Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Studies on flavonoid metabolism. Metabolism of (+)-[14C]catechin in the rat and guinea pig

1. The fate of (+)-[U-(14)C]catechin and (+)-[ring A-(14)C]catechin has been studied in the guinea pig and rat. 2. (+)-[U-(14)C]Catechin was shown to give rise to labelled phenolic acids, labelled phenyl-gamma-valerolactones and (14)CO(2). 3. (+)-[ring A-(14)C]-Catechin did not give rise to labelled phenolic acids, but labelled phenyl-gamma-valerolactones were detected together with a higher proportion of (14)CO(2). 4. Administered [(14)C]delta-(3-hydroxyphenyl)-gamma-valerolactone gave rise to labelled m-hydroxyphenylpropionic acid in the rat whereas administered [(14)C]m-hydroxyphenylpropionic acid gave rise to a compound yielding labelled m-hydroxybenzoic acid on hydrolysis. 5. The distribution of radioactivity in the urine and faeces of (+)-[(14)C]catechin-fed animals is described; a high proportion of residual radioactivity was found in urine that had been exhaustively extracted with diethyl ether.     

Reactive oxygen species and nitric oxide affect cell wall metabolism in tobacco BY-2 cells

The effects of hydrogen peroxide (H2O2), nitric oxide (NO), and a combination of both on the metabolism of cell wall polysaccharides were studied in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY-2) suspension cultured cells in the presence of D-[U-14C]glucose or D-[U-14C]galactose as radioactive tracers. We found that the radiolabelling of newly synthesised total cell wall polysaccharides (pectins, hemicelluloses and alpha-cellulose), buffer-soluble polysaccharides, and membrane-associated polysaccharides decreased under the influence of exogenous systems generating H2O2 and NO. However, when the total amount of newly synthesised cell wall polysaccharides was calculated as a percentage of the total cellular radioactivity (ethanol-soluble pool plus the homogenate of ethanol-insoluble material), all treatments showed negligible effects in the presence of D-[U-14C]glucose or D-[U-14C]galactose as tracers. This occurred because the treatments generating H2O2, NO and H2O2 plus NO caused a marked decrease in the concentration of the ethanol-soluble pool as well as in the total radioactivity found in the homogenate of the ethanol-insoluble material. Most of the radioactivity taken up by the cells was evolved as 14CO2 during the respiratory processes. A qualitative and quantitative characterisation of the ethanol-soluble pool showed that radioactive UDP-sugars in BY-2 suspension cultured cells were differentially reduced by all treatments. Therefore, the decrease of the newly synthesised cell wall polysaccharides seems to be strictly dependent on the reduction of the UDP-sugars pool.     

Biosynthesis of an unusual amino acyl moiety contained in bestatin

The biosynthetic pathway of an unusual amino acyl [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl (AHP)] moiety which is contained in bestatin has been studied by testing the incorporation of potential precursors. L-[U-14C]-Phenylalanine, L-[U-14C]leucine, and [U-14C]acetic acid were efficiently incorporated into bestatin, but the radioactivity of L-[1-14C]phenylalanine, [1-14C]glyoxylic acid, and [14C]oxalic acid were not incorporated. Incorporation of acetic acid into 1- and 2-carbon of the AHP moiety was confirmed by incorporation of [13C]acetic acid. Thus, the AHP moiety was shown to be biosynthesized from L-phenylalanine and two carbon atoms of acetic acid, accompanied by decarboxylation of the phenylalanine.     

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

The lipogenic role of leucine in animal skeletal muscles

After incubation of rat, pig and cattle skeletal muscle homogenates with [U-14C]leucine, 80.4%, 37.0% and 57.0% of radioactivity was found in the proteins, 9.4%, 58.7% and 40.9% in the lipids, and 10.2%, 4.3% and 2.1% in 14CO2. This suggests that along-side with utilization in protein synthesis, leucine plays an essential role in lipid synthesis in muscle tissues of agricultural animals. The contribution of [U-14C]leucine to lipogenesis with substrates is greater than that of [U-14C]acetate and [U-14C]glucose in cattle skeletal muscles in vitro and greater than that of [U-14C]acetate in pig muscle. The CO2 production during oxidation of the [U-14C]leucine carbohydrate chain is higher than that during [U-14C]glucose and [U-14C]palmitate oxidation in skeletal muscles of rat and pig. In skeletal muscles of all animal species under study [U-14C]acetate is oxidized far more intensively than the other substrates tested.     

Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of [14C]glucose into fat and turnover of newly synthesized fat

1. Lipogenesis was studied in vivo by giving mice 250mg. meals of [U-(14)C]glucose and measuring the disposition and incorporation of label. About 48% of the (14)C dose was eliminated as (14)CO(2) in the first 2hr. At 60min. after administration, 1.0, 1.9 and 11.9% of the dose was recovered as liver glycogen, liver fatty acid and carcass fatty acid respectively. Of the [(14)C]glucose converted into fat in the epididymal pads about 90% was present as glyceride fatty acid and 10% as glyceride glycerol. 2. Hepatic synthesis of fatty acid was depressed by dietary fat to a much greater extent than was synthesis outside the liver. Both feeding with fat and starvation decreased the proportion of the label taken up by adipose tissue present as fat (triglyceride) and increased the proportion of triglyceride label present as glyceride glycerol. These results are consistent with the hypothesis that the primary action of both these conditions in decreasing fat synthesis is to inhibit synthesis of fatty acids. 3. Turnover of body fat labelled in vivo from [U-(14)C]glucose was estimated from the decline in radioactivity measured over the first 24hr. of the experiment. The half-life of liver and extrahepatic fatty acids (excluding epididymal fat) was 16hr. and 3 days respectively. In contrast, no measurable decrease in radioactivity of the fatty acids of epididymal fat was observed for 7 days after administration of the [U-(14)C]glucose.     

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.     

Utilization of pyruvate and pyruvate precursors by normal and carcinogen-altered rat tracheal epithelial cells in culture

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen-altered cells derived from dimethylbenz(a)anthracene-exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate-supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three- to four-fold higher incorporation of radioactivity from [2-14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U-14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two-fold greater glucose consumption and two-fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two-fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U-14C]glutamine or [U-14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7-fold higher utilization of [1-14C]glucose along the oxidative arm of the pentose cycle in the normal cells.     

Metabolism of (3-(14)C)tryptophan and (2-(14)C)alanine in cattle tissues

In the experiments involving incubation of the liver, brain cortex, muscle and adipose tissues homogenates with [3-14C] tryptophan for an hour 43.2-89.3% of the label was found in proteins, 7.2-47.2%--in lipids, 2.6-9.4%--in CO2. Following incubation of the above-mentioned tissue homogenates with [2-14C] alanine, proteins, lipids and CO2 contain 28.8-49.3%; 22.6-31.9% and 21.6-49.3% of radioactive label, respectively. Radioactivity of lipids synthesized by the homogenates of the investigated tissues from [3-14C] tryptophan and [2-14C] alanine is 23.5-63.5 and 21.1-56.0%, respectively, the radioactivity of CO2 being 1.4-5.1 and 9.3-11.8% of the above-mentioned compounds synthesized from [1-14C] acetate. The results obtained testify to the considerable contribution of [3-14C] tryptophan and [2-14C] alanine to protein synthesis as well as to their involvement in the substrate supply of lipogenesis and energetic processes in various organs and tissues of cattle.     

Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported (Huang, M., and Veech, R.L. (1982) J. Biol. Chem. 257, 11358-11363). The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.     

The small-scale production of [U-14C]acetylene from Ba14CO3: application to labeling of ammonia monooxygenase in autotrophic nitrifying bacteria

A small-scale method has been adapted from an established procedure for the generation of [U-14C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba14CO3 with excess barium metal to produce Ba14C2. The BaC2 is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO3 ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U-14C]Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent Mr of 25,000-27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography.     

A radio-gas chromatographic method for determining the specific radioactivity of glycolic acid in -14C-labeled leaf tissue.

A method for the extraction and quantitative determination of both the mass and radioactivity of glycolic acid from -14C-labeled leaf tissue is described. The recoveries of both mass and radioactivity from standard [1-14C]glycolic acid solutions averaged 98 percent, and recovery of radioactivity added to plant samples as [1-14C]glycolic acid was over 90 percent after the complete procedure. The method was reliable with total samples containing as little as 130 nmol of glycolic acid. The mass of glycolic acid recovered from sunflower leaf tissue was proportional to the amount of tissue extracted. In experiments with different plant material, the amount of glycolic acid varied between 530 and 1120 nmol/dm-2 of leaf tissue. The specific radioactivity of the glycolic acid in sunflower leaf tissue during photosynthesis in -14CO(2) was never more than 20 percent of the specific radioactivity of the -14CO(2) supplied.     

Studies on the enzymic N-acylation of amino sugars in the sheep colonic mucosa

1. d-[2-(14)C]Glucose, [2-(14)C]acetate, hydroxy[3-(14)C]pyruvate, [3-(14)C]pyruvate and [U-(14)C]glycine were incorporated by surviving scrapings of sheep colonic mucosal tissue into glycoprotein. 2. d-[2-(14)C]Glucose, [2-(14)C]acetate, incorporated hydroxy-[3-(14)C]pyruvate and [3-(14)C]pyruvate resulted in labelling of each of the monosaccharide residues of the glycoprotein, namely N-glycollylneuraminic acid, N-acetylneuraminic acid, galactose, fucose, glucosamine and galactosamine. [U-(14)C]Glycine was incorporated as glycyl and seryl residues of the glycoprotein. 3. Despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollylneuraminic acid and N-acetylneuraminic acid were 8.5 and 5.2% by weight of the glycoprotein respectively) the corresponding ratio of the radio-active labelling from d-[2-(14)C]glucose in N-glycollylneuraminic acid to that in N-acetylneuraminic acid was 1.00:7.27 (expressed as percentages of the total radioactivity in the glycoprotein). Neutral sugar, hexosamine and N-acetylneuraminic acid residues of the mucoprotein were each labelled to a similar extent. 4. Similarly, the ratio of the radioactivity in N-glycollylneuraminic acid to that in N-acetylneuraminic acid in the mucoprotein from tissue incubations with [2-(14)C]-acetate was 1.0:4.0. 5. Both [2-(14)C]acetate and [2-(14)C]glucose with whole tissue led to labelling of the N-glycollyl substituent and of the main nonose skeleton of the N-glycollylneuraminic acid. In whole-tissue incubations, [3-(14)C]pyruvate was also a precursor of radioactive N-glycollylneuraminic acid. 6. Hydroxy[3-(14)C]-pyruvate and [U-(14)C]glycine caused labelling of the carbohydrate and peptide residues of the glycoprotein, but did not give rise to labelling in the N-glycollylneuraminic acid residues. 7. With a wide variety of possible N-glycollyl precursors (fructose 6-phosphate, hydroxypyruvate, glycollate and chemically synthesized glycollyl-CoA) biosynthesis of N-glycollylglucosamine was not observed in cell-free preparations.     

An improved procedure for the synthesis of 14C-labeled phosphatidylserine from cerebral phosphatidic acid

A complete procedure to prepare a highly labeled phosphatidyl-L-[U-14C]serine possessing the same fatty acid composition of brain phospholipids is reported. CDP-diglyceride was synthesized by reaction between phosphatidic acid and CMP-morpholidate as the dicyclohexylcarboxamidium salt. The reaction between CDP-diglyceride and L-[U-14C]serine to produce the labeled phosphatidylserine was catalyzed by the CDP-diglyceride: L-serine phosphatidyl transferase (EC 2.7.8.8) from E. coli. A selective inhibition of phosphatidylserine decarboxylase activity, present as contaminant in the enzyme extract, was introduced in order to avoid a low yield of product. Traces of phosphatidylethanolamine (about 1%) were easily removed by preparative thin-layer chromatography. The yield of the labeled product was as high as 87% and it specific radioactivity was 170 mCi/mmol.     

Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes.

The pattern of incorporation of radioactivity from [1-14C]acetate and [2-14C]acetate into the polyprenyl side-chain of ubiquinones in bacteria (Azotobacter vinelandii, Pseudomonas sesami, Escherichia coli and Rhodopseudomonas capsulata) was studied. For this purpose, a new degradation method involving a modified Barbier-Wieland reaction of laevulinic acid was developed, and used along with the iodoform reaction. Both C-1 and C-2 of acetate were incorporated exclusively into C-2 of laevulinic acid suggesting that the well-known pathway through acetoacetyl-CoA ('acetoacetate pathway') was not operative in these bacteria. An alternative pathway ('acetolactate pathway'), starting with pyruvate and acetaldehyde as the distal precursors, and utilizing the reactions of leucine and valine metabolism, was postulated. It was also postulated that C-1 of acetate is incorporated not directly, but after oxidation to CO2. The pattern of incorporation of radioactivity from [U-14C]valine, [U-14C]alanine and NaH14CO3 into the side-chain of ubiquinone of R. capsulata was in agreement with the operation of the 'acetolactate pathway'.     

Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis

A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.