Correlation between human erythrocyte aldehyde dehydrogenase activity and sensitivity to alcohol

Young healthy Japanese men were given 0.48 g ethanol/kg body weight orally. Those responding with a marked increase in heart rate after alcohol also exhibited facial flushing and had higher acetaldehyde levels than those not responding, in spite of similar blood alcohol levels. The activity of aldehyde dehydrogenase in erythrocytes was found tocorrelate significantly (r=?0.73, p<0.01) with the increase in heart rate after alcohol drinking. It is suggested that erythrocyte aldehyde dehydrogenase activity affects or reflects blood acetaldehyde levels and physiological response to alcohol, and may prove useful as a marker for alcohol sensitivity in Orientals.     

Molecular genetics of human aldehyde dehydrogenase.

Four non-allelic genes, which encode four different aldehyde dehydrogenase (ALDH) isozymes, have been cloned and characterized at the present time. The coding nucleotide sequences, and organization of introns and exons of these genes have been elucidated. The ALDH1 gene encodes the major cytosolic ALDH1 existing in the liver and other tissues. The genetic deficiency of this isozyme was found at a low frequency (< 10%) in both Caucasians and Orientals. The deficiency and alcohol sensitivity character are inherited together in one large Caucasian family examined. The ALDH1 gene contains two hormone response elements in its upstream 5' region. The ALDH2 gene encodes the major liver mitochondrial ALDH2 which has a very low Km for acetaldehyde. The atypical ALDH2(2) allele is common (about 30%) in Orientals; and subjects with ALDH2(2) allele, both homozygous and heterozygous status, lack ALDH activity. These individuals are alcohol sensitive and have a markedly reduced risk in developing alcoholism and alcoholic liver diseases. The ALDH3 gene produces a cytosolic ALDH3 isozyme existing in the stomach and liver carcinoma but hardly in normal liver. The ALDH3 locus is polymorphic in Orientals and presumably other populations. The ALDH5 gene, which is expressed in testes and liver, is highly polymorphic in both Caucasians and Orientals. The variation of these two loci may affect the development of alcohol-related problems.     

Distribution of disulfiram and its chief metabolites over erythrocyte cell membranes and inactivation of erythrocyte aldehyde dehydrogenase activity

The distribution of disulfiram (Antabuse over erythrocyte cell membranes and the inhibitory action of the parent drug and its metabolites on a disulfiram sensitive erythrocyte isozyme of aldehyde dehydrogenase (ALDH) was investigated in intact and haemolyzed human erythrocytes. These studies showed that not only disulfiram but also its bis(diethyldithiocarbamato) copper complex (Cu(DDC)2) were distributed over the erythrocyte cell membranes. In addition, disulfiram was the only substance examined that inactivaed erythrocyte ALDH, a reaction which was dependent on the concentration of disulfiram added.     

Inactivation of aldophosphamide by human aldehyde dehydrogenase isozyme 3

Tumors resistant to chemotherapeutic oxazaphosphorines such as cyclophosphamide often overexpress aldehyde dehydrogenase (ALDH), some isozymes of which catalyze the oxidization of aldophosphamide, an intermediate of cyclophosphamide activation, with formation of inert carboxyphosphamide. Since resistance to oxazaphosphorines can be produced in mammalian cells by transfecting them with the gene for human ALDH isozyme 3 (hALDH3), it seems possible that patients receiving therapy for solid tumors with cyclophosphamide might be protected from myelosuppression by their prior transplantation with autologous bone marrow that has been transduced with a retroviral vector causing overexpression of hALDH3. We investigated whether retroviral introduction of hALDH3 into a human leukemia cell line confers resistance to oxazaphosphorines. This was examined in the polyclonal transduced population, that is, without selecting out high expression clones. hALDH3 activity was 0.016 IU/mg protein in the transduced cells (compared with 2x10(-5) IU/mg in untransduced cells), but there was no detectable resistance to aldophosphamide-generating compounds (mafosfamide or 4-hydroperoxycyclophosphamide). The lack of protection was due, in part, to low catalytic activity of hALDH3 towards aldophosphamide, since, with NAD as cofactor, the catalytic efficiency of homogeneous, recombinant hALDH3 for aldophosphamide oxidation was shown to be about seven times lower than that of recombinant hALDH1. The two polymorphic forms of hALDH3 had identical kinetics with either benzaldehyde or aldophosphamide as substrate. Results of initial velocity measurements were consistent with an ordered sequential mechanism for ALDH1 but not for hALDH3; a kinetic mechanism for the latter is proposed, and the corresponding rate equation is presented.     

Inhibition of human erythrocyte and leukocyte aldehyde dehydrogenase activities by diethylthiocarbamic acid methyl ester. An in vivo metabolite of disulfiram

The inhibitory effects of diethylthiocarbamic acid methyl ester (DTC-Me), an in vivo metabolite of disulfiram (Antabuse), on the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activities in human erythrocytes and leukocytes were studied. ALDH assays were performed by incubating intact isolated blood cells in the presence of different concentrations of DTC-Me, using 3,4-dihydroxy-phenylacetaldehyde, the aldehyde derived from dopamine, as the substrate. DTC-Me was more selective as inhibitor of the leukocyte ALDH activity (which resembles the liver "mitochondrial" low Km ALDH), whereas both disulfiram and diethyldithiocarbamic acid, the reduced monomer of disulfiram, were more selective for the erythrocyte ALDH (which is similar to the "cytosolic" high-Km ALDH). Diethylthiocarbamic acid, the free acid of DTC-Me, was less potent than DTC-Me, and caused similar inactivation of the erythrocyte and leukocyte ALDH activities. The inhibition of ALDH by DTC-Me could not be completely restored by extensive dilution of intact or sonicated blood cell samples, which indicated that ALDH was irreversibly inhibited. Since the inhibition patterns with DTC-Me agrees with the previously reported patterns of inhibition of the high-Km and low-Km isozymes after the administration of disulfiram, the results suggest that DTC-Me might be the active in vivo inhibitory metabolite of disulfiram.     

Differences in the kinetic properties and sensitivity to inhibitors of human placental, erythrocyte, and major hepatic aldehyde dehydrogenase isoenzymes

(i) The characteristics of the major human hepatic isoenzymes of aldehyde dehydrogenase (ALDH), ALDH I and ALDH II, were compared with the ALDH activities found in human placenta and erythrocytes, (ii) In human liver biopsies, the Km of ALDH I was approximately 7 mumol/L whereas it was 32 mumol/L for ALDH II. The Vmax for ALDH I was 2-3 times greater than the ALDH II Vmax. Human liver ALDH I and II also differed in their sensitivity in inhibitors. Namely, ALDH I was less sensitive to disulfiram than the ALDH II isoenzyme. (iii) ALDH activity in human placenta and erythrocytes was much lower than in liver tissue. Kinetic data showed that placental ALDH isoenzyme had a high Km (in the millimolar range) and increased its activity raising the pH from 7.4 to 8.8, more than the hepatic ALDH I and ALDH II isoenzymes did. Erythrocyte ALDH activity presented a dual component; the smaller one was characterized by a low Km (micromolar range), whereas most of the ALDH activity showed a high Km (millimolar range). (iv) Placental ALDH was resistant to nitrefazole inhibition and was inhibited by disulfiram in a manner similar to the hepatic ALDH I isoenzyme; erythrocyte ALDH was more sensitive to the inhibitory action of disulfiram and nitrefazole. (v) It is concluded that erythrocyte and placental ALDH isoenzymes are different from the hepatic ALDH I and ALDH II forms. It is also suggested that placental and erythrocyte ALDH isoenzymes are different high-Km isoenzymes.     

Gamma-oxosulfones as aldehyde dehydrogenase inhibitors

γ-Oxosulfones as Inhibitors of Aldehyde Dehydrogenase The inhibition of aldehyde dehydrogenase by the γ-oxosulfones 1 has been studied. The rates of decomposition of the sulfones to sulfinic acids and alkenes are correlated with the inhibiting effects against aldehyde dehydrogenase.     

Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes

Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O?-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min?1 · kg?1, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H?1?O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (～5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O?-benzylguanine was within ～80% of the observed total clearance in humans after intravenous administration (15 ml · min?1 · kg?1), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.     

Non-P450 aldehyde oxidizing enzymes: the aldehyde dehydrogenase superfamily

BACKGROUND: Aldehydes are highly reactive molecules. While several non-P450 enzyme systems participate in their metabolism, one of the most important is the aldehyde dehydrogenase (ALDH) superfamily, composed of NAD(P)+-dependent enzymes that catalyze aldehyde oxidation. OBJECTIVE: This article presents a review of what is currently known about each member of the human ALDH superfamily including the pathophysiological significance of these enzymes. METHODS: Relevant literature involving all members of the human ALDH family was extensively reviewed, with the primary focus on recent and novel findings. CONCLUSION: To date, 19 ALDH genes have been identified in the human genome and mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sj?gren-Larsson syndrome, type II hyperprolinemia, gamma-hydroxybutyric aciduria and pyridoxine-dependent seizures. ALDH enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. Finally, ALDH enzymes display multiple catalytic and non-catalytic functions including ester hydrolysis, antioxidant properties, xenobiotic bioactivation and UV light absorption.     

Identification of human liver mitochondrial aldehyde dehydrogenase as a potential target for microcystin-LR

Microcystins (MCs) are hepatotoxins produced by a variety of freshwater cyanobacteria. The toxicity of these hepatotoxins is a severe health issue for both humans and livestock; MCs have been implicated in the development of liver cancer, necrosis, and even deadly intrahepatic bleeding. Microcystin-LR (MC-LR) is the MC variant most commonly encountered in a contaminated aquatic system. Thus far, MC-LR has only been shown to target the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) and it is still unknown whether MC-LR can bind and inhibit any other protein targets inside the cell. To find potential MC-LR targets, we screened a phage display library for peptide ligands that specifically recognize MC-LR. Using these peptide sequences as guides, we performed a series of bioinformatics analyses revealing that MC-LR binds human liver aldehyde dehydrogenase 2 (ALDH2) at residues 447-451. We confirmed MC-LR binding of ALDH2 via automated docking computation, which yielded results matching our experimental and bioinformatics analyses. ALDH2 dysfunction may lead to aldehyde-induced reactive oxygen species (ROS) generation and, in turn, apoptosis. Therefore, ALDH2 could potentially be a target of MC-LR associated with the process of ROS-induced apoptosis. Our current study presents a new approach to the study of interactions of biological molecules by combining phage display technology with computational methods.     

线粒体醛脱氢酶对心脏保护作用的研究进展

线粒体醛脱氢酶(ALDH2)是醛脱氢酶的亚型之一,具有脱氢酶和酯酶等多种酶功能。体内乙醇、氨基酸、生物胺、维生素、类固醇及脂质的代谢过程中会产生众多醛类物质。ALDH2在辅助因子NAD(P)+的参与下将醛类物质脱氢成为相应的羧酸,对减轻醛类物质对机体的毒性作用具有重要意义。ALDH2发挥酯酶功能则不需要辅助因子,可将羧酸酯或者其他酸转化为相应的羧酸和醇。近年的研究表明,ALDH2酶活性的下降将加重酒精、缺血等多种因素引起的心肌损伤和促进硝酸甘油耐受的发生,因此针对ALDH2开发和研制特异性激动剂,将为心脏疾病的药物防治提供新思路。     

Metabolism of malondialdehyde by rat liver aldehyde dehydrogenase

Mammalian liver contains a group of pyridine nucleotide linked aldehyde dehydrogenases [E.C. 1.2.1.3] which are present in high specific activity and possess wide substrate specificities. Malondialdehyde (MDA), a difunctional three-carbon aldehyde thought to be toxic, is generated during membrane lipid peroxidation in hepatocytes. The role of aldehyde dehydrogenase (ALDH) in the metabolism of MDA was tested in vitro with subcellular fractions and semipurified cytosolic preparations from rat livers. The cytosolic fraction accounted for virtually all of the MDA (50 microM) metabolizing activity observed in the postnuclear supernatant fraction. The rate of MDA disappearance was relatively low in the mitochondrial fraction and was not detectable in reaction mixtures which contained microsomes. Rat liver cytosol contained two ALDHs with MDA metabolizing activity. These enzymes were separated by DEAE-cellulose ion exchange chromatography and had apparent Km values of 16 microM and 128 microM for malondialdehyde. Mitochondria contained an ALDH enzyme with lower affinity (Km of 7.3 mM with NAD+) for malondialdehyde. These data show that rat liver contains at least three ALDH enzymes which oxidize malondialdehyde.     

Activation of aldehyde dehydrogenase at physiological temperatures.

The release of NADH from the enzyme.NADH complexes was rate limiting at 37 degrees, for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase. Marked substrate activation was observed at this temperature as was activation by p-(chloromercuri)benzoate. Activation of enzymic activity may be of importance in vivo.     

Role of mitochondrial aldehyde dehydrogenase in nitrate tolerance

Glyceryl trinitrate (GTN) is used in the treatment of angina pectoris and cardiac failure, but the rapid onset of GTN tolerance limits its clinical utility. Research suggests that a principal cause of tolerance is inhibition of an enzyme responsible for the production of physiologically active concentrations of NO from GTN. This enzyme has not conclusively been identified. However, the mitochondrial aldehyde dehydrogenase (ALDH2) is inhibited in GTN-tolerant tissues and produces NO2- from GTN, which is proposed to be converted to NO within mitochondria. To investigate the role of this enzyme in GTN tolerance, cumulative GTN concentration-response curves were obtained for both GTN-tolerant and -nontolerant rat aortic rings treated with the ALDH inhibitor cyanamide or the ALDH substrate propionaldehyde. Tolerance to GTN was induced using both in vivo and in vitro protocols. The in vivo protocol resulted in almost complete inhibition of ALDH2 activity and GTN biotransformation in hepatic mitochondria, indicating that long-term GTN exposure results in inactivation of the enzyme. Treatment with cyanamide or propionaldehyde caused a dose-dependent increase in the EC50 value for GTN-induced relaxation of similar magnitude in both tolerant and nontolerant aorta, suggesting that although cyanamide and propionaldehyde inhibit GTN-induced vasodilation, these inhibitors do not affect the enzyme or system involved in tolerance development to GTN. Treatment with cyanamide or propionaldehyde did not significantly inhibit 1,1-diethyl-2-hydroxy-2-nitrosohydrazine-mediated vasodilation in tolerant or nontolerant aorta, indicating that these ALDH inhibitors do not affect the downstream effectors of NO-induced vasodilation. Immunoblot analysis indicated that the majority of vascular ALDH2 is present in the cytoplasm, suggesting that mitochondrial biotransformation of GTN by ALDH2 plays a minor role in the overall vascular biotransformation of GTN by this enzyme.     

Differences in kinetic characteristics and in sensitivity to inhibitors between human and rat liver alcohol dehydrogenase and aldehyde dehydrogenase

1. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that liver alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected in both human and rat liver. The human isoenzymes corresponded to the ALDH-I and ALDH-II type. 3. In the rat, one isoenzyme had low Km and showed similar activity than in human liver but differed in their sensitivity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.     

Bioactivation of pentaerythrityl tetranitrate by mitochondrial aldehyde dehydrogenase

Mitochondrial aldehyde dehydrogenase (ALDH2) contributes to vascular bioactivation of the antianginal drugs nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN), resulting in cGMP-mediated vasodilation. Although continuous treatment with GTN results in the loss of efficacy that is presumably caused by inactivation of ALDH2, PETN does not induce vascular tolerance. To clarify the mechanisms underlying the distinct pharmacological profiles of GTN and PETN, bioactivation of the nitrates was studied with aortas isolated from ALDH2-deficient and nitrate-tolerant mice, isolated mitochondria, and purified ALDH2. Pharmacological inhibition or gene deletion of ALDH2 attenuated vasodilation to both GTN and PETN to virtually the same degree as long-term treatment with GTN, whereas treatment with PETN did not cause tolerance. Purified ALDH2 catalyzed bioactivation of PETN, assayed as activation of soluble guanylate cyclase (sGC) and formation of nitric oxide (NO). The EC(50) value of PETN for sGC activation was 2.2 ± 0.5 μM. Denitration of PETN to pentaerythrityl trinitrate was catalyzed by ALDH2 with a specific activity of 9.6 ± 0.8 nmol · min(-1) · mg(-1) and a very low apparent affinity of 94.7 ± 7.4 μM. In contrast to GTN, PETN did not cause significant inactivation of ALDH2. Our data suggest that ALDH2 catalyzes bioconversion of PETN in two distinct reactions. Besides the major denitration pathway, which occurs only at high PETN concentrations, a minor high-affinity pathway may reflect vascular bioactivation of the nitrate yielding NO. The very low rate of ALDH2 inactivation, presumably as a result of low affinity of the denitration pathway, may at least partially explain why PETN does not induce vascular tolerance.     

醛脱氢酶2与人类疾病的关系及其小分子激动剂研究

醛脱氢酶2（ALDH2）是人体内重要的抗氧化应激损伤因子之一，而较高比例的东亚人携带ALDH2失活突变基因。与ALDH2密切相关的疾病有很多，如心血管疾病、神经退行性疾病和肝脏疾病等。近期研究还发现ALDH2与铁死亡也有联系。正因如此，ALDH2逐渐成为上述相关疾病治疗的潜在靶点，研究者报道了其多个类型的小分子激动剂，展现出一定的应用前景。本文重点介绍ALDH2的结构、功能、与人类疾病的关系以及其激动剂的研究进展。