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Glutathione and antioxidants protect microsomes against lipid peroxidation and enzyme inactivation
1Department of Food Science and Technology, University of California, Davis 95616.
Abstract:
The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 microM iron as ferric sulfate and 50 microM ascorbate, ALDH, glucose-6-phosphatase (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathione S-transferase and nicotinamide adenine dinucleotide phosphate-cytochrome c reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected and N,N'-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 microM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.
Insights
Oxidative stress from iron and ascorbate causes lipid peroxidation and inactivates key liver enzymes like aldehyde dehydrogenase (ALDH). Antioxidants protect against this damage, highlighting lipid peroxyl radicals
Area of Science:
- Biochemistry
- Toxicology
- Cellular Biology
Background:
- Lipid peroxidation is a known mechanism of cellular damage.
- Enzyme function is critical for hepatic (liver) metabolism.
- The specific roles of oxidative stress and lipid peroxidation in enzyme inactivation require further elucidation.
Purpose of the Study:
- To investigate the link between lipid peroxidation and enzyme inactivation in rat liver microsomes.
- To determine if prior inactivation of aldehyde dehydrogenase (ALDH) affects the inactivation of other enzymes.
- To assess the role of 4-hydroxynonenal (4-HN) and lipid peroxyl radicals in oxidative damage to microsomal enzymes.
Main Methods:
- Incubation of rat hepatic microsomes with iron (ferric sulfate) and ascorbate.
- Measurement of enzyme levels (ALDH, G6Pase, Cyt-P450) and thiobarbituric acid-reactive substances (TBARS) as a marker of lipid peroxidation.
- Assessment of protection by antioxidants (reduced glutathione, N,N'-diphenyl-p-phenylenediamine, butylated hydroxytoluene) and the effect of ALDH inhibition.
Main Results:
- Iron and ascorbate caused rapid, concurrent decreases in ALDH, G6Pase, and Cyt-P450 levels, alongside increased TBARS.
- Antioxidants significantly protected against both lipid peroxidation and enzyme inactivation.
- ALDH was more susceptible to inactivation than G6Pase, serving as a sensitive marker for oxidative stress. 4-HN showed a minor role in enzyme inactivation.
Conclusions:
- Lipid peroxyl radicals are crucial mediators of microsomal enzyme inactivation during oxidative stress.
- Aldehyde dehydrogenase (ALDH) is a sensitive indicator of oxidative damage in hepatic microsomes.
- Chain-breaking antioxidants effectively prevent oxidative damage to microsomal enzymes.