Biochemical Analyses

5.1. Measurement of hydrogen peroxide

5.1.1. Method

H2 0 2-generated Fe^"^, according to the method of Michaels and Hunt (1978) as modified by Jiang et al. (Jiang et a/. 1990; W olff, 1994). Ferrous ion (Fe?"^), relatively stable to autoxidation in dilute H2SO4, can be oxidised by H2O2 to yield ferric ion (Fe^^). Xylenol orange binds the latter forming a complex with a maximum absorbance at 560 nm, the colour yield being highly enhanced by sorbitol.

5.1.2. In chemically defined studies

Briefly, 50 samples were added to 950 yîL xylenol orange reagent (consisting of 250 /xM ammonium ferrous sulphate, 100 /xM xylenol orange, and 100 mM sorbitol in 25 mM H2SO4) and incubated for 30 minutes at room temperature. Absorbance at 560 nm of the Fe^^-xylenol orange complex was calibrated against standard concentrations of H2O2. The standard H2O2 solution was calibrated using e = 39.4 M'^cm'^ at 240 nm (Nelson and Kiesow, 1972). Under the conditions of quinone-thiol incubation used, the measurement of H2O2 formed, using the xylenol orange assay, compared closely with those of O2 uptake as measured using an oxygen electrode.

5.1.3. In liver slices

5 .1.3.1. Principle

Since H2O2 does not accumulate within slices to an extent to permit direct isolation and characterisation, generation of H2O2 in liver slices was detected by measuring irreversible inhibition of endogenous catalase catalytic activity by 3-amino-l ,2,4-triazole (A M T ) (Cohen and Hochstein, 1964). Briefly, catalase catalytic activity (2H2O2 -» 2H2O + O2) results from the formation of an active catalase-H2 0 2 complex I and proceeds by the reduction o f complex I by a second molecule of H2O2 (Aebi, 1984). To irreversibly inhibit this activity, A M T must react with complex I thus requiring the presence of H2O2 (Margoliash and Novogrodsky, 1958). This mechanism serves as a detection system for H2O2: when irreversible inhibition of catalase by A M T is observed, it may be inferred that H2O2 had been present in the test system. Inhibition of catalase was measured by a discontinuous method using the xylenol orange assay described in § I I . 5 .1 .2 ., as in Ou and W o lff (1993).

5.1.3.2. Measurement

Slices were prepared as described in § I I . 4 .2 ., put into the Ringer solution (other components were added or not as indicated) at room temperature and the experiment started by placing the flasks into an incubator bath at 37°C under 95% oxygen with shaking (90 strokes/min). After 2 hours, the slices were taken out of the first flask and reincubated in 5 mL fresh Ringer solution (other components were added or not as indicated) for a further 2 hours. When catalase activity was measured at 2 hr, 50 mM A M T was added in the first incubation medium; when it was measured at 4 hr, 50 mM A M T was added in the second incubation medium only. At the end of the incubation, slices were washed in phosphate-buffer saline (PBS) to remove any traces o f A M T prior to transfer into tubes containing 2 mL ice-cold PBS with 100 juM DETAPAC and homogenized for 10 s using an ultra-turrax homogeniser at full speed. Homogenates were then diluted to 200 fig liver (wet weight)/mL with PBS containing 150 fiM H2O2, mixed and incubated for 10 minutes at room temperature. Catalase activity was determined by reference to the concentration of H2O2 remaining at the end of the incubation period. 50 fiL aliquot of H2 0 2-exposed homogenate was added to 950 fiL of the xylenol orange reagent (which also halted H2O2 consumption by catalase) and the assay carried out as described in § I I . 5 .1 .2 .. Vials were centrifuged (12,000 g x 3 min) to remove flocculated material before reading the supernatant at 560 nm. Catalase activity was calculated in terms of H2O2 consumed per minute. Under the conditions used, all incubations contained residual H2O2 and H2O2 consumption was approximately linear over the 1 0 minutes observed.

5.2. Measurement of GSH

5.2.1. Method

The concentration of GSH was measured using o-phthalaldehyde (OPT) (Cohn and Lyle, 1966; Hissin and H ilf, 1976). GSH was shown by these authors to react specifically with OPT at pH 8.0, by reference to the reaction of OPT with other low molecular weight thiols and non-thiol compounds. This was confirmed in our hands, with cysteine and NAC (data not shown). However, as with other non-enzymatic assay

systems, a high specificity is not guaranteed for biological samples. Indeed, some other biological thiols have been reported to fluoresce when reacting with OPT (Jocelyn and Kamminga A ., 1970; Brigelius et aZ. 1983). Yet GSH being the most abundant non­ protein thiol in the liver, the OPT method is here a useful analytical tool for GSH.

5.2.2. In chemically defined studies

10 /xL sample aliquots were added to 1.90 mL 100 mM sodium phosphate buffer pH 8.0 containing 100 fxM DETAPAC, to avoid metal-catalysed autoxidation. 100 fiL of o-phthalaldehyde (1 mg/ mL in methanol) were then added and rapidly mixed. Fluorescence was measured (Ex, 340 nm; Em, 420 nm) after incubation for 30 minutes at room temperature in the dark, with reference to a GSH standard.

5.2.3. In liver slices

Samples were kept on ice as much as possible to hinder metal-catalysed autoxidation of thiols until reaction with OPT. Aliquots of 5% TCA slice-homogenate supernatants (see § I I . 4 .2 .) were diluted 30 times in 100 mM sodium phosphate pH 8.0 containing 100 ^M DETAPAC (to avoid metal-catalysed autoxidation). 100 /xL of those diluted aliquots were added to 1.8 mL 100 mM sodium phosphate buffer pH 8.0 containing 100

fxM DETAPAC. 100 fiL of o-phthalaldehyde (1 mg/ mL in methanol) were then added. Fluorescence was measured (Ex, 340 nm; Em, 420 nm) after incubation for 30 minutes at room temperature in the dark, with reference to a GSH internal standard.

When combined studies of catalase activity (§ II.5 .1 .3 .) and GSH content were carried out, 1 mL of slice homogenate in PBS/DETAPAC (see § I I . 5 .1 .3 .2 .) was vortex-mixed with 110 jLtL 50% TCA containing 100 /xM DETAPAC, centrifuged at 830 g for 10 minutes and the supernatant assessed for its GSH content as described above.

5.3. Measurement of GSSG

The concentration of GSSG in quinone-thiol incubation mixtures was monitored by measuring the oxidation of NADPH by glutathione reductase at 340 nm (Akerboom and Sies, 1981),