Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

Effect of Escherichia coli endotoxin on ascorbic acid transport in isolated adrenocortical cells

The active uptake of ascorbic acid by isolated rat adrenocortical cells increases with ascorbic acid concentration, depends on time and calcium, and is inhibited by ACTH concentrations required for maximal steroidogenesis. Lipopolysaccharide of Escherichia coli 0111:B4 modifies the ascorbic acid uptake in a calcium-dependent manner. At low calcium concentrations, lipopolysaccharide exerts a stimulatory effect on ascorbic acid transport and at high concentrations lipopolysaccharide produces a dose-dependent inhibitory effect. This inhibition of the ascorbic acid transport by the endotoxin can alter the ascorbic acid accumulation in the adrenal gland during endotoxin shock.     

Decreased levels of ascorbic acid in lung following exposure to ozone

Mice were exposed to concentrations of 20, 40 and 200 ppm ozone in air for 30 min. Ozone exposure decreased lung ascorbic acid levels and increased lung weight by up to 50% in a dose related manner. On incubation in Krebsphosphate solution, lung slices from mice exposed to 200 ppm ozone released a smaller fraction of their content of ascorbic acid into the medium than did lung slices from control mice, suggesting that there was a preferential loss of extracellular ascorbic acid during ozone exposure. These results are consistent with the proposed function of ascorbic acid as an extracellular antioxidant in lungs.     

Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B.

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.     

Osteoclast response to low extracellular sodium and the mechanism of hyponatremia-induced bone loss

Our recent animal and human studies revealed that chronic hyponatremia is a previously unrecognized cause of osteoporosis that is associated with increased osteoclast numbers in a rat model of the human disease of the syndrome of inappropriate antidiuretic hormone secretion (SIADH). We used cellular and molecular approaches to demonstrate that sustained low extracellular sodium ion concentrations ([Na(+)]) directly stimulate osteoclastogenesis and resorptive activity and to explore the mechanisms underlying this effect. Assays on murine preosteoclastic RAW 264.7 cells and on primary bone marrow monocytes both indicated that lowering the medium [Na(+)] dose-dependently increased osteoclast formation and resorptive activity. Low [Na(+)], rather than low osmolality, triggered these effects. Chronic reduction of [Na(+)] dose-dependently decreased intracellular calcium without depleting endoplasmic reticulum calcium stores. Moreover, we found that reduction of [Na(+)] dose-dependently decreased cellular uptake of radiolabeled ascorbic acid, and reduction of ascorbic acid in the culture medium mimicked the osteoclastogenic effect of low [Na(+)]. We also detected downstream effects of reduced ascorbic acid uptake, namely evidence of hyponatremia-induced oxidative stress. This was manifested by increased intracellular free oxygen radical accumulation and proportional changes in protein expression and phosphorylation, as indicated by Western blot analysis from cellular extracts and by increased serum 8-hydroxy-2'-deoxyguanosine levels in vivo in rats. Our results therefore reveal novel sodium signaling mechanisms in osteoclasts that may serve to mobilize sodium from bone stores during prolonged hyponatremia, thereby leading to a resorptive osteoporosis in patients with SIADH.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae

There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; high-affinity uptake somehow depends on the presence of hexose kinases (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983; L. F. Bisson and D. G. Fraenkel, J. Bacteriol. 155:995-1000, 1983). We report here on the effect of culture conditions on the level of high-affinity uptake. The high-affinity component was low during growth in high concentrations of glucose (100 mM), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase. The higher level of uptake was found in growth on low concentrations of glucose (0.5 mM) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol. These results suggest that some component of high-affinity uptake is repressible by glucose. A shift from medium with 100 mM glucose to medium with 5 mM glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required. Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide. The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves. These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed. Apparent similarities between glucose uptake in yeast and animal cells are noted.     

Cell growth and the regulation of prostaglandin synthesis

Prostaglandin (PG) synthesis was determined in human embryo lung fibroblasts (HELF) during active, slowed and nongrowing phases. Bradykinin and ascorbic acid were used to induce PG synthesis. The cells were also exposed to arachidonic acid, a PG precursor. During active growth, PGE₂ synthesis in response to stimulation by either bradykinin or ascorbic acid was low. As growth slowed the cellular response changed. During quiescence bradykinin and ascorbic acid stimulated PG production markedly while the conversion of free arachidonic acid to PGE₂ also increased markedly. This change in response by quiescent cells was not due to an increase in cell density. When growing and quiescent cells at the same cell density were compared, the growing cells showed very little response to bradykinin while the quiescent cells were very responsive. The change in response was also not due to any differences in arachidonic acid concentrations in the culture medium during growth and non-growth.     

Fatty acid profiles of cells from the insect cell line iprl-21 (Spodoptera frugiperda) and of the tissue culture medium after repeated use

Summary When IPL-1 medium was used for three serial incubations of cells of the IPRL-21 insect cell line (Spodoptera frugiperda, J. E. Smith) at least 23 fatty acids were identified from the media and/or from the cells. During the first incubation only negligible changes occurred in the total fatty acid content of the medium, but after the second and third incubations the total content decreased. Seven of the 23 fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids) comprised 92% of the total fatty acid content, but the specific concentrations varied after each 7-day incubation. During the first incubation, the concentration of the monoene fatty acids increased, and the concentration of the more highly unsaturated fatty acids decreased. During the second and third serial incubations, the specific concentrations of all fatty acids decreased, with the exception of palmitoleic acid. These changes in the total fatty acid content and in the specific concentration of individual fatty acids in the cell indicated uptake of fatty acids from the medium and/or cellular lipid biosynthesis. The fatty acid content of the cells differed during the active growth phase and the stationary phase.     

Histochemical studies on the developing adrenal gland of Gallus domesticus

Summary The distribution of adrenaline, noradrenaline, aliesterases and non-specific cholinesterases in the cortical and medullary cells and that of ascorbic acid in the cortex have been studied histochemically in sections of adrenal glands from embryonic, juvenile and adult chicken. Both the catecholamines are secreted by the embryonic medulla from the 11th day of incubation but noradrenaline is the more abundant of the two hormones at all stages and it is secreted by the majority of chromaffin cells. There is a tendency for the adrenaline-secreting cells to predominate in the subcapsular layer of the medulla. Both types of chromaffin cells reveal considerable cholinesterase activity consistently from the second half of incubation period onwards.A high concentration of aliesterases and ascorbic acid are developed and maintained in the cortical cords from the time the cortex begins secretory activity, namely, the 10-day incubation stage. Lower concentrations of cholinesterases are also present in the cells of the cortex. The cords of the peripheral zone of cortex show higher concentrations of both the enzymes and ascorbic acid than those of the central zone.From a thesis submitted to McGill University, Montreal, Canada in 1963 in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The work was done during tenure of a Canadian Commonwealth Scholarship.     

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.     

Ethylenediaminetetraacetic acid (EDTA) does not increase iron uptake or ferritin synthesis by Caco-2 cells

Ethylenediaminetetraacetic acid (NaFe-EDTA) is a chelator capable of binding a wide variety of metals, with a high affinity constant for Fe(3+). NaFe-EDTA has been extensively studied and validated as an excellent choice for iron fortification programs and extensive research has demonstrated its high bioavailability specially for cereal based foods. To further evaluate the usefulness of this compound we performed iron uptake experiments with EDTA using the Caco-2 cell system. Cells were incubated in PBS at pH 5.5 or 7.0, containing or not ascorbic acid. Different sources of EDTA, different concentrations of NaFe-EDTA and the inclusion of another iron compound as electrolytic iron, were tested. Also, the ferritin content of Caco-2 cells 24h after 1h incubation with iron compounds was evaluated. Except for the addition of ascorbic acid, under the experimental conditions used, Caco-2 cells were not capable of obtaining iron from NaFe-EDTA. Furthermore, iron uptake from electrolytic iron was inhibited when Na(2) or K(2)-EDTA were included. Ferritin determinations to Caco-2 cells evaluated 24h after 1h incubation periods, showed that NaFe-EDTA did not induce new ferritin synthesis, since iron did not enter the cells. Further studies are required to evaluate incorporation of iron from NaFe-EDTA to a common iron pool and the requirements for iron uptake by Caco-2 cells.     

Transition of Chemolithotroph Ferrobacillus ferrooxidans to Obligate Organotrophy and Metabolic Capabilities of Glucose-Grown Cells

Transition of chemolithotrophic Ferrobacillus ferrooxidans to organotrophy occurred after 60 hr of incubation in an organic medium. Three distinct phases, based on metabolic activities of cells, were observed during the course of transition. Conversion of cellular nutrition to organotrophy resulted in a gradual loss of Fe(2+) oxidation and cessation of CO(2) fixation. These changes were concomitant with a rapid increase in uptake of glucose and phosphate during the latter part of transition period. The outcome of transition was governed by the pH of the medium, temperature of incubation, availability of oxygen, age of the chemolithotrophic cells, and the type of energy and carbon source available to the bacterium. Presence or absence of p-aminobenzoic acid and Fe(2+) ions did not influence transition of cells. A defined medium containing glucose, mineral salts, and p-aminobenzoic acid at pH 2.5 was found to be most suitable for transition and for culture of heterotrophic convertants. Maximum growth rate of the heterotrophic cells was attained with vigorous aeration at 35 C. The bacterium could be cultured on a variety of organic compounds, including complex organic media, provided they were used in low concentrations. Serological studies on autotrophic cells and the heterotrophic convertant have shown a definite antigenic relationship between the two cell types.     

The effects of glucose on ascorbic acid uptake in heart endothelial cells: Possible pathogenesis of diabetic angiopathies

Glucose in concentrations of 20 mg% (or greater) significantly inhibited ¹⁴C-labelled ascorbic acid (1.25 mg%) uptake in endothelial cells in the presence of insulin (1600 ωU/ml). The absence of insulin also significantly reduced ascorbic acid uptake. Furthermore, this reduction could be exacerbated by glucose (40, 160 mg%) but not equimolar concentrations of fructose. Increased ascorbic acid concentrations (two-fold) in the absence of insulin (1) significantly enhanced uptake, and (2) reversed the inhibition by glucose. These findings support earlier reports that ascorbic acid uptake into the cell may be compromised by decreased insulin and/or increased extracellular glucose levels. Since previous animal studies have correlated experimental ascorbic acid deficiencies with atherogenic processes (presumably by altering glycosaminoglycan metabolism), the postulation that the “diabetic condition” (low insulin, hyperglycemia) accelerates the cellular changes leading to atherosclerosis by impairing ascorbic acid uptake into the vascular endothelium, may now be supported.     

INFLUENCE OF FERULIC ACID ON MINERAL DEPLETION AND UPTAKE OF 86Rb BY PAUL'S SCARLET ROSE CELL-SUSPENSION CULTURES

The influence of 10^-4 m ferulic acid on mineral depletion and ion uptake in sterile cultures of Paul's Scarlet rose was examined. The effect of ferulic acid on the rate of depletion of Mg²⁺, Ca²⁺, K⁺, P, Fe³⁺, Mn²⁺, and Mo³⁺ from the medium during the 14-day growth cycle varied with the age of the cells and the ion under consideration. In general, rates of uptake were higher than control rates in older cells and less than control rates in cells 3–5 days old. The degree of inhibition of uptake of ⁸⁶Rb also varied with age. Young (4–5 day) cells showed approximately 50% inhibition at high concentrations of RbCl (system 2) and approximately 25% inhibition at low concentrations of RbCl (system 1). In contrast, the rate of ⁸⁶Rb uptake in 10-day cells was not significantly altered by incubation in ferulic acid.     

Putrescine uptake by the cellular slime mould dictyostelium discoideum.

1. Rapid labelling occurs when myxamoebae of Dictyostelium discoideum strain AX2 are incubated with [1,4-14C]putrescine. Labelling is energy-dependent. 2. The label enters a pool from which rapid exchange with extracellular putrescine does not occur, and labelling is believed to represent uptake into the cells. 3. The concentration-dependence of putrescine uptake indicates that a number of systems are involved, at least one of which is saturable, with a Km of 9.1 micro M-putrescine. At high putrescine concentrations the overall uptake process is non-saturable. 4. Significant metabolism of putrescine and loss of intracellular putrescine to the medium only occurred when cells were incubated with millimolar concentrations of extracellular putrescine. 5. Putrescine uptake was inhibited by diamines, polyamines, bivalent metal ions and omega-aminocarboxylic acids. 6. The ability to take up putrescine at low concentrations decreased during starvation of myxamoebae. 7. The results are interpreted in terms of a model for putrescine uptake involving adsorptive pinocytosis at low concentrations and fluid-phase pinocytosis at high concentrations.     

Morphological and biochemical alterations of oomycete fish pathogen <Emphasis Type="Italic">Saprolegnia parasitica</Emphasis> as affected by salinity,ascorbic acid and their synergistic action

Vegetative growth of Saprolegnia parasitica decreased by increasing the concentration of NaCl and ascorbic acid. Under these conditions, the morphological features of the vegetative hyphae were distinguishable from those used as controls. NaCl and ascorbic acid in combination improved the tolerance of S. parasitica to high levels of salinity. Sporangial formation, release and proliferation were very sensitive to even lower levels of salinity. For instance, at 0.03 M NaCl sporangia formation was rarely observed. Ascorbic acid alone had a little effect on sporangial formation and release, but when combine with NaCl the developmental processes were improved. Reduction of numbers and plasmolysis of oogonia were found at various NaCl concentrations, whereas ascorbic acid stimulated the formation of these reproductive organs at low concentrations. The synergistic effect of NaCl and ascorbic acid improved and overcomed the symptoms of oogonial plasmolysis. Protease activity of S. parasitica was significantly reduced at all NaCl concentrations, whilst ascorbic acid significantly increased and inhibited it at low concentrations and at moderate and high concentrations, respectively. The combination of these compounds reduced protease activity at all tested concentrations with significant difference at the highest concentration. The total free amino-acids content of S. parasitica mycelia was significantly reduced at all the NaCl concentrations, whereas ascorbic acid significantly increased it at low but inhibited it at higher concentrations. The combination of NaCl and ascorbic acid significantly increased the accumulation of free amino-acids at low and moderate concentrations, but decreased them at high concentrations. Total protein content was reduced at all tested concentrations of NaCl and ascorbic acid had also similar effect. However, the combined effect of NaCl and ascorbic acid significantly enhanced and reduced total protein content at low and high concentrations, respectively. Treatments with NaCl induced proline accumulation in S. parasitica, which paralleled the salt concentration.     

Recycling of vitamin C by a bystander effect

Human cells transport dehydroascorbic acid through facilitative glucose transporters, in apparent contradiction with evidence indicating that vitamin C is present in human blood only as ascorbic acid. On the other hand, activated host defense cells undergoing the oxidative burst show increased vitamin C accumulation. We analyzed the role of the oxidative burst and the glucose transporters on vitamin C recycling in an in vitro system consisting of activated host-defense cells co-cultured with human cell lines and primary cells. We asked whether human cells can acquire vitamin C by a "bystander effect" by taking up dehydroascorbic acid generated from extracellular ascorbic acid by neighboring cells undergoing the oxidative burst. As activated cells, we used HL-60 neutrophils and normal human neutrophils activated with phorbol 12 myristate 13-acetate. As bystander cells, we used immortalized cell lines and primary cultures of human epithelial and endothelial cells. Activated cells produced superoxide anions that oxidized extracellular ascorbic acid to dehydroascorbic acid. At the same time, there was a marked increase in vitamin C uptake by the bystander cells that was blocked by superoxide dismutase but not by catalase and was inhibited by the glucose transporter inhibitor cytochalasin B. Only ascorbic acid was accumulated intracellularly by the bystander cells. Glucose partially blocked vitamin C uptake by the bystander cells, although it increased superoxide production by the activated cells. We conclude that the local production of superoxide anions by activated cells causes the oxidation of extracellular ascorbic acid to dehydroascorbic acid, which is then transported by neighboring cells through the glucose transporters and immediately reduced to ascorbic acid intracellularly. In addition to causing increased intracellular concentrations of ascorbic acid with likely associated enhanced antioxidant defense mechanisms, the bystander effect may allow the recycling of vitamin C in vivo, which may contribute to the low daily requirements of the vitamin in humans.     

Ascorbic acid transport and accumulation in human neutrophils

The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake.     

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases.

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 mm pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mm, than of higher concentrations (1.0 mm) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K_m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.