Blood acetaldehyde concentration gradient between hepatic and antecubital venous blood in ethanol-intoxicated alcoholics and controls*

After ethanol (0.8 g kg-1 body weight orally) significant concentrations of acetaldehyde (2-20 mumol 1(-1] were found in hepatic venous blood of moderately intoxicated non-alcoholic male Caucasians in spite of the absence of detectable levels (less than 2 mumol 1(-1] in simultaneously taken antecubital blood. In thirteen chronic alcoholics the elevation of blood acetaldehyde was more constant in the hepatic than in the peripheral vein. Fructose infusion caused a marked elevation of acetaldehyde both in the hepatic and peripheral vein of four controls, but not of four alcoholics, who eliminated ethanol about 50% faster than controls. The rate of disappearance of acetaldehyde from sampled and in vitro incubated hepatic venous blood was similar to that observed after addition of acetaldehyde in vitro to ethanol-free control blood (2 nmol ml-1 min-1 at 20 mumol 1(-1) acetaldehyde; Km about 30 mumol 1(-1]. Uptake of acetaldehyde in blood was calculated to explain maximally 30-40% of the concentration gradient between central and peripheral blood.     

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.     

The Role of Acetaldehyde in Mediating the Deleterious Effect of Ethanol on Pyridoxal 5′-Phosphate Metabolism

Previous studies in vivo and with isolated perfused rat livers have suggested that the deleterious effect of ethanol on hepatic pyridoxal 5'-phosphate metabolism is mediated by acetaldehyde. Inasmuch as acetaldehyde has no effect on the synthesis of pyridoxal phosphate, it has also been postulated that acetaldehyde accelerates pyridoxal phosphate degradation by displacing this coenzyme from binding proteins, which protect it against hydrolysis. To test these hypotheses, studies have been performed with isolated rat hepatocytes, subcellular fractions of rat liver, and human erythrocytes. Ethanol oxidation lowered the pyridoxal phosphate content of isolated liver cells when acetaldehyde oxidation was inhibited by either disulfiram or prior treatment of rats with cyanamide. Additions of 7.5 mM acetaldehyde alone at 40-min intervals to cell suspensions decreased hepatic pyridoxal phosphate content only slightly because acetaldehyde was rapidly metabolized. However, when acetaldehyde oxidation and reduction were inhibited by cyanamide treatment and by 4-methyl-pyrazole and isobutyramide, respectively, a 40% decrease in hepatic pyridoxal phosphate content was observed in 80 min of incubation.In equilibrium dialysis experiments, acetaldehyde, 7.5 and 15 mM, displaced protein-bound pyridoxal phosphate in undialyzed hepatic cytosol and in hemolysate supernate containing added pyridoxal phosphate. In the presence of alkaline phosphatase, acetaldehyde accelerated the degradation of pyridoxal phosphate in dialyzed hemolysate supernate and hepatic cytosol with added pyridoxal phosphate. Acetaldehyde also inhibits tyrosine aminotransferase. The kinetics of inhibition were mixed competitive-noncompetitive with respect to pyridoxal phosphate. These observations support the hypothesis that the deleterious effect of ethanol oxidation on pyridoxal phosphate metabolism is mediated at least in part by acetaldehyde which displaces this coenzyme from protein binding, thereby enhancing its degradation.     

Ethanol and acetaldehyde directly inhibit testicular steroidogenesis

The effects of ethanol and acetaldehyde on testicular steroidogenesis were examined. We found that ethanol markedly inhibited the gonadotropin-stimulated production of testosterone in enzymatically dispersed cell preparations of the testes of adult rats. The effects of ethanol on testicular steroidogenesis appeared to be noncompetitive since testosterone production could not be restored to nondrug-treated levels even by extremely high concentrations of gonadotropin. Acetaldehyde also inhibited testicular steroidogenesis in vitro but was between 1000 and 4000 times more effective than ethanol. As little as 50 microM acetaldehyde was effective in suppressing testicular steroidogenesis, whereas much higher (200 mM) concentrations of ethanol were required. Our results further demonstrated that cell viability was unaffected by incubation with very high concentrations of ethanol and acetaldehyde, indicating that the two drugs did not simply irreversibly impair the ability of the dispersed cells to appropriately respond to stimulation by gonadotropins. These results suggest that ethanol directly inhibits testicular steroidogenesis, but that acetaldehyde is much more potent.     

Impaired oxygen utilization. A new mechanism for the hepatotoxicity of ethanol in sub-human primates.

The role of oxygenation in the pathogenesis of alcoholic liver injury was investigated in six baboons fed alcohol chronically and in six pair-fed controls. All animals fed alcohol developed fatty liver with, in addition, fibrosis in three. No evidence for hypoxia was found, both in the basal state and after ethanol at moderate (30 mM) or high (55 mM) levels, as shown by unchanged or even increased hepatic venous partial pressure of O2 and O2 saturation of hemoglobin in the tissue. In controls, ethanol administration resulted in enhanced O2 consumption (offset by a commitant increase in splanchnic blood flow), whereas in alcohol fed animals, there was no increase. At the moderate ethanol dose, the flow-independent O2 extraction, measured by reflectance spectroscopy on the liver surface, tended to increase in control animals only, whereas a significant decrease was observed after the high ethanol dose in the alcohol-treated baboons. This was associated with a marked shift in the mitochondrial redox level in the alcohol-fed (but not in control) baboons, with striking rises in splanchnic output of glutamic dehydrogenase and acetaldehyde, reflecting mitochondrial injury. Increased acetaldehyde, in turn, may aggravate the mitochondrial damage and exacerbate defective O2 utilization. Thus impaired O2 consumption rather than lack of O2 supply characterizes liver injury produced by high ethanol levels in baboons fed alcohol chronically.     

Method of acetaldehyde measurement with minimal artifactual formation in red blood cells and plasma of actively drinking subjects with alcoholism.

After alcohol consumption, a substantial amount of acetaldehyde that is reversibly bound to protein and nonprotein components of the red blood cells circulates in the blood and could cause extrahepatic toxicity. However, acetaldehyde measurement in human red blood cells is hampered by considerable ex vivo artifactual formation as a result of nonenzymatic oxidation of ethanol during protein precipitation. To eliminate this source of artifactual formation, free and reversibly bound acetaldehyde were trapped with semicarbazide from red blood cell hemolysates, and both the stroma and the hemoglobin were sequentially removed by centrifugation and ion-exchange chromatography in carboxymethyl Sephadex, respectively. The eluted semi-carbazone was dissociated with perchloric acid, and the acetaldehyde that was released in the protein-free supernatants was measured by head-space gas chromatography. Maximal retention of hemoglobin by carboxymethyl Sephadex and complete recovery of acetaldehyde and ethanol were achieved at a pH of 5.3. The artifactual formation decreased from 2.62 +/- 0.32 mumol of acetaldehyde per millimole of ethanol in the initial hemolysates to 1.38 +/- 0.20 mumol after removal of the stroma and to a level that is comparable to measurements in plasma (0.09 +/- 0.02 mumol) after removal of both the stroma and the hemoglobin. In 12 actively drinking subjects with alcoholism, with blood ethanol levels that ranged between 9 and 81 mmol/L, the concentrations of acetaldehyde in red blood cells (11.50 +/- 1.46 mumol/L; range: 7.5 to 22 mumol/L) were minimally affected by blood ethanol levels and were three times as high as those in the plasma (3.74 +/- 1.49 mumol/L).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of zinc deficiency on ethanol metabolism and alcohol and aldehyde dehydrogenase activities

Alcohol dehydrogenase, low Km and high Km mitochondrial and microsomal aldehyde dehydrogenase, and in vivo ethanol elimination rates were determined in five groups of male Sprague-Dawley rats given liquid diets, as follows: control (C), control plus 5% ethanol (CE), pair-fed control and zinc-deficient (PC-ZnD), zinc-deficient (ZnD), and zinc-deficient plus 5% ethanol (ZnDE). Rats fed CE had decreased liver and serum zinc content. The animals given ZnD diets had an even more dramatic decrease in their tissue zinc concentrations and displayed marked growth retardation. The in vivo blood ethanol elimination rate was increased in animals fed ethanol, and this increase was accompanied by increased alcohol and aldehyde dehydrogenase activities. There was a significant decrease in the ethanol elimination rate in rats given ZnD and ZnDE diets. Alcohol dehydrogenase activities in rats with deficient zinc levels also were decreased, and there were no changes in acetaldehyde dehydrogenase activities. Our results suggest that the metabolism of ethanol to acetaldehyde is impaired in zinc deficiency, but acetaldehyde to acetate conversion appears normal.     

Liver Transplantation: Present Results and Problems

Mechanisms of the hepatotoxicity of ethanol are reviewed, including effects resulting from alcohol dehydrogenase (ADH) mediated excessive hepatic generation of NADH and acetaldehyde. Gastric ADH explains first-pass metabolism by ethanol; its activity is low in alcoholics and in females and is decreased by some commonly used drugs. In addition to ADH, ethanol can be oxidized by liver microsomes: studies over the last 25 years have culminated in the molecular elucidation of the ethanol-inducible cytochrome P-450 (2E1) which causes metabolic tolerance to ethanol and to various commonly used medications, enhanced degradation of testosterone and vitamin A (with vitamin A depletion) and selective hepatic perivenular toxicity. The latter results from free radical generation and activation of various xenobiotics, causing increased vulnerability of the heavy drinker to the toxicity of industrial solvents, anaesthetic agents, commonly prescribed drugs, over-the-counter analgesics, chemical carcinogens and even nutritional factors such as vitamin A and β-carotene. Furthermore, induction of the microsomal pathway contributes to increased acetaldehyde generation which promotes GSH depletion and lipid peroxidation and other toxic effects. Nutritional deficits may affect the toxicity of ethanol and acetaldehyde, as illustrated by the depletion in glutathione, ameliorated by S-adenosyl-L-methionine. Other ‘supemutrients’ include polyenylphosphatidylcholine, shown to correct the alcohol-induced hepatic phosphatidylcholine depletion and to prevent alcoholic cirrhosis in non-human primates.     

Biochemical basis for alcohol-induced liver injury

Chronic ethanol ingestion leads to hepatocellular injury and alcoholic liver disease (ALD) only if multiple factors combine to favor centrilobular hepatocellular hypoxia. It is hypothesized that these factors include a shift in the redox state, the induction of the microsomal ethanol oxidizing system (MEOS), a high blood alcohol level (BAL), a high polyunsaturated fat diet and episodic decreased O2 supply to the liver. The shift in the redox state favors a low cellular pH, decreased fatty acid oxidation and increased triglyceride formation. The increased MEOS activity increases O2 consumption and portal-central O2 gradient as well as favors acetaldehyde toxic effects including retention of hepatic lipids and export proteins causing cell swelling. The resultant increase in the concentration of acetaldehyde and lactate may stimulate fibrosis as they stimulate collagen synthesis in vitro. The resultant fatty liver narrows the sinusoids slowing sinusoid blood flow. The combination of events reduces available O2 leading to decreased levels of ATP and cellular pH making the liver vulnerable to episodes of systemic hypoxia. The role of membrane changes are reviewed, i.e., 1) membrane fluidity as related to changes in the species of phospholipids, 2) mitochondrial function as related to the changes in the lipid environment of the electron transport chain, and 3) linoleic acid-prostaglandin metabolism. Acute ethanol in vitro has been shown to affect liver cell metabolism regulation by triggering and increasing protein phosphorylation through the Ca2+-phospholipase C pathway. A high fat diet enhances the liver injury caused by chronic ethanol ingestion.     

Impaired acetaldehyde metabolism in partially hepatectomized rats

Two-thirds hepatectomy in rats resulted in elevated blood ethanol and acetaldehyde levels as compared to those of sham operated and CCl4-induced toxic injured rats. The acetaldehyde/ethanol ratio increased also. Although the liver mass regenerated within 3 days, ethanol metabolism remained disturbed. Mitochondrial aldehyde dehydrogenase activity was significantly diminished only following partial hepatectomy. The results suggest that abnormal ethanol and especially acetaldehyde metabolism in partially hepatectomized rats is not due simply to reduced liver tissue but to a diminished aldehyde dehydrogenase activity in the remaining tissue.     

Vitamin B6 metabolism in chronic alcohol abuse The effect of ethanol oxidation on hepatic pyridoxal 5''-phosphate metabolism.

Individuals with chronic alcohol abuse frequently exhibit lowered plasma levels of pyridoxal 5'-phosphate, the coenzyme form of vitamin B6. Because the liver is the primary source of this coenzyme in plasma and also the principal organ that oxidizes ethanol, the effect of ethanol on hepatic pyridoxal phosphate metabolism was studied in the rat. The chronic feeding of ethanol (36 percent of the total dietary calories) for 6 wk significantly decreased the hepatic pyridoxal phosphate content both in animals given a sufficient amount of vitamin B6 in their diet and in those rendered vitamin B6 deficient. In isolated perfused livers, the addition of 18 mM ethanol lowered the pyridoxal phosphate content of livers from vitamin B6-sufficient animals and deceased the net synthesis of pyridoxal phosphate from pyridoxine by the livers of vitamin B6-deficient animals. Ethanol also diminished the rate of release of pyridoxal phosphate into the perfusate by the livers of vitamin B6-deficient rats. These effects of ethanol, in vitro, were abolished by 4-methyl pyrazole, an inhibitor of alcohol dehydrogenase. Thus the derangement of pyridoxal phosphate metabolism produced by ethanol is dependt upon its oxidation. These data support previous findings whic indicate that acetaldehyde is the responsible agent which acts by accelerating the degradation of intracellular pyridoxal phosphate.     

Methanol--an up-to-now neglected constituent of all alcoholic beverages. A new biochemical approach to the problem of chronic alcoholism

Alcoholism is usually understood as ethanolism. There is some evidence that its oxidation product acetaldehyde may condense with endogenous amines to form tetrahydroisoquinoline (TIQ) and - tetrahydro-beta-carboline (THBC) alkaloids which ultimately might be responsible for addiction. In most animal experiments pure ethanol solutions were fed, but chronic alcoholics prefer normal alcoholic beverages, and it is widely ignored that all these beverages without exception also contain methanol. Its metabolite formaldehyde is a much more potent reaction partner for TIQ and THBC formation than acetaldehyde. As our findings in chronic alcoholics proved that these persons in contrast to healthy subjects are able to oxidize methanol despite high ethanol levels, there must be a continuous leakage of formaldehyde. And it seems possible that methanol plays a more significant role in the pathophysiology and possibly the etiology of chronic alcoholism than ethanol.     

The effect of acute, chronic, and prenatal ethanol exposure on insulin sensitivity

Ethanol has been considered as a lifestyle factor that may influence the risk of type 2 diabetes mellitus. In healthy adults, acute ethanol consumption results in insulin resistance. Acute ethanol consumption causes insulin resistance selectively in skeletal muscle by an indirect mechanism. Possible mediators include triglycerides (TGs), catecholamines, acetaldehyde, alterations in insulin binding, and hepatic insulin sensitizing substance (HISS). Recent studies in rats showed that acute administration of ethanol causes insulin resistance in a dose-dependent manner that is secondary to the blockade of insulin-induced HISS release. Chronic ethanol consumption may improve insulin sensitivity, but the results from the randomized controlled trials are mixed. Differences in ethanol dose, consumption period, and abstention period may account for the discrepant results. Epidemiological studies have suggested that the relationship between ethanol and insulin sensitivity is either an inverted U-shape or a positive linear relationship. Future randomized controlled trials should consider the dose of ethanol and the duration of ethanol consumption and abstention in the experimental design. Chronic prenatal and postnatal (nursing) ethanol exposure results in insulin resistance that is secondary to the absence of HISS release/action with the HISS-independent insulin action and insulin-like growth factor-1 (IGF-1)-mediated glucose disposal action remaining unimpaired. The impaired HISS release may be related to a reduction in hepatic glutathione (GSH) levels. The effect of chronic ethanol consumption on HISS has not been evaluated.     

Ethanol decreases plasma sulphydryls in man: effect of disulfiram

Based on animal experiments, interactions of ethanol and its metabolites with sulphydryls have been implicated in the toxicity of ethanol, but acute effects of ethanol on sulphydryls have not been documented in man. Plasma free glutathione and cysteine were therefore measured following the administration of 0.2 g kg-1 ethanol to normal healthy volunteers and chronic alcoholics on disulfiram, where the effects of high concentrations of acetaldehyde can be observed. In both groups, plasma glutathione decreased shortly following ethanol, and a sustained decreased in glutathione was seen in the subjects on disulfiram. In patients on disulfiram, but not the healthy controls, plasma cysteine decreased significantly. The decrease in plasma cysteine was correlated to the rise in acetaldehyde, suggesting that cysteine, but not glutathione, forms an adduct with acetaldehyde in man. We conclude that even moderate doses of ethanol may disturb the sulphydryl homeostasis and could interfere with biologically important processes that depend on sulphydryl groups.     

Determination of acetaldehyde in human blood using thiourea to inhibit ethanol interference

Determination of acetaldehyde in human blood by the semicarbazide method has been studied. An artefactual production of acetaldehyde from ethanol in blood and plasma of about 20 mumol/l was observed at concentrations of ethanol of about 60 mmol/l. This artefact was reduced to less than 1 mumol/l after addition of thiourea. The presumably non-enzymatic production of acetaldehyde from ethanol was demonstrated by the release of 3H from (1R)-[3H]ethanol added to the blood immediately after sampling. The results demonstrate that oxidation of ethanol is the major cause of the artefactual acetaldehyde formation. In human volunteers, metabolising ethanol, very low concentrations of acetaldehyde were found by the modified method, which includes thiourea.     

Diphenhydramine and the calcium carbimide-ethanol reaction: a placebo-controlled clinical study

The effect of diphenhydramine on the cyanamide-ethanol reaction was evaluated in a double-blind, controlled clinical study. Seven healthy subjects ingested 50 mg calcium carbimide at 4 hours and 100 mg diphenhydramine or placebo at 2 hours before a 0.2 gm/kg iv infusion of ethanol. Blood acetaldehyde and blood ethanol analyses were performed together with recordings of blood pressure, pulse rate, and flushing intensity during the hour after ethanol infusion. Diphenhydramine increased the mean ethanol AUC but did not influence blood acetaldehyde levels. Antihistamine reduced the flushing response by 40% and decreased the pulse rate from 40 minutes onward after ethanol infusion subsequent to calcium carbamide dosing. Blood pressure was not significantly influenced by ethanol at the calcium carbimide dose we used.     

Effects of acute ethanol administration on the hepatic xanthine dehydrogenase/oxidase system in the rat

Administration of ethanol (5 g/kg p.o.) to female Sprague-Dawley rats resulted in conversion of a portion of hepatic xanthine dehydrogenase to xanthine oxidase 12 hr after treatment. Conversion was partly reversed in vitro by treatment of hepatic 100,000 X g supernatant with dithiothreitol, whereas pretreatment of rats with pyrazole (100 mg/kg i.p.) prevented conversion 18 hr after ethanol administration. Incubation of acetaldehyde with rat liver supernatant at 37 degrees C converted xanthine dehydrogenase to xanthine oxidase in a dose-dependent manner, whereas incubation of ethanol with rat liver supernatant did not lead to conversion. Acetaldehyde-induced conversion in vitro was reversed by treatment with dithiothreitol, and was partially blocked by addition of equimolar concentrations of reduced glutathione. These data suggest that biotransformation of ethanol is required for conversion of hepatic xanthine dehydrogenase to xanthine oxidase. Because xanthine oxidase utilizes molecular oxygen to produce superoxide radical, ethanol-induced conversion of xanthine dehydrogenase to xanthine oxidase could contribute to the enhanced lipid peroxidation reported previously after administration of a single dose of ethanol.     

Effect of ethanol and acetaldehyde on intracellular protease activities in human liver, brain and muscle tissues in vitro

The effect of ethanol and acetaldehyde on the activity of a range of intracellular cytoplasmic and lysosomal proteolytic enzymes has been determined in human brain, liver and skeletal muscle tissues in vitro. There was a substantial degree of inhibition for most protease types in all tissues if sufficiently high concentrations of ethanol (10%, v/v; 1.7 mol/L) or acetaldehyde (1%, v/v; 0.17 mol/L) were used in the assay media. However, it was concluded that direct inhibition of proteases in vivo by ethanol or acetaldehyde is improbable, at the concentrations of these agents likely to pertain in vivo, and that any effect of these agents on intracellular protein catabolism must occur via a more subtle biochemical mechanism.     

Acetaldehyde,microbes, and cancer of the digestive tract

Excessive alcohol consumption and heavy smoking are the main risk factors of upper digestive tract cancer in industrialized countries. The association between heavy drinking and cancer appears to he particularly prominent in Asian individuals who have an inherited deficient ability to detoxify the first metabolite of ethanol oxidation, acetaldehyde. Alcohol itself is not carcinogenic. However, according to cell culture and animal experiments acetaldehyde is highly toxic, mutagenic, and carcinogenic. In addition to somatic cells, microbes representing normal human gut flora are also able to produce acetaldehyde from ethanol. After the ingestion of alcoholic beverages, this results in high local acetaldehyde concentrations in the saliva, gastric juice, and the contents of the large intestine. In addition, microbes may produce acetaldehyde endogenously without alcohol administration. This review summarizes the epidemiological, genetic, and biochemical evidence supporting the role of locally produced acetaldehyde in the pathogenesis of digestive tract cancer. Special emphasis is given to those factors that regulate local acetaldehyde concentration in the contents of the gastrointestinal tract. The new evidence presented in this review may open a microbiological approach to the pathogenesis of digestive tract cancer and may have an influence on future preventive strategies.