Search

Objectives: We have assessed the effect of elevated concentrations of hydroxyphenylpyruvic acid (HPPA), hydroxyphenyllactic acid (HPLA) and tyrosine, on a range of chemistry tests in serum and urine to explore the potential for chemical interference on routine laboratory analyses in patients with alkaptonuria (AKU) treated with nitisinone and similarly implications for patients with hereditary tyrosinemia type 1 (HT-1)., Materials and Methods: HPPA, HPLA and tyrosine were added separately to pooled serum from subjects without AKU in a range of assays with Roche Modular chemistries. Effects on urine were assessed by changes in urine strip chemistries after mixing a positive control urine with various amounts of the test compounds and reading on a Siemens urine strip meter., Results: No significant effect (p > 0.1) was observed up to 225 μmol/L of HPPA and HPLA, and up to 5000 μmol/L tyrosine, on any of the serum-based assays including those with peroxidase-coupled reaction systems of enzymatic creatinine, urate, total cholesterol, HDL cholesterol and triglyceride. Both the monohydroxy HPPA, and the dihydroxy homogentisic acid (HGA), at increased urine concentrations typical of nitisinone-treated AKU and non-treated AKU respectively, did however show marked negative interference in strip assays for glucose and leucocytes; i.e. those with peroxide-linked endpoints. The effect of increased HPLA was less marked., Conclusions: In patients with AKU or on nitisinone treatment and HT-1 patients on nitisinone, urine strip chemistry testing should be used sparingly, if at all, to avoid false negative reporting. It is recommended that urine assays should be organised with a suitable specialist laboratory., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Loxoprofen is a prodrug that exerts strong analgesic and anti-inflammatory effects through its active trans-alcohol metabolite, which is produced in the liver by carbonyl reductase. Previous metabolic studies have evaluated loxoprofen, but its sulfate and taurine conjugates have not yet been studied. We characterized the metabolomic profile of loxoprofen in rat plasma, urine, and feces using high-resolution mass spectrometry. We identified 17 metabolites of loxoprofen in the three different biological matrices, 13 of which were detected in plasma and feces and 16 in urine. Amongst these metabolites, two novel taurine conjugates (M12 and M13) and two novel acyl glucuronides (M14, M15) were identified for the first time in rats. In addition, we detected three novel sulfate conjugates (M9, M10, and M11) of loxoprofen. Further study of these metabolites of loxoprofen is essential in order to assess their potency and toxicity., (© 2019 John Wiley & Sons, Ltd.)

The focus of the present study is on in vitro and in vivo metabolite identification of ambrisentan (AMBR) a selective endothelin type - A (ETA) receptor antagonist using quadruple time-of-flight mass spectrometry (QTOF/MS). in vitro metabolism study was conducted by incubating AMBR in rat liver microsomes (RLM), rat and human liver S9 fractions. In vivo study was carried out through the collection of urine, faeces and plasma samples at various time points after oral administration of AMBR in suspension form at a dose of 25 mg/kg to six male Sprague - Dawley (SD) rats. The samples were prepared using an optimized sample preparation techniques involving protein precipitation (PP), freeze liquid extraction (FLE) and solid phase extraction (SPE). The extracted samples were further concentrated and analyzed by developing a sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method. A total of seventeen metabolites were identified in in vivo samples which includes hydroxyl, demethylated, demethoxylated, hydrolytic, decarboxylated, epoxide and glucuronide metabolites. Most of the metabolites were observed in faeces and urine matrices and few were observed in the plasma matrix. Only ten metabolites were identified in in vitro study which was commonly observed in in vivo study. The detailed structural elucidation of all the metabolites was done using UHPLC/QTOF/MS/MS in combination with accurate mass measurements. The toxicity profile of AMBR and its metabolites were predicted using TOPKAT software. In addition, a mass spectrometric method was developed for the detection and characterization of GSH-trapped reactive epoxide metabolitein human liver S9 fraction supplemented with glutathione (GSH) as trapping agent., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Loxoprofen is a non-steroidal anti-inflammatory drug of the 2-arylpropionic acid type, which has used to treat musculoskeletal disorders in the horse racing industry. However, it has also used illicitly to mask clinical signs of inflammation and pain in racehorses. Thus, its accurate analysis has become an important issue in horse doping laboratories. In this study, an analytical method of loxoprofen was developed as tert-butyldimethylsilyl (TBDMS) derivative by gas chromatography-mass spectrometry (GC-MS). Characteristic fragment ions of [M-15], [M-57], and [M-139] permitted the accurate and selective detection of loxoprofen. Under optimal conditions, this method showed good linearity (r ≥ 0.999) in the range of 10-500 ng/mL, repeatability (% relative standard deviation = 5.6-8.5), and accuracy (% relative error = - 0.3-0.9) with a detection limit of 1.0 ng. When applied to the analysis of loxoprofen in tablet and patch products, loxoprofen was positively identified as TBDMS derivative by GC-MS. The present method provided rapid and accurate determination of loxoprofen in patch and tablet products. Levels of loxoprofen were highest in equine urine at 0.5 and 1 h after oral administration with single dose (3 mg/kg) to three horses, and then rapidly reduced to below the lower limit of quantification at 24 h. Therefore, the present method will be useful for the pharmacokinetic study and doping tests for loxoprofen and other similar acidic drugs in horses.

Background: In severe falciparum malaria metabolic acidosis and acute kidney injury (AKI) are independent predictors of a fatal outcome in all age groups. The relationship between plasma acids, urine acids and renal function was investigated in adult patients with acute falciparum malaria., Methods: Plasma and urinary acids which previously showed increased concentrations in proportion to disease severity in patients with severe falciparum malaria were quantified. Patients with uncomplicated malaria, sepsis and healthy volunteers served as comparator groups. Multiple regression and multivariate analysis were used to assess the relationship between organic acid concentrations and clinical syndromes, in particular AKI., Results: Patients with severe malaria (n = 90), uncomplicated malaria (n = 94), non-malaria sepsis (n = 19), and healthy volunteers (n = 61) were included. Univariate analysis showed that both plasma and creatinine-adjusted urine concentrations of p-hydroxyphenyllactic acid (pHPLA) were higher in severe malaria patients with AKI (p < 0.001). Multiple regression analysis, including plasma or creatinine-adjusted urinary acids, and PfHRP2 as parasite biomass marker as independent variables, showed that pHPLA was independently associated with plasma creatinine (β = 0.827) and urine creatinine (β = 0.226). Principal component analysis, including four plasma acids and seven urinary acids separated a group of patients with AKI, which was mainly driven by pHPLA concentrations., Conclusions: Both plasma and urine concentrations of pHPLA closely correlate with AKI in patients with severe falciparum malaria. Further studies will need to assess the potential nephrotoxic properties of pHPLA.

Scope: Most studies on the role of whole grain for health rely on self-reported intake data, which are prone to measurement errors. There is a need for dietary biomarkers that can provide an objective measure of intake. Alkylresorcinols (AR) and their main metabolites 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA) have been proposed as biomarkers for whole grain (WG) wheat and rye intake., Methods and Results: The medium-term reproducibility and relative validity of four putative urinary AR metabolites (3,5-dihydroxycinnamic acid (DHCA), 5-(3,5-dihydroxyphenyl) pentanoic acid (DHPPTA), 2-(3,5-dihydroxybenzamido)acetic acid (DHBA-glycine) and 3,5-dihydroxycinnamic acid amide (DHCA-amide)) as biomarkers for WG intake were investigated. Three-day weighed food records and 24-h urine samples from two occasions 2-3 months apart were obtained from 69 Swedish adults. WG intake was calculated and urinary AR metabolites were analyzed. The medium-term reproducibility determined for DHCA, DHPPTA, and DHBA-glycine varied from moderate-to-excellent (intra-class correlation coefficient = 0.63-0.85). Moreover, DHCA and DHPPTA excretion correlated well with self-reported total WG intake (r = 0.55, p < 0.001 and r = 0.42, p < 0.001, respectively)., Conclusion: DHCA or DHPPTA excretion in 24-h urine might be a suitable medium- to long-term biomarker of WG wheat and rye intake. These findings need to be confirmed in populations with low and infrequent WG intake., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Alkylresorcinols (AR) are phenolic lipids present in the bran of wheat and rye. Plasma AR and their urinary metabolites may be suitable biomarkers of whole-grain (WG) wheat and rye consumption. The objective of this study was to examine plasma AR and urinary AR metabolites in response to WG wheat consumption., Methods: In a randomized crossover study, 19 subjects (10 males, 9 females; BMI 22.0 kg/m(2); age 26 years) incorporated either 3 servings (48 g) or 6 servings (96 g) of WG wheat daily into their regular diet for 1 week. Subjects completed a 2-week washout period, abstaining from all WG consumption, before each intervention. Fasting blood and 24-h urine were collected before and after each intervention. Plasma AR homologues (C19:0, C21:0, C23:0) were quantified by GC-MS after diethyl ether and solid phase extraction and derivatization. Urinary AR metabolites [3,5-dihydroxybenzoic acid and 3-(3,5-dihydroxyphenyl)-propanoic acid] were determined using HPLC with electrochemical detection after enzymatic deconjugation and ethyl acetate extraction., Results: Urinary total AR metabolites were significantly higher after 6 compared with 3 servings of WG wheat (56 vs. 32 μmol/day, P < 0.001). This dose-response relationship was independent of age, sex, energy intake, and baseline urinary AR metabolite concentration. Plasma total AR tended to be higher after 6 compared with 3 servings of WG wheat (103.0 vs. 86.9 nmol/L), but this difference was not significant (P = 0.42)., Conclusion: The results suggest that urinary AR metabolites from 24-h urine collections may be useful as biomarkers of compliance in intervention studies of WG wheat.

Autism spectrum disorders (ASDs) are a group of mental illnesses highly correlated with gut microbiota. Recent studies have shown that some abnormal aromatic metabolites in autism patients are presumably derived from overgrown Clostridium species in gut, which may be used for diagnostic purposes. In this paper, a GC/MS based metabolomic approach was utilized to seek similar biomarkers by analyzing the urinary information in 62 ASDs patients compared with 62 non-ASDs controls in China, aged 1.5-7. Three compounds identified as 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), 3-hydroxyphenylacetic acid (3HPA), and 3-hydroxyhippuric acid (3HHA) were found in higher concentrations in autistic children than in the controls (p < 0.001). After oral vancomycin treatment, urinary excretion of HPHPA (p < 0.001), 3HPA (p < 0.005), and 3HHA (p < 0.001) decreased markedly, which indicated that these compounds may also be from gut Clostridium species. The sensitivity and specificity of HPHPA, 3HPA, and 3HHA were evaluated by receiver-operating characteristic (ROC) analysis. The specificity of each compound for ASDs was very high (>96%). After two-regression analysis, the optimal area under the curve (AUC, 0.962), sensitivity (90.3%), and specificity (98.4%) were obtained by ROC curve of Prediction probability based on the three metabolites. These findings demonstrate that the measurements of the three compounds are strong predictors of ASDs and support the potential clinical utility for identifying a subgroup of ASDs subjects.

Alkylresorcinols (ARs) are phytochemicals mainly associated with rye/wheat bran. Plasma ARs and their plasma and urine metabolites are considered as biomarkers for whole-grain rye/wheat intake. However ARs metabolite day and night variations have not been studied in prostate cancer patients yet. We investigated ARs metabolites 3, 5-dihydroxy-benzoic acid (DHBA), and 3-(3, 5-dihydroxyphenyl)-1-propanoic acid (DHPPA) in urine and plasma in prostate cancer patients and in control group. DHPPA in 12-h overnight urine correlated with the intake of rye bread and bread fiber across short time periods (3 days). Plasma DHPPA concentration was significantly greater in the prostate cancer group than in the control group. DHPPA and DHBA excretion was significantly higher in the overnight urine than in day urine in the prostate cancer group but not in the control group. DHPPA concentration in plasma in the prostate cancer group did not depend on the intake of rye bread in the previous day, suggesting an impaired metabolism of ARs metabolites in the prostate cancer group. The results of this study suggest DHPPA in 12-h overnight urine as a biomarker to estimate the intake of rye bread and bread fiber.

Biomarkers of dietary intake are prominent tools in nutritional research. The alkylresorcinol metabolites 3,5-dihydroxybenzoic acid (3,5-DHBA) and 3-(3,5-dihydroxyphenyl)propanoic acid (3,5-DHPPA) have been proposed as exposure biomarkers of whole-grain (WG) wheat and rye intake. However, the profile of alkylresorcinol metabolites is not fully understood. The aim of this study was to investigate the metabolism of alkylresorcinols in mice and in humans, while further determining urinary pharmacokinetics of the novel alkylresorcinol metabolites to explore their potential as biomarkers of WG wheat intake. Utilization of the liquid chromatography-mass spectrometry approach resulted in 10 alkylresorcinol metabolites identified in mice and in humans, including 3 phenolic acids and 7 of their phase II conjugates. Among them, 2 novel metabolites were discovered: 5-(3,5-dihydroxyphenyl)pentanoic acid (3,5-DHPPTA) and 2-(3,5-dihydroxybenzamido)acetic acid (3,5-DHBA glycine). The structures of these 2 metabolites were confirmed by comparing with authentic standards synthesized in-house. In the pharmacokinetic study, a group of 12 volunteers consumed a polyphenolic-restricted diet for 4 d before ingesting WG wheat bread containing 61 mg of alkylresorcinols. Urine samples were collected for 32 h, and alkylresorcinol metabolites were quantified with HPLC-coulometric electrode array detection. The mean urinary excretion rates and mean apparent half-life of 3,5-DHPPTA, 3,5-DHBA glycine, 3,5-DHBA, and 3,5-DHPPA at each time point were determined. Our results suggest that 3,5-DHPPTA and 3,5-DHBA glycine may be used in combination with 3,5-DHBA and 3,5-DHPPA as potential biomarkers to increase the accuracy of recording WG wheat and rye intake in epidemiologic studies. Further validation of 3,5-DHPPTA and 3,5-DHBA glycine as potential biomarkers is warranted.

The aim of this study was to investigate the relation between the etiology of late-onset childhood autism and anaerobic bacteria. Thirty children diagnosed with autistic disorder and control group have been included in the study. 3-(3-hydroxy phenyl)-3-hydroxypropionic acid (HPHPA) excretion rates which is a metabolic product of the genus Clostridium, were measured via mass spectrometry-gas chromatography (MS-GC) method from urine samples. When the assayed average HPHPA values compared with each group, a statistically significant difference was found (p < 0.05). Data obtained from this study support the existence of a significant correlation between autism etiology and anaerobic bacteria.

Scope: Non-targeted urine metabolite profiling has not been previously exploited in the field of whole grain (WG) products. WG products, particularly rye, are important elements in a healthy Nordic diet. The aim of this study was to identify novel urinary biomarkers of WG rye bread (RB) intake in a randomised crossover study with RB versus refined wheat bread (WB)., Methods and Results: UPLC-QTOF/MS metabolite profiling was applied to urine from a 2 × 4 wk crossover intervention with RB versus WB in 20 subjects. Sixteen metabolites were revealed as major contributing biomarkers. The most discriminative metabolite after the cereal intervention was identified as 3-(3,5-dihydroxyphenyl)-1-propanoic acid sulphate, which was excreted to a higher extent after the RB versus WB intervention. Other alkylresorcinol metabolites were identified, as well as enterolactone glucuronide, azelaic acid, 2-aminophenol sulphate and its benzoxazinoid precursor 2,4-dihydroxy-1,4-benzoxazin-3-one. Our study also suggests that nitrogen-containing metabolites are other major markers. However, other methodologies will be needed to elucidate their final structure., Conclusion: The present non-targeted metabolite profiling proved to be a useful approach to identify major urine metabolites discriminating RB intake from that of white wheat bread. Once validated these markers could help evaluate compliance to healthy Nordic diets., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) is key in tyrosine catabolism. Inhibition of HPPD results in tyrosinemia and increased urinary excretion of 3 phenylketones: 4-hydroxyphenylpyruvate (HPPA), 4-hydroxyphenyllactate (HPLA), and 4-hydroxyphenylacetate (HPAA). A previous study involving administration of a novel HPPD inhibitor to dogs resulted in detection of ketonuria in treated animals using urine dipsticks read by reflectance photometry. Dipstick-positive results were suspected to be false because high concentrations of urinary phenylketones have been reported to react with ketone test fields of urine dipsticks, but visual confirmation was not performed., Objective: The purpose of this study was to determine which of the 4- hydroxyphenolic acids produced by HPPD inhibition react with ketone test fields of 3 commercially available urine dipsticks., Methods: Canine urine samples were prepared with HPPA, HPLA, HPAA, and lithium acetoacetate (positive control) at 6 concentrations. Unmodified urine samples were used as negative controls. All samples were tested for ketones using Combur 10 Test M dipsticks read by a Miditron dipstick analyzer. Urinalysis was also performed by visually inspecting ketone test fields on the Combur 10 Test M, Multistix 10 SG, and Aution 10 EA dipsticks., Results: Urine samples containing HPPA were positive for ketones with Combur 10 Test M dipsticks read by the Miditron analyzer and produced a red–brown color change in ketone test fields of all 3 dipsticks. Urine samples containing HPLA and HPAA were negative by all methods., Conclusion: The phenylketone HPPA reacts with ketone test fields of 3 commercially available urine dipsticks, producing a red–brown color change that may be misinterpreted as positive for ketones by reflectance photometry.

A compound identified as 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) was found in higher concentrations in urine samples of children with autism compared to age and sex appropriate controls and in an adult with recurrent diarrhea due to Clostridium difficile infections. The highest value measured in urine samples was 7500 mmol/mol creatinine, a value 300 times the median normal adult value, in a patient with acute schizophrenia during an acute psychotic episode. The psychosis remitted after treatment with oral vancomycin with a concomitant marked decrease in HPHPA. The source of this compound appears to be multiple species of anaerobic bacteria of the Clostridium genus. The significance of this compound is that it is a probable metabolite of m-tyrosine (3-hydroxyphenylalanine), a tyrosine analog which depletes brain catecholamines and causes symptoms of autism (stereotypical behavior, hyperactivity, and hyper-reactivity) in experimental animals.

Background: Hereditary tyrosinemia type 1 (HT1; MIM 276700) is caused by mutations in the fumarylaceto-acetate hydrolase (FAH) gene, and is the most severe disorder associated with the tyrosine catabolic pathway. HT1 is a very rare disorder and no genetically confirmed case of HT1 in Korea has yet been reported. In this study, we present a Korean neonate with clinical and biochemical features of HT1., Methods: A female neonate was admitted to our hospital for further work-up of an abnormal newborn screening test. We analyzed amino acids and organic acids in the patient's blood and urine. To confirm the presence of the genetic abnormality, all the coding exons of the FAH gene and the flanking introns were amplified by polymerase chain reaction (PCR)., Results: The patient's newborn screening test revealed increased concentrations of methionine and tyrosine. Subsequent urine organic acid analysis showed increased urinary excretion of 4-hydroxyphenyllactate, 4-hydroxyphenylpyruvate, succinate, and succinylacetone. Gap-PCR and sequence analysis of the FAH gene revealed a homozygous large deletion mutation encompassing exons 12-14. The patient's parents were not consanguineous but were heterozygous carriers of the same mutation., Conclusions: The patient had a novel, large deletion mutation of FAH and is the first report of genetically confirmed HT1 in Korea.

In vivo and in vitro metabolism of scopolamine is investigated using a highly specific and sensitive liquid chromatography-mass spectrometry (LC-MSn) method. Feces, urine, and plasma samples are collected individually after ingestion of 55 mg/kg scopolamine by healthy rats. Rat feces and urine samples are cleaned up by a liquid-liquid extraction and a solid-phase extraction procedure (C18 cartridges), respectively. Methanol is added to rat plasma samples to precipitate plasma proteins. Scopolamine is incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubating solution are extracted with ethyl acetate. Then these pretreated samples are injected into a reversed-phase C18 column with mobile phase of methanol-ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MSn system. Identification and structural elucidation of the metabolites are performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MSn spectra with those of the parent drug. The results reveal that at least 8 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, and hydroxyscopolamine N-oxide) and the parent drug exist in feces after administering 55 mg/kg scopolamine to healthy rats. Three new metabolites (tetrahydroxyscopolamine, trihydroxy-methoxyscopolamine, and dihydroxy-dimethoxyscopolamine) are identified in rat urine. Seven metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, and hydroxyscopolamine) and the parent drug are detected in rat plasma. Only 1 hydrolyzed metabolite (scopine) is found in the rat intestinal flora incubation mixture, and 2 metabolites (aposcopolamine and norscopolamine) are identified in the homogenized liver incubation mixture.

Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0-3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 microl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3M Na(2)SO(4), pH 2.0-3.5; organic membrane: 95 microl of 1-hexanol; acceptor solution: 4.0 microl of 0.1M NH(3)/NH(4)(+) with pH 11.1; donor solution temperature: 45-50 degrees C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1-5000 microg/l with r(2)>0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 microg/l (based on S/N=3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.

Naveglitazar [LY519818; benzenepropanoic acid, alpha-methoxy-4-[3-(4-phenoxyphenoxy)propoxy], (alpha-S)-] is a nonthiozolidinedione peroxisome proliferator-activated receptor alpha-gamma dual, gamma-dominant agonist that has shown glucose-lowering potential in animal models and in the clinic. Studies have been conducted to characterize the disposition, metabolism, and excretion of naveglitazar in mice, rats, and monkeys after oral and/or i.v. bolus administration. After oral administration of [(14)C]naveglitazar, naveglitazar was well absorbed and moderately metabolized in all species evaluated, with total recoveries of radioactivity ranging from 90 to 96%. Naveglitazar was the most abundant peak observed in circulation at C(max), representing 68 to 81% of the total radioactivity in plasma. The most prominent metabolite observed in circulation was the R-enantiomer of naveglitazar, LY591026, which is formed via enzymatic chiral inversion. para-Hydroxy naveglitazar and the sulfate conjugate of para-hydroxy naveglitazar were also observed in circulation in most species, especially in the monkey. The metabolic pathways observed include enzymatic chiral inversion, aromatic hydroxylation, oxidative dehydrogenation, and/or various phase II conjugation pathways. Naveglitazar was highly bound to plasma proteins among the species examined (>99%), and binding was independent of concentration. Biliary excretion was recognized as the most prominent excretion pathway in bile duct-cannulated rats (79 of the 96% recovered), producing an acyl glucuronide conjugate of naveglitazar and a sulfate and glucuronide diconjugate of para-hydroxy naveglitazar, which were shown to be reversible. The primary excretory pathway observed in mice and monkeys was via the feces. In summary, naveglitazar was well absorbed, moderately metabolized, and excreted via the feces in mice, rats, and monkeys.

The effect of pretreatment (i.e., oral administration of loxoprofen for 3 consecutive days followed by a 7-day washout) on the pharmacokinetics and metabolism of the drug was studied in humans. In a control study, a Loxonin tablet (60 mg as loxoprofen anhydrous) was administered orally to 6 healthy male Korean subjects. In a pretreatment study, a Loxonin tablet was administered orally to the subjects once daily for 3 consecutive days. On the 10(th) day, a Loxonin tablet was administered orally to the subjects, and the concentrations of loxoprofen and the trans- and cis-alcohol metabolites in the plasma and urine were measured as a function of time. Using this pretreatment, the area under the curve (AUC) of the trans-alcohol metabolite of loxoprofen in the plasma, but not those of loxoprofen and the cis-alcohol metabolite, was increased (1.5-fold, p < 0.05), leading to increased contribution of the trans-alcohol metabolite to the total urinary recovery of loxoprofen (1.3-fold, p < 0.05). The urinary recovery of total metabolites, which was largely (> 90%) comprised of conjugate metabolites, was also increased as a result of the pretreatment (1.5-fold, p < 0.05). These results indicate that stereoselective reduction to trans-alcohol metabolites as well as the phase II metabolism of loxoprofen may be increased by such a pretreatment in human subjects., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:973-979, 2002)

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.

We determined urinary galactose and 4-hydroxyphenyllactic acid (4HPLA) in 4338 of 5-day-old newborns using a newly developed GC-MS screening method. Fifty-two infants were chemically diagnosed as having transient galactosuria based upon elevated urinary galactose levels (4.78-30.53 mg/mg creatinine, control 1.10 +/- 0.89 mg/mg creatinine). These infants did not excrete galactitol or galactonic acid into the urine, which is typical of hereditary galactosemia. Nearly 40% of the transient galactosuria was associated with immature infants (low birth weight or borne before 37 gestational weeks). Immature hepatic function is one explanation for neonatal transient galactosuria, but heterozygotes or the carriers of galactose degradation enzyme deficiencies were also suspected in some of the newborns, judging from the comparisons of urinary galactose and 4HPLA excretion between neonates and patients with galactosemia.

Orally active nonpeptide antagonists of endothelin (ET) receptors may prove beneficial in the treatment of cardiovascular and renal disease. The pharmacodynamics and pharmacokinetics of these drugs are not sufficiently known, and practical methods for their analysis have not been developed. We describe a simple, sensitive, and reproducible radioreceptor assay (RRA) for LU135252, a selective antagonist of the ETA receptor, using porcine aortic smooth muscle membranes as the acceptor and 125I-endothelin-1 as the ligand. With methanol extraction of plasma and urine samples, recovery of LU135252 ranged from 79% to 91% at 60-1000 nmol/L. The logit-log transformed calibration curves constructed with LU135252 added to plasma or to urine were linear (r = 0.993 +/- 0.005, n = 11) in the range from 18.7 to 2400 nmol/L. The detection limit with plasma- and urine-based calibration curves was 19 nmol/L. The interassay coefficient of variation was 12.6% at 70 nmol/L (n = 9) and 6.5% at 590 nmol/L (n = 9). Endothelin-1 did not interfere in the RRA at pathophysiologically and clinically relevant concentrations [up to 15 pmol/L (40 pg/ mL)]. When LU135252 was added to plasma, the signal was completely stable after storage for 1 week at 4 degrees C, although there was a modest loss of the signal after 24 h at room temperature. The practical performance of this RRA was then tested in plasma samples obtained from (a) rats after a single oral administration of LU135252, (b) from coronary-ligated rats chronically treated with LU135252, and (c) in plasma and urine samples obtained from dogs during intrarenal infusion of LU135252.

We describe biochemical and clinical features of 11 subjects (ages, 1.2-84 years, nine females and two males) with transient 5-oxoprolinuria (0.6-23.6 mol/mol of creatinine, reference range <0.07). A variety of conditions preceded the onset of acidosis, and all had taken acetaminophen (paracetamol), although in therapeutic amounts in most subjects. Metabolic acidosis was documented in nine subjects, and all had an increased anion gap and abnormal liver functions. 5-Oxoproline was the major urinary organic acid in five subjects, whereas the rest had more complex profiles comprising 5-oxoproline and other organic acids, such as lactate, 3-hydroxybutyrate, and 4-hydroxyphenyl lactate. The 5-oxoproline was predominantly of the L-configuration. One subject died during an acidotic episode, and the rest recovered with no apparent long-term ill effects. Urinary 5-oxoproline was within the reference range in six subjects that were re-tested after the anion gap normalized. These findings suggest that acetaminophen, in association with other unidentified factors, is involved in the development of this condition through a mechanism of depletion of liver glutathione stores.

Two high-performance liquid chromatographic methods for the stereoselective determination of R,S-2-[4-(3-methyl-2-thienyl)-phenyl]propionic acid (R,S-MTPPA), a new anti-inflammatory agent, and its taurine conjugates (R,S-MTPPA-TAU) in dog urine have been developed and validated. The urine samples were subjected to solid extraction or TLC preparation, then R,S-MTPPA and R,S-MTPPA-TAU were separated on Chiralcel OD and Chiral AGP columns, respectively, with ultraviolet absorbance detection at 272 nm. The dose-response relationships for enantiomers of R,S-MTPPA and R,S-MTPPA-TAU were linear in the concentration ranges of 0.5-50 (r>0.9993) and 5-200 microg/ml (r>0.9982), respectively. Recoveries of all tested enantiomers from dog urine were roughly 90% within the above concentration ranges. Intra- and inter-day reproducibilities were sufficient for metabolic studies. These methods were applied to stereoselective determination of the enantiomers in dog urine after administration of either S- or R-MTPPA.

The disposition of S-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (CAS 155680-07-2, S-MTPPA, code: M-5011) was studied after oral administration to rats, dogs and monkeys using the 14C-labeled drug. After oral dosing, S-MTPPA was well absorbed from the gastrointestinal tract, to the extent of 97.7% in rats. The concentration of S-MTPPA in rat plasma reached a peak (Cmax: 13.07 micrograms/ml) at 15 min (tmax) after dosing and declined with a half-life (t1/2) of 2.5 h. The values of the parameters tmax, Cmax and t1/2 for dogs were 30 min, 26.2 micrograms/ml and 7.0 h, and those for monkeys were 15 min, 12.8 micrograms/ml and 3.0 h, respectively. The radioactivity was widely distributed in tissues and almost completely excreted in urine and feces within 48 h after oral administration to rats. The excretion of radioactivity in bile, urine and feces within 48 h after oral administration of 14C-S-MTPPA to bile duct-cannulated rats amounted to 75.0, 18.6 and 1.4% of the dose, respectively. The drug was metabolized mainly by oxidation of the thiophenyl moiety and by glucuronidation of the carboxyl group in rats and monkeys. The major urinary and fecal metabolite in dogs was identified as the taurine conjugate of MTPPA.

Capillary electrophoresis (CE) equipped with a diode-array detector have been used to determine diagnostic metabolites occurring in urine of patients with various diseases. The urine samples were injected directly onto the CE instrument without any pretreatment. Identification of abnormal metabolites was based on relative migration times and characteristic diode-array spectra. The CE-method readily diagnosed alkaptonuria, neuroblastoma and liver failure due to galactosemia. The simple and automated CE method appears to be suitable for implementation as a screening test in analytical systems aimed at diagnosis of metabolic disorders.

A TLC method for the simultaneous analysis of vanillylmandelic, homovanillic and 5-hydroxyindole-3-acetic acids in urine is described. The sample is cleaned up through a cyano minicolumn and extracted with diethyl ether. The acids are resolved by high-performance TLC, visualized by Folin Ciocalteau reagent and quantificated by densitometry at 600 nm with beta-(4-hydroxy-3-phenyl) acetic acid as the internal standard.

A sensitive gas chromatographic procedure with nitrogen-phosphorus detection was developed for the quantitative determination of 3-cyano-3,3-diphenylpropionic acid, the acidic metabolite of the narcotic analgesic bezitramide (Burgodin). Gas chromatography with mass spectrometric detection was used for confirmation. An internal standard, 5-cyano-5,5-diphenylvaleric acid, was synthesized and purified in our laboratory. The compound was extracted from 5 mL of hydrolyzed urine with n-hexane-ethyl acetate (85:15, v/v). Derivatization of the extract with an ethereal diazomethane solution was performed immediately before chromatographic analysis. Under the described conditions, the quantitation limit for 3-cyano-3, 3-diphenylpropionic acid in urine was 10 ng/mL. The extraction recovery was 82%. The calibration graph was linear over the concentration range from 10 to 500 ng/mL; at a 100-ng/mL concentration, within-day and day-to-day percent coefficients of variation of 1.9 and 3.7%, respectively, were obtained. Urine samples from 16 persons suspected of using bezitramide were analyzed, and they revealed metabolite concentrations that ranged from 10.5 to 88 ng/mL.

CS-670(I), being developed as a non-steroidal anti-inflammatory agent, is a racemic prodrug. It has been found to be readily metabolized to active metabolites: trans and unsaturated mono-ols (trans-OH, unsaturated-OH). We report here a method for the quantitative determination of the eight diol stereoisomers excreted in urine after administration I. The diols were well separated and quantitated using capillary column GC-MS after a rather simple derivatization with diazomethane-trifluoroacetic anhydride. Sex differences in rats and species differences between rats and mice were observed in the metabolism of I: the trans-diols originating from trans-OH were predominantly excreted in male and female rat urine but the excretion rate was greater in the male rats; the cis-diols originating from cis mono-ol (cis-OH) were the major urinary metabolites in mice. The hydroxy groups were mainly introduced at the respective equatorial hydrogen atoms at the 4'-carbon of trans-OH and the 5'-carbon of cis-OH. The 4'- and 5'-hydroxy groups in the diols were in the cis conformation with respect to the original 2'-hydroxy group. As approximately 9% of the trans-diols were excreted in urine after administration of cis-OH to rats, the chiral inversion from cis-OH to trans-OH was suggested to occur through the saturated ketone intermediate.

The metabolism of [3-14C/phenyl-2H5] cinnamic acid was investigated in rats and mice at a dose level of 2.5 mmol/kg body weight. Recoveries of the 14C dose were between 92 and 98% with most (82-90%) present in the 0-24 hr urine samples. Urinary metabolites were identified by their chromatographic properties and mass spectra. In both species the major metabolite was hippuric acid, which is also an endogenous urinary component. Several minor metabolites, 3-hydroxy-3-phenylpropionic acid, benzoic acid and benzoyl glucuronide, were found in both species. Two, acetophenone and cinnamoylglycine, the glycine conjugate of cinnamic acid, could be positively identified only in mouse urine. The effect of dose size on the urinary excretion of 14C-cinnamic acid metabolites was studied over the dose range 0.0005 to 2.5 mmol/kg. In the rat the pattern of metabolite excretion was very similar over the whole dose range with slight increases in the proportion of the dose excreted as minor metabolites as dose size increased. In the mouse the excretion of cinnamoylglycine was more important at the lowest dose level and decreased in relative importance as dose size increased. Changes in the other metabolites were similar to those seen in the rat.

The metabolism of trans-[3-14C]cinnamaldehyde was investigated in male and female Fischer 344 rats and CD1 mice at doses of 2 and 250 mg/kg body weight given by ip injection and in males at 250 mg/kg by oral gavage. Some 94% of the administered dose was recovered in the excreta in 72 hr in both species with most (75-81%) present in the 0-24-hr urine. Less than 2% of the administered dose was found in the carcasses at 72 hr after dosing. Urinary metabolites were identified by their chromatographic characteristics. In both species the major urinary metabolite was hippuric acid accompanied by 3-hydroxy-3-phenylpropionic acid, benzoic acid and benzoyl glucuronide. The glycine conjugate of cinnamic acid was formed to a considerable extent only in the mouse. The oxidative metabolism of cinnamaldehyde essentially follows that of cinnamic acid, by beta-oxidation analogous to that of fatty acids. Apart from the metabolites common to cinnamic acid and cinnamaldehyde, 7% of 0-24-hr urinary 14C was accounted for by two new metabolites in the rat and three in the mouse, which have been shown in other work to arise from a second pathway of cinnamaldehyde metabolism involving conjugation with glutathione. The excretion pattern and metabolic profile of cinnamaldehyde in rats and mice are not systematically affected by sex, dose size and route of administration. The data are discussed in terms of their relevance to the safety evaluation of trans-cinnamaldehyde, particularly the validity or otherwise of extrapolation of toxicity data from high to low dose.

The concentration of several phenolic acids and alcohols was measured in urine from germ-free and specific pathogen-free (SPF) rats before and after inoculation with faecal microorganisms, and from conventional rats before and after gut sterilization. The rate of excretion of benzoic acid, phenylacetic acid, and m- and p-hydroxyphenylpropionic acid in the germ-free animals was markedly increased after inoculation. Some acids showed no increase, including the endogenously generated homovanillic, vanilmandelic and p-hydroxyphenyllactic acids. Most others sought showed a small but significant increase. Some of the compounds excreted by the germ-free animals may have been in the food pellets, either as such or as precursors. The pattern was somewhat different in the SPF rats. The excretion of p-hydroxyphenylpropionic, p-hydroxyphenylacetic and m-hydroxyphenylacetic acids was initially much higher than in the germ-free animals and their excretion decreased after inoculation, presumably because of an altered pattern of gut flora. This work quantifies the effect of gut flora in the formation of some of the more important phenolic acids found in rat urine.

A study was conducted to investigate the relationship between vitamin C intake and the rate of tyrosine catabolism in premature babies. A 13C tyrosine breath test was developed for the measurement of tyrosine catabolism. Premature babies were randomly allocated to receive a daily intake of vitamin C which ranged from 8 to 100 mg/kg body weight, for 5 days. Tyrosine catabolism was measured at the beginning and the end of this period. Daily intakes of vitamin C of 20 mg/kg or more elicited a greater increase in tyrosine catabolism over 5 days than 8 mg/kg/day. The magnitude of the difference, in terms of percentage of tyrosine metabolised, was, however, small and of doubtful biological significance. Vitamin C intakes above 20 mg/kg/day had no further measurable effect on the catabolism of tyrosine.

A new metabolite of felbamate, isolated from the polar metabolite fraction of a human urine sample, was identified as 3-carbamoyloxy-2-phenylpropionic acid (CPPA) by electron impact and CI/MS of the methyl ester in the isolated fraction. Its presence in the unconjugated free form in two different human urine samples was confirmed by HPLC comparison of retention times with authentic synthetic CPPA and by on-line negative ion HPLC thermospray tandem mass spectrometric techniques. The amount of CPPA in 0-4 hr postdose human urine was estimated to be approximately 12% of the drug-derived substances present in the urine. Some CPPA was also found to be present in dog urine.

The urinary excretion of metabolites of orally administered phenylpropionic acid (PPA) in 72 children, aged 2 days to 16 years, thought to be at-risk of medium acyl CoA dehydrogenase deficiency has been studied. Forty had presented as Reye Syndrome, 9 as a Reye-like syndrome and 24 were sibs of decreased RS, sibs of RLS cases or sibs of infants who had died suddenly and without explanation where an autopsy revealed the presence of very heavy fatty infiltration of the liver. These studies demonstrated that PPA metabolites are maximally excreted during the 3 hours following the oral load and that this urine collection should be diagnostic. PPA loading is a relatively simple, safe test which is part of the investigation of a patient suspected of having an inborn error of metabolism.

A stereospecific high-performance liquid chromatographic method was developed for the assay of pirprofen enantiomers in rat plasma and urine. Following addition of internal standard (ketoprofen) and acidifier (L-ascorbic acid) to biological fluids, pirprofen was extracted into an isopropanol-isooctane (5:95) mixture. Diastereomers of pirprofen enantiomers, which were formed using L-leucinamide, were separated on a reversed-phase column with ultraviolet detection at 275 nm using 0.06 M KH2PO4-acetonitrile-triethylamine (64:36:0.1) as mobile phase. The limit of quantitation was 0.1 microgram/ml for each enantiomer, based on 100 microliters of rat plasma. No spontaneous oxidation of pirprofen to its pyrrole metabolite occurred during sample preparation and analysis. In three female rats which were dosed with 10 mg/kg racemic pirprofen orally, plasma concentrations of the enantiomers could be followed for 24 h. Pirprofen enantiomers in plasma were virtually unconjugated, and negligible concentrations of pyrrole metabolites were observed. Less than 10% of the total dose was recovered in urine as intact drug and its glucuroconjugates. The assay was found suitable for the study of the pharmacokinetics of pirprofen enantiomers in the rat.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.

The metabolism of the anticholinergic drug aprophen was studied in rats after oral administration via stomach intubation. beta-Hydroxyethylaprophen, a major urinary metabolite of aprophen, was isolated and identified by normal-phase high-performance liquid chromatography and electron ionization mass spectrometry. More than 22% of the parent drug was recovered and quantified over a 72-h collection period. Results show that 2,2-diphenylpropionic acid, another major metabolite of aprophen which lacks anticholinergic properties, was also isolated and identified in this study. Experiments are currently underway to synthesize and test the anticholinergic properties of beta-hydroxyethylaprophen in mammals.

A simple, sensitive and selective method for the determination of ximoprofen and its keto and hydroxy metabolites in human urine has been developed using high-performance liquid chromatography in the reversed-phase mode. The limit of reliable determination of ximoprofen and each of its metabolites in urine is about 1 microgram/ml (4 nmol/ml). The method has been applied to urine samples obtained from human volunteers after administration of single intravenous doses of 30 mg of ximoprofen and about 70% dose was accounted for in terms of these compounds and their glucuronic acid conjugates.

The enantiomers of 2-phenylpropionic acid and four congeneric anti-inflammatory drugs were separated as their diastereoisomeric amides with S-(-)-1-(naphthen-1-yl)ethylamine by high-performance liquid chromatography using a silica-packed radial compression cartridge. The order of elution of the diastereoisomeric amides was always R, S or -, S before S,S or +,S. The conditions for the derivatization, using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide as coupling agent, were optimized, and it was found that the addition of 1-hydroxybenzotriazole rendered the reaction quantitative. Good calibration curves were obtained for the quantitation and determination of the enantiomeric composition of 2-phenylpropionic acid in urine, and the application of the method to the study of the metabolism of this acid in vivo is described.

Plasma and urine concentrations of 2-(3-chloro-4[3-pyrrolinyl]phenyl) propionic acid, pirprofen, a new nonsteroidal anti-inflammatory compound, are described for normal male volunteers receiving one or more doses of the drug. Orally administered pirprofen is rapidly and almost completely absorbed from the gastrointestinal tract, resulting in maximum plasma levels in 1 to 2 hr. Mean peak levels are 23 microng/ml after an oral pirprofen dose of 200 mg; lower doses given proportionally lower levels. Administration 1 hr after a meal slightly delays the peak plasma level, but the extent of absorption is not affected significantly. Administration of pirprofen, 150 mg, 4 times daily, or 200 mg, 3 times daily, results in nearly identical plasma levels at steady-state. Pirprofen has an apparent elimination half-life of about 7 hr. The results obtained from a 200-mg pirprofen-14C dose indicate that excretion of the drug occurs primarily by the renal route in the form of metabolites and is essentially complete within 24 hr. In urine, less than 5% of the administered dose is accounted for as unchanged drug.

A study on chickens was conducted to investigate whether or not: a) excess dietary tyrosine increases the content of tyrosine metabolites in plasma and excreta, b) these elevations of tyrosine metabolites are presented by increasing dietary protein level or supplementing with ascorbic acid (AA), and c) urine is a major excretory route of tyrosine metabolites. Chicks fed a 10% protein diet with excess tyrosine developed external foot lesions accompanied by retarded growth and depressed feed intake. These adverse effects were alleviated by elevating dietary protein level or supplementing with AA. Excreta and plasma of chicks fed the 10% protein diet contained small or undetectable amounts of free tyrosine, 4-hydroxyphenylpyruvate (4-HPP), 4-hydroxyphenylacetate (4-HPL), and 4-hydroxyphenylacetate (4-HPA), while these metabolites were markedly increased by the addition of excess tyrosine to the 10% protein diet. From the results with colostomized cocks, the major source of 4-HPP, 4-HPL, and 4-HPA excreted by chicks fed a tyrosine excess diet was considered more likely to be of urinary than fecal origin. Elevated contents of tyrosine and its metabolites in plasma were partially counteracted by increasing dietary protein level or AA supplementation. In excreta, elevated contents of tyrosine and its metabolites caused by excess tyrosine were reduced by increasing dietary protein level and supplementing with AA when expressed in the proportion of tyrosine intake. These results suggest that the beneficial effects of increased dietary protein level and supplementation with AA are related to enhanced ability of chicks to degrade excessively ingested tyrosine.