Neal W. Cornell

Enzymatic measurement of ethanol or NAD in acid extracts of biological samples

An enzymatic method for the measurement of ethanol has been developed to permit analyses with unneutralized acid extracts of blood, liver, cell suspensions, or other biological materials. Components of the assay mixture include NAD, yeast alcohol dehydrogenase, tris(hydroxymethyl)aminomethane (Tris), and lysine. Tris is a trapping agent for the reaction product, acetaldehyde. Lysine is used to maintain the pH at 9.7 where oxidation of ethanol is quantitative and most rapid, even when as much as 0.2 ml of 0.5 N HClO4 is added. Lysine also causes the reaction to be 2 to 4 times faster than it is when either glycine or 2-amino-2-methyl-1-propanol is used as the buffer. The assay is linear up to an ethanol concentration of 0.125 mM in the reaction mixture and is complete by 4 min. By substituting ethanol for NAD in the reagents, the assay performs equally well in measuring NAD.

Properties of alcohol dehydrogenase and ethanol oxidation in vivo and in hepatocytes

Studies in this laboratory have been concerned with testing the properties of alcohol dehydrogenase (ADH) in vitro as predictors of ethanol oxidation in the rat in vivo. With the kinetic constants for the extracted enzyme determined under physiological conditions (pH 7.3, ionic strength = 0.25, 38 degrees C), it was possible to predict rates of ethanol elimination in the rat in vivo within +/- 15%. The results indicate that the level of ADH is the major rate determining factor and that physiological levels of free cytosolic NADH have a minor influence (less than or equal to 20%) on the rate of ethanol oxidation in vivo. Those conclusions are supported by results with isolated hepatocytes which, when incubated without other substrates, oxidize ethanol at 1/3 the rate in vivo. Under that condition, titrations with 4-pentylpyrazole show that ADH is not rate determining, and acceleration of NADH reoxidation stimulates ethanol removal. When hepatocyte incubations are supplemented with substrates, ethanol oxidation proceeds at rates similar to those in vivo, and the rates are, as in vivo, determined largely by the cellular content of ADH.

The inhibition of alcohol dehydrogenase in vitro and in isolated hepatocytes by 4-substituted pyrazoles

As a means of comparing the functional properties of an enzyme in dilute solution in vitro with those for the same enzyme acting in its normal cellular environment, a study was conducted with 4-substituted pyrazoles as inhibitors of rat liver alcohol dehydrogenase in vitro and ethanol oxidation in isolated rat hepatocytes. Inhibitor constants (Kis) for the same set of pyrazole derivatives were also determined for human liver alcohol dehydrogenase. The best-fitting equations were derived to relate the Kis to the chemical nature of substituents. These quantitative structure-activity relationships show that pyrazoles with stronger electron-withdrawing substituents are weaker inhibitors both for the enzyme in vitro and, to an equal extent, for ethanol oxidation by intact cells. Inhibitor effectiveness is also dependent on substituent hydrophobicity, but, while increasing hydrophobicity makes stronger inhibitors of the enzyme in vitro, it can diminish the effectiveness in vivo by decreasing permeability through the cell membrane. A structure-activity analysis of published Kis for pyrazoles acting against human pi-ADH indicates that its active site differs from those in other alcohol dehydrogenases.

Rapid fractionation of cell suspensions with the use of brominated hydrocarbons

Isolated hepatocytes can be rapidly separated from the suspending medium using an inexpensive, easily constructed centrifuge tube and a water immiscible liquid which has a density high enough to preclude the medium but low enough to permit sedimentation of cells. For that purpose, mixtures of either bromododecane or bromodecane with dodecane work well and have viscosities that are 30 to 50 times lower than that for the widely used silicon oils. As tests of the method, ATP, urea, and chloride were determined in suspensions and in cell and medium fractions. All of the ATP in cell suspensions was recovered in the cell fraction while most of the urea was in the medium. Chloride was about 10 mm in fresh hepatocytes, but, after 10 min at 38°C, the intracellular level of this ion returned to that found for the liver in vivo, 40–50 mm.

Inhibition by 5-(tetradecyloxy)-2-furoic acid of fatty acid and cholesterol synthesis in isolated rat hepatocytes.

Fatty acid and cholesterol synthesis in isolated rat hepatocytes were strongly inhibited by 5-(tetradecyloxy)-2-furoic acid. With either3H2O or [2-14C]acetate as the labeled precursor, the concentrations of inhibitor causing 50% decrease in fatty acid and cholesterol synthesis were, respectively, <0.005 mM and 0.020 mM. At 0.1 mM inhibitor, citrate concentration in cells from fed rats was increased by 75%; lactate and pyruvate concentrations were decreased by 30%; ethanol oxidation was decreased by 20%; with cells from starved rats, the mitochondrial [NAD+]/[NADH] was decreased. Other parameters were unaffected. Both its potency and its specificity indicate that 5-(tetradecyloxy)-2-furoic acid will be useful in studies on the regulation of lipid biosynthesis.

Induction of cytochrome P-450 by alcohols and 4-substituted pyrazoles: Comparison of structure-activity relationships

A comparison was made between 4-substituted pyrazoles and short-chain alcohols as inducers of cytochrome P-450. A quantitative structure-activity analysis of the data led to the following equations: (I) Pyrazoles: Log 1/C = 0.85 (+/- 0.21) Log P + 1.93 (+/- 0.38), r = 0.970 (II) Alcohols: Log 1/C = 0.78 (+/- 0.14) Log P + 1.46 (+/- 0.13), r = 0.988 where C is the concentration that caused a 50% increase in cytochrome P-450, is the partition coefficient between octanol and water, and r is the correlation coefficient. The results suggest that induction of cytochrome P-450 by these compounds depends on hydrophobicity alone. Electronic and steric factors have insignificant roles.

Association of ATP citrate lyase with mitochondria

Abstract (1) The association of ATP citrate lyase with mitochondria was studied with isolated rat hepatocytes and mitochondria. (2) When hepatocytes were treated with digitonin, about 25% of the lyase activity was released like a mitochondrial enzyme. (3) The effect of temperature on release of lyase from hepatocytes was different from that on the release of other cytosolic or mitochondrial enzymes. (4) The fraction of total hepatic lyase in mitochondrial preparations made with exogenous MgCl 2 was 30 times greater than that for a cytosolic marker enzyme, phosphoglycerate kinase. (5) Lyase substrates enhanced the release of the enzyme both from hepatocytes and from isolated mitochondria. (6) The metabolic significance of association of ATP citrate lyase with mitochondria is discussed. (7) Data obtained in the course of these experiments indicate that less than 3% of adenylate kinase is cytosolic.

THE ROLE OF ALCOHOL DEHYDROGENASE IN GOVERNING RATES OF ETHANOL METABOLISM IN RATS

There are conflicting reports concerning the role of alcohol dehydrogenase (ADH) in determining the rate of ethanol metabolism in vivo. We have measured the Vmax of rat liver ADH in vitro and have compared it with published rates of ethanol metabolism in rats in vivo, and with the rate of ethanol oxidation in isolated rat hepatocytes. The published rates of ethanol metabolism in vivo (3. 32 ± 0.14 μmol/min/g liver) can be accounted for by ADH operating at 65% of its maximum velocity (5. 0 μmol/min/g). Hepatocytes supplied with 5 mM pyruvate oxidise ethanol at a rate of 2.97 μmol/min/g wet weight cells, which is similar to the rates observed in vivo. In the absence of added substrates, isolated hepatocytes metabolise ethanol at only one third of the maximum rate, so that in this instance the level of ADH is not rate limiting. Acetaldehyde does not accumulate, so that the concentration of this metabolite is not influencing the rate of ethanol metabolism. In hepatocytes without substrate, free cytosolic NAD+ and NADH levels probably determine the rate of ethanol metabolism. However, in rats in vivo, and in isolated hepatocytes supplied with pyruvate, the major rate-determining factor for ethanol oxidation is the level of ADH activity.

Mechanism responsible for aminooxyacetate and glycolate stimulation of ethanol oxidation by isolated hepatocytes

Abstract Aminooxyacetate (H 2 NOCH 2 CO 2 − ) is known to inhibit ethanol oxidation by blocking transamination reactions required for the transport of reducing equivalents from the cytosol to the mitosol. Ethanol oxidation is stimulated, however, when high concentrations of aminooxyacetate are used. This paradoxical stimulation of ethanol oxidation by aminooxyacetate has been resolved. Aminooxyacetate is metabolized to glycolate and glyoxylate. Glycolate stimulates ethanol oxidation in part by providing H 2 O 2 for catalase-mediated ethanol oxidation. Evidence was also found, however, for a glycolate-glyoxylate shuttle for the transport of reducing equivalents between the cytosol and the peroxisomes. d,l -2-Hydroxy-3-butynoate, an inhibitor of peroxisomal glycolate oxidation, blocks the stimulation of ethanol oxidation caused by glycolate or aminooxyacetate. The stimulation of ethanol oxidation by these compounds shows partial sensitivity to 4-pentylpyrazole or 3-aminotriazole, indicating that both alcohol dehydrogenase (EC 1.1.1.1) and catalase (EC 1.11.1.6) are involved. Glyoxylate stimulates ethanol oxidation, and its conversion to glycolate is dependent on ethanol. Although it appears that a glycolate-glyoxylate shuttle for reoxidation of cytosolic NADH can be readily established in vitro , no evidence was found for the existence of this shuttle in vivo . Ethyl hydrazinoacetate has been proposed as a substitute for aminooxyacetate in metabolic studies; however, enzymatic hydrolysis of this compound to give ethanol limits its usefulness as an inhibitor of ethanol oxidation. 2-Aminooxypropionate does not show the paradoxical stimulation of ethanol oxidation. Its effectiveness in inhibiting aspartate aminotransferase (EC 2.6.1.1) is similar to that for aminooxyacetate. Thus, 2-aminooxypropionate appears to be a good alternative to aminooxyacetate in metabolic studies in vivo , in perfused liver or in isolated cells.

Alcohol Dehydrogenase in Cultured Human Skin Fibroblasts Human Fibroblast Alcohol Dehydrogenase

Studies of ethanol oxidation and other metabolic pathways in humans are often limited by the availability of a reproducible test material. Because of this we have tested human fibroblasts for ethanol metabolism and alcohol dehydrogenase content. Seven different cell lines have been studied and found to contain an enzymatic activity identified as alcohol dehydrogenase by the following criteria: it is NAD+-dependent, the Km for ethanol is like human liver, it is completely inhibited by 25 microM 4-pentylpyrazole. The fibroblast activity was analyzed by isoelectric focusing and found to contain several isozymes also present in the human liver sample. In addition, fibroblasts contain 2 major isozymes which migrate anodally to any isozymes previously reported in human liver. Thus, fibroblasts appear to be useful material for comparing enzymatic aspects of ethanol metabolism in alcoholics and nonalcoholics.