 0.021 for M20, respectively. CONCLUSIONS A bioanalytical method to quantify LY3214996, abemaciclib and its M2 and M20 metabolites is successfully developed and validated. The method is currently applied to evaluate plasma pharmacokinetics and CNS penetration of LY3214996 and abemaciclib in recurrent glioblastoma patients in an ongoing Phase 0 clinical trial (NCT04391595).

Development and Validation of HPLC/UV-Spectrophotometric Procedures for Metronidazole Quantitative Determination

2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Klimenko Lina Yu ◽  
Shkarlat Galyna L ◽  
Shovkova Zoia V ◽  
Yaremenko Vitaliy D ◽  
Shpychak Oleg S
Keyword(s):  
Quantitative Determination ◽  
Sodium Hydroxide Solution ◽  
Gradient Elution ◽  
Calibration Model ◽  
Column Temperature ◽  
Medical Laboratories ◽  
Accuracy And Precision ◽  
Development And Validation ◽  
Gradient Elution Mode ◽  
Potential Object

Metronidazole is the most popular representative of antiprotozoal medicines from the group of 5-nitroimidazoles. Metronidazole blocks the enzymes of alcohol dehydrogenase and acetaldehyde dehydrogenase, therefore when its joint taking with alcohol it is observed the strong intoxication syndrome and fatal poisonings too. Therefore metronidazole can be a potential object of chemical toxicological investigations. The purpose of our paper is to develop HPLC/UV-procedure of metronidazole quantification with application of the system of HPLC-analyzer MiLiChrome® A-0230 implemented in practice of forensic medical laboratories in Russia and Ukraine and carry out step-by-step validation of the developed procedure. Chromatographic conditions: Eluent A (0.2 M LiClO4 – 0.005 M HClO4) and Eluent B (acetonitrile) wereused as the mobile phase components; HPLC microcolumn Ø2×75 mm and ProntoSIL 120-5-C18 AQ, 5 μm were used as an analytical column; temperature was 40°С; flow rate was 100 μl/min; gradient elution mode was from 5% to 100% Eluent B for 40 min, then 100% Eluent B for 3 min; detection was performed at 277 nm. Retention time for metronidazole is 5.95 min. Since metronidazole is easy soluble and stable enough in the solutions of diluted alkalis 0.001 M sodium hydroxide solution has been proposed for preparation of the solutions in developing HPLC/UV-procedure of metronidazole quantification. Validation of the procedure has been carried out in the variants of the method of calibration curve and method of standard by such parameters as in process stability, linearity/calibration model, accuracy and precision within 3 different analytical runs using different batches of reagents and different glassware; experiments have been performed by three different analysts. New procedure of metronidazole quantitative determination by the method of HPLC/UV has been developed. Its validation has been carried out and acceptability for application has been shown.

Nonalcoholic Fatty Liver Disease and Vitamin E - A Promising Relationship?

2019 ◽  
Vol 70 (1) ◽  
pp. 78-83
Author(s):  
Alexandra Totan ◽  
Daniela Gabriela Balan ◽  
Daniela Miricescu ◽  
Radu Radulescu ◽  
Iulia Ioana Stanescu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Vitamin E ◽  
Therapeutic Potential ◽  
Plga Nanoparticles ◽  
Diet Group ◽  
Normal Diet ◽  
Oxidative Stress Biomarkers ◽  
Promising Alternative ◽  
Nonalcoholic Fatty Liver ◽  
High Caloric Diet

Oxidative stress (OS) plays an important role in NAFLD molecular mechanism. Nanoencapsulation represents a novel strategy to enhance therapeutic potential of conventional drugs. Our study analyses the encapsulated vitamin E effect on lipid metabolism and oxidative stress biomarkers in NAFLD rats. Animals were divided into 3 groups : G1 - the normal diet group; G2- the high caloric diet group ; G3 - high-caloric diet group receiving PLGA-vit E, 50 mg / kg. Serum advanced human oxidative protein (AOPP), total antioxidant capacity (TAC) and vitamin E were analysed using ELISA technique. Our results showed significant increase of G2 GPT, ALP, GGT, TG, glucose, TC and AOPP, versus G1 ( P [ 0.05) and a significant decrease of G2 serum TAC and vitamin E versus G1 results ( p = 0.01 and 0.01). Vitamin E nanoparticles (G3) caused a significant increase of TAC and significant decrease of serum AOPP, versus G2 (p [ 0.01). Results showed a significant reduction of GPT, GGT, ALP, TG and total cholesterol ( p [0.05) in G3 versus G2. PLGA nanoparticles should be considered an attractive and promising alternative to improve the bioavailability and biological activity of vitaminE.

Methyl jasmonate delays the latency to anoxic convulsions by normalizing the brain levels of oxidative stress biomarkers and serum corticosterone contents in mice with repeated anoxic stress

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Abayomi Ololade Adelaja ◽  
Oluwafemi Gabriel Oluwole ◽  
Oritoke Modupe. Aluko ◽  
Solomon Umukoro
Keyword(s):  
Oxidative Stress ◽  
Blood Glucose ◽  
Methyl Jasmonate ◽  
Brain Injuries ◽  
Antioxidant Property ◽  
Oxidative Stress Biomarkers ◽  
Serum Corticosterone ◽  
Stress Agent ◽  
Cellular Antioxidants ◽  
The Brain

AbstractObjectivesRepeated exposure to anoxic stress damages the brain through cortisol-mediated increases in oxidative stress and cellular-antioxidants depletion. Thus, compounds with antioxidant property might confer protection against anoxic stress-induced brain injuries. In this study, we further examined the protective effect of methyl jasmonate (MJ), a potent anti-stress agent against anoxic stress-induced convulsions in mice.MethodsThirty-six male Swiss mice randomized into six groups (n=6) were given MJ (25, 50 and 100 mg/kg, i.p.) or vehicle (10 mL/kg, i.p.) 30 min before 15 min daily exposure to anoxic stress for 7 days. The latency(s) to anoxic convulsion was recorded on day 7. The blood glucose and serum corticosterone levels were measured afterwards. The brains were also processed for the determination of malondialdehyde, nitrite, and glutathione levels.ResultsMethyl jasmonate (MJ) delayed the latency to anoxic convulsion and reduced the blood glucose and serum corticosterone levels. The increased malondialdehyde and nitrite contents accompanied by decreased glutathione concentrations in mice with anoxic stress were significantly attenuated by MJ.ConclusionsThese findings further showed that MJ possesses anti-stress property via mechanisms relating to the reduction of serum contents of corticosterone and normalization of brain biomarker levels of oxidative stress in mice with anoxic stress.

scholarly journals Oxidative Stress Biomarkers in Urine of Metal Carpentry Workers Can Be Diagnostic for Occupational Exposure to Low Level of Welding Fumes from Associated Metals

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3167
Author(s):  
Flavia Buonaurio ◽  
Maria Luisa Astolfi ◽  
Daniela Pigini ◽  
Giovanna Tranfo ◽  
Silvia Canepari ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Occupational Exposure ◽  
Oxidation Products ◽  
Control Group ◽  
Welding Fumes ◽  
Oxidative Stress Biomarkers ◽  
Limit Values ◽  
Stress Biomarkers ◽  
Specificity And Sensitivity ◽  
The Impact

Urinary concentrations of 16 different exposure biomarkers to metals were determined at the beginning and at the end of a working shift on a group of workers in the metal carpentry industry. Five different oxidative stress biomarkers were also measured, such as the oxidation products of RNA and DNA metabolized and excreted in the urine. The results of workers exposed to metals were compared to those of a control group. The metal concentrations found in these workers were well below the occupational exposure limit values and exceeded the mean concentrations of the same metals in the urine of the control group by a factor of four at maximum. Barium (Ba), mercury (Hg), lead (Pb) and strontium (Sr) were correlated with the RNA oxidative stress biomarker, 8-oxo-7, 8-dihydroguanosine (8-oxoGuo), which was found able to discriminate exposed workers from controls with a high level of specificity and sensitivity. The power of this early diagnostic technique was assessed by means of the ROC curve. Ba, rubidium (Rb), Sr, tellurium (Te), and vanadium (V) were correlated with the level of the protein oxidation biomarker 3-Nitrotyrosine (3-NO2Tyr), and Ba, beryllium (Be), copper (Cu), and Rb with 5-methylcytidine (5-MeCyt), an epigenetic marker of RNA damage. These effect biomarkers can help in identifying those workers that can be defined as “occupationally exposed” even at low exposure levels, and they can provide information about the impact that such doses have on their health.

scholarly journals Oxidative stress in common variable immunodeficiency

2021 ◽  
Vol 19 ◽  
pp. 205873922110024
Author(s):  
Sevgen Tanir Basaranoglu ◽  
Sukru Cekic ◽  
Emine Kirhan ◽  
Melahat Dirican ◽  
Sara S. Kilic
Keyword(s):  
Oxidative Stress ◽  
Common Variable Immunodeficiency ◽  
Chronic Inflammatory Demyelinating Polyneuropathy ◽  
Infectious Complications ◽  
Clinical Syndrome ◽  
University Hospital ◽  
Unknown Etiology ◽  
Healthy Controls ◽  
Oxidative Stress Biomarkers ◽  
Common Variable

Common variable immunodeficiency (CVID) is a heterogenous group of immunologic disorders of unknown etiology. Alterations of the normal cellular balance due to an increase in reactive oxygen species and/or decrease in antioxidant defense may lead to increased oxidative stress. We aimed to evaluate the levels of oxidative stress biomarkers in patients with CVID who had different presentations. We investigated the serum catalase (CAT), erythrocyte superoxide dismutase (SOD), erythrocyte reduced glutathione as antioxidants and serum malondialdehyde levels as lipid peroxidation marker in patients with CVID in Uludag University Hospital Department of Pediatric Allergy and Immunology’s outpatient clinics. In the analysis, there were 21 patients and 27 matched healthy controls. The median levels of CAT in patients with CVID was significantly lower than in healthy controls ( p = 0.04). Among the patients with CVID, 19% had autoimmune disease, one had Sjögren’s syndrome, one had autoimmune alopecia, one had juvenile rheumatoid arthritis, and one had chronic inflammatory demyelinating polyneuropathy. Patients with autoimmune complications had significantly lower CAT levels compared to the ones without autoimmune diseases ( p = 0.03). The patients without non-infectious complications (NICs) had lower SOD levels than the patients with NICs ( p = 0.05). The analysis of oxidative stress markers in the patients with CVID suggested a series of abnormalities in the anti-oxidant system. The clinical syndrome associations may be a useful tool for future studies to set prediction markers for the prognosis of patients with CVID.

scholarly journals The Antioxidant Effect of Curcumin and Rutin on Oxidative Stress Biomarkers in Experimentally Induced Periodontitis in Hyperglycemic Wistar Rats

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1332
Author(s):  
Gilda M. Iova ◽  
Horia Calniceanu ◽  
Adelina Popa ◽  
Camelia A. Szuhanek ◽  
Olivia Marcu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Diabetic Rats ◽  
Gingival Tissue ◽  
Control Group ◽  
Albino Rats ◽  
Oxidative Stress Biomarkers ◽  
Significant Difference ◽  
The Impact ◽  
Experimentally Induced

Background: There is a growing interest in the correlation between antioxidants and periodontal disease. In this study, we aimed to investigate the effect of oxidative stress and the impact of two antioxidants, curcumin and rutin, respectively, in the etiopathology of experimentally induced periodontitis in diabetic rats. Methods: Fifty Wistar albino rats were randomly divided into five groups and were induced with diabetes mellitus and periodontitis: (1) (CONTROL)—control group, (2) (DPP)—experimentally induced diabetes mellitus and periodontitis, (3) (DPC)—experimentally induced diabetes mellitus and periodontitis treated with curcumin (C), (4) (DPR)—experimentally induced diabetes mellitus and periodontitis treated with rutin (R) and (5) (DPCR)—experimentally induced diabetes mellitus and periodontitis treated with C and R. We evaluated malondialdehyde (MDA) as a biomarker of oxidative stress and reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG and catalase (CAT) as biomarkers of the antioxidant capacity in blood harvested from the animals we tested. The MDA levels and CAT activities were also evaluated in the gingival tissue. Results: The control group effect was statistically significantly different from any other groups, regardless of whether or not the treatment was applied. There was also a significant difference between the untreated group and the three treatment groups for variables MDA, GSH, GSSG, GSH/GSSG and CAT. There was no significant difference in the mean effect for the MDA, GSH, GSSG, GSH/GSSG and CAT variables in the treated groups of rats with curcumin, rutin and the combination of curcumin and rutin. Conclusions: The oral administration of curcumin and rutin, single or combined, could reduce the oxidative stress and enhance the antioxidant status in hyperglycemic periodontitis rats.

scholarly journals Characterization of Differentially Expressed Circulating miRNAs in Metabolically Healthy versus Unhealthy Obesity

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 321
Author(s):  
Susana Rovira-Llopis ◽  
Rubén Díaz-Rúa ◽  
Carmen Grau-del Valle ◽  
Francesca Iannantuoni ◽  
Zaida Abad-Jimenez ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Metabolism ◽  
Insulin Signalling ◽  
Cross Sectional Study ◽  
Differentially Expressed ◽  
Circulating Mirnas ◽  
Oxidative Stress Biomarkers ◽  
Cross Sectional ◽  
Metabolically Healthy Obese ◽  
Metabolically Healthy

Obese individuals without metabolic comorbidities are categorized as metabolically healthy obese (MHO). MicroRNAs (miRNAs) may be implicated in MHO. This cross-sectional study explores the link between circulating miRNAs and the main components of metabolic syndrome (MetS) in the context of obesity. We also examine oxidative stress biomarkers in MHO vs. metabolically unhealthy obesity (MUO). We analysed 3536 serum miRNAs in 20 middle-aged obese individuals: 10 MHO and 10 MUO. A total of 159 miRNAs were differentially expressed, of which, 72 miRNAs (45.2%) were higher and 87 miRNAs (54.7%) were lower in the MUO group. In addition, miRNAs related to insulin signalling and lipid metabolism pathways were upregulated in the MUO group. Among these miRNAs, hsa-miR-6796-5p and hsa-miR-4697-3p, which regulate oxidative stress, showed significant correlations with glucose, triglycerides, HbA1c and HDLc. Our results provide evidence of a pattern of differentially expressed miRNAs in obesity according to MetS, and identify those related to insulin resistance and lipid metabolism pathways.

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