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Fatty infiltration of the liver is a common finding in alcoholic patients, but the mechanisms responsible for the accumulation of hepatic fat are not clear (2). In the present investigation, an attempt was made to clarify this relationship by studying the effect of ethanol on fatty acid metabolism in rat liver in vitro (3). MATERIALS AND METHODSSeventy-one male Sprague-Dawley rats weighing from 250 to 400 g were maintained on Purina Laboratory Chow, except for a 24 hour period immediately preceding the experiment. During this period, they were given 36.5 g glucose per kg together with 7.5 g ethanol per kg or, in the experiment shown in Table III, 50 g per kg fructose, administered by means of 5 gastric tube feedings of equal volume. Approximately 3 hours after the last gastric feeding, the animals were decapitated. The liver was quickly removed, and slices were prepared in the cold with a Stadie-Riggs slicer (4). The C14-labeled 1 and unlabeled substrates were dissolved in 5 ml isotonic KrebsRinger bicarbonate buffer, pH 7.4 (6), and the final concentration of the substrates was expressed as millimoles per liter. and the radioactivity counted in a Packard liquid scintillation counter. Randomized liver slices (approximately 0.5 g) were suspended in the incubation mixture in the main compartment of incubation flasks (8). The flasks were sealed with serum caps, gassed five minutes with a mixture of 95 per cent 02 and 5 per cent CO2, and then incubated for 3 hours at 37.50 C in a Dubnoff shaking water bath. Rat epididymal fat pads (0.5 to 1.0 g) were incubated in the same manner. At the end of the incubation, 0.2 ml of 10 N sulfuric acid was injected with a needle through the serum cap into the main compartment, and 1 ml of alkaline hyamine (9) into the center well. The, flasks were then shaken for 30 minutes in an ice bath to trap the evolving CO2 in the hyamine. The hyamine-C"4O. solution' was transferred into a volumetric flask and made up to 5 ml with toluene; 2 ml of this solution was mixed with 16 ml of toluene containing 0.4 per cent DPO and 0.005 per cent POPOP (9), and the radioactivity was measured in a Packard liquid scintillation counter. To calculate the disintegrations per minute (dpm), the obtained counts were multiplied by the efficiency of the counter which was determined separately for each experiment with an internal C"4 standard. C 40-2 was expressed in dpm per gram of wet tissue or per milligram of tissue nitrogen. The incubated tissue from each flask was homogenized in the incubation mixture, and an aliquot of this homogenate was used for determination of total nitrogen by the micro-Kjeldahl method (10). The rest of the homogenate was transferred to a screw-capped bottle, to which 0.4 ml of 90 per cent (wt/vol) potassium hydroxide per ml of homogenate was added. This mixture was saponified by autoclaving for 1 hour at 120°C. After cooling, ethanol was added to obtain a final concentration of 50 per cent. The mixture was vigorously shaken with 20 ml petroleum ether for 10 minutes to remove nonsa...

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A B S T R A C T The quantitative relationship between the catabolism of heme and the formation of bilirubin and carbon monoxide (CO) was studied in untreated rats and in animals treated with phenobarbital or the porphyrogenic drug, allylisopropylacetamide (AIA). A novel metabolic chamber permitting continuous collection of the bile and breath was utilized for quantitation of bilirubin-"C and '4CO after the administration of hematin-"C or glycine-1'C.After intravenous infusion of hematinY`C, control and phenobarbital-treated rats produced equimolar amounts of labeled bilirubin and CO; a minor fraction of the infused radioactivity appeared in the bile in other metabolites. The equimolar relationship in the formation of bilirubin and CO was also observed after pulselabeling with glycine-2-1`C; in phenobarbital-treated rats both metabolites were formed at an increased rate as compared to controls. By contrast, AIA treatment reduced the fractional conversion of hematin-1`C to bilirubin and CO; a major fraction of the infused radioactivity appeared in the bile in metabolites other than bilirubin. In addition, in AIA-treated animals the molar CO/bilirubin recovery ratio was consistently greater than 1.0. Comparable results were obtained in AIAtreated rats after pulse-labeling with glycine-2-1'C. These findings suggest that (a) in control and phenobarbital-treated rats infused hematin and heme formed in the liver are converted predominantly to bilirubin and CO, appearing in equimolar amounts; only a minor fraction of the hematin is degraded to other metabolites; (b) treatment with phenobarbital results in a proportional increase in the formation of both bilirubin and CO, reflecting increased heme synthesis and degradation in the liver; and (c) treatment with the porphyrogenic This work was presented in part at

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