Pattern Of Metabolite Formation Research Articles

Single versus multiple metabolite quantification of in vitro starch digestion: A comparison for the case of pulse cotyledon cells

Different quantification methods for in vitro amylolysis were compared for individual chickpea and lentil cotyledon cells (ICC) as a relevant case study. For the first time, much-applied spectrophotometric methods relying on the quantification of certain functional groups (i.e., DNS, GOPOD) were compared to chromatographic quantification of starch metabolites (HPLC-ELSD). The estimated rate constant and linked initial rates of amylolysis were highly correlated for DNS, GOPOD, and HPLC-ELSD. However, absolute amylolysis levels depended on the applied method and sample-specific metabolite formation patterns. Multiresponse modelling was employed to further investigate HPLC-ELSD metabolite formation patterns. This delivered insight into the relative importance of different amylolysis reactions during in vitro digestion of pulse ICC, proving that maltotriose and maltose formation determined the overall amylolysis rate in this case. Multiresponse reaction rate constants of maltotriose and maltose formation were highly correlated to single response amylolysis rate constants (and initial rates) obtained for all three quantification methods.

Effect of Probiotic Administration on Serum Tryptophan Metabolites in Pediatric Type 1 Diabetes Patients.

Type 1 diabetes (T1D) is characterized by anomalous functioning of the immuno regulatory, tryptophan-catabolic enzyme indoleamine 2,3 dioxygenase 1 (IDO1). In T1D, the levels of kynurenine—the first byproduct of tryptophan degradation via IDO1—are significantly lower than in nondiabetic controls, such that defective immune regulation by IDO1 has been recognized as potentially contributing to autoimmunity in T1D. Because tryptophan catabolism—and the production of immune regulatory catabolites—also occurs via the gut microbiota, we measured serum levels of tryptophan, and metabolites thereof, in pediatric, diabetic patients after a 3-month oral course of Lactobacillus rhamnosus GG. Daily administration of the probiotic significantly affected circulating levels of tryptophan as well as the qualitative pattern of metabolite formation in the diabetic patients, while it decreased inflammatory cytokine production by the patients. This study suggests for the first time that a probiotic treatment may affect systemic tryptophan metabolism and restrain proinflammatory profile in pediatric T1D.

Abstract 2933: Application of a mouse model humanized for the major pathways of drug disposition in anticancer drug development and use

Abstract A vast number of targeted anticancer drugs are being developed in the pharmaceutical industry whose efficacy will only become fully realized through their combination with other established or novel anti-tumor agents. This is particularly the case because combination chemotherapy is a major approach being taken to overcome the rapid onset of drug resistance which current dosing regimens can cause. This has raised the conundrum of which of the large number of possible drug combinations, which may involve combinations of more than two drugs, have the greatest chance of success. It is not possible to test all the possible combinations by clinical trial alone so more informative preclinical models are badly needed. Nearly all targeted anti-tumor agents are substrates for the cytochrome P450-dependent monooxygenase system. The testing of novel drug regimens in mice is severely compromised by the major species differences in this enzyme system both in catalytic function, in the pattern of metabolite formation and the regulation by transcription factors such as CAR and PXR. In order to circumvent this problem we have created a mouse model where thirty four murine P450’s have been deleted from the mouse genome and substituted for the major enzymes involved in drug disposition in man ie CYP1A1, CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP3A7. The mice have also been humanized for the transcription factors CAR and PXR. CYP3A4 and CYP2D6 are expressed off the human promoters. We report the validation the utility of this model by studying the in vivo metabolism and disposition of model drugs and the metabolism and disposition of the EGFR inhibitor, osimertinib and the BRAF inhibitor, dabrafenib. We show that the use of this model allows accurate prediction of clinically observed drug exposures, in the generation of human metabolites and drug/drug interactions. This model has therefore great potential for the development of combination therapies involving complex drug regimens and in the design of clinical trials targeted at overcoming drug resistance. Citation Format: Colin J. Henderson, Nico Scheer, Yury Kapelyukh, Aileen McLaren, Kenneth MacLeod, Anja Rode, De Lin, C Roland Wolf. Application of a mouse model humanized for the major pathways of drug disposition in anticancer drug development and use [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2933.

Metabolism of the carcinogen alpha-asarone in liver microsomes

Alpha-asarone (1) is a naturally occurring phenylpropene found in several plants, e.g. Acorus calamus. 1-containing plant materials and essential oils thereof are used for flavoring foods and in many phytopharmaceuticals. 1 has been claimed to have positive pharmacological effects, however, it is carcinogenic in male mice (liver) and probably genotoxic. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rat, bovine, porcine, and human origin using HPLC-DAD and LC-ESI-MS/MS and derived kinetic data on the metabolite formation.The main metabolic pathway was the side-chain hydroxylation leading to (E)-3′-hydroxyasarone (2). Epoxidation of 1 presumably led to (E)-asarone-1′,2′-epoxide (4) which instantly hydrolyzed to form erythro- and threo-configured diols (5b+5a). As a minor reaction O-demethylation of 1 was observed. The metabolite formation showed little species-specific differences with the exception of porcine liver microsomes for which the formation of diols 5b+5a exceeded the formation of alcohol 2. The kinetic parameters imply a dependence of the pattern of metabolite formation from substrate concentration. On the basis of our results and earlier findings we hypothesize the genotoxic epoxide 4 being the ultimate carcinogen metabolically formed from 1.

Hepatic metabolism of carcinogenic β-asarone.

β-Asarone (1) belongs to the group of naturally occurring phenylpropenes like eugenol or anethole. Compound 1 is found in several plants, e.g., Acorus calamus or Asarum europaeum. Compound 1-containing plant materials and essential oils thereof are used to flavor foods and alcoholic beverages and as ingredients of many drugs in traditional phytomedicines. Although 1 has been claimed to have several positive pharmacological effects, it was found to be genotoxic and carcinogenic in rodents (liver and small intestine). The mechanism of action of carcinogenic allylic phenylpropenes consists of the metabolic activation via cytochrome P450 enzymes and sulfotransferases. In vivo experiments suggested that this pathway does not play a major role in the carcinogenicity of the propenylic compound 1 as is the case for other propenylic compounds, e.g., anethole. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rats, bovines, porcines, and humans using (1)H NMR, HPLC-DAD, and LC-ESI-MS/MS techniques. We synthesized the majority of identified metabolites which were used as reference compounds for the quantification and final verification of metabolites. Microsomal epoxidation of the side chain of 1 presumably yielded (Z)-asarone-1',2'-epoxide (8a) which instantly was hydrolyzed to the corresponding erythro- and threo-configurated diols (9b, 9a) and the ketone 2,4,5-trimethoxyphenylacetone (13). This was the main metabolic pathway in the metabolism of 1 in all investigated liver microsomes. Hydroxylation of the side chain of 1 led to the formation of three alcohols at total yields of less than 30%: 1'-hydroxyasarone (2), (E)- and (Z)-3'-hydroxyasarone (4 and 6), with 6 being the mainly formed alcohol and 2 being detectable only in liver microsomes of Aroclor 1254-pretreated rats. Small amounts of 4 and 6 were further oxidized to the corresponding carbonyl compounds (E)- and (Z)-3'-oxoasarone (5, 7). 1'-Oxoasarone (3) was probably also formed in incubations with 1 but was not detectable, possibly due to its rapid reaction with nucleophiles. Eventually, three mono-O-demethylated metabolites of 1 were detected in minor concentrations. The time course of metabolite formation and determined kinetic parameters show little species-specific differences in the microsomal metabolism of 1. Furthermore, the kinetic parameters imply a very low dependence of the pattern of metabolite formation from substrate concentration. In human liver microsomes, 71-75% of 1 will be metabolized via epoxidation, 21-15% via hydroxylation (and further oxidation), and 8-10% via demethylation at lower as well as higher concentrations of 1, respectively (relative values). On the basis of our results, we hypothesize that the genotoxic epoxides of 1 are the ultimate carcinogens formed from 1.

Terrestrial Field Dissipation of Diclosulam at Four Sites in the United States

The soil dissipation of diclosulam was studied using 14C-labeled and nonradiolabeled material in Mississippi, North Carolina, Georgia, and Illinois between 1994 and 1997. The test substance was preemergence broadcast applied at target rates of 35 and 37 g ai x ha(-1) for the 14C-labeled and the nonradiolabeled studies, respectively. The degradation of diclosulam was rapid with half-lives ranging from 13 to 43 days at the four sites. Rapid degradation rates and the increasing sorption to soil over time resulted in low persistence and mobility of this compound. Metabolite formation and dissipation in the field reflected observations of photolysis, hydrolysis, and aerobic soil metabolism studies in the laboratory. The rapid field dissipation rates, metabolite formation patterns, and sorption characteristics obtained in these field studies were consistent with the laboratory data generated for diclosulam, and reflect the multiple concurrent degradation mechanisms occurring in the field.

Anaerobic degradation of atrazine and metolachlor and metabolite formation in wetland soil and water microcosms.

The half-lives, degradation rates, and metabolite formation patterns of atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] were determined in an anaerobic wetland soil incubated at 24 degrees C for 112 d. At 0, 7, 14, 28, 42, 56, and 112 d, the soil and water were analyzed for atrazine and metolachlor, and their major metabolites. The soil oxidation-reduction potential reached -200 mV after 14 d. Degradation reaction rates were first-order for atrazine in anaerobic soil and for metolachlor in the aqueous phase. Zero-order reaction rates were best fit for atrazine in the aqueous phase and metolachlor in anaerobic soil. In anaerobic soil, the half-life was 38 d for atrazine and 62 d for metolachlor. In the aqueous phase above the soil, the half-life was 86 d for atrazine and 40 d for metolachlor. Metabolites detected in the anaerobic soil were hydroxyatrazine and deethylatrazine for atrazine, and relatively small amounts of ethanesulfonic acid and oxanilic acid for metolachlor. Metabolites detected in the aqueous phase above the soil were hydroxyatrazine, deethylatrazine, and deisopropylatrazine for atrazine, and ethanesulfonic acid and oxanilic acid for metolachlor. Concentrations of metabolites in the aqueous phase generally peaked within the first 25 d and then declined. Results indicate that atrazine and metolachlor can degrade under strongly reducing conditions found in wetland soils. Metolachlor metabolites, ethanesulfonic acid, and oxanilic acid are not significantly formed under anaerobic conditions.

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae.

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers' yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h(-1) under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h(-1) and 0.40 h(-1). Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.

Field Dissipation of Cloransulam-methyl at Four Sites in the U.S. Soybean Market

The soil dissipation of cloransulam-methyl was studied using 14C-labeled and non-radiolabeled material in Wisconsin, Indiana, Mississippi, and North Carolina between 1993 and 1995. The test substance was pre-emergence broadcast-applied at target rates of 50 and 61.5 g ai ha-1 for the 14C-labeled and the non-radiolabeled studies, respectively. Bromide was used to track water movement at the NC and WI sites. The degradation of cloransulam-methyl was rapid and best characterized by a two-compartment model resulting in initial-rate half-lives ranging from 2.5 to 4.8 days at the MS, IN, and WI sites and by a first-order degradation model at the NC site with a half-life of 11.2 days. The rapid dissipation rates, metabolite formation patterns, and sorption characteristics obtained in this field study were consistent with the existing laboratory data generated for cloransulam-methyl. Rapid degradation rates and the increasing sorption to soil over time resulted in low persistence and mobility of this compound. Keywords: Cloransulam-methyl; triazolopyrimidine sulfonanalide; soil dissipation

Sterol-side chain cleavage by fusants ofAlkaligenes fœcalis andArthrobacter oxydans

Protoplasts of a strain ofA. foecalis incapable of utilizing beta-sitosterol as carbon source for growth were fused with protoplasts ofA. oxydans - a strain capable of complete degradation of beta-sitosterol. Five fusants showing morphology and pattern of transformation of C-19 steroids identical toA. Foecalis were selected. Analysis of the fermentation broth containing beta-sitosterol showed that the fusants were capable of utilizing beta-sitosterol for growth but their pattern of metabolite formation from beta-sitosterol was different from that ofA. oxydans. The study revealed that the protoplast fusion technique could be used for intergeneric transfer of genetic determinants linked to partial cleavage of beta-sitosterol side chain toA. foecalis fromA. oxydans.

The effect of pregnancy on the metabolism of noradrenaline in reproductive organs of the rabbit.

The metabolites of (-)-3H-noradrenaline (1.2 mumol l-1) formed by uteri from non-pregnant mature rabbits were, in descending order of importance, 3H-DOPEG greater than 3H-MOPEG greater than 3H-NMN greater than 3H-VMA (3H-DOMA not measured). The pattern of metabolite formation per unit mass of tissue was markedly different in uteri from 28 day pregnant rabbits due to a fourfold increase in 3H-NMN formation and a fourfold decrease in 3H-DOPEG formation. The effects of cocaine indicated that 3H-NMN was largely extraneuronal in origin and 3H-DOPEG was both neuronal and extraneuronal in origin. The pattern of metabolite formation in pregnant rabbit uteri closely resembled the reported pattern in uteri from ovariectomized rabbits treated with 17 beta-oestradiol and progesterone. In the rabbit oviduct and ovary, the patterns of metabolite formation, and their modification by cocaine, indicated that (-)-3H-noradrenaline was metabolised mainly by intraneuronal deamination in the oviduct and mainly by extraneuronal O-methylation in the ovary. Pregnancy did not affect (-)-3H-noradrenaline metabolism in the oviduct, but decreased the metabolism in the ovary.

Influence of airborne particulate on the metabolism of benzo[a]pyrene in the isolated perfused lung.

Benzo[a]pyrene (BaP), a ubiquitous potent carcinogen, has been associated with the increased incidence of human bronchiogenic carcinoma in occupational and urban settings. A detailed knowledge of the rate and pattern of metabolite formation and factors affecting their formation is essential for understanding the mechanism of action of BaP in the lung. An isolated perfused New Zealand rabbit lung preparation was used to investigate the effects of a crude airborne particulate mixture on the metabolism of BaP. [14C]BaP with and without crude air particulate (CAP) was administered intratracheally to an isolated perfused lung (IPL) preparation after intratracheal pretreatment of the whole animal with CAP and/or BaP, or intraperitoneal pretreatment of the whole animal with BaP. BaP and its metabolites were extracted from perfusing blood at 6 time points up to 180 min after administration of [14C]BaP to the IPL. BaP and its metabolites were also extracted from lung tissue, washout fluid, aveolar macrophages, and trachea bronchi at the end of the perfusion at 180 min. Patterns of BaP metabolites were determined by chromatographic techniques and liquid scintillation counting. Particulate pretreatment of the whole animal or administration of the particulate to the IPL altered BaP metabolism by the perfusing lung. Particulate pretreatment of the whole animal resulted in increases in the total rates of appearance of metabolites of BaP in the blood (ng/g lung . h), while particulate administration to the IPL resulted in decreases in the total rate of appearance of metabolites of BaP in the blood and negated the effects of pretreatments. Coadministration of particulate with BaP to the IPL with and without particulate pretreatment of the whole animal, or BaP administration to the IPL preceded by particulate pretreatment of the whole animal, enhanced dihydrodiol formation and depressed formation of water-soluble materials. This is important because dihydrodiol formation is considered part of the active pathway of BaP carcinogenicity. These data suggest that pulmonary particulate exposure in the presence of BaP results in the initial increased production of dihydrodiols of BaP that may be further metabolized to compounds believed to be the ultimate carcinogenic form(s) of BaP.

Anti-inflammatory drugs in experimental atherosclerosis: Part 4. Inhibition of atherosclerosis in vivo and thromboxane synthesis and platelet aggregation in vitro

Groups of New Zealand white male rabbits were fed atherogenic diets containing 1% cholesterol. The diets of experimental groups were supplemented additionally with either aspirin, phenylbutazone, mefenamic acid, flufenamic acid, oxyphenylbutazone or aminopyrine. Blood cholesterol and phospholipids were measured at 3–4 week intervals. After 12 weeks the animals were sacrificed and the severity of atherosclerosis in the thoracic aorta was measured. In separate experiments, rabbit platelets were incubated with each of the drugs individually and conversion of [ l4C] arachidonic acid to thromboxanes and related compounds was assayed. Inhibition of collagen and arachidonic acid-induced platelet aggregation by each drug was also measured. All drugs inhibited thromboxane synthesis and platelet aggregation in varying degrees with flufenamate and aspirin being most and aminopyrine least effective. The pattern of metabolite formation from [ 14C]arachidonate was consistent with a block in the cyclooxygenase reaction. Phenylbutazone, flufenamic acid and oxyphenylbutazone produced significant reductions in atherosclerotic plaque formation without major changes in blood cholesterol levels or blood cholesterol-phospholipid ratios. Aspirin and aminopyrine were ineffective. The results indicate that the effectiveness of anti-inflammatory drugs as inhibitors of thromboxane synthesis and platelet aggregation in vitro does not afford a sufficient predictive index of their anti-atherogenicity in vivo. The significance of these findings is discussed in terms of the possible involvement of cyclooxygenase derivatives in atherogenesis.

Prostaglandin metabolism during circulatory shock

The rates of metabolic degradation and the patterns of metabolite formation of tritium-labeled prostaglandins E 2 and F 2α were assessed in vitro in tissues obtained from normal rabbits and from rabbits subjected to hemorrhagic or endotoxic shock. Normal rabbit tissues metabolized prostaglandin E 2 at the following rates: renal cortex 479 ± 34, liver 389 ± 95, and lung 881 ± 93 pmol of PGE 2 metabolized/mg soluble protein per min at 37°C (mean ± S.E.). Prostaglandin F 2α metabolism proceeded in normal animal tissues at rates of 477 ± 39, 324 ± 95, and 633 ± 69 pmol of PGF 2α metabolized/mg soluble protein per min for renal cortex, liver and lung, respectively. There were no significant differences between these rates of PGE 2 and PGF 2α metabolism when compared to rates in tissues obtained from animals subjected to either hemorrhagic or endotoxic shock. In addition, no significant differences were observed between the rate of PGE 2 metabolism and that of PGF 2α metabolism for any tissue. However, the lung was able to metabolize PGE 2 and PGF 2α significantly more rapidly than the liver, and to degrade PGE 2 at a significantly greater rate than the renal cortex. Although slightly different patterns of metabolite production were observed between lung and kidney homogenates, only the liver metabolized prostaglandins almost exclusively to more polar metabolites. While hemorrhagic or endotoxic shock induced slight changes in the patterns of PGE 2 metabolite formation in all three tissues studied, PGF 2α metabolite formation patterns were not significantly altered by circulatory shock. Thus, prostaglandin metabolism is not significantly impaired during the first 2 h of hemorrhagic or endotoxic shock in rabbit tissues. Therefore, impairment of prostaglandin metabolism is not the major factor responsible for the early increase in circulating prostaglandin concentrations in these forms of shock.

Metabolic studies of tetrabenazine, a psychotropic drug in animals and man

Following administration of tetrabenazine (2-oxo-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo[a]quinolizine) to animals or man, 9 metabolites of the drug were detected in the urine by thin layer chromatography. Five of these compounds were found unconjugated and 4 conjugated with glucuronic acid. The structures of all 5 unconjugated metabolites and of 2 aglucones Were established either by comparison in thin layer chromatography with synthetic compounds or by NMR mass spectroscopy and elemental analysis of metabolites isolated by column chromatography on silicic acid. The main steps of biological degradation of the drug are: reduction of the keto group at C 2 oxidation at position 2′ of the isobutyl side chain selective ether cleavage at C 9, followed by conjugation of the phenols to glucuronic acid. The same pattern of metabolite formation was observed in the rabbit, in the dog and in man. In all three species the glucuronides represent the prevailing form of excretion of the drug in the urine. A general scheme for the biological degradation of tetrabenazine is suggested. A description of the synthesis of several metabolites is given.