Application of a biosensor for monitoring of ethanol

An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method.     

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.     

Alcohols inhibit adipocyte basal and insulin-stimulated glucose uptake and increase the membrane lipid fluidity

Benzyl alcohol and ethanol, at aqueous concentrations that cause local anesthesia of rat sciatic nerve, affect structural and functional properties of rat adipocytes. The data strongly suggest that structurally-intact membrane lipids are required for the proper cellular uptake of glucose and for the physiologic response of adipocytes to insulin. The structure of adipocyte membrane lipids was examined with the spin label method. Isolated adipocyte ‘ghost’ membranes were labeled with the 5-nitroxide stearate spin probe I(12,3). Order parameters that are sensitive to the fluidity of the lipid environment of the incorporated probe were calculated from ESR spectra of labeled membranes. Benzyl alcohol and ethanol dramatically increased the fluidity of the adipocyte ghost membrane, as indicated by decreases in the polarity-corrected order parameter S. This concentration-dependent fluidization commenced at approx. 10 mM benzyl alcohol and progressively increased at all higher concentrations tested (up to 107 mM). S decreased approx. 5.7% at 40 mM benzyl alcohol, a change in S comparable in magnitude to that induced by a 6°C increase in the incubation temperature. Benzyl alcohol and ethanol inhibited basal glucose uptake in adipocytes and uptake maximally stimulated by insulin. Temperature-induced increases in membrane fluidity, detected with 1(12,3), that closely paralleled the fluidity effects of alcohols were associated only with increases in basal and insulin-stimulated glucose uptake. The contention that the membrane lipid fluidity plays a role in insulin action needs further study.     

Investigation and evaluation of a method for determination of ethanol with the SIRE Biosensor P100, using alcohol dehydrogenase as recognition element

A new method for rapid determination of ethanol was developed, using alcohol dehydrogenase as recognition element for the SIRE (sensors based on injection of the recognition element) Biosensor, which is an amperometric biosensor. The method was simple, fast, accurate, specific and cost-effective. The recognition element solution used was stable at least for 24 h in room temperature, and at least one month when lyophilised. The optimal potential versus the silver wire electrode, the optimal pH of the buffer and the optimal temperature of the water bath was determined to be +950 mV, 8.1 and 308 K, respectively. The optimal concentrations of alcohol dehydrogenase, BSA and NAD(+) were determined to be 200 U/ml, 20 mg/ml and 15 mM, respectively. The total analysis time was between 50 s and 4 min per analysis, depending on the concentration range. The linear range was 0-12.5 mM. The detection limit was less than 0.1 mM. The repeatability (%R.S.D.) was 3-5% (n=10). The reproducibility was 5-8% (n=5). Methanol gave no signal at all, but higher alcohols, such as propanol, pentanol and hexanol, gave significant signals, decreasing with increasing length of the carbon chain. The price for one measurement was calculated to be 0.052 euro. The results from measurements with the biosensor were compared to those from an established analysis kit for ethanol. The results correlated well (R(2)=0.9874). The concentration of ethanol in different alcoholic beverages was investigated and correlated well with the concentrations given by the manufacturers.     

Development of a disposable ethanol biosensor based on a chemically modified screen-printed electrode coated with alcohol oxidase for the analysis of beer

A disposable amperometric biosensor for the measurement of ethanol has been developed. It comprises a screen-printed carbon electrode doped with 5% cobalt phthalocyanine (CoPC-SPCE), and coated with alcohol oxidase; a permselective membrane on the surface acts as a barrier to interferents. The measurement of ethanol is based on the signal produced by H2O2, the product of the enzymatic reaction. Optimisation studies were performed using amperometry in stirred solution and the magnitude of the signal was found to be dependent on pH, enzyme loading, type of membrane and applied potential. The same technique was used to evaluate the biosensor for the determination of ethanol in samples. The results obtained compared well with the manufacturers specifications. In order to test the possibility of using the devices in the field, chronoamperometry was also used to determine ethanol in the same beer samples. The precision and recovery data again indicated that the biosensor should give reliable results under the conditions described.     

Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.     

An enzyme electrode for on-line determination of ethanol and methanol

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.     

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.     

Studies on canine gastric antrum smooth muscle: preparation and characterization of a plasma membrane enriched fraction

A method is described for preparation of large amounts of a plasma membrane (PM) enriched fraction from the smooth muscle of dog antrum. It consists of preparing microsomes, treating them with ATP + EGTA + Mg, centrifuging in 30% sucrose and then centrifuging the resulting supernatant in 15% sucrose to yield the plasma membrane enriched fraction P6. The subcellular fractions obtained at various steps during purification were characterized by: 5'-nucleotidase and phosphodiesterase I as plasma membrane markers; cytochrome c oxidase as an inner mitochondrial marker; NADPH-cytochrome c reductase as a putative endoplasmic reticulum marker; electron microscopy; polyacrylamide sodium dodecyl sulfate slab gel electrophoresis. The distribution of ATP-dependent and independent Ca uptake in presence and absence of azide and the effect of 5 mM oxalate or 25 mM phosphate on this uptake was also examined. The fraction P6 consists of mostly smooth surface vesicles 164.3 +/- 7.2 nm in diameter, has an exclusion volume of 9.7 microL/mg for [3H]inulin and 11.1 microL/mg for [3H]sucrose. P6 is maximally enriched in the ATP-dependent azide-insensitive Ca-uptake capacity and as compared with the postnuclear supernatant (S1) it shows a very small percent stimulation by oxalate and phosphate. The ATP-dependent Ca uptake by the P6 fraction occurs optimally at pH 7.0-7.4 and is much larger than the ATP-independent Ca uptake. At pH 7.1, the ATP-dependent Ca uptake occurs with a Km of 0.27 microM and a Hill coefficient greater than 2 for Ca2+. Half maximum binding of Ca2+ occurred at 300 microM Ca2+. Ca ionophores A23187 and ionomycin inhibited the ATP-dependent Ca uptake, and if added after the uptake, these caused a release of the accumulated Ca2+. From these and other data it is concluded that this PM preparation contains a Ca transport system which can lead to formation of greater than 1000-fold Ca2+ concentration gradient across the vesicle membrane in 1 min when extravesicular Ca2+ concentration is 0.3 microM. Thus this preparation is an extremely useful material for studying the mechanism of action of the Ca pump in smooth muscle plasma membrane.     

Significant pathways of hepatic ethanol metabolism.

Rat liver microsomes oxidized ethanol two to three times faster than propanol when incubated with either an NADPH- or an H2O2-generating system. In addition, solubilized, purified microsomal subfractions were found to contain protein with an electrophoretic mobility identical to rat liver catalase on SDS polyacrylamide gels, suggesting that the separation of catalase from cytochrome P-450 and other microsomal components may not be feasible. These data support the postulate that catalase is responsible for NADPH-dependent microsomal ethanol oxidation. Direct read-out techniques for pyridine nucleotides, the catalase-H2O2 complex, and cytochrome P-450 were utilized to evaluate the specificity of inhibitors of alcohol dehydrogenase (4-methylpyrazole; 4 mM) and catalase (aminotriazole; 1.0 g/kg) qualitatively in perfused rat livers. 4-Methylpyrazole and aminotriazole are specific inhibitors for alcohol dehydrogenase and catalase, respectively, under these conditions. Neither inhibitor nor a combination of them altered the mixed function oxygen of p-nitroanisole to p-nitrophenol as observed by oxygen uptake and product formation. When ethanol utilization was measured over the concentration range 20-80 mM in perfused liver, a concentration dependence was observed. At low concentrations of ethanol, ethanol oxidation was almost totally abolished by 4-methylpyrazole; however, the contribution of 4-methylpyrazole-insensitive ethanol uptake increased as a function of ethanol concentration. At 80 mM ethanol, ethanol utilization was nearly 50% methylpyrazole-insensitive. This portion of ethanol oxidation, however, was abolished by aminotriazole. The data indicate that alcohol dehydrogenase and catalase-H2O2 are responsible for hepatic ethanol oxidation. At low ethanol concentrations (less than 20 mM), alcohol dehydrogenase is predominant; however, at higher ethanol concentrations (up to 80 mM), the contribution of catalase-H2O2 to overall ethanol utilization is significant. No evidence that the endoplasmic reticulum is involved in ethanol metabolism in the perfused liver emerged from these studies.     

Development of disposable lipid biosensor for the determination of total cholesterol

A prototype chronoamperometric biosensor for the determination of total cholesterol was developed that consists of a homemade potentiostat and disposable strips immobilized with Fe(3)O(4), cholesterol oxidase (ChOx), and cholesterol esterase (ChE). The principle of sensing cholesterol is based on the detection of reduction signal of hydrogen peroxide generated in two enzymatic reactions. The co-immobilization of ChE and ChOx allows the sensor to detect both concentrations of esterified and free cholesterol. The effects of biosensor on catalyst, enzymes, applied potential, and buffer pH was investigated, and the operation conditions were optimized. The detection of cholesterol can be accomplished in one step, a 10 microL of sample was dropped onto the area of sensing strip and the reduction signal was obtained at an applied potential of -200 mV (vs. Ag/Ag(+)). The pre-reaction time was set at 15s before applying potential on the strip and the sampling time was 5s. The sensing device displays a linear response over the range of 100-400mg/dL (R(2)=0.999) for cholesteryl oleate. The coefficient variation was determined as 5.06% (N=20) for 100mg/dL cholesteryl oleate and the detection limit is 19.4 mg/dL (S/N=3). The probable interferences in bio-matrix were selected to test the selectivity and no significant response was observed in the biosensor.     

Increase in ovarian blood volume during ovulation in the gonadotropin-primed immature rat

This study quantifies ovarian blood volume in Wistar rats by measuring the optical density (414 nm) of hemoglobin in ovarian extracts and comparing this measurement to the optical density of known amounts of whole blood. Immature rats were primed with pregnant mare's serum gonadotropin (PMSG), 10 IU s.c., at 23 days of age. On Day 25, the ovulatory process was initiated by human chorionic gonadotropin (hCG), 10 IU s.c., and ova began to appear in the oviducts 10 h later. At 2-h intervals, the ovaries were extirpated and homogenized in 1.0 ml of 0.05 M tris (hydroxymethyl)aminomethane buffer (pH 7.4) for 30 s. Homogenates were centrifuged for 20 min and the supernatant fluids were analyzed with a Gilford RESPONSE UV/VIS spectrophotometer. The hemoglobin in these ovarian extracts had the same peak absorbance of 414 nm characteristic of oxyhemoglobin in whole blood taken by cardiac puncture of the rats. There was a linear relationship between the absorbance and the volume of whole blood in the samples. The volume of blood per ovary from groups of 8 rats was 0.60 +/- 0.07 microL at 0 h after hCG. The volume increased to 1.37 +/- 0.26 microL at 4 h after hCG and reached a peak of 4.55 +/- 0.72 microL at 10 h. Indomethacin treatment (0.3-10.0 mg/rat, s.c.) partially inhibited this 7-fold increase in ovarian blood volume. In conclusion, the increase in ovarian blood volume during ovulation may reflect the vasodilation and hyperemia that are characteristic of inflamed tissues.     

Immobilization of tyrosinase and alcohol oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol) with pyrrole

Immobilization of tyrosinase and alcohol oxidase is achieved in the copolymer of pyrrole with vinyl alcohol with thiophene side groups (PVATh-co-PPy) which is a newly synthesized conducting polymer. PVATh-co-PPy/alcohol oxidase and PVATh-co-PPy/tyrosinase electrodes are constructed by the entrapment of enzyme in conducting copolymer matrix during electrochemical copolymerization. For tyrosinase and alcohol oxidase enzymes, catechol and ethanol are used as the substrates, respectively. Kinetic parameters: maximum reaction rates (V(max)) and Michaelis-Menten constants (K(m)) are obtained. V(max) and K(m) are found as 2.75 micromol/(minelectrode) and 18 mM, respectively, for PVATh-co-PPy/alcohol oxidase electrode and as 0.0091micromol/(minelectrode) and 40 mM, respectively, for PVATh-co-PPy/tyrosinase electrode. Maximum temperature and pH values are investigated and found that both electrodes have a wide working range with respect to both temperature and pH. Operational and storage stabilities show that although they have limited storage stabilities, the enzyme electrodes are useful with respect to operational stabilities.     

Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation

A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).     

Cytoplasmic changes in primary cultured adult mouse sensory neurons induced by ethanol and acetaldehyde treatments.

Primary cultured sensory neurons prepared from adult mice were maintained for 8 days in vitro. Such cultures were exposed to either a range of ethanol concentrations (50-300 mM) or acetaldehyde (0.5-2 mM) in serum-free medium for up to 24 h. Treated neuronal cultures, together with untreated controls in both the presence and absence of serum, were prepared for transmission electron microscopy. Nuclear morphology was not changed following treatment with either substance at the doses studied. A number of changes were observed, however, in the cytoplasm of neurons, and these were intensified by an increase in concentration and the length of exposure. Acetaldehyde induced effects at a much lower concentration than was required to induce a response with ethanol. Myelin lamellae loosely wound around dense granular core material appeared in multivesicular bodies at low doses. The prevalence of these increased with concentrations of 100 mM ethanol and 1 mM acetaldehyde; the numbers of lamellae in each myelin figure also increased but the core material was less prominent. Electron-dense bodies were also evident at higher dosages together with evidence of vacuolation of the endoplasmic reticulum and Golgi complexes. Mitochondrial profiles similar to those in untreated neurons persisted throughout the exposure periods. The generation of these inclusions may reflect a mechanism of membrane turnover, both of internal systems and cell membrane cycling, as a response to alcohol and aldehyde treatment.     

Investigation of the effect of clinically relevant interferents on glucose monitoring using near-infrared spectroscopy

Near infrared spectroscopy (NIR) is a promising technique for continuous blood glucose monitoring for diabetic patients. Four interferents, at physiological concentrations, were introduced to study how the glucose predictions varied with a standard multivariate calibration model. Lactate and ethanol were found to interfere strongly with the glucose predictions unless they were included in the calibration models. Lactate was mistaken for glucose and gave erroneously high glucose predictions, with a dose response of 0.46 mM/mM. The presence of ethanol resulted in too low glucose predictions, with a dose response of −0.43 mM/mM. Acetaminophen, a known interferent in the glucose monitoring devices used for diabetes management today, was not found to be an interferent in NIR spectroscopy, nor was caffeine. Thus, interferents that may appear in high concentrations, such as ethanol and lactate, must be included in the calibration or model building of future NIR-based glucose measurement devices for diabetes monitoring.     

Enzyme microgels in packed-bed bioreactors with downstream amperometric detection using microfabricated interdigitated microsensor electrode arrays.

In this article, we describe the use of pH- responsive hydrogels as matrices for the immobilization of two enzymes, glucose oxidase (GOx) and glutamate oxidase (GlutOx). Spherical hydrogel beads were prepared by inverse suspension polymerization and the enzymes were immobilized by either physical entrapment or covalent immobilization within or on the hydrogel surface. Packed-bed bioreactors were prepared containing the bioactive hydrogels and these incorporated into flow injection (FI) systems for the quantitation of glucose and monosodium glutamate (MSG) respectively. The FI amperometric detector comprised a microfabricated interdigitated array within a thin-layer flow cell. For the FI manifold incorporating immobilized GOx, glucose response curves were found to be linear over the concentration range 1.8-280 mg dL(-1) (0.1-15.5 mM) with a detection limit of 1.4 mg dL(-1) (0.08 mM). Up to 20 samples can be manually analyzed per hour, with the hydrogel-GOx bioreactor exhibiting good within-day (0.19%) precision. The optimized FI manifold for MSG quantitation yielded a linear response range of up to 135 mg dL(-1) (8 mM) with a detection limit of 3.38 mg dL(-1) (0.2 mM) and a throughput of 30 samples h(-1). Analysis of commercially produced soup samples gave a within-day precision of 3.6%. Bioreactors containing these two physically entrapped enzymes retained > 60% of their initial activities after a storage period of up to 1 year.     

Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).     

Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking

Two kinds of bioelectronic gas sensors (bio-sniffer) incorporating alcohol oxidase (AOD) and aldehyde dehydrogenase (ALDH) were developed for the convenient analysis of ethanol and acetaldehyde in expired gas, respectively. The sniffer devices for gaseous ethanol and acetaldehyde were constructed by immobilizing enzyme on electrodes covered with filter paper and hydrophilic PTFE membrane, respectively. The AOD and ALDH sniffers were used in the gas phase to measure ethanol vapor from 1.0 to 500 ppm, and acetaldehyde from 0.11 to 10 ppm covering the concentration range encountered in breath after alcohol consumption. Both bio-sniffers displayed good gas selectivity which was attributed to the substrate specificity of the relevant enzymes (AOD and ALDH) as gas recognition material. From the results of physiological application, the bio-sniffers could monitor the concentration changes in breath ethanol and acetaldehyde after drinking. The ethanol and acetaldehyde concentrations in expired air from ALDH2 [-] (aldehyde dehydrogenase type 2 negative) subjects were higher than that of the ALDH2 [+] (positive) subjects. The results indicated that the lower activity of ALDH2 induced an adverse effect on ethanol metabolism, leading to ethanol and acetaldehyde remaining in the human body, even human expired air.     

New insights on the mechanism of the alcohol-induced increase in portal blood flow

Acute administration of ethanol increases portal blood flow by 40-60%. This increase in blood flow compensates for the increase in O2 consumption that follows alcohol intake and may play a protective role against hypoxic hepatocellular necrosis. We have investigated the mechanism of this hemodynamic effect of ethanol in the rat using the labeled microsphere technique. We ruled out a direct role of systemic glucagon and of acetaldehyde in mediating the increase in portal flow. However, the increase in flow is maximal at a blood ethanol concentration of 3.5 mM, corresponding to that required to achieve the Vmax of alcohol dehydrogenase, and is suppressed by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Alcohol ingestion results in zonal liver hypoxia and in increases in acetate, both of which have been shown to increase the levels of adenosine, a potent vasodilator, in blood and tissues. Ethanol produces a 400% increase in arterial adenosine. Adenosine infusion leads to a dose-dependent increase in portal blood flow of up to 100%, an effect that is suppressed by administration of 8-phenyltheophylline, an antagonist of adenosine at A1 and A2 receptors. Similarly, the ethanol-induced increase in portal blood flow is fully suppressed by 8-phenyltheophylline. In conclusion, adenosine appears to play an important role in the mechanism by which ethanol increases portal blood flow.