Binding Of Acetaldehyde Research Articles

Covalent binding of acetaldehyde to hepatic proteins during ethanol oxidation.

Acetaldehyde production and the radiolabeling of hepatic proteins were determined in rat liver slices incubated with 14C-ethanol (10 mmol/L). Significant labeling of hepatic proteins occurred in the presence of protein synthesis inhibitors, indicating that, under these conditions, the radiolabeling of protein did not occur via de novo protein synthesis. Additional experiments indicated that the major source of protein-bound radioactivity derived from 14C-ethanol oxidation was the formation of 14C-acetaldehyde adducts with proteins. This conclusion was made from observations that pyrazole, an inhibitor of ethanol oxidation and, therefore, acetaldehyde formation, decreased radiolabeling of protein, whereas cyanamide, which elevated hepatic acetaldehyde levels, markedly increased the labeling of protein. Furthermore, L-cysteine, which can bind acetaldehyde and, therefore, act as an acetaldehyde trap, substantially reduced protein-bound radioactivity. It was also demonstrated that acetaldehyde formed both stable and unstable adducts with hepatic proteins and that unstable adducts may undergo conversion to form stable adducts during incubation.

Aldehyde dehydrogenase activity as the rate-limiting factor for acetaldehyde metabolism in rat liver

The velocity of acetaldehyde metabolism in rat liver may be governed either by the rate of regeneration of NAD from NADH through the electron transport system or by the activity of aldehyde dehydrogenase (ALDH). Measurements of oxygen consumption revealed that the electron transport system was capable of reoxidizing ALDH-generated NADH much faster than it was produced and hence was not rate-limiting for aldehyde metabolism. To confirm that ALDH activity was the rate-limiting factor, low- K m ALDH in slices or intact mitochondria was partially inhibited by treatment with cyanamide and the rate of acetaldehyde metabolism measured. Any inhibition of low- K m ALDH resulted in a decreased rate of acetaldehyde metabolism, indicating that no excess of low- K m ALDH existed. Approximately 40% of the metabolism of 200 μ m acetaldehyde in slices was not catalyzed by low- K m ALDH. Fifteen of this 40% was catalyzed by high- K m ALDH. A possible contribution by aldehyde oxidase was ruled out through the use of a competitive inhibitor, quinacrine. Acetaldehyde binding to cystosolic proteins may account for the remainder. By measuring acetaldehyde accumulation during ethanol metabolism, it was also established that low- K m ALDH activity was rate-limiting for acetaldehyde oxidation during concomitant ethanol oxidation.

Enhancement of acetaldehyde-protein adduct formation by l-ascorbate

The effect of l-ascorbate on the binding of [ 14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 m m) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 μ m. An ascorbate concentration as low as 0.5 m m was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly- l-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.

Acetaldehyde adducts with proteins: Binding of [ 14C]acetaldehyde to serum albumin

Acetaldehyde, the immediate oxidation product of ethanol metabolism, was assessed for its ability to bind covalently to a purified protein in solution. Bovine serum albumin (BSA) 2 2 Abbreviations used: BSA, bovine serum albumin; TCA, trichloroacetic acid; PTA, phosphotungstic acid. was used as the model protein incubated in the presence of 0.2 m m [ 14C]acetaldehyde at pH 7.4 and at 37 °C. Acetaldehyde formed both stable and unstable adducts with serum albumin. Unstable adducts were identified following stabilization with the reducing agent sodium borohydride. We examined both types of binding using trichloroacetic acid precipitation, gel filtration, and dialysis as means to separate bound from free acetaldehyde. All three methods of analysis gave comparable results except that the number of stable acetaldehyde adducts with albumin were significantly lower following separation by dialysis. The effects of l-cysteine, l-lysine, and reduced glutathione were assessed for their abilities as competitive reagents to decrease binding of [ 14C]acetaldehyde to BSA. Addition of cysteine caused a rather dramatic concentration-dependent reduction in [ 14C]acetaldehyde binding to BSA when compared to that caused by lysine which displayed a relatively mild effect on covalent binding. The presence of glutathione caused a concentration-dependent decrease in protein-bound radioactivity that was stronger than that by lysine but not as effective as cysteine. The ability of each reagent to reverse the formation of preformed acetaldehyde adducts with BSA was also examined. Only l-cysteine effectively decreased the number of unstable acetaldehyde adducts with BSA while stable binding of acetaldehyde remained essentially unaffected by any of the three reagents. These results indicate that acetaldehyde can covalently bind to protein and form unstable as well as stable adducts.

Acetaldehyde adducts of phospholipids.

Incubation of rat liver microsomes with acetaldehyde followed by reduction with borohydride of the Schiff bases formed leads to the formation of the N-ethyl derivatives of phosphatidylethanolamine and phosphatidylserine. Proof for this structure came from a comparison of these microsomal derivatives, and the nitrogenous bases derived therefrom by acid hydrolyses, with synthetic N-ethylphosphatidylethanolamine, N-ethylphosphatidylserine, N-ethylethanolamine, and N-ethylserine. Modification of membrane phospholipids by covalent binding of acetaldehyde to form Schiff bases may perturb some of the biochemical processes associated with membranes.

Binding of acetaldehyde to rat liver microsomes: Enhancement after chronic alcohol consumption

Summary Rat hepatic microsomal preparations were assessed for their capacity to bind exogenous 14C-acetaldehyde and 14C-acetaldehyde formed endogenously from 14C-ethanol in the microsomal ethanol-oxidizing system (MEOS). Exogenous acetaldehyde was found to bind to rat liver microsomes. Blocking of the free amino groups and thiol groups with site specific reagents (pyridoxal 5′-phosphate and p-hydroxymercuribenzoate) reduced the binding of acetaldehyde. Acetaldehyde formed endogenously from ethanol also bound to hepatic microsomes. This was increased after chronic alcohol consumption, in association with enhanced MEOS activity. The binding of acetaldehyde produced by MEOS was significantly greater than that of an equivalent amount of exogenous acetaldehyde. Thus, acetaldehyde produced in situ may exert local toxic effects on the endoplasmic reticulum.

Liver aldehyde oxidase: A form of xanthine oxidase

Pig liver aldehyde oxidase is not specific for aldehydes, but in addition catalyzes the oxidation at reduced rates of hypoxanthine, xanthine and DPNH. The sites of binding of acetaldehyde and hypoxanthine by the enzyme appear to be different. Flavin is a component common to the pathways of oxidation of acetaldehyde and hypoxanthine. Quinacrine inhibits the enzymic catalysis by preventing the reduction of the flavin. The heme component present in preparations of the enzyme does not appear to be involved in the enzymic catalysis.