Detection of GSH Conjugates

The detection, identification, and quantitative analysis of GSH conjugates have advanced with time as new analytical technologies and techniques become available (Baillie and Davis, 1993). GSH conjugates are chemically and thermally unstable, and they are very polar. Therefore, analysis using gas chromatography (GC)-MS requires... 

Many tissues are capable, at least vitro, of conjugation of xenobiotics with GSH (l)j the liver is assumed to be the most active, especially with Ingested xenobiotics, however GSH transferase activity has been detected in the Intestinal mucosa (6). The interorgan translocation of GSH conjugates formed in the intestinal... 

The application of an Orbitrap platform was also described for this purpose. In this case, one can use the excellent full scan sensitivity of the high-resolution accurate mass measurement in both the survey and data-dependent scan modes in order to identify the reactive intermediates and then provide data for their structure assignment [23], A labeled form of GSH 2 1 ratio of 5mM glutathione and I3( V5N Gly glutathione was used. The high mass accuracy in the survey scan can be used to detect the GSH adduct. The method was successfully applied for the detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlo-pidine, and mifepristone. 

FIGURE 15.8 Work flow of (a) PI-EPI with polarity switching and (/>) pMRM-EPI for the detection and structural characterization of GSH conjugates, (c) Work flow of NL-EPI or pMRM—EPI with polarity switching for the detection and structural characterization of NAC adducts, (d) Work flow of pMRM-EPI with polarity switch for the detection and structural characterization of dGSH adducts. 

In vivo intermediate in mammals by the detection of two of its derivatives,, the glutathione (GSH) conjugate and its further metabolites formed by an initial carbamylation reaction (W) (see below) and 2-chloroacrolein detected in the microsome-NADPH system and derived from the rearrangement-elimination reaction sequence discussed above (6). Sulfallate also yields 2-chloroacrolein in the microsome-NADPH system, presumably by -CH2 hydroxylation (22) on analogy with the metabolism of EPTC shown previously. 

An active glutathione S-transferase system was detected in the onion enzyme system when it was assayed with [ C]PCNB and GSH (9.). An initial rate of 14 nmol product/mg protein/hr was observed and a yield of 18 was obtained in 17 hr. HPLC indicated that S-(PCP)GSH was the only major conjugated product of this reaction. This was consistent with the Iji vivo studies with onion that showed that S-(PCP)GSH was the dominant GSH conjugate formed. In contrast, an enzyme from pea produced S-(PCP)GSH, S-(TCNP)GSH, and what appeared to be two isomeric S,S -(TCP)diGSH conjugates (6 ). 

Lipid peroxidation/oxidative stress (Fig. 7.36). Lipid peroxidation products (e.g., malondialdehyde) have been detected, and GSH is depleted seemingly by oxidation rather than conjugation, and prior depletion of GSH increases the toxicity. NADPH is... 

Paracetamol is a widely used analgesic, which causes liver necrosis and sometimes renal failure after overdoses in many species. The half-life is increased after overdoses because of impaired conjugation of the drug. Toxicity is due to metabolic activation and is increased in patients or animals exposed to microsomal enzyme inducers. The reactive metabolite (NAPQI) reacts with GSH, but depletes it after an excessive dose and then binds to liver protein. Cellular target proteins for the reactive metabolite of paracetamol have been detected, some of which are enzymes that are inhibited. Therefore, a number of events occur during which ATP is depleted, Ca levels are deranged, and massive chemical stress switches on the stress response. 

Typically, the mass window is set for +/—10 mDa. The purpose of using a narrow mass window is to provide high selectivity for GSH conjugate detection and to exclude false positives from nominal mass endogenous components. 

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