Comparison of Oxidative Stress Levels in Healthy Children and Children with Allergic Rhinitis.

Background/aim: Allergic rhinitis (AR) is characterized by chronic inflammation of the nasal mucosa. Under the influence of exogenous factors - allergens, reactive oxygen species (ROS) are released during cellular metabolism. They induce a series of pathological changes in the mucosa. Oxidative stress is а result of an imbalance between the production of ROS and the ability to neutralize them. The aim of this study is to compare the levels of oxidative stress between healthy children and children with allergic rhinitis. Material and methods: A total number of 60 children were included (30 healthy children and 30 children with AR). The oxidative stress index was determined by using the FRAS 5 (Free Radical Analytical System) Bravo system. Demographic characteristics, medical history, children's living conditions and eating habits were obtained from the questionnaire. Anthropometric measurements and the absolute number of eosinophils in the peripheral smear were performed on each child. Results: This study showed high oxidative stress index and a significantly higher value of the absolute number of eosinophils in the peripheral smear in children with AR in comparison to healthy children (p<0.05). The group of children with AR had more atopic characteristics and was more exposed to passive smoking than healthy children. Conclusion: Compared to healthy children, children with AR have a high index of oxidative stress, despite of the very high mean value of the concentration of water-soluble antioxidants in serum (PAT test) in the group of children with AR.

Effect of Supplemental Antioxidant-Based Therapy on the Oxidative Stress Level in COVID-19 Patients.

Background: COVID-19 is a disease in several stages starting with virus replication to dysregulation in immune system response, organ failure and recovery/death. Our aim was to determine the effect of Ganoderma lucidum, lycopene, sulforaphane, royal jelly and resveratrol extract on markers of oxidative stress, inflammation, routine laboratory analyses and duration of symptoms in COVID-19 patients. Methods: The oxidative stress parameters and interleukines 6 and 8 (IL-6, IL-8), tumor necrosis factor alpha (TNF-α) were determined in order to estimate the antioxidant and the anti-inflammatory effect of the product using a spectrophotometric and a magnetic bead-based multiplex assay in serum of 30 patients with mild form of COVID-19. Results: Statistically significant differences were obtained for all investigated parameters between the treated patients and the control group. Moreover, significant differences were observed for leukocytes, neutrophil to leukocyte ratio and iron. The average duration of the symptoms was 9.4±0.487 days versus 13.1±0.483 days in the treatment and the control group, respectively (p=0.0003). Conclusion: Our results demonstrated the promising effect of Ge132+NaturalTM on reducing the oxidative stress and the IL-6, IL-8 and TNF-α levels, and symptoms duration in COVID-19 patients. The evidence presented herein suggest that the combination of Ganoderma lucidum extract, lycopene, sulforaphane, royal jelly and resveratrol could be used as a potent an adjuvant therapy in diseases accompanied by increased oxidative stress and inflammation.

Hematological Findings and Alteration of Oxidative Stress Markers in Hospitalized Patients with SARS-COV-2.

Background/aim: Hematological parameters are the starting point in COVID-19 severity classification. The aim of this study was to analyze oxidative stress in hospitalized COVID-19 patients and to determine its association with D-dimer, neutrophil to lymphocyte ratio (NLR), and platelets to lymphocyte ratio (PLR) as markers for disease progression. Materials and methods: 52 patients with moderate and severe forms of COVID-19 were enrolled. A hematological and coagulation profile was performed for each patient. PAT (total antioxidant power, iron-reducing) and d-ROMs (plasma peroxides) were determined in serum at admission and 7 days after hospitalization. Results: The severe group presented parameters that indicated a poor prognosis. Patients that recovered had a significant reduction in d-ROM (t-test, p<0.01) and improvement in oxidative stress index (t-test, p<0.05). Patients that died had significantly decreased PAT (p<0.01) resulting in an increase in oxidative stress. Except for d-ROM vs PLR in both groups and d-ROM vs D-dimer in the severe group, a good correlation between oxidative stress parameters and D-dimer, PLR, and NLR was demonstrated (p<0.01). Conclusion: Our results show that oxidative stress markers can be used as a tool for disease progression in COVID-19. This analysis is easily accessible and affordable in addition to conventional hematological parameters performed for severity classification.

Comparative, Single-Dose, 2-Way Cross-Over Bioavailability Study of Two Olanzapine 10 Mg Tablet Formulations in Healthy Volunteers Under Fasting Conditions.

Objectives: Olanzapine is an atypical antipsychotic that is approved across Europe, the USA, and in many other countries for oral treatment of schizophrenia and acute manic episodes in patients with bipolar disorder as well as for maintenance therapy to prevent recurrence in responders. The objective of the present study was to compare the pharmacokinetics of two 10 mg tablet formulations of Olanzapine following a single oral dose in healthy volunteers under fasting conditions, as per the European Medicine Agency (EMA) guidelines to grant marketing authorization. Methods: This study was a randomized, open-label, two-treatment, two-period, two-sequences, single-dose, cross-over design with a washout period of 14 days. Both the test and the reference products were administered as 10 mg tablets with 240 mL of water after an overnight fast in each study period. A total of twenty blood samples were collected before dosing and within 144 hours after drug administration. Adverse events were monitored, recorded, and evaluated by investigators throughout the study. Results: Of the 24 healthy adult male subjects enrolled, all of them completed both study periods. The geometric mean ratio 90% confidence intervals (CI) for fasting Cmax, AUC0-t, and AUC0-infinity were 94.83-113.71%, 95.04-105.69% and 95.94-107.00%, respectively. The 90% CI for the ratios of the three primary pharmacokinetic parameters (using log-transformed data) were within the range of 80-125%, meeting the regulatory criteria for bioequivalence. Conclusions: The generic Olanzapine was bioequivalent to the reference formulation. It was well tolerated and provides an acceptable alternative to the reference drug.

Presentation of cytokine profile in relation to oxidative stress parameters in patients with severe COVID-19: a case-control pilot study.

Introduction: COVID-19 can be worsened by hyper-production of cytokines accompanied by increased level of oxidative stress. The aim of this study was to investigate the correlation between a set of cytokines and the markers of the oxidative stress. Methods: The levels of cytokines IL-2, IL-4, IL-6, IL8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α, MCP-1 and EGF were determined by using High Sensitivity Evidence Investigator™ Biochip Array technology. The oxidative stress parameters (d-ROM, PAT, OS index) were measured in serum on FRAS5 analytical photometric system. Results: IL-6, IL-8, IL-10, VEGF, MCP-1 and EGF were significantly higher (p<0.05) in the patients with severe COVID-19 with increased levels of IL-2, IFN-g, TNF-a and IL-1α. The d-ROM, OS index, and PAT were significantly higher (p<0.05) in severe COVID-19 patients. IL-6 demonstrated the strongest correlation with all of the markers of the oxidative stress, d-ROM (r=0.9725, p=0.0001), PAT (r=0.5000, p=0.0001) and OS index (r=0.9593, p=0.012). Similar behavior was evidenced between IFN-g and d-ROM (r=0.4006, p=0.0001), PAT (r=0.6030, p=0.0001) and OS index (r=0.4298, p=0.012). Conclusion: The oxidative stress markers show good correlation with the tested cytokines which can be measured at the beginning of the disease in a primary care setting to predict the course of COVID-19.

Vitamin D levels and oxidative stress markers in patients hospitalized with COVID-19.

BACKGROUND: COVID-19 is characterized by the presence of oxidative stress. Vitamin D status has been reviewed as one of the factors that may affect disease severity. The aim of this study was to assess the relationship between serum vitamin D levels, oxidative stress markers and disease severity in hospitalized COVID-19 patients. METHODS: Vitamin D levels were measured in 33 patients with COVID-19. The total antioxidant power and plasma peroxides were determined in serum. RESULTS: Severe COVID-19 patients have lower vitamin D levels (18.39 ± 2.29 ng/mL vs. 28.47 ± 3.05 ng/mL, p < .05) and higher oxidative stress compared to the moderate group. When divided according to serum vitamin D levels, significantly higher values of LDH (604.8 ± 76.98 IU/mL vs. 261.57 ± 47.33 IU/mL) and D-dimer (5978 ± 2028ng/mL vs. 977.7 ± 172 ng/mL) were obtained in the group with vitamin D below 30 ng/mL, followed with significantly higher levels of plasma peroxides (d-ROMs: 414.9 ± 15.82 U.Carr vs. 352.4 ± 18.77 U.Carr; p < .05) and oxidative stress index (OSI: 92.25 ± 6.60 vs. 51.89 ± 6.45; p < .001). CONCLUSION: The presented data provide a justification to consider vitamin D as an important factor that could ameliorate disease severity through its anti-inflammatory and antioxidant effects.

Evaluation of oxidative stress markers in hospitalized patients with moderate and severe COVID-19.

Background. Clinical evidence suggests increased oxidative stress in COVID-19 patients and this worsened redox status could potentially contribute to the progression of the disease. Objectives. To investigate the oxidative stress we have measured oxidative stress parameters, namely, PAT (total antioxidant power, iron reducing) and d-ROMs (plasma peroxides). Additionally we have investigated their correlation with the most frequently used clinical parameters CRP, LDH, and NLR in serum from moderate and severe COVID-19 patients hospitalized in a tertiary hospital. Methods. PAT and d-ROMs were determined by analytical photometric metric method in serum from 50 hospitalized patients. For each of them, two samples were collected and analyzed immediately after collection seven days apart. Results. All patients at admission had a much higher value for plasma peroxides and a significant correlation between oxidative stress parameters and CRP, LDH, and NLR. (p<0.05), except for OS index (OSI) vs CRP in the severe group. At discharge, plasma peroxides were reduced and OSI was improved in the moderate group. Conclusion. We consider that using OSI at the beginning of COVID-19 disease presents a valuable starting point for the general assessment of oxidative stress and hence enabling a better triage of the patients in terms of disease severity.

A New Solid-Phase Extraction Method for Determination of Pantoprazole in Human Plasma Using High-Performance Liquid Chromatography.

BACKGROUND: A new simple, selective and accurate high-performance liquid chromatographic (HPLC) method utilising solid-phase extraction for the determination of pantoprazole in human plasma samples has been developed. AIM: The purpose of this paper was developing a new HPLC method suitable for the determination of pantoprazole in plasma samples, which enables simple and rapid isolation and concentration of the analysed drug. METHODS: The chromatographic separation was accomplished on a LiChroCart LiChrospher 60 RP select B column using a mobile phase composed of 0.2 % (V/V) water solution of triethylamine (pH 7) and acetonitrile (58:42, V/V) followed by UV detection was at 280 nm. The solid-phase extraction method using LiChrolut RP-18 (200 mg, 3 ml) was applied to the obtained good separation of investigated drug from endogenous plasma components. Best results were achieved when plasma samples were buffered with 0.1 mol/L KH2PO4 (pH 9) before extraction, eluted and reconstituted with acetonitrile and 0.001 mol/L NaOH after extraction, respectively. RESULTS: The standard calibration curves showed good linearity within the range of 25.0-4000.0 ng/mL with a correlation coefficient greater than 0.996. Retention times of pantoprazole and internal standard, lansoprazole was 4.1 and 6.0 min respectively. The limit of quantification was 25.0 ng/mL. For intra- and inter-day precision relative standard deviations ranged from 4.2 to 9.3%. The relative errors for stability investigations were ranged from 0.12 to -10.5%. CONCLUSION: This method has good precision and accuracy and was successfully applied to the pharmacokinetic and bioequivalence study of 40 mg pantoprazole in healthy volunteers.

Importance of 6-Thioguanine Nucleotide Metabolite Monitoring in Inflammatory Bowel Disease Patients Treated with Azathioprine.

The active metabolite of azathioprine, 6-thioguanine nucleotide (6-TGN) is the main component responsible for the immunosuppressive effect in treatment of inflammatory bowel disease (IBD). The aim of this study was to assess the correlation between the concentration of 6-thioguanine nucleotide and disease activity, azathioprine-related adverse effects and time duration of treatment in patients with inflammatory bowel disease. Thirty-four patients were included in this study. Type of disease, gender, time duration of therapy and adverse effects were recorded. Metabolite concentration was determined by high performance liquid chromatography. Twenty-one percent of patients have experienced an adverse effect, with leucocytopenia most commonly occurring (42.9%). More adverse effects were registered when patients were treated with azathioprine in a period of less than 3 months in comparison to the group of patients that have been under therapy between 3-12 months and more than 12 months (p˂0.05). Most of the patients that presented any adverse effect had high 6-TGN concentration (>450 pmol/8x108 Er). The mean value of 6-TGN metabolite concentration in IBD patients treated with azathioprine was 437.46 pmol/8x108 Er ± 198.82 pmol/8x108. The time duration of azathioprine treatment did not have any significant impact on the achieved 6-TGN concentration (p>0.05).Twenty patients (58.9%) had achieved remission after therapy initiation with azathioprine. More alertness is recommended to clinicians towards patients in the first 3 months of the therapy. Our study demonstrated that higher 6-TGN concentration is associated with azathioprine toxicity.

High Performance Liquid Chromatographic Method for Direct Determination of Diazepam in Whole Blood and Serum - Optimization of Solid-Phase Extraction Method.

Herein, we present a simple and rapid high performance liquid chromatographic (HPLC) method with UV-detection for the direct determination of diazepam in whole blood and serum that can be used for monitoring diazepam levels in clinical samples analysis. The isolation of diazepam and the internal standard bromazepam from serum and whole blood samples was performed using solid phase extraction method with RP select B cartridges. The analytes were separated employing a reversed phase C8 column with a mobile phase composed of 0.1 % (V/V) triethylamine in water (pH 3.5) and acetonitrile (63:37, V/V). UV detection was carried out at 240 nm. Linearity was achieved in the range from 10.0-1000.0 ng/ml for serum and whole blood. The method was applied to spiked and real biological samples after an oral administration of 10 mg diazepam. In conclusion, the proposed method is simple, rapid and provides efficient clean-up of the complex biological matrix and high recovery of diazepam.

Determination of Acyclovir in Human Plasma Samples by HPLC Method with UV Detection: Application to Single-Dose Pharmacokinetic Study.

BACKGROUND: The aim of this study is estimation of pharmacokinetic parameters: Cmax, tmax, t1/2, AUC0-t and AUC0-∞ with the two-way analysis of variance, single observation (ANOVA) for two preparations containing acyclovir. OBJECTIVE: In order to evaluate pharmacokinetic study of acyclovir, method for quantitative determination of acyclovir in human plasma should be simple, rapid and reproducible. Therefore, the method is developed, validated and applied for analysis of acyclovir in plasma samples obtained from healthy volunteers. MATERIAL AND METHODS: High performance liquid chromatographic (HPLC) method with UV-detection for the determination of acyclovir in human plasma is presented. This method involves protein precipitation with 20 % (V/V) perchloric acid. The chromatographic separation was accomplished on a reversed phase C8 column with a mobile phase composed of 0.1 % (V/V) triethylamine in water (pH 2.5). No internal standard is required. UV detection was set at 255 nm. The method was successfully applied for the evaluation of pharmacokinetic profiles of acyclovir tablets in 24 healthy volunteers. RESULTS: The validation results shows that proposed method is rugged, precise (RSDs for intra- and inter-day precision ranged from 1.02 to 8.37 %) and accurate (relative errors are less than 6.66 %). The calibration curve was linear in the concentration range of 0.1-2.0 µg/ml and the limit of quantification was 0.1 µg/ml. The Cmax, tmax and AUCs for the two products were not statistically different (p>0.05), suggesting that the plasma profiles generated by Zovirax were comparable to those produced by acyclovir manufactured by Jaka 80 company. CONCLUSION: Good precision, accuracy, simplicity, sensitivity and shorter time of analysis of the method makes it particularly useful for processing of multiple samples in a limited period of time for pharmacokinetic study of acyclovir.

HPLC method for determination of verapamil in human plasma after solid-phase extraction.

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm x 4 mm I.D., 5 microm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH2PO4 with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10-500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.

Development of an HPLC method for the determination of nifedipine in human plasma by solid-phase extraction.

Nifedipine, a dihydropyridine calcium channel antagonist, is widely used in the treatment of hypertension and other cardiovascular disorders. A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for determination of nifedipine in human plasma samples. A series of studies were conducted in order to investigate the effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers, and to develop a convenient and easy-to-use method for quantitative analysis of nifedipine. The method involves solid-phase extraction on C18 cartridges. The chromatographic separation was accomplished on a Lichrocart Lichrospher 60 RP selectB column with a mobile phase composed of 0.020 mol/L KH2PO4 (pH 4.8) and acetonitrile (42:58, v/v). UV detection was set at 240 nm. The calibration curve was linear in the concentration range of 5.0-200.0 ng/mL for nifedipine in plasma and the limit of quantification was 5.0 ng/mL.

Sample preparation and RPHPLC determination of diuretics in human body fluids.

This article describes reverse phase high-performance liquid chromatography (RPHPLC) methods for determination of diuretics in different human body fluids (whole blood, plasma, serum or urine). Sample preparation procedures, including solid-phase extraction, liquid-liquid extraction, dilution, precipitation as well as automated RPHPLC procedures, are discussed in order to present the advantages and disadvantages of each type of sample preparation. Also, values of analytical recovery of each procedure used for sample preparation are summarized. The most important RPHPLC parameters (detection mode, stationary phase, mobile phase, sensitivity, etc.) are also summarized and discussed.

Development of solid-phase extraction method and its application for determination of hydrochlorothiazide in human plasma using HPLC.

A high-performance liquid chromatographic method was developed, validated and applied for the determination of hydrochlorothiazide in human plasma. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of hydrochlorothiazide and internal standard were investigated. The method involves solid-phase extraction on RP-select B cartridges followed by isocratic reversed-phase chromatography on a Hibar Lichrospher 100 RP-8 column with UV detection at 230 nm. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. Limit of quantification was 10 ng mL(-1). The method has been implemented to monitor hydrochlorothiazide levels in patient samples.

Development of an HPLC method for the determination of ranitidine and cimetidine in human plasma following SPE.

A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for the determination of ranitidine and cimetidine in plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of investigated drugs were investigated. Sample preparation was carried out by adding an internal standard, famotidine, and the clean-up procedure was accomplished using solid-phase extraction (SPE). This method uses ultraviolet detection, the separation used a Lichrocart Lichrospher 60 RP-select B column and the mobile phase consisted of 0.2% triethylamine (TEA), 0.04 mol l(-1) KH2PO4 at pH 6.8 and 14% acetonitrile. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma. The method has been implemented to monitor ranitidine levels in clinical samples.

High-performance liquid chromatographic determination of diltiazem in human plasma after solid-phase and liquid-liquid extraction.

A selective, sensitive, and accurate high-performance liquid chromatographic method for determination of diltiazem in plasma samples has been developed and validated. The effects of mobile phase composition, buffer concentration, mobile phase pH, and concentration of organic modifiers on retention of diltiazem and internal standard were investigated. Solid-phase and liquid-liquid extraction were examined and proposed for isolation of the drug and elimination of endogenous plasma interferences. A 5 microm Lichrocart Lichrospher 60 RP-select B chromatographic column was used; the mobile phase was acetonitrile-0.025 mol L(-1) KH(2)PO(4) (pH 5.5), 35:65 ( v/ v) at a flow-rate of 1.5 mL min(-1). The detection wavelength was 215 nm. The calibration plots were linear in the concentration range 20.0-500.0 ng mL(-1). The method has been implemented to monitor diltiazem levels in patient samples.

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography.

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.