scholarly journals The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose

1984 ◽  
Vol 222 (2) ◽  
pp. 437-446 ◽  
Author(s):  
C Vind ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Maximal Activity ◽  
Isolated Hepatocytes ◽  
Single Compartment ◽  
Dehydrogenase Reaction ◽  
Order Of Magnitude ◽  
Rate Limiting

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.

scholarly journals ВПЛИВ ЗАМІЩЕННОГО НІКОТИНАМІДУ ТА ЙОГО МОЖЛИВИХ МЕТАБОЛІТІВ НА АКТИВНІСТЬ ФЕРМЕНТІВ ОБМІНУ ЕТАНОЛУ

2020 ◽  
Vol 144 (2) ◽  
pp. 98-104
Author(s):  
О. В. Кислова
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aldehyde Dehydrogenase ◽  
Ethanol Oxidation ◽  
The Body ◽  
Inhibition Constant ◽  
Ethanol Metabolism ◽  
Dehydrogenase Reaction ◽  
Metabolism Enzymes

To study the influence of N-phenyl-N-(1-cyclopropylethyl)nicotinamide and its possible metabolites: hydrochlorides of N-(1-cyclopropylethyl)amine and N-phenyl-N-(1-cyclopropylethyl)amine - on the activity of  main ethanol oxidation enzymes in vitro and kinetic nature of their interaction. The studies were carried out using alcohol dehydrogenase and aldehyde dehydrogenase of rat liver subcellular fractions, which were obtained by differential centrifugation. The enzyme activity was determined spectrophotometrically. The kinetic nature of alcohol dehydrogenase and isozyme form of aldehyde dehydrogenase  interaction with substituted nicotinamide was investigated in the concentration range of 25-100 μM. The research results were processed by the Lineweaver-Burk method. Studies have shown that N-phenyl-N-(1-cyclopropylethyl)nicotinamide is able to reduce the rate of the reverse alcohol dehydrogenase reaction of acetaldehyde reduction to ethanol in the presence of NADH by 46% with an inhibition constant 53 μM. The activity of soluble mitochondrial aldehyde dehydrogenase was suppressed by 50% with an inhibition constant 108 μM. The kinetic nature of the substituted nicotinamide interaction with enzymes at saturating concentrations of the reaction cofactors NADH and NAD+ is quite complex. Allosteric effects can play a significant role in enzymatic activity. Possible metabolites of the compound - hydrochlorides of N-(1-cyclopropylethyl)- and N-phenyl-N-(1-cyclopropylethyl)amine – didn`t significantly influence on ethanol metabolism enzymes activity. A new inhibitor of the rate of the reverse alcohol dehydrogenase reaction and the activity of soluble mitochondrial isozyme form of aldehyde dehydrogenase, which lead to the accumulation of acetaldehyde in the body, has been discovered. N-phenyl-N-(1-cyclopropylethyl)nicotinamide can be used as a potential antialcohol sensitizing drug after research in vivo.

scholarly journals Hydrogen transfer between ethanol molecules during oxidoreduction in vivo

1985 ◽  
Vol 229 (2) ◽  
pp. 315-322 ◽  
Author(s):  
T Cronholm
Keyword(s):  
Alcohol Dehydrogenase ◽  
Isotope Effect ◽  
Hydrogen Transfer ◽  
Ethanol Oxidation ◽  
Internal Standard ◽  
Ethanol Metabolism ◽  
Acetaldehyde Oxidation ◽  
Extensive Formation ◽  
Rate Limiting

Rates of exchange catalysed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected in rats with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by g.l.c.-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labelling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labelled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. The results indicated that both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high as or higher than the net ethanol oxidation. Thus, the rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociation of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde.

scholarly journals Rapid Elimination of Blood Alcohol Using Erythrocytes: Mathematical Modeling and In Vitro Study

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Yuliya G. Alexandrovich ◽  
Elena A. Kosenko ◽  
Elena I. Sinauridze ◽  
Sergey I. Obydennyi ◽  
Igor I. Kireev ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Ethanol Oxidation ◽  
Alcohol Intoxication ◽  
Limiting Factors ◽  
Blood Alcohol ◽  
Cell Recovery ◽  
Optimal Method ◽  
Metabolic System ◽  
Rate Limiting

Erythrocytes (RBCs) loaded with alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD) can metabolize plasma ethanol and acetaldehyde but with low efficiency. We investigated the rate-limiting factors in ethanol oxidation by these enzymes loaded into RBCs. Mathematical modeling and in vitro experiments on human RBCs loaded simultaneously with ADH and ALD (by hypoosmotic dialysis) were performed. The simulation showed that the rate of nicotinamide-adenine dinucleotide (NAD+) generation in RBC glycolysis, but not the activities of the loaded enzymes, is the rate-limiting step in external ethanol oxidation. The rate of oxidation could be increased if RBCs are supplemented by NAD+ and pyruvate. Our experimental data verified this theoretical conclusion. RBCs loaded with the complete system of ADH, ALD, NAD+, and pyruvate metabolized ethanol 20–40 times faster than reported in previous studies. The one-step procedure of hypoosmotic dialysis is the optimal method to encapsulate ADH and ALD in RBCs after cell recovery, encapsulation yield, osmotic resistance, and RBC-indexes. Consequently, transfusion of the RBCs loaded with the complete metabolic system, including ADH, ALD, pyruvate, and NAD+ in the patients with alcohol intoxication, may be a promising method for rapid detoxification of blood alcohol based on metabolism.

scholarly journals A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates

1973 ◽  
Vol 131 (2) ◽  
pp. 261-270 ◽  
Author(s):  
F. M. Dickinson ◽  
G. P. Monger
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Ternary Complexes ◽  
Kinetics Of Oxidation ◽  
Yeast Alcohol Dehydrogenase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Binary Complexes ◽  
Rate Limiting ◽  
Kinetics Of

1. The kinetics of oxidation of ethanol, propan-1-ol, butan-1-ol and propan-2-ol by NAD+ and of reduction of acetaldehyde and butyraldehyde by NADH catalysed by yeast alcohol dehydrogenase were studied. 2. Results for the aldehyde–NADH reactions are consistent with a compulsory-order mechanism with the rate-limiting step being the dissociation of the product enzyme–NAD+ complex. In contrast the results for the alcohol–NAD+ reactions indicate that some dissociation of coenzyme from the active enzyme–NAD+–alcohol ternary complexes must occur and that the mechanism is not strictly compulsory-order. The rate-limiting step in ethanol oxidation is the dissociation of the product enzyme–NADH complex but with the other alcohols it is probably the catalytic interconversion of ternary complexes. 3. The rate constants describing the combination of NAD+ and NADH with the enzyme and the dissociations of these coenzymes from binary complexes with the enzyme were measured.

scholarly journals Pathways of reducing equivalents in hepatocytes from rats. Estimation of cytosolic fluxes by means of 3H-labelled substrates for either A- or B-specific dehydrogenases

1987 ◽  
Vol 243 (3) ◽  
pp. 625-630 ◽  
Author(s):  
C Vind ◽  
A Hunding ◽  
N Grunnet
Keyword(s):  
Experimental Data ◽  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Least Squares ◽  
Oxidation Rate ◽  
Isolated Hepatocytes ◽  
Glycerol Phosphate ◽  
Hydrogen Atoms ◽  
Best Fit ◽  
Glycerol Phosphate Dehydrogenase

The metabolism of [2-3H]lactate and [2-3H]glycerol was studied in isolated hepatocytes from fed rats. In order to estimate the rate of equilibrium between the 4A and 4B hydrogen atoms of NADH, we compared the flow of 3H from [2-3H]lactate and [2-3H]glycerol, the oxidations of which are catalysed by A- and B-type dehydrogenases, respectively. Hepatocytes were incubated with lactate, glycerol and ethanol and tracer amounts of [2-3H]lactate or [2-3H]glycerol and the labelling rates of lactate, ethanol, glucose and glycerol phosphate were determined. The data were used to calculate the oxidation rate of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase, triosephosphate dehydrogenase and glycerol phosphate dehydrogenase. The rates were calculated by obtaining the best fit of a model to the experimental data by using a least-squares procedure. The results support our model and suggest that the fluxes through various dehydrogenases are sufficient to equilibrate the 4A and 4B hydrogen atoms of cytosolic NADH. The validity of the metabolic models used was evaluated by comparison of rates of NADH oxidation catalysed by cytosolic dehydrogenases as calculated by two different models.

scholarly journals Heterogeneity of the sn-glycerol 3-phosphate pool in isolated hepatocytes, demonstrated by the use of deuterated glycerols and ethanol

1984 ◽  
Vol 224 (3) ◽  
pp. 731-739 ◽  
Author(s):  
T Cronholm ◽  
T Curstedt
Keyword(s):  
Mass Spectrometry ◽  
Alcohol Dehydrogenase ◽  
Organic Acids ◽  
Ethanol Oxidation ◽  
Isolated Hepatocytes ◽  
Female Rats ◽  
Dihydroxyacetone Phosphate ◽  
Deuterium Excess ◽  
Relative Contribution ◽  
Glycerol Uptake

Hepatocytes were isolated from female rats and incubated with [1,1,3,3-2H4]glycerol or [2-2H]glycerol. The deuterium excess in phosphatidylcholines, sn-glycerol 3-phosphate and other organic acids was determined by g.l.c./mass spectrometry. The unlabelled fraction of the major phosphatidylcholines decreased exponentially, and the turnover was not changed by the presence of ethanol. The relative contribution of the two deuterated glycerols was about the same in the major phosphatidylcholine as in sn-glycerol 3-phosphate, indicating that formation by acylation of dihydroxyacetone phosphate is insignificant. [1,1,3,3-2H4]Glycerol had lost deuterium to a larger extent when it was incorporated in the phosphatidylcholine than when it was incorporated in sn-glycerol-3-phosphate, indicating that the phosphatidylcholines are formed from a separate pool of sn-glycerol 3-phosphate. Deuterium at C-2 was transferred between sn-glycerol 3-phosphate molecules to about 25%. Ethanol decreased the extent of deuterium transfer, the extent of glycerol uptake and the loss of deuterium at C-1 and C-3 in sn-glycerol 3-phosphate. The results indicate that the oxidation to dihydroxyacetone phosphate was inhibited by the NADH formed during ethanol oxidation. [2-2H]Glycerol also labelled an alcohol dehydrogenase substrate, malate and lactate, indicating oxidation of sn-glycerol 3-phosphate in the cytosol. The two acids appeared to be formed in reductions with different pools of NADH.

scholarly journals Inhibition of microsomal oxidation of ethanol by pyrazole and 4-methylpyrazole in vitro Increased effectiveness after induction by pyrazole and 4-methylpyrazole

1986 ◽  
Vol 239 (3) ◽  
pp. 671-677 ◽  
Author(s):  
D E Feierman ◽  
A I Cederbaum
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Liver Microsomes ◽  
Chronic Ethanol ◽  
Ethanol Metabolism ◽  
Ethanol Treatment ◽  
Previously Treated ◽  
Kinetics Of ◽  
Oxidation Of Ethanol

Pyrazole and 4-methylpyrazole, which are inhibitors of alcohol dehydrogenase, were also found to be effective inhibitors of the oxidation of ethanol by liver microsomes (microsomal fractions) in vitro. Ethanol oxidation by microsomes from rats previously treated for 2 or 3 days with either pyrazole or 4-methylpyrazole appeared to be especially sensitive to inhibition in vitro by pyrazole or 4-methylpyrazole. The kinetics of inhibition by pyrazole or 4-methylpyrazole in all microsomal preparations were mixed, as the Km for ethanol was elevated while Vmax was lowered. However, Ki values for pyrazole (about 0.35 mM) and especially 4-methylpyrazole (about 0.03-0.10 mM) were much lower than those found with the saline controls (about 0.7-1.1 mM). In contrast, Ki values for dimethyl sulphoxide as an inhibitor of microsomal ethanol oxidation were similar in all microsomal preparations. Pyrazole and 4-methylpyrazole reacted with microsomes to produce type II spectral changes whose magnitude increased after treatment with either pyrazole or 4-methylpyrazole. Thus the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole appears to be associated with increased interactions with the cytochrome P-450 isoenzyme(s) induced by these compounds. These isoenzymes have properties similar to those of the isoenzyme induced by chronic ethanol treatment. Therefore, caution is needed in the use of pyrazole or 4-methylpyrazole to assess pathways of ethanol metabolism, especially after chronic ethanol treatment, since these agents, besides inhibiting alcohol dehydrogenase, are also effective inhibitors of microsomal ethanol oxidation.

scholarly journals Inhibition of catalase-dependent ethanol metabolism in alcohol dehydrogenase-deficient deermice by fructose

1987 ◽  
Vol 248 (2) ◽  
pp. 415-421 ◽  
Author(s):  
J A Handler ◽  
B U Bradford ◽  
E B Glassman ◽  
D T Forman ◽  
R G Thurman
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Metabolism ◽  
Beta Oxidation ◽  
Low Concentrations ◽  
H2o2 Generation ◽  
Order Of Magnitude ◽  
Rate Limiting ◽  
Cytochrome P 450 ◽  
Generating System ◽  
Stimulation Of

Hepatic microsomal fractions from ADH (alcohol dehydrogenase)-negative deermice incubated with an NADPH-generating system metabolized butanol and ethanol at rates around 10 nmol/min per mg. In contrast, cytosolic catalase from ADH-negative deermouse liver oxidized ethanol, but not butanol, when incubated with an H2O2-generating system. Thus butanol is oxidized by cytochrome P-450 in microsomal fractions, but not by cytosolic catalase, in tissues from ADH-negative deermice. In perfused livers from ADH-negative deermice, rates of ethanol uptake at low concentrations of ethanol (1.5 mM) were about 60 mumol/h per g, yet butanol (1.5 mM) uptake was undetectable (less than 4 mumol/h per g). At higher concentrations of alcohol (25-30 mM), rates of ethanol uptake were about 80 mumol/h per g, whereas rates of butanol uptake were only about 9 mumol/h per g. Because rates of butanol metabolism via cytochrome P-450 in deermice were more than an order of magnitude lower than rates of ethanol uptake in livers from ADH-negative deermice, it is concluded that ethanol uptake by perfused livers from ADH-negative deermice is catalysed predominantly via catalase-H2O2. In support of this conclusion, rates of H2O2 generation, which are rate-limiting for the peroxidation of ethanol by catalase, were about 65 mumol/h per g in livers from ADH-negative deermice, values similar to rates of ethanol uptake of about 60 mumol/h per g measured under identical conditions. Rates of ethanol uptake by perfused livers from ADH-positive, but not from ADH-negative, deermice were increased by about 50% by infusion of fructose. Thus it is concluded that the stimulation of hepatic ethanol uptake by fructose is dependent on the presence of ADH. Unexpectedly, fructose decreased rates of ethanol metabolism and H2O2 generation by about 60% in perfused livers from ADH-negative deermice, probably by decreasing activation of fatty acids and thus diminishing rates of peroxisomal beta-oxidation.

scholarly journals Intermediates of peroxisomal β-oxidation. A study of the fatty acyl-CoA esters which accumulate during peroxisomal β-oxidation of [U-14C]hexadecanoate

1990 ◽  
Vol 270 (1) ◽  
pp. 175-180 ◽  
Author(s):  
K Bartlett ◽  
R Hovik ◽  
S Eaton ◽  
N J Watmough ◽  
H Osmundsen
Keyword(s):  
Lactate Dehydrogenase ◽  
Chain Length ◽  
Full Range ◽  
Fatty Acyl ◽  
Acetyl Coa ◽  
Low Concentrations ◽  
High Concentrations ◽  
Rate Limiting

1. 14C-labelled fatty acyl-CoA esters resulting from β-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of β-oxidation now resembling chain-shortening of hexadecanoate by two cycles of β-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal β-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal β-oxidation.

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