Functional Compartmentation of Acetaldehyde Oxidation in Rat Liver*

SUMMARY Perfused rat liver and suspensions of isolated rat liver cells have been used to study the influence of the transaminase inhibitors aminooxyacetate and DL-cycloserine on the rate of acetaldehyde utilization, and the effects of acetaldehyde on the state of reduction of cytosolic and mitochondrial pyridine nucleotides. In the presence of 4-methylpyrazole to inhibit acetaldehyde reduction to ethanol, acetaldehyde removal was constant over the range from 0.1 to 0.4 mhr at a rate of approximately 400 pmoles per g dry weight per hour. Between 0.4 and 10 mM acetaldehyde, the rate of acetaldehyde uptake increased by 60% with increasing acetaldehyde concentrations, with a half-maximum increment of uptake being achieved at about 1 mM acetaldehyde. DL-Cycloserine had no effect on acetaldehyde uptake at low concentrations of acetaldehyde but almost completely inhibited the stimulation of uptake observed at high concentrations. Neither or,-cycloserine nor aminooxyacetate had any inhibitory effect on the reduction of mitochondrial pyridine nucleotides induced by acetaldehyde. At high concentrations


SUMMARY
Perfused rat liver and suspensions of isolated rat liver cells have been used to study the influence of the transaminase inhibitors aminooxyacetate and DL-cycloserine on the rate of acetaldehyde utilization, and the effects of acetaldehyde on the state of reduction of cytosolic and mitochondrial pyridine nucleotides.
In the presence of 4-methylpyrazole to inhibit acetaldehyde reduction to ethanol, acetaldehyde removal was constant over the range from 0.1 to 0.4 mhr at a rate of approximately 400 pmoles per g dry weight per hour. Between 0.4 and 10 mM acetaldehyde, the rate of acetaldehyde uptake increased by 60% with increasing acetaldehyde concentrations, with a half-maximum increment of uptake being achieved at about 1 mM acetaldehyde.
DL-Cycloserine had no effect on acetaldehyde uptake at low concentrations of acetaldehyde but almost completely inhibited the stimulation of uptake observed at high concentrations. Neither or,-cycloserine nor aminooxyacetate had any inhibitory effect on the reduction of mitochondrial pyridine nucleotides induced by acetaldehyde. At high acetaldehyde concentrations (3 to 4 mM) pyruvate stimulated acetaldehyde uptake, but little effect was observed at low acetaldehyde concentrations (0.3 mM). Acetaldehyde uptake was inhibited by fatty acids, P-hydroxybutyrate, and amobarbital, and stimulated by acetoacetate. Oxidation of endogenous fatty acid was diminished by acetaldehyde. These data indicate that oxidation of acetaldehyde by rat liver occurs almost entirely in the mitochondrial compartment when the mean arterial-venous acetaldehyde concentration is below about 0.4 mM. At higher acetaldehyde concentrations, oxidation occurs also in the cytosol, and reducing equivalents generated in the cytosol by a high K, NADlinked aldehyde dehydrogenase are transported to the mitochondria mainly by the malate-aspartate cycle. It may be concluded that acetaldehyde generated during ethanol metabolism is oxidized to acetate predominantly in the mito-* This work was supported by Grants AM-15520 and AA-90292 from the United States Public Health Service.
chondria, so that only 1 eq of NADH is generated in the cytosol per mole of ethanol oxidized via alcohol dehydrogenase.
A wide range of enzymes capable of oxidizing acetaldehyde to acetate in liver have been described.
One type comprising a variety of flavoprotein oxidases has a low substrate specificity and affinity for aldehydes, and probably plays a minor role in acetaldehyde oxidation under physiological conditions (l-4). Racker (5) described the purification of a nonspecific NADlinked aldehyde dehydrogenase from beef liver with a high affinit.y toward acetaldehyde (Michaelis constant less than 10m5 M). NAD-linked aldehyde dehydrogenases with high affinity toward acetaldehyde have since been isolated and characterized from liver of a number of species (6-13) as well as from other organs such as kidney and brain (6,14,15). Subcellular distri-.bution studies of the aldehyde dehydrogenase activity of rat liver have shown that the cell supernatant contains at least two NAD-dependent aldehyde dehydrogenases which differ in their substrate specificities and physical properties (16,17) in addition to one or more enzymes associated with the mitochondrial fraction (14,(18)(19)(20)(21)(22).
Clear evidence for the heterogeneity of the cytoplasmic enzymes has been obtained by Deitrich et al. (17,23), who found that one of the soluble NAD-dependent aldehyde dehydrogenases was induced lo-fold without any change of the kinetic characteristics by treatment of genetically selected rats with phenobarbital.
The soluble aldehyde dehydrogenases of rat liver (16, 17) appear to have apparent K, values for acetaldehyde 2 to 3 orders of magnitude higher than those of the corresponding enzymes from beef (5, 9), horse (II), or human (7, 8) liver.
In contrast to the earlier studies of Btittner (6) who found that most of the aldehyde dehydrogenase activity of rat liver was extramitochondrial, more recent studies (22)(23)(24)(25) have shown that about 80% of the total activity is in the mitochondrial fraction. However, the mitochondrial NADdependent aldehyde dehydrogenase activity appears to be heterogeneous. Marjanen (24) reported an apparent K, for acetaldehyde of below 10 PM, while Grunnet (22) found two K, values for acetaldehyde; one below 1 PM and one about 1 mM. Mitochondrial fractionation studies by Tottmar et al. (26) suggest that an enzyme with a relatively high K, for acetaldehyde (1 to 2 mM) similar to that of supernatant aldehyde dehydrogenase is located on the outer membrane while a second enzyme in the matrix has an apparent K, for acetaldehyde in the region of 1 pM.
These latter authors also found considerable activity of the high K, enzyme in the microsomal fraction of rat liver, and less than 5y0 of total activity in the soluble fraction. Isolated liver mitochondria oxidize acetaldehyde and other aldehydes when added at low concentrations, although high concentrations are inhibitory (E-22). Acetaldehyde also inhibited the oxidation of pyruvate by mitochondria from a number of tissues (19). Previous studies using surface fluorometric techniques to monitor changes in the oxidation-reduction state of pyridine nucleotides in the cytosolic and mitochondrial spaces of bloodfree perfused livers (27, 28) have shown that after ethanol addition a rapid reduction of pyridine nucleotides occurred in both spaces. These results were interpreted as indicating a rapid transport of reducing equivalents from cytosol to mitochondria by the malate-aspartate cycle. Furthermore, metabolic balances describing the interaction of ethanol oxidation with gluconeogenesis from alanine were calculated on the basis of the production of 2 eq of NADH in the cytosol per mole of ethanol utilized (28). The recent studies on the cellular distribution of aldehyde dehydrogenases quoted above, together with further studies of the effects of ethanol and acetaldehyde on cellular pyridine nucleotide oxidation-reduction changes in perfused rat liver (29) suggest that the mitochondrial oxidation of acetaldehyde predominates at low concentrations, and that the cytosolic enzyme is functionally operative only at acetaldehyde concentrations much higher than those of 100 to 200 PM reported in uivo (30-33) or in the effluent fluid of perfused livers (34) after ethanol administration, The present study was initiated in order to clarify the respective roles of the cytosolic and mitochondrial aldehyde dehydrogenases for acetaldehyde metabolism in the intact liver cell. Use of DL-CyClOSerine to inhibit extramitochondrial transaminase reactions (35, 36) associated with the malate-aspartate cycle has established that the reduction of mitochondrial pyridine nucleotides by acetaldehyde was not affected by this inhibitor and that uptake of acetaldehyde by isolated liver cells was also unaffected at acetaldehyde concentrations below 0.4 mu. On the other hand, competition for acetaldehyde oxidation was exerted by compounds such as fatty acids or /3-hydroxybutyrate which generate reducing equivalents directly in the mitochondria.
It is evident, therefore, that the various metabolic effects associated with ethanol oxidation by the liver must be interprkted in terms of the subcellular compartmentation of the two reductive steps of ethanol metabolism to acetate.

EXPERIMENTAL PROCEDURES
Animals-Male albino rats (Holteman) 180 to 220 g in weight were fasted for 20 to 28 hours prior to liver perfusion studies or isolation of hepatocytes.

RESULTS
A comparison of the flavin and pyridine nucleotide fluorescence responses to brief periods of ethanol and acetaldehyde infusion in perfused rat liver is shown in Fig. 1 to acetoacetate in the effluent fluid of perfused rat livers after infusion of acetaldehyde. Methylpyrasole (50 PM) and aminooxyacetate (0.2 mM) were added at the times indicated and infusion continued to produce the concentrations shown in the arterial fluid. tion of acetaldehyde to ethanol via alcohol dehydrogenase (29). A change of the flavin nucleotide fluorescence reflects changes of the oxidation-reduction state of the mitochondrial pyridine nucleotide pool as documented elsewhere (29,38,44), while a change of the pyridine nucleotide fluorescence represents the sum of contributions of oxidation-reduction changes of pyridine nucleotides in both the cytosolic and mitochondrial compartments.
In the experiments shown in Figs. 1 and 2, lactate (1 mM), pyruvate (0.2 II~M), @-hydroxybutyrate (0.2 n@, and acetoacetate (0.3 mM) were infused into the arterial fluid flowing to the liver. Studies have shown that at the concentrations used a rapid equilibration of lactate and pyruvate and of &hydroxybut,yrate and acetoacetate occurred with their respective dehydrogenases and the intracellular pyridine nucleotide pools during one passage through the liver (29). Measurements of the ratios of lactate to pyruvate and P-hydroxybutyrate to acetoacetate in the effluent fluid, therefore, provide an ancillary method for estimating the pyridine nucleotide oxidation-reduction potentials in the separate cytosolic and mitochondrial spaces (45). Both ethanol and acetaldehyde caused a prompt reduction of flavin and pyridine nucleotides, but a marked increase in the size of the pyridine nucleotide fluorescence response was observed with acetaldehyde relative to that obtained with ethanol.
On the basis of these results alone it could be predicted that acetaldehyde would cause a larger reduction of cytosolic pyridine nucleotides than ethanol.
However, direct measurements of the lactate to pyruvate ratio in the effluent fluid from the liver in companion experiments ( Fig. 2) failed to confirm this interpretation.
The data show that 1 m acetaldehyde in the presence of methylpyrazole increased the lactate to pyruvate ratio to a value of about 20 compared with an increase to 50 after addition of a similar concentration of ethanol. The lack of effectiveness of low concentrations of acetaldehyde compared with ethanol in raising the lactate to pyruvate ratio in perfused liver has also been reported by Lindros et al. (34). An alternative explanation may be advanced that NADH bound to cytosolic acetaldehyde dehydrogenase exhibits a large horescence enhancement.
This phenomenon has recently been observed with an acetaldehyde dehydrogenase isozyme purified from horse liver (46).
Figs. I and 2 show that acetaldehyde infusion in the presence of aminooxyacetate as transaminase inhibitor (47) produced essentially identical changes of flavin and pyridine nucleotide fluorescence and increase of the /3-hydroxybutyrate to acetoacetate ratio as in the absence of aminooxyacetate.
Addition of aminooxyacetate itself caused an increased state of reduction of total pyridine nucleotides and a slight oxidation of flavin nucleotides, indicating pyridine nucleotide reduction in the cytosol but oxidation in the mitochondria.
These changes correlated with an increase of the lactate to pyruvate ratio from 6 to 18 and a decrease of the &hydroxybutyrate to acetoacetate ratio from 0.48 to 0.42. Since aminooxyacetate largely abolishes the increased reduction of mitochondrial pyridine nucleotides observed after ethanol addition (48-50) it may be concluded that reducing equivalents are not generated at an appreciable rate in the cytosol by oxidation of acetaldehyde at low concentrations (cf. Ref. 29). However, it is evident from the increase of the lactate to pyruvate ratio after acetaldehyde addition that an interaction occurs between cytosolic acetaldehyde dehydrogenase and lactate dehydrogenase to increase the cytosolic NADH: NAD ratio.
Further studies using nn-cycloserine as transaminase inhibitor (Fig. 3), also showed that it was without effect on the increase of the fi-hydroxybutyrate to acetoacetate ratio obtained upon addition of 2 mM acetaldehyde to the perfused rat liver.
Aminooxyacetate and cycloserine inhibit transaminases by reacting with the pyridoxal phosphate form of the enzyme to form a relatively stable analogue of the normal enzyme substrate complex (see Ref. 36 for references).
a-Ketoglutarate accelerates the rate of inhibition with aspartate aminotransferase, while glutamate and aspartate protect the enzyme.
Kinetic studies with isolated aspartate aminotransferase, measured in the direction of oxalacetate formation in a coupled reaction with malate dehydrogenase, showed that at relatively low aspartate concentrations (1 to 2 mM) aminooxyacetate was a rather better in- hibitor than DL-cycloserine as judged by concentrations for half-maximum inhibition of 0.07 and 0.2 mu, respectively, obtained with the two inhibitors.
D-Cycloserine was found to be much less inhibitory than the DL mixture on isolated aspartate aminotransferase.
Aminooxyacetate at concentrations of 0.5 to 1 mM completely inhibited aspartate formation by rat liver mitochondria incubated with 1 mM malate and 10 mu glutamate under conditions of State 3 respiration, whereas DL-cycloserine was ineffective at concentrations up to 10 mM. Aminooxyacetate, therefore, inhibits both cytosolic and mitochondrial transaminases in the intact cell, whereas cycloserine inhibits only the cytosolic transaminases.
This conclusion was corroborated by experiments with liver cells incubated with 10 mM DL-cycloserine or 0.5 mM aminooxyacetate, which were centrifuged and aspartate aminotransferase activity measured in the supernatant after ultrasonic disruption of the resuspended cells. Very little activity was found in cells incubated with aminooxyacetate, but up to 40% of the total activity was found in cells incubated with DL-cycloserine.
In order to investigate the relative proportions of acetaldehyde metabolized in the cytosol and mitochondria in greater detail, rates of acetaldehyde uptake at different concentrations of acetaldehyde were measured in the presence and absence of DLcycloserine using isolated rat liver cells. The concentration of the cell suspension was varied along with the initial acetaldehyde concentration so that only about half of the acetaldehyde was removed during the incubation at each acetaldehyde concentration.
Up to six samples were removed over a period of 30 or 60 min in order to calculate a mean rate for an average acetaldehyde concentration. Fig. 4A shows data from a series of experiments performed in the absence of methylpyrazole. Both acetaldehyde removal and ethanol production were measured, and the rates shown are calculated values for acetaldehyde oxidation after correction of the acetaldehyde uptake for ethanol formation.
From the latter measurements a r/',,, of 1220 pmoles per g dry weight per hour for the rate of ethanol forma-4929 icetaldehyde uptake ( -:ontro1 % tion from acetaldehyde and an apparent K, for acetaldehyde of 2 mM could be calculated, which is somewhat higher than that previously estimated by Lindros et al. (34). The data illustrated in Fig. 4A for acetaldehyde metabolism in the absence and presence (Fig. 4B) of methylpyrazole, where ethanol production was negligible, show a relatively high rate of acetaldehyde oxidation at mean acetaldehyde concentrations as low as 0.1 mM. This rate was not affected by the presence of 10 mu DL-cycloserine.
Above about 0.4 mM acetaldehyde, an increased rate of acetaldehyde oxidation was observed which was sensitive to inhibition by DL-cycloserine.
For the second phase of acetaldehyde oxidation, acetaldehyde concentrations of 1.5 and 1.1 mM may be calculated for half-maximum increases of acetaldehyde oxidation from the data obtained in the absence and presence of methylpyrazole, respectively. The presence of methyl pyrazole had the effect of diminishing the rate of acetaldehyde oxidation (corrected for ethanol formation) as previously observed by Lindros et al. (34). The data shown in Fig. 4 provide substantial support to the conclusions reached in another paper (29) that at low acetaldehyde levels (up to 0.4 mM) commensurate with those obtained during ethanol oxidation, acetaldehyde oxidation proceeds almost entirely via a low K, mitochondrial dehydrogenase. At higher acetaldehyde concentrations, oxidation by cytosolic acetaldehyde dehydrogenase (apparent K, 1 to 2 mM) is superimposed on the mitochondrial oxidation, and transport of reducing equivalents from cytosol to mitochondria proceeds mainly by the malate-aspartate cycle. Table I shows that with relatively high acetaldehyde concentrations (3 to 4 mM) added to suspensions of liver cells in the presence of 0.2 mu propionylpyrazole to inhibit alcohol dehydrogenase activity, the rate of acetaldehyde uptake was strongly increased by addition of pyruvate as a trapping system for cytosolic NADH.
On the other hand, addition of substrates which generate reducing equivalents directly in the mitochondria, such as octanoate, oleate, or @-hydroxybutyrate, all caused a substantial inhibition of acetaldehyde uptake, while addition of acetoacetate stimulated acetaldehyde uptake. In Experiment 3 of Table I, addition of acetaldehyde decreased ketone body formation from oleate from 384 to 311 pmoles per g dry weight per hour and increased the ratio of @-hydroxybutyrate to acetoacetate from 3.0 f 0.2 to 8.7 f 1.3. Addition of amobarbital (Amytal) which inhibits electron transport between NADH dehydrogenase and cytochrome b also caused a severe inhibition of acetaldehyde uptake. When low (0.3 to 0.5 mM) initial acetaldehyde concentrations were used (Table II), the cont.rol rate of acetaldehyde uptake was lower (cf. Fig. 4), and pyruvate had a much smaller stimulatory effect on uptake, particularly at a mean acetaldehyde concentration of 0.2 mM. Oleate and /3-hydroxybutyrate were strongly inhibitory, while acetoacetate addition almost doubled acetaldehyde uptake. These results show a competition for mitochondrial NAD between acetaldehyde dehydrogenase and other mitochondrial dehydrogenases.
On the other hand, relatively high acetalde- hyde concentrations are required to show interactions with cytosolic NAD-linked dehydrogenases. The effects of acetaldehyde infusion on oxygen uptake and 14COZ production from [UJ4]oleate added in tracer amounts was investigated in perfused rat livers supplied with either 0.45 mu pyruvate (Fig. 5) or 0.45 mM I,(+)-lactate (Fig. 6) in the arterial fluid. Methylpyrazole (50 PM) was also infused to inhibit reduction of acetaldehyde to ethanol.
Addition of substrate stimulated cell respiration and oxidation of endogenous fatty acids, the latter effect being judged from the 2-to a-fold stimulation of '4COz production. This finding is in accordance with previous indirect estimates based on calculations from metabolic balance studies of the effect of lactate (51) and pyruvate (52) on endogenous fatty acid oxidation.
Infusion of 1 mM acetaldehyde for 10 min inhibited the generation of 14C02 with both substrates, but had opposite effects on the rate of respiration; this being stimulated in the presence of pyruvate but inhibited transiently in the presence of lactate.
Infusion of 0.2 mu aminooxyacetate caused a small increase of 14C02 production, probably as compensation for inhibition of endogenous ureogenesis. It produced no effect on oxygen consumption in the presence of pyruvate but an inhibition of oxygen consumption to the endogenate rate in the presence of lactate.
These changes 045mM  correlated with a lack of inhibitory effect of aminooxyacetate on by the fall of r4C02 production and small stimulation of respiragluconeogenesis from pyruvate but an almost complete inhibition (Fig. 6). Uptake of acetaldehyde was 70% higher with tion of gluconeogenesis from lactate (Ref. 53 and Table III).
pyruvate than with lactate as substrate, presumably as a result A second lo-min infusion of 1 mM acetaldehyde in the presence partly of dismutation between lactate and acetaldehyde deof aminooxyacetate again inhibited i4C02 production. With hydrogenases in the cytosol, and partly to increased mitochonpyruvate as substrate, the inhibitory effect of acetaldehyde on drial oxidation since the rate of electron transport was higher r4C02 production was the same with or without aminooxyacetate with pyruvate as substrate.
Aminooxyacetate had little effect also present.
However, with lactate as substrate the inhibitory on acetaldehyde uptake in the presence of pyruvate, but in the effect of acetaldehyde was smaller in the presence than the ab-presence of lactate a 50% stimulation of acetaldehyde uptake sence of aminooxyacetate, and a small stimulation of respiration was observed. This correlated with an increase of respiration was observed in contrast to an inhibition in the absence of amino- (Fig. 6), suggesting that prior to acetaldehyde addition the liver oxyacetate.
was substrate-depleted. Table III shows further metabolic changes induced by acetaldehyde in a series of liver perfusions following the same experimental protocol as those shown in Figs. 5 and 6. Acetaldehyde stimulated gluconeogenesis from pyruvate by about 407& increased pyruvate uptake by 20$,, and the lactate to pyruvate ratio 2.6-fold, but had no significant effect on lactate production. Uptake of acetaldehyde was stimulated about 15% by pyruvate addition.
It is evident that acetaldehyde oxidation occurred partly in the mitochondrial compartment, thereby competing for oxidation with endogenous fatty acids, and partly in the cytosol, causing a stimulation of gluconeogenesis by increasing the availability of reducing equivalents. The increased oxygen consumption (Fig. 5)  Indirect evidence based on the steady state level of reduction of pyridine nucleotides in the mitochondria and cytosolic compartments in the intact rat liver suggested that at low acetaldehyde concentrations (e.g. below 0.5 mM) reducing equivalents were generated directly in the mitochondria, while at higher concentrations, acetaldehyde was also both reduced by alcohol dehydrogenase to ethanol and oxidized by acetaldehyde dehydrogenase to acetate in the cytosol (29). The present results, made possible by the use of isolated liver cells to measure rates of acetaldehyde uptake over a wide range of concentrations, fully support this conclusion.
Further insight into the compartmentation of acetaldehyde oxidation has been gained by the use of aminooxyacetate and nn-cycloserine to inhibit the transamination steps of the malate-aspartate cycle (54). A number of previous studies with rat kidney cortex and liver (44,53,(55)(56)(57)(58)(59)(60)(61) have established that transamination via aspartate aminotransferase is involved obligatorily in gluconeogenesis from lactate and in the transfer of excess reducing equivalents from the cytosol to mitochondria, although the quantitative contribution of the malate-aspartate cycle to the over-all flux of reducing equivalents is in dispute (60, 61). The rationale behind the use of these inhibitors in the present experiments was that oxidation of acetaldehyde in the cytosol should be inhibited to the extent that the malate-aspartate cycle contributes to the removal of reducing equivalents into mitochondria, while oxidation of acetaldehyde in the mitochondria should be unaffected. Although considerable circumspection must be exercised in the interpretation of data using these inhibitors due to the possibility of Schiff-base formation between aminooxyacetate and acetaldehyde, no notable differences could be detected between the effects of aminooxyacetate and nL-cycloserine at concentrations of substrate and inhibitors used in the present experiments.
Furthermore, clear evidence of inhibitory effects in the intact liver cell was obtained with both aminooxyacetate (Fig. 2, Fig. 6, and Table III) and m-cycloserine (Figs. 3 and 4) in the presence of acetaldehyde.
It may be concluded, therefore, that no major artifact is introduced by use of either inhibitor under carefully selected conditions.
The finding that m-cycloserine at acetaldehyde concentrations below about 0.4 mM had no effect on acetaldehyde uptake by isolated liver cells but suppressed the increased uptake observed at higher acetaldehyde concentrations is strongly suggestive of a dual location for acetaldehyde oxidation.
The high degree of inhibition by or,-cycloserine at elevated acetaldehyde concentrations indicates that reducing equivalents generated in the cytosol were transferred to the mitochondria for reoxidation largely by the malate-aspartate cycle. Furthermore, when alcohol dehydrogenase is active, ethanol formation and acetaldehyde uptake are greatly stimulated by increasing acetaldehyde concentrations (29,34).
The apparent K, of 1 .l mu for the cycloserineinhibited acetaldehyde uptake in the presence of methylpyrazole agrees closely with the apparent K, for the cytosolic aldehyde dehydrogenase (16,17,22,25,26). The data in Fig. 4 show that the total activity of the cytosolic acetaldehyde dehydrogenase appears to be at least as great as that of the mitochondrial enzyme, despite reports that the over-all NAD-linked aldehyde dehydrogenase activity is largely mitochondrial (22-25). Direct mitochondrial oxidation of acetaldehyde is evidenced by the inabilit,y of either aminooxyacetate or nL-cycloserine to inhibit mitochondrial NADH generation and acetaldehyde uptake at low acetaldehyde concentrations.
Since acetaldehyde levels do not rise above 0.2 mM during ethanol metabolism (30-34), it is clear that when acetaldehyde is generated from ethanol its oxidation is purely a mitochondrial function in rat liver. However, this might be a species peculiarity since the apparent K, for hepatic cytosolic NAD-linked aldehyde dehydrogenases is much lower in other species than in the rat (5, 7-9, 11).
Direct mitochondrial oxidation of acetaldehyde has important implications with regard to the regulation of ethanol metabolism. Notably, mutual competitive inhibition between acetaldehyde and fatty acids for oxidation appears to be at the level of mitochondrial NAD-linked dehydrogenases rather than at the step of transport of reducing equivalents into mitochondria.
Likewise, amobarbital inhibition of ethanol metabolism (27,48,62) is probably accounted for by an indirect inhibition of mitochondrial acetaldehyde oxidation as a result of decreased NADH reoxidation by the electron transport chain with secondary feedback via alcohol dehydrogenase to ethanol uptake.
The present data also illustrate inhibition of hepatic endogenous fatty acid oxidation by acetaldehyde, which is similar to that observed by ethanol.' Presumably the suppression of citric acid cycle activity by ethanol (28, 63-67) is partially accounted for by competition between acetaldehyde dehydrogenase and citric acid cycle dehydrogenases for mitochondrial NAD.
The very high affinity of the mitochondrial aldehyde dehydrogenase for acetaldehyde indicates that the acetaldehyde concentration is unlikely to limit ethanol oxidation, as previously suggested by Krebs (68). Regulation of the hepatic rate of ethanol uptake appears to be affected both 1 J. R. Williamson, K. Ohkawa, I. V. Deaciuc, K. 0. Lindros, and R. Parrilla, manuscript in preparation.
by the rate of translocation of reducing equivalents generated from alcohol dehydrogenase into mitochondria (48). as well as by the rate of NADH reoxidation in the electron transport chain (34,44).