Activation tagging identifies Arabidopsis transcription factor AtMYB68 for heat and drought tolerance at yield determining reproductive stages.

Heat stress occurring at reproductive stages can result in significant and permanent damage to crop yields. However, previous genetic studies in understanding heat stress response and signaling were performed mostly on seedling and plants at early vegetative stages. Here we identify, using a developmentally defined, gain-of-function genetic screen with approximately 18 000 Arabidopsis thaliana activation-tagged lines, a mutant that maintained productive seed set post-severe heat stress during flowering. Genome walking indicated this phenotype was caused by the insertion of 35S enhancers adjacent to a nuclear localized transcription factor AtMYB68. Subsequent overexpression analysis confirmed that AtMYB68 was responsible for the reproductive heat tolerance of the mutant. Furthermore, these transgenic Arabidopsis plants exhibited enhanced abscisic acid sensitivity at and post-germination, reduced transpirational water loss during a drought treatment, and enhanced seed yield under combined heat and drought stress during flowering. Ectopic expression of AtMYB68 in Brassica napus driven either by 35S or by heat-inducible promoter recapitulated the enhanced reproductive heat stress and drought tolerance phenotypes observed in the transgenic Arabidopsis. The improvement to heat stress is likely due to enhanced pollen viability observed in the transgenic plants. More importantly, the transgenic canola showed significant yield advantages over the non-transgenic controls in multiple locations, multiple season field trials under various drought and heat stress conditions. Together these results suggest that AtMYB68 regulate plant stress tolerance at the most important yield determining stage of plant development, and is an effective target for crop yield protection under current global climate volatility.

Investigation of intestinal elimination and biliary excretion of ibuprofen in hyperglycemic rats.

An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion in the rat. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg, i.v.). The intestinal perfusion medium contained 250 µM (±)-ibuprofen. An isocratic high-performance liquid chromatography method with UV-visible detection was developed to quantitate ibuprofen in the intestinal perfusate, while a gradient method was applied to quantitate ibuprofen and ibuprofen-ß-d-glucuronide in the bile. The limit of quantitation of ibuprofen was found to be 0.51 µM in the perfusate of the small intestine. In the bile, the limit of quantitation of ibuprofen and ibuprofen-ß-d-glucuronide was 4.42 and 10.3 µM, respectively. Unconjugated ibuprofen and ibuprofen-ß-d-glucuronide were detected in the bile; however, no ß-d-glucuronide of ibuprofen could be detected in the intestinal perfusate. The results indicate that experimental diabetes can cause a decrease in the disappearance of ibuprofen from the small intestine. Excretion of both ibuprofen and ibuprofen-ß-d-glucuronide decreased to the bile in experimental diabetes. The results can be explained by the results of molecular biological studies indicating streptozotocin-initiated alterations in the intestinal and hepatic transport processes.

Toxicokinetic Study of a Gastroprotective Dose of Capsaicin by HPLC-FLD Method.

BACKGROUND: A low dose of capsaicin and its natural homologs and analogs (capsaicinoids) have shown to prevent development of gastric mucosal damage of alcohol and non-steroid anti-inflammatory drugs. Based on this experimental observation, a drug development program has been initiated to develop per os applicable capsaicin containing drugs to eliminate gastrointestinal damage caused by non-steroid anti-inflammatory drugs. METHODS: As a part of this program, a sensitive and selective reverse-phase high-performance liquid chromatography-based method with fluorescence detection has been developed for quantification of capsaicin and dihydrocapsaicin in experimental dog's plasma. RESULTS: The method was evaluated for a number of validation characteristics (selectivity, repeatability, and intermediate precision, LOD, LOQ, and calibration range). The limit of detection (LOD) was 2 ng/mL and the limit of quantification (LOQ) was 10 ng/mL for both capsaicin and dihydrocapsaicin. The method was used for analysis of capsaicin and dihydrocapsaicin in the plasma samples obtained after per os administration of low doses (0.1, 0.3, and 0.9 mg/kg bw) of Capsaicin Natural (USP 29) to the experimental animals. CONCLUSIONS: The obtained results indicated that the administered capsaicinoids did not reach the general circulation.

Reagent-induced asymmetric induction in addition reaction of reduced glutathione onto bis-Mannich chalcones.

The stereochemistry of non-enzyme catalyzed nucleophilic addition of GSH to 4'-hydroxychalcone 1 and its bis-Mannich derivative 2 was investigated at different pH values (pH 3.2, 6.1, 7.4, and 8.0). The stereochemical outcome of the reactions was evaluated by HPLC-UV-Vis method. Under strongly acidic conditions (pH 3.2), an unexpected diastereoselective addition of GSH onto the bis-Mannich derivative 2 was observed. Such a selectivity could not be observed in the similar reaction of 2 with N-acetylcysteine. The observed stereoselectivity can be rationalized by ion-pair formation between the protonated Mannich nitrogens and the deprotonated GSH(glutamate)-carboxylate. To the best of our knowledge, this is the first example of reagent-induced asymmetric induction in Michael-type additions of thiols.

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat.

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 µm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC-DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5-50 µg/mL. After liquid-liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC-MS. The method was linear over the concentration range 0.25-5.0 µg/mL. Two hydroxylated metabolites of salicylic acid (2,5-dihydroxybenzoic acid and 2,3-dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3-DHB, 72.5% for 2,5-DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.

Phenolic compounds in Hungarian acacia, linden, milkweed and goldenrod honeys.

Honey is a valuable source of nutrients, minerals and phenolic compounds. Phenolic acids and flavonoids are associated with health benefits of honey and can serve as markers for distinguishing honey types. This study aimed at determining the phenolic profile of four Hungarian unifloral honeys that were not analyzed previously. After verifying their botanical origin with melissopalynological analysis, total reducing capacity was determined with Folin-Ciocalteau method, and phenolic composition was analyzed with HPLC-DAD-MS. From the 25 phenolic substances examined, pinobanksin was the most abundant, followed by chrysin, p-hydroxybenzoic acid and galangin. Quercetin and p-syringaldehyde were detected only in acacia honey, which contained higher levels of chrysin and hesperetin compared to the other three honeys. Milkweed and linden honeys displayed higher levels of caffeic, chlorogenic, ferulic and p-coumaric acids compared to acacia and goldenrod honeys. Taxifolin may serve as a unique marker compound of milkweed honey. Goldenrod honey contained the highest level of syringic acid. Principal component analysis supported the indicator role of polyphenols in honey identification, discriminating clearly the four unifloral honeys. Our results suggest that phenolic profiles may be useful to find markers of honey's floral origin, but geographical origin can strongly influence the composition of characteristic compounds.

Simultaneous fatal poisoning of two victims with 4F-MDMB-BINACA and ethanol.

PURPOSE: Methyl-2-(1-(4-fluorobutyl)-1H-indazole-3-carboxamido)-3,3-dimethylbutanoate (4F-MDMB-BINACA) is a newly emerging synthetic cannabinoid receptor agonists (SCRA) first described in 2018 in both Europe and the United States. Two fatal cases are reported caused by simultaneous consumption of 4F-MDMB-BINACA and ethanol. METHODS: The victims were brothers who were both found deceased after consuming 4F-MDMB-BINACA and ethanol. Post-mortem toxicological analyses of blood and urine were carried out by supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) and headspace gas chromatography with flame ionization detection (HS-GC-FID). RESULTS: The concentration of 4F-MDMB-BINACA in the postmortem blood was 2.50 and 2.34 ng/mL, and blood alcohol concentration was 2.11 and 2.49 g/L, respectively. CONCLUSION: According to the reported cases and reviews of the scientific literature, concurrent ethanol consumption should amplify the toxicity of SCRAs. The threshold SCRA concentration for fatal overdose can be estimated ng/mL level (0.37-4.1 ng/mL according to the reported cases) in cases in which 1.5-2.5 g/L of ethanol is present in the blood.

Fatal Overdose with the Cannabinoid Receptor Agonists MDMB-4en-PINACA and 4F-ABUTINACA: A Case Report and Review of the Literature.

A case of a 26-year-old male who died from consuming synthetic cannabinoid receptor agonists MDMB-4en-PINACA and 4F-ABUTINACA is reported. MDMB-4en-PINACA and 4F-ABUTINACA are potent synthetic cannabinoid receptor agonists (SCRAs). This is the first detailed reporting of MDMB-4-en-PINACA and 4F-ABUTINACA associated fatality, which can help the routine forensic work. The scientific literature on the symptoms associated with these substances are evaluated, along with the pharmacological properties and possible mechanism of death. A forensic autopsy was performed according to Recommendation No. R (99)3 of the Council of Europe on medico-legal autopsies. Histological samples were stained with hematoxylin and eosin (HE). Complement component C9 immunohistochemistry was applied to all heart samples. Toxicological analyses were carried out by supercritical fluid chromatography coupled with tandem mass spectrometry (SFC-MS/MS) and headspace gas chromatography with a flame ionization detector (HS-GC-FID). The literature was reviewed to identify reported cases of MDMB-4en-PINACA and 4F-ABUTINACA use. Autopsy findings included brain edema, internal congestion, petechial bleeding, pleural ecchymoses, and blood fluidity. Toxicological analyses determined 7.2 ng/mL of MDMB-4en-PINACA and 9.1 ng/mL of 4F-ABUTINACA in the peripheral blood. MDMB-4en-PINACA and 4F-ABUTINACA are strong, potentially lethal SCRA, and their exact effects and outcome are unpredictable.

Lethal case of myocardial ischemia following overdose of the synthetic cannabinoid ADB-FUBINACA.

The serious adverse effects of synthetic cannabinoids (SCB), the lack of human pharmacological data on SCBs, and the increasing number of SCBs with diverse structures are growing public health concerns. A fatal case of myocardial ischemia after ADB-FUBINACA overdose is reported. A 41-year-old male died after consuming a brown, powder-like drug. Autopsy revealed pallor in the left ventricle of the subendocardial two-third of the myocardium, and histological examination revealed early signs of myocardial ischemia: few wavy myocardial fibers, contraction band necrosis in the subendocardial region, and patchy subendocardial complement component 9 (C9) positivity. Toxicological analysis detected a high concentration of the indazole carboxamide derivative SCB ADB-FUBINACA (peripheral blood: 105 ng/mL) and a low concentration of the synthetic cathinone (SC) derivative stimulant N-ethylpentylone (NEP). The literature concerning ADB-FUBINCA overdoses is reviewed, and the possible mechanism of death and the cardiac effects of SCBs are discussed. Effects of SCBs are unpredictable, but they are potentially cardiotoxic, capable causing arrhythmias, cardiac hypertrophies, and myocardial ischemia. The cardiotoxicity of SCBs can be attributed to vasospasms, decreased myocardial contractility, and increased cardiac workload and oxygen demand. Based on the autopsy, histology, and toxicology, it could be reasonably suggested, that ADB-FUBINACA have been a significant contributor to the myocardial ischemia seen in histology. The mechanism of death was likely fatal arrhythmia induced by the patchy myocardial ischemia. Due to the low concentration of NEP, it's role in the fatal outcome is improbable.

Functionally active TRPA1 ion channel is downregulated in peptidergic neurons of the Edinger-Westphal nucleus upon acute alcohol exposure.

Introduction: The centrally projecting Edinger-Westphal nucleus (EWcp) contributes to the control of alcohol consumption by its urocortin 1 (UCN1) and cocaine- and amphetamine-regulated transcript (CART) co-expressing peptidergic neurons. Our group recently showed that the urocortinergic centrally projecting EWcp is the primary seat of central nervous system transient receptor potential ankyrin 1 (TRPA1) cation channel mRNA expression. Here, we hypothesized that alcohol and its metabolites, that pass through the blood-brain barrier, may influence the function of urocortinergic cells in centrally projecting EWcp by activating TRPA1 ion channels. We aimed to examine the functional activity of TRPA1 in centrally projecting EWcp and its possible role in a mouse model of acute alcohol exposure. Methods: Electrophysiological measurements were performed on acute brain slices of C57BL/6J male mice containing the centrally projecting EWcp to prove the functional activity of TRPA1 using a selective, potent, covalent agonist JT010. Male TRPA1 knockout (KO) and wildtype (WT) mice were compared with each other in the morphological studies upon acute alcohol treatment. In both genotypes, half of the animals was treated intraperitoneally with 1 g/kg 6% ethanol vs. physiological saline-injected controls. Transcardial perfusion was performed 2 h after the treatment. In the centrally projecting EWcp area, FOS immunohistochemistry was performed to assess neuronal activation. TRPA1, CART, and urocortin 1 mRNA expression as well as urocortin 1 and CART peptide content was semi-quantified by RNAscope in situ hybridization combined with immunofluorescence. Results: JT010 activated TRPA1 channels of the urocortinergic cells in acute brain slices. Alcohol treatment resulted in a significant FOS activation in both genotypes. Alcohol decreased the Trpa1 mRNA expression in WT mice. The assessment of urocortin 1 peptide immunoreactivity revealed lower basal urocortin 1 in KO mice compared to WTs. The urocortin 1 peptide content was affected genotype-dependently by alcohol: the peptide content decreased in WTs while it increased in KO mice. Alcohol exposure influenced neither CART and urocortin 1 mRNA expression nor the centrally projecting EWcp/CART peptide content. Conclusion: We proved the presence of functional TRPA1 receptors on urocortin 1 neurons of the centrally projecting EWcp. Decreased Trpa1 mRNA expression upon acute alcohol treatment, associated with reduced neuronal urocortin 1 peptide content suggesting that this cation channel may contribute to the regulation of the urocortin 1 release.

Illicit Drugs as a Potential Risk to the Aquatic Environment of a Large Freshwater Lake after a Major Music Festival.

The present study strengthens the view that residues of drugs of abuse may become widespread surface water contaminants following a local music festival. Overall, 10 illicit drugs were detected from the aquatic environment after the festival; cocaine and 3,4-methylenedioxymethamphetamine were present in the highest concentrations. The presence of illicit drugs and their metabolites over 3 monitored festival yr suggested that consumption of these drugs was temporally linked with events. Weather conditions seriously influenced detection of contaminants deriving from events at the lakeshore. Most of the illicit drugs retained their pharmacological activities, with a potentially adverse impact on wildlife. Environ Toxicol Chem 2021;40:1491-1498. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

New psychoactive versus conventional stimulants - a ten-year review of casework in Hungary.

New psychoactive stimulants appeared in Hungary in 2010 as in several other European countries. We present our findings from cases where new psychoactive and conventional stimulants (we listed amphetamine, methamphetamine and MDMA as conventional drugs) have been detected in biological specimens between 2010 and 2019. MATERIALS: Biological samples (including urine, blood and body tissues), sent to the Department of Forensic Medicine, University of Pécs, South-West Hungary, in the period 2010-2019. METHOD: High performance liquid chromatography coupled with diode array detection (HPLC-DAD); supercritical fluid chromatography coupled to tandem mass spectrometry (SFC-MS/MS). RESULTS: During the nine-year period between 2010 and 2019, we found new stimulants in 973 (21.1%) cases, and conventional stimulants in 658 (14.2%) cases (out of 4604 analyses -100%- of samples sent to the laboratory for toxicology screening). 594 (12.9%) of all cases were post mortem analyses. The new drugs we've detected could be classified into three groups based on their chemical structure: cathinones (in 960 from our cases), substituted phenethylamines (8), and tryptamines (5). The most frequently identified new psychoactive stimulants were (in the order of decreasing frequency): pentedrone (262), mephedrone (188), N-ethylhexedrone (126), methylenedioxypyrovalerone (MDPV; 98), α-pyrrolidinopentiophenone (alpha-PVP; 93), 4-CMC (35). CONCLUSION: The new substances were detected in highest proportion in 2011; by 2018, the number of conventional drugs exceeded the new stimulants in our cases. According to the data of the Hungarian seizures, the decrease was predictable: from 2015, the seizures of traditional stimulants exceeded the seizures of new stimulants. In 2019 the new stimulants were dominated again among the detected substances in the samples.

Interdisciplinary review for correlation between the plant origin capsaicinoids, non-steroidal antiinflammatory drugs, gastrointestinal mucosal damage and prevention in animals and human beings.

BACKGROUND: The plant origin capsaicinoids (capsaicin, dihydrocapsaicin, norcapsaicin, dihydrocapsaicin, homocapsaicin, homodihydrocapsaicin) are well known and used as nutritional additive agents in the every day nutritional practice from the last 9,500 years; however, we had have a very little scientifically based knowledge on their chemistry, physiology and pharmacology in animal observations, and in humans up to the mid-twentieth century. Our knowledge about their chemistry, physiology, pharmacology entered to be scientifically based evidence from the year 1980, dominantly in animal observations. The human observations with capsaicin (capsaicinoids), in terms of good clinical practice, have been started only in the last 10-year period (from 1997) in randomized, prospective, multiclinical studies. The name of "capsaicin" used only in the physiological and pharmacological research both in animal experiments and in human observation. The "capsaicin" (as a "chemically" used natural compound) modifies the so-called capsaicin-sensitive afferent nerves depending on their applied doses. AIMS: The specific action of capsaicin (capsaicinoids) on sensory afferent nerves modifying gastrointestinal (GI) function (under very specific conditions) offers a possibility for the production of an orally applicable drug or for other drug combinations, which can be used in the human medical therapy. The production of new drug is based on the critical interdisciplinary review of the results obtained with capsaicinoids. MATERIALS AND METHODS: This paper gives an interdisciplinary and critical overview on the chemical, physiological, pharmacological and toxicological actions of the natural origin capsaicinoids (from the point of drug production) under conditions of acute, subacute and chronic administration in animal experiments and human observations, toxicology, pharmacokinetics). This interdisciplinary review covers the following main chapters: (1) physiological and pharmacological research tool by capsaicin in the animals and human beings, (2) capsaicin research in animals (including the acute, subacute toxicology and chronic toxicology metabolism, genotoxicology), (3) capsaicin observation with capsaicin in human beings. CONCLUSION: (1) The capsaicin used in the physiological and pharmacological observations (in animals and human beings) chemically represents different chemical compounds, which can be obtained from the plants (paprika, chilli, etc.), (2) capsaicinoids are able to modify the capsaicin-sensitive afferent nerves, which have principle roles in the defence of different organs (including the gastrointestinal tract [against the different chemicals, heat, strech, chemical millieu-induced damage], (3) the application of capsaicin (capsaicinoids) can be repeated for the beneficial effects on the gastrointestinal tract as those in animal experiments. After this interdisciplinary and critical review, this paper demonstrates the well-planned research pathways of the discoveries of capsaicinoids from plant chemistry, via physiology, pharmacology and toxicology in animal experiments and human observations.

Spatiotemporal variations of pharmacologically active compounds in surface waters of a summer holiday destination.

The release of pharmacologically active compounds (PhACs) into aquatic ecosystems poses an environmental risk resulting in a chronic exposure of non-target organisms. A great variety of PhACs, of generally low concentrations, and the complicated sample preparation, makes circumstantial the accurate detection and quantification. Additionally, there is little information published about the spatiotemporal variation of the PhAC load in a larger catchment area utilised for touristic purposes. In addition to the natural biotic and abiotic changes, the seasonal variation of tourism also has a dramatic impact on water quality and the natural ecosystem in larger catchment areas. Therefore, our aim was to develop a reliable solid-phase extraction (SPE)-supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) method for simultaneous multi-residue analysis of drugs to reveal the spatiotemporal changes in the PhAC contaminations in the waters of an important touristic region, the catchment area of the largest shallow lake in Central Europe, Lake Balaton (Hungary). The environmental application of the developed method revealed 69 out of the traced 134 chemical compounds, including 15 PhACs, which were detected from natural waters for the first time. Wastewater treatment plant (WWTP) loads have a major role in the PhAC contamination of the studied area; at the same time, the mass tourism-induced PhAC contamination was also detectable. Furthermore, the impact of tourism was indicated by elevated concentrations of recreational substances (e.g., caffeine and illicit drugs) in the touristic season affecting the water quality of this important summer holiday destination.

Antioxidant and antimicrobial properties of randomly methylated ß cyclodextrin - captured essential oils.

Free essential oils and their active components have a low physiochemical stability and low aqueous solubility which limit their applications as food preservatives and in packaging industry. The aim of this study was to characterize the physicochemical properties, antioxidant activities and antimicrobial activity of randomly methylated ß cyclodextrin (RAMEB) encapsulated thyme oil, lemon balm oil, lavender oil, peppermint oil and their active components that include thymol, citral, linalool, menthol and borneol. Inclusion complex formation of essential oils (EOs) and RAMEB were evaluated by several methods. Antioxidant capacities of RAMEB-EOs/components were reported to be more stable than free EOs/components (P < 0.05). Rapid SYBR green I/propidium iodide live/dead microbial cellular discrimination assay for Schizosaccharomyces pombe, Escherichia coli and Staphylococcus aureus showed similar results when compared with flow cytometry analysis (P < 0.01) suggesting that our novel microplate fluorescence method could be applied for the fast live/dead microbial discrimination in antimicrobial assays.

[Analysis of microalbuminuria with immunonephelometry and high performance liquid chromatography. Evaluation of new criteria]. / Immunnefelometria és nagy teljesítményu folyadékkromatográfia a microalbuminuria vizsgálatában. Ujonnan javasolt határértékek vizsgálata.

INTRODUCTION: Hypertension as well as type 2 diabetes mellitus is a major factor in population mortality. Both diseases damage the endothelium, the early sign of which is microalbuminuria, which can be screened by dipstick and can be diagnosed by using immuno-based and high performance liquid chromatography methods. Using high performance liquid chromatography, the non-immunoreactive albumin can be detected as well. AIMS: The authors aimed at the examination of albuminuria in the case of immunonephelometrically negative patients with high performance liquid chromatography, in diabetic and hypertensive and non-diabetic hypertensive populations. The authors also wanted to compare the present (albumin-creatinine ratio: male: > or =2.5 mg/mmol, female: > or =3.5 mg/mmol) and a new criteria of the Heart Outcomes Prevention Evaluation study (patients without diabetes: immunological method, > or =0.7 mg/mmol; high performance liquid chromatography, > or =3.1 mg/mmol; individuals with diabetes: immunological method, > or =1.4 mg/mmol; high performance liquid chromatography, > or =5.2 mg/mmol) of microalbuminuria. METHODS: Examination of fresh urines of 469 microalbuminuria negative patients by dipstick were performed by immunonephelometry. Patients, who were microalbuminuria negative by immunonephelometry as well, were further analyzed by high performance liquid chromatography using the Accumintrade mark Kit, based on size-exclusion chromatography. RESULTS: Three times higher albuminuria were found with high performance liquid chromatography than with immunonephelometry. The intraindividual coefficient of variation did not differ in the two methods (37 +/- 31% vs. 40 +/- 31%, p = 0.869; immunonephelometry vs. high performance liquid chromatography; mean +/- standard deviation). Using the present criteria for microalbuminuria, 43% of immunonephelometrically negative patients proved to be microalbuminuric by high performance liquid chromatography. Using the new criteria of the Heart Outcomes Prevention Evaluation study, the rate of microalbuminuria positivity among the immunonephelometrically negative patients decreased to 14.5% by high performance liquid chromatography and the decrease in the number of microalbuminuria positive cases by high performance liquid chromatography could be observed mainly in the diabetic and hypertensive group (49% vs. 7.5%), while slighter decrease could be observed in the non-diabetic hypertensive group (37% vs. 26.5%). Applying the traditional criteria, the strongest predictor was the male gender by the logistic regression analysis. In 28% of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established using high performance liquid chromatography. CONCLUSIONS: Almost in one-third of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established by high performance liquid chromatography for which diagnosis three constitutive urine examinations are still needed. New criteria determined by the Heart Outcomes Prevention Evaluation study can be used neither in case of diabetic and hypertensive patients, nor in the case of non-diabetic hypertensive patients. The gender as the most important predictor of microalbuminuria cannot be ignored.

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid.

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.

Oxidation of Hydroxy- and Dihydroxybenzoic Acids Under the Udenfriend's Conditions. An HPLC Study.

BACKGROUND: Non-enzymatic hydroxylation of aromatic compounds to the respective phenolic derivatives is a possible metabolic pathway of xenobiotics. The formed metabolites can undergo consecutive oxidative reactions with free radicals to form potential toxic molecules. OBJECTIVE: Development of HPLC methods to separate, identify and quantitate the main products formed from salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid under in vitro hydroxylation conditions (Udenfriend's system). METHOD: An RP-HPLC-UV-Vis method was developed to separate salicylic acid and isomeric dihydroxybenzoic acids formed in the Udenfriend's system. Confirmation of structures of the oxidized products of salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was performed by HPLC-ESI-MS/MS method. RESULTS: The HPLC-UV-Vis method was evaluated for a number of validation characteristics (selectivity, repeatability and intermediate precision, LOD, LOQ and calibration range). It was found that oxidation of salicylic acid resulted in the formation of 2,3- and 2,5-dihydroxybenzoic acids. Furthermore, the hydroxylated metabolites can be further metabolized under the Udenfriend's conditions. CONCLUSION: The results give evidence for possible involvement of the oxidized metabolites of salicylic acid in the development of biological action of salicylates at the site of inflammation, where high hydroxyl radical level can be detected.

Interactions of zearalenone and its reduced metabolites α-zearalenol and ß-zearalenol with serum albumins: species differences, binding sites, and thermodynamics.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species. ZEN mainly appears in cereals and related foodstuffs, causing reproductive disorders in animals, due to its xenoestrogenic effects. The main reduced metabolites of ZEN are α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL). Similarly to ZEN, ZELs can also activate estrogen receptors; moreover, α-ZEL is the most potent endocrine disruptor among these three compounds. Serum albumin is the most abundant plasma protein in the circulation; it affects the tissue distribution and elimination of several drugs and xenobiotics. Although ZEN binds to albumin with high affinity, albumin-binding of α-ZEL and ß-ZEL has not been investigated. In this study, the complex formation of ZEN, α-ZEL, and ß-ZEL with human (HSA), bovine (BSA), porcine (PSA), and rat serum albumins (RSA) was investigated by fluorescence spectroscopy, affinity chromatography, thermodynamic studies, and molecular modeling. Our main observations are as follows: (1) ZEN binds with higher affinity to albumins than α-ZEL and ß-ZEL. (2) The low binding affinity of ß-ZEL toward albumin may result from its different binding position or binding site. (3) The binding constants of the mycotoxin-albumin complexes significantly vary with the species. (4) From the thermodynamic point of view, the formation of ZEN-HSA and ZEN-RSA complexes are similar, while the formation of ZEN-BSA and ZEN-PSA complexes are markedly different. These results suggest that the toxicological relevance of ZEN-albumin and ZEL-albumin interactions may also be species-dependent.

Pharmacokinetic interaction of diosmetin and silibinin with other drugs: Inhibition of CYP2C9-mediated biotransformation and displacement from serum albumin.

Diosmin and silibinin (SIL) are polyphenolic compounds which are the active components of several drugs and dietary supplements. After the oral administration of diosmin (flavonoid glycoside), only its aglycone diosmetin (DIO) reaches the systemic circulation. Both DIO and SIL form complexes with serum albumin and are able to inhibit several cytochrome P450 enzymes. Therefore, it is reasonable to hypothesize that these polyphenols may displace some drugs from serum albumin and inhibit their biotransformation, potentially leading to the disruption of drug therapy. In this study, the inhibitory action of DIO and SIL on CYP2C9-catalyzed metabolism of diclofenac to 4'-hydroxydiclofenac was examined, using warfarin as a positive control. Furthermore, interaction of DIO and SIL with human and bovine serum albumins as well as the displacement of warfarin from albumin by DIO and SIL were tested, employing steady-state fluorescence spectroscopy, fluorescence anisotropy, ultrafiltration, and molecular modeling. It is demonstrated that DIO and SIL are potent inhibitors of CYP2C9 enzyme and are able to displace the Site I ligand warfarin from human serum albumin. Because DIO and SIL may interfere with the pharmacokinetics of several drugs through both ways, we need to consider the potentially hazardous consequences of the consumption of diosmin or SIL together with other drugs.