Glutathione detection

In some cases. Phase I metabolites may not be detected, owing to their instability or high chemical reactivity. The latter type are often electrophilic substances, called reactive intermediates, which frequently react non-enzymically as well as enzymically with conjugating nucleophiles to produce a Phase II metabolite. A common example of this type is the oxidative biotransformation of an aromatic ring and conjugation of the resulting arene oxide (epoxide) with the tripeptide glutathione. Detection of metabolites derived from this pathway often points to the formation of precursor reactive electrophilic Phase I metabolites, whose existence is nonetheless only inferred. 

Smith, W.E., Reglinski, J., Hoey, S., Brown, D.H. and Sturrock, R.D. (1990) Changes in glutathione in intact erythrocytes during incubation with penicillamine as detected by proton spin-echo NMR spectroscopy. Inorganic Chemistry, 29, 5190-5196. 

GSHPx, CAT and SOD, which normally protect cells from free-radical damage have not been detected in aqueous humour. It has therefore been su ested that damage by free radicals and hydrogen peroxide to the anterior segment is prevented by a non-enzymatic extracellular oxidoreduction system involving a constant supply of reduced glutathione to the aqueous fluid from the ciliary epithelium, cornea and lens (Riley, 1983). 

Vandeberg, P. J. and Johnson, D. C., Pulsed electrochemical detection of cysteine, cystine, methionine, and glutathione at gold electrodes following their separation by liquid chromatography, Anal. Chem., 65, 2713, 1993. 

Simple etching of the capillary end served to decouple the electrophoretic current from that of amperometric detection, permitting quantitation of attomole levels of catecholamines from brain microdialyzates.24 A postcolumn reactor using bromine generated electrochemically in situ has been used in the detection of peptide thiols, such as glutathione and cysteine, separated by capillary electrophoresis.25... 

According to a Macherey-Nagel application note [35], a mixture of 20 ng each of (i)-cysteine, (L)-glutathione, and (L)-penicillamine was resolved in less than 12 min by HPLC. The method used a Nucleosil 100-5SA column (15 cm x 4.6 mm i.d.) with aqueous 4.5 g/L ammonium citrate-6 g/L phosphoric acid at pH 2.2 as the mobile phase (eluted at 1 mL/min) and electrochemical detection at a gold electrode polarized at +800 mV. 

Figure 6 HPLC separation of a 4.0-nmol mixture of blood thiols detected by CL. (1) IV-acetyl-L-cysteine (2) reduced glutathion (3) cysteine (4) methionine (5) oxidized glu-tathion. (From Ref. 95, with permission.)...

Spin trapping has been widely used for superoxide detection in various in vitro systems [16] this method was applied for the study of microsomal reduction of nitro compounds [17], microsomal lipid peroxidation [18], xanthine-xanthine oxidase system [19], etc. As DMPO-OOH adduct quickly decomposes yielding DMPO-OH, the latter is frequently used for the measurement of superoxide formation. (Discrimination between spin trapping of superoxide and hydroxyl radicals by DMPO can be performed by the application of hydroxyl radical scavengers, see below.) For example, Mansbach et al. [20] showed that the incubation of cultured enterocytes with menadione or nitrazepam in the presence of DMPO resulted in the formation of DMPO OH signal, which supposedly originated from the reduction of DMPO OOH adduct by glutathione peroxidase. 

Dieckhaus, C.M. et al. 2005. Negative ion tandem mass spectrometry for the detection of glutathione conjugates. Chem. Res. Toxicol. 18 630. 

However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme. 

---