Human brain: aldehyde dehydrogenase activity and isozyme distribution in different areas

Three human post-mortem brains were dissected into seventeen areas and assayed for aldehyde dehydrogenase (EC 1.2.1.3) activity employing two assay systems: one at 68 microM and another at 13.6 mM propionaldehyde. The levels of activity with 68 microM propionaldehyde were significantly higher in cerebellum and putamen. The same brain areas were also examined by isoelectric focusing. By this procedure two distinct bands of aldehyde dehydrogenase activity (the cytoplasmic E1 and mitochondrial E2) could be readily visualized in cerebellum and putamen while other brain areas contained mainly the mitochondrial E2 isozyme.     

Subcellular distribution of aldehyde dehydrogenase activities in human liver

The subcellular distributions of aldehyde dehydrogenase activities towards acetaldehyde have been determined in wedge-biopsy samples of human liver. A form with Km values of less than 1 microM and 285 microM towards acetaldehyde and NAD+ respectively was present in the mitochondrial fraction. This enzyme had no detectable activity towards N-tele-methylimidazole acetaldehyde, the aldehyde derived from the oxidation of N-tele-methylhistamine. The activity in the cytosol was more sensitive to inhibition by disulfiram and had Km values of 270 microM and 25 microM for acetaldehyde and NAD+, respectively. It was active towards N-tele-methylimidazole acetaldehyde with a Km value of 2.5 microM and a maximum velocity that was 40% of that determined with acetaldehyde. Both these cytosolic activities had alkaline pH optima.     

Determination of aldehyde dehydrogenase isozyme activity in human liver

As acetaldehyde (Ac-CHO) has been implicated as a cause of alcoholic liver injury, accurate knowledge concerning changes in the Ac-CHO oxidizing system in human liver is essential for the understanding of the pathogenesis. However, an assay system for aldehyde dehydrogenase (ALDH: EC 1.2, 1.3) isozymes in human biological material has not yet been established. In the present study, the assay systems for human liver ALDH isozyme activity were analyzed. In human red blood cells, in which only one type of ALDH isozyme, high Km ALDH, is present, a maximum activity was observed at a substrate concentration of over 300 microM. In human liver of the usual type in which ALDH I (low Km isozyme) was not deficient, the activity reached a first plateau at 12 microM Ac-CHO after which the activity started to increase again at 20 microM Ac-CHO and continued to increase until 5.0 mM Ac-CHO. In the liver of the unusual type, which is deficient in low Km ALDH, activity was not detected at Ac-CHO concentrations lower than 10 microM. These results indicate that the optimum substrate concentrations for the determination of ALDH isozymes are 12 microM for low Km, 300 microM for high Km and over 1 mM for very high Km ALDH isozymes. The maximum activities of these three isozymes in the liver were obtained at a pH ranging between 9.0-9.5 and at an NAD concentration of over 500 microM. From these results, it is concluded that the assay system of Blair and Bodley is applicable for the determination of ALDH isozyme activity in human biological material with the exception of determining Km values.     

Methodological aspects of aldehyde dehydrogenase assay by spectrophotometric technique

Aldehyde dehydrogenase (ALDH) activity was assayed spectrophotometrically by measuring the increase in delta A at 340 nm, as a criteria of NAD conversion to NADH in the presence of propionaldehyde. The effect of pH and substrate(s) concentration of nonenzymatic increase in absorbance at 340 nm was studied. Results indicate that the increase in absorbance at 340 nm is not entirely due to NAD conversion to NADH. It was observed that nonenzymatic interaction of NAD and aldehyde could as well result in increase in absorbance at 340 nm. The magnitude of the nonenzymatic contribution towards increase in absorbance at 340 nm is found to be pH, substrate(s) conc., and time dependent. Further, the observed nonenzymatic reaction product was found to be different from that of NADH as confirmed by u.v. spectral characteristics (lambda max. 346 nm) and its inability to activate NADH/NADPH-dependent glutathione reductase. Based on these findings, a final assay method comprising a substrate blank consisting of NAD and aldehyde, and the assay pH of 7.4 is recommended for measuring the ALDH activity. Further, under these experimental conditions the Km value of human RBC ALDH was found to be 0.59 mM for propionaldehyde substrate.     

Inactivation of aldehyde dehydrogenase in intact rat liver mitochondria by dopamine

Aldehyde dehydrogenase (EC 1.2.1.3) within isolated rat liver mitochondria was inactivated by incubation with dopamine. Concurrent with this inactivation, incorporation of radioactivity from 14C-labelled dopamine into three mitochondrial matrix proteins (subunit mol.wt. = 120,000; 54,000; 20,000 daltons) occurred. The 54,000 mol.wt. protein also interacted with antihuman mitochondrial aldehyde dehydrogenase antibody. Inactivation of aldehyde dehydrogenase by dopamine occurred more readily in females than in males. Use of monoamine oxidase (EC 1.4.3.4) inhibitors (deprenyl and clorgyline) partially protected against inactivation. Monoamine oxidase catalyzed conversion of to 3,4-dihydroxyphenylacetaldehyde only partially accounted for aldehyde dehydrogenase activity loss.     

Aldehyde dehydrogenase content and composition of human liver

Human liver contains only four proteins which catalyze dehydrogenation of acetaldehyde; two of these are tetrameric with MW of 220,000 and are structurally related. These enzymes were purified previously to homogeneity and are now known as the cytoplasmic E1 and mitochondrial E2. The other two proteins do not appear to be structurally related to E1 and E2. The recently isolated E4 enzyme is a dimer of MW of ca. 175,000; the E3 may be a polymorphic enzyme. The Enzyme Commission classification for E1 and E2 is EC 1.2.1.3, that for E4 is at present uncertain since its Michaelis constants for short chain aldehydes are high, making it unlikely that these would be its natural substrates. The relationship between E3 and E4 is also uncertain. Employing a suitably designed assay, E1 and E2 are assayed as "low Km" enzymes while E3 and E4 are assayed as "high Km" enzymes. Therefore by employing such an assay, combined with electrofocusing procedure, an assessment of enzyme content and composition of aldehyde dehydrogenase in human liver can be made.     

Acetaldehyde metabolism by brain mitochondria from UChA and UChB rats

The acetaldehyde (AcH) oxidizing capacity of total brain homogenates from the genetically high-ethanol consumer (UChB) appeared to be greater than that of the low-ethanol consumer (UChA) rats. To gain further information about this strain difference, the activity of aldehyde dehydrogenase (AIDH) in different subcellular fractions of whole brain homogenates from naive UChA and UChB rat strains of both sexes has been studied by measuring the rate of AcH disappearance and by following the reduction of NAD to NADH. The results demonstrated that the higher capacity of brain homogenates from UChB rats to oxidize AcH when compared to UChA ones was because the UChB mitochondrial low Km AIDH exhibits a much greater affinity for NAD than that of the UChA rats, as evidenced by four- to fivefold differences in the Km values for NAD. But the dehydrogenases from both strains exhibited a similar maximum rate at saturating NAD concentrations. Because intact brain mitochondria isolated from UChB rats oxidized AcH at a higher rate than did mitochondria from UChA rats only in state 4, but not in state 3, this strain difference in AIDH activity might be restricted in vivo to NAD disposition.     

Catalase mediated conversion of cyanamide to an inhibitor of aldehyde dehydrogenase

A minor pathway for cyanamide metabolism catalyzed by catalase is responsible for the conversion of cyanamide to an inhibitor of aldehyde dehydrogenase. Catalase itself is also inhibited by cyanamide. Both the activation of cyanamide by catalase and the inhibition of catalase by cyanamide were blocked in vivo by ethanol pretreatment, suggesting that these two processes are closely linked. Like other catalase oxidation reactions, the catalase mediated activation of cyanamide was inhibited by 3-amino-1,2,4-triazole in vivo and sodium azide in vitro. The relative formation of the active cyanamide metabolite was assessed in vitro by following the loss of yeast aldehyde dehydrogenase activity with time. Inhibition of the yeast enzyme by activated cyanamide was dependent on NAD+ or NADP+, a requirement not fulfilled by NADH or NADPH. Although H2O2 inhibited yeast aldehyde dehydrogenase in vitro and cyanamide inhibited hepatic catalase in vivo, the possible in hepatic H2O2 concentration following cyanamide administration does not account for the effects of cyanamide on ethanol metabolism. While the cyanamide activating enzyme has been identified as catalase, the reaction products of this reaction and, in particular, the structure of the active metabolite involved in the inhibition of aldehyde dehydrogenase remain unknown.     

Detection of aldehyde dehydrogenase deficiency in Chachi Indians,Ecuador

The activity of rnitochondrial aldehyde dehydrogenase (ALDH2) was tested by isoelectric focusing of hair root extracts from 50 Chachi Indians (Ecuador). Quality of extracts and the intactness of cytoplasmic and mitochondrial enzymes were ascertained by assaying; of phosphoglucomutase (PGM) and malate dehydrogenase (MDH) in the same extracts. Three of the 39 successfully assayed Chachi Indian samples showed virtual absence of the ALDH2 band on the isoelectropherogram, and the control enzymes were stained normally in these subjects. These data confirm the existence of a mitochondrial ALDH deficiency among South American Indians. The molecular origin of the ALDH2 deficiency in this population is unknown.     

Use of an "acetaldehyde clamp" in the determination of low-KM aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells.

The high-affinity (K(M)<1 microM) mitochondrial class 2 aldehyde dehydrogenase (ALDH2) metabolizes most of the acetaldehyde generated in the hepatic oxidation of ethanol. H4-II-E-C3 rat hepatoma cells have been found to express ALDH2. We report a method to assess ALDH2 activity in intact hepatoma cells that does not require mitochondrial isolation. To determine only the high-affinity ALDH2 activity it is necessary to keep constant low concentrations of acetaldehyde in the cells to minimize its metabolism by high-K(M) aldehyde dehydrogenases. To maintain both low and constant concentrations of acetaldehyde we used an "acetaldehyde clamp," which keeps acetaldehyde at a concentration of 4.2+/-0.4 microM. The clamp is attained by addition of excess yeast alcohol dehydrogenase, 14C-ethanol, and oxidized form of nicotinamide adenine dinucleotide (NAD(+)) to the hepatoma cell culture medium. The concentration of 14C-acetaldehyde attained follows the equilibrium constant of the alcohol dehydrogenase reaction. Thus, 14C-acetate is generated virtually by the low-K(M) aldehyde dehydrogenase activity. 14C-acetate is separated from the culture medium by an anionic resin and its radioactivity is determined. We showed that (1) acetate production is linear for 120 min, (2) addition of 160 microM cyanamide to the culture medium leads to a 75%-80% reduction of acetate generated, and (3) ALDH2 activity is dependent on cell-to-cell contact and increases after cells reach confluence. The clamp system allows the determination of ALDH2 activity in less than one million H4-II-E-C3 rat hepatoma cells. The specificity and sensitivity of the "acetaldehyde clamp" assay should be of value in evaluation of the effects of new agents that modify Aldh2 gene expression, as well as in the study of ALDH2 regulation in intact cells.     

Developmental profile of hepatic alcohol and aldehyde dehydrogenase activities in long-sleep and short-sleep mice

Ethanol is metabolized primarily in the liver by a cytosolic alcohol dehydrogenase (ADH). The product, acetaldehyde, is metabolized to acetate by nonspecific aldehyde dehydrogenases (AHD). Mouse liver contains five major constitutive AHD isoenzymes: mitochondrial high Km (AHD-1), mitochondrial low Km (AHD-5), cytosolic high Km (AHD-7), cytosolic low Km (AHD-2) and microsomal high Km (AHD-3). The Long-Sleep (LS) and Short-Sleep (SS) mice differ in their sleep time response to ethanol as early as 10 days of age, and this difference increases with increasing age. Age- and genotype-related differences in metabolism could account for the pattern of responses seen in these mice. We measured the activity of hepatic ADH and the five AHD isoenzymes in LS and SS mice from 3 days of age to adulthood to determine if there were differences in the developmental profiles of these enzyme activities. We found no sex differences in the developmental profile of either ADH or AHD, and the LS and SS mice have nearly identical ADH and AHD activities with the possible exception of the high Km mitochondrial enzyme activity between days 3 and 6, and the low Km mitochondrial enzyme between days 28 and 32. Thus, it appears that differences in ethanol or acetaldehyde metabolism do not contribute significantly to the differential sensitivity to ethanol between young LS and SS mice or to the differential sensitivity between young and adult mice.     

ISOENZYMES OF HUMAN LIVER ALCOHOL DEHYDROGENASE AND ALDEHYDE DEHYDROGENASE IN ALCOHOLIC AND NON-ALCOHOLIC SUBJECTS

Liver biopsy samples from 45 patients 21 of whom were alcoholic,were used to study the isoenzymes of alcohol dehydrogenase andaldehyde dehydrogenase. The isoenzymes were separated by polyacrylamidegel isoelectric focusing in the pH range 9–11 for alcoholdehydrogenase and 3.5–8.5 for aldehyde dehydrogenase andthe gels were specifically stained for enzyme activity. Up to5 forms of aldehyde dehydrogenase and 8 forms of alcohol dehydrogenasecould be separated by these procedures. Although the isoenzymepatterns differed considerably between individuals there wereno consistent differences between alcoholics and non-alcoholics,neither was there any correlation with severity of the liverdisease. These results do not support the view that an individual'scomplement of these isoenzymes is an important determining factorin alcoholism.     

Characterization of alpha-ketobutyrate metabolism in rat tissues: effects of dietary protein and fasting

The oxidative decarboxylation of alpha-ketobutyrate was studied in rat tissue preparations. Decarboxylation was confined to the mitochondrial fraction and required coenzyme A, NAD, TPP and FAD for optimal activity in solubilized preparations. The pH optimum for this reaction in liver was 7.8, somewhat higher than that reported for other alpha-keto acid dehydrogenases. An apparent Km of 0.63 mM for alpha-ketobutyrate was determined for the rat liver system. Competition by other alpha-keto acids at 10 mM concentrations inhibited enzyme activity up to 75%. Tissue distribution of alpha-ketobutyrate dehydrogenase activity relative to liver activity was (in percent): liver, 100; heart, 127; brain, 63; kidney, 57; skeletal muscle, 38; and small intestine, 7. Total liver alpha-ketobutyrate dehydrogenase was decreased by 40% after a 24-hour fast. Similar results were found for kidney and heart activity. alpha-Aminobutyrate-pyruvate aminotransferase activity in liver or kidney was not affected by fasting; however, it was induced in liver by 50% after feeding a 40% casein diet for 10 days compared to rats fed a 20% casein diet. Increasing the dietary casein content from 6 through 40% of the diet resulted in about a fivefold increase in liver alpha-ketobutyrate dehydrogenase activity. The substantial extrahepatic capacity for alpha-ketobutyrate metabolism makes it unlikely that a loss of liver function results in an inability to metabolize alpha-ketobutyrate. Whether alpha-ketobutyrate is decarboxylated by a specific enzyme or by an already characterized complex such as pyruvate dehydrogenase or the branched-chain keto acid dehydrogenase remains to be established.     

Effects of benzodiazepines on aldehyde dehydrogenase activity

The reported ability of benzodiazepines to increase human erythrocyte aldehyde dehydrogenase (ALDH) activity and reverse the disulfiram-induced inhibition of ALDH was reexamined. When ALDH activity assays were carried out spectrophotometrically on a hemoglobin-free lysate of human erythrocytes with propionaldehyde as substrate, addition of diazepam (10 μmol/1) did not affect the enzyme activity. When assays were carried out on intact or hemolysed erythrocytes using high performance liquid chromatographic technique with 3,4-dihydroxyphenylacetaldehyde as substrate, no significant increase in erythrocyte ALDH activity was found in the presence of chlordiazepoxide, oxazepam, diazepam, or desmethyldiazepam in the concentration range 1–20 μmol/1. Rather, a significant decrease (about 50%) in activity was obtained when lysed cells were incubated with 20 μmol/1 chlordiazepoxide. Diazepam inhibited the rat liver mitochondria! low Km ALDH activity by about 50%. Disulfiram inhibited the ALDH activity almost completely in assays on human erythrocyte or rat liver mitochondrial ALDH. The ALDH activity was not regained by the subsequent addition of diazepam, nor was the effect of disulfiram reduced when diazepam was added prior to disulfiram. In an alcoholic subject who was followed during onset of disulfiram (Antabuse) therapy, the concurrent use of diazepam did not prevent a rapid decline in blood ALDH activity. The present results suggest that benzodiazepines do not increase ALDH activity in vitro, nor interfere with the inhibition of ALDH by disulfiram.     

Aldehyde dehydrogenase and monoamine oxidase in rat liver mitochondria

Monoamine oxidase (EC 1.4.3.4) and aldehyde dehydrogenase (EC 1.2.1.3) activities were compared in the liver mitochondria of male and female rats. Monoamine oxidase activity using benzylamine as a substrate was significantly higher in males as compared with females: 1.45 versus 0.74 mumols/mg mitochondrial protein/hr, respectively. Monoamine oxidase activity using tyramine as a substrate and aldehyde dehydrogenase activity were the same in males and females. Monoamine oxidase-tyramine and aldehyde dehydrogenase activities did not vary with the different phases of the estrous cycle in the female but the activity of monoamine oxidase-benzylamine did; rats in the proestrous phase had the highest activity and those in the estrous phase had the lowest.     

Properties of rat retina alcohol dehydrogenase

The rat eye fraction, including retina, pigment epithelium and choroid, contains an alcohol dehydrogenase (ADH) isoenzyme that is not present in rat liver. Starch gel electrophoresis of retina ADH shows an anodic band that can be visualized by activity staining, using either ethanol or pentanol as substrates. Ethanol is a poor substrate (Km: 336 mM, at pH 10.0) for the purified retina ADH which prefers long chain, 2-unsaturated and aromatic alcohols. The enzyme has a pH optimum of 10.0 for ethanol oxidation and it is inhibited by 4-methylpyrazole (KI: 10 microM). Electrophoretic and kinetic properties clearly differentiate the retina ADH from the hepatic cathodic ADH isoenzymes and from an anodic chi-ADH-like form that we have also detected in rat liver. At the pH and ethanol concentrations found "in vivo," retina ADH can oxidize ethanol to an appreciable extent. The subsequent production of acetaldehyde and redox change may be responsible for visual disorders during alcohol intoxication.     

Niacin deficiency and cancer in women.

A new interest in the relationship between niacin and cancer has evolved from the discovery that the principal form of this vitamin, NAD, is consumed as a substrate in ADP-ribose transfer reactions. Poly(ADP-ribose) polymerase, an enzyme activated by DNA strand breaks, is the ADP-ribosyltransferase of greatest interest with regard to effects on the niacin status of cells since its Km for NAD is high, and its activity can deplete NAD. Studies of the consequences of DNA damage in cultured mouse and human cells as a function of niacin status have supported the hypothesis that niacin may be a protective factor that limits carcinogenic events. To test this hypothesis in humans, we used a biochemical method based on the observation that as niacin nutriture decreases, NAD readily declines and NADP remains relatively constant. This has been demonstrated in both fibroblasts and in whole blood from humans. Thus, we use “niacin number,” (NAD/NAD+NADP) × 100% from whole blood, as a measure of niacin status. Healthy control subjects showed a mean niacin number of 62.8 +/? 3.0 compared to 64.0 for individuals on a niacin-controlled diet. Analyses of women in the Malmö Diet and Cancer Study showed a mean niacin number of 60.4 with a range of 44 to 75. The distribution of niacin status in this population was nongaussian, with an unpredictably large number of individuals having low values.     

Enzyme analyses of Bulinus africanus group snails (Mollusca: Planorbidae) from Tanzania.

38 population samples of snails of the Bulinus africanus group, collected from three separate areas of Tanzania, have been examined. Enzymes in crude digestive gland extracts of individual snails have been analysed by isoelectric focusing in polyacrylamide gels. The enzymes studied were: malate dehydrogenase (MDH); phosphoglucomutase (PGM); glucosephosphate isomerase (GPI); acid phosphatase (AcP) and hydroxybutyrate dehydrogenase (HBDH). Samples of B. nasutus were clearly differentiated from other species and enzyme differences were apparent between samples from the lake and coastal areas. Similarly, although clear distinctions could not always be made, samples of B. africanus, B. globosus and B. ugandae were characterized by their enzyme types. Individual variation was detected within populations and the significance of enzyme polymorphisms in relation to identification has been considered. No correlation was found between snail enzyme type and susceptibility to Schistosoma haematobium or S. bovis.     

The effect of dietary guar gum on the activities of some key enzymes of carbohydrate and lipid metabolism in mouse liver

The effects of a 100 g/kg substitution of guar gum on the body-weight gain, food consumption and faecal dry weight of mice fed on a high-sucrose diet and on the activities of hepatic glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (EC 1.1.1.40), ATP-citrate (pro-3S)-lyase (EC 4.1.3.8), 6-phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40) and fructose-1, 6-bisphosphatase (EC 3.1.3.11) were studied. Guar gum had no effect on body-weight gain or food consumption but increased faecal dry weight. Guar gum increased the activities of glucose-6-phosphate dehydrogenase, malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) and 6-phosphofructokinase expressed on a wet-liver-weight basis. Guar gum increased the activities of glucose-6-phosphate dehydrogenase, malate dehydrogenase (oxaloacetate-decarboxylating)(NADP+), ATP-citrate (pro-3S)-lyase and 6-phosphofructokinase expressed on a liver-protein basis. Guar gum increased the activities of glucose-6-phosphate dehydrogenase and malate dehydrogenase (oxaloacetate-decarboxylating)(NADP+) expressed on a body-weight basis. These results suggest that guar gum increases the flux through some pathways of hepatic lipogenesis when mice are fed on high-sucrose diets.     

Basis of the stereospecific preference of porcine kidney fibroblasts for D-2-hydroxy-4-methylthiobutanoic acid as a source of methionine

In previous studies we have found that porcine kidney fibroblasts will grow in medium containing D-2-hydroxy-4-methylthiobutanoic acid (D-methionine hydroxy analogue, D-MHA) as the sole source of methionine but not in medium containing the L-isomer (L-MHA) alone. The fibroblasts have been found to have both D-2-hydroxy acid dehydrogenase (EC 1.1.99.6), which uses D-MHA as substrate (Km = 6.0 mM) and L-2-hydroxy acid oxidase (EC 1.1.3.1), which uses L-MHA as substrate (Km = 7.1 mM). These two activities should make it possible for the fibroblasts to grow on either isomer. Only one protein band with L-2-hydroxy acid oxidase activity can be detected with enzyme-specific staining of protein profiles obtained after polyacrylamide gel electrophoresis. The enzyme L-2-hydroxy acid oxidase from porcine kidney has properties that are different from the two porcine isozymes reported previously by others. Passage of DL-[14C]MHA at tracer levels into the porcine kidney fibroblasts in culture is reduced to 31% of control in the presence of 3.75 mM D-MHA, 86% of control with 3.75 mM L-MHA and 65% with 3.75 mM D-lactate but is not affected by up to 3.75 mM L-lactate. It appears that the transport specificity is the basis for the growth promotion of kidney fibroblasts by the D-isomer of MHA as opposed to L-MHA when each is used as the sole source of methionine.