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Peptide YY 3–36-amide (PYY 3–36 ) is a peptide hormone, which is known to decrease appetite and food-intake by activation of the Y 2 receptor. The current studies were designed to identify the metabolites of PYY 3–36 in mini-pig and rhesus monkey. Plasma samples were analyzed by high resolution LC–MS (and MS/MS) in order to unambiguously identify the metabolites of PYY 3–36 . In summary, the metabolism of PYY 3–36 was similar in mini-pig and rhesus monkey. Several metabolites were identified and PYY 3–34 was identified at the highest levels in plasma. In addition, mini-pigs were also dosed with PYY 1–36 -amide, PYY 3–35 , PYY 3–34 and [ N- methyl 34Q]-PYY 3–36 -amide in order to investigate the mechanisms by which PYY was metabolized. PYY 3–35 was rapidly converted to PYY 3–34 whereas dosing of PYY 3–34 to mini-pigs only showed circulating degradation products at low levels, i.e., PYY 3–34 was metabolically more stable than PYY 3–36 and PYY 3–35 . [ N- methyl 34Q]-PYY 3–36 -amide was hypothesized to be stable toward cleavage between 34Q and 35R and after i.v. administration to mini-pigs, one major cleavage product was identified as [ N- methyl 34Q]-PYY 3–35 . Overall, this showed that cleavage between 35R and 36Y was possible as well as between 34Q and 35R (as shown for PYY 3–35 ), which indicated that metabolism of PYY 3–36 to PYY 3–34 may be a two-step process. PYY 1–36 was also dosed to mini-pigs, which showed that PYY 1–36 was metabolized in the C-terminal as PYY 3–36 . The overall degradation pattern of PYY 1–36 was more complex due to the simultaneous enzymatic degradation in the N-terminal to form PYY 2–34/36 and PYY 3–34/36 . In vitro incubations with heparin stabilized plasma showed that PYY 3–36 was degraded with a half-life of 175 min, whereas incubations with PYY 3–35 (half-life of 6 min) showed a rapid formation of PYY 3–34 . In conclusion, the present studies showed that PYY 3–36 underwent enzymatic degradation in the C-terminal part and that the major circulating metabolite was PYY 3–34 . Furthermore, it may be a sequential two-step process leading to the formation of PYY 3–35 and subsequently the metabolically more stable PYY 3–34 . [ABSTRACT FROM AUTHOR]

Objective: Paeoniae Radix Rubra (PRR) is a commonly used traditional Chinese medicine with the effects of clearing away heat, cooling the blood, and relieving blood stasis. To 1) elucidate the metabolites and metabolic pathways of PRR and its 14 main constituents in mice and 2) reveal the possible origins of the known effective forms of PRR and their isomers, the metabolism of PRR in mice was systematically studied for the first time. Methods: PRR and its 14 constituents were administered to mice by gavage once a day for seven consecutive days, respectively. All urine and feces were collected during the 7 days of dosing, and blood was collected at 1 h after the last dose. Metabolites were detected and identified using high performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS n ). Results: In total, 23, 16, 24, 17, 18, 30, 27, 17, 22, 17, 33, 3, 8, 24, and 31 metabolites of paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorin, hydroxybenzoylpaeoniflorin, benzoyloxypaeoniflorin, galloylpaeoniflorin, lactiflorin, epicatechin gallate, catechin gallate, catechin, ellagic acid, 3,3'-di- O -methylellagic acid, methylgallate, and PRR were respectively identified in mice; after eliminating identical metabolites, a total of 195 metabolites remained, including 8, 11, 25, 17, 18, 30, 27, 17, 21, 17, 1, 2, 8, 20, and 20 newly identified metabolites, respectively. The metabolic reactions of PRR and its 14 main constituents in mice were primarily methylation, hydrogenation, hydrolysis, hydroxylation, glucuronidation, and sulfation. Conclusion: We elucidated the metabolites and metabolic pathways of PRR and its 14 constituents (e.g., paeoniflorin, catechin, ellagic acid, and gallic acid) in mice and revealed the possible origins of the 10 known effective forms of PRR and their isomers. The findings are of great significance to studying the mechanism of action and quality control of PRR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Lv, Zhang, Shi, Guo, Xu, Wang, Chen, Xiang, Xu, Wang and Cai.)

ABSTRACT A highly sensitive and specific LC-MS/MS method was developed to investigate the in vivo bio-transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative-ion mode. The assay procedure involves a simple liquid-liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP-C18 column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De-glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Highlights: [•] The diverse laccase-catalyzed oxidation reactions with small organics are classified into two types: catabolic and anabolic. [•] These bifunctional actions are readily found in laccase-driven in vivo metabolic pathways. [•] Biological functions of the in vivo metabolisms can inspire discovery of new application areas for in vitro bifunctional reactions of laccases and small organics. [Copyright &y& Elsevier]

Abstract: Type 2 diabetes patients exhibit a reduction in oxidative muscle fibres and an increase in glycolytic muscle fibres. In this study, we investigated whether both genetic and non-genetic factors influence the mRNA expression levels of three myosin heavy chain (MHC) genes represented in different fibre types. Specifically, we examined the MHC7 (slow-twitch oxidative fibre), MHCIIa (fast-twitch oxidative fibre) and MHCIIx/d (fast-twitch glycolytic fibre) genes in human skeletal muscle. We further investigated the use of MHC mRNA expression as a proxy to determine fibre-type composition, as measured by traditional ATP staining. Two cohorts of age-matched Swedish men were studied to determine the relationship of muscle mRNA expression of MHC7, MHCIIa, and MHCIIx/d with muscle fibre composition. A classical twin approach, including young and elderly Danish twin pairs, was utilised to examine if differences in expression levels were due to genetic or environmental factors. Although MHCIIx/d mRNA expression correlated positively with the level of type IIx/d muscle fibres in the two cohorts (P <0.05), a relatively low magnitude of correlation suggests that mRNA does not fully correlate with fibre-type composition. Heritability estimates and genetic analysis suggest that the levels of MHC7, MHCIIa and MHCIIx/d expression are primarily under non-genetic influence, and MHCIIa indicated an age-related decline. PGC-1α exhibited a positive relationship with the expression of all three MHC genes (P <0.05); meanwhile, PGC-1β related positively with MHCIIa expression and negatively with MHCIIx/d expression (P <0.05). While MHCIIa expression related positively with insulin-stimulated glucose uptake (P <0.01), MHCIIx/d expression related negatively with insulin-stimulated glucose uptake (P <0.05). Our findings suggest that the expression levels of the MHC genes are associated with age and both PGC-1α and PGC-1β and indicate that the MHC genes may to some extent be used to determine fibre-type composition in human skeletal muscle. [Copyright &y& Elsevier]

Abstract: High-performance liquid chromatography with diode-array detection (HPLC–DAD) and tandem mass spectrometry (HPLC–MS/MS) was used for separation and identification of metabolites in rat urine, bile and plasma after oral administration of rhubarb decoction. Based on the proposed strategy, 91 of the 113 potential metabolites were tentatively identified or characterized. Besides anthraquinones metabolites, gallic acid, (−)-epicatechin and (+)-catechin metabolites were also detected and characterized in these biological samples. Our results indicated that glucuronidation and sulfation were the main metabolic pathways of anthraquinones, while methylation, glucuronidation and sulfation were the main metabolic pathways of gallic acid, (−)-epicatechin and (+)-catechin. Phase I reactions (e.g., hydroxylation and reduction) played a relatively minor role compared to phase II reactions in metabolism of phenolic compounds of rhubarb decoction. The identification and structure elucidation of these metabolites provided essential data for further pharmacological and clinical studies of rhubarb and related preparations. Moreover, the results of the present investigations clearly indicated the relevance and usefulness of the combination of chromatographic, spectrophotometric, and mass-spectrometric analysis to detect and identify metabolites. [ABSTRACT FROM AUTHOR]

Ginsenoside Re is one of the major the bioactive triterpene saponins in ginseng root, a well-known adaptogen in traditional Chinese medicine. It is believed that the lead compound may be further developed into a promising new drug for preventing hypertension and cardiovascular disease. To better understand the pharmacological activities of the component, an investigation of its in vivo metabolism was necessary. In the present study, a high-performance liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-TOF-MS/MS) has been applied to discover and identify the metabolites of ginsenoside Re in rat urine following intravenous and oral administration of the component, respectively. The rat urine samples were collected and pretreated through C18 solid-phase extraction cartridges prior to analysis. Negative electrospray ionization mass spectrometry was used to discern ginsenoside Re and its possible metabolites in urine samples. The metabolites were identified and tentatively characterized by means of comparing molecular mass, retention time, and fragmentation pattern of the analytes with those of the parent compound, ginsenoside Re. As a result, eleven and nine metabolites together with Re were detected and identified in rat urine collected after intravenous and oral administration, respectively. A possible metabolic pathway of ginsenoside Re was also investigated and proposed. Oxidation and deglycosylation were found to be the major metabolic processes of the constituent in rat. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Type 2 diabetes is characterized by hyperlipidemia, hyperinsulinemia, and insulin resistance. The aim of this study was to investigate whether acute hyperlipidemia-induced insulin resistance in the presence of hyperinsulinemia was due to defective insulin signaling. Hyperinsulinemia (∼300 mU/I) with hyperlipidemia or glycerol (control) was produced in cannulated male Wistar rats for 0.5, I h, 3 h, or 5 h. The glucose infusion rate required to maintain euglycemia was significantly reduced by 3 h with lipid infusion and was further reduced after 5 h of infusion, with no difference in plasma insulin levels, indicating development of insulin resistance. Consistent with this finding, in vivo skeletal muscle glucose uptake (31%, P < 0.05) and glycogen synthesis rate (38%, P < 0.02) were significantly reduced after 5 h compared with 3 h of lipid infusion. Despite the development of insulin resistance, there was no difference in the phosphorylation state of multiple insulin-signaling intermediates or muscle diacylglyceride and ceramide content over the same time course. However, there was an increase in cumulative exposure to long-chain acyl-CoA (70%) with lipid infusion. Interest- ingly, although muscle pyruvate dehydrogenase kinase 4 protein content was decreased in hyperinsulinemic glycerol-infused rats, this decrease was blunted in muscle from hyperinsulinemic lipid-infused rats. Decreased pyruvate dehydrogenase complex activity was also observed in lipid- and insulin-infused animals (43%). Overall, these results suggest that acute reductions in muscle glucose metabolism in rats with hyperlipidemia and hyperinsulinemia are more likely a result of substrate competition than a significant early defect in insulin action or signaling. [ABSTRACT FROM AUTHOR]

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Midazolam is extensively metabolized and is eliminated in urine predominantly as conjugates of 1′-hydroxymidazolam. • Midazolam is known to undergo N-glucuronidation in vitro by UGT1A4. • In vitro 1′-hydroxymidazolam is O-glucuronidated by UGT2B4 and UGT2B7, and N-glucuronidated by UGT1A4. WHAT THIS STUDY ADDS • N-glucuronide of midazolam has been quantified in human urine, indicating for the first time that this route of metabolism occurs in vivo. • Metabolism of 4-hydroxymidazolam has been compared with that of 1′-hydroxymidazolam in vitro. • This study provides further evidence, in vitro and in vivo, of the importance of N-glucuronidation in the metabolism of midazolam and its metabolites. AIMS Midazolam (MDZ) is a benzodiazepine used as a CYP3A4 probe in clinical and in vitro studies. A glucuronide metabolite of MDZ has been identified in vitro in human liver microsome (HLM) incubations. The primary aim of this study was to understand the in vivo relevance of this pathway. METHODS An authentic standard of N-glucuronide was generated from microsomal incubations and isolated using solid-phase extraction. The structure was confirmed using proton nuclear magnetic resonance (NMR) and 1H-13C long range correlation experiments. The metabolite was quantified in vivo in human urine samples. Enzyme kinetic behaviour of the pathway was investigated in HLM and recombinant UGT (rUGT) enzymes. Additionally, preliminary experiments were performed with 1′-OH midazolam (1′-OH MDZ) and 4-OH-midazolam (4-OH MDZ) to investigate N-glucuronidation. RESULTS NMR data confirmed conjugation of midazolam N-glucuronide (MDZG) standard to be on the α-nitrogen of the imidazole ring. In vivo, MDZG in the urine accounted for 1–2% of the administered dose. In vitro incubations confirmed UGT1A4 as the enzyme of interest. The pathway exhibited atypical kinetics and a substrate inhibitory cooperative binding model was applied to determine Km (46 µM, 64 µM), Vmax (445 pmol min−1 mg−1, 427 pmol min−1 mg−1) and Ki (58 µM, 79 µM) in HLM and rUGT1A4, respectively. From incubations with HLM and rUGT enzymes, N-glucuronidation of 1′-OH MDZ and 4-OH MDZ is also inferred. CONCLUSIONS A more complete picture of MDZ metabolism and the enzymes involved has been elucidated. Direct N-glucuronidation of MDZ occurs in vivo. Pharmacokinetic modelling using Simcyp™ illustrates an increased role for UGT1A4 under CYP3A inhibited conditions. [ABSTRACT FROM AUTHOR]

Some major metabolic pathways of 3,4-methylenedioxymethamphetamine (MDMA) have been shown to be dependent on cytochrome P450 (CYP) isozymes by in vitro studies. The aim of this study was to clarify the roles of these CYP enzymes for in vivo metabolism of MDMA with respect to two pathways using rats: N-demethylation of MDMA to 3,4-methylenedioxyamphetamine (MDA) and O-demethylenation of MDMA to 3,4-dihydroxymethamphetamine (HHMA)followed by O-methylation to 4-hydroxy-3-methoxymethamphetamine (HMMA). Rats were pretreated with phenobarbital (PB, 80 mg/kg i.p.) or β-naphthoflavone (BNF, 80 mg/kg i.p.) once a day for 3 days before administration of MDMA (10 mg/kg i.p.). Metabolic changes were monitored by measuring the urinary excretion of MDMA and its metabolites. Twenty-four hours after MDMA administration, MDA in rat urine was significantly decreased by 43% and 70%, and HMMA was significantly increased by 33% and 64% in urine samples from PB-pretreated and BNF-pretreated rats, respectively, as compared with the control values. Testosterone 6β-hydroxylase (CYP3A), pentoxyresorufin O-dealkylase (CYP2B1), ethoxyresorufin O-deethylase (CYP1A1), and methoxyresorufin O-demethylase (CYP1A2) activities were increased 2–6 fold in both PB-pretreated and BNF-pretreated rat liver microsomes sampled at 24 h after MDMA administration as compared with the control values. These results suggest that PB-induced and BNF-induced CYP enzymes have inhibitory effects on N-demethylation of MDMA to MDA in vivo in rats. If HHMA is the precursor of HMMA in rats, there is a possibility that the O-demethylenation of MDMA to HHMA is increased by the induced CYP enzymes. The decreased urinary concentration of MDMA and very low percent recoveries of MDA, HMMA, and (4-hydroxy-3-methoxyphenyl)acetone (HMPA) in the inducer-pretreated groups suggest that other metabolic pathways of MDMA exist and are activated under the present experimental conditions. [ABSTRACT FROM AUTHOR]

The title of this commentary contains a frequently asked question whenever someone presents or proposes a prodrug strategy that releases formaldehyde as a result of bioconversion of a prodrug to parent drug. Formaldehyde, a highly water-soluble one-carbon molecule, is endogenous to cells, tissues, and body fluids. Although formaldehyde is generated and incorporated into essential metabolic processes by the human body, exposure to large amounts of formaldehyde vapor can irritate the nasal mucosa and may potentially be carcinogenic. It also gives a positive Ames test. Metabolism of both endogenous and exogenous formaldehyde involves rapid oxidation to formic acid catalyzed by glutathione dependent and independent dehydrogenases in the liver and erythrocytes. Balancing this rapid detoxification pathway is endogenous formation from normal metabolic processes and exogenous formaldehyde input, resulting in approximately 0.1 mM systemic levels. The possibility that formaldehyde released upon bioconversion of prodrugs might induce toxicity has been repeatedly stated, but no convincing evidence for this perceived toxicity has been documented in experimental studies. Therefore, as pharmaceutical chemists and not as toxicologists, we present our perspective on the apparent concern with release of formaldehyde as a by-product of in vivo bioconversion of selective prodrugs, and suggest that in comparison to the total amount of daily endogenous formaldehyde production from metabolism, and exogenous exposure from food and the environment, the amount generated by prodrugs is minute and is unlikely to cause any systemic toxicity in humans. Such an argument does not preclude formaldehyde-based toxicity assessment of a prodrug. Instead, it reduces the risk that in vivo liberation of formaldehyde will cause undue toxicity. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:4184–4193, 2008 [ABSTRACT FROM AUTHOR]

Abstract: Two new phenylacetylene derivatives, 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)indoline 8 and 5-((4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)phenyl)ethynyl)-1H-indole 14, targeting β-amyloid (Aβ) plaques have been prepared. In vitro binding carried out in tissue homogenates prepared from postmortem AD brains with [125I]IMPY (6-iodo-2-(4′-dimethylamino-)phenyl-imidazo[1,2-a]pyridine) as the radioligand indicated good binding affinities (K i =4.0 and 1.5nM for 8 and 14, respectively). Brain penetration of the corresponding radiofluorinated ligands, evaluated in the normal mice, showed good initial brain penetration (4.50 and 2.43% ID/g (injected dose/gram) for [18F]8 and [18F]14 at 2min after injection) with moderate to low washout rates from the brain (1.71% ID/g at 2h and 2.10% ID/g at 3h, respectively). Autoradiography and homogenate binding studies demonstrated the high specific binding of [18F]14 to the Aβ plaques; however, [18F]8 showed low specific binding. These preliminary results identified that indolylphenylacetylene, 14, may be a good lead for further structural modification to develop a useful Aβ plaque imaging agent. [Copyright &y& Elsevier]

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471-E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (-10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS 160 (Akt substrate of 160 kDa), glycogen synthase kinase-3β] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport. [ABSTRACT FROM AUTHOR]

Metabolic pathways of the oxime K-48 have been elucidated by means of in vitro and in vivo experiments. K-48 exposure to rat liver microsomal fraction resulted in the formation of a hydroxylated derivative, in addition to a small molecular fragment. The in vivo metabolism in rats was investigated after intramuscular administration of 50 μmol oxime. K-48 was present in the rat serum in unchanged form. Similarly, the analysis of rat cerebrospinal fluid indicated the sole occurrence of unchanged K-48. In contrast, unchanged K-48 was not found in the rat urine, where only the metabolite generated by epoxidation on the alkyl chain connecting the two pyridinium rings was present. The presence of unchanged K-48 in the serum and cerebrospinal fluid facilitates quantitative determination using HPLC separation and ultraviolet absorbance detection. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Peroxisome proliferator activated receptor-γ coactivator-1β ( PGC-1β, also known as PPARGC1B) expression is reduced in skeletal muscle from patients with type 2 diabetes mellitus and in elderly subjects. Ala203Pro, a common variant in the PGC-1β gene is associated with obesity. The aim of this study was to investigate whether the PGC-1β Ala203Pro polymorphism influences the age-related decline in skeletal muscle PGC-1β expression, in vivo metabolism and markers for muscle fibre type composition. The PGC-1β Ala203Pro polymorphism was genotyped in 110 young (age 28.0 ± 1.9 years) and 86 elderly (age 62.4 ± 2.0 years) twins and related to muscle PGC-1β expression, in vivo metabolism and markers for fibre type composition. Insulin-stimulated non-oxidative glucose metabolism (NOGM; p = 0.025) and glycolytic flux rate (GF; p = 0.026) were reduced in young Ala/Ala carriers compared with carriers of a 203Pro allele. In addition, a regression analysis, correcting for covariates, showed that the PGC-1β 203Pro allele was positively related to insulin-stimulated NOGM and GF in the young twins. While muscle expression of PGC-1β was reduced in elderly compared with young carriers of the Ala/Ala genotype ( p ≤ 0.001), there was no significant age-related decline in PGC-1β expression in carriers of the 203Pro allele ( p ≥ 0.4). However, a regression analysis, correcting for covariates, showed that only age was significantly related to muscle PGC-1β expression. Finally, PGC-1β expression correlated positively with markers for oxidative fibres in human muscle. This study suggests that young carriers of a PGC-1β 203Pro allele have enhanced insulin-stimulated glucose metabolism and may be protected against an age-related decline in PGC-1β expression in muscle. [ABSTRACT FROM AUTHOR]

In vivo and in vitro metabolisms of anisodamine were investigated using a highly specific and sensitive LC-MSn method. Feces, urine, and plasma samples were collected individually after ingestion of 25 mg/kg anisodamine to healthy rats. Rat feces and urine samples were cleaned up by a liquid-liquid extraction and a solid phase extraction procedure (C18 cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Anisodamine was incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubation solution were extracted with ethyl acetate. Then, these pretreated in vivo and in vitro samples, were injected into a reversed-phase C18 column with mobile phase of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by an on-line MSn system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (ΔM), retention times and full scan MSn spectra with those of the parent drug. Fifteen new metabolites (aponoranisodamine, apoanisodamine, methoxyanisodamine, hydroxy-methoxyanisodamine, trihydroxyanisodamine, dimethoxyanisodamine, dihydroxy-methoxyanisodamine, tetrahydroxyanisodamine, hydroxy-dimethoxyanisodamine, trihydroxy-methoxyanisodamine, dihydroxy-dimethoxyanisodamine, tetrahydroxy-methoxyanisodamine, trihydroxy-dimethoxyanisodamine, dihydroxy-trimethoxyanisodamine, and hydroxy-tetramethoxyanisodamine) were identified in rat urine after ingesting anisodamine. Seven metabolites (nor-6β-hydroxytropine, 6β-hydroxytropine, tropic acid, aponoranisodamine, apoanisodamine, noranisodamine, and anisodamine N-oxide) and the parent drug were detected in rat feces. Six metabolites (nor-6β-hydroxytropine, 6β-hydroxytropine, tropic acid, apoanisodamine, hydroxyanisodamine, and anisodamine N-oxide) and the parent drug are detected in rat plasma. Only apoanisodamine was detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (6β-hydroxytropine and tropic acid) and the dehydrated metabolite of anisodamine were found in the rat intestinal flora incubation mixture. [ABSTRACT FROM AUTHOR]

Abstract: Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so-called indirect pathway. The operation of both pathways depends on glucose availability. Below 0.5mM glucose (calculated intracellular concentration), the indirect route accounts for 90% of polysaccharide formation, while the direct pathway supports 70% of total glucose incorporation when administered glucose is above 1.5mM. A sigmoidal curve was obtained for the direct pathway with n H =2.04, and half saturation was reached at 2.6mM glucose. The curve for the indirect route presented an n H of 1.15 and an S 0.5 of 0.9mM glucose. [Copyright &y& Elsevier]

Abstract: Magnetic resonance spectroscopy (MRS) has been utilized to study several metabolic pathways in vivo and in live tissues in vitro non-invasively. Despite its inherited lack of sensitivity, its application has extended all the way to in situ human tissues and organs since proper technical advancements were devised. Examples of its application described here demonstrate the value of in vivo MRS as a technique that determines parameters of mitochondrial dysfunction directly and indirectly which could be of value for the diagnosis, prognosis, and follow-up of mitochondrial disorders. [Copyright &y& Elsevier]

A method for the simultaneous determination of hydrogen cyanide (HCN) and aliphatic nitriles using manual headspace (HS) gas chromatography (GC) with a capillary porous polymer column GS-Q and a nitrogen–phosphorus detector is described. With a HS incubation at 50°C for 30 min and a GC temperature at 180°C, HCN and volatile nitriles [acetonitrile, acrylonitrile (VCN), propionitrile, isobutyronitrile] were well separated and could be detected within 7 min with a detection limit of 0.7–2.4 ng/ml in blood samples. The HS-GC method was used in an in vivo study of VCN metabolism. VCN was administered orally (at nearly one-half its LD50) to rats, and heart blood and urine were sampled. Blood concentrations of HCN and VCN were measured by HS-GC, and plasma and urinary thiocyanate concentrations were measured by the König colorimetric method. Blood levels of HCN and VCN peaked 1.5 h after VCN administration, at which time the cyanide level (about 0.7 μg/ml) is close to the fatal level. HCN levels were observed to be at almost background levels at 10 h, although 50 ng/ml VCN was still detectable. The plasma thiocyanate level increased, reaching a peak (about 30 μg/ml) at 5 h. The cumulative urinary thiocyanate amount gradually increased, and at 10 h more than 1 mg thiocyanate was excreted into the urine. It is therefore possible to clarify the cause of cyanide poisoning using HS-GC analysis, when someone has taken volatile nitriles. [ABSTRACT FROM AUTHOR]

Two analytical methods have been evaluated for quantitative determination of de-conjugated chrysene metabolites in fish bile. High performance liquid chromatography-fluorescence (HPLC-F) and gas chromatography–mass spectrometry (GC–MS) were compared regarding instrumental and overall limits of detection (LOD) as well as recoveries for the following nine chrysene compounds: 1-, 2,- 3-, 4- and 6-hydroxychrysene (1-, 2-, 3-, 4- and 6-OH-chr), 1,2-dihydroxy-1,2-dihydrochrysene (1,2-DHD-chr), 3,4-dihydroxy-3,4-dihydrochrysene (3,4-DHD-chr), 5,6-dihydroxy-5,6-dihydrochrysene (5,6-DHD-chr) and chrysene. Instrumental LODs were comparable for the two methods whereas the overall LOD was better for HPLC-F. Recoveries varied per chrysene compound for both HPLC-F (62–107%) and GC–MS (48–124%). In vivo formed chrysene metabolites were studied in the bile of Atlantic cod (Gadus morhua) exposed to chrysene (1 mg/kg) via intra-peritoneal (i.p.) and inter-muscular (i.m.) injection. Total amounts of chrysene metabolites were three times higher in i.p. compared to i.m. exposed cod bile, but the relative distribution of determined metabolites was very similar. 1,2-DHD-chr was the most prominent metabolite in de-conjugated bile and constituted more than 88% of the total chrysene metabolites. Additional chrysene metabolites formed were 3,4-DHD-chr and 1-, 2-, 3- and 4-OH-chr. K-region chrysene metabolites (oxidation at carbons 5 and 6) were not detected and seem to be a less favoured biotransformation route. The two methods were applied and evaluated for analysis of chrysene metabolites in two bile reference materials (BCR 720 and 721) and a limited number of field exposed cods. [Copyright &y& Elsevier]

Phosphorus Magnetic Resonance Spectroscopy (31P-MRS) has been utilized to study energy, carbohydrate, and phospholipid metabolism in vitro and in vivo in live tissues non-invasively. Despite its lack of sensitivity, its application has extended to in situ human tissues and organs since proper signal localization was devised. Follow-up of phosphocreatine in neuromuscular diseases and schizophrenia and follow-up of phospholipid-related molecules in tumors are described here to demonstrate the value of 31P-MRS as an imaging technique to determine in vivo markers of disease and in the diagnosis, prognosis, and follow-up of human diseases. [ABSTRACT FROM AUTHOR]

Background: Myocardial lipid metabolism appears abnormal in dilated cardiomyopathy (DCM). A dual-tracer approach with two different fatty acid analogs may allow us to observe such alteration in vivo. 15-(Ortho-123I-phenyl)-pentadecanoic acid (oPPA) and 15-(para-123I-phenyl)-pentadecanoic acid (pPPA) have similar kinetics in circulation, diffusion, and transport. However, pPPA in normal myocardium undergoes beta-oxidation and may also be lost from myocardial cells through back-diffusion; oPPA is hardly catabolized and normally retained mainly in the cytosolic lipid pool. Use of both pPPA and oPPA in the dual-tracer approach focuses observation on the turnover of myocardial lipids (with pPPA) that is scaled against loss of fatty acid through back-diffusion into circulation (with oPPA).Methods and Results: Fifteen patients with idiopathic DCM and five control subjects were given oPPA and pPPA sequentially for dynamic planar scintigraphy. Uptake and elimination rates were determined for both substrates from three myocardial regions per individual; the corresponding six elimination rate constants and the three differences between them were analyzed for significant alterations in patients from control values. At least 66% of the patients had a significant alteration in myocardial lipid turnover in three types of patterns: (1) increased beta-oxidation, (2) decreased beta-oxidation, and (3) increased back-diffusion, in part associated with decreased beta-oxidation. Even with the limited number of patients and control subjects, the pattern of abnormality of lipid turnover in DMC appeared to be consistent individually but heterogeneous in the patient group. Moreover, a highly significant increase in beta-oxidation was observed for the posterolateral region of the myocardium compared with the anteroseptal and apical regions in control subjects and patients.Conclusion: The dual-tracer approach uncovered in vivo that in at least two thirds of the patients with DCM myocardial lipid turnover was significantly altered compared with control values. [ABSTRACT FROM AUTHOR]

Analysis by gas chromatography (GC) of the volatile compounds present in the urine from rats administered dioxane, a hepatic carcinogen to this species, revealed a major metabolite. The appearance of the metabolite was pH-dependent, undetectable at high pH; reacidification of the urine sample brought about the reappearance of the metabolite. The amount excreted was dose-dependent and time-dependent, reaching a maximum between 20 and 28 h after dioxane administration. Diethylene glycol administered to rats gave rise to the same metabolite. When isolated and purified from lyophilized urine by preparative GC, the metabolite exhibited an intense carbonyl band at 1750 cm in the infrared spectrum. Nuclear magnetic resonance spectrum showed two triplets and one singlet with equal intensity at δ 3.85, 4.48 and 4.37, respectively. GC-mass spectrometric studies indicated a parent peak at m/e 102. The metabolite was identified as p-dioxane-2-one. Synthetic reference compound exhibited identical IR, NMR, and GC-mass spectra as the metabolite. The tentative pathway and the biological significance of dioxane metabolism are discussed. [ABSTRACT FROM AUTHOR]

The metabolism of sphingolipids in the central nervous system (CNS) has been studied in adult rats by intraventricular administration of fluorescent ceramide (CER). Rats were sacrificed at various time points post inoculation and the fluorescence of CER, cerebrosides (CB), sulfatides (SULF) and sphingomyelin (SPM) was determined in the CNS myelin and in the pellet, containing mainly microsomes, obtained by Norton myelin preparation. The incorporation of fluorescence was more in the pellet than in the myelin at all times studied. Initially the fluorescence present in the pellet was prevalently due to untransformed CER but an increase of fluorescent products with time was observed. CB was the main product up to 2 h post inoculation, then it decreased with concomitant increase of fluorescent SULF. In the myelin we did not observe differences in incorporation and transformation of fluorescent CER with time: CB was the main fluorescent product at all times studied. At 0.5 h post inoculation the fluorescence, observed by fluorescence microscope, was located in the cell lining the ventricles while after 24 h it appeared also in the paraventricular areas. [ABSTRACT FROM AUTHOR]