Erythrocyte Aldehyde Dehydrogenase Activity Research Articles

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.

Determination of Erythrocyte Aldehyde Dehydrogenase Activity by High-Performance Liquid Chromatography

An ion-paired high-performance liquid chromatographic method was developed for the determination of erythrocyte aldehyde dehydrogenase activity. This method does not require pretreatment to remove hemoglobin from the hemolysate before enzyme reaction is initiated. In addition, the advantage with this high-performance liquid chromatographic method is that erythrocyte aldehyde dehydrogenase activity can be measured using whole blood without either separation or washing of erythrocytes. Therefore, it can be easily used in the determination of erythrocyte aldehyde dehydrogenase activity, which is an indication of excess alcohol consumption.

Diazepam- and chlordiazepoxide-mediated increases in erythrocyte aldehyde dehydrogenase activity and its possible implications

Erythrocyte ALDH activity was assayed in alcoholic ( n = 70) and nonalcoholic ( n = 40) subjects. In general, alcoholics without any prior medications ( n = 57) were found to have a decreased ALDH activity (mean ± SD: 3.38 ± 1.7 mU; p < 0.001) as compared to control group (5.10 ± 1.57 mU). However, a group of alcoholics who were detoxified with benzodiazepines ( n = 13) prior to blood collection for enzyme assay were found to have higher ALDH activity (4.92 ± 2.46 mU; p < 0.05) as compared to alcoholics who were not detoxified. In vitro experiments demonstrated that both diazepam (DZM) and chlordiazepoxide (CDP) could activate the ALDH. The magnitude of enzyme activation by DZM and CDP appear to correlate with their relative potency of tranquilizing effect. Further, the observed ability of DZM to reverse the inhibition of ALDH mediated by disulfiram may explain the biochemical basis of the reported ability of benzodiazepines (BDZ) to reduce the intensity of disulfiram ethanol reaction (DER).

Atherosclerosis and acetaldehyde metabolism in blood

Acetaldehyde elimination in blood homogenates and erythrocyte aldehyde dehydrogenase (ALDH) activity were studied in 64 patients operated before the age of 60 years because of symptomatic stenosis of aorta, iliac, or carotid arteries and in 38 healthy controls. The disappearance of acetaldehyde in blood homogenates was biphasic. Patients showed an enhanced elimination of acetaldehyde during the second phase (30-60 min), as compared to controls (T1/2 of acetaldehyde was 103 +/- 47 and 198 +/- 93 min, respectively, P less than 0.001). No correlation was found between ALDH activity and acetaldehyde elimination rate. Acetaldehyde elimination in blood homogenates and [14C]acetaldehyde binding to plasma proteins, hemoglobin, and erythrocyte membranes were studied in 10 patients with atherosclerotic disease and in 12 healthy controls. There was a significant correlation between unstable binding of [14C]acetaldehyde to plasma proteins and the half-life of acetaldehyde in the elimination test (p = 0.74, P less than 0.005). Fractionation of plasma proteins after incubation with [14C]acetaldehyde revealed no difference between patients and controls in the distribution of radioactivity. The binding of [14C]acetaldehyde to hemoglobin or erythrocyte membranes did not differ between patients and controls. These results indicate that patients with angiopathy and an enhanced acetaldehyde elimination in blood have reduced binding of acetaldehyde to plasma proteins. As unstable binding of acetaldehyde to proteins is known to involve free amino groups of amino acid residues, modification of these residues in atherosclerotic disease is conceivable.

Erythrocyte aldehyde dehydrogenase activity in alcoholics and non-alcoholics and its value as a Biochemical marker in alcoholism

Erythrocyte aldehyde dehydrogenase (ALDH) activity in a normal reference population from North India was determined from results of 22 healthy subjects (12 male and 10 female). We compared these data with results of 19 alcoholic patients (13 male and 6 female). The mean erythrocyte ALDH activity of male and female alcohol abusers was 16.2% and 20.9% lower than the mean values for healthy subjects, respectively (P<0.001 and <0.01). It is proposed that erythrocyte ALDH activity may be a useful clinical chemical marker of alcohol abuse and alcoholism.

Dose-effect relationship of disulfiram in human volunteers. II: A study of the relation between the disulfiram-alcohol reaction and plasma concentrations of acetaldehyde, diethyldithiocarbamic acid methyl ester, and erythrocyte aldehyde dehydrogenase activity.

The study was designed to elucidate the basic pharmacological and biochemical effects of the disulfiram dose (Antabus) provoking disulfiram-alcohol reaction (DAR) in 52 human volunteers after ethanol challenge. Disulfiram was given daily in increasing doses (1, 100, 200, and 300 mg) in successive 14 day periods, with ethanol challenge at the end of each period, until a DAR was achieved. Irrespective of dose (except the 1 mg dose), the DAR was always accompanied by almost complete inactivation (about 97%) of aldehyde dehydrogenase (ALDH) activity in erythrocytes, plasma concentrations of diethyldithiocarbamic acid methyl ester (Me-DDC) in the range of 8-472 nmol/l and accumulated plasma concentrations of acetaldehyde in the range of 7-197 mumol/l. In four of the volunteers, the cardiovascular effects of the DAR were recorded as a decrease in diastolic blood pressure (14-47 mmHg) and an increase in pulse rate (9-40 beats/min.), accompanied by a two- to fourfold increase in the plasma concentrations of adrenaline and noradrenaline. The enzyme kinetics of ALDH in erythrocytes were regularly analysed in eight volunteers during DSF intake. In addition to the expected decrease in oxidizing capacity, the Km values were also impaired, which suggests that the inhibitor is implicated in an active site directed reaction.

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 K m , of ALDH I was approximately 7 μmol/L whereas it was 32 μ mol/L for ALDH II. The V max , for ALDH I was 2–3 times greater than the ALDH II V max . 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 K m (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 K m (mumolar range), whereas most of the ALDH activity showed a high K m (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- K m isoenzymes.

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.

Inhibition of erythrocyte aldehyde dehydrogenase activity and elimination kinetics of diethyldithiocarbamic acid methyl ester and its monothio analogue after administration of single and repeated doses of disulfiram to man

The elimination kinetics of the methyl esters of diethyldithiocarbamic acid (Me-DDC) and its monothio analogue (Me-DTC) has been compared in ten alcoholic patients after a single oral dose of 400 mg disulfiram (Antabuse). Erythrocyte aldehyde dehydrogenase (ALDH) activity was continuously followed in the subjects until complete inactivation. The relation between individual steady-state concentrations of Me-DTC in plasma, blood acetaldehyde concentration and the dose of disulfiram sufficient to give the disulfiram alcohol reaction was investigated in 12 healthy volunteers treated with increasing doses of disulfiram and then challenged with ethanol. The mean peak plasma concentration of Me-DDC occurred at 1.8 h and its plasma half-life was 6.3 h. The corresponding values for Me-DTC were 3.3 h and 11.2 h, respectively. This suggests oxidative formation of Me-DTC from Me-DDC. In alcoholics with a rapid decrease in erythrocyte ALDH activity (in less than 5 days), the mean peak plasma concentration of Me-DTC was 278 nmol.l-1, whereas the peak concentration was below the detection limit in subjects with a prolonged inactivation time (7-20 days). The healthy volunteers were divided into three groups of four subjects, with clinically valid disulfiram alcohol reactions at 100, 200, and 300 mg doses of disulfiram, respectively. The mean plasma concentrations of Me-DTC at steady-state were proportional to the disulfiram doses given, compared within groups at different doses, and between groups at equal and different optimal doses of the drug. The concentrations of Me-DTC were significantly increased, as compared above, whereas the blood concentrations of acetaldehyde were not significantly increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human erythrocyte aldehyde dehydrogenase

Human erythrocyte aldehyde dehydrogenase was purified to homogeneity. The enzyme exhibited a single band of activity on starch gel electrophoresis and on isoelectric focusing. It was a tetramer with an estimated molecular weight of 230,000 daltons and an isoelectric point of 5.0. Its pH optimum of 8.5, Michaelis-Menten constant for acetaldehyde of 46 μM, and high sensitivity to noncompetitive inhibition by disulfiram resembled human liver cytosolic aldehyde dehydrogenase. Low concentrations of magnesium (5–10μM) resulted in enhancement of erythrocyte aldehyde dehydrogenase activity, whereas higher physiological concentrations of magnesium resulted in uncompetitive inhibition of enzyme activity. Magnesium inhibited the enzyme activity by increasing the binding of NADH to the enzyme as had been found to be the case for the inhibitory effect of magnesium on the human liver cytosolic enzyme. Erythrocyte aldehyde dehydrogenase may metabolize small amounts of acetaldehyde escaping the liver during ethanol metabolism and protect extrahepatic tissues from acetaldehyde toxicity.

Effect of acute alcohol administration on erythrocyte aldehyde dehydrogenase activity in man.

The acute oral administration of ethanol to normal subjects resulted in an increase in erythrocyte aldehyde dehydrogenase activity. Incubation of blood with ethanol at 37 degrees C for 2 hr also increased the enzyme activity. By contrast, addition of ethanol directly to the enzyme after its partial purification had no effect on the enzyme activity. The increase in erythrocyte aldehyde dehydrogenase activity following acute ethanol administration is directly opposite to the effect of chronic ethanol consumption in decreasing the enzyme activity in alcoholics. The mechanism for this effect is unknown but may be related to alterations in the erythrocyte membrane and its interaction with the enzyme.

Chlorpropamide-alcohol flush reaction and isoenzyme profiles of alcohol dehydrogenase and aldehyde dehydrogenase.

To investigate the enzymatic basis of the chlorpropamide-alcohol flush reaction (CPAF) we compared the isoenzyme profiles of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from liver biopsies, and of ALDH alone from erythrocytes and leucocytes, in CPAF-positive and CPAF-negative subjects. No differences were seen in ADH or ALDH phenotypes, or in the relative activities of the isoenzymes, between the two groups before chlorpropamide was given; in particular, no subjects showed the 'null' ALDH phenotype that is associated with the alcohol flush reaction in oriental subjects. There was a significant decrease in erythrocyte ALDH activity after 7 days' treatment with chlorpropamide in CPAF-positive individuals but no such difference was seen in CPAF-negative subjects. These results indicate that CPAF has a different enzymatic basis from the alcohol flush reaction of oriental subjects and suggest that in CPAF-positive subjects erythrocyte ALDH may be particularly susceptible to inhibition by chlorpropamide.

Erythrocyte aldehyde dehydrogenase activity in alcoholic subjects and its value as a marker for hepatic aldehyde dehydrogenase in subjects with and without liver disease.

Erythrocyte aldehyde dehydrogenase activity was assayed in actively drinking alcoholics, patients with alcoholic liver disease who claimed to be abstaining, patients with non-alcoholic liver disorders and normal controls. Hepatic cytosolic aldehyde dehydrogenase was also assayed in the majority of the subjects. Actively drinking alcoholics had significantly lower erythrocyte aldehyde dehydrogenase activity than controls (P less than 0.01) but abstaining alcoholic liver disease and non-alcoholic liver disorder subjects did not. There was a significant correlation between erythrocyte and hepatic cytosolic aldehyde dehydrogenase activity in the control group (r = 0.94, P less than 0.05) but not in the other study groups.

Erythrocyte aldehyde dehydrogenase activity in alcoholism.

Erythrocyte aldehyde dehydrogenase activity was determined in 44 chronic alcoholic patients within 18-36 hr after discontinuation of chronic alcohol intake and in 20 nonalcoholic controls. The enzyme activity was decreased to 4.98 +/- 0.52 mlU/mg of protein in the alcoholics as compared with a value of 8.25 +/- 1.29 mlU/mg of protein in the controls (p less than 0.05). The level of the enzyme activity did not correlate significantly with the daily quantity of alcohol consumption or the degree of liver injury reflected in elevations of serum aspartate aminotransferase. Repeat determination in 23 of the alcoholics after 2 weeks of supervised abstinence in an inpatient unit resulted in an increase in the enzyme activity to control levels. These findings show that the decreased activity of erythrocyte aldehyde dehydrogenase which occurs in association with alcohol ingestion is not an inherent characteristic of alcoholism.

Disulfiram and erythrocyte aldehyde dehydrogenase inhibition.

During disulfiram therapy erythrocyte aldehyde dehydrogenase (ALDH) was fully inhibited. The time for total loss of erythrocyte ALDH activity ranged from 36 to 120 hr. In contrast to the 85% recovery of in vitro disulfiram-inhibited ALDH activity, this in vivo disulfiram-ALDH inhibition could not be reversed by mercaptoethanol. It is proposed that the in vivo and in vitro mechanisms of ALDH inhibition by disulfiram differ. Erythrocyte ALDH activity can be readily monitored to determine patient compliance and is an accessible model for investigations of in vivo mechanisms of drug inhibition. Because the disulfiram-inhibited erythrocyte ALDH is not regenerated until new erythrocytes are made (120 days), a significant portion of the extrahepatic acetaldehyde metabolic capacity remains inhibited for long periods after disulfiram is discontinued. Thus, the recidivistic patient who discontinues disulfiram and waits several days (to regenerate liver ALDH activity) before drinking will be exposed to even higher ethanol-derived blood acetaldehyde levels than usual, which may induce further alcohol-associated organ damage and alcohol dependence.

Effects of disulfiram and its reduced metabolite, diethyl-dithiocarbamate on aldehyde dehydrogenase of human erythrocytes

Inhibition of human erythrocyte aldehyde dehydrogenase (ALDH) activity was studied using disulfiram and its reduced metabolite, diethyldithiocarbamate (DDC). The enzyme was rapidly inactivated by disulfiram and the inhibition was protected by reduced glutathione (GSH), in a concentration dependent manner when the enzyme premixed with GSH was reacted with disulfiram. Higher reactivity of the thiol group of the enzyme than that of GSH to disulfiram was suggested from the observation that half of the enzyme activity was inhibited when the ratio of disulfiram to GSH was 1:10. Although DDC alone showed no inhibitory effect on the enzyme, inactivation was mediated by a low concentration of heme-containing peroxidases, but not by met-hemoglobin. Under this condition, the inhibition potential was not protected, even with a high concentration of GSH. The constant reoxidation system of DDC is probably directly related to the enzyme inactivation.

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.