Platelet monoamine oxidase activity in subgroups of alcoholics and controls: results from the Collaborative Study on the Genetics of Alcoholism.

OBJECTIVE: Platelet monoamine oxidase (MAO) B activity levels were evaluated to determine whether low platelet MAO activity is a marker for alcoholism, correlates of alcoholism (e.g., cigarette smoking), or a subtype of alcoholism. METHODS: Adult women (n = 788) and men (n = 685) participating in the Collaborative Study on the Genetics of Alcoholism study were evaluated with a semistructured interview, and blood samples were obtained for determination of platelet MAO activity using tryptamine (0.1 mM) as substrate. DSM-III-R alcohol-dependent individuals were subgrouped using four currently available methods (e.g., two variations of the type 1/type 2 scheme, primary versus secondary typology, type A/type B dichotomy). RESULTS: In the overall sample, subjects' gender, cigarette smoking status, and the Collaborative Study on the Genetics of Alcoholism site at which their platelets were prepared explained 22% of the variance in platelet MAO activity levels, and multivariate analysis showed that carrying a broad diagnosis of alcohol dependence did not uniquely explain any additional variance in platelet MAO activity levels. Furthermore, within each of the alcoholic subgrouping methods tested, there were no significant differences in platelet MAO activity for type 1 versus type 2, type A versus type B, or primary versus secondary alcoholics. CONCLUSIONS: Cigarette smoking and male gender are associated with decreased platelet MAO activity levels. After considering these factors, a diagnosis of alcohol dependence does not predict any additional variance in MAO-B activity. Phenotypes of alcoholics (e.g., type 1 versus type 2, type A versus type B, primary versus secondary) do not differ in platelet MAO activity. The results suggest that decreased platelet MAO activity is not a trait marker of alcoholism or one of its subtypes; but, rather, is a state marker of cigarette smoking.

Rat mitochondrial aldehyde dehydrogenase polymorphism and major histocompatibility complex RT1.A phenotypes are not associated with alcohol drinking in P and NP rats.

Previous studies revealed polymorphisms in mitochondrial aldehyde dehydrogenase (ALDH2) and the major histocompatibility complex RT1.A that appeared in association with alcohol drinking preference in the alcohol-preferring (P) and alcohol-nonpreferring (NP) rat lines. To determine the strength of these associations, the P and NP lines were crossed, and cosegregation studies were performed in the F2 progeny. The ALDH2Q allele and the ALDH2R allele, found in the P and NP lines, respectively, were found not to be associated with the high and low drinking animals in the F2 progeny. Kinetic studies with ALDH2Q and ALDH2R isozymes purified from the mitochondria of P and NP rat livers, respectively, showed that the polymorphism would not be expected to be associated with altered aldehyde metabolism. The association between RT1.A and alcohol preference (RT1.Auu with high and RT1.AII with low alcohol intake) also could not be confirmed in the segregating F2 progeny of the P x NP intercross.

Kinetic characterization of two classes of dog liver alcohol dehydrogenase isoenzymes.

In order to relate the catalytic properties of alcohol dehydrogenase (ADH), the rate-limiting enzyme for alcohol metabolism, with the pharmacokinetics of ethanol elimination in vivo, the multiple molecular forms of dog liver ADH were purified and their steady state kinetics investigated. Two different classes of ADH forms were identified by starch gel electrophoresis: the class I isoenzymes migrate to the cathode and the class II forms migrate to the anode. Three different patterns of the cathodic class I isoenzymes were identified in different liver specimens. Three molecular forms were observed for patterns A and C, and five for B. The two classes of isoenzymes were separated by affinity chromatography and purified by column chromatography. The three predominant class I isoenzymes, A1, B2, and C1, in type A, B, and C livers, respectively, were isolated by high performance cation-exchange chromatography. The steady state kinetic constants of the A1, B2, and C1 isoenzymes are similar, but differ substantially from those of the class II enzyme. The class II enzyme is much less sensitive to pyrazole inhibition, Ki = 2 mM, than the class I forms, Ki = 0.6 microM. Methanol is not a substrate for the class II enzyme, whereas it is oxidized by the class I isoenzymes. The class I isoenzymes exhibit a lower Km and substrate inhibition Ki for ethanol, 0.4 and 160 mM, respectively, than values for the class II enzyme, 10 and 610 mM, respectively. The properties of class I and II dog liver ADH are similar to those of the respective isoenzymes purified from human and monkey liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Human liver alcohol dehydrogenase: purification and kinetic characterization of the beta 2 beta 2, beta 2 beta 1, alpha beta 2, and beta 2 gamma 1 "Oriental" isoenzymes.

Four alcohol dehydrogenase isoenzymes with "atypical" pH optima for ethanol oxidation at 8.8 were isolated from Japanese livers with the homozygous ADH2 2-2 and the heterozygous ADH2 2-1 phenotypes. Agarose gel isoelectric focusing patterns after dissociation--recombination of three isoenzymes purified from the homozygous livers indicate that they are beta 2 beta 2, alpha beta 2, and beta 2 gamma 1. A fourth isoenzyme, purified from livers with the heterozygous phenotype by agarose-hexane--AMP affinity chromatography, was identified as beta 2 beta 1 by dissociation-recombination studies. The kinetic properties of the three heterodimers, beta 2 beta 1, alpha beta 2, and beta 2 gamma 1, are intermediate between those of the respective homodimers, suggesting that the two subunits act independently. Product inhibition studies indicate that beta 2 beta 2 obeys an ordered sequential mechanism, as do the alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2 homodimers which have the "typical" pH optimum for ethanol oxidation at pH 10.0-10.5. The kinetic constants of beta 2 beta 2 differ substantially from those of the other homodimers. At pH 7.5, the Vmax for ethanol oxidation of beta 2 beta 2 is 5-40 times higher than that of alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. The Km and Ki values of beta 2 beta 2 for NAD+ and NADH are also considerably higher than those of the other homodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Human liver alcohol dehydrogenase: ADH Indianapolis results from genetic polymorphism at the ADH2 gene locus.

New molecular forms of human liver alcohol dehydrogenase (ADH), collectively designed ADH Indianapolis (ADHInd), were recently discovered in 29% of liver specimens from Black Americans [Bosron, W. F., Li, T.-K., and Vallee, B. L. (1981). Proc. Natl. Acad.Sci. USA 77:5784]. Three different ADHInd phenotypes have now been identified by starch gel electrophoresis, and four ADHInd enzyme forms isolated by affinity and ion-exchange chromatography. The most cathodic ADHInd form has a single pH optimum at 7.0 for ethanol oxidation and is a homodimer of a newly discovered subunit, as evidenced by dissociation--recombination studies. The remaining three purified ADHInd forms have dual pH optima for ethanol oxidation at 7.0 and 10.0 and generate two new bands on starch gel electrophoresis after dissociation-recombination. They appear to be heterodimers of this new subunit with the known subunits, alpha, beta 1, and gamma 1. Based on the occurrence of these four ADHInd isozymes and isozymes containing beta 1 subunits in the homogenate supernatants of 135 livers, we conclude that ADHInd results from polymorphism at the ADH2 locus, with the variant ADH2Ind allele coding for the beta Ind subunit. The frequency of ADH2Ind was 0.16 in Black Americans. The frequency of the ADH31 and ADH32 alleles also differed in these two populations.

Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase.

Ten, electrophoretically distinct, molecular forms of alcohol dehydrogenase have been isolated from a single human liver by affinity and ion-exchange chromatography. The starch gel electrophoresis patterns after the dissociation-recombination of the forms are consistent with the hypothesis that they arise from the random combination of alpha, beta 1, gamma 1, and gamma 2 subunits into six heterodimeric and four homodimeric isoenzymes. Large differences in kinetic properties are observed for the homodimeric isoenzymes, alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. At pH 7.5, the Km value of beta 1 beta 1 for ethanol is 0.049 mM and that of alpha alpha is 4.2 mM. Forms gamma 1 gamma 1 and gamma 2 gamma 2 do not obey Michaelis-Menten kinetics at pH 7.5 but exhibit negative cooperativity with Hill coefficients of 0.54 and 0.55 and [S]0.5 values of 1.0 and 0.63 mM, respectively. However, all isoenzymes display Michaelis-Menten kinetics for ethanol oxidation at pH 10.0 with Km values ranging from 1.5 to 3.2 mM. The maximum specific activity of beta 1 beta 1 is considerably lower than that of the other three homodimers at both pH 7.5 and 10.0. The Km values of the four homodimers for NAD+ at pH 7.5 range from 7.4 to 13 microM and those for NADH, from 6.4 to 33 microM. Ki values for NADH range from 0.19 to 1.6 microM. At pH 7.5, the kinetic properties of alpha alpha and beta 1 beta 1, prepared in vitro from dissociated and recombined alpha beta 1, are similar to those of the native homodimers. The forms gamma 1 gamma 1 and gamma 2 gamma 2, prepared from dissociated and recombined alpha gamma 1 and beta 1 gamma 2, respectively, exhibit negative cooperativity with Hill coefficients that are similar to those seen with the respective native homodimers.