GSH assay

Until most recently, GSH assay involving neutral loss of 129 has mostly been carried out using triple-quadmpole mass spectrometers operated under nominal mass conditions. This approach suffers from low sensitivity because both... 

Kinetic analysis of GST reaction curve can predict Am for GSH over 4.0 pmol/L, but there are no sufficient data for analyses at GSH below 3.0 pmol/L after optimization of GST activity for complete conversion of GSH at 5.0 pmol/L within 6.0 min, reaction curve within 5.0 min for GSH at 5.0 pmol/L can be used for kinetic analysis of reaction curve to predict Am. With the optimized GST activity for reaction within 5.0 min, the Hnear range for GSH assay is from 1.5 pmol/L to over 90.0 pmol/L by the integration strategy while it is from 4.0... 

In reviewing the classical methods of glutathione (GSH) assay, this paper will be concerned with (1) a consideration of current methods, (2) descriptions of the methods, and (3) a discussion of the relative value of the different methods as related to the needs of modern research. 

GSH Assay. Cellular GSH was measured by tt fiuorometric asstty (20). Typically, cell lysates were diluted with O.IM potassium phosphate buffer, pH 8.0, and proteins were precipitated with trichloroacetic add. A 100 pi sanq>le then mixed with 100 pi of o-phthaldialdehyde (10 mg in 10 ml of methanol) and 1.8 ml of O.IM potassium phosphate buffer 8.0) containing 5 mM EDTA. The solution was incubated at room tenq>erature for 15 min, and formation of GSH-phthalaldehyde conjugate was then measured by a fluorescence spectrometer (HitacU Model F-2000). The conjugate was exdted at a wavelengA of 350 nm, and the fluorescence emission was measured at 420 nm. 

The glutathione (GSH) level was assayed by the method of Sedlak and Lindsay (ref. 14), using Ellman s reagent (5,5-dithio-bis-(2-nitrobenzoic acid). Calibration was carried out parallely to each series, using reduced glutathione as a standard. 

A battery of different biochemical quantitative assays was applied to many different tissues and species. DNA damage and lipid peroxidation assays measure the direct impact of genotoxics and oxidant pollutants [16,17] whereas alteration of GSH levels in liver is a marker for oxidative stress [18]. Mercury and other heavy metals are known to induce metallothionein levels in different tissues although this effect is variable in different species and organs [19-22]. 

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 ). 

An analogous assay using a radiolabeled soft nucleophile would also be required to complement the hard nucleophile radiolabeled cyanide trapping assay. Investigations into radiolabeled glutathione have proved unsuccessful since the material is unstable due to cross-reactions induced by beta radiation. Alternate soft nucleophiles such as cysteine, N-acetyl cysteine and P-mercaptoethanol all have promise as radiolabeled substances for quantitative trapping experiments since they are more stable than GSH and equally nucleophilic, although clearly these would not be substrates for GST. 

Corn seeds were germinated between moist paper towels and were then treated with 5 uM CDAA either 1 or 2.5 days prior to assay for GSH af 6 days after planting (Table V). Using the reagent DTNB [2,2-dithiobis (2-nitrobenzoic acid)], root GSH contents were assayed spectrophotometrical ly as previously described (20). 

CDAA also elevated GSH-S -transferase activity in corn roots or shoots (Table VI) when assayed spectrophotometrical ly with CDNB (l-chloro-2,4-dinitrobenzene) (20). However, in this case, treatment with CDAA for 2.5 days produced the greatest increase in enzyme activity in the roots of 6-day-old corn seedlings. 

Although CDAA pretreatments elevated both GSH and GSH-S.-transferase activity (as assayed by CDNB), it was essential to determine whether these effects would actually result in greater CDAA metabolism. For these studies, we examined [14C]CDAA metabolism in vitro and in excised corn root tips [detailed methods described by Ezra et. al. (20)]. The in vitro assay revealed a significant level of non-enzymatic [A C] CDAA... 

Although methods have been devised for the measurement of GSH utilization by the enzyme, and of H202 consumption, these are difficult and have now largely been replaced by an assay based on that of Paglia and Valentine (1967) in which the product GSSG is used to drive the oxidation of NADPH+H+ catalysed by glutathione reductase ... 

Assay conditions for a range of different substrates are set out in Table 6.1 substrate and GSH concentrations given are final concentrations in the complete reaction mixture. 

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