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Trimethylamine N-oxygenation and N-demethylation in rat liver microsomes
1National Institute of Public Health, Praha, Czech Republic.
Abstract:
The in vitro oxidation of trimethylamine (TMA) to TMA N-oxide (TMAO) and dimethylamine (DMA) was studied in rat liver microsomes. Pretreatment of rats with phenobarbital, 3-methylcholanthrene, ethanol or pregnenolone 16 alpha-carbonitrile had little or no effect on the liver microsomal metabolism of TMA to TMAO or DMA. Changing the atmosphere in the incubation vessel from 20% oxygen/80% nitrogen (air) to 100% oxygen had a selective stimulatory effect on the N-oxygenation of TMA but did not affect TMA N-demethylation. In addition, the Km for TMA N-demethylation was 5-fold higher than for the N-oxygenation reaction. The results of these studies suggest that the enzyme systems responsible for N-demethylation and N-oxygenation are different and that they are under different regulatory control. Carbon monoxide (CO/O2 = 80/20) had little or no inhibitory effect on either the N-demethylation or N-oxygenation of TMA by liver microsomes from control or pregnenolone 16 alpha-carbonitrile-treated rats. Additional studies indicated that methimazole, an inhibitor of FAD-containing monooxygenase (FMO), was a potent inhibitor of TMA oxidation. Preincubation of liver microsomes from control or pregnenolone 16 alpha-carbonitrile-treated rats at 37 degrees for 10 min without NADP(H) (a procedure that irreversibly inactivated FMO activity) resulted in > 95% inhibition of TMA N-demethylation and N-oxygenation, and this inhibition was prevented by including a NADPH-generating system in the preincubation medium (a procedure for preventing the thermal inactivation of FMO activity). The data suggest that FMOs are the major enzymes responsible for N-demethylation and N-oxygenation of TMA in rat liver microsomes.
Insights
Rat liver microsomes metabolize trimethylamine (TMA) via N-demethylation and N-oxygenation. FAD-containing monooxygenases (FMOs) are identified as the key enzymes responsible for both TMA metabolic pathways.
Area of Science:
- Biochemistry
- Pharmacology
- Enzymology
Background:
- Trimethylamine (TMA) is metabolized in the liver through N-demethylation to dimethylamine (DMA) and N-oxygenation to TMA N-oxide (TMAO).
- Understanding the enzymes involved in TMA metabolism is crucial for comprehending its physiological and toxicological roles.
Purpose of the Study:
- To investigate the in vitro oxidation pathways of trimethylamine (TMA) in rat liver microsomes.
- To identify the specific enzyme systems responsible for TMA N-demethylation and N-oxygenation.
- To explore the regulatory control mechanisms governing these metabolic pathways.
Main Methods:
- Studied in vitro oxidation of TMA to TMAO and DMA using rat liver microsomes.
- Examined the effects of various pretreatments (phenobarbital, 3-methylcholanthrene, ethanol, pregnenolone 16 alpha-carbonitrile) on TMA metabolism.
- Investigated the impact of altered oxygen concentrations and carbon monoxide on TMA metabolism.
- Utilized methimazole as an inhibitor of FAD-containing monooxygenase (FMO).
- Assessed the role of FMOs by studying the effects of preincubation with and without a NADPH-generating system.
Main Results:
- Pretreatments with common enzyme inducers had minimal impact on TMA metabolism.
- Increased oxygen concentration selectively stimulated TMA N-oxygenation but not N-demethylation.
- The Michaelis constant (Km) for TMA N-demethylation was significantly higher than for N-oxygenation, suggesting distinct enzyme systems.
- Methimazole potently inhibited TMA oxidation.
- Irreversible inactivation of FMO activity by preincubation led to >95% inhibition of both TMA N-demethylation and N-oxygenation, which was prevented by a NADPH-generating system.
Conclusions:
- The N-demethylation and N-oxygenation of TMA in rat liver microsomes are mediated by distinct enzyme systems under different regulatory control.
- FAD-containing monooxygenases (FMOs) are the primary enzymes responsible for both TMA N-demethylation and N-oxygenation in rat liver microsomes.