Gene expression profiling of systems involved in the metabolism and the disposition of xenobiotics: comparison between human intestinal biopsy samples and colon cell lines.

Intestinal cell lines are used as in vitro models for pharmacological and toxicological studies. However, a general report of the gene expression spectrum of proteins that are involved in the metabolism and the disposition of xenobiotics in these in vitro systems is not currently available. To fill this information gap, we systematically characterized the expression profile of 377 genes encoding xenobiotic-metabolizing enzymes, transporters, and nuclear receptors and transcription factors in intestinal mucosa (ileum, ascending colon, transverse colon, descending colon, and rectum) from five healthy subjects and in five commonly used intestinal cell lines (Caco-2, C2BBe1, HT29, T84, and FHC). For this, we performed a quantitative real-time reverse transcription-polymerase chain reaction analysis using TaqMan low-density arrays and analyzed the results by different statistical approaches: Spearman correlation coefficients, hierarchical clustering, and principal component analysis (PCA). A large variation in gene expression spectra was observed between intestinal cell lines and intestinal tissues. Both hierarchical clustering and PCA showed that two distinct clusters are visible, of which one corresponds to all cultured cell lines and the other to all intestinal biopsies. The best agreement between human tissue and the representative cell line was observed for human colonic tissues and HT29 and T84 cell lines. Altogether, these data demonstrated that gene expression profiling represents a new valuable tool for investigating in vitro and in vivo expression level correlation. This study has pointed out interesting expression profiles for various colon cell lines, which will be useful for choosing the appropriate in vitro model for pharmacological and toxicological studies.

Impact of tryptophan metabolism on the vulnerability to alcohol-related blackouts and violent impulsive behaviours.

AIMS: We examined (1) the association of SLC6A4 genotypes and alcohol dependence (AD) in a sample of alcoholics; (2) the validity of lifetime occurrence of blacked-out violent impulsive behaviour (BOVIB) during binge drinking bouts as a criterion for subtyping AD patients and (3) a mechanistic hypothesis for BOVIB involving tryptophan-2,3-dioxygenase (TDO) activity. METHODS: Three common polymorphisms of the SLC6A4 gene (5-HTTLPR, A/G SNP of LPR region and VNTR in intron 2) were genotyped. An oral tryptophan (Trp) load (OTL) was administered to a sample of patients seeking help for AD. BOVIB history and psychological status were screened by BOVIB-Q, depression (BDI), anxiety (BAI, STAI) and personality (TCI) questionnaires. During the 7 h following Trp load, serum kynurenine (Kyn) and Trp were monitored. RESULTS: BOVIB+ patients showed significantly higher scores on depression, anxiety and character scales but no significant association was found between SLC6A4 polymorphisms and BOVIB. Patients with a history of BOVIB (BOVIB+ subgroup) differed from those exempt from such episodes (BOVIB- subgroup) for TDO activity response to OTL assessed by the Kyn:Trp ratio (P = 0.043) and the slope of concentration increase ratio (SCIR) of serum Kyn (P = 0.043). CONCLUSIONS: Put together, these findings support the validity of the BOVIB criterion to differentiate a sub-group of vulnerable AD subjects and suggest that OTL may help to concurrently define a specific endophenotype.

Indoleamine 2,3-dioxygenase activity of acute myeloid leukemia cells can be measured from patients' sera by HPLC and is inducible by IFN-gamma.

The enzyme indoleamine 2,3-dioxygenase (IDO) converts tryptophan to kynurenine, blocking T-cell activation and inducing immunosuppression. In patients with acute myeloid leukemia (AML), the serum kynurenine/tryptophan ratio (Kyn/Trp) was raised, suggesting a higher IDO activity than in healthy people. Patients with higher Kyn/Trp ratios showed lower survival. IDO activity was also detected in AML cells after exposure to IFN-gammain vitro, suggesting that the higher Kyn/Trp ratio in serum of AML patients might have resulted from stimulated leukemic blast cells. Thus, in AML, the activity of IDO can be easily monitored, providing a tool for future clinical testing of IDO-blocking drugs.

Quantitative determination of glyphosate in human serum by 1H NMR spectroscopy.

The determination and quantification of glyphosate in serum using (1)H NMR spectroscopy is reported. This method permitted serum samples to be analysed without derivatization or any other sample pre-treatment, using 3-trimethylsilyl 2,2',3,3'-tetradeuteropropionic acid (TSP-d(4)) as a qualitative and quantitative standard. Characterization of the herbicide N-(phosphonomethyl)glycine was performed by analysing chemical shifts and coupling constant patterns. Quantification was performed by relative integration of CH(2)-P protons to the TSP-d(4) resonance peak. The method was tested for repeatability (n=5) and yielded coefficients of variation of 1% and 3%, respectively: detection and quantification limits were also determined and were 0.03 and 0.1mmol/L, respectively. The method was applied to the quantification of glyphosate in a case of acute poisoning.

[Purple urine bag syndrome: a case report]. / A propos d'un cas du syndrome des urines violettes.

The authors report a case of dark purple urine in a woman with bowel obstruction and bilateral percutaneous nephrostomy urinary diversion for 30 years. This colour was due to the presence of high urinary concentrations of 3-indoxyl sulphate due to the enzymatic activity of Providencia rettgeri. A favourable course was observed in response to antibiotics.

Major metabolites of zolpidem: expeditious synthesis and mass spectra.

An expeditious route to the two major metabolites of Zolpidem-and readily applicable to the synthesis of the drug-was established via a cyclization reaction between a 2-aminopyridine and a suitable alpha-bromoacetophenone. The structures of the target compounds were confirmed from a 2D (1)H-(15)N NMR correlation. Their mass spectra contribute to a reliable toxicological identification of the drug in the case of drug-facilitated crimes.

Analysis of ingested material and urine by GC-MS and 1H NMR spectroscopy: poisoning of an adult with adulterated soda.

The purpose of this work is to characterize chemical compounds added to an ingested soda by (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy and by gas chromatography-mass spectrometry in the electron impact mode. A second point was to highlight possible metabolic disturbances by considering urinary profile. Without any pretreatment, dimethylphtalate, 2-butanone, and 2,2,4-trimethylpentanediol diisobutyrate were found in the adulterated soda. Quantitative analysis was performed by relative integration of peak areas. Huge quantities of 2,2,4-trimethylpentanediol diisobutyrate and dimethylphtalate were found in the oily layer. 2-Butanone, which is miscible in water, was found in the two phases as well as small quantities of dimethylphtalate. The urine sample was collected on hospital admission and was also analyzed by (1)H NMR spectroscopy. The major abnormal compound found was 1,2-propanediol. Other disturbances concerned endogenous metabolites such as 2-ketoglutaric acid, lactic acid, and betaine.

1H NMR spectroscopy and GC-MS analysis of alpha-chloralose. Application to two poisoning cases.

Alpha-chloralose, a compound widely used as a rodenticide and in the control of bird pests, is readily available. Two cases of intentional poisoning are reported. Both patients became comatose and presented hypersialorrhea and myoclonal crises in the legs. They were discharged from hospital after several days. As clinical signs of alpha-chloralose poisoning lack specificity, anamnesis might be difficult, particularly in the case of delayed diagnosis. Toxicological analysis is therefore critical, and this article reports the investigation of serum and urine samples by gas chromatography-mass spectrometry (GC-MS) in the electron-impact mode, and by 1H nuclear magnetic resonance (1H NMR) spectroscopy. Non-hydrolyzed urinary samples and those hydrolyzed by beta-glucuronidase were taken into consideration. After acetylation, GC-MS analysis was based on characteristic mass-to-charge ratio values of 272 for alpha-chloralose and 206 for beta-hydroxyethyltheophylline, which was used as internal standard. Characterization of alpha-chloralose species by 1H NMR spectroscopy was performed taking two parameters into account: chemical shift and coupling-constant values. Without any pretreatment, 1H NMR spectroscopy revealed the presence of free (5.50 and 6.15 ppm) and conjugated forms of alpha-chloralose by characteristic resonances of H1 and chloral-type protons, respectively. Quantitative analysis was performed by relative integration of peak areas. Serum alpha-chloralose showed concentrations below the quantitation limit of both methods. In urine samples, the free chemical species rapidly decreased. GC-MS analysis revealed the predominence of conjugation after a beta-glucuronidase hydrolysis step. 1H NMR analysis directly showed that on admission of the first patient, average urinary concentrations were 1.73 mmol/L (535 mg/L) for the free form and 13.72 and 6.25 mmol/L for the two conjugated forms. A later enzymatic treatment confirmed the total concentration of alpha-chloralose chemical species. Analysis of alpha-chloralose in urine by either GC-MS or 1H NMR spectroscopy methods proved to be comparable.

Detection and quantitation of xenobiotics in biological fluids by 1H NMR spectroscopy.

NMR spectroscopic investigation can be applied to a large variety of xenobiotics in acute poisoning cases (therapeutic agents, pesticides, solvents, alcohols). In a salicylate poisoning case, the three major metabolites of acetylsalicylic acid--salicylic, salicyluric, and gentisic acids--have been detected in crude urine. Valproic acid as glucuronoconjugated form was identified in urine samples from two poisoned patients. Paraquat (Gramoxone) was identified by its two aromatic signals at 8.49 and 9.02 ppm and quantitated in urine of two acutely poisoned patients (985 and 500 micromol/L). In an intentional poisoning case with tetrahydrofuran, this compound was characterized by its resonances at 1.90 and 3.76 ppm, and quantitated at 11.3 and 11.8 mmol/L in serum and urine samples, respectively. Methanol, ethylene glycol, and the corresponding metabolites formate and glycolate were detected in the same spectrum of serum samples from three poisoned patients. Detection and quantitation of many exogenous and endogenous compounds could be achieved by 1H HMR spectroscopy in biological fluids without any hypothesis on the chemical species.

1H NMR urine analysis as an effective tool to detect creatine supplementation.

Creatine is one of the main compounds in muscular energetic metabolism leading to phosphocreatine to maintain high ATP levels. Creatine is found in blood and excreted in small amounts in urine. Creatine supplementation and athletic performances are supposed to be correlated, particularly in intensive and intermittent efforts. After oral creatine supplementation, a 1H nuclear magnetic resonance (NMR) spectroscopy method was developed for its direct analysis, without any pretreament of urine samples. This method can be used to detect any supplementation of creatine, a substance prohibited in France. The detection limit is 10 micromol/L (1.31 mg/L) and analysis is performed in 10 min. After a single oral supplementation of 2.1 g to three subjects, a kinetic investigation reveals a maximum concentration of 20 mmol/L (2.62 g/L), observed between 1 and 6 h after ingestion. This procedure was used to test 13 urine specimens obtained from bodybuilders. From the concentrations measured (range: 0.41 to 10.30 mmol/L, 54 to 1350 mg/L), the doping practices of at least nine athletes could be observed. Creatine is not often analyzed in hospital laboratories. This paper documents how easily creatine can be determined and quantitated by 1H NMR spectroscopy.