Biotransformation Reactions Research Articles

Biotransformation of the Novel Myeloperoxidase Inhibitor AZD4831 in Preclinical Species and Humans.

We report herein an in-depth analysis of the metabolism of the novel myeloperoxidase inhibitor AZD4831 ((R)-1-(2-(1-aminoethyl)-4-chlorobenzyl)-2-thioxo-2,3-dihydro-1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one) in animals and human. Quantitative and qualitative metabolite profiling were performed on samples collected from mass balance studies in rats and humans. Exposure of circulating human metabolites with comparable levels in animal species used in safety assessment were also included. Structural characterization of 20 metabolites was performed by liquid chromatography high-resolution mass spectrometry, and quantification was performed by either 14C analysis using solid phase scintillation counting or accelerator mass spectrometry and, where available, authentication with synthesized metabolite standards. A complete mass balance study in rats is presented, while data from dogs and human are limited to metabolite profiling and characterization. The metabolism of AZD4831 is mainly comprised of reactions at the primary amine nitrogen and the thiourea sulfur, resulting in several conjugated metabolites with or without desulfurization. A carbamoyl glucuronide metabolite of AZD4831 (M7) was the most abundant plasma metabolite in both human healthy volunteers and heart failure patients after single and repeated dose administration of AZD4831, accounting for 75%-80% of the total drug-related exposure. Exposures to M7 and other human circulating metabolites were covered in rats and/or dogs, the two models most frequently used in the toxicology studies, and were also highly abundant in the mouse, the second model other than rat used in carcinogenicity studies. The carbamoyl glucuronide M7 was the main metabolite in rat bile, while a desulfurized and cyclized metabolite (M5) was abundant in rat plasma and excreta. SIGNIFICANCE STATEMENT: The biotransformation of AZD4831, a novel myeloperoxidase inhibitor inhibiting xanthine derivative bearing thiourea and primary aliphatic amine functions, is described. Twenty characterized metabolites demonstrate the involvement of carbamoylation with glucuronidation, desulfurization, and cyclization as main biotransformation reactions. The carbamoyl glucuronide was the main metabolite in human plasma, likely governed by a significant species difference in plasma protein binding for this metabolite, but this and other human plasma metabolites were covered in animals used in the toxicity studies.

Identification and characterization of pathogenicity-related genes of Rhizoctonia solani AG3 during tobacco infection.

Tobacco target spot disease is caused by a ubiquitous soil-borne phytopathogen Rhizoctonia solani; the pathogenic mechanisms underlying the effects of R. solani remain unclear. Deeper understanding of the functional responses to R. solani during host plant infection would help identify the molecular mechanisms essential for successful host invasion. In this study, we performed global transcriptional analysis of R. solani during various stages (12, 24, 48, 72, 96, and 120h) of tobacco infection via an RNA sequencing method, while utilizing the pathosystem model R. solani AG3-tobacco (Nicotiana tabacum L.). After R. solani inoculation, the number of differentially expressed genes of R. solani differed at the various time points. Moreover, several gene ontology and Kyoto encyclopedia of genes and genomes pathways were unique in different infection stages, especially with respect to the genes involved in plant cell wall degradation and catalysis of biotransformation reactions, such as the pectin metabolic process and pectin catabolic process. The overexpressing-PD8 N. benthamiana plants enhanced the susceptibility to R. solani. In addition, we found that large amounts of reactive oxygen species (ROS) were generated in tobacco after infected by R. solani. R. solani encoding FAD/NAD binding oxidoreductase and peroxidase gene family to eliminating ROS and counteract oxidative stress. Moreover, Perox3 was validated that can enhance the ability of scavenging ROS by co-injecting. Overall, our findings show that pectin-degrading enzymes and cytochrome P450 genes are critical for plant infection. These results provide comprehensive insights into R. solani AG3 transcriptome responses during tobacco invasion.

Efficient Synthesis and In Vitro Hypoglycemic Activity of Rare Apigenin Glycosylation Derivatives.

Apigenin is a natural flavonoid with significant biological activity, but poor solubility in water and low bioavailability limits its use in the food and pharmaceutical industries. In this paper, apigenin-7-O-β-(6″-O)-d-glucoside (AG) and apigenin-7-O-β-(6″-O-succinyl)-d-glucoside (SAG), rare apigenin glycosyl and succinyl derivatives formed by the organic solvent-tolerant bacteria Bacillus licheniformis WNJ02 were used in a 10.0% DMSO (v/v) system. The water solubility of SAG was 174 times that of apigenin, which solved the application problem. In the biotransformation reaction, the conversion rate of apigenin (1.0 g/L) was 100% at 24 h, and the yield of SAG was 94.2%. Molecular docking showed that the hypoglycemic activity of apigenin, apigenin-7-glucosides (AG), and SAG was mediated by binding with amino acids of α-glucosidase. The molecular docking results were verified by an in vitro anti-α-glucosidase assay and glucose consumption assay of active compounds. SAG had significant anti-α-glucosidase activity, with an IC50 of 0.485 mM and enhanced glucose consumption in HepG2 cells, which make it an excellent α-glucosidase inhibitor.

Immobilization of cytochrome P450 enzymes onto magnetic beads: An approach to drug metabolism and biocatalysis

Cytochrome P450 (CYP, P450) presents a wide range of applicability in drug metabolism studies and in biocatalysis as a great alternative to synthesizing compounds. Nonetheless, their lack of stability is one of their major drawbacks. Aiming the possibility of enhancing the catalytic activity and promoting higher stability, liver microsomal fractions have been immobilized on magnetic beads (Mb). The immobilized procedure was modulated by using rat liver microsomal fractions (RLM-Mb). The optimal condition achieved was further employed for the immobilization of the human microsomal fractions on magnetic beads (HLM-Mb). In vitro metabolism assays were conducted using albendazole (ABZ) as a model drug, and the formation of albendazole sulfoxide (ABZSO) was monitored. Biotransformation reactions applying the produced HLM-Mb were examined for the best temperature to increase the production of metabolites and their reuse cycles. A kinetic study was carried out for HLM-Mbs monitoring the production of ABZO by the oxidation reaction of ABZ by CYP3A4. The Km value was 25.6 µmol L−1 and Vmax was 121.0 µmol L−1. Inhibition assays were conducted in the presence of ketoconazole and the production of ABZSO decreased by 46.8 ± 2.5%. Enzymatic activities for CYP2C9 and CYP2D6 on HLM-Mbs were evaluated by monitoring the hydroxylation reactions of diclofenac and bufuralol as substrates. The immobilization of CYP P450 on magnetic beads increased not only the production of ABZSO metabolites but also the stability of CYP. The use of HLM-Mbs jointly with immobilized glucose-6-phosphate dehydrogenase (G6PDH-Mbs) as a unique dual generator system to produce NADPH has established the one-pot conditions for biocatalysis in a greener approach with reuse of the biocatalyst (G6PDH-HLM-Mbs). To this end, the herein reported HLM-Mbs and G6PDH-HLM-Mbs are excellent analytical tools to be explored either in biocatalysis reactions or in in vitro metabolism studies.

Biotransformation of dihydrocapsaicin by human intestinal fungi and the inhibitory effects of metabolites against LSD1

Dihydrocapsaicin is the main bioactive component in Capsicum plants, which is widely used in China and India as a food drug and additive. In this study, the biotransformation of dihydrocapsaicin was performed using four cultivated human intestinal fungal strains in vitro. Eight metabolites, including seven previously undescribed metabolites (1 and 3−8) and one known analog (2), were obtained. Numerous spectroscopic data, such as NMR and HRESIMS, were collected to determine their structures. Based on the structures of the dihydrocapsaicin metabolites, the main biotransformation reactions were revealed to be hydroxylation, alcohol oxidation, and lactylation. In particular, the lactylation of hydroxyl groups is mainly mediated by Rhizopus oryzae R2701. In addition, metabolite 1 showed significant inhibitory effect on lysine-specific demethylase 1 (LSD1) (IC50 1.99 μM). Therefore, the biotransformation of dihydrocapsaicin by intestinal fungi afforded various derivatives, which were important resources for developing LSD1 inhibitors and potential application in cancer treatment.

Evaluation of Antioxidant Activity and Biotransformation of Opuntia Ficus Fruit: The Effect of In Vitro and Ex Vivo Gut Microbiota Metabolism.

Opuntia ficus-indica biological effects are attributed to several bioactive metabolites. However, these actions could be altered in vivo by biotransformation reactions mainly via gut microbiota. This study assessed gut microbiota effect on the biotransformation of O. ficus-indica metabolites both in vitro and ex vivo. Two-time aliquots (0.5 and 24 h) from the in vitro assay were harvested post incubation of O. ficus-indica methanol extract with microbial consortium, while untreated and treated samples with fecal bacterial culture from the ex vivo assay were prepared. Metabolites were analyzed using UHPLC-QTOF-MS, with flavonoid glycosides completely hydrolyzed in vitro at 24 h being converted to two major metabolites, 3-(4-hydroxyphenyl)propanoic acid and phloroglucinol, concurrent with an increase in the gallic acid level. In case of the ex vivo assay, detected flavonoid glycosides in untreated sample were completely absent from treated counterpart with few flavonoid aglycones and 3-(4-hydroxyphenyl)propanoic acid in parallel to an increase in piscidic acid. In both assays, fatty and organic acids were completely hydrolyzed being used as energy units for bacterial growth. Chemometric tools were employed revealing malic and (iso)citric acids as the main discriminating metabolites in vitro showing an increased abundance at 0.5 h, whereas in ex vivo assay, (iso)citric, aconitic and mesaconic acids showed an increase at untreated sample. Piscidic acid was a significant marker for the ex vivo treated sample. DPPH, ORAC and FRAP assays were further employed to determine whether these changes could be associated with changes in antioxidant activity, and all assays showed a decline in antioxidant potential post biotransformation.

Talaromyces purpurogenus MRS-F13 catalyzed biotransformation of (-)-verbenone to (-)-10-hydroxyverbenone and their anti-inflammatory activity profile

Verbenone is a natural monoterpene present as an essential component in rosemary oil from Rosmarinus officinalis, Verbena triphylla and Eucalyptus globule. Microbial biotransformation of monoterpenoids to value-added products has immense industrial potential. This study involves the biotransformation of (-)-verbenone to (-)-10-hydroxyverbenone by a fungus i.e. Talaromyces purpurogenus strain MRS-F13 in fermentation broth during the exponential phase. Biotransformation reaction has been successfully demonstrated at 15 L scale in the bioreactor with 70% conversion within 96 h. The anti-inflammatory activity investigations revealed that (-)-verbenone and its biotransformed product exhibited moderate inhibition of TNF-α and nitric oxide, whereas (-)-10-hydroxyverbenone presented an improved anti-oxidant activity. Highlights Talaromyces purpurogenus catalysed biotransformation of (-)-verbenone to (-)-10-hydroxyverbenone has been demonstrated. Approximately 70% bioconversion was obtained in 96 h in submerged culture at 15L bioreactor scale. Biotransformed product (-)-10-hydroxyverbenone has shown improved anti-inflammatory activity without cytotoxicity.

Biotransformation of asiatic acid by Cunninghamella echinulata and Circinella muscae to discover anti-neuroinflammatory derivatives

In this study, the biotransformation of asiatic acid by Cunninghamella echinulata CGMCC 3.970 and Circinella muscae CGMCC 3.2695 was investigated. Scaled-up biotransformation reactions yielded eight metabolites. Their structures were established based on extensive NMR and HR-ESI-MS data analyses and four of them are new compounds. C. echinulata could catalyze the regioselecitve hydroxylation, carbonylation and lactonization to yield five metabolites. C. muscae could selectively catalyze hydroxylation, acetylation and glycosylation to yield four products. Furthermore, all the identified metabolites were evaluated for their anti-neuroinflammatory activities in LPS-induced BV-2 cells. Most metabolites displayed pronounced inhibitory effect on nitric oxide (NO) production. The results suggested that biotransformed derivatives of asiatic acid might be served as potential neuroinflammatory inhibitors.

Estradiol, Estrone and Ethinyl Estradiol Metabolism Studied by High Resolution LC-MS/MS Using Stable Isotope Labeling and Trapping of Reactive Metabolites.

Biotransformation reactions that xenobiotics undergo during their metabolism are crucial for their proper excretion from the body, but can also be a source of toxicity, especially in the case of reactive metabolite formation. Unstable, reactive metabolites are capable of covalent binding to proteins, and have often been linked to liver damage and other undesired side effects. A common technique to assess the formation of reactive metabolites employs trapping them in vitro with glutathione and characterizing the resulting adducts by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Some endogenous compounds, however, can interfere with xenobiotic metabolites of interest, making the analysis more difficult. This study demonstrates the usefulness of isotope-labeled compounds to detect and elucidate the structures of both stable metabolites and trapped adducts of three estrogen analogs using an untargeted LC-MS/MS workflow. The metabolism of estradiol, estrone and ethinyl estradiol was investigated. Unlabeled and deuterated versions of these three compounds were incubated with human or rat liver microsomes in the presence of two different trapping agents, namely glutathione and N-acetylcysteine. The detection of closely eluting deuterated peaks allowed us to confirm the formation of several known metabolites, as well as many previously uncharacterized ones. The structure of each adduct was elucidated by the detailed analysis of high-resolution MS/MS spectra for elucidating fragmentation pathways with accurate mass measurements. The use of isotopic labeling was crucial in helping confirm many metabolites and adduct structures, as well as removing endogenous interferences.

Comprehensive Biotransformation Analysis of Phenylalanine-Tyrosine Metabolism Reveals Alternative Routes of Metabolite Clearance in Nitisinone-Treated Alkaptonuria

Metabolomic analyses in alkaptonuria (AKU) have recently revealed alternative pathways in phenylalanine-tyrosine (phe-tyr) metabolism from biotransformation of homogentisic acid (HGA), the active molecule in this disease. The aim of this research was to study the phe-tyr metabolic pathway and whether the metabolites upstream of HGA, increased in nitisinone-treated patients, also undergo phase 1 and 2 biotransformation reactions. Metabolomic analyses were performed on serum and urine from patients partaking in the SONIA 2 phase 3 international randomised-controlled trial of nitisinone in AKU (EudraCT no. 2013-001633-41). Serum and urine samples were taken from the same patients at baseline (pre-nitisinone) then at 24 and 48 months on nitisinone treatment (patients N = 47 serum; 53 urine) or no treatment (patients N = 45 serum; 50 urine). Targeted feature extraction was performed to specifically mine data for the entire complement of theoretically predicted phase 1 and 2 biotransformation products derived from phenylalanine, tyrosine, 4-hydroxyphenylpyruvic acid and 4-hydroxyphenyllactic acid, in addition to phenylalanine-derived metabolites with known increases in phenylketonuria. In total, we observed 13 phase 1 and 2 biotransformation products from phenylalanine through to HGA. Each of these products were observed in urine and two were detected in serum. The derivatives of the metabolites upstream of HGA were markedly increased in urine of nitisinone-treated patients (fold change 1.2–16.2) and increases in 12 of these compounds were directly proportional to the degree of nitisinone-induced hypertyrosinaemia (correlation coefficient with serum tyrosine = 0.2–0.7). Increases in the urinary phenylalanine metabolites were also observed across consecutive visits in the treated group. Nitisinone treatment results in marked increases in a wider network of phe-tyr metabolites than shown before. This network comprises alternative biotransformation products from the major metabolites of this pathway, produced by reactions including hydration (phase 1) and bioconjugation (phase 2) of acetyl, methyl, acetylcysteine, glucuronide, glycine and sulfate groups. We propose that these alternative routes of phe-tyr metabolism, predominantly in urine, minimise tyrosinaemia as well as phenylalanaemia.

Characterization of the metabolism of aloin A/B and aloesin in rats using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Aloin A/B and aloesin are the major bioactive constituents in Aloe vera, with diverse pharmacological activities, including anti-bacterial, anti-tumour, anti-inflammatory and intestinal regulation. However, the in vivo metabolism of aloin A/B and aloesin is still unclear. In this study, the metabolic processes of aloin A/B and aloesin in rats were investigated using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and MetaboLynx™ software with the mass defect filter technique. Based on the proposed method, the prototype components of three compounds were all detected in rat plasma, urine and feces. Meanwhile, 25 aloin A/B metabolites (six phase I, three phase II, 16 phase I combined with phase II) and three aloesin metabolites (two phase I and one phase II) were detected in rats after oral administration of aloin A, aloin B and aloesin, and the main biotransformation reactions were hydroxylation, oxidation, methylation, acetylation and glucuronidation. In addition, aloin A and aloin B can be transformed into each other in vivo and the metabolic profiles of aloin A and aloin B are identical. These results provide essential data for further pharmaceutical research and clinical application of aloin A/B and aloesin.

Acinetobacter baumannii, Klebsiella pneumoniae and Elizabethkingia miricola isolated from wastewater have biodegradable activity against fluoroquinolone

Ciprofloxacin (CIP) and levofloxacin (LEV), widely used fluoroquinolone antibiotics, are often found in sewage from the sewage treatment plants and marine environment. In this study, CIP and LEV biodegrading bacterial consortia were obtained from industrial wastewater. Microorganisms in these consortia were identified as Acinetobacter baumannii (A. baumannii), Klebsiella pneumoniae (K. pneumoniae) and Elizabethkingia miricola (E. miricola). The impacts of the critical operating parameters on the elimination of CIP and LEV by bacterial consortia have been investigated and optimized to achieve the maximum levels of CIP and LEV biodegradation. Using liquid chromatography with tandem mass spectrometry (LC-MS-MS), possible degradation pathways for CIP and LEV were suggested by analyzing the intermediate degradation products. The role of the enzymes fluoroquinolone-acetylating aminoglycoside (6′-N-acetyltransferase) and cytochrome P450 (CYP450) in the breakdown of fluoroquinolones (FQs) was investigated as well. According to our findings, various biodegradation mechanisms have been suggested, including cleavage of piperazine ring, substitution of F atom, hydroxylation, decarboxylation, and acetylation, as the main biotransformation reactions. This study discovers the ability of non-reported bacterial strains to biodegrade both CIP and LEV as a sole carbon source, providing new insights into the biodegradation of CIP and LEV.Graphical abstract

Wine residues extracts modulating in vitro metabolic syndrome

Metabolic syndrome is considered a worldwide epidemic health problem. The manifestations include obesity, dyslipidemias, elevation of arterial blood pressure, and insulin resistance. The wine waste is composed mainly of skin, seed and grape pomace, and is a source of phenolic compounds. Previous studies have demonstrated that grape phenolic compounds have antioxidants and anti-inflammatory properties, acting directly in the pathogenic process of several diseases. In order to improve the bioavailability and bioactivity of these compounds, enzymatic biotransformation reactions were developed.The aimed of this work was to evaluate whether phenolic extracts produced with wine waste by biotransformation with Tannase (tannin acyl hydrolase), were able to attenuate the metabolic syndrome manifestations like oxidative stress and reduce the inflammatory parameters in vitro assays.Four different extracts were produced and the non-citotoxic concentrations of samples used (100, 200 and 500 μg/mL) were determined by MTT assay. The inflammatory response was induced in macrophages (RAW 264.7) culture using LPS (Lipopolysaccharide). The biotransformed extracts were able to reduce the TNF-α cell culture supernatant concentration by about 44%; and the IL-6 secretion was reduced by 82%. All the extracts demonstrate efficacy in reduction of intracellular reactive oxygen species (ROS) production and the same was observed for NO (nitric oxide) concentration in the cell culture supernatant after the administration of extracts. These results indicated the strong potential of employing phenolic wine waste extracts on the modulation of metabolic syndrome parameters, use that also brings environmental and socioeconomic advantages, since it brings an alternative for the use of residues.

Fungal biotransformation of carvone and camphor by Aspergillus flavus and investigation of cytotoxic activities of naturally obtained essential oils

In this study, the biotransformation of carvone and camphor by Aspergillus flavus and the products were investigated. The biotransformation reaction of carvone by A. flavus resulted in the production of neodihydrocarveol, dihydrocarvone, 2-cyclohexene-1-one,2-methyl-5-(1-methylethenyl), limonene-1,2-diol, trans-p-mentha-1(7),8-dien-2-ol, p-menth-8(10)-ene-2,9-diol, and the biotransformation reaction of camphor resulted in the production of 2 -campholenic acid, 2-cyclohexene-1-one,2-hydroxy-4,4,6,6-tetramethyl, α-campholene aldehyde. The naturally produced essential oils by biotransformation of carvone and camphor were observed to be cytotoxic to breast cancer cells while no significant inhibition was seen in the healthy cell line. Additionally, biotransformation products had the highest inhibition (74%) against aflatoxin B1. The bioactivities of biotransformation products are promising, and they can be further investigated for their therapeutic potential as active agents.

Biochemical and Functional Characterization of an Iron-Containing Alcohol Dehydrogenase from Thermococcus barophilus Ch5.

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH641 and Tba ADH547). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH547. Herein, we present evidence that Tba ADH641 possesses two activities for ethanol oxidization and acetaldehyde reduction at high temperature, capable of using NAD(H) and NADP(H) as coenzyme. Biochemical data show that Tba ADH641 possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH547 and other iron-containing ADH homologues. However, Tba ADH641 and Tba ADH547 display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH641 displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH547. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH641, which has not been described to date, are necessary for enzyme activity, thus augmenting our understanding on catalytic mechanism of iron-containing ADH. Overall, our work demonstrates that Tba ADH641 is an iron-containing ADH with novel features, which is distinct from Tba ADH547, thus providing a potential biocatalyst for biotransformation reaction.

Application of moving bed biofilm reactor - nanofiltration - membrane bioreactor with loose nanofiltration hollow fiber membranes for synthetic roxithromycin-containing wastewater treatment: Long-term performance, membrane fouling and microbial community

The present study operated the novel moving bed biofilm reactor-nanofiltration-membrane bioreactor (MBBR-NF-MBR) with loose polyamide NF membranes for the first time to treat roxithromycin (ROX) wastewater. Results showed that both MBBR-NF-MBRs achieved superior COD removal of 98.4% and 97.2% and excellent removal of ROX at 74.1% and 65.5%, respectively. The main membrane fouling mechanism was reversible fouling caused by the combination of abundant polysaccharides, proteins and Ca-P precipitates, which could be effectively removed by acidic cleaning. Sorption and biodegradation were the main removal routes of ROX in MBBR. Partial retention of loose NF membrane contributed to microbial metabolism and increased microbial diversity, especially the genera Hyphomicrobium in attached biofilm, which was reasonable for ROX removal. The cleavage of cladinose, demethylation, phosphorylation and β-oxidation in macrolactone ring were the main biotransformation reactions of ROX. This study provides novel insights for micropollutants wastewater treatment by using loose NF membrane in MBR.

Enzymatic Synthesis of Novel and Highly Soluble Puerarin Glucoside by Deinococcus geothermalis Amylosucrase.

Puerarin (daidzein-8-C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties; however, the poor solubility of puerarin limits its applications. To resolve this poor solubility, Deinococcus geothermalis amylosucrase (DgAS) was used to modify puerarin into more soluble derivatives. The results showed that DgAS could biotransform puerarin into a novel compound: puerarin-4′-O-α-glucoside. The biotransformation reaction was manipulated at different temperatures, pH values, sucrose concentrations, reaction times, and enzyme concentrations. The results showed that the optimal reaction condition was biotransformed by 200 μg/mL DgAS with 20% (w/v) sucrose at pH 6 and incubated at 40 °C for 48 h, and the optimal production yield was 35.1%. Puerarin-4′-O-α-glucoside showed 129-fold higher solubility than that of puerarin and, thus, could be further applied for pharmacological use in the future.

Stereoisomers in sports drug testing: Analytical strategies and applications

Analytics employed in modern doping controls are designed to cover an extensive range of rather diverse classes of substances, all of which are banned in sport according to the list of prohibited substances and methods of doping, resulting from their potential to be performance-enhancing and/or harmful to health. Many of these bioactive substances or their metabolites are chiral, which are comprehensively characterized and, if appropriate analytical approaches are applied, can be clearly identified. In sports drug testing, the enantiomeric composition of relevant compounds is not considered in all instances, although differences of isomers concerning their biological activity have been established. To date, the separation of stereoisomers in doping controls is only applied for selected target compounds, but with the development of efficient chiral chromatographic stationary phases, the added value of information on e.g. racemic shifts during the metabolic biotransformation reactions of drugs has been recognized. The immense variability of the substance classes represents however a major challenge, especially because both ‘classic’ doping agents belonging to the category of lower molecular mass molecules (e.g. stimulants, β2-agonists, betablockers, corticoids, etc.) as well as larger molecules from the category of peptides and proteins necessitate consideration. In the present (mini)review, the current status of analytical techniques in the field of doping control analysis of stereoisomers is highlighted and critically reviewed.

Single-well push-pull tests evaluating isobutane as a primary substrate for promoting in situ cometabolic biotransformation reactions.

A series of single-well push-pull tests (SWPPTs) were performed to investigate the efficacy of isobutane (2-methylpropane) as a primary substrate for in situ stimulation of microorganisms able to cometabolically transform common groundwater contaminants, such as chlorinated aliphatic hydrocarbons and 1,4-dioxane (1,4-D). In biostimulation tests, the disappearance of isobutane relative to a nonreactive bromide tracer indicated an isobutane-utilizing microbial community rapidly developed in the aquifer around the test well. SWPPTs were performed as natural drift tests with first-order rates of isobutane consumption ranging from 0.4 to 1.4day-1. Because groundwater contaminants were not present at the demonstration site, isobutene (2-methylpropene) was used as a nontoxic surrogate to demonstrate cometabolic activity in the subsurface after biostimulation. The transformation of isobutene to isobutene epoxide (2-methyl-1,2-epoxypropane) illustrates the epoxidation process previously shown for common groundwater contaminants after cometabolic transformation by alkane-utilizing bacteria. The rate and extent of isobutene consumption and the formation and transformation of isobutene epoxide were greater in the presence of isobutane, with no evidence of primary substrate inhibition. Modeled concentrations of isobutane-utilizing biomass in microcosms constructed with groundwater collected before and after each SWPPT offered additional evidence that the isobutane-utilizing microbial community was stimulated in the aquifer. Experiments in groundwater microcosms also demonstrated that the isobutane-utilizing bacteria stimulated in the subsurface could cometabolically transform a mixture of co-substrates including isobutene, 1,1-dichloroethene, cis-1,2-dichloroethene, and 1,4-D with the same co-substrate preferences as the bacterium Rhodococcus rhodochrous ATCC strain 21198 after growth on isobutane. This study demonstrated the effectiveness of isobutane as primary substrate for stimulating in situ cometabolic activity and the use of isobutene as surrogate to investigate in situ cometabolic reactions catalyzed by isobutane-stimulated bacteria.

MOLECULAR CHARACTERIZATION AND LIPOLYTIC ACTIVITY OF YARROWIA LIPOLYTICA ISOLATED FROM YOGHURT CREAM

Objective: Yarrowia lipolytica is an aerobic dimorphic yeast that produces various metabolites such as enzymes, organic acids, proteins, surfactants and has industrial potential in some biotransformation and bioremediation reactions. To be a preliminary study to industrial productions, in present study it was aimed to isolate Y. lipolytica strain from yoghurt cream, perform its molecular characterization and investigate the lipolytic activity. Material and Method: A total of 10 samples were taken from the homemade yoghurt cream obtained by fermented milk from local dairy producers in Ankara. Yeast-Peptone-Glycerol (YPG) broth and Yarrowia lipolytica Distinctive (YLD) agar media were used for isolation of the species. Species-level identification was carried out by ITS-Polymerized chain reaction analysis. Lipolytic activity was determined with Rhodamine-B/Olive Oil Agar nutrient medium procedure. Result and Discussion: Yeast colonies were isolated from homemade yoghurt creams by growing in YPG broth. Yeasts were stained with Gram staining method and Gram-positive stained ones were inoculated on YLD agar. At the end of the incubation period, the brown pigmented colony growing on YLD agar was selected as Yarrowia species. According to the molecular characterization results the brown pigmented colony was identified as Yarrowia lipolytica with a total of 358 bases, 100% sequence matching ratio and 100% similarity ratio and the strain was determined as lipase positive. The development of the Y. lipolytica strain isolated in our study as a producer culture that can be used in industrial production should be supported by further studies to benefit from its various properties.