Artificial intelligence scoring of liver biopsies in a phase II trial of semaglutide in nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Artificial intelligence-powered digital pathology offers the potential to quantify histological findings in a reproducible way. This analysis compares the evaluation of histological features of NASH between pathologists and a machine-learning (ML) pathology model. APPROACH AND RESULTS: This post hoc analysis included data from a subset of patients (n=251) with biopsy-confirmed NASH and fibrosis stage F1-F3 from a 72-week randomized placebo-controlled trial of once-daily subcutaneous semaglutide 0.1, 0.2, or 0.4 mg (NCT02970942). Biopsies at baseline and week 72 were read by 2 pathologists. Digitized biopsy slides were evaluated by PathAI's NASH ML models to quantify changes in fibrosis, steatosis, inflammation, and hepatocyte ballooning using categorical assessments and continuous scores. Pathologist and ML-derived categorical assessments detected a significantly greater percentage of patients achieving the primary endpoint of NASH resolution without worsening of fibrosis with semaglutide 0.4 mg versus placebo (pathologist 58.5% vs. 22.0%, p < 0.0001; ML 36.9% vs. 11.9%; p =0.0015). Both methods detected a higher but nonsignificant percentage of patients on semaglutide 0.4 mg versus placebo achieving the secondary endpoint of liver fibrosis improvement without NASH worsening. ML continuous scores detected significant treatment-induced responses in histological features, including a quantitative reduction in fibrosis with semaglutide 0.4 mg versus placebo ( p =0.0099) that could not be detected using pathologist or ML categorical assessment. CONCLUSIONS: ML categorical assessments reproduced pathologists' results of histological improvement with semaglutide for steatosis and disease activity. ML-based continuous scores demonstrated an antifibrotic effect not measured by conventional histopathology.

Urinary nucleic acid oxidation product levels show differential associations with pharmacological treatment in patients with type 2 diabetes.

The relationship between RNA and DNA oxidation and pharmacological treatment has not been systematically investigated in patients with type 2 diabetes (T2D). We aimed to investigate the association between pharmacological treatments and levels of urinary markers of nucleic acid oxidation in T2D patients. Vejle Diabetes Biobank cohort data was nested into nationwide registry data. Multiple logistic regression was used to associate drug usage with risk of high (above median) RNA and DNA oxidation. Data from 2664 T2D patients (64% male, age range: 25-75) were included. Questionnaire-validated lipid lowering drug use was associated with low RNA oxidation (Odds ratio, OR 0.71, 95% CI: [0.59-0.87]). Insulin and non-specific antidiabetic drugs were associated with low DNA oxidation (insulin: OR 0.60, 95% CI [0.49-0.73]). Oral antidiabetics were associated with high DNA oxidation and RNA oxidation (OR 1.30, 95% CI [1.10-1.53] and OR 1.26, 95% CI [1.07-1.29]). Our findings indicate that diabetes-related drugs are associated with RNA and DNA oxidation and further studies are required to determine causality in T2D patients.

Identification and quantification of isoguanosine in humans and mice.

Isoguanine (2-hydroxyadenine), considered to be a non-natural nucleobase has, however, been shown to occur in the croton bean, butterfly wings and a mollusk. For the first time, to the best of our knowledge, we report the identification of isoguanosine (2-hydroxyadenosine), the ribonucleoside, in humans and mouse. Isoguanosine is identified and quantified in RNA from mouse liver samples and in human urine and cerebrospinal fluid. Isoguanine could not be detected as the 2'-deoxyribonucleoside in mouse liver DNA. It could be speculated that the source of isoguanosine was formation from adenosine during oxidative stress in the body. However, the urinary concentrations of isoguanosine and the levels in the liver found here by using isotope dilution liquid chromatography-tandem mass spectrometry are identical to or exceed those of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. Guanine is the nucleobase that is oxidized the easiest, so it appears spectacular that the levels of isoguanosine are higher than the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine. It also appears intriguing that it was only possible to detect the ribonucleoside isoguanosine and not the 2'-deoxyribonucleoside. These observations could indicate that the isoguanosine found is not formed by oxidative stress and could have biological functions.

Exposure to antazoline-naphazoline eye drops during pregnancy and the risk of congenital malformations: a Danish nationwide cohort study.

PURPOSE: To investigate whether exposure to antazoline-naphazoline eye drops in the first trimester of pregnancy was associated with an increased risk of malformations in humans. METHODS: All women giving live birth between 1997 and 2011 in Denmark were included in this nationwide cohort study. All women redeeming at least one prescription of antazoline-naphazoline eye drops during the first 84 days of pregnancy were identified. Logistic regression was used to estimate the odds ratios of malformations among exposed offspring compared to non-exposed offspring. RESULTS: We identified 977 706 births between 1997 and 2011. A total of 3061 women (0.32%) were exposed to antazoline-naphazoline eye drops in the first trimester of pregnancy. The rate of congenital malformations was 3.0% (n = 93) in exposed offspring and 3.5% (n = 33 594) in unexposed offspring. First-trimester exposure to antazoline-naphazoline was not associated with major congenital malformations overall (odds ratio: 0.88, 95% confidence interval: 0.71-1.09) or with any specific major malformation. The number of redeemed prescriptions was unchanged during all trimesters of pregnancy as compared to before and after pregnancy (p < 0.05). CONCLUSION: Exposure to antazoline-naphazoline eye drops in the first trimester of pregnancy appears not to be associated with increased teratogenic risk.

Increased oxidation of RNA despite reduced mitochondrial respiration after chronic electroconvulsive stimulation of rat brain tissue.

Major depressive disorder (MDD) affects 350 million people worldwide and is a serious socio-economic burden. The most efficient treatment of MDD is electroconvulsive therapy (ECT), which has been shown to influence the oxidative status believed to be part of the pathophysiology of MDD. We investigated the effects of chronic electroconvulsive stimulation (ECS) on mitochondrial respiration and mitochondrial hydrogen peroxide production, RNA oxidation, and the content of mitochondria in the piriform cortex of the rat. We found reductions of mitochondrial respiration in respiratory states 2 and 3 by 33% and 32%, respectively, and a 23% reduction in electron transfer capacity. RNA oxidation, as measured by 8-oxo-7,8-dihydroguanosine, was increased by 58%, while mitochondrial production of H2O2 was unaffected. The increased oxidative stress may thus be ascribed to extra-mitochondrial sources.

RNA oxidation and iron levels in patients with diabetes.

AIM: The urinary biomarker for oxidative stress to RNA, 8-oxo-7,8-dihydro-guanosine (8-oxoGuo) is associated with mortality in patients with type 2 diabetes. Iron has also been linked to diabetes. In individuals with untreated hereditary iron overload it has been observed that 8-oxoGuo was higher compared to controls. In the current study, we hypothesized that 8-oxoGuo was associated with diagnosis of diabetes, and that iron confounded this association. METHODS: Participants from a general Danish population were included in the study (n = 3567). UPLC-MS/MS method was used for 8-oxoGuo (nmol/mmol creatinine) measurement in spot urine. Iron biomarkers included total plasma iron, ferritin, transferrin saturation (TS) and transferrin. RESULTS: 8-oxoGuo was 17% higher in diabetes patients (n = 208) compared to non-diabetes controls. Unadjusted logistic regression model showed an odds ratio of diabetes of 1.38 (95%CI:1.21-1.57, P < 0.0001) per unit increase of 8-oxoGuo. When the model was adjusted for possible confounders the odds ratio was 1.09 (95%CI:0.94-1.26, P = 0.24). When additional adjustment was performed including ferritin, TS, or transferrin, respectively, the OR were 1.14 (95%CI:0.97-1.33, P = 0.09), 1.10 (95%CI: 0.95-1.28, P = 0.18), and 1.17 (95%CI:1.01-1.38, P = 0.04). CONCLUSIONS: Our study indicates that 8-oxoGuo is higher in diabetes patients. The lack of association between 8-oxoGuo and diabetes in the adjusted model may be due to the cross-sectional design including post-treatment bias. Our data did not show consistent effect of all iron biomarkers in relation to diabetes. Most likely, the iron biomarkers were affected by inflammation thus not reflecting true iron levels.

Iron induced RNA-oxidation in the general population and in mouse tissue.

Iron promotes formation of hydroxyl radicals by the Fenton reaction, subsequently leading to potential oxidatively generated damage of nucleic acids. Oxidatively generated damage to RNA, measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) in urine, is increased in patients with genetic iron overload, which have led us to test the hypothesis that high iron status, assessed by iron biomarkers and genetic disposition, increases urinary excretion of 8-oxoGuo. In a general Danish population study we used a Mendelian randomization design with HFE genotypes as a proxy for iron status and supplemented with ex vivo experiments in mice muscle tissue exposed to iron(II) sulfate to attempt to clarify this hypothesis. The biomarkers ferritin, transferrin, and transferrin saturation (TS) were associated with 8-oxoGuo (in linear univariable and multivariable regression analyses: P < 0.001). Mendelian randomization indicated a causal pathway between genetically elevated iron biomarkers (assessed by ferritin and TS) and high levels of 8-oxoGuo. The ex vivo experiments showed a monotonically increase in 8-oxoGuo with increased iron concentration (ANOVA: P = 0.0008) that was prevented with iron chelation (P = 0.01). Our results indicate a causal relationship between iron biomarkers and 8-oxoGuo. Furthermore, the ex vivo experiment shows a mechanistic link between iron and 8-oxoGuo formation. Both iron overload and the biomarker 8-oxoGuo have been linked to e.g. diabetes, which merits future studies to investigate if iron induced 8-oxoGuo is involved in disease development.

Cardiovascular and All-Cause Mortality Risk Associated With Urinary Excretion of 8-oxoGuo, a Biomarker for RNA Oxidation, in Patients With Type 2 Diabetes: A Prospective Cohort Study.

OBJECTIVE: Cardiovascular mortality risk remains high among patients with type 2 diabetes. Oxidative stress indicated by high urinary excretion of the biomarker for RNA oxidation, 8-oxo-7,8-dihydroguanosine (8-oxoGuo), is associated with an increased risk of death in newly diagnosed and treated patients. We assessed whether 8-oxoGuo is associated with specific cardiovascular and all-cause mortality risk. RESEARCH DESIGN AND METHODS: Urinary biomarkers for nucleic acid oxidation were measured in a cohort of patients with type 2 diabetes aged ≥60 years (n = 1,863), along with biochemical measurements, questionnaire findings, and Central Person Registry information to estimate the hazard ratios (HRs) for log2-transformed RNA oxidation using Cox regression. RESULTS: During the 5-year follow-up, 173 of 1,863 patients had died (9.3%), including 73 patients who died of cardiovascular disease (42.2%). Doubling of RNA oxidation was associated with an HR of all-cause mortality of 2.10 (95% CI 1.63-2.71; P < 0.001) and an HR of cardiovascular death of 1.82 (95% CI 1.20-2.77; P = 0.005) after multiple adjustments. The 5-year absolute risks (ARs) of all-cause mortality (AR 13.9 [95% CI 10.8-17.0] vs. AR 6.10 [95% CI 4.00-8.30]) and cardiovascular mortality (AR 5.49 [95% CI 3.44-7.55] vs. AR 3.16 [95% CI 1.59-4.73]) were approximately two times higher in the highest quartile of RNA oxidation than in the lowest quartile. CONCLUSIONS: We conclude that high RNA oxidation is associated with all-cause and cardiovascular mortality risk in patients with type 2 diabetes. Targeting oxidative stress via interventions with long-term follow-up may reveal the predictive potential of the biomarker 8-oxoGuo.

The effect of empagliflozin on oxidative nucleic acid modifications in patients with type 2 diabetes: protocol for a randomised, double-blinded, placebo-controlled trial.

INTRODUCTION: Cardiovascular disease is the leading cause of morbidity and mortality in patients with type 2 diabetes (T2D). Although glycaemic control reduces microvascular complications, the effect of intensive treatment strategies or individual drugs on macrovascular diseases is still debated. RNA oxidation is associated with increased mortality in patients with T2D. Inspired by animal studies showing effect of a sodium-glucose cotransporter-2 (SGLT-2) inhibitor (empagliflozin) on oxidative stress and a recent trial evaluating empagliflozin that demonstrated improved cardiovascular outcomes in patients with T2D at high risk of cardiovascular events, we hypothesise that empagliflozin lowers oxidative stress. METHODS AND ANALYSIS: In this randomised, double-blinded and placebo-controlled study, 34 adult males with T2D will be randomised (1:1) to empagliflozin or placebo once daily for 14 days as add-on to ongoing therapy. The primary endpoints will be changes in 24-hour urinary excretion of 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) determined before and after intervention (by ultra-performance liquid chromatography tandem mass-spectrometry). Additionally, fasting levels of malondialdehyde (MDA) will be determined in plasma before and after intervention (by high-performance liquid chromatography). Further, the plasma levels of iron, transferrin, transferrin-saturation, and ferritin are determined to correlate the iron metabolism to the markers of oxidative modifications. ETHICS AND DISSEMINATION: The study protocol has been approved by the Regional Committee on Biomedical Research Ethics (approval number H-16017433), the Danish Medicines Agency, and the Danish Data Protection Agency, and will be carried out under the surveillance and guidance of the GCP unit at Bispebjerg Frederiksberg Hospital, University of Copenhagen in compliance with the ICH-GCP guidelines and in accordance with the Declaration of Helsinki. The results of this study will be presented at national and international conferences, and submitted to a peer-reviewed international journal with authorship in accordance with Internation Committee of Medical Journal Editors (ICMJE) Recommendations state. TRIAL REGISTRATION: Study name: EMPOX; Pre-results: clinicaltrials.gov (NCT02890745). Protocol version 5.1 - August, 2016.

The effect of smoking on the urinary excretion of 8-oxodG and 8-oxoGuo in patients with type 2 diabetes.

Over the past decades, attention has been paid to understanding the impact of oxidative stress and related modifications of DNA and RNA on various human health risks. A recent meta-analysis comprising 1915 smokers and 3462 non-smokers found a significantly higher level of DNA oxidation measured as urinary 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) excretion in smokers compared with non-smokers in a healthy population. We aimed to investigate if an increased urinary excretion of 8-oxodG in smokers versus never smokers and former smokers could be verified in a population with type 2 diabetes. Additionally, we measured RNA oxidation levels through urinary excretion of 8-oxo-7, 8-dihydroguanosine (8-oxoGuo). Our study included urinary samples from 2721 type 2 diabetic patients, analyzed using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Logistic regression was used to examine the relationship between daily smokers (n = 462) versus former (n = 1341) and never smokers (n = 918) regarding the RNA and DNA oxidation, respectively. We did not find any significant effect of smoking on urinary excretion of 8-oxodG or 8-oxoGuo in our study. Due to a sparse study area, it is still too early to draw any conclusions on smoking and RNA-oxidation. Regarding DNA oxidation, our study suggests that the effect of smoking seen in healthy populations might be attenuated in patients with type 2 diabetes.

Clarithromycin, trimethoprim, and penicillin and oxidative nucleic acid modifications in humans: randomised, controlled trials.

AIMS: In vitro studies have demonstrated that formation of reactive oxygen species (ROS) contributes to the effect of bactericidal antibiotics. The formation of ROS is not restricted to bacteria, but also occurs in mammalian cells. Oxidative stress is linked to several diseases. This study investigates whether antibiotic drugs induce oxidative stress in healthy humans as a possible mechanism for adverse reactions to the antibiotic drugs. METHODS: This study contains information from two randomised, controlled trials. Participants underwent 1 week treatment with clarithromycin, trimethoprim, phenoxymethylpenicillin (penicillin V), or placebo. Oxidative modifications were measured as 24-h urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and plasma levels of malondialdehyde before and after treatment as a measurement of DNA oxidation, RNA oxidation, and lipid peroxidation, respectively. RESULTS: Clarithromycin significantly increased urinary excretion of 8-oxodG by 22.0% (95% confidence interval (CI): 3.6-40.4%) and 8-oxoGuo by 14.9% (95% CI: 3.7-26.1%). Further, we demonstrated that trimethoprim significantly lowered urinary excretion of 8-oxodG by 21.7% (95% CI: 5.8-37.6%), but did not influence urinary excretion of 8-oxoGuo. Penicillin V did not influence urinary excretion of 8-oxodG or 8-oxoGuo. None of the antibiotic drugs influenced plasma levels of malondialdehyde. CONCLUSION: Clarithromycin significantly increases oxidative nucleic acid modifications. Increased oxidative modifications might explain some of clarithromycin's known adverse reactions. Trimethoprim significantly lowers DNA oxidation but not RNA oxidation. Penicillin V had no effect on oxidative nucleic acid modifications.

Markers of oxidative stress in obese men with and without hypertension.

OBJECTIVES: The aim of our study was to investigate if the 24-hour excretion of the urinary markers for oxidative stress to DNA and RNA, measured as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanosine (8-oxoGuo), respectively, were increased in obese individuals with or without hypertension compared to lean controls. METHODS: A total of 63 obese hypertensive men (obeseHT), 40 obese normotensive men (obeseNT) and 27 lean normotensive men (leanNT) were included in the study. Body mass index (BMI) was between 20.0 and 24.9 kg/m2 in leanNT participants and ≥30 kg/m2 in obese participants. Hypertension was defined as a mean 24-hour systolic ambulatory blood pressure (AMBP) ≥ 130 mmHg or a mean 24-hour diastolic AMBP ≥80 mmHg and normotension as mean 24-hour AMBP <130/80 mmHg. Twenty-four hour urinary 8-oxoGuo and 8-oxodG excretion (nmol/24 h) were measured by a validated liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS). RESULTS: Urinary 8-oxoGuo excretion was (median and [interquartile range]) 30.8 [27.8-32.2] nmol/24 h in leanNT, 36.8 [31.3-40.2] nmol/24 h in obeseNT and 40.6 [31.7-48.5] nmol/24 h in obeseHT. The difference was statistically significant (p = .002) and post hoc tests showed a significant difference between leanNT and obeseHT (p = .001) as well as obeseNT (p = .002), whereas the two obese groups did not differ (p = .6). No statistically significant differences in 8-oxodG concentrations were observed between the three groups (p = .3). CONCLUSION: The measurement of urinary excretion of 8-oxoGuo suggests that obesity in men, but not hypertension, is associated with increased oxidative damage to RNA.

Simvastatin and oxidative stress in humans: A randomized, double-blinded, placebo-controlled clinical trial.

Simvastatin reduces the blood concentration of cholesterol by inhibiting hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis, and thereby reduces the risk of cardiovascular disease. In addition, simvastatin treatment leads to a reduction in fluxes in mitochondrial respiratory complexes I and II and might thereby reduce the formation of reactive oxygen species, which have been implicated in the pathogenesis of arteriosclerosis. Therefore, we hypothesized that simvastatin may reduce oxidative stress in humans in vivo. We conducted a randomized, double-blinded, placebo-controlled study in which subjects were treated with either 40mg of simvastatin or placebo for 14 days. The endpoints were six biomarkers for oxidative stress, which represent intracellular oxidative stress to nucleic acids, lipid peroxidation and plasma antioxidants, that were measured in urine and plasma samples. A total of 40 participants were included, of which 39 completed the trial. The observed differences between simvastatin and placebo groups in the primary outcomes, DNA and RNA oxidation, were small and nonsignificant (p=0.68), specifically, 3% in the simvastatin group compared to 7.1% in the placebo group for DNA oxidation and 7.3% in the simvastatin group compared to 3.4% in the placebo group. The differences in biomarkers related to plasma were not statistically significant between the treatments groups, with the exception of total vitamin E levels, which, as expected, were reduced in parallel with the reduction in plasma cholesterol. In healthy young male volunteers, short-term simvastatin treatment, which considerably reduces cholesterol, does not lead to a clinically relevant reduction in a panel of measures of oxidative stress. Whether simvastatin has effects on oxidative stress in diseased populations, such as diabetes or hemochromatosis, where oxidative stress is prominent, is unknown but seems unlikely.

Birth outcomes after exposure to mebendazole and pyrvinium during pregnancy - A Danish nationwide cohort study.

Mebendazole and pyrvinium are anthelmintics used to treat infections with pinworms, a common infection in children. Other indications for treatment with mebendazole are infections with soil-transmitted helminths. These infections are rare in Denmark, but affect more than 1.5 billion people worldwide. Limited safety data of anthelmintics during pregnancy exists and the purpose of this study was to investigate the association between exposure to mebendazole or pyrvinium during pregnancy and the adverse pregnancy outcomes: congenital malformations, stillbirths, neonatal mortality and small for gestational age. The Danish Fertility Database was used to identify all births in Denmark from 1997 to 2007. Maternal exposure to anthelmintics was identified through The Danish Prescription Registry. Of 713667 births, 2567 mothers redeemed a prescription for mebendazole; 1588 for pyrvinium. Logistic regression analysis adjusted for potential confounders. We found no association between exposure to mebendazole and major congenital malformations (OR = 0.7 (CI 95% 0.5-1.1)) or other negative birth outcomes and we found no association between exposure to pyrvinium and major congenital malformations (OR = 0.8 (CI 95% 0.4-1.5)) or other negative birth outcomes. No increased risk was found of having negative birth outcomes after exposure at any trimester during pregnancy.

Diclofenac/misoprostol during early pregnancy and the risk of miscarriage: a Danish nationwide cohort study.

INTRODUCTION: Misoprostol can be used in the prevention of gastric ulcer in treatment with diclofenac and is used in rheumatic diseases. Since misoprostol causes contractions of the uterus, it can also be used to induce abortions when administrated vaginally. The aim of the study was to investigate if early pregnancy exposure to oral diclofenac/misoprostol was associated with miscarriage. METHOD: We conducted a nationwide cohort study identifying all registered pregnancies in Denmark from 1997 to 2011. All births were identified using the Medical Birth Registry, and all records of induced abortion and miscarriage were from the National Hospital Register. Data on drug use were from the National Prescription Register. Cox proportional hazard regression models were used to calculate the hazard of miscarriage in women exposed to diclofenac/misoprostol in early pregnancy. RESULT: We identified 1,338,824 pregnancies (970,491 births, 142,147 miscarriages, 226,145 induced abortions). One hundred sixty-six were exposed to diclofenac/misoprostol in the early pregnancy of which 28.3 % (47) ended up in a miscarriage compared to 10.6 % among unexposed. The adjusted hazard ratio of having a miscarriage after exposure to diclofenac/misoprostol in the first trimester was 3.6 (CI 95 % 2.6-4.9). CONCLUSION: We found an increased risk of miscarriage after exposure to diclofenac/misoprostol during the early pregnancy. Women in the fertile age should not be treated with the combination of diclofenac/misoprostol if other options were available.

Exposure to topical chloramphenicol during pregnancy and the risk of congenital malformations: a Danish nationwide cohort study.

PURPOSE: To investigate whether exposure to topical chloramphenicol in the first trimester of pregnancy is associated with congenital malformations. METHODS: The authors conducted a nationwide cohort study including all women giving live birth between 1997 and 2011 in Denmark. All women redeeming at least one prescription of chloramphenicol eye drops or eye ointment during the first 84 days of pregnancy were identified. Logistic regression was used to estimate the odds ratios of malformations among exposed women compared to non-exposed women. RESULTS: 966 372 births between 1997 and 2011 were included. A total of 6024 women were exposed to topical chloramphenicol in the first trimester. The rate of congenital malformations was 3.50% among offspring of exposed mothers and 3.49% among unexposed. Exposure to topical chloramphenicol in the first trimester was not associated with major congenital malformations (adjusted odds ratio = 1.06, 95% CI 0.91-1.22) or specific major malformations. The number of redeemed prescriptions decreased significantly during pregnancy as compared to before and after pregnancy (p < 0.0001). CONCLUSION: In this study, we found no association between dispensing of chloramphenicol eye drops or eye ointment in the first trimester of pregnancy and major congenital malformations. This is in accordance with a previous study analysing the risk of systemic chloramphenicol.

Diurnal variation of urinary markers of nucleic acid oxidation.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) are biomarkers of oxidative stress with clinical potential in a variety of diseases. As part of their clinical validation, this study aimed to investigate whether the urinary excretion of 8-oxodG and 8-oxoGuo undergoes diurnal variation and to evaluate the validity of 6-hour sampling as well as creatinine corrected spot urine sampling. METHODS: A total of 23 healthy study subjects collecting their 24-h urine in four fractions covering 6 hours each. Urinary 8-oxodG and 8-oxoGuo levels were quantified using a modified version of UPLC-MS/MS. RESULTS: No significant difference in excretion levels between the 12-h diurnal and 12-h nocturnal state or between the four 6-h periods during the day was found for either biomarker. A strong linear relationship between the excretion levels in each of the 6-h periods and the 24-h excretion level was shown for both biomarkers. Creatinine correction of the 6-h levels reduced the biological variation of the excretion levels and weakened the linear relationship with the uncorrected 24-h excretion level for both biomarkers. The correlations were strengthened when the 24-h excretion level was expressed per kg body weight. CONCLUSION: The results showed that 8-oxodG and 8-oxoGuo did not undergo diurnal variation in the study population overall and hence that the time of sampling is not crucial. Furthermore, 6-h sampling can be used as a substitute for 24-h sampling, and creatinine corrected sampling may be rational due to the reduction in biological variation of the biomarkers and the reasonable correlation with body weight-adjusted 24-h levels.