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Guinea pig ethanol metabolism as well as distribution and activities of ethanol metabolizing enzymes were studied. Alcohol dehydrogenase (ADH; EC 1.1.1.1) is almost exclusively present in liver except for minor activities in the cecum. All other organ tissues tested (skeletal muscle, heart, brain, stomach, and testes) contained only negligible enzyme activities. In fed livers, ADH could only be demonstrated in the cytosolic fraction (2.94 μmol/g liver/min at 38 °C) and its apparent Km value of 0.42 m m for ethanol as substrate is similar to the average Km of the human enzymes. Acetaldehyde dehydrogenase (ALDH; EC 1.2.1.3) of guinea pig liver was measured at low (0.05 m m ) and high (10 m m ) acetaldehyde concentrations and its subcellular localization was found to be mainly mitochondrial. The total acetaldehyde activity in liver amounts to 3.56 μmol/g/ min. Fed and fasted animals showed similar zero-order alcohol elimination rates after intraperitoneal injection of 1.7 or 3.0 g ethanol/kg body wt. The ethanol elimination rate of fed animals after 1.7 g ethanol/kg body wt (2.59 μmol/g liver/min) was inhibited by 80% after intraperitoneal injection of 4-methylpyrazole. Average ethanol elimination rates in vivo after 1.7 g/kg ethanol commanded only 88% of the totally available ADH activity in fed guinea pig livers. Catalase (EC 1.11.1.6), an enzyme previously implicated in ethanol metabolism, is of 3.4-fold higher activity in guinea pig (10,400 U/g liver) than in rat livers (3,100 U/g liver), but 98% inhibition by 3-amino-1,2,4-triazole did not significantly alter ethanol elimination rates. After ethanol injection, fed and fasted guinea pigs reacted with prolonged hyperglycemia.

Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities. From 4 × 10 8 to 5 × 10 8 cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.

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 NAD 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 NAD 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 NAD 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.

Kupffer and endothelial cells of rat liver were purified by recirculating collagenase perfusion, metrizamide gradient, and counterflow elutriation. Every step of the isolation procedure was followed by light microscopy and histochemistry. The final fractions of purified sinusoidal cells were examined in their isolated state by transmission and scanning electron microscopy. The scope of this manuscript is the detailed documentation and discussion of all technical aspects and problems of a rather new isolation technique, as well as the detailed description of the purified sinusoidal cells of the liver by scanning electron microscopy. Broader acceptance of this isolation procedure should lead to the specific characterization of biochemical and immunological functions of these cells and elucidate their pathophysiological significance.

Extramitochondrial malic enzyme (EC 1.1.1.40) of rat liver and skeletal muscle is shown for the first time to be subject to significant inhibition by acetyl CoA. The liver enzyme seems to be slightly more sensitive to inhibition than the muscle enzyme, and inhibition can only be observed at malate concentrations below Vmax. Since acetyl CoA is a metabolite of ethanol (acetate) oxidation in liver and muscle, its inhibitory effect on malic enzyme could explain (a) the prolonged disequilibrium of the liver malic enzyme reaction after acute ethanol injection in rats (1), and (b) the inhibition of alanine release from perfused rat hindquarters in the presence of acetate, the major metabolite of ethanol in the peripheral vascular system (2).

The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate. Calcium ions (1.3 m m ) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca2+ and NH4+ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.

A simplified procedure was developed for isolation of intact, hormone-sensitive liver cells in a high and reproducible yield. These cells produce glucose from various precursors at rates comparable to those achieved in isolated perfused liver. Glucagon enhanced glucose synthesis from pyruvate, dihydroxyacetone, fructose, or xylitol more effectively at low than at high substrate concentration. At high pyruvate concentrations (>2 mM), glucagon or adenosine 3′:5′-cyclic monophosphate (0.1 mM) exerts a curious inhibition of gluconeogenesis that can be reverted to stimulation on addition of ethanol. It is suggested that glucagon and cyclic AMP inhibit pyruvate dehydrogenase and thus limit the supply of reducing equivalents needed for glucose formation. Supporting evidence for hormonal control of pyruvate dehydrogenase in isolated liver cells is provided by the fact that glucagon decreases and insulin increases decarboxylation of [ 1 - 14 C]pyruvate. Calcium salts (1.3 mM) enhance glucose formation from pyruvate but greatly enhance the inhibition exerted by the divalent cationophore, A23187. Inhibition by glucagon of glucose synthesis from pyruvate is additive with the effects of A23187 + Ca ++ . However, with dihydroxyacetone as substrate, glucagon partially reverses the inhibition exerted by A23187 + Ca ++ . The results are consistent with glucagon effecting an inhibition of pyruvate dehydrogenase and a stimulation of hexosediphosphatase activities.

Perfusion of 14C-(ring)-parathion or 14C-(ring)-paraoxon with blood through isolated, intact rat livers resulted in the rapid degradation of these insecticides. Degradation was negligible in the absence of rat liver (controls), thus demonstrating the capacity of the liver per se to effectively degrade these compounds. Of the total radiocarbon recovered after liver perfusion with [14C]parathion, 33 % could be attributed to unchanged [14C]parathion (similarly distributed between the liver and the blood) while 67.9 % was degraded to water soluble compounds and 2.5% was converted to organic soluble paraoxon and traces of p-nitrophenol. Nearly all of the [14C]paraoxon, however, was degraded by the intact rat liver, resulting in water soluble products that amounted to 98.5% of the total radiocarbon recovered. Unexplained losses of radiocarbon with the perfusion apparatus used were lower in the presence of rat liver which degraded the insecticides to more water soluble compounds. The water soluble degradation products produced from [14C]parathion and [14C]paraoxon were non-toxic to mosquito larvae (Aedes aegypti L.). These ring-labelled products were found to be conjugated p-nito-phenol. Nearly all of the water soluble radiocarbon was located in the perfused blood, while only small amounts (1.8 to 3.0% of recovered) were excreted via the bile or were associated with the liver tissue (1.3 to 1.8 % of recovered).

Guinea pig nonparenchymal hepatic cells were isolated by enzymatic digestion and subsequent separation on a 17.5% metrizamide gradient. Endothelial cell and Kupffer cell-enriched fractions were separated by centrifugal elutriation. Viability of both cell fractions was approximately 80%. Endothelial cells were cultured on a substratum of guinea pig liver collagen and 1% gelatin (1:1). Freshly isolated and cultured sinusoidal endothelial cells contained Factor VIII R:antigen, angiotensin I converting enzyme activity, and they synthesized prostaglandins characteristic of other endothelial cells. Sieve plates were identified in both freshly isolated and cultured cells. Fresh endothelial cells and Kupffer cells formed Fc receptor-mediated rosettes with IgG-opsonized sheep red blood cells, but cultured endothelial cells did not. Only Kupffer cells demonstrated Fc and C3 receptor-mediated phagocytosis. These methods for isolating and culturing sinusoidal endothelial cells should permit further functional assessment of endothelial cells and their interrelationship with other sinusoidal lining cells.

The pancreatic hormone, glucagon, stimulates the net uptake of inorganic 32 P in vivo into rat liver and its incorporation into proteins of microsomes, mitochondrial membranes, and lysosomes. Incorporation into cytosolic proteins was enhanced only slightly by glucagon. More than 95% of the protein-bound phosphate is present as phosphoserine. Both the radioactivity and the total amount of protein-bound phosphate are increased after injection of glucagon. Glucagon treatment enhanced 32 P incorporation into the alcohol-ether soluble lipids of mitochondria but did not alter the relative distribution of 32 P in various phospholipid species.

Studies were performed in our laboratory to investigate certain aspects of pyruvate translocation into rat liver mitochondria, which is still a matter of controversy. Even though evidence has been collected in recent years showing that monocarboxylate uptake in mitochondria is not a controlled and carrier-limited translocation [ 1, 21, Papa et al. [3] reported recently that pyruvate is transported through a specific translocator into mitochondria. The method selected for our study was the technique of rapid centrifugation (with or without silicone layer), which was established as the method of choice for many carboxylic acid [l] and adenine nucleotide [ 1,4] transport and exchange studies. Mitochondrial terminology such as “uptake” and “transport” (translocation of a substrate from one side of the membrane to the other) usually does not distinguish precisely between a translocation through a membrane system or a simple “surface limited” or “bulk phase limited” [5] adsorption of anions or cations on proteins and lipids. For these transport studies, we prepared different denatured, metabolically and structurally destroyed mitochondria as a control to normal mitochondria. We present evidence that it is not possible to justify the kinetic treatment [3] of the data obtained with the method of rapid centrifugation as representing active transport. Adsorption of pyruvate to mitochondrial proteins and/or lipids rather than specific transport to the matrix space appears to account for the binding of pyruvate by rat liver mitochondria.

Exposed thiol groups do not appear to be related to the binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro. Treating stripped rough endoplasmic reticulum with GSSG did not diminish binding of polyribosomes, suggesting that binding in vitro has no correlation with the inhibition of protein synthesis in vitro reported by Kosower et al. (1971). Thiol reagents, which are known to dissociate ribosomes, did not significantly decrease binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum. Denaturing the protein of 32P-labelled polyribosomes or stripped rough endoplasmic reticulum of liver or hepatoma with heat, trichloroacetic acid, or HClO4 did not alter the binding in vitro. Therefore, the practice of measuring the binding of 32P-labelled polyribosomes to stripped rough endoplasmic reticulum in vitro (Shires et al., 1971b) is an unsuitable indicator of biological significance in the intact cell.