Microbes and mucosa in the regulation of intracolonic acetaldehyde concentration during ethanol challenge

— Aims: The bacteriocolonic pathway for ethanol oxidationleads to high intracolonic levels of carcinogenic acetaldehyde.The respective roles of colonic mucosal cells and gut florain the regulation of intracolonic acetaldehyde concentrationare not known. Disulfiram inhibits hepatic acetaldehyde oxidationand may have an effect on colonic mucosal cells. On the otherhand, metronidazole treatment leads to overgrowth of acetaldehyde-producingaerobic flora in the large intestine. The aim of this studywas to characterize by means of disulfiram and metronidazolethe contribution of colonic mucosal cells and intracolonic microbesto the regulation of intracolonic acetaldehyde concentrationduring ethanol oxidation in rats. Methods: Forty male Wistarrats were used. Three groups of 10 rats each received metronidazole,disulfiram, or both for 5 days, and a fourth group of 10 ratsserved as controls and did not receive any premedication. Faecalsamples were taken for the ALDH (aldehyde dehydrogenase) determinationbefore the injection of ethanol, after which all rats receivedethanol (1.5 g/kg) 2 h prior to taking samples from blood, liver,colonic mucosa and colonic contents. Results: Disulfiram decreasedsignificantly hepatic and colonic mucosal ALDH activities, andresulted in increased blood and intracolonic acetaldehyde levels.In disulfiram-treated rats, mean intracolonic acetaldehyde levelwas 8-fold higher than that in the blood. Metronidazole inhibitedonly colonic mucosal high K_M ALDH and increased intracolonic,but not blood, acetaldehyde levels. Faecal ALDH activity wasnot detectable in any of the groups. Conclusions: This studydemonstrates that during ethanol challenge, intracolonic acetaldehydelevel is regulated not only by intracolonic microbes, but alsoby colonic mucosal cells.     

INVOLVEMENT OF GENETIC POLYMORPHISM OF ALCOHOL AND ALDEHYDE DEHYDROGENASES IN INDIVIDUAL VARIATION OF ALCOHOL METABOLISM

The involvement of genetic polymorphism at the alcohol dehydrogenase2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci in determiningblood acetaldehyde levels and the rate of ethanol eliminationafter ethanol intake was investigated. Sixty-eight healthy subjectsingested 0.4 g of ethanol per kg of body weight over 10 min.Blood acetaldehyde levels scarcely increased in the subjectshomozygous for ALDH2^*I, regardless of their ADH2 genotypes (ADH2^*1/^*1,ADH2^*1/^*2 and ADH2^*2/^*2). The acetaldehyde levels in the subjectswith the ALDH2^*1/^*2 heterozygote increased to 23.4 µMon average, and no significant differences were observed betweenthe three ADH2 genotype groups. Subjects homozygous for ALDH2^*2showed very high levels of blood acetaldehyde, and the averagevalue was 79.3 µM. The values of Widmark's ß₆₀(mg/ml/hr)and ethanol elimination rate (mg/kg/br) showed significant differencesamong the three ALDH2 genotypes, and in decreasing order thevalues were ALDH2^*1/^*1, ALDH2^*1/^*2, ALDH2^*2 However, no significantdifferences were seen among the ADH2 genotypes.     

EFFECT OF ACUTE AND CHRONIC ETHANOL ADMINISTRATION ON THE TRANSPORT OF THIAMINE FROM PLASMA TO DIFFERENT BRAIN REGIONS OF THE RAT

The effect of ethanol (4.7 g/kg body wt intragastrically asa single dose or once daily for 35 days) on the transport ofthiamine from plasma to four brain regions (cerebellum, cerebralcortex, pons and medulla) was studied in albino rats. Animalswere given an intravenous injection of labelled thiamine witha sampling procedure which allowed the determination of regionalblood flow and tissue thiamine uptake. Regional blood flow wasfound to be enhanced after acute, but non chronic, ethanol administration.The magnitude of increase ranged from 13 to 35% depending onthe brain region being considered. Thiamine was transferredfrom plasma to cerebral tissue by a saturable process with anon-saturable component prevailing at thiamine concentrationsabove 10–15 µM. Three main modifications in thethiamine transport were found as a result of ethanol treatment:a reduction in affinity for the carrier (K_m increased), an increasein maximal transport rate (J_max and an increase in non-saturablediffusion (K_D constant increased). The effects were more pronouncedafter acute ethanol administration. As a consequence of thesemodifications both acute and chronic ethanol treatment causedan increase in thiamine transport rate at high plasma concentrations.On the contrary, at low (physiological) plasma concentrations,thiamine transport was little increased by acute ethanol administrationand virtually unaffected by chronic ethanol intoxication.     

ALTERATION OF ACETALDEHYDE METABOLISM IN CARBON TETRACHLORIDE-INTOXICATED RAT LIVER: ANALYSIS USING LIVER PERFUSION SYSTEM

The hepatic metabolism of acetaldehyde in carbon tetrachloride(CCl₄)-intoxicated rats was studied using a non-recirculatinghaemoglobin-free liver-perfusion system. Acetaldehyde uptakeby the liver from acutely CCl₄-treated animals (4.16 mmol/kg,i.p.) at 24 hr after the treatment was not significantly altered,whereas that by the liver from chronically CCl₄-treated animals(2.08 mmol/kg,i.p., twice a week, for 8–12 weeks) wasdecreased by approximately 50% when it was determined in thepresence of 0.01–5 mM acetaldehyde. In liver from ratschronically intoxicated with CCl₄, the following important biochemicalchanges were observed: (1) The activity of low K_m aldehyde dehydrogenase(ALDH) in hepatic mitochondria was decreased by approximately75%. (2) The basal levels of the lactate/pyruvate (cytosolic[NADH]/[NAD⁺]) ratio as well as the ß-hydroxybutyrate/acetoacetate(mitochondrial [NADH]/[NAD⁺]) ratio were elevated by more than2-fold. (3) Mitochondrial NADH oxidation was also reduced byapproximately 35% of the control level. (4) The basal levelof hepatic oxygen uptake was attenuated by approximately 50%,and the infusion of acetaldehyde (0.01–5.0 mM) causeda further decrease in the uptake. (5) The rate of ethanol productionfrom acetaldehyde by the catalytic action of alcohol dehydrogenasewas found to be unaltered when low concentrations of acetaldehyde(0.01–0.2 mM) were used, whereas a significant suppressionof the rate of ethanol production was detected in the presenceof high concentrations of acetaldehyde (0.6–5 mM). Thesedata suggest that the changes in activity of the lowK_m mitochondrialacetaldehyde dehydrogenase and those in mitochondrial NADH oxidationcoupled with mitochondrial respiration may, at least in part,play important roles in the decreased hepatic acetaldehyde metabolismobserved in chronically CCl₄-treated rats.     

AGE-RELATED DIFFERENCES IN BLOOD ETHANOL PARAMETERS AND SUBJECTIVE FEELINGS OF INTOXICATION IN HEALTHY MEN

Healthy men within 4 age groups, 20–29, 30–39, 40–49and 50–59 years (N = 12 per group), drank 0.68 g/kg ethanolas neat whisky after fasting overnight. The drink was finishedwithin 20 min and the concentrations of ethanol in capillaryblood were determined at 30/60 min intervals for 7 hr. At thetime of blood sampling, the men were asked to estimate theirfeelings of intoxication according to an arbitrary scale onwhich the score 10 indicated ‘tipsy’ or a ‘Littlehigh’. The time course of blood ethanol concentrationwas similar in all 4 groups with the curve for 50–59-year-oldmen on the highest level and 20–29-year-old men lowest.Subjective intoxication scores were significantly less in theyoungest age group and the maximum score for each subject wascorrelated with age (r = 0.45, P << 0.01). Total bodywater (TBW) was not correlated with age directly (r = 0.17,P > 0.05) but when it was expressed as percent of body weighta strong negative correlation was found (r = –0.77, P> 0.001). The mean total body water estimated from ethanoldilution was 46.7 ± 4.11. (+ SD, N = 48) and this correspondsto 58% of mean body weight of the men. The distribution volumeof ethanol/kg body weight (V_d) decreased with ageing (r = –0.59,P < 0.001) being 0.72, 0.71, 0.68 and 0.66 l/kg on averagein the 20–29, 30–39, 40–49 and 50–59-year-oldage groups respectively. The reduction of V_d corresponds toan age-related decrease in the percentage of body water andan increase in the percentage of body fat. This results in elevatedblood ethanol concentrations in older individuals for a constantdose/kg body weight. The absorption of ethanol from the gutand its rate of disappearance from blood were not markedly influencedby age. The lower subjective feelings of intoxication reportedby younger men may indicate that age-related differences inacute functional tolerance to ethanol or different thresholdvalues of blood ethanol may exist before an effect is felt.     

MEASURING AND REPORTING THE CONCENTRATION OF ACETALDEHYDE IN HUMAN BREATH

Most of the acetaldehyde generated during the metabolism ofethanol becomes tightly bound to endogenous molecules such ashaemoglobin, amino acids and certain phospholipids. Free acetaldehydepasses the blood-brain bamer and traces of this toxic metaboliteare excreted through the lungs and can be detected in the expiredair. The blood/air partition coefficient of acetaldehyde at34 %C, the average temperature of endexpired air, is about 190:1.Because of various problems associated with measuring acetaldehydein blood samples, several research groups have instead investigatedthe analysis of acetaldehyde in breath which offers an indirectand alternative approach for clinical and research purposes.However, care is needed when interpreting the results of breathacetaldehyde measurements, because of the possibility of localformation from microflora inhabiting the upper airways and mouth.The concentration of acetaldehyde exhaled in breath after drinkingalcohol demonstrates large inter-individual differences dependingon various genetic (racial) and environmental factors. Moreover,acetaldehyde is an endogenous metabolite and even without drinkingany alcohol the concentrations expelled in breath span from0.2 to 0.6 nmol/l, with higher levels observed in smokers andabstinent alcoholics. Breath acetaldehyde concentration reachedbetween 5 and 50 nmol/l in European subjects who drank a moderatedose of ethanol (0.4–0.8 g/kg), with the highest valuesseen in smokers. The concentration of breath acetaldehyde inJapanese subjects after drinking alcohol reached between 200and 500 nmol/l at the peak. These much higher levels followbecause a large proportion of Orientals (40–50%) inheritan inactive form of the low K_m mitochondrial isoenzyme of aldehydedehydrogenase (ALDH₂). The highest concentrations of breathacetaldehyde were seen in healthy Caucasians who drank a smalldose of alcohol (0.25 g/kg) after taking the alcohol-sensitizingdrug calcium carbimide, which blocks the action of ALDH isozymes.During the most intense acetaldehyde-flush reaction, breathacetaldehyde reached between 200 and 1300 nmol/l, but even theseabnormally high concentrations did not interfere with the analysisof ethanol in breath by means of non-specific infrared analyserscurrently used in many countries for testing drinking drivers.     

CHARACTERISTICS OF ALDEHYDE DEHYDROGENASES OF CERTAIN AEROBIC BACTERIA REPRESENTING HUMAN COLONIC FLORA

We have proposed the existence of a bacteriocolonic pathwayfor ethanol oxidation resulting in high intracolonic levelsof toxic and carcinogenic acetaldehyde. This study was aimedat determining the ability of the aldehyde dehydrogenases (ALDH)of aerobic bacteria representing human colonic flora to metabolizeintracolonically derived acetaldehyde. The apparent Michaelisconstant (K_m) values for acetaldehyde were determined in crudeextracts of five aerobic bacterial strains, alcohol dehydrogenase(ADH) and ALDH activities of these bacteria at conditions prevailingin the human large intestine after moderate drinking were thencompared. The effect of cyanamide, a potent inhibitor of mammalianALDH, on bacterial ALDH activity was also studied. The apparentK_m for acetaldehyde varied from 6.8 (NADP⁺ -linked ALDH of Escherichiacoli IH 13369) to 205 µM (NAD⁺ -linked ALDH of Pseudomonasaeruginosa IH 35342), and maximal velocity varied from 6 nmol/min/mg(NAD⁺ -linked ALDH of Klebsiella pneumoniae IH 35385) to 39nmol/min/mg (NAD⁺ -linked ALDH of Pseudomonas aeruginosa IH35342). At pH 7.4, and at ethanol and acetaldehyde concentrationsthat may be prevalent in the human colon after moderate drinking,ADH activity in four out of five bacterial strains were 10–50times higher than their ALDH activity. Cyanamide inhibited onlyNAD⁺ -linked ALDH activity of Pseudomonas aeruginosa IH 35342at concentrations starting from 0.1 mM. We conclude that ALDHsof the colonic aerobic bacteria are able to metabolize endogenicacetaldehyde. However, the ability of ALDHs to metabolize intracolonicacetaldehyde levels associated with alcohol drinking is ratherlow. Large differences between ADH and ALDH activities of thebacteria found in this study may contribute to the accumulationof acetaldehyde in the large intestine after moderate drinking.ALDH activities of colonic bacteria were poorly inhibited bycyanamide. This study supports the crucial role of intestinalbacteria in the accumulation of intracolonic acetaldehyde afterdrinking alcohol. Individual variations in human colonic floramay contribute to the risk of alcohol-related gastrointestinalmorbidity.     

ETHANOL AND PHOSPHATIDYLETHANOL REDUCE THE BINDING OF [3H]INOSITOL 1,4,5-TRISPHOSPHATE TO RAT CEREBELLAR MEMBRANES

In this study, we have analysed the effect of ethanol and phosphatidylethanol,a unique phospholipid formed only in the presence of ethanol,on the binding of [³H]inositol 1,4,5-trisphosphate to rat cerebellarmembranes. Rats were intraperitoneally injected daily with 3g of ethanol/kg body weight for different periods of time. Repeatedadministration of ethanol induced a reduction in the bindingcapacity (B_max) without affecting the affinity constant (K_d).A significant 32% reduction was observed after 21 days of exposure(from control B_max values of 25±3 pmol/mg and K_d valuesof 9±2 nM). In an in-vitro assay, phosphatidylethanol(500 µM) and phosphatidic acid (500 µM), but noother phospholipids tested, induced a reduction in B_max (39%and 43%, respectively). The observed effect displayed by phosphatidylethanolwas not due to its degradation to phosphatidic acid or otherphospholipids. The results emphasize the importance of examiningphosphatidylethanol (PEth) as a possible mediator of the effectsof ethanol on cellular processes. However, the role of PEthin the observed effect of long-term ethanol exposure still needsfurther consideration.     

ETHANOL INDUCES HETEROGENEOUS REDUCTION OF CYTOCHROME aa3 WITHIN PERFUSED RAT LIVER LOBULE ASSESSED USING MICROSPECTROSCOPY

The redox state of cytochrome aa₃ was measured at microspots(20µm diameter) within the lobule of perfused rat livers,using reflectance microspectroscopy, and the effect of ethanolinfusion on sublobular distribution of the redox states wasevaluated. A sigmoidal relationship was observed between oxygendelivery and the reduction of cytochrome aa₃ in both the periportaland pericentral regions of the liver lobule when the influentO₂ concentration was decreased in a graduated manner. This sigmoidalcurve was shifted to the more reduced state by ethanol infusion,with ethanol (25–100mM) increasing the degree of cytochromeaa₃ reduction in a dose-dependent manner according to the distancefrom the periportal region along a sinusoid. This increase wasspatially heterogeneous within a liver lobule. These data indicatethat ethanol accelerates cytochrome aa₃ reduction, with a distinctgradient of reduction along sinusoids but a heterogeneous distributionwithin the liver lobule.     

IDENTIFICATION AND CHARACTERISATION OF ALCOHOL-INDUCED FLUSHING IN CAUCASIAN SUBJECTS

The prevalence of the alcohol-flushing reaction was assessedin a group of healthy Caucasian medical students (200) by self-reportingand was found to occur in approximately 50% of female and 8%of male subjects. In most of the alcohol flushers there wereother family members similarly affected. The presence of thisside-effect after a small quantity of alcohol did not necessarilydecrease the amount of alcohol consumed. A test dose of ethanol(0.4 g/kg body weight) confirmed the presence of the alcohol-inducedflushing, which was of much shorter duration and intensity thanthat of the Oriental alcohol-induced flusher, as measured bylaser Doppler velocimetsy, and was not associated with highcirculating concentrations of acetaldehyde. Topical administrationof 5 M acetaldehyde showed an enhanced erythema in Caucasianflushers compared to non-flushing controls. This effect wasnot observed with topical ethanol. Low erythrocyte ALDH₁ activitywas found in all Caucasians (n = 30) who showed the alcohol-inducedflushing reaction.     

Cholinergic nerves mediate acetaldehyde action in the gastrointestinal tract

The regulation mechanism of inhibition of intestinal ethanol absorption induced by high acetaldehyde (AcH) concentration in blood was investigated. We used atropine (AT), atropine methylbromide (ATMB), pirenzepine (PI), bethanechol (BE) and pilocarpine (PL) with or without cyanamide (CY; a potent inhibitor of aldehyde dehydrogenase, which induces high AcH concentration in blood). The K(a) (absorption rate constant) value after the CY-alone pretreatment was significantly lower than that in controls. In the high AcH-induced cases, the values of K(a) in AT and ATMB pretreatments were similar to controls, but the value of K(a) in PI pretreatment was lower than that in controls. The values of K(a) in the case of BE pretreatment with and without high AcH levels were lower than in controls. The K(a) value in the PL with CY was significantly lower than that with CY alone. However, its action was blocked by ATMB pretreatment. These results suggest that high blood AcH concentrations inhibit intestinal ethanol absorption through the peripheral cholinergic nerves via muscarinic receptors, except for the muscarinic M(1) receptor, compared to other subtypes of muscarinic receptors.     

EFFECTS OF SEED SAPONINS OF THEA SINENSIS L. (RYOKUCHA SAPONIN) ON ALCOHOL ABSORPTION AND METABOLISM

We evaluated the effects of the seed saponins of Thea sinensisL. on alcohol absorption and metabolism in rats and mice. Anethanolic extract from the seeds of T. sinensis was orally administeredto the rats 1 hr before or 0.5 hr after administration of ethanol(2 g/kg), and the blood ethanol assayed 0.5, 1, 2, 3, and 4hr after ethanol administration. The ethanol level decreasedafter both pie- and post- administration of the extract. Theextract was further purified to obtain a saponin fraction whichwas orally administered to mice 1 hr before ethanol administration.Blood, liver, and stomach were obtained 0, 1, 3, and 6 hr afterethanol administration, and the ethanol, acetaldehyde, acetate,and acetone concentrations in each specimen were measured byhead space gas chromatography. The saponin fraction decreasedthe ethanol levels in the blood and liver but increased thatin the stomach five-fold over the control level, suggestinginhibition of alcohol absorption. The ethanol disappearancetime from the blood was shortened, suggesting the promotionof alcohol disappearance. The acetate and acetone levels wereunaffected. However, the acetaldehyde level decreased in theblood, liver, and stomach. The decreases in the ethanol andacetaldhyde levels in the liver suggested the protective effectsof the seed saponins on the liver. The saponins did not directlyinhibit hepalic alcohol dehydrogenase activity. The seed saponinsof T. sinensis seem to suppress alcohol absorption by slowinggastric emptying and by inhibiting absorption across the cellmembranes of the digestive tract.     

EFFECT OF CHRONIC ETHANOL ADMINISTRATION ON THIAMINE TRANSPORT IN MICROVILLOUS VESICLES OF RAT SMALL INTESTINE

The effect of long-term ethanol administration on the membranemechanism of thiamine entry in rat enterocytes was investigatedby using microvillous vesicles of small intestine. Experimentswere carried out in three groups of Wistar albino rats of bothsexes (290–400 g of initial body wt). Group I did notreceive any treatment, group II received 4.7 g of ethanol/kgbody wt (as a 50% hydroalcoholic solution) daily by gastricgavage for 35 days and group III (pair-fed controls) receiveda daily solution of saccharose (isoenergetic with the dose ofethanol administered to group II) by gastric gavage for 35 days.Ethanol or saccharose were administered in the morning and astandard diet was given throughout the treatment period. Allanimals were killed by decapitation 24 hr after the last administration,when the blood level of ethanol was virtually zero. Microvilloussmall intestinal vesicles were incubated with ³H-thiamine (³H-T)at 25°C and the amount of ³H-T taken up was measured bya rapid filtration method. Compared with data obtained in groupsI and III, chronic ethanol administration was found to inducea statistically significant decrease in ³H-T vesicular uptakeat 4 sec (determined at ³H-T concentrations ranging from 0.12to 7.5 µM) and a decrease in the apparent J_max (maximaltransport rate) value of the saturable component, without affectingthe apparent K_m (half-saturation concentration) value. These results indicate that in rats chronic ethanol administrationmay impair the intestinal absorption of thiamine by reducingthiamine entry into the enterocyte.     

THE ACUTE EFFECTS OF ETHANOL AND ACETALDEHYDE ON THE SYNTHESIS OF MIXED AND CONTRACTILE PROTEINS OF THE JEJUNUM

An investigation was made into the acute effects of ethanoland acetaldehyde with or without enzyme inhibitors of alcoholdehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase(cyanamide) on fractional rates of protein synthesis of mixedand contractile proteins of the jejunum. Ethanol decreased thefractional rates of mixed and contractile protein synthesis(i.e. k_o defined as the percentage of tissue protein renewedeach day) by -25%. Pretreatment with 4-methylpyrazole followedby treatment with ethanol further reduced mixed and contractilek_o by -30%, when compared with saline plus saline and 4-methylpyrazoleplus saline groups. The greatest reductions in k_o of mixed andcontractile proteins occurred with cyanamide pretreatment followedby ethanol treatment: mixed and contractile protein k_o in thecyanamide plus ethanol group decreased by -60% when comparedwith saline plus saline and cyanamide plus saline groups, whereask_o decreased by -45% when compared with the saline plus ethanolinjected group. Acetaldehyde treatment alone caused no significantinhibition of protein synthesis. However, 4-methylpyrazole pretreatmentplus acetaldehyde treatment significantly reduced mixed andcontractile k_o by -20% when compared with the saline group,and by -15% when compared with the 4-methylpyrazole plus salineand saline plus acetaldehyde groups. These data show that ethanolalone and perhaps high levels of acetaldehyde may be responsiblefor the inhibition of intestinal protein synthesis and relatedpathological derangements, e.g. motility disturbances due toloss of contractile proteins.     

ANTI-ACETALDEHYDE ADDUCT ANTIBODIES GENERATED BY ETHANOL-FED RATS REACT WITH REDUCED AND UNREDUCED ACETALDEHYDE-MODIFIED PROTEINS

We have previously shown that rats fed ethanol for prolongedperiods generate antibodies reactive with proteins modifiedby acetaldehyde in vitro. In this report we demonstrate thatthese antibodies react with two groups of adducts: those formedwhen acetaldehyde reacts with proteins at 37°C for 24 hr(‘unreduced’ adducts) and those formed by a 1 hrincubation followed by the addition of sodium cyanoborohydride(a reducing agent specific for Schiff bases) to the reactionmixture (reduced adducts). These data suggest that adducts fromboth of these groups are formed in vivo as a result of ethanolingestion by rats.     

PROBLEMS INVOLVED IN THE DETERMINATION OF ENDOGENOUS ACETALDEHYDE IN HUMAN BLOOD

Little is known about the possible existence of endogenous acetaldehydein human blood. This has partly been due to analytical difficultiespreventing accurate determination of blood acetaldehyde levelswith and without the presence of ethanol. In the present studythe possible existence of endogenous acetaldehyde in human bloodwas investigated with headspace gas chromatography using threedifferent procedures for the treatment of samples: (1) no treatmentof whole blood, cells, or plasma (direct headspace method),(2) hemolysation, and (3) perchloric acid (PCA) precipitation,prior to the headspace determinations. In in vilro experiments,with the direct headspace and the hemolysation methods, higheracetaldehyde peaks were obtained depending on the headspaceincubation time, temperature and ethanol concentration. Bothmethods displayed about the same values of acetaldehyde in bloodcells, ranging between 0 and 40 µM, at incubation timesbetween 15 and 180 min, an incubation temperature of 65°C,and ethanol concentrations less than 5 µM. Less acetaldehydeformation (0–15 µM) was obtained with PCA precipitatesof whole blood and cell components. Very low acetaldehyde levels(0–1 µM) were obtained in the supernatants withoutprecipitates from either whole blood or cells after headspaceequilibration. Substantially less acetaldehyde was formed inplasma preparations than with whole blood and cell fractions.In human experiments, the disturbance of endogenous or exogenousethanol was minimized by separating and washing the blood cellsfollowed by PCA treatment. No differences in acetaldehyde concentrationswere observed in blood samples taken before, during, or afterethanol intoxication (1.5 g/kg dose) of four healthy non-alcoholicvolunteers. The present results demonstrated substantial artefactualacetaldehyde formation during the analytical procedures, withthe PCA method being least problematic. No evidence was obtainedfor the release of acetaldehyde possibly bound during ethanolintoxication. Whether real endogenous acetaldehyde, especiallyin a bound form, was present initially and then released duringthe present conditions, remains also to be proven.     

ALDEHYDE DEHYDROGENASE ACTIVITY AND ACETATE PRODUCTION BY AEROBIC BACTERIA REPRESENTING THE NORMAL FLORA OF HUMAN LARGE INTESTINE

We have recently proposed the existence of a bacteriocolonicpathway for ethanol oxidation, i.e ethanol is oxidized by alcoholdehydrogenase of intestinal bacteria resulting in high intracoloniclevels of reactive and toxic acetaldehyde. This study was aimedto examine aldehyde dehydrogenase (ALDH) activity, acetaldehydeconsumption and production of acetate by aerobic bacteria (n=27),representing the normal human colonic flora. Most bacterialstrains did not show any membrane-associated aldehyde dehydrogenase,but possessed marked cytosolic NADP⁺- and NAD⁺-dependent aldehydedehydrogenase activity, ranging from 155 nmol of NAD(P)H produced/min/mgof protein to zero with acetaldehyde as substrate. NADP⁺-linkedALDH activity was significantly higher than NAD⁺-linked activityin most of the tested bacteria. In addition, aerobic bacteriametabolized acetaldehyde effectively in vitro and this couldbe inhibited by cyanamide in nearly half of the tested strains.Production of acetate from acetaldehyde ranged from 2420 nmol/10⁹colony-forming units to almost negligible. In conclusion, manyhuman aerobic colonic bacteria possess significant aldehydedehydrogenase activity and can, consequently, produce acetatefrom acetaldehyde in vitro at least under the partially aerobicconditions proposed to prevail on the colonic mucosal surface.Individual variation in the capability of colonic flora to removetoxic acetaldehyde may be one factor regulating intracolonicacetaldehyde levels, as well as the rate of bacteriocolonicpathway for ethanol oxidation.     

DIFFERENT AFFINITY OF CORTICAL GHB BINDING SITES IN SARDINIAN ALCOHOL-PREFERRING (sP) AND-NON PREFERRING (sNP) RATS

Specific gamma-hydroxybutyric acid (GHB) binding sites in corticalmembranes of selectively bred alcohol-preferring sP and alcohol-nonpreferring sNP rats were compared using [2,3³H]GHB ligand. ThesP rat line showed an increased affinity (-40% lower K_d) ofboth the high- and low-affinity sites in comparison with thesNP line. No significant difference in GHB receptor density(B_max) was detected between the two rat lines. The results raisethe possibility that differences in GHB binding sites may playa role in the genetic predisposition to ethanol preference inour rat line.     

TIME COURSE AND GENETIC VARIATION IN THE REGULATION OF CALCIUM CHANNEL ANTAGONIST BINDING SITES IN RODENT TISSUES DURING THE INDUCTION OF ETHANOL PHYSICAL DEPENDENCE AND WITHDRAWAL

Physical dependence was induced by ethanol inhalation in maleSprague-Dawley rats and, in parallel experiments, in two linesof mice (WSR and WSP) genetically selected for differentialseverity of ethanol withdrawal. In dependent rats [³H]nitrendipinebinding sites were significantly increased in cerebral cortex,cardiac and smooth muscle (vas deferens). Cerebral corticalmembranes were the first to show an increase, the B_max for nitrendipinebinding rising sharply after 3–4 days of ethanol administration,whereas binding sites in the other tissues increased after 5–6days. Nitrendipine binding affinity in all tissues was consistentlyreduced immediately preceding the rise in B_max to a new steadystate, but then returned to control values. Between 6 and 10days of ethanol exposure there was no further increase in theB_max for nitrendipine binding, and on removal of ethanol, thenumbers of nitrendipine binding sites fell precipitously tocontrol levels within 24 h of withdrawal. In the geneticallyselected mice, the up-regulation of nitrendipine binding sitesin cardiac membranes was significantly greater in the WSP line.This correlates with severity of physical signs of withdrawaland parallels previous results obtained in brain. The resultsare consistent with an increase in the synthesis and membraneinsertion of dihydropyridine sensitive calcium channel proteinsin several tissues during the induction of ethanol dependenceand suggest that in the brain this change may play a role inthe ethanol withdrawal syndrome.     

ETHANOL-INDUCIBLE CYTOCHROME P-450 ACTIVITY AND INCREASE IN ACETALDEHYDE BOUND TO MICROSOMES AFTER CHRONIC ADMINISTRATION OF ACETALDEHYDE OR ETHANOL

Chronic ethanol consumption results in acetaldehyde adduct formationwith proteins such as haemoglobin and liver proteins in vivo.Our purpose was to study the binding of acetaldehyde to livermicrosomal proteins, a site of ethanol oxidation via cytochromeP-450 (especially P-450 II E1), after chronic administrationof ethanol or acetaldehyde for 21 days to rats. The liver microsomaloxidation of 1-butanol by the ethanol-inducible P-450 also wasexamined. Acetaldehyde bound to liver microsomal proteins washigher in ethanol-fed rats compared with acetaldehyde-treatedrats (0.735 vs 0.413 nmol/mg of protein respectively). The biotransformationof n-butanol to butyraldehyde by liver microsomes was increased(by 136%) in ethanol-fed rats vs controls, whereas in acetaldehyde-treatedrats this increase was much lower (only 27%). However, in thislast group, a significant negative relationship between thequantity of acetaldehyde bound to microsomal proteins and themonooxygenase-catalyzed transformation of butanol by liver microsomeswas demonstrated (r = –0.79, P < 0.01). These resultssuggest that proteins of liver microsomes are a target for acetaldehydebinding during ethanol oxidation and such adduct formation couldimpair the oxidative properties of the alcohol-inducible cytochromeP-450.