Kathryn E. Crow

Intracellular localisation and properties of aldehyde dehydrogenases from sheep liver

Abstract 1. 1. The distribution of aldehyde dehydrogenases in sheep liver was studied. Activity was found in the cytoplasm, mitochondria and microsomes. 2. 2. Cross-contamination of activities from different subcellular fractions, during the isolation procedures used, was shown to be insignificant. Accordingly, the level of aldehyde dehydrogenase activity found in each fraction should reflect the distribution pattern in vivo. 3. 3. Aldehyde dehydrogenases from the cytoplasm and mitochondria were isolated and some of their catalytic properties examined. The results show that the enzymes from the two fractions are not identical.

Studies on possible mechanisms for the interaction between cyanamide and aldehyde dehydrogenase

Abstract Cyanamide is known to interfere with the metabolism of alcohol by decreasing the activity of aldehyde dehydrogenase in vivo , thereby leading to an accumulation of acetaldehyde following the ingestion of ethanol. We have studied various mechanisms for the chemical interaction between aldehyde dehydrogenase and cyanamide (or some of its possible metabolites). Cyanamide was shown to react under physiological conditions with amino and thiol groups, forming guanidino and isothiouronium compounds respectively. However, it was found that the enzyme is not appreciably affected in vitro by high concentrations of cyanamide. Dicyandiamide and the aminoethylisothiouronium ion (AET) also have no effect in vitro . It was postulated that the pathway of in vivo enzyme modification by cyanamide may involve thiourea (formed by breakdown of isothiouronium compounds) and formamidine disulphide (an oxidation product of thiourea). However, although the disulphide is a moderately-effective inactivator of aldehyde dehydrogenase in vitro , administration of thiourea or AET to rats does not result in any significant loss of aldehyde dehydrogenase activity in either the cytoplasm or the mitochondria

Determination of acetaldehyde in blood using automated distillation and fluorometry

Abstract A sensitive enzymic method for the determination of acetaldehyde in human blood has been developed. The method may be operated in a semiautomated or fully automated mode and involves continuous-flow distillation of samples with fluorometry. Levels of acetaldehyde between 0.5 and 20 μmol/liter in blood may be determined, using either yeast or sheep liver aldehyde dehydrogenases.

Factors influencing rates of ethanol oxidation in isolated rat hepatocytes

The stimulation of ethanol oxidation by fructose which has frequently been observed in isolated hepatocytes was found to occur only in unsupplemented cells. In the presence of other substrates (lactate, pyruvate) which accelerate ethanol oxidation, fructose had no additional effect. Acceleration of ethanol oxidation by fructose was not directly related to the ATP demand created by fructose. The effects of fructose on ethanol oxidation rates were not due to changes in acetaldehyde concentration. In cells from fed animals, acetaldehyde concentrations rose as high as 200 microM in some incubations, and therefore became a significant factor limiting ethanol oxidation rates. In hepatocytes isolated from starved rats incubated with pyruvate, where acetaldehyde concentrations were very low, (1-2 microM) it was possible to assess the effect of changes in [lactate]/[pyruvate] (and hence free cytosolic NADH) on rates of ethanol oxidation. The results showed that the increase in free cytosolic [NADH] usually found during ethanol oxidation in vivo would inhibit rates of ethanol clearance by a maximum of 20%.

Rat liver mitochondrial malate dehydrogenase: Purification, kinetic properties, and role in ethanol metabolism

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.

Human liver cytosolic malate dehydrogenase: Purification, kinetic properties, and role in ethanol metabolism☆

Cytosolic malate dehydrogenase from human liver was isolated and its physical and kinetic properties were determined. The enzyme had a molecular weight of 72,000 +/- 2000 and an amino acid composition similar to those of malate dehydrogenases from other species. The kinetic behaviour of the enzyme was consistent with an Ordered Bi Bi mechanism. The following values (microM) of the kinetic parameters were obtained at pH 7.4 and 37 degrees C: Ka, 17; Kia, 3.6; Kb, 51; Kib, 68; Kp, 770; Kip, 10,700; Kq, 42; Kiq, 500, where a, b, p, and q refer to NADH, oxalacetate, malate, and NAD+, respectively. The maximum velocity of the enzyme in human liver homogenates was 102 mumol/min/g wet wt of liver for oxalacetate reduction and 11.2 mumol/min/g liver for malate oxidation at pH 7.4 and 37 degrees C. Calculations using these parameters showed that, under conditions in vivo, the rate of NADH oxidation by the enzyme would be much less than the maximum velocity and could be comparable to the rate of NADH production during ethanol oxidation in human liver. The rate of NADH oxidation would be sensitive to the concentrations of NADH and oxalacetate; this sensitivity can explain the change in cytosolic NAD+/NADH redox state during ethanol metabolism in human liver.

Acetaldehyde and Acetate Production during Ethanol Metabolism in Perfused Rat Liver

It has been reported that, in isolated perfused liver metabolizing 16 or 32 mM ethanol, as much as 60% of acetaldehyde formed from ethanol left the liver unmetabolized (Lindros et al., 1972). Krebs (1969) found that sufficient acetaldehyde was formed during perfusion with 10 mM ethanol to bring the alcohol dehydrogenase reaction into equilibrium. By contrast, Williamson et al. (1969) found that only trace amounts of acetaldehyde were released by livers metabolising 10 mM ethanol. Eriksson et al. (1975) have claculated that, in vivo, less that 5% of acetaldehyde formed from ethanol leaves the liver un-metabolized. This is so even in the presence of ethanol concentrations as high as 50 μmol/g wet wt. liver (Eriksson and Sippel, 1977).

Acetaldehyde Levels in Peripheral Venous Blood and Breath of Human Volunteers

If the extrahepatic metabolism of acetaldehyde in humans is similar to that which occurs in rats (1-6), venous blood acetaldehyde concentrations may not reflect those in potentially sensitive organs such as the brain. It is important to obtain estimates of the levels of acetaldehyde in blood (a) leaving the liver, in order to determine the maximum toxic potential of acetaldehyde, and (b) presented to the brain, since current theories proposing a role for acetaldehyde in the development of addiction to ethanol involve its interaction with components of the central nervous system (7,8).

Breath Acetaldehyde Levels after Ethanol Consumption

Breath acetaldehyde levels in human subjects have been determined in the past by the separate collection of breath prior to analysis. Stowell et al. (1979) trapped acetaldehyde in a semicarbazide solution before enzymatic analysis while Forsander et al. (1974) used a cold water trap prior to analysis by gas chromatography. Although trapping of acetaldehyde from breath allows it to be concentrated, the disadvantages of such methods are that the recovery procedures are laborious.

Effects of ethanol treatment and castration on liver alcohol dehydrogenase activity

Induction of alcohol dehydrogenase (ADH) activity by chronic ethanol treatment and castration has previously been reported to occur in Sprague-Dawley rats. In the present study, no induction was found following chronic ethanol treatment and only a low level of induction was found with castration. However the activity of ADH was high in control animals compared with those used in other studies. The activity of ADH in control animals was not decreased by testosterone administration, which has been shown to reverse induction of the enzyme produced by chronic ethanol treatment or castration in other studies. It is concluded that the male Sprague-Dawley rat is not necessarily a suitable animal for the study of ADH induction by chronic ethanol treatment and that further unknown factors must be identified before the regulation of ADH activity in vivo is fully understood.