Molecular Characterization and Mutational Analysis of Clarithromycin- and Levofloxacin-Resistance Genes in Helicobacter pylori from Gastric Biopsies in Southern Croatia.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

Young Women's Attitudes and Behaviors in Treatment and Prevention of UTIs: Are Biomedical Students at an Advantage?

We wanted to investigate whether students who study within biomedical fields (i.e., medicine, pharmacy science) differ from those whose studies are not connected to the biomedical field in terms of their attitudes and behaviors related to urinary tract infections (UTIs). This was a cross-sectional survey-based study conducted among 392 female students, of whom 243 attended a biomedical school and 149 (38.0%) attended a non-biomedical school, using a previously published tool. The survey was distributed as an online link via student representatives at different faculties. Only 22 (5.6%) of women felt that they could not recognize a UTI. A greater proportion of biomedical students wiped front to back, while significantly more non-biomedical students chose cotton underwear and avoided daily sanitary pads compared to biomedical students. As many as 215 (54.8%) women stated that they used cranberry preparations. Biomedical students showed greater awareness about possible resistance to repeated treatment (p = 0.002) and greater knowledge of possible interactions of antibiotics (p < 0.001). This study reveals that young women are confident in recognizing an UTIs, are open to alternative treatments, and would consider UTI management in a pharmacy setting. However, it reveals that there might be gaps in their knowledge regarding antibiotic resistance risks, possible interactions, and efficacy of available preparations, as participants from the group of biomedical students showed greater knowledge and different behaviors.

Registered Drug Packs of Antimicrobials and Treatment Guidelines for Prostatitis: Are They in Accordance?

The aim of this study was to analyze if registered drug packs of antibiotics are in accordance with national guidelines for prostatitis treatment regard to the amount of drug units.; Methods: Croatian, UK (NICE), Australian, Spanish and Slovenian national guidelines were analyzed in this study. Results: Comparing treatment guidelines with registered drug packs resulted in perfect accordance only for drug packs registered in the UK with the NICE guidelines, where even split-pack dispensing is possible. Interestingly, when comparing drug packs registered in the UK with treatment proposed in the national guidelines of Croatia, Italy, Spain, Australia, USA and Slovenia, they matched almost perfectly. In other investigated countries, registered drug packs' national guidelines' analysis showed mismatch in 25-100% of recommendations (Italy and Slovenia, respectively). Conclusions: Mismatch between registered drug packs that are dispensed to patients and treatment guidelines may result in excess units of antimicrobials that may be misused by the patient in the future, or excess antimicrobials may become unnecessary waste, further promoting antimicrobial resistance. Greater accordance of registered drug packs of antimicrobials with treatment guidelines may lower rates of antimicrobials misuse.

Acute Liver Failure after Treatment with Rivaroxaban for Aortic Thrombosis Associated with COVID-19 Infection and Methylenetetrahydrofolate Reductase Gene Polymorphism (C677T).

Coronavirus disease-2019 (COVID-19) has become associated with prothrombotic state that could lead to severe arterial thrombotic complications. In the case of severe COVID-19 infection, hepatic dysfunction has been observed in more than 50% of patients. In this article, we present a case of aortic thrombosis associated with COVID-19 infection and methylenetetrahydrofolate reductase gene polymorphism (C677T) treated with rivaroxaban resulting in acute liver failure with fatal outcome.

Comparison of the Impact of Insulin Degludec U100 and Insulin Glargine U300 on Glycemic Variability and Oxidative Stress in Insulin-Naive Patients With Type 2 Diabetes Mellitus: Pilot Study for a Randomized Trial.

BACKGROUND: There is an ongoing discussion about possible differences between insulin degludec (IDeg-100) and glargine U300 (IGlar-300). There is little data and head-to-head comparison of IDeg-100 and IGlar-300 regarding their simultaneous impact on glycemic variability and oxidative stress in patients with type 2 diabetes mellitus (T2DM). OBJECTIVE: In our randomized, open-label, crossover study, we compared the impact of IDeg-100 and IGlar-300 on glycemic variability and oxidative stress in insulin-naive patients with T2DM. METHODS: We recruited a total of 25 adult patients with T2DM (7 females) whose diabetes was uncontrolled (HbA1c ≥7.5%) on two or more oral glucose-lowering drugs; a total of 22 completed the study. Mean age was 57.3 (SD 6.99) years and duration of diabetes was 9.94 (SD 5.01) years. After the washout period, they were randomized alternately to first receive either IDeg-100 or IGlar-300 along with metformin. Each insulin was administered for 12 weeks and then switched. At the beginning and end of each phase, biochemical and oxidative stress parameters were analyzed. On 3 consecutive days prior to each control point, patients performed a 7-point self-monitoring of blood glucose profile. Oxidative stress was assessed by measuring thiol groups and hydroperoxides (determination of reactive oxygen metabolites test) in serum. RESULTS: IGlar-300 reduced mean glucose by 0.02-0.13 mmol/L, and IDeg-100 reduced glucose by 0.10-0.16 mmol/L, with no significant difference. The reduction of the coefficient of glucose variation also did not show a statistically significant difference. IGlar-300 increased thiols by 0.08 µmol/L and IDeg-100 increased thiols by 0.15 µmol/L, with no significant difference (P=.07) between them. IGlar-300 reduced hydroperoxides by 0.040 CARR U and IDeg-100 increased hydroperoxides by 0.034 CARR U, but the difference was not significant (P=.12). CONCLUSIONS: The results of our study do not show a significant difference regarding glycemic variability between patients receiving either insulin IDeg-100 or IGlar-300, although IGlar-300 showed greater dispersion of data. No significant difference in oxidative stress was observed. In a larger study, doses of insulins should be higher to achieve significant impact on glycemic parameters and consequently on glycemic variability and oxidative stress. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04692415; https://clinicaltrials.gov/ct2/show/NCT04692415.

Helicobacter pylori Infection in Croatian Population: Knowledge, Attitudes and Factors Influencing Incidence and Recovery.

Basic and clinical knowledge about Helicobacter pylori infections has been improved in the past. However, the translation of this knowledge into public health intervention has remained poor. A survey based cross-sectional study was performed to assess the factors regarding the H. pylori infection in the general population. The survey was conducted using a previously developed questionnaire, adapted for the population in Croatia. Respondents (N = 1131) had a good knowledge score with a median of 4 out of 5 correct answers (interquartile range: 2-4). Senior participants had a lower frequency of high knowledge answers about H. pylori (43.1%) compared to younger (56.1%) and middle-aged participants (51.5%, p = 0.014). Rural participants had a higher frequency of low knowledge answers compared to urban and suburban ones (21.7% vs. 9.5% and 9.4%, p = 0.011). Only 315 participants (27.9%) were screened for the H. pylori infection, despite high support for the screening programs among the untested (74.7%) and tested (85.7%). Habits of smoking (p = 0.036) and coffee drinking (p = 0.008) were associated with more symptoms after eradication therapy. Further education is needed for the groups at risk for H. pylori infection, especially to raise the awareness of the importance of screening programs. More research is warranted to assess the effects of dietary changes on therapy outcomes.

Did an introduction of CONSORT for abstracts guidelines improve reporting quality of randomised controlled trials' abstracts on Helicobacter pylori infection? Observational study.

OBJECTIVE: To determine abstracts' adherence to the Consolidated Standards of Reporting Trials for Abstracts (CONSORT-A) statement and to explore the factors associated with reporting quality. DESIGN: An observational study. SETTING: Abstracts of randomised controlled trials published between 2010 and 2019, found searching the MEDLINE database. PARTICIPANTS: A total of 451 abstracts of the clinical trials on Helicobacter pylori infections were included. PRIMARY AND SECONDARY OUTCOME MEASURES: Abstracts' reporting quality was determined by assessing their adherence to 17-item CONSORT-A checklist, with overall score being calculated as the sum of items that were adequately reported for each abstract. Additional factors that might influence the reporting quality of the abstracts were analysed, with univariate and multivariate linear regression used to determine how those factors influenced the overall reporting quality. RESULTS: Included abstracts had an overall median quality score of 8/17 (IQR 7-9). Large proportions of abstracts adequately reported interventions, participants, objectives, numbers randomised and conclusions (97.1, 99.3, 89.1. 94.7 and 98.4% of abstracts, respectively). Trial design, randomisation, blinding and funding were severely under-reported with only 8.0, 2.7, 11.0 and 2.0% of abstracts reporting each item. Overall quality scores for H. pylori abstracts were higher in association with CONSORT-A endorsement (B=5.698; 95% CI 1.781 to 9.615), pharmacological interventions (B=4.063; 95% CI 0.224 to 7.902), multicentre settings (B=5.057; 95% CI 2.370 to 7.743), higher numbers of participants (B=3.607; 95% CI 1.272 to 5.942), hospital settings (B=4.827; 95% CI 1.753 to 7.901) and longer abstracts (B=3.878; 95% CI 0.787 to 6.969 for abstracts with 251-300 words and B=7.404; 95% CI 3.930 to 10.878 for abstracts with more than 300 words). CONCLUSIONS: The overall reporting quality of abstracts was inadequate. The endorsement of CONSORT-A guidelines by more journals might improve the standards of reporting.

Characteristics and clinical outcomes of patients with acute gastrointestinal bleeding related to anticoagulant or antiplatelet therapy: a retrospective study.

AIM: To investigate the demographic characteristics, endoscopic and laboratory findings, comorbidities and mortality rate of patients with gastrointestinal bleeding related to anticoagulant or antiplatelet therapy. METHODS: We reviewed the records of patients admitted for gastrointestinal bleeding to the Intensive Care Unit of the Department of Gastroenterology, University Hospital Split, between 2015 and 2019. The characteristics and clinical outcomes of patients taking anticoagulant/antiplatelet therapy were analyzed. RESULTS: The study enrolled 1367 patients, 434 (31.7%) of whom received anticoagulant/antiplatelet therapy (mean age 74.9±10.7 years; 64.3% men). The most frequently prescribed drug was acetylsalicylic acid (56.7%), the most common bleeding site was the stomach (41.3%), and the most prevalent cause of bleeding was ulcer (61.6%). Patients taking anticoagulant/antiplatelet therapy who died had significantly higher creatinine (P=0.011) and lower albumin (P=0.015). In the multivariate analysis, the factors that negatively affected survival were older age, higher creatinine, and lower albumin. Patients taking anticoagulant/antiplatelet therapy had slightly lower in-hospital mortality (8.3%) compared with other patients (10.3%). CONCLUSION: Although anticoagulant/antiplatelet therapy increases the risk of gastrointestinal bleeding, it does not directly affect the outcome, which is mainly determined by age and comorbidities.

Randomized Clinical Trial Comparing Concomitant and Tailored Therapy for Eradication of Helicobacter pylori Infection.

As high clarithromycin resistance (>20%) in the Split-Dalmatia region of Croatia hinders the treatment of H. pylori infection, the primary objective of this study was to compare concomitant quadruple with the tailored, personalized therapy as first-line eradication treatment of H. pylori. In an open-label, randomized clinical trial, 80 patients with H. pylori infection were randomly assigned to either concomitant (esomeprazole 40 mg, amoxicillin 1 gr, metronidazole 500 mg, clarithromycin 500 mg, twice daily for 14 days) or tailored therapy in accordance with the results of the antimicrobial susceptibility testing. Eradication status was assessed 4 weeks after treatment. Eradication rates were significantly higher in tailored group than in concomitant group both in intention-to-treat (70 vs. 92.5%, p = 0.010) and per-protocol (87.5 vs. 100%, p = 0.030) analysis in the setting of increasing antibiotic resistance (clarithromycin 37.5%, metronidazole 17.5%, dual resistance 10%). Adverse effects were more frequent in the concomitant group (32.5 vs. 7.5%, p = 0.006). Tailored therapy achieves higher eradication with a lower adverse events rate. With the increasing resistance of H. pylori strains to antibiotic treatment, eradication regimes with such characteristics should be strongly considered as a reasonable choice for first-line treatment.

The differences between insulin glargine U300 and insulin degludec U100 in impact on the glycaemic variability, arterial stiffness and the lipid profiles in insulin naïve patients suffering from type two diabetes mellitus - outcomes from cross-over open-label randomized trial.

BACKGROUND AND AIMS: Diabetes mellitus type two is one of the major cardiovascular risk factors. Treatment of diabetes can reduce this risk, but the treatment options differ a lot in their risk-reducing capabilities. We compared the impact of insulin degludec (IDeg-100) and insulin glargine U300 (IGlar-300) on cardiovascular risk parameters - glycaemic variability (GV), arterial stiffness and lipid parameters - in insulin naive patients with DMT2. METHODS: To 23 individuals who previously had uncontrolled DMT2 on two or more oral antidiabetic drugs, IGlar-300 and IDeg-100 were applied for 12 weeks and then switched in a cross over design manner. Prior and after of each insulin phase, we analysed biochemical parameters,7-point SMBG profile over three days and arterial stiffness which was assessed indirectly by measuring the augmentation index (AIx) on the principles of applanation tonometry. RESULTS: There were no significant differences between IGlar-300 and IDeg-100 regarding reduction of mean glucose values and coefficient of variation (CV). Both insulins insignificantly reduced AIx for standardised pulse of 75 beats/min and without differences between them. IGlar-300 and IDeg-100 reduced triglycerides and increased HDL with no significant difference between the two insulins. IGlar-300 increased the total cholesterol level and IDeg-100 decreased total cholesterol, but without statistically significant difference. IGlar-300 increased LDL level by 0.508 mmol/L and IDeg-100 decreased LDL by 0.217 mmol/L, with statistically significant difference (p = 0.0215). CONCLUSIONS: This study did not show significant difference between IGlar-300 and IDeg-100 regarding glycaemic parameters and augmentation index using the same dose of 0.2 IU/kg for both insulins, but it has revealed possible differences in impact on lipid profile. TRIAL REGISTRATION: Clinicaltrials.gov, NCT04692415 . Retrospectively registered on December 31th 2020.

Adherence to Maastricht V/Florence consensus report for the management of Helicobacter pylori infection among primary care physicians and medical students in Croatia: A cross-sectional study.

BACKGROUND: Helicobacter pylori (H pylori) eradication is becoming increasingly difficult. The aim of our study was to determine the knowledge of current guidelines and attitude in the diagnosis and treatment of H pylori infection in primary care physicians (PCPs) and medical students in Croatia. MATERIALS AND METHODS: A study was conducted among PCPs and medical students to evaluate adherence to Maastricht V consensus guidelines. Questionnaire was distributed by e-mail to 2338 PCPs offices in Croatia and to the medical students from the University of Split School of Medicine. Responses were collected electronically from June 22 to August 22, 2020. RESULTS: Two hundred forty-nine PCPs and 169 medical students were included in the study. Bismuth or non-bismuth-based quadruple therapy as first-line treatment for H pylori was the choice of 4.8% of PCPs and 13% of students, while 66.3% PCPs and 79.9% students would choose clarithromycin-based triple therapy. Bismuth-based quadruple therapy was the most preferred second line of treatment for 45.4% of PCPs and 34.9% of students. Only 2.8% PCPs and 7.1% of students would correctly recommend first and second line of treatment for H. pylori infection. A larger proportion of students than PCPs would prefer C13-urea breath test (50.3% vs 6.4%). Only 59.0% PCPs would treat for H pylori in all patients including the asymptomatic ones. Students more frequently recognized the link between H pylori and gastric cancer compared with PCPs (92.9% vs 73.5%). CONCLUSIONS: Primary care physicians and medical students' knowledge of H pylori guidelines are insufficient in Croatia and ask for additional training.

Randomised clinical trial comparing concomitant and hybrid therapy for eradication of Helicobacter pylori infection.

BACKGROUND: The primary objective of this study was to compare concomitant and hybrid therapy in the first line eradication treatment of Helicobacter pylori infection in Split-Dalmatia County, Croatia, in which clarithromycin resistance is above 20%. The secondary objective of the study was to determine and compare compliance and adverse events rate between these therapeutic protocols. MATERIALS AND METHODS: In an open-label, randomised clinical trial 140 patients total with H. pylori infection were randomly assigned to either concomitant (esomeprazole 40 mg, amoxicillin 1 g, metronidazole 500 mg, clarithromycin 500 mg, twice daily for 14 days) or hybrid (esomeprazole 40 mg and amoxicillin 1 g twice daily during 14 days with adding metronidazole 500 mg and clarithromycin 500 mg twice daily, in the last 7 days,) treatment group. RESULTS: Eradication rates for concomitant group and hybrid therapy group were 84.1% (58/69) and 83.1% (59/71) respectively in the intention-to-treat analysis and 96.7% (58/60) and 95.2% (59/62) in per-protocol analysis. There was no significant difference between the groups (ITT analysis: P = 0.878; PP analysis: P = 0.675). Adverse events were more frequent in the concomitant group (33.3% vs 18.3%, P = 0.043). There was no difference among groups regarding compliance rate. CONCLUSION: Hybrid therapy has similar eradication rate as concomitant therapy, with lower adverse events rate. In the era of increasing antibiotic resistance, eradication regime with less antibiotic's usage, as hybrid therapy, should be reasonable first line treatment choice for H. pylori infection. Clinical Trials, gov: NCT03572777.

Fatal Hepatitis-Associated Aplastic Anemia in a Young Male.

Hepatitis-associated aplastic anemia is a rare syndrome in which bone marrow failure occurs within weeks to 1 year after attack of acute hepatitis. Studies suggest that cytotoxic T lymphocytes play a central role in bone marrow destruction, but the exact etiology remains unknown. Bone marrow transplantation or immunosuppressive therapy are primary curative options. We present a case of a young male who was admitted to the Department of Gastroenterology and Hepatology for acute hepatitis of an unknown cause. Liver biopsy revealed extensive inflammatory process with hepatocyte necrosis. Forty days later, new onset pancytopenia was identified. Bone marrow biopsy showed severe hypocellularity, and he was diagnosed with severe hepatitis-associated aplastic anemia. Treatment with cyclosporine was initiated, but with inadequate response, and pretransplant evaluation was started. Due to severe neutropenia, following alveotomy procedure, the patient developed deep neck infection with consequent airway obstruction. Despite urgent treatment, his condition deteriorated to sepsis with lethal outcome.

Correlation of registered drug packs with Maastricht V/Florence Consensus Report and national treatment guidelines for management of Helicobacter pylori infection.

High prevalence of Helicobacter pylori infection, the complexity for its treatment, poor correlation of registered drug packs or poor patient adherence to the treatment may contribute to antibiotic resistance and healthcare costs. The aim of the present study was to investigate whether registered drug packs are in accordance with European and national guidelines for H pylori eradication with reference to the number of drug units. In this study, we considered treatment options for the management of H pylori infection recommended by the Maastricht V/Florence Consensus Report and by national guidelines in the United Kingdom (UK), Croatia, Italy and Slovenia for adults. Drugs proposed by the guidelines were identified in national drug databases in July of 2019. When considering correlation for 10-day treatment regimens, drug packs registered in Croatia could not be matched with recommendations for sequential therapies. A number of proposed treatments could not be matched due to small variety of drug packs in Croatia. Drug packs registered in the UK more often matched recommended 14-day treatment regimens and national guidelines. With reference to European guidelines, 10-day treatments could more frequently be matched in Italy and in Slovenia. Furthermore, results of this study indicate that there is smaller variety in drug pack sizes registered in Croatia and Slovenia when compared to UK and Italy. Considering poor correlation of drug packs with treatment guidelines for H pylori, adherence to antimicrobial treatment and proper disposal of antimicrobials is warrant. Discussing adherence to antimicrobial treatment with patients should be introduced as a standard of patient care and education.

Maturational Changes of Delta Waves in Monozygotic and Dizygotic Infant Twins.

AIMS: To compare developmental changes of delta 1 (0.5-2.0 Hz) and delta 2 (2.25-3.75 Hz) power spectra between healthy monozygotic (MZ) and dizygotic (DZ) twin pairs and among MZ and DZ twin groups during active/REM (AS/REM) and quiet/NREM (QS/NREM) sleep stages at 38th, 46th, and 52nd weeks of postmenstrual age (PMA). MATERIALS AND METHODS: Electroencephalography (EEG) recordings were analyzed using fast Fourier transforms. Differences in the developmental changes of delta power within twin pairs and between twin groups were estimated by calculating mean absolute differences of relative spectral values in delta 1 (0.5-2 Hz) and delta 2 (2.25-3.75 Hz) frequencies. RESULTS: A review of electrodes showed that relative delta 1 power decreased, whereas delta 2 power increased from 38th toward 52nd week of PMA regardless of zygosity, sleep stages, and electrode position. Twin groups did not significantly differ (P > .05) in within-pair MZ and DZ similarity for delta 1 and delta 2 power spectra; similarity between MZ twin partners for delta 1 and delta 2 power spectra was as high as that of DZ twin partners on each electrode position, sleep stage, and period of measurement. CONCLUSIONS: Developmental changes of delta 1 and delta 2 power spectra occurred equally in MZ and DZ twin groups during AS and QS sleep stages at 38th, 46th, and 52th PMA. The rhythm of EEG maturation evidenced by the maturation of delta 1 and delta 2 power spectra was not dependent on zygosity.

Signet Ring Carcinoma of the Appendix Presenting as Crohn's Disease in a Young Male.

Primary signet ring cell carcinoma is a rare event in surgery. It looks like acute appendicitis and it is difficult to diagnose it on clinical grounds alone. The diagnosis is always confirmed by histopathology of a surgically removed appendix. A young man, 22 years old, presented with vomiting, diarrhea, and cramps in his abdomen without abdominal tenderness (mild abdominal discomfort in the right lower abdominal quadrant without signs of peritoneal irritation) during the previous month. The first endoscopic results showed only changes of mucosa that could be attributed to endoscopic and clinical representation of Crohn's disease. A few days after the initiation of the therapy with aminosalicylates and corticosteroids, the patient went into ileus and was transferred to the Department of Surgery, where he underwent an emergency right-sided hemicolectomy with resection of the transversal colon and forming of an ileostoma. The first pathohistological diagnosis was pseudomembranous colitis. Because the patient's condition was deteriorating, a revision of the pathohistological diagnosis was done. After careful revision and extensive sampling, a signet ring cell carcinoma arising in the appendix with infiltration of the ileocecal region was found. Immunohistochemically, tumor cells were positive for CDX-2 CK7, CK20, CK19, and carcinoembryonic antigen and negative for chromogranin A. Sixteen isolated lymph nodes were negative. Although the patient had a disease that was localized to the appendix and ileocecal region with no apparent distal metastasis, his clinical condition was worsening rapidly and he died after 2 months. This case shows the aggressive biological behavior of the appendix signet ring cell carcinoma. Scrupulous histopathological examination of the appendix is an obligatory procedure. Elimination of the signet ring cell carcinoma from other carcinoma subtypes is of special importance as it has an exceptionally poor prognosis and is generally diagnosed in its advanced stages.

Diagnosis of Helicobacter pylori infection : A short review.

Helicobacter pylori infections represent an important factor in the pathogenesis of chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma. The recently published Maastricht V/Florence consensus report indicated that the urea breath test using 13 C urea still remains the best non-invasive test to diagnose H. pylori infections with high sensitivity and specificity. Among the stool antigen tests, the ELISA monoclonal antibody test is a rational option. Effective therapy should be based only on susceptibility testing in regions with documented high clarithromycin resistance (>15%). Advanced high-resolution endoscopic technologies enable increased diagnostic accuracy for detection of H. pylori infections.

Acute application of antioxidants protects against hyperoxia-induced reduction of plasma nitrite concentration.

We investigated the effects of acute intake of antioxidants on hyperoxia-induced oxidative stress, reduction of plasma nitrite and change in arterial stiffness. Twelve healthy males randomly consumed either placebo or an oral antioxidant cocktail (vitamin C, 1000 mg; vitamin E, 600 IU; alpha-lipoic acid, 600 mg). Every therapy was consumed once, a week apart, in a cross-over design, 30 min before the experiment. The volunteers breathed 100% normobaric oxygen between 30th and 60th min of 1-h study protocol. Plasma levels of nitrite, lipid peroxides (LOOH) and vitamin C, arterial stiffness (indicated by augmentation index, AIx) and arterial oxygen (Ptc O2 ) pressure were measured before and after hyperoxia. Exposure to oxygen caused a similar increase of Ptc O2 in both placebo and antioxidants groups, confirming comparable exposure to hyperoxia (438 ± 100 versus 455 ± 83 mm Hg). Vitamin C was increased in the antioxidants group confirming successful application of antioxidants (69 ± 14 versus 57 ± 15 µm). Hyperoxia resulted in increased AIx and LOOH and decreased nitrite in placebo (-32 ± 11 versus -47 ± 13%, 72 ± 7 versus 62 ± 6 µm H2 O2 and 758 ± 184 versus 920 ± 191 nm, respectively), but not in the antioxidants group (-42 ± 13 versus -50 ± 13%, 64 ± 9 versus 61 ± 8 µm H2 O2 and 847 ± 156 versus 936 ± 201 nm, respectively). The acute intake of selected antioxidants was effective in preserving bioavailabity of ˙NO and vascular function, against hyperoxia-induced oxidative stress.

LC-MS/MS and GC-MS/MS measurement of plasma and urine di-paracetamol and 3-nitro-paracetamol: proof-of-concept studies on a novel human model of oxidative stress based on oral paracetamol administration.

Paracetamol (acetaminophen) is a widely used safe analgesic drug when administered at therapeutic doses. Given the chemical reactivity of its phenolic group towards electrophilic species, we assumed that detection of paracetamol metabolites distinctly different from its known phase I metabolite N-acetyl-p-benzoquinone imine (NAPQI) and the phase II glucuronic, sulfuric and mercapturic acids in biological samples upon oral administration of paracetamol (e.g., a 500-mg tablet) may represent a novel model of oxidative stress in humans. Such potential paracetamol metabolites are di-paracetamol and 3-nitro-paracetamol, in analogy to the well-investigated endogenous biomarkers di-tyrosine and 3-nitro-tyrosine. Di-paracetamol and 3-nitro-paracetamol are known to be formed both by enzymatic and non-enzymatic routes. In the present work we report on mouse and human pilot studies on the formation and appearance of di-paracetamol and 3-nitro-paracetamol in blood of mice intraperitoneally administered paracetamol, as well as in plasma and urine samples of healthy subjects who received a 500-mg paracetamol tablet or placebo. For the analysis of di-paracetamol and 3-nitro-paracetamol in plasma and urine samples, analytes were extracted by solvent extraction with ethyl acetate and subsequently analyzed by LC-MS/MS without and with derivatization with pentafluorobenzyl bromide. GC-MS/MS was used to detect 3-nitro-paracetamol and quantify paracetamol as pentafluorobenzyl derivatives. Our studies indicate that di-paracetamol and 3-nitro-paracetamol appear in plasma and urine when paracetamol is given orally to healthy humans at the therapeutic dosage of 5-7 mg/kg. The molar ratio of di-paracetamol to paracetamol in urine was determined to be 1:535 in the paracetamol group and 1:6844 in the placebo group; the molar ratio of 3-nitro-paracetamol to paracetamol in urine was determined to be 1:199 in the paracetamol group and 1:8657 in the placebo group. Our studies suggest that a fraction of circulating and excretory di-paracetamol and 3-nitro-paracetamol may be formed artefactually during sample workup including derivatization. Further studies based on the quantitative determination of di-paracetamol and 3-nitro-paracetamol in biological samples by LC-MS/MS and/or GC-MS/MS using stable-isotope labeled analogues as internal standards are warranted to test the utility of paracetamol as a probe of oxidative stress in animals and in humans in health and disease.