Biomedical subjects

T K Li

Publications and source records attributed to T K Li.

At least 253 records · Page 14Linked to original sources

PubMed1986

Studies on an animal model of alcoholism.

Past and ongoing studies indicate that the selectively bred P line of rats satisfies virtually all the suggested criteria for an animal model of alcoholism. They attain pharmacologically active levels of BAC and develop tolerance and physical dependence with voluntary oral ethanol ingestion, while in the free-feeding state. Ethanol is positively reinforcing to the P rats and consumption appears to be directed by the post-ingestive, pharmacological effects of ethanol, as revealed by the intragastric self-administration studies. Some interesting differences between the P and the NP lines have been uncovered. They differ in the content of serotonin in several brain regions and they respond differently to ethanol. The P rats develop acute tolerance to sedative-hypnotic doses of ethanol more rapidly than do the NP rats, and they exhibit stimulation with low doses of ethanol. These differences suggest hypotheses on mechanisms underlying alcohol-seeking behavior which can now be tested experimentally. It should be emphasized, however, that the described findings are the product of but a single genetic experiment. Clearly, replication is needed, and we are currently doing this, using a better defined, heterogeneous stock of rats. This one experiment, however, has demonstrated the feasibility of developing animal models of alcoholism and offers hope that the genetic and biological basis of alcohol-seeking behavior can be explored in the laboratory. The screening and testing of pharmacological agents able to deter alcohol-seeking behavior is an obvious practical application of this model.

Alcohol Drinking↗

PubMed1985

Central nervous system control of alcohol dehydrogenase activity in rats.

The activity of alcohol dehydrogenase is higher in the livers of female rats than that in male rats. Stereotaxic lesions of the anterior periventricular hypothalamus of male rats increased the activity of alcohol dehydrogenase by about 50 percent; larger lesions which destroyed many of the major hypothalamic nuclei increased the enzyme activity 100 percent and abolished the sexual difference in alcohol dehydrogenase activity. The changes in enzyme activity were independent of changes in plasma thyroxine, testosterone and estradiol levels. Hypothalamic lesions in female rats had no effect on the enzyme activity. The sexual difference in alcohol dehydrogenase activity in rats is under hypothalamic control and does not appear to be mediated directly by the plasma level of androgens or estrogens.

Alcohol Dehydrogenase↗

PubMed1985

Expression of alcohol dehydrogenase in primary monolayer cultures of rat hepatocytes.

With the use of an extensively modified Leibovitz-15 medium, the alcohol dehydrogenase activity of hepatocytes prepared from male rats was successfully maintained in primary culture at the level observed in freshly isolated hepatocytes. Enzyme activity was higher in freshly isolated cells from female rats than from male rats, but it fell to the level characteristic of the male animals after four days in culture. The levels of activity of the cells in culture from both sexes were unaffected by treatment with estrogens or androgens. The results suggest that the sex-determined differences in alcohol dehydrogenase activity in rats do not arise from direct effects of gonadal steroids on the liver.

Alcohol Dehydrogenase↗

PubMed1985

Genetically determined differences in liver alcohol dehydrogenase activity in male rats.

Alcohol dehydrogenase (EC 1.1.1.1) activity was measured in liver extracts from one outbred and three inbred strains of rats. Strain-specific differences in enzyme activity were observed in the adult male rats. The differences appeared as the animals reached puberty. Studies on the enzyme purified from Sprague-Dawley and ACI rats indicate that the enzymes in these strains are identical and that the difference in activity found in liver extracts is due to differences in the amount of enzyme present. Genetic crosses between Sprague-Dawley and ACI rats suggest that the liver content of alcohol dehydrogenase is controlled by an autosomal regulatory locus with the characteristics of a temporal gene.

Alcohol Oxidoreductases↗

PubMed1985

Cloning and sequencing of cDNA encoding the complete mouse liver alcohol dehydrogenase.

The main ethanol-active alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) in mouse liver (ADH-AA) is similar in catalytic and molecular properties to horse liver ADH-EE and to the human class I ADHs. We have isolated cDNA clones encoding the entire mouse liver enzyme plus flanking regions. A mixture of 16 different oligonucleotides, each 14 bases long, was used to screen a liver cDNA library made from a DBA/2J mouse. A strongly hybridizing clone was found and identified as an ADH-encoding cDNA by partial DNA sequencing. This clone was used as a probe to identify others. Two overlapping cDNA clones together contained the entire protein-encoding region plus 100 nucleotides of the 5' noncoding region and 133 nucleotides of the 3' noncoding region culminating in a short poly(dA) tail. The amino acid sequence of the mouse liver enzyme deduced from this cDNA closely resembles that of horse liver ADH-E: 316 of 374 residues are identical, and 29 of the differences are conservative substitutions. The 5' region of this cDNA is interesting: the AUG that initiates the ADH polypeptide is preceded by an AUG that would encode the first amino acid of a tripeptide. Presumably termination of this tripeptide is followed by reinitiation at the AUG immediately preceding the sequence of the mature ADH polypeptide.

Alcohol Dehydrogenase↗

PubMed1985

The measurement of plasma vitamin B6 compounds: comparison of a cation-exchange HPLC method with the open-column chromatographic method and the L-tyrosine apodecarboxylase assay.

A cation-exchange high-performance liquid chromatographic (HPLC) method was found to be comparable to the open-column (OCC) method for measuring six different B6 compounds in human plasma and the L-tyrosine apodecarboxylase (LTD) assay for pyridoxal-P (PLP). Plasma samples were obtained from 9 subjects before and after 7 days of pyridoxine (PN) supplementation. PLP, pyridoxal (PL) and 4-pyridoxic acid (4-PA) were the major B6 compounds in plasma and the only compounds which increased after supplementation. The coefficients of correlation between any 2 of the 3 methods in measuring plasma PLP were greater than 0.93, and between HPLC and OCC in quantifying PL and 4-PA were 0.82 and 0.63, respectively. With the low plasma levels of pyridoxamine-P, PN and pyridoxamine, the results from OCC were consistently higher than those from HPLC. However, recoveries of spiked B6 compounds in plasma by these methods were between 84 to 105 percent for all the 5 vitamers and 4-PA.

Adult↗

PubMed1985

Genetics of alcoholism.

Great progress has been made by research on the contribution genetic factors make to a vulnerability toward alcoholism. Animal studies have demonstrated the importance of genetics in ethanol preference and levels of consumption, and human family, twin, and adoption research have revealed a 4-fold higher risk for offspring of alcoholics, even if they were adopted out at birth. The work presented in this symposium reviews the ongoing search for genetic trait markers of a vulnerability toward alcoholism. Dr. Li has used both animal and human research to demonstrate the possible importance of the genetic control of enzymes involved in ethanol metabolism and has worked to help develop an animal model of alcoholism. The possible importance of subgroups with different levels of predisposition toward alcoholism is emphasized by Dr. Cloninger. An overview of the studies of sons of alcoholics, given by Dr. Schuckit, reveals the potential importance of a decreased intensity of reaction to ethanol as part of a predisposition toward alcoholism and discusses the possible impact of some brain waves and ethanol metabolites to an alcoholism vulnerability. Dr. Deitrich reviews interrelationships between studies of animals and humans in the search for factors involved in a genetic vulnerability toward alcoholism. Taken together, these presentations underscore the importance of genetic factors in alcoholism, review animal and human research attempting to identify markers of a vulnerability, and reveal the high level of interaction between human and animal research.

Adoption↗

PubMed1985

Purification and characterization of human liver beta 1 beta 1, beta 2 beta 2 and beta Ind beta Ind alcohol dehydrogenase isoenzymes.

Mammalian alcohol dehydrogenases (ADH) are dimeric molecules and electrophoretically distinct isoenzymes appear in human liver. They arise from the combination of different subunits produced at five separate gene loci and polymorphic subunits are produced at two of the loci. Three different homodimeric alloenzymes, beta 1 beta 1, beta 2 beta 2 and beta Ind beta Ind, that are assigned to the ADH2 gene have been purified to homogeneity, and the study of their kinetic properties has revealed striking differences among them. Whereas the pH-optimum for ethanol oxidation of beta 1 beta 1 is 10.0-10.5, that of beta 2 beta 2 is 8.5-8.8 and that of beta Ind beta Ind is 7.0-7.2. The Km of beta 1 beta 1 for ethanol at pH 7.5 is low, 49 microM, in contrast to values for beta 2 beta +2 and beta Ind beta Ind, 0.94 and 64 mM. Km and Ki values for NAD are 7.4 and 90 microM for beta 1 beta 1, 180 and 340 microM for beta 2 beta 2 and 490 and 1300 microM for beta Ind beta Ind. The activity of beta 1 beta 1 is stimulated by 40 mM NaCl but that of beta 2 beta 2 is not. Vmax values for ethanol oxidation range from 9.2 min-1 for beta 1 beta 1 to 400 and 560 min-1 for beta 2 beta 2 and beta Ind beta Ind. Both beta 1 beta 1 and beta 2 beta 2 are inactivated by iodoacetate with the formation of one S-carboxymethylcysteine per subunit; however, the second-order rate constant for inactivation of beta 1 beta 1 is four times that of beta 2 beta 2. These differences in the properties of beta 1 beta 1 and beta 2 beta 2 are consistent with the recently reported substitution of His in beta 2 for Arg 47 in beta 1 at the anion binding center of the active site. The active site sequence of the beta Ind subunit has not yet been determined.

Alcohol Oxidoreductases↗

PubMed1985

Relationship between body store of vitamin B6 and plasma pyridoxal-P clearance: metabolic balance studies in humans.

Factors that regulate the clearance of plasma pyridoxal-P (PLP) are unknown. Four volunteers were given a diet supplying approximately 12 mumol pyridoxine (PN) per day. The pharmacokinetics of plasma PLP clearance were measured in these subjects before and after 4 weeks of intravenous PN supplementation (122 mumol/day). Urinary B6 excretion, mainly as 4-pyridoxic acid (4-PA), increased progressively after initiation of PN supplementation until a new steady state was reached on day 10 of supplementation, whereupon greater than 93% of the daily injected PN could be recovered in the urine. Hence, urinary excretion is almost the sole route for vitamin B6 elimination. Fasting plasma PLP concentration increased with supplementation and also reached a new steady state at this time. When supplementation was terminated, urinary B6 excretion decreased in 5 days to an amount only slightly higher than that before supplementation. This amount was maintained for 2 months. By comparison, plasma PLP decreased more slowly and remained considerably higher than the presupplementation level for the rest of the study. These data confirm that urinary 4-PA excretion is a better indicator of B6 intake than is plasma PLP content, whereas plasma PLP content is a better indicator of the body store of the vitamin. Plasma clearance and volume of distribution of PLP decreased significantly after supplementation, but half-life t 1/2 did not change. Plasma clearance of PLP, therefore, is dependent on the vitamin B6 status of an individual.

Adult↗

PubMed1984

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)

Alcohol Dehydrogenase↗

PubMed1984

Intragastric self-infusion of ethanol by ethanol-preferring and -nonpreferring lines of rats.

An ethanol-preferring line of rats, developed by selective breeding, consumed as much as 9.4 +/- 1.7 grams of ethanol per kilogram of body weight per day through intragastric self-infusions, yielding blood ethanol concentrations of 92 to 415 milligrams per 100 milliliters. By contrast, the ethanol- nonpreferring line self-administered only 0.7 +/- 0.2 gram per kilogram per day. These findings indicate that the reinforcing effect of ethanol is postabsorptive and is not mediated by the drug's smell or taste. Hence the ethanol-preferring line of rats may be suitable animal model of alcoholism.

Alcohol Drinking↗

PubMed1984

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity.

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The Km values for ethanol and NAD+, approximately 0.4 and 0.03 mM, respectively, of enzyme purified from both strains are similar. Moreover, the Ki for NADH, 1 microM, the pH optimum for ethanol oxidation, 10.5, and the Vmax for ethanol oxidation, 160 min-1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.

Alcohol Dehydrogenase↗

PubMed1984

Polymorphism of human liver alcohol dehydrogenase: identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing.

Liver homogenate-supernatants from most Japanese exhibit an "atypical" pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the "typical" pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with beta 2 subunits while typical livers contain isozymes with beta 1 subunits, both produced by the ADH2 gene. Because it is difficult to differentiate the atypical ADH2 2-2 phenotype from the ADH2 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The beta beta isozymes in A1 and A2 livers were purified. Type A1 livers contained a single beta beta isozyme with an atypical pH-rate profile; it was designated beta 2 beta 2. Three beta beta isozymes were isolated from A2 livers, two of which corresponded to beta 1 beta 1 and beta 2 beta 2. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated beta 2 beta 1. Type A1 livers are, therefore, the homozygous ADH2 2-2 phenotype, and type A2 livers, the heterozygous ADH2 2-1 phenotype. The ADH2 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH2 2-1 phenotype, in 31%. Accordingly, the frequency of ADH2(2) was 0.68.

Adult↗

PubMed1984

Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme.

Alcohol Dehydrogenase↗

PubMed1984

Clearance and metabolism of plasma pyridoxal 5'-phosphate in the dog.

This study was undertaken to characterize the metabolic fate of plasma pyridoxal 5'-phosphate (pyridoxal-P), the role of various organs in mediating its degradation, and the contribution of urinary excretion. Anesthetized dogs underwent sham operation, hepatectomy, nephrectomy, or combined surgical removal of the stomach, small intestine, and the spleen; however, the kinetics of plasma pyridoxal-P clearance after the intravenous administration of 2.5 mg of this B6 vitamin were not altered. In anesthetized sham-operated dogs and in unanesthetized dogs, pyridoxal (product of pyridoxal-P hydrolysis) but not 4-pyridoxic acid (oxidation product of pyridoxal) accumulated in plasma and urine after pyridoxal-P administration. Urinary excretion of pyridoxal-P was negligible and it could not account for the rapid clearance of pyridoxal-P from the plasma. The rate of pyridoxal-P hydrolysis mediated by plasma alkaline phosphatase and the cellular elements in whole blood in vitro was also too slow to account for the rapid disappearance of plasma pyridoxal-P in vivo. These results indicate that (1) normally, the overall capacity of the body to hydrolyze circulating pyridoxal-P in plasma is so large that removal of the liver, kidneys, or the intestinal tract and spleen has little or no effect on the rate of plasma pyridoxal-P decay; and (2) plasma pyridoxal-P decay normally proceeds by way of hydrolysis to form pyridoxal. Because pyridoxal is not further oxidized by the liver and the amount of pyridoxal excreted in the urine accounts for less than 25% of the pyridoxal that can be derived from the injected pyridoxal-P load, the preponderant metabolic fate of plasma pyridoxal-P most likely involves its hydrolysis to pyridoxal and then the uptake of pyridoxal by extrahepatic tissues.

Alkaline Phosphatase↗

PubMed1983

Steady-state kinetic properties of purified rat liver alcohol dehydrogenase: application to predicting alcohol elimination rates in vivo.

The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.

Alcohol Dehydrogenase↗

PubMed1983

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.

Alcohol Dehydrogenase↗

PubMed1983

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.

Alcohol Dehydrogenase↗