Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.

Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes.

Zanthoxylum piperitum (DC), a potential feeding deterrent for mammals: studies with Microtus ochrogaster (Wagner).

Total extract from the fruit of Szechuan pepper (Zanthoxylum piperitum DC), the volatile components of the extract and a non-volatile fraction containing alkylamides (NVA fraction) are feeding deterrents for rats. The present study investigated the effectiveness of these natural repellents in prairie voles (Microtus ochrogaster Wagner). Two-choice feeding trials were conducted during which food-deprived voles were offered choices between oat-bran wafers. In Experiment 1, 10 voles were given three sets of feeding trials, each 2 h long. Baseline consumption was established during the first set of two trials by offering a choice between two oat-bran wafers dipped in ethanol, the control solvent. During the second set of two trials the voles were given a choice between an oat-bran wafer dipped in ethanol and a wafer dipped in Zanthoxylum extract. During the third set the voles were given a choice between a wafer served on top of a screened dish containing a sample of ethanol and a wafer served on top of a dish containing a sample of extract. In this manner the voles were exposed to volatile compounds emanating from the extract but could not contact it. Wafers dipped in extract were almost completely avoided. The volatile components of extract also significantly reduced food intake. In Experiment 2, habituation to the volatile constituents of extract was examined in 16 Zanthoxylum-naïve voles. Baseline consumption was established by offering two wafers served on top of screened dishes containing ethanol. This was followed by twelve tests during which a choice between a wafer served above a sample of ethanol and a wafer served above a sample of extract was given. The voles failed to habituate to the volatile components of extract, consistently consuming less of the wafers served above extract. In Experiment 3 a dose-response curve to Zanthoxylum extract was established, using 12 stimulus-naive voles. After baseline consumption was established, the animals were given two tests each, presenting a choice between a control wafer and a wafer dipped in a dilution of extract (0.001-100 g liter(-1)). Only concentrations of 10 and 100 g liter(-1) reduced food intake. In Experiment 4 the effects of the non-volatile fraction of extract were compared to those of whole extract. Vegetable oil was used as solvent. Eight stimulus-naïve voles were given two tests with a choice between an oil-dipped and an extract-dipped wafer. A second group of eight voles received two tests with a choice between an oil-dipped and NVA-dipped wafer. Extract-dipped wafers were avoided, but the NVA fraction had no effect on food consumption.

Analysis of plant sterol and stanol esters in cholesterol-lowering spreads and beverages using high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy.

Plant sterol and stanol esters were separated on a Luna hexyl-phenyl column using a gradient of acetonitrile (90-100%) in water. The eluted compounds were detected by atmospheric pressure chemical ionization (APCI)-mass spectroscopy (MS) in the positive mode. Sterol and stanol esters produced [M + H - HOOCR](+) ions. Application of the hyphenated technique-LC-MS-allowed differentiation between a number of esters of sitosterol, campesterol, stigmasterol, and (tentatively) avenasterol, as well as sitostanol and campestanol esters. With cholesteryl decanoate used as the internal standard, the method showed good linearity, precision, and reproducibility. The method required minimal sample pretreatment and can be applied to samples with high water content (juices) as well as samples with high oil content (margarine spreads). The method could be useful for the analysis of sterol and stanol esters in fortified food products.