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Biomedical subjects

E Baraona

Publications and source records attributed to E Baraona.

At least 37 records · Page 2Linked to original sources
PubMed1995

Upstream structure of human ADH7 gene and the organ distribution of its expression.

The ADH7 gene encoding human class IV (sigma) alcohol dehydrogenase (ADH) was cloned from a Caucasian genomic DNA library and its upstream structure was determined. Moreover, the organ distribution of its expression was examined. Northern hybridization analysis with a specific probe for sigma-ADH showed that expression of the gene is organ specific rather than ubiquitous, and occurs in the stomach but not in the liver. The lack of CG rich sequence and presence of TATA and CCAAT boxes in the upstream region of ADH7 may reflect the organ specific expression. The findings that this region lacks hepatocyte nuclear factor and has only one CCAAT enhancer binding protein consensus site may account for the fact that this gene is not expressed in the liver. The upstream region had sequences which are compatible with a glucocorticoid response element, a metal binding factor-1, and an active gene regulatory protein-1, suggesting that sex hormones, zinc, and retinoic acid may be involved in the regulation of the expression of this gene.

Alcohol Dehydrogenase↗
PubMed1995

Comparison of the ADH7 gene structure in Caucasian and Japanese subjects.

The ADH 7 gene, encoding the sigma-alcohol dehydrogenase isozyme, was cloned from a Caucasian genomic DNA library. Comparison of the nucleotide sequence of its exon 7 with that of an ADH 7 previously cloned from a Japanese subject revealed a substitution of the glycine-287 in the Caucasian sigma isozyme with valine in the Japanese. Since a possible mutation at this site could account for ethnic differences in the gastric activity of this isozyme, the frequency of this change was examined in both races. The exon 7 of the ADH 7 was amplified by PCR from 7 Caucasian and 7 Japanese genomic DNA and applied to restriction fragment length polymorphism analysis, using Ava II to digest the sequence encoding glycine-287 and Mae III to digest that encoding valine-287. Regardless of ethnicity, all PCR amplicons were digested by Ava II and none by Mae III, suggesting that the ethnic difference in the 287 amino acid may represent an uncommon mutation, which does not account for the high frequency of minimal activity of the sigma-alcohol dehydrogenase in the Japanese stomach compared to those of non-Orientals.

Alcohol Dehydrogenase↗
PubMed1995

Significant increase of blood alcohol by cimetidine after repetitive drinking of small alcohol doses.

To assess effects of repetitive alcohol drinking and pre-existing first-pass metabolism on the cimetidine-induced increase in blood alcohol concentrations, 20 healthy men (aged 20 to 40) of varied ethnicity and consuming less than 60 g alcohol per week underwent baseline quantitation of first-pass metabolism of alcohol. This was followed by oral administration of 0.6 g/kg ethanol given postprandially in 3 to 4 drinks spread over 135 min, before and after cimetidine (400 mg twice a day for 7 days). Blood alcohol concentrations were determined by breath analysis. First-pass metabolism was quantified by applying Michaelis-Menten kinetics to blood alcohol curves after intravenous or oral administration of equal alcohol doses. At baseline, 15 subjects had a substantial first-pass metabolism (over one sixth of the dose); their alcohol levels increased with repeated doses with a mean peak of 27 +/- 3 mg/dl before and 39 +/- 5 after cimetidine (P < 0.01), an effect much greater and longer than after a single alcohol dose. Three subjects exceeded 50 mg/dl, the legal limit for driving in several countries. By contrast, in the five subjects with minimal first-pass metabolism, cimetidine did not increase alcohol levels. Thus, under conditions mimicking social drinking, cimetidine increased blood alcohol to concentrations known to impair psychomotor skills and they persisted at those levels over prolonged periods of time. In a minority of subjects, no such interaction was found, but their first-pass metabolism at baseline was absent or minimal and thus no inhibition by the drug was to be expected.

Adult↗
PubMed1994

Molecular cloning of human class IV alcohol dehydrogenase cDNA.

A cDNA encoding a new type of alcohol dehydrogenase was cloned from a human stomach cDNA library. PCR amplification of 5'-stretch human stomach lambda gt11 library, using degenerate inosine-containing oligonucleotide probes compatible with peptide sequences of human sigma-ADH, resulted in a single product. Subsequently, internal non-degenerate primers were constructed according to the sequences occurring in the product. By PCR with combinations of these new primers and lambda gt11 forward and reverse primers, fragments of the cDNA containing its 5' and 3' ends were amplified. The full length cDNA sequence has 1125 nucleotides with a 72% similarity to those of human class I ADH. The polypeptide sequence, predicted from the cDNA, corresponds to 373 amino acids with a high degree of similarity (96%) to fragments of sigma-ADH previously reported. Northern hybridization analysis with the specific probe for the mRNA of this protein showed that it is expressed in the human stomach but not in the liver. These data indicate that the cDNA we cloned is that of human class IV ADH.

Alcohol Dehydrogenase↗
PubMed1994

Collagen-acetaldehyde adducts in alcoholic and nonalcoholic liver diseases.

Alcoholic and, to a lesser extent, nonalcoholic patients with liver disease have serum antibodies to acetaldehyde-protein adducts produced in vitro. These antibodies presumably reflect the presence of adducts in the liver, but the protein that triggers this immune response has not been identified. To study this, we measured the reactivity of cytosolic proteins to rabbit IgG developed against a P-450 2E1-acetaldehyde adduct, isolated from alcohol-fed rats, that recognizes acetaldehyde-modified epitopes in proteins. Adducts were determined on Western blots by scanning densitometry of antibody-linked alkaline phosphatase activity in 4 normal livers and in needle biopsy specimens from subjects with liver disease, 17 alcoholic and 14 nonalcoholic. In all livers, except for a normal one, we found a reactive protein of at least 200 kD, similar to the collagen-acetaldehyde adduct we reported to be markedly increased in rats with experimentally induced cirrhosis. The immunostaining intensity in the alcoholic patients with liver disease was eightfold (p < 0.01) and that in nonalcoholic patients with liver disease was fourfold, greater (p < 0.02) than the weak staining in normal livers; it correlated with the degree of inflammation and serum AST or gamma-glutamyl transpeptidase activities. The adduct was reproduced on incubation of normal cytosolic proteins with 2.5 mmol/L acetaldehyde, whereas higher concentrations yielded many additional adducts; the adduct also reacted with IgG antibody to rat collagen type I and disappeared after digestion with collagenase, suggesting that the target protein is a form of collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1994

First pass metabolism of ethanol.

The human stomach has both low and high K(m) ADH isozymes, resulting in significant ethanol metabolism in gastric cells in vitro, and decreased bioavailability of ethanol (first pass metabolism: FPM) in vivo. Intraduodenal or intraportal infusion of amounts of ethanol equivalent to those emptied into the duodenum or disappearing from pylorus-ligated stomachs produced significantly higher blood levels than intragastric administration, whereas portal ligation had no effect, documenting the role of gastric ethanol metabolism in vivo. This "protective barrier" against the systemic effects of ethanol disappears after gastrectomy and is partly lost in the alcoholic because of accelerated gastric emptying and decreased gastric ADH activity, respectively. The latter is also lower in women than in men, at least below the age of 50. Some ADH isozymes require a relatively high ethanol concentration for optimal activity; therefore, the concentration of alcoholic beverages affects the amount metabolized. Fasting strikingly decreases FPM, most likely because of accelerated gastric emptying, resulting in shortened exposure of ethanol to gastric ADH and its more rapid intestinal absorption. Commonly used drugs, such as aspirin, acetaminophen and some H2-blockers decrease gastric ADH activity in vitro and produce increased blood alcohol levels in vivo, particularly at a low alcohol dose, equivalent to social drinking. Effects at higher ethanol dosage are still the subject of controversy. However, not all subjects display significant FPM, and published negative reports with H2 blockers do not specify whether first pass metabolism was present to begin with; some of the negative investigations also used dilute concentrations of alcohol, shown to minimize gastric metabolism. Thus, the stomach can metabolize amounts of ethanol of clinical relevance, an effect which is attenuated by various drugs, resulting in increased blood levels.

Adult↗
PubMed1993

First-pass metabolism of ethanol is predominantly gastric.

Oral consumption of alcohol results in much lower blood alcohol concentrations (BACs) than does the same dose administered intravenously, suggesting significant first-pass metabolism (FPM). The questions remain, however, (1) whether this difference truly represents FPM or simply reflects slower absorption of alcohol, and (2) if there is FPM, is it mainly of gastric or hepatic origin. To study this, rats were given the same dose alcohol (1 g/kg) by either intragastric intubation or by intravenous, intraportal, and intraduodenal infusions at a rate that mimicked the loss of alcohol from the stomach. Higher BAC levels after intravenous than intragastric alcohol indicated true FPM. Higher levels after intraportal or intraduodenal infusions (in fact, comparable to those obtained with the intravenous route) demonstrated negligible FPM when the route of delivery bypassed the stomach, yet included the liver. Furthermore, rats that had developed portosystemic shunts after ligation of the portal ven exhibited blood alcohol curves and FPM equivalent to those of sham-operated controls, indicating that FPM is not dependent on first-pass flow through the liver, but reflects gastric metabolism. The absence of significant hepatic FPM is attributable to the saturation of hepatic alcohol dehydrogenase by recirculating alcohol, resulting in no appreciable increase in metabolism secondary to newly absorbed alcohol. Finally, the in vivo gastric metabolism of alcohol in pylorus-ligated rats was demonstrated by significantly lower BACs when alcohol was administered intragastrically than when an amount identical to that lost from the ligated stomach was given intraportally. Thus, the lower BACs with oral as opposed to intravenous alcohol are not simply a consequence of slow absorption, but result from FPM occurring predominantly in the stomach.

Alcohol Dehydrogenase↗
PubMed1993

Alcohol consumption enhances fatty acid omega-oxidation, with a greater increase in male than in female rats.

Because ethanol inhibits mitochondrial fatty acid oxidation, with substantial accumulation of fatty acids in the livers of female (but not male) rats, and induces microsomal activities, we assessed possible changes in omega-oxidation. To study this, we pair-fed 24 male and 24 female littermate rats of the same age liquid diets containing 36% of energy either as ethanol or as additional carbohydrate for 4 wk. In controls, the microsomal omega-hydroxylation of lauric acid was 28% greater in female than in male rats (p < 0.05). Ethanol feeding significantly increased this activity in both genders (p < 0.01), but the rise in male rats (89%) was significantly higher than that in female rats (24%). This activity was unaffected by the presence of ethanol in the assay. The effects of ethanol were associated with increases in the content of cytochrome P-450 4A1 (as assessed in Western blots by the reactivity against a sheep antibody against P-450 4A1), and more so in male than in female rats. Despite possible competition by ethanol with the hydroxy fatty acid oxidation to dicarboxylic acids through alcohol dehydrogenase, suberic and sebacic acids accumulated significantly in the livers of alcohol-fed male rats. These effects of ethanol and gender on omega-oxidation paralleled those on the hepatic cytosolic fatty acid-binding protein and fatty acid esterification previously reported in similarly treated rats. Dicarboxylic acid products of omega-oxidation have been incriminated as mediators of similar effects by other drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effects of ethanol on prostanoid production by liver fat-storing cells.

Fat-storing cells participate in the development of alcoholic liver disease. To study possible effects of ethanol on prostaglandin metabolism by fat-storing cells, we isolated them from normal rat liver. Cultured fat-storing cells produced substantial amounts (DNA, about 2 ng/micrograms every 24 hr) of prostaglandin E2 and prostaglandin I2 (measured as 6-keto prostaglandin F1 alpha) but no significant amounts of prostaglandin F2 alpha. This production was markedly enhanced by the addition of ethanol in concentrations likely to occur in the blood during alcohol consumption. We confirmed the presence of class 1 alcohol dehydrogenase activity and isoenzymes in the cytosol of cultured fat-storing cells in their second passage. The stimulatory effect of ethanol was inhibited by 4-methylpyrazole (an alcohol dehydrogenase inhibitor), exaggerated by disulfiram (an aldehyde dehydrogenase inhibitor) and reproduced by concentrations of acetaldehyde likely to occur in the liver. Thus, our results indicate that fat-storing cells produce vasodilatory prostaglandins and that this production is enhanced by the acetaldehyde that results from the oxidation of ethanol catalized by alcohol dehydrogenase present in these cells.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Acetaldehyde adducts and autoantibodies against VLDL and LDL in alcoholics.

Alcohol consumption markedly increases the hepatic output of very low density lipoprotein (VLDL), whereas it decreases the resulting low density lipoprotein (LDL) levels and apolipoprotein B. As ethylation of apoB-lysine renders LDL immunogenic and accelerates their clearance, and as alcoholics develop antibodies against acetaldehyde-protein adducts, we searched for antibodies against lipoproteins. We measured serum IgG, IgA, and IgM titers against VLDL, LDL and high density lipoprotein (HDL) in 10 non-alcoholics and 35 recently drinking alcoholics by ELISA assay. Alcoholics had higher IgG titers than non-alcoholics against VLDL and LDL; these were higher with VLDL than LDL or HDL. Using VLDL and LDL (but not HDL) from alcoholics gave the greatest response. There was no difference in IgA and IgM reactivity. To search for acetaldehyde adducts, we measured the reactivity of VLDL, LDL, HDL, and residual serum proteins against a rabbit anti P4502E1-acetaldehyde adduct IgG, which recognizes the adducts but not the unmodified proteins (except for P4502E1). ApoB-containing lipoproteins from alcoholics (and to a lesser extent non-lipoprotein proteins) reacted with anti-adduct IgG more strongly than those of non-alcoholics. The difference was striking for VLDL, less for LDL, and not detectable for HDL. This suggests that acetaldehyde reacts with apoB prior to its secretion from the liver and that the altered VLDL are partially removed prior to their conversion to LDL. In conclusion, alcoholics develop acetaldehyde adducts in apoB-containing lipoproteins, particularly VLDL. The immune response to these neoantigens could result in accelerated clearance of VLDL and LDL and decreased conversion of VLDL to LDL.

Acetaldehyde↗
PubMed1993

Acetaldehyde-collagen adducts in N-nitrosodimethylamine-induced liver cirrhosis in rats.

Increased acetaldehyde levels have been found in non-alcoholic liver diseases and an acetaldehyde-collagen adduct has been reported in rats with CCl4-induced cirrhosis. In cytosol and microsomes of rats with cirrhosis produced by N-nitrosodimethylamine, a similar acetaldehyde-protein adduct of approximately 200 kD was recognized by rabbit IgG raised against either an in vitro produced hemocyanin-acetaldehyde adduct or an in vivo occurring P4502E1-acetaldehyde adduct isolated from alcohol-fed rats, as well as by anti-rat collagen (I) IgG. Its immune complexes contained 3 proteins that reacted with the anti-collagen IgG and were digested by collagenase: 2 proteins with molecular weights similar to procollagens alpha 1 and alpha 2, and a beta 1,2(I)-like protein which was readily produced by in vitro modification of cytosol with acetaldehyde.

Acetaldehyde↗
PubMed1992

Effects of ranitidine on blood alcohol levels after ethanol ingestion. Comparison with other H2-receptor antagonists.

OBJECTIVE: To determine whether the H2-receptor antagonist, ranitidine, which is a potent inhibitor of gastric alcohol dehydrogenase activity in vitro, increases the bioavailability of orally administered ethanol (0.3 g/kg of body weight) and to compare the resulting blood alcohol concentrations with those of two other H2-antagonists, cimetidine and famotidine, the latter of which does not inhibit gastric alcohol dehydrogenase. DESIGN: For each of the H2-receptor antagonists, a different group of subjects was used. In each group, a paired design was adopted with each subject serving as his own control. SETTING: Hospital laboratory. SUBJECTS: Normal, healthy men aged 24 to 46 years. INTERVENTION: Eight men were treated for 1 week with ranitidine (300 mg/d), six with cimetidine (1000 mg/d), and six with famotidine (40 mg/d). MEASURES: Peak blood alcohol concentrations, areas under the blood alcohol curve, first-pass metabolism, and bioavailability of orally consumed ethanol. RESULTS: Relative to baseline, ranitidine increased the mean peak concentration and the area under the curve of blood alcohol concentrations by 34% (P less than .05) and 41% (P less than .01), respectively. First-pass metabolism of ethanol was decreased from 70 +/- 10 to 31 +/- 9 mg/kg of body weight, with a corresponding increase in ethanol bioavailability of 79.6% to 92.6%. By comparison, cimetidine had even a greater effect on blood alcohol levels, while famotidine had no significant effects. CONCLUSION: Patients treated with ranitidine or cimetidine should be warned of possible functional impairments after consumption of amounts of ethanol considered safe in the absence of such therapy.

Adult↗
PubMed1992

Effect of omeprazole on gastric first-pass metabolism of ethanol.

Some commonly used H2-receptor antagonists affect gastric first-pass metabolism of ethanol and lead to unexpectedly high blood alcohol concentrations after consumption of alcohol. To investigate whether omeprazole--a substituted benzimidazole recently approved for clinical use--has a similar harmful effect, we administered a moderate dose of ethanol (0.3 g/kg body wt) orally to seven normal volunteers before and after one week of omeprazole administration (20 mg daily). No significant effect of the drug was found on either mean peak blood alcohol concentrations or on areas under the blood alcohol curve; neither did these parameters differ significantly before or after an acute dose of omeprazole (13.2 mg/kg body wt) in rats, whether ethanol (0.25 g/kg body wt) was administered intragastrically or intravenously. In vitro, omeprazole in concentrations likely to occur in the gastric lumen (0.01-1.0 mM), did not affect gastric alcohol dehydrogenase activity of humans or rats. Thus omeprazole does not affect gastric first-pass metabolism of ethanol and can be considered as a safe choice for the treatment of patients who do not refrain from alcohol consumption during therapy.

Adult↗
PubMed1992

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)

Acetaldehyde↗
PubMed1991

Effects of ethanol consumption on bioactivation and hepatotoxicity of N-nitrosodimethylamine in rats.

To study the effects of ethanol on the hepatotoxicity of N-nitrosodimethylamine (NDMA), 5 mg NDMA/kg body weight was injected intraperitoneally 3 times a week for 6 weeks into rats pair-fed liquid diets containing 36% of energy either as ethanol or as additional carbohydrates. Another group of rats was pair-fed with the same diets but injected with saline instead of NDMA. Co-administration of ethanol and NDMA produced much higher elevations of serum alanine and aspartate aminotransferase and glutamic dehydrogenase activities than the administration of either agent alone. The combined treatment also slightly increased focal necrosis, whereas other liver lesions (steatosis and fibrosis) and the functional impairment of mitochondrial respiration were not affected significantly. Microsomal low Km NDMA demethylation, as well as NDMA denitrosation, were inhibited markedly by incubation with an antibody against P450IIE1, suggesting the involvement of this alcohol-inducible P450 in both NDMA bioactivation reactions. The addition of ethanol inhibited P450-dependent demethylation and denitrosation of NDMA in liver microsomes, whereas both activities were enhanced markedly by chronic ethanol administration. At ethanol concentrations similar to those prevailing in the blood of alcohol-fed animals at the time of NDMA administration, hepatic microsomal demethylation and denitrosation remained significantly higher in ethanol-fed rats given NDMA than in controls. Our results suggest that bioactivation plays a critical role in the hepatotoxicity of NDMA and its aggravation by chronic alcohol consumption.

Alanine Transaminase↗
PubMed1991

Effects of H2-receptor antagonists on gastric alcohol dehydrogenase activity.

Inhibition of gastric alcohol dehydrogenase (ADH) activity by cimetidine results in elevated blood levels of ethanol after moderate consumption. To search for alternative H2-blockers lacking such an effect, we compared cimetidine, ranitidine, nizatidine, and famotidine. They inhibited rat gastric ADH noncompetitively, with a Ki for ethanol oxidation of 0.68 mM for cimetidine, 0.5 mM for ranitidine, 1 mM for nizatidine, and 4.5 mM for famotidine. These concentrations are higher than therapeutic plasma levels, but intracellular concentrations in the gastric mucosa (assessed with [3H]cimetidine and [14C]famotidine) were at least 10- and 2-fold greater than in the blood, respectively. These results suggests that, given at therapeutic doses in vivo, the degree of inhibition by cimetidine and ranitidine should be significant and comparable, that by nizatidine should be smaller, and that by famotidine should be negligible. These drugs also exerted either mixed or competitive inhibition of rat hepatic ADH, but the effects of cimetidine and famotidine were observed at concentrations unlikely to occur in vivo. Thus, in alcoholics and in social drinkers who require treatment with H2-receptor antagonists, famotidine might be preferable to the other H2 blockers tested.

Alcohol Dehydrogenase↗