Methods and Results of

Pivetta et al 2006

Pivetta et al used mouse livers to conduct their experiments. Subsequent spectroscopic studies were used to measure the inhibitory effects of acetaldehyde on the three enzymes via the concentration of NADPH, which is oxidized in the enzymatic reactions.

Methods

They determined the activity by spectroscopy studies. The scientists pre –incubated either ebselen or GR and GPx for "30 min at 25°C, using different acetaldehyde concentrations (0, 30, 100, and 300µM),” in a 50mM phosphate buffer at pH 7.0. Then tert-butyl peroxide was added. Both enzymes attempt to convert this peroxide to a less biologically harmful compound. Because NADPH is consumed in the reaction catalyzed by both of these enzymes, the activity of the enzymes can be measured by the decline in NADPH concentration, seen at 340nm. GPx is also measured indirectly by NADPH concentration. The thiol-peroxidase activity of ebselen was measured in a similar manner.

To test this effect, L.A. Pivetta et al "quickly removed, put on ice and homogenized in eight volumes of a 25mM HEPES buffer, pH 7.0" the livers of 2 month old male Swiss albino mice.

Results

Graphs from the paper on how acetaldehyde affected various enzyme activities showed that pre-incubation of acetaldehyde with GR and GPx caused no direct results. Furthermore, thiols, such as dithioerythritol and glutathione (GSH), were also not directly affected by acetaldehyde. However, when in a medium with acetaldehyde, non-protein thiols, representing GSH, from the rat livers were oxidized. (Pivetta) Furthermore, even though acetaldehyde did not considerably oxidize GSH by itself or with glutathione-S-transferase, the presence of glutamyltranspeptidase (GGT) brought about this reaction.

It was also thought that perhaps acetaldehyde affected the nucleophilic selenol groups produced in the reaction between ebselen and GSH. Acetaldehyde was found to have no such effect. Therefore, the oxidation of various thiols is reliant upon other liver components for its harmful oxidative effect; GGT may be one such enzyme involved in this pathway. As the reactions to metabolize alcohol rely on the electrons donated by thiol compounds like GSH, the diminishing levels of thiol constituents in the liver with excess alcohol ingestion may be related to GGT’s activity. (Pivetta)

Read on to see the conclusions of Pivetta et al's experiment.

Figure 13: A Mouse: the Experimentee

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Figure 14: Pre-Incubation Results

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