Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate

Summary Rats received a single intravenous injection with liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP). This treatment resulted in the elimination of macrophages in spleen and liver within 2 days. Macrophages ingest the liposomes and are destroyed by the drug, which is released from the liposomes after disruption of the phospholipid bilayers under the influence of lysosomal phospholipases. Repopulation of macrophages in spleen and liver was studied at different time intervals after treatment. Macrophages in the liver (Kupffer cells) and red pulp macrophages in the spleen were the first cells to reappear, followed by marginal metallophilic macrophages and marginal-zone macrophages in the spleen. Different markers of the same cell did not reappear simultaneously. On the other hand, the same marker (recognized by the monoclonal antibody ED2) reappeared much more rapidly in the liver than in the spleen. The present results in the rat were different from those earlier obtained in the mouse. Red pulp macrophages were the first cells and marginal zone macrophages were the last cells to repopulate the spleen in both rodents after treatment with Cl2MDP liposomes. However, there was much more overlap in the repopulation kinetics of splenic macrophage subpopulations in the rat, when compared with the mouse.     

Transfer and reoxidation of reducing equivalents as the rate-limiting steps in the oxidation of ethanol by liver cells isolated from fed and fasted rats

A study was made of factors regulating the oxidation of ethanol in liver cells isolated from fed and fasted rats. The rate of ethanol oxidation was greater in liver cells from fed rats than from fasted rats. Inhibitors of the malate-aspartate shuttle decreased the rate of ethanol oxidation, suggesting that this shuttle contributes to the reoxidation of cytosolic NADH produced during the oxidation of ethanol. The greater inhibition of ethanol oxidation by antimycin than by rotenone suggests that the α-glycerophosphate shuttle also plays an important role in transporting reducing equivalents. The components of the malate-aspartate and α-glycerophosphate shuttles stimulated ethanol oxidation to a greater extent in liver cells from fasted rats than those from fed rats, suggesting that in the fasted state, ethanol oxidation is regulated by the intracellular concentrations of substrate shuttle components which transfer reducing equivalents into the mitochondria. Therefore, uncoupling agents, which stimulate oxygen consumption, do not stimulate ethanol oxidation, and concentrations of antimycin which depress oxygen uptake are much less effective in decreasing ethanol oxidation. By contrast, in liver cells from fed rats, the rate of ethanol oxidation was increased by uncoupling agents. Such stimulation was not observed when cells were prepared in the absence of albumin, probably due to leakage of shuttle substrates which leads to abnormally low intracellular levels. Indeed, when the shuttle substrates were added back to these preparations, uncouplers were effective in stimulating the rate of ethanol oxidation beyond the stimulation produced by the shuttle substrates alone. Thus, under conditions of sufficient intracellular levels of the intermediates of the substrate shuttles, ethanol oxidation is regulated by the capacity of the mitochondrial respiratory chain to reoxidize reducing equivalents generated by the alcohol dehydrogenase reaction.     

Lymphocyte expression in transgenic trout by mouse immunoglobulin promoter/enhancer

Two groups of transgenic rainbow trout (Oncorhynchus mykiss, Walbaum) have been produced and compared. One group harbored the reporter gene of chloramphenicol acetyltransferase (CAT) associated with mouse immunoglobulin (Ig) promoter/enhancer (pUCL-CAT-E). The other group carried the same reporter gene under the control of the cytomegalovirus promoter/enhancer (pCMV-CAT). Slot blot analysis of DNA from blood cells and other tissues from pUCL-CAT-E fish showed variation of copy number between the major tissues but not between red and white blood cells. Southern blot analysis indicated that multiple copies organized in concatemers were incorporated into the genome. The pCMV-CAT fish had a pronounced expression of CAT in both white and red blood cells. In contrast, activity of CAT was found in the white blood cells of all pUCL-CAT-E fish but not in their red blood cells. Expression in white blood cells was found preferentially in sIg⁺ cells, indicating that B cells are the major expressors. High expression was also found in spleen and kidney, but the activity found in thymocytes was equal to the background level. Analysis of some major tissues showed high white blood cell expression associated with low tissue expression, except that liver (known to contain lymphoid tissue in fish) was higher. Thus the regulatory elements of the Ig gene from mouse induce a tissue-specific expression in fish.     

Erythropoietin responses and physical characterization of erythroid progenitor cells in Rauscher virus infected BALB/c mice.

Infection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU-E) increase exponentially up to 800-fold that of normal levels by the third week of infection. In vitro these CFU-E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU-E from normal mice. Numbers of CFU-E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU-E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU-E colonies at concentrations which did not support growth of normal marrow BFU-E. When compared to normal, CFU-E found in RLV-infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU-E were more homogeneous (modal density 1.0695 g/cm3) than CFU-E from normal spleen. Analysis of physical properties of CFU-E and the nonhemoglobinized erythroblast-like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter. Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast-like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU-E.     

The effects of surgical stress and short-term fasting on protein synthesis in vivo in diverse tissues of the mature rat.

1. We measured fractional rates of protein synthesis, capacities for protein synthesis (i.e. RNA/protein ratio) and efficiencies of protein synthesis (i.e. protein-synthesis rate relative to RNA content) in fasted (24 or 48 h) or fasted/surgically stressed female adult rats. 2. Of the 15 tissues studied, fasting caused decreases in protein content in the liver, gastrointestinal tract, heart, spleen and tibia. There was no detectable decrease in the protein content of the skeletal muscles studied. 3. Fractional rates of synthesis were not uniformly decreased by fasting. Rates in striated muscles, uterus, liver, spleen and tibia were consistently decreased, but decreases in other tissues (lung, gastrointestinal tract, kidney or brain) were inconsistent or not detectable, suggesting that, in many tissues in the mature rat, protein synthesis was not especially sensitive to fasting. 4. In fasting, the decreases in fractional synthesis rate resulted from changes in efficiency (liver and tibia) or from changes in efficiency and capacity (heart, diaphragm, plantaris and gastrocnemius). In the soleus, the main change was a decrease in capacity. 5. Surgical stress increased fractional rates of protein synthesis in diaphragm (where there were increases in both efficiency and capacity) by about 50%, in liver by about 20%, in spleen by about 40%, and possibly also in the heart. In liver and spleen, capacities were increased. In other tissues (including the skeletal muscles), the fractional rates of protein synthesis were unaffected by surgical stress.     

Splenic storage volume in the unanesthetized resting beagle

Determination of the dog's splenic storage volume without surgical procedures requires measurement of both circulating and total red cell volumes. The estimation of circulating red cell volume by radioisotope techniques is impeded by the rapid uptake of tagged cells in the spleen. The circulating cell volume might be calculated from plasma volume and large vessel hematocrit, provided that the latter is corrected for the unequal distribution of red cells in the circulatory system. However, the correction factor can only be estimated in the splenectomized dog. We describe here a method to determine the factor in the intact dog, "physiologically splenectomized" by the severe exercise. The values obtained by this method slightly exceed those in the resting dog, as shown by studies in splenectomized exercising beagles in which splenic function was simulated by infusion of packed cells. The method was tested in beagles exercised by swimming and treadmill running and it was concluded that in the unanesthetized resting beagle about one-third of all erythrocytes is stored in the spleen. Labeled cells are equilibrated with about one-half of the splenic storage volume within 10 min after their injection. During maximal exertion the mean increase in large vessel hematocrit was 38.6 +/- 3.3%, the mean decrease in plasma volume 13.6 +/- 1.7% and the mean increase in plasma osmolarity 2.8 +/- 0.9% (percentages of control values).     

Effect of cold-acclimation on oxygen uptake and glucose production of perfused duckling liver

The control of hepatic metabolism by substrates and hormones was assessed in perfused liver from young Muscovy ducklings. Studies were performed in fed or 24-h fasted 5-week-old thermoneutral (25 degrees C; TN) or cold-acclimated ducklings (4 degrees C; CA) and results were compared with those obtained in rats. Basal oxygen uptake of perfused liver (LVO2) was higher after cold acclimation both in fed (+65%) and 24-h fasted (+29%) ducklings and in 24-h fasted rats (+34%). Lactate (2 mM), the main gluconeogenic substrate in birds, similarly increased LVO2 in both TN and CA ducklings and the effect was larger after fasting. Both glucagon and norepinephrine dose-dependently increased LVO2 in ducklings and rats, but cold acclimation did not improve liver response and liver sensitivity to norepinephrine in ducklings was even reduced in CA animals. Liver contribution to glucagon-induced thermogenesis in vivo was estimated to be 22% in TN and 12% in CA ducklings. Glucagon stimulated gluconeogenesis from lactate in duckling liver and the stimulation was 2.2-fold higher in CA than in TN fasted birds. These results indicate a stimulated hepatic oxidative metabolism in CA ducklings but hepatic glucagon-induced thermogenesis (as measured by LVO2) was not improved. A role of the liver is suggested in duckling metabolic acclimation to cold through an enhanced hepatic gluconeogenesis under glucagon control.     

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions.

1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.     

Tissue distribution and cellular distribution of liposomes encapsulating muramyltripeptide phosphatidyl ethanolamide

In previous studies it was shown that administration of liposome-encapsulated MTPPE (LE-MTPPE) led to resistance againstKlebsiella pneumoniae infection. To get more insight in the cell types that are involved in this by LE-MTPPE induced antibacterial resistance, the tissue distribution of liposomes encapsulating MTPPE and the distribution over the cells in the main target organs were investigated. After intravenous injection of the liposomes in mice a substantial amount was recovered from liver and spleen and a smaller amount from the lung. In the liver 83% of the liposomes was taken up by the macrophages. In the spleen also most liposomes were taken up by macrophages of the red and white pulp as well as by dendrocytes. The liver and spleen were also the organs in which, after intravenous inoculation,K. pneumoniae was trapped. It was observed that cells containing LE-MTPPE often had not taken up bacteria. Most bacteria, about 73%, were found in cells not containing liposomes. The capacity of the liposome-containing cells to take up bacteria did not change with time. This suggests that the by LE-MTPPE immunostimulating effect is due to the production of cytokines by the cells that take up LE-MTPPE. These cytokines might stimulate other cells to the killing of bacteria.     

Cell types and interactions in the spleen of the dogfish Scyliorhinus canicula L.: an electron microscopic study

The cell composition of the spleen of the dogfish, Scyliorhinus canicula L. was investigated by electron microscopy. It comprised areas of red and white blood cells. The red cells were observed in various stages of development and the spleen is probably the main erythropoietic organ, it is likely that thrombocytes are also produced in the spleen. The presence of plasma cells, and the appearance of rosette-like contacts between lymphocytes and phagocytic cells, probably macrophages, indicated that immunological processes were taking place. Destruction of effete blood cells, primarily erythrocytes, was indicated by the presence of macrophages containing residues of ingested blood cells.     

Possible involvement of 4-hydroxynonenal in splenocyte regulated liver regeneration

Liver regeneration is a complex, systemic process regulated by humoral and cellular mechanisms. Inflammatory response to the extensive tissue damage, as in partial hepatectomy, plays important role during regeneration. Hence, it is assumed that the spleen might play a role in systemic inflammatory response involved in liver regeneration. On the other hand, liver damage and consequential regeneration are often associated with oxidative stress and lipid peroxidation. One of the end products of lipid peroxidation, 4-hydroxynonenal (HNE), is nowadays considered not only as a "second toxic messenger of free radicals" but also as a growth-regulating factor. We therefore studied in vitro interactions of the HNE-treated murine liver cells and autologous spleen cells. The spleen cells supported recovery of liver cells from the HNE cytotoxicity although spleen cells themselves exerted cytotoxic effects against the proliferating liver cells that were not treated with HNE. Our results imply that the cytokines secreted by activated immunocompetent cells may be responsible for the observed recovery of the HNE-damage liver cells, suggesting that HNE might be an important factor regulating cellular and cytokine mediated mechanisms of liver regeneration control.     

Ester composition of carnitine in the perfusate of liver and in the plasma of donor rats

When the carnitine pool of fed rats was labelled with tritium, in non-recirculating perfusate of their liver 44% of acid-soluble 3H activity was identified as free carnitine and 47% as short-chain acylcarnitine. Of the latter component acetylcarnitine accounted for 30% and propionylcarnitine for 10% of total acid-soluble. In plasma the contribution of short-chain acylcarnitines to total carnitine in fed, fasted and diabetic rats was 15.6%, 43.1% and 48.0%, respectively. Recirculating perfusion of livers from the same animals revealed that livers from fed rats released short-chain acylcarnitines as much as 56.2% of total and this proportion did not increase further in the other two groups. At the same time, ketone bodies in the perfusate increased gradually in the fed, fasted and diabetic group, paralleling the plasma ketone levels. Although liver supplies the organism with carnitine the increment of plasma short-chain acylcarnitines seen in ketosis is not a result of some extra output by the liver.     

The effect of transferrin saturation on internal iron exchange

Radioiron was introduced into the intestinal lumen to evaluate absorption, injected as nonviable red cells to evaluate reticuloendothelial (RE) processing of iron, and injected as hemoglobin to evaluate hepatocyte iron processing. Redistribution of iron through the plasma was evaluated in control animals and animals whose transferrin was saturated by iron infusion. Radioiron introduced into the lumen of the gut as ferrous sulfate and as transferrin-bound iron was absorbed about half as well in iron-infused animals, and absorbed iron was localized in the liver. The similar absorption of transferrin-bound iron suggested that absorption of ferrous iron occurred via the mucosal cell and did not enter by diffusion. The decrease in absorption was associated with an increase in mucosal iron and ferritin content produced by the iron infusion. An inverse relationship (r = -0.895) was shown between mucosal ferritin iron and absorption. When iron was injected as nonviable red cells, it was deposited predominantly in reticuloendothelial cells of the spleen. Return of this radioiron to the plasma was only 6% of that in control animals. While there was some movement of iron from spleen to liver, this could be accounted for by intravascular hemolysis. Injected hemoglobin tagged with radioiron was for the most part taken up and held by the liver. Some 13% initially localized in the marrow in iron-infused animals was shown to be storage iron unavailable for hemoglobin synthesis. These studies demonstrate the hepatic trapping of absorbed iron and the inability of either RE cell or hepatocyte to release iron in the transferrin-saturated animal.     

Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.     

Distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells.

Antiviral mechanisms by which natural killer (NK) cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of C57BL/6 mice were measured, revealing different mechanisms of control in different organs. Three days postinfection, MCMV titers in the spleens of perforin 0/0 mice were higher than in those of perforin +/+ mice, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers and not in spleen titers. Depletion of gamma interferon (IFN-gamma) in C57BL/6 mice by injections with monoclonal antibodies to IFN-gamma resulted in an increase of viral titers in the liver but not in the spleen. Analyses using IFN-gamma-receptor-deficient mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that in a recipient environment where IFN-gamma cannot exert its effects, the depletion of NK cells caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-gamma has the ability to induce a variety of cells to produce nitric oxide, and administrating the nitric oxide synthase inhibitor N(omega)-monomethyl-L-arginine into MCMV-infected C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. Taken together, these data suggest that in C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, while in the liver the production of IFN-gamma by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-lr locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver.     

Prevention of fasting-mediated bone marrow atrophy by leptin administration

Leptin is an adipokine that regulates body weight. In the current study, we demonstrate that continuous injection of leptin prevents the lymphocyte reduction observed in fasted mice, especially the immature B cell populations in the bone marrow. Although leptin administration reduced apoptotic cells in the bone marrow of fasted mice, it did not prevent glucocorticoid-mediated apoptosis in vitro. Bone marrow atrophy has also been shown in the leptin receptor-deficient db/db mice. In order to investigate the mechanisms underlying these processes, we transplanted bone marrow cells from db/db or control (+m/+m) mice into C.B-17/lcr-scid/scid mice. We found that the spleen and bone marrow B cell populations were completely reconstituted when db/db and +m/+m cells were transplanted into scid mice. Our findings suggest that direct interactions between leptin and bone marrow cells are not essential for the development of B cells in a metabologically normal environment.     

Glucose homeostasis during the early stages of liver regeneration in fasted rats

Kinetic studies with [2-3H]glucose in vivo and gluconeogenic activity measurements in vivo and in vitro were performed in 70% hepatectomized rats submitted to fasting, which represents an extra burden for glucose synthesis but does not impair liver regeneration. Rates of glucose replacement, under steady-state conditions, 14 and 24 h postoperatively, did not differ in partially hepatectomized fasted rats and sham-operated controls. Phosphoenolpyruvate carboxykinase activities increased more rapidly during fasting in remnant livers than in intact livers from controls. Rates of incorporation of 14C from alanine into circulating glucose in hepatectomized rats were already maximal 14 h after surgery, whereas in controls they continued to augment. The maximal rates after partial hepatectomy could not be surpassed by performing the operation in diabetic animals. It is concluded that the relatively high blood sugar levels during fasting in hepatectomized rats do not depend on a reduced peripheral utilization of glucose, but only on a rapid increase in the gluconeogenic activity. The data suggest that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.