Development and validation of a simultaneous quantitative analytical method for two Alternaria toxins and their metabolites in plasma and urine using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Much more attention has been paid to the contamination of Alternaria toxins because of food contamination and the threat to human health. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the prototypical alternariol, alternariol monomethylether, and the metabolites 4-oxhydryl alternariol, and alternariol monomethylether 3-sulfate ammonium salt of Alternaria toxins. The positive samples were used as matrix samples to optimize the different experimental conditions. 0.01% formic acid solution and acetonitrile were used as the mobile phase, and analytes were scanned in negative electron spray ionization under multiple reaction monitoring, and quantitative determination by isotope internal standard method. Application of this method to samples of human plasma and urine showed the detection of the above analytes. The results showed that the recoveries were from 80.40% to 116.4%, intra-day accuracy was between 0.6% and 8.0%, and inter-day accuracy was between 1.1% and 12.1%. The limit of detection of the four analytes ranged from 0.02 to 0.6 µg/L in urine, and 0.02 to 0.5 µg/L in plasma, respectively. Thus, the developed method was rapid and accurate for the simultaneous detection of analytes and provided a theoretical basis for the risk assessment of Alternaria toxins for human exposure.

Early pregnancy metabolites predict gestational diabetes mellitus: implications for fetal programming.

BACKGROUND: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders. OBJECTIVE: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy. STUDY DESIGN: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus. RESULTS: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection. CONCLUSION: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity.

Investigation on the urinary excretion kinetics of three atractylenolides from crude and processed Atractylodis rhizoma extracts in rats by UPLC-MS/MS.

Atractylodis rhizoma is a frequently-used traditional Chinese medicine in clinical practice, which have the effect of eliminating dampness and tonifying spleen. And after being processed with wheat bran, the dryness of A. rhizoma is reduced, and the function of tonifying spleen is enhanced. Atractylenolides are the major bioactive components of A. rhizoma, including atractylenolide I (AI), atractylenolide Ⅱ (AⅡ) and atractylenolide Ⅲ (AⅢ). The present study aimed to develope a new UPLC-MS/MS method for simultaneous quantification of three atractylenolides in rat urine, and applied to the excretory kinetics in Sprague-Dawley rats after oral administration of crude and processed A. rhizoma extracts. Analytes and internal standard were detected without interference in the multiple reaction monitoring (MRM) mode with positive electrospray ionization. The excretory kinetics parameters were calculated by a urine drug analysis model of drug and statistics (DAS) 3.2.8 software. The t1/2 and Ke of three atractylenolides had no significant difference between crude and processed A. rhizoma, but the recovery accumulative excretion of them in processed A. rhizoma were apparently higher than the crude ones (p<0.05, p<0.01). The results showed that only a small amount of atractylenolides excreted in urine and processing A. rhizoma with wheat bran by stir frying could promote the urinary excretion of them.

Human Gut Microbiota Metabolism of Dietary Sesquiterpene Lactones: Untargeted Metabolomics Study of Lactucopicrin and Lactucin Conversion In Vitro and In Vivo.

SCOPE: Gut microbiota converts dietary phytochemicals into metabolites and modulates their health effects. The microbial metabolism of dietary terpenoids, as the sesquiterpene lactones of leafy vegetables, is unknown. METHODS AND RESULTS: In vitro fermentation of lactucopicrin, lactucin, and romaine lettuce with gut microbiota from independent donors, show their extensive metabolism through untargeted metabolomics of the fecal incubations. Dehydroxylations and double bond hydrogenations are the main catabolic reactions. Isomers of dihydrolactucopicrin, tetrahydrolactucopicrin, and deoxylactucin, are observed after lactucopicrin metabolism. Tetrahydrolactucin and hexahydrolactucin are also found after lactucin metabolism. Lettuce fermentation shows similar metabolic conversions. Phase II conjugates of most of these metabolites are detected in the urine of healthy volunteers after escarole salad intake. Glucuronides, and sulfates, of dihydrolactucopicrin, tetrahydrolactucopicrin, dihydrolactucin, and deoxylactucin, are detected in the urine although with large inter-subject variability. CONCLUSION: This is the first report on the gut microbiota metabolism of sesquiterpene lactones in humans, and one of the first reports to describe that dietary terpenoids of widely consumed leafy vegetables are extensively catabolized by human gut microbiota. A large inter-subject variation in the metabolism of sesquiterpene lactones also reflects differences in gut microbiota composition. It suggests that inter-individual differences in their health effects should be expected.

Kinetic profile and urinary excretion of phenyl-γ-valerolactones upon consumption of cranberry: a dose-response relationship.

Cranberries are a rich source of poly(phenols), mainly monomeric and oligomeric flavan-3-ols. However, information on the appearance of their main circulating microbial metabolites, namely phenyl-γ-valerolactones and phenylvaleric acid, is lacking despite its relevance to understanding the health effects attributed to cranberries. The aim of this study was to evaluate the absorption, metabolism and urinary excretion of cranberry flavan-3-ols through the targeted analysis of phenyl-γ-valerolactones and their related phenylvaleric acids, considering also their potential as biomarkers of flavan-3-ol intake and inter-individual variability in their appearance in plasma and urine. A six-arm acute crossover, randomized, double-blinded, controlled intervention trial was performed in ten healthy males who consumed a cranberry juice drink (375, 716, 1131, 1396, 1741 mg of total flavan-3-ols) or an isocaloric control drink with one-week washout. Plasma and urine were analyzed by UHPLC-ESI-QqQ-MS/MS and 22 compounds were identified. Glucuronide and sulfate conjugates of 5-(3',4'-dihydroxyphenyl)-γ-valerolactone were the main circulating and excreted metabolites after cranberry juice intake, with glucuronidation appearing to be the most favorable conjugation route. These compounds reached maximum plasma concentration at about 4-6 h. Plasma and urinary concentrations of the sum of the metabolites increased in relation to the amounts of cranberry flavan-3-ols provided by the drink, showing a clear and linear dose-dependent relationship and underscoring their potential as biomarkers of flavan-3-ol intake. A high inter-individual variability in circulating and urinary metabolite levels was observed and, interestingly, some subjects seemed to display a greater efficiency in metabolizing flavan-3-ols and producing phenyl-γ-valerolactones.

Development of a simple and rapid LC-MS/MS method for the simultaneous quantification of five Alternaria mycotoxins in human urine.

Alternaria mycotoxins, such as tenuazonic acid (TeA), altenuene (ALT), alternariol (AOH), tentoxin (TEN) and alternariol monomethyl ether (AME) are frequently found in foods and may pose a potential risk to human health. Human biomonitoring can help measure our exposure to these mycotoxins, and help us determine if the exposure is changing over time. In this study, a simple liquid-liquid extraction sample preparation procedure followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of five Alternaria mycotoxins in human urine. High recoveries (92.7-103.2%) were obtained for all the tested mycotoxins with relative standard deviations (RSDs, %) of less than 6.4%. The limits of quantification (LOQs) for the analytes in urine ranged from 0.001 to 0.05 ng/mL. The method was successfully applied to investigate the levels of five Alternaria mycotoxins from 135 volunteers. In all of the samples, at least one Alternaria mycotoxin was detected. TeA, AME and AOH were the predominant Alternaria mycotoxins, and the detection rates were 85.9%, 96.3% and 51.9%, respectively.

Monomeric Flavanols Are More Efficient Substrates for Gut Microbiota Conversion to Hydroxyphenyl-γ-Valerolactone Metabolites Than Oligomeric Procyanidins: A Randomized, Placebo-Controlled Human Intervention Trial.

SCOPE: The majority of ingested flavanols reach the colon where they are catabolized by the microbiota to form hydroxyphenyl-γ-valerolactones (HGVLs). It is not known if the HGVLs are catabolic products of monomeric (epi)catechins (EPC), oligomeric procyanidins (OPCs), or both. Using data from a randomized, double-blind, placebo-controlled crossover trial the relative contributions of catechins and OPC to the bioavailable pool of HGVLs are estimated. METHODS AND RESULTS: Participants ingested an apple extract once daily for 28 days that delivered the following: i) 70 mg EPC and 65 mg OPC (low dose EPC), ii) 140 mg EPC and 130 mg OPC (high dose EPC), iii) 6 mg EPC and 130 mg OPC (OPC), and iv) a placebo control. Urine is collected over a 24-h period before and after treatments. The median urinary excretion of HGVLs after ingestion of the high dose EPC is tenfold higher than that excreted after ingestion of the OPC that provided an equivalent dose of PC. Approximately 22% of catechins are converted to HGVLs in contrast to PC, for which there is limited conversion. CONCLUSION: Monomeric catechins are efficiently converted to derived HGVLs that are absorbed and excreted in human urine, whereas oligomeric PCs are much less efficiently converted.

A sensitive LC-MS/MS method to determine ginkgolide B in human plasma and urine: application in a pharmacokinetics and excretion study of healthy Chinese subjects.

1. Ginkgolide B (GB), the most active of the ginkgolides, has been developed as a new drug for the treatment of vascular insufficiency; however, the pharmacokinetics of GB remain unclear. Here, we investigated the pharmacokinetics and urine excretion properties of GB in healthy Chinese subjects administered single- and multiple-dose injectable GB based on a new LC-MS/MS method.2. GB pharmacokinetics were found to be dose-dependent from 20 to 60 mg. GB reached a steady state by day 6 with once-daily dosing at 40 mg. Systemic exposure to GB, as characterised by AUC0-∞, indicated accumulation following repeated once-daily dosing for seven consecutive days. The mean urinary cumulative excretion rate of GB in response to 20, 40, and 60 mg GB was 41.9 ± 18.5%, 32.9 ± 12.2%, and 43.9 ± 8.5%, respectively.3. Dose-proportional pharmacokinetics of GB were observed after intravenous administration in healthy subjects. A gradual reduction in the volume of distribution and slight change in mean resistance time led us to conjecture the limited accumulation of GB based on distribution equilibrium in vivo.4. This comprehensive study of the clinical pharmacokinetics of GB will provide useful information for its application and further development.

Profiling Vaccinium macrocarpon components and metabolites in human urine and the urine ex-vivo effect on Candida albicans adhesion and biofilm-formation.

The aim of this work was to profile, by using an HPLC-MS/MS method, cranberry compounds and metabolites found in human urine after ingestion of a highly standardized cranberry extract (Anthocran®). Two different strategies were adopted for the data analysis: a targeted and an untargeted approach. These strategies allowed the identification of 42 analytes including cranberry components, known metabolites and metabolites hitherto unreported in the literature, including six valerolactones/valeric acid derivatives whose presence in urine after cranberry consumption has never been described before. Absolute concentrations of 26 over 42 metabolites were obtained by using pure available standards. Urine collected at different time points after the last dosage of Anthocran® were tested on the reference strain C. albicans SC5314, a biofilm-forming strain. Fractions collected after 12 h were found to significantly reduce the adhesion and biofilm formation compared to the control (p < 0.05). A similar effect was then obtained by using Anthocran™ Phytosome™, the lecithin formulation containing 1/3 of standardized cranberry extract and formulated to enhance the absorption of the cranberry components. The urinary profile of cranberry components and metabolites in the urine fractions collected at 1 h, 6 h and 12 h after the last capsule intake were then reproduced by using the pure standards at the concentration ranges found in the urine fraction, and tested on C. albicans. Only the mixture mimicking the urinary fraction collected at 12 h and containing as main components, quercetin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone was found effective thus confirming the ex-vivo results.

Simultaneous determination of seven effective components of Tripterygium glycosides in human biological matrices by ultra performance liquid chromatography-triple quadrupole mass spectrometry.

This paper developed a novel, sensitive, and selective ultra-performance liquid chromatography-triple quad mass spectrometry method to simultaneously determine seven effective constituents (triptolide, triptophenolide, celastrol, wilforgine, wilforine, wilfordine and wilfortrine) of Tripterygium glycosides (GTW) in human serum and urine. The chromatographic separation was performed on the C18 column using an ammonium acetate buffer solution-acetonitrile (both containing 0.1% formic acid) in a gradient program with a flow rate of 0.3 mL/min. Monitoring reaction mode was applied to target compounds quantitative analysis in the positive electrospray ionization (ESI) mode. The analysis process took 11 min in total. This method was fully validated with a linear range of 1-200 ng/mL for triptolide, 0.4-80 ng/mL for celastrol, 0.1-20 ng/mL for triptophenolide, wilforgine, wilforine, wilfordine, and wilfortrine. The intra-day and inter-day accuracy and precision of the target compounds all met the 15% criterion in both serum and urine. Extraction recovery, matrix effect, and dilution integrity were also validated. The short-term and long-term stability results indicated that all the constituents were stable in human serum and urine under the investigated storage conditions. 10 patients' specimens were collected and analyzed. Most of the compounds exhibited the tendency of higher concentration in urine than that in serum. The concentration that was detected in the serum and in the urine of alkaloids showed a positive-correlation property. This is the first time that triptophenolide was quantified in human bio-matrices. The method is feasible for multi-components therapeutic monitoring or pharmacokinetics study in clinical pharmaceutical research of Tripterygium glycosides.

Resorcylic acid lactones in urine samples of slaughtered animals resulting from potential feed contamination with zearalenone.

Resorcylic Acid Lactones, including zeranol, anabolics listed in the group A4 of Directive 96/23/EC, are banned in Europe for use in animals since 1985. Zeranol, after administration to animals, is metabolized to taleranol and zearalanone. It can also naturally occur in the urine due to conversion of zearalenone that may be present in animal feed. In 2010-2017, in Poland, 7746 animal samples were tested for zeranol residues within the official monitoring program. In 13, zeranol was detected after screening. Re-examinations confirmed resorcylic acid lactones in six samples. The recommendations state that only the presence of zeranol and/or taleranol gives the basis for non-compliance. Confirmation should cover the entire profile of six resorcylic acid lactones. In case of detection, the relationship ratio should be verified. Following the proposed criteria, it could be concluded that zeranol detected in urine samples in Poland originated from contamination of feed with mycotoxin, not from illegal use.

[Determination of coriatin and corianin in plasma and urine using ultra-performance liquid chromatography-triple quadrupole mass spectrometry].

An ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) method has been developed for the determination of coriatin and corianin in plasma and urine, which are the biomarkers of poisoning caused by Coriaria sinica Maxim. Plasma and urine samples were extracted and purified using a solid supported liquid/liquid extraction method. Chromatographic separation was performed on a Cortecs C18 column (100 mm×2.1 mm, 1.6 µm) using a gradient elution of methanol and water. Coriatin and corianin were detected using negative electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode and quantified via a matrix working standard curve internal standard method; florfenicol was used as the internal standard. The assay was linear in the calibration range of 0.03-5.0 µg/L for coriatin and 0.3-50 µg/L for corianin in plasma, and 0.1-10 µg/L and 1-100 µg/L for coriatin and corianin in urine, respectively. The average recoveries were 86.2%-110% for coriatin and corianin in plasma and urine with relative standard deviations of 5.1%-14.6% (n=6). The limits of detection (S/N=3) for coriatin and corianin were 0.01 µg/L and 0.1 µg/L in plasma, and 0.03 µg/L and 0.3 µg/L in urine, respectively. The method is simple, sensitive and accurate for the determination of coriatin and corianin in plasma and urine for toxicological purposes.

Full 1 H and 13 C NMR spectral assignment of conjugated valerolactone metabolites isolated from urine of black tea consumers by means of SPE-prepLC-MS-LC-MS-NMR.

The health benefits of black tea have been linked to polyphenol metabolites that target specific modes of action in the human body. A major bottleneck in unravelling the underlying mechanisms is the preparative isolation of these metabolites, which hampers their structural elucidation and assessment of in vitro bioactivity. A solid phase extraction (SPE)-preparative liquid chromatography (prepLC)-MS-LC-MS-NMR workflow was implemented for preparative isolation of conjugated valerolactone metabolites of catechin-based polyphenols from urine of black tea consumers. First, the urine was cleaned and preconcentrated using an SPE method. Subsequently, the clean urine concentrate was injected on a preparative LC column, and conjugated valerolactones were obtained by MS-guided collection. Reconstituted fractions were further separated on an analytical LC column, and valerolactone fractions were collected in an MS-guided manner. These were reconstituted in methanol-d4 and identified and quantified using 1D and 2D homo- and hetereonuclear NMR experiments (at a field strength of 14.1 T), in combination with mass spectrometry. This resulted in the full spectral 1 H and 13 C NMR assignments of five conjugated valerolactones. These metabolites were collected in quantities of 8-160 µg and purities of 70-91%. The SPE-prepLC-MS-LC-MS-NMR workflow is suitable for isolating metabolites that occur at sub-µM concentrations in a complex biofluid such as urine. The workflow also provides an alternative for cumbersome and expensive de novo synthesis of tea metabolites for testing in bioactivity assays or for use as authentic analytical standards for quantification by mass spectrometry.

Resorcylic Acid Lactones in Urine Samples of Croatian Farm Animals.

Metabolic transformation of zearalenone (ZEA), a mycotoxin which can contaminate both food and feed, results in the formation of five metabolites, one of them being zeranol (α-ZAL), which can be abused in farm animals as a growth promoter. To the best of our knowledge, there is no analytical method that can distinguish whether α-ZAL is present in an animal urine sample as a result of ZEA biotransformation or as a result of anabolic abuse. This study aimed at monitoring resorcylic acid lactones (RALs) concentration in urine of farm animals over several years. Six hundred and three cattle and pig urine samples were collected on farms in different Croatian regions and analyzed for RAL presence. Based on the testing results, all RAL-positive samples were considered to be consequential to feed contamination. The difference in primary ZEA metabolites' ratio (α-zearalenol/ß-zearalenol) was observed between cattle (0.03-0.41) and pig (2.05-17.39) urine samples. If the animals are treated with α-ZAL and fed on ZEA-contaminated feed, α-ZAL and taleranol found in their organisms could come from two sources, so that the reliability of the statistical model might be questionable. Based on these findings, there exists the need for improving the approach to the distinction between α-ZAL abuse and ZEA feed contamination.

First insights into Alternaria multi-toxin in vivo metabolism.

Alternaria mycotoxins frequently contaminate agricultural crops and may impact animal and human health. However, data on mammalian metabolism and potential biomarkers of exposure for human biomonitoring (HBM) are scarce. Here, we report the preliminary investigation with respect to metabolism and excretion of Alternaria toxins in Sprague Dawley rats. Four animals were housed in metabolic cages for 24 h after gavage administration of an Alternaria alternata culture extract containing ten known toxins. LC-MS/MS analysis of 17 Alternaria toxins in urine and fecal samples allowed to gain first insights regarding xenobiotic metabolism and excretion rates. Alternariol (6-10%), alternariol monomethyl ether (AME, 6-7%) and tenuazonic acid (up to 55%) were recovered in urine and fecal samples (9%, 87%, 0.3%, respectively), while perylene quinones administered at comparatively high levels, were either determined at very low levels (up to 0.5% altertoxin I in urine and 15% in feces; 0.2% alterperylenol in urine and 3% in feces) or not at all (altertoxin II, stemphyltoxin III). AME-3-sulfate, which was not present in the administered extract, was determined in urine, representing up to 23% of the AME intake. Critical evaluation of the applied sample preparation protocol and LC-MS/MS analysis revealed interesting preliminary results and information crucial for improving follow-up experiments.

Multiresidue Determination of the Anabolic-Agent Residues Steroids, Stilbenes, and Resorcylic Acid Lactones in Bovine Urine by GC-MS/MS with Microwave-Assisted Derivatization.

In this work, a GC-MS/MS method was developed for the determination of anabolic-agent residues in bovine urine. The optimized sample preparation was as follows: enzymatic hydrolysis by ß-glucuronidase-sulfatase enzyme from Helix pomatia for 16 h at 37.5 °C, liquid-liquid extraction with diethyl ether, solid-phase extraction with HLB and aminopropylsilane cartridges, and microwave-assisted derivatization using 25 µL of MSTFA/NH4I/ethanethiol and full microwave power for 2 min. The method was validated according to Decision 657/2002/EC, Codex Alimentarius, and Manual da Garantia da Qualidade Analítica guidelines. The acceptability criteria for quantitative analysis were met for α-ethinylestradiol, α-nandrolone, ß-estradiol, ß-zearalanol, ß-zearalenol, drostanolone, ethisterone, dienestrol, diethylstilbestrol, hexestrol, megestrol, methyltestosterone, and zearalenone. The analytes α-zearalenol, α-zearalanol, and norethandrolone were validated for qualitative analysis.

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

Human pharmacokinetics of ginkgo terpene lactones and impact of carboxylation in blood on their platelet-activating factor antagonistic activity.

Terpene lactones are a class of bioactive constituents of standardized preparations of Ginkgo biloba leaf extract, extensively used as add-on therapies in patients with ischemic cardiovascular and cerebrovascular diseases. This investigation evaluated human pharmacokinetics of ginkgo terpene lactones and impact of their carboxylation in blood. Human subjects received oral YinXing-TongZhi tablet or intravenous ShuXueNing, two standardized ginkgo preparations. Their plasma protein-binding and platelet-activating factor antagonistic activity were assessed in vitro. Their carboxylation was assessed in phosphate-buffered saline (pH 7.4) and in human plasma. After dosing YinXing-TongZhi tablet, ginkgolides A and B and bilobalide exhibited significantly higher systemic exposure levels than ginkgolides C and J; after dosing ShuXueNing, ginkgolides A, B, C, and J exhibited high exposure levels. The compounds' unbound fractions in plasma were 45-92%. Apparent oral bioavailability of ginkgolides A and B was mostly >100%, while that of ginkgolides C and J was 6-15%. Bilobalide's bioavailability was probably high but lower than that of ginkgolides A/B. Terminal half-lives of ginkgolides A, B, and C (4-7 h) after dosing ShuXueNing were shorter than their respective values (6-13 h) after dosing YinXing-TongZhi tablet. Half-life of bilobalide after dosing the tablet was around 5 h. Terpene lactones were roughly evenly distributed in various body fluids and tissues; glomerular-filtration-based renal excretion was the predominant elimination route for the ginkgolides and a major route for bilobalide. Terpene lactones circulated as trilactones and monocarboxylates. Carboxylation reduced platelet-activating factor antagonistic activity of ginkgolides A, B, and C. Ginkgolide J, bilobalide, and ginkgo flavonoids exhibited no such bioactivity. Collectively, differences in terpene lactones' exposure between the two preparations and influence of their carboxylation in blood should be considered in investigating the relative contributions of terpene lactones to ginkgo preparations' therapeutic effects. The results here will inform rational clinical use of ginkgo preparations.

[Clinical and biochemical aspects of pathogenesis of urolithiasis].

AIM: To investigate the role of infection in the pathogenesis of urolithiasis using chromatography mass spectrometry analysis. MATERIALS AND METHODS: The study analyzed clinical and laboratory data of 316 urolithiasis patients hospitalized between February 2005 and January 2015. All patients underwent a comprehensive clinical examination, including laboratory tests (hematological and biochemical blood tests, clinical and bacteriological tests of urine) and chromatography mass spectrometry analysis urine and blood. The laboratory testing was carried out both during the patients hospital stay and outpatient follow-up. RESULTS: We analyzed the biological material for the presence of characteristic ions. Urine samples of 316 urolithiasis patients were found to contain activators of "cooperative sensitivity." Moreover, there was a significant increase in the concentration of signaling compounds of the "cooperative sensitivity" of microorganisms in patients with complicated urolithiasis in comparison with the control indices (lactones-0.006 plus/minus 0.0004 mmol/L, normal values less than 0.002, quinolones 0.004 plus/minus 0.0003 mmol/l, normal values - less than 0.002 and furan esters - 0.005 plus/minus 0.0004, normal values less than 0.002). Threshold values of the activators of "cooperative sensitivity" demonstrated the readiness of the microbial community to initiate an inflammatory process. The presence of activators such as lactones, quinolones and furan esters in the samples of urolithiasis patients predisposes to the activation of pathogenic genes in a large group of microorganisms, including gram positive and gram negative species. DISCUSSION: In our opinion, to improve the quality of diagnostic, treatment and preventive measures in patients with different types of stone formation, it is advisable to use chromatography mass spectrometry analysis, which allows determination of priority clinical and laboratory indicators. CONCLUSION: The data on the role of infection in the pathogenesis of urolithiasis obtained by chromatographic methods suggest the possibility of using the indicators of the activators of the "cooperative sensitivity" of microbes in patients with various forms of urolithiasis to assess the disease severity.

No Association between Mycotoxin Exposure and Autism: A Pilot Case-Control Study in School-Aged Children.

Evaluation of environmental risk factors in the development of autism spectrum disorder (ASD) is needed for a more complete understanding of disease etiology and best approaches for prevention, diagnosis, and treatment. A pilot experiment in 54 children (n = 25 ASD, n = 29 controls; aged 12.4 ± 3.9 years) screened for 87 urinary mycotoxins via liquid chromatography-tandem mass spectrometry to assess current exposure. Zearalenone, zearalenone-4-glucoside, 3-acetyldeoxynivalenol, and altenuene were detected in 9/54 (20%) samples, most near the limit of detection. No mycotoxin/group of mycotoxins was associated with ASD-diagnosed children. To identify potential correlates of mycotoxin presence in urine, we further compared the nine subjects where a urinary mycotoxin was confirmed to the remaining 45 participants and found no difference based on the presence or absence of mycotoxin for age (t-test; p = 0.322), gender (Fisher's exact test; p = 0.456), exposure or not to selective serotonin reuptake inhibitors (Fisher's exact test; p = 0.367), or to other medications (Fisher's exact test; p = 1.00). While no positive association was found, more sophisticated sample preparation techniques and instrumentation, coupled with selectivity for a smaller group of mycotoxins, could improve sensitivity and detection. Further, broadening sampling to in utero (mothers) and newborn-toddler years would cover additional exposure windows.