Adaptive Effects of Intermittent Hypoxia Training on Oxygen-Dependent Processes as a Potential Therapeutic Strategy Tool.

BACKGROUND/AIMS: Important benefits of intermittent hypoxic training (IHT) have emerged as an effective tool for enhancing adaptive potential in different pathological states, among which acute hypoxia dominates. Therefore, the aim of our study was to evaluate the mechanisms related to the effects of the nitric oxide system (nitrites, nitrates, carbamide, and total polyamine content) on ADP-stimulated oxygen consumption and oxidative phosphorylation in heart and liver mitochondria and biomarkers of oxidative stress in the blood, heart, and liver of rats exposed to the IHT method and acute hypoxia and treated with the amino acid L-arginine (600 mg/kg, 30 min) or the NO synthase inhibitor L-NNA (35 mg/kg, 30 min) prior to each IHT session. METHODS: We analysed the modulation of the system of oxygen-dependent processes (mitochondrial respiration with the oxygraphic method, microsomal oxidation, and lipoperoxidation processes using biochemical methods) in tissues during IHT in the formation of short-term and long-term effects (30, 60, and 180 days after the last IHT session) with simultaneous administration of L-arginine. In particular, we investigated how mitochondrial functions are modulated during intermittent hypoxia with the use of oxidation substrates (succinate or α-ketoglutarate) in bioenergetic mechanisms of cellular stability and adaptation. RESULTS: The IHT method is associated with a significant increase in the production of endogenous nitric oxide measured by the levels of its stable metabolite, nitrite anion, in both plasma (almost 7-fold) and erythrocytes (more than 7-fold) of rats. The intensification of nitric oxide-dependent pathways of metabolic transformations in the energy supply processes in the heart and liver, accompanied by oscillatory mechanisms of adaptation in the interval mode, causes a probable decrease in the production of urea and polyamines in plasma and liver, but not in erythrocytes. The administration of L-arginine prior to the IHT sessions increased the level of the nitrite-reducing component of the nitric oxide cycle, which persisted for up to 180 days of the experiment. CONCLUSION: Thus, the efficacy of IHT and its nitrite-dependent component shown in this study is associated with the formation of long-term adaptive responses by preventing the intensification of lipoperoxidation processes in tissues due to pronounced changes in the main enzymes of antioxidant defence and stabilisation of erythrocyte membranes, which has a pronounced protective effect on the system of regulation of oxygen-dependent processes as a whole.

Aminopyrine breath test predicts liver-related events and death in HCV-related cirrhosis on SVR after DAA therapy.

BACKGROUND & AIMS: In patients with hepatitis C virus (HCV)-related advanced cirrhosis, the effects of sustained virological response (SVR) by direct antiviral agents (DAAs) on decompensation and liver deaths are less clearcut, since up to 30% of patients do not improve, and no predictors of outcome have been identified. We used 13 C-aminopyrine breath test (ABT) to assess whether its changes can predict liver-related outcomes after DAA treatment in patients with HCV cirrhosis. METHODS: Fifty consecutive patients with HCV cirrhosis were enrolled. Patients were included if they had Child A cirrhosis at risk for decompensation - defined as Child A6 (N = 22, 44%) or previous decompensation (N = 7, 14%) - or Child B cirrhosis (N = 21, 42%) eligible for DAA-based antiviral therapy. ABT was performed at baseline and 12 weeks after the end of antiviral therapy. Patients received sofosbuvir-based regimens. RESULTS: Aminopyrine breath test was available for all 50 patients at baseline. The 120' cumulative dose was directly associated at regression analysis only with albumin levels (P = .001). ABT was available at follow-up week 12 for 41 patients (FUW12), all with SVR, and followed for a median of 25.2 months (range 12.2-32.1 months). Lower Ʌ ABT - defined as changes of 120' cumulative dose from FUW12 to baseline - (HR 0.97, 95% CI 0.94-0.99; P = .02) and FUW12 hepatic encephalopathy (HR 19.0, 95% CI 1.16-310.3; P = .03) were the only independent predictors of liver events/death at multivariate Cox regression analysis. The AUC of Ʌ ABT was good (0.87, 95% CI 0.75-0.97), with a delta ≥0% well discriminating patients at lower vs patients at higher risk of liver-related events/death (P < .001). CONCLUSIONS: In patients with advanced HCV cirrhosis who achieve SVR with DAA, Ʌ ABT assists in assessing the residual likelihood of liver-related events and deaths after viral cure.

Simultaneous determination of aminopyrine and antipyrine in porcine muscle, milk, and eggs using liquid chromatography with tandem mass spectrometry.

The concentrations of residual aminopyrine and antipyrine in porcine muscle, milk, and egg samples were analyzed using liquid chromatography with tandem mass spectrometry after undergoing a series of sample pretreatment steps. Owing to an ion suppression effect, matrix-matched calibrations were used for analyte quantitation with determination coefficients (R(2) ) ≥ 0.9931. The recovery rates for aminopyrine and antipyrine in various matrices at two spiking levels (5 and 10 ng/g) fell in the range of 60.96-68.87 and 61.87-66.99%, respectively. Meanwhile, the intra- and inter-day precisions (expressed as relative standard deviation) were 1.02-12.95 and 1.71-5.50%, respectively. The method's detection limit (1 ng/g) was very low, thus enabling the detection of low residue levels. The applicability of the developed method was demonstrated with actual market samples and none of the tested analytes was detected in any of the samples.

Critical appraisal of 13C breath tests for microsomal liver function: aminopyrine revisited.

As liver diseases are a major health problem and especially the incidence of metabolic liver diseases like non-alcoholic fatty liver disease (NAFLD) is rising, the demand for non-invasive tests is growing to replace liver biopsy. Non-invasive tests such as carbon-labelled breath tests can provide a valuable contribution to the evaluation of metabolic liver function. This review aims to critically appraise the value of the (13) C-labelled microsomal breath tests for the evaluation of metabolic liver function, and to discuss the role of cytochrome P450 enzymes in the metabolism of the different probe drugs, especially of aminopyrine. Although a number of different probe drugs have been used in breath tests, the perfect drug to assess the functional metabolic capacity of the liver has not been found. Data suggest that both the (13) C(2) -aminopyrine and the (13) C-methacetin breath test can play a role in assessing the capacity of the microsomal liver function and may be useful in the follow-up of patients with chronic liver diseases. Furthermore, CYP2C19 seems to be an important enzyme in the N-demethylation of aminopyrine, and polymorphisms in this gene may influence breath test values, which should be kept in mind when performing the (13) C(2) -aminopyrine breath test in clinical practice.

Curiosities in drug metabolism.

1. It is inevitable that during some xenobiotic biotransformation studies, a certain metabolite or degradation product arises of which the identity is uncertain, the route of formation is ambiguous, or it is just a plain mystery. 2. The following communication draws attention to three drugs reported in the literature, chlorphentermine, phenothiazine and aminopyrine, where after many years of investigation there still exists uncertainty over some of their metabolites. Noticeably, these three examples probably involve (potential) interaction of a nitrogen centre within the drug molecule. 3. It is hoped that the resurrection and assemblage of these data will offer interesting reading and that these examples may prove sufficiently intriguing to motivate further exploration and some resolution of these lingering concerns.

Developing an ultrasensitive immunochromatographic assay for authentication of an emergent fraud aminopyrine in herbal tea.

Aminopyrine is a nonsteroidal anti-inflammatory drug only for medical purposes, however, it has been illegally added in traditional Chinese herbal teas for fraud activity recently. In this study, a specific antibody against aminopyrine with IC50 of 3.00 ng/mL was obtained for the first time by a rational hapten design. Furthermore, an ultrasensitive gold nanoparticles immunochromatographic assay (AuNPs-ICA) for determination of aminopyrine based on a portable reader was firstly developed, with cut-off value of 100.00 ng/mL, limit of detection (LOD) of 4.80 ng/mL and limit of quantification (LOQ) of 5.71 ng/mL for herbal tea, respectively. The recovery rates ranged from 93.21 % to 105.61 %, with inter-assay coefficient of variation (CV) from 1.08 % to 3.82 %. Additionally, 24 blind samples were examined simultaneously by AuNPs-ICA and LC-MS/MS, demonstrating a good consistency for each other. The proposed AuNPs-ICA is an ultrasensitive and reliable tool for on-site surveillance screening of fraud additives in herbal tea.

[Determination of dimethyl sulfate genotoxic impurities in tertiary amine drugs by ultra-high performance liquid chromatography-tandem mass spectrometry].

Dimethyl sulfate is an important chemical raw material that is widely used in the synthesis of drugs, dyes, spices, and pesticides. The highly toxic and corrosive dimethyl sulfate residue in medicines is harmful to the human body, and hence, the residue level should be strictly controlled. Traditional detection methods use high-purity acetonitrile and anhydrous as the solvents, which limits the choice of detection solvents and degrades the versatility and accuracy of detection. Therefore, a simple and accurate method for the determination of dimethyl sulfate residues is urgently needed. Dimethyl sulfate is usually detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with pyridine as the methylation substrate. In this study, a new method for the detection of dimethyl sulfate was established using tertiary amines such as aminophenazone, which has many advantages over pyridine, as the methylation substrate. For example, the hybrid orbital and electron cloud of the N atom are different, resulting in stronger nucleophilicity of aminophenazone. High temperatures that are detrimental to the stability of dimethyl sulfate are not required when using aminophenazone, and the aliphatic quaternary ammonium salt product is more stable, with good stability, low interference, good ionization properties, and high response. The separation was performed on a Waters Atlantis HILIC C18 column (100 mm×2.1 mm, 3.0 µm) using a mobile phase consisting of 10 mmol/L ammonium acetate solution-0.1% formic acid methanol solution (50∶50, v/v) at a flow rate of 0.3 mL/min. The column temperature was set at 40 ℃, and the sample size was 1 µL. Dimethyl sulfate was determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes. Dimethyl sulfate showed good linear relationships within the range of 0.9935 to 7.9480 ng/mL (r=0.9997). The limit of detection and limit of quantification for dimethyl sulfate were 0.50 ng/mL and 1.15 ng/mL, respectively. The recoveries (n=3)of dimethyl sulfate were 94.9% to 106.4%. The relative standard deviations (RSDs) were 1.44% to 5.51%. The RSD of the methylated aminophenazone peak area was 4.32%, indicating good stability of the reaction product. Dimethyl sulfate genotoxic impurities were not detected in 9 batches of aminophenazone, caffeine, and tegafur samples, which indicated that the drug manufacturers paid attention to the control of these impurities. The proposed method is advantageous over the existing techniques in terms of the better ion peak shape and higher molecular weight, without interference from other fragments. The method is specific, sensitive, simple, rapid, and accurate, and it can be used for the determination of dimethyl sulfate genotoxic impurities in aminophenazone and other medicines.

Non-Alcoholic Steatohepatitis Decreases Microsomal Liver Function in the Absence of Fibrosis.

The incidence of non-alcoholic fatty liver disease (NAFLD) is rising across the globe, with the presence of steatohepatitis leading to a more aggressive clinical course. Currently, the diagnosis of non-alcoholic steatohepatitis (NASH) is based on histology, though with the high prevalence of NAFLD, a non-invasive method is needed. The 13C-aminopyrine breath test (ABT) evaluates the microsomal liver function and could be a potential candidate. We aimed to evaluate a potential change in liver function in NASH patients and to evaluate the diagnostic power of ABT to detect NASH. We performed a retrospective analysis on patients suspected of NAFLD who underwent a liver biopsy and ABT. 440 patients were included. ABT did not decrease in patients with isolated liver steatosis but decreased significantly in the presence of NASH without fibrosis and decreased even further with the presence of significant fibrosis. The predictive power of ABT as a single test for NASH was low but improved in combination with ALT and ultrasonographic steatosis. We conclude that microsomal liver function of patients with NASH is significantly decreased, even in the absence of fibrosis. The ABT is thus a valuable tool in assessing the presence of NASH; and could be used as a supplementary diagnostic tool in clinical practice.

Non-alcoholic fatty liver disease causes dissociated changes in metabolic liver functions.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a major health concern affecting 25% of the world's population. It is generally held that a fatty liver does not influence liver function, but quantitative measurements of metabolic liver functions have not been systematically performed. We aimed to study selected hepatocellular metabolic functions in patients with different stages of NAFLD. METHODS: Twenty-five non-diabetic, biopsy-proven NAFLD patients [12 with simple steatosis; 13 with non-alcoholic steatohepatitis (NASH)] and ten healthy controls were included in a cross-sectional study. Hepatocyte cytosolic function was assessed by the galactose elimination capacity (GEC), mitochondrial-cytosolic metabolic capacity by the functional hepatic nitrogen clearance (FHNC), microsomal function by the aminopyrine breath test, and excretory liver function by indocyanine green (ICG) elimination. RESULTS: GEC was 20% higher in NAFLD than in controls [3.15 mmol/min (2.9-3.41) vs. 2.62 (2.32-2.93); P = 0.02]. FHNC was 30% lower in NAFLD [23.3 L/h (18.7-28.9) vs. 33.1 (28.9-37.9); P = 0.04], more so in simple steatosis [19.1 L/h (13.9-26.2); P = 0.003] and non-significantly in NASH [27.9 L/h (20.6-37.8); P = 0.19]. Aminopyrine metabolism was 25% lower in simple steatosis [8.9% (7.0-10.7)] and 50% lower in NASH [6.0% (4.5-7.5)] than in controls [11.9% (9.3-12.8)] (P < 0.001). ICG elimination was intact. CONCLUSIONS: The hepatocellular metabolic functions were altered in a manner that was dissociated both by different effects on different liver functions and by different effects of different stages of NAFLD. Thus, NAFLD has widespread consequences for metabolic liver function, even in simple steatosis.

Analytical challenges in the confirmative identification of dipyrone as an adulterant in illicit drug samples.

Dipyrone is an analgesic and antipyretic drug that is sometimes encountered as an adulterant in illicit drug samples, particularly illicit fentanyl containing samples. It undergoes thermal decomposition to aminopyrine and 4-methylaminoantipyrine during analysis via gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). During analysis via high pressure liquid chromatography (HPLC) and high pressure liquid chromatography-mass spectrometry (HPLC-MS), it undergoes hydrolytic decomposition solely to 4-methylaminoantipyrine. Given that mass spectrometry is a widely used confirmatory analytical technique, these instabilities present challenges for the forensic chemist seeking to confirm the presence of dipyrone. Studies were conducted to determine rigorous confirmative protocols for the identification of dipyrone in multicomponent illicit drug samples.

Transformation of aminopyrine in the presence of free available chlorine: Kinetics, products, and reaction pathways.

Aminopyrine (AMP) has been frequently detected in the aquatic environment. In this study, the transformation mechanism of AMP by free available chlorine (FAC) oxidation was investigated. The results showed that FAC reacted with AMP rapidly, and a 74% elimination was achieved for 1.30 µM AMP after 2 min at 14.08 µM FAC dose. AMP chlorination was strongly pH-dependent, and its reaction included second- and third-order kinetic processes. Three active FAC species, including chlorine monoxide (Cl2O), molecular chlorine (Cl2), and hypochlorous acid (HOCl), were observed to contribute to AMP degradation. The intrinsic rate constants of each FAC species with neutral (AMP0) and cation (AMP+) species were obtained by kinetic fitting. Cl2O exhibited the highest reactivity with AMP0 (kAMP0, Cl2O = (4.33 ± 1.4) × 109 M-1s-1). In addition, Cl2 showed high reactivity (106-107 M-1s-1) in the presence of chloride, compared with HOCl (kAMP+, HOCl = (5.73 ± 0.23) × 102 M-1s-1, kAMP0, HOCl = (9.68 ± 0.96) × 102 M-1s-1). At pH 6.15 and 14.08 µM FAC dose without chloride addition, the contribution of Cl2O reached to the maximum (33.3%), but in the whole pH range, HOCl was the main contributor (>66.6%) for AMP degradation. The significance of Cl2 was noticeable in water containing chloride. Moreover, 11 transformation products were identified, and the main transformation pathways included pyrazole ring breakage, hydroxylation, dehydrogenation, and halogenation.

Eyelid skin as a potential site for drug delivery to conjunctiva and ocular tissues.

The feasibility of topical application onto the (lower) eyelid skin to deliver hydrophilic and lipophilic compounds into the conjunctiva and ocular tissues was evaluated by comparing with conventional eye drop application. Skin permeation and the concentration of several model compounds, and skin impedance were determined utilizing eyelid skin from hairless rats, as well as abdominal skin in the same animals for comparison. In vitro static diffusion cells were used to assess the skin permeation in order to provide key insights into the relationship between the skin sites and drugs. The obtained results revealed that drug permeation through the eyelid skin was much higher than that through abdominal skin regardless of the drug lipophilicity. Specifically, diclofenac sodium salt and tranilast exhibited approximately 6-fold and 11-fold higher permeability coefficients, respectively, through eyelid skin compared with abdominal skin. Histomorphological evaluation and in vivo distribution of model fluorescent dyes were also examined in the conjunctiva and skin after eyelid administration by conventional microscope and confocal laser scanning microscope analyses. The result revealed that eyelid skin has a thinner stratum corneum, thereby showing lower impedance, which could be the reason for the higher drug permeation through eyelid skin. Comparative evaluation of lipophilic and hydrophilic model compounds administered via the eyelid skin over 8h revealed stronger fluorescence intensity in the skin and surrounding tissues compared with eye drop administration. These results suggested that the (lower) eyelid skin is valuable as a prospective site for ophthalmic medicines.

A phase II study of sunitinib in advanced hepatocellular carcinoma.

BACKGROUND: In 2007, sorafenib was the first drug able to improve overall survival in patients with advanced hepatocellular carcinoma. AIM: In 2005 we designed a phase II study to assess safety and efficacy of sunitinib. METHODS: This is a single arm, open-label, single-centre phase II trial. Eligibility criteria were advanced hepatocellular carcinoma; no prior chemotherapy, performance status 0-1; and Child≤B8. The treatment schedule was 50mg each day orally, 4 weeks on, 2 weeks off. RESULTS: Between 10/2007 and 10/2010, 34 patients were enrolled. A significant worsening of liver functional reserve after sunitinib was observed. Grade 3/4 adverse effects occurred in 80% of patients and included fatigue (47%), nausea (15%), liver failure (15%), encephalopathy (12%) and upper gastrointestinal bleeding (12%). Six patients (18%) died within 60 days of enrolment. A partial response was observed in 4 patients (12%). Median time to tumour progression was 2.8 months and median overall survival was 5.8 months. CONCLUSION: A dose of 50mg/d induces a high rate of severe adverse events. Toxicity remains a key concern also at the dose of 37.5mg/d. However, sunitinib is able to induce a prolonged response in some patients. Positron Emission Tomography/Computed Tomography scans may select good responders.

Two or more synchronous combination of noninvasive tests to increase accuracy of liver fibrosis assessement in chronic hepatitis C; results from a cohort of 446 patients.

BACKGROUND: The prediction of fibrosis is an essential part of the assessment and management of patients with chronic liver disease. Non-invasive tests (NITs) have a number of advantages over the traditional standard of fibrosis assessment by liver biopsy, including safety, cost-effectiveness, and widespread accessibility. OBJECTIVES: The aim of this study was to determine the accuracy of certain biomarkers and transient elastography (TE) alone or in combination to predict the stage of liver fibrosis in chronic hepatitis C (CHC). Also, we examined whether the combination of certain biomarkers and TE could increase the diagnostic accuracy of liver fibrosis assessment. PATIENTS AND METHOD: A total of 446 patients who were previously diagnosed with CHC were included in the study. In the study group, 6 blood-based scores (APRI, Forns, Fib-4, Hepascore, FibroTest, and Fibrometer) were calculated, and TE was performed to validate the stage of fibrosis, compared with liver biopsy (LB) as the standard. RESULTS: Significant fibrosis (F ≥ 2) was predicted with an AUROC of 0.727, 0.680, 0.714, 0.778, 0.688, 0.797, and 0.751 for the APRI, Forns, Fib-4, FibroTest, Hepascore, and Fibrometer scores and TE (Fibroscan), respectively. Severe fibrosis (F ≥ 3) was predicted, with AUROCs ranging between 0.705 and 0.811 for Hepascore and Fibrometer, respectively. Of the biomarkers, Fibrometer had the highest AUROC value in predicting both significant and severe fibrosis. The combination of APRI or FIB-4 with Fibrometer increased the diagnostic accuracy for significant fibrosis (from 69.07 to 82.27 for APRI, P = 0.001 and from 57.74 to 81.33, P = 0.001 for Fib-4). Combining APRI or Fib-4 with TE also increased the diagnostic accuracy (from 69.07 to 80.70%, P = 0.001 for APRI and from 57.74 to 81.33%, P = 0.001 for Fib-4) for significant fibrosis. The association that included Fibrotest was also reliable for the improvement of diagnostic accuracy. These combinations were more accurate or the assessment of severe fibrosis. CONCLUSIONS: The synchronous association between a simple, inexpensive score and a complex but expensive score or TE increases the diagnostic accuracy of non-invasive methods for the assessment of liver fibrosis stage.

Effect of methomyl on hepatic mixed function oxidases in rats.

OBJECTIVE: To study the effect of the methomyl on mixed function oxidase system in rats. MATERIALS AND METHODS: The effect of the methomyl on mixed function oxidase was studied using different dosages, durations and sex. Microsomes were isolated using the calcium precipitation method. The levels of cytochrome P(450) , and cytochrome b(5) were determined using extinction coefficient of 91 and 85 mM(-1) respectively. The activities of drug metabolizing enzymes, hemoglobin content, liver function enzymes, and serum cholinesterase activity were assayed by using standard methods. RESULTS: Intraperitoneal administration of methomyl (4 mg/kg body weight) showed significant decrease in the level of cytochrome P(450) , and the activities of aminopyrine N-demethylase and aniline hydroxylase on the third day of the treatment. Methomyl (4 mg/kg) treatment of old male rat and adult female rat also showed a decrease in the level of cytochrome P(450) , and aminopyrine N-demethylase activity. The serum samples from methomyl treated rats (male and female), when analyzed for alanine aminotransferase (SGPT) and aspartate aminotransferase (SGOT) as markers of the liver toxicity, showed significant increase in the activity. The activities of SGPT and SGOT were significantly higher in the treated rats (2 and 4 mg/kg) than in the control group. A significant decrease in the level of hemoglobin and serum cholinesterase activity was observed, when there was an increase in the dose level. A significant increase was observed in alkaline phosphatase activity at all dose levels. CONCLUSION: Methomyl influences mixed function oxidase and creates abnormality of liver functions in the rats. This effect depends on the dose and duration of methomyl.