The liver is an organ site for the release of inosine metabolized by non-glycolytic pig red cells

The metabolic energy source used by the pig red cell, which is unable to metabolize blood-borne glucose, was examined. Potential physiological substrates include adenosine, inosine, ribose, deoxyribose, dihydroxyacetone and glyceraldehyde, of which inosine was previously implicated. A net ATP synthesis by red cells occurs during in situ perfusion through the adult miniature pig liver. HPLC analysis of the perfusate revealed the presence primarily of inosine and hypoxanthine. Inosine production by the liver was 0.015 mumol/g per min. Moreover, red cells maintain ATP when suspended in a balanced salt medium during a 6 h incubation at 38 degrees C, in which inosine is continuously infused to give an external concentration of no more than 3 mumol/l, mimicking its plasma level. Inosine consumption under these infusion conditions was 56 nmol/ml cell per h, which is two orders of magnitude lower than when inosine is present in millimolar concentration. The total red cell inosine consumption of 9.63 mumol/h is much less than the total liver inosine production of 212 mumol/h. These findings suggest that the liver is an organ site elaborating inosine, and that maintenance of a 3 mumol/l inosine in plasma is sufficient to meet the energy requirements of the pig red cells.     

Evidence for early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells

Various mitogens activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent fibroblasts. Increased synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP) may precede or underlie these activations, but little direct evidence has been presented for this notion, due to lack of suitable analytical methods. To preferentially label intracellular ribose phosphate and quantitatively follow metabolic flux through PRPP into nucleotides, we prepared [ribosyl-14C]inosine and used it as a tracer. Evidence showed the validity of this method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure of PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that epidermal growth factor plus insulin stimulates the metabolic flux through PRPP. Similar extents of stimulation were induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone, suggesting the presence of an unknown signaling pathway common to these mitogens. This system is highly useful for studies of the mechanisms that stimulate in situ activity of PRPP synthetase.     

Entry of acetyl-L-carnitine into biosynthetic pathways

[1-14C]Acetyl-L-carnitine was injected into fed and fasted mice and the time-course of distribution of 14C in various tissues and tissue components was determined. The major product was 14CO2. However, considerable quantities of radioactivity were localized in liver with much smaller quantities in heart, brain, skeletal muscle and kidney. Most of the 14C in liver was located in the fatty acids of phospholipids and triacylglycerols within 10 min after injection. The data demonstrate that the acetyl moiety of acetylcarnitine rapidly enters lipid biosynthetic pools in liver.     

METABOLISM OF d-[U-14C]RIBOSE IN RAT TISSUES

Abstract— d -[U-¹⁴C]Ribose injected subcutaneously into the rat enters the blood, liver and brain. At 30 min after injection 40-70 per cent of the radioactivity in the brain was found in amino acids and only 2-6 per cent in free sugars. In contrast, free sugars (mainly glucose) and carboxylic acids accounted for most of the radioactivity in liver and blood. Evidence for the entry of [U-¹⁴C]ribose into the brain was obtained by intracarotid or intravenous injection of [U-¹⁴C]ribose after interrupting the blood supply to the liver and kidney. Under these conditions the radioactivity in the brain was found in amino acids, carboxylic acids and ribose; no significant amount of [¹⁴C]glucose was detected in brain or heart. It is concluded that ribose is metabolized directly in vivo in the brain. d -[U-¹⁴C]Ribose was metabolized also by brain slices in vitro to form ¹⁴C-labelled amino acids and carboxylic acids; the rate was equivalent to the utilization of 0.65 μ mol of ribose/g/h. The specific radioactivity of glutamine and of γ -aminobutyrate was similar to or higher than that of glutamate in the brain. These results are discussed in the context of metabolic compartments.     

Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

Rate of concentration and digestion of radioactive growth hormone preparations injected in rats,as measured by the amount and nature of radioactivity in the tissues

Summary The distribution of radioactivity in the tissues of the rat has been established after the administration of radioactive bovine growth hormone preparations.Bovine growth hormone was used either transformed in to a¹⁴C-guanidinated derivative, which was fully active, of labeled with less than 1 mole per mole of¹²⁵I.The tissue radioactivity distribution curves obtained belong to two different categories: in kidney, liver and spleen there is an early concentration which attains a maximum in 15 minutes after the injection of the hormone, and rapidly declines. In heart, skeletal muscle, pancreas, intestine, bone and fat, the radioactivity increases gradually and a steady-state is reached after 30 to 60 minutes.Kidney is the organ where the highest concentration of radioactivity occurs. However, muscle accumulates more than 60% of the initial doses after 2 hours. Very little radioactivity appears in the urine, in this period.Similar results have been obtained with pharmacological or physiological doses of the labeled hormones.Blood plasma does not degrade the injected hormone but kidney, liver and muscle rapidly produce radioactive fragments soluble in 10% trichloro-acetic acid.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70^th birthday.     

Heterogeneity of glycogen synthesis upon refeeding following starvation.

1. Starvation of rats for 40 hr decreased the body weight, liver weight and blood glucose concentration. The hepatic and skeletal muscle glycogen concentrations were decreased by 95% (from 410 mumol/g tissue to 16 mumol/g tissue) and 55% (from 40 mumol/g tissue to 18.5 mumol/g tissue), respectively. 2. Fine structural analysis of glycogen purified from the liver and skeletal muscle of starved rats suggested that the glycogenolysis included a lysosomal component, in addition to the conventional phosphorolytic pathway. In support of this the hepatic acid alpha-glucosidase activity increased 1.8-fold following starvation. 3. Refeeding resulted in liver glycogen synthesis at a linear rate of 40 mumol/g tissue per hr over the first 13 hr of refeeding. The hepatic glycogen store were replenished by 8 hr of refeeding, but synthesis continued and the hepatic glycogen content peaked at 24 hr (approximately 670 mumol/g tissue). 4. Refeeding resulted in skeletal muscle glycogen synthesis at an initial rate of 40 mumol/g tissue per hr. The muscle glycogen store was replenished by 30 min of refeeding, but synthesis continued and the glycogen content peaked at 13 hr (approximately 50 mumol/g tissue). 5. Both liver and skeletal muscle glycogen synthesis were inhomogeneous with respect to molecular size; high molecular weight glycogen was initially synthesised at a faster rate than low molecular weight glycogen. These observations support suggestions that there is more than a single site of glycogen synthesis.     

Serum and organ creatine phosphokinase alterations in exercise.

Rats that swam for 3 h showed a 6-fold increase in serum creatine phosphokinase (SCPK) activity which declined to control values within 7 h after swimming. Of the excess SCPK, 77% was BB isoenzyme; the remainder was mainly MM with traces of MB. Kidney, liver, brain and lung contain mainly BB (50-80%) and only a trace of MB (0-7%). Heart CPK was composed of little BB (8%) and more MB (28%) and MM (64%). Skeletal muscle CPK was almost entirely MM. CPK activity is highest in skeletal muscle, intermediate in heart and brain and lowest in kidney, liver and lung. It is suggested that skeletal muscle and heart are not involved in CPK release in swimming, and kidney, liver and brain may be sites of release.     

Studies on nitrosamine metabolism I. Subcellular distribution of radioactivity in tumor-susceptible tissues of RFM mice following administration of [14C] dimethylnitrosamine

The distribution of radioactivity in tumor-susceptible (liver and lung) and non-tumor-susceptible (heart, forestomach, and esophagus) tissues of male RFM mice was investigated at timed intervals following a single intragastric administration of ¹⁴C-labeled DMN.³ The greatest amount of radioactivity was associated with the tumor-susceptible tissues—liver and lung. At 15 min, the relative amount of radioactivity in the homogenates of heart, forestomach, esophagus, livers and lung was 1, 2, 3, 10, and 70, respectively. The AS components of lung contained about six times as much radioactivity as the liver 15 min after administration; at 16 hr, the level of radioactivity had decreased and was equal in amount. The AI components of both tumor-susceptible tissues incorporated much less radioactivity than the AS components, indicating that only a small amount of methyl label is covalently bound to cellular macromolecules. The amount or radioactivity in the AI components ranged from 2–34% in the lung and from 11–33% in the liver. In the lung C-fraction the range of radioactivity was 75–89% for the AS components and 52–74% for the AI components. The radioactivity in the AS components of liver C-fraction ranged from 50–89%, and from 52–68% for the AI components. The results suggest differences in the affinity, transport, and/or metabolism of DMN between liver and lung, as well as between tumor-susceptible and non-tumor-susceptible tissues.     

Post mortem glycogenolysis is a combination of phosphorolysis and hydrolysis

1. Glycogen, glucose, lactate and glycogen phosphorylase concentrations and the activities of glycogen phosphorylase a and acid 1,4-alpha-glucosidase were measured at various times up to 120 min after death in the liver and skeletal muscle of Wistar and gsd/gsd (phosphorylase b kinase deficient) rats and Wistar rats treated with the acid alpha-glucosidase inhibitor acarbose. 2. In all tissues glycogen was degraded rapidly and was accompanied by an increase in tissue glucose and lactate concentrations and a lowering of tissue pH. In the liver of Wistar and acarbose-treated Wistar rats and in the skeletal muscle of all rats glycogen loss proceeded initially very rapidly before slowing. In the gsd/gsd rat liver glycogenolysis proceeded at a linear rate throughout the incubation period. Over 120 min 60, 20 and 50% of the hepatic glycogen store was degraded in the livers of Wistar, gsd/gsd and acarbose-treated Wistar rats, respectively. All 3 types of rat degraded skeletal muscle glycogen at the same rate and to the same extent (82% degraded over 2 hr). 3. In Wistar rat liver and skeletal muscle glycogen phosphorylase was activated soon after death and the activity of phosphorylase a remained well above the zero-time level at all later time points, even when the rate of glycogenolysis had slowed significantly. Liver and skeletal muscle acid alpha-glucosidase activities were unchanged after death. 4. The decreased rate and extent of hepatic glycogenolysis in both the gsd/gsd and acarbose-treated rats suggests that this process is a combination of phosphorolysis and hydrolysis. 5. Glycogen was purified from Wistar liver and skeletal muscle at various times post mortem and its structure investigated. Fine structural analysis revealed progressive shortening of the outer chains of the glycogen from both tissues, indicative of random, lysosomal hydrolysis. Analysis of molecular weight distributions showed inhomogeneity in the glycogen loss; in both tissues high molecular weight glycogen was preferentially degraded. This material is concentrated in lysosomes of both skeletal muscle and liver. These results are consistent with a role for lysosomal hydrolysis in glycogen degradation.     

Effect of AMP-Deaminase 3 Knock-Out in Mice on Enzyme Activity in Heart and Other Organs

Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3 -/- mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%–30%. No change of AMPD activity was observed in the skeletal muscle and the liver.     

Physiological significance of catalase and glutathione peroxidases,and in vivo peroxidation,in selected tissues of the toadDiscoglossus pictus (Amphibia) during acclimation to normobaric hyperoxia

1. Various parameters related to oxidative stress were measured in adult Discoglossus pictus acclimated for 15 days to either normoxia or hyperoxia (PO2 = 710 mmHg). 2. Total weight of the toads and total and relative wet weight of liver, kidneys, lungs and heart were not changed by hyperoxic acclimation. 3. In vivo tissue peroxidation increased in lung, decreased in skeletal muscle, and was not changed in liver, kidney, heart and skin after hyperoxic exposure. 4. Hyperoxic acclimation increased catalase activities in the lung, liver, kidney and heart but not in skeletal muscle and skin. 5. Liver showed higher GSH-peroxidase activity with cumene-OOH than with H2O2 as substrate, whereas lung, skeletal muscle and skin presented similar GSH-peroxidase activities with both substrates. 6. GSH-peroxidase activities did not change between hyperoxic and normoxic animals in liver, lung, skeletal muscle and skin. 7. These results show that catalase, not GSH-peroxidase, is the principal H2O2 detoxifying enzyme involved in the adaptation of D. pictus to hyperoxia.     

Effects of a cafeteria diet and starvation of global 14C(U)-glucose disposal

The label distribution in control and cafeteria-diet fed rats, either in basal conditions or after 24 hours of food deprivation, 10 minutes after the i.v. injection of carrier-free D-14C-(U)-glucose, has been studied. The radioactivity recovered in the different fractions of liver, kidney, heart, striated muscle and white adipose tissue showed comparable patterns of change with starvation in both dietary groups. Most of the radioactivity was found in the free amino acid fraction as well as in proteins, with significant proportions also in lipid and liver glycogen. However, most of the label was lost due to its oxidation, remaining in the combined indicated tissues 10-20% of the injected label. On the whole, cafeteria rats consumed more glucose than controls, the lowest oxidation corresponding to the starved-control group. The amount of glucose oxidized by cafeteria rats was actually comparable to that of fed controls. The availability of other energetic sources--i.e. lipid--allows for an increased glucose utilization in cafeteria rats, even in the starved state.     

The distribution and comparison of glutathione, glutathione reductase and glutathione S-transferase in various camel tissues

Extracts prepared from liver, kidney, lung and brain of camel contain glutathione, glutathione S-transferase and glutathione reductase. Liver had the highest level of glutathione (218.7 mumol/g wet weight) whereas brain had the lowest level (66.4 mumol/g wet weight). The highest activity for glutathione reductase was found in the kidney (2.6 mumol/min/mg protein) while the lowest activity was found in the lung (0.9 mumol/min/mg protein). Glutathione S-transferase activity was the highest in liver (4.2 mumol/min/mg protein) and the lowest in brain (1 mumol/min/mg protein). Purified glutathione S-transferases from lung, kidney, brain and liver were similar in their molecular size, subunit composition as well as immuno-reactivity and showed some differences in their response to heat and inhibitors.     

Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.     

Different copy numbers of apparently identically deleted mitochondrial DNA in tissues from a patient with Kearns-Sayre syndrome detected by PCR

An apparently identical deletion of 4.977 bp in length (position 8,483-13,459) was detectable in the mitochondrial DNA from skeletal muscle, heart muscle, kidney, and liver of a patient with Kearns-Sayre syndrome. The proportion of deleted genome varied from 60% for the skeletal muscle to 15% for heart muscle and kidney, and was below 5% in the liver. The mtDNA heteroplasmy of the liver was only detectable after amplification by PCR. In skeletal and heart muscle histochemical and immunocytochemical findings concerning cytochrome c oxidase were in good correlation with the proportion of deleted mitochondrial DNA.     

Plasma clearance and tissue distribution of radiolabeled leptin in the chicken

Leptin is an adipose and liver tissue-derived secreted protein in chickens that has been implicated in the regulation of food intake and whole-body energy balance. In this study, the metabolic clearance and tissue uptake of leptin were examined in the chicken (Gallus gallus). Four-week-old broiler males were infused with (125)I-labeled mouse leptin. Chromatography of radiolabeled leptin in plasma produced two peaks, one at 16 kDa (free leptin) and a free iodine peak. No leptin binding protein in blood was detected. Leptin was cleared with a half-life estimate of 23 min. In order to investigate the tissue distribution and uptake of radiolabeled leptin, multiple tissues were removed from infused birds at 15 and 240 min post-infusion, and trichloroacetic acid (TCA)-precipitable radioactivity was determined. The amounts of radioactivity at 15 min post-infusion in the tissues in rank order were: kidney, testis, lung, spleen, heart, liver, small and large intestine, gizzard, pancreas, bursa, leg and breast muscle, adrenals, and brain. A slightly different pattern of distribution was observed at 240 min post-infusion. We conclude from these studies that unlike mammals, no circulating leptin binding protein is present in chickens. Leptin is metabolized and cleared very rapidly from blood by the kidney.     

The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver.

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1--30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3--17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the 'defined' pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.     

Early events in the initiation of ammonia formation in kidney

Experiments were designed to examine the early events in the initiation of glutamate deamination in kidney. Perfused kidneys from methionine sulfoximine-treated rats formed ammonia from [15N]glutamate via the purine nucleotide cycle. The turnover of the 6-amino group of adenine nucleotides to yield ammonia occurred at the rate of 0.30 mumol/g of kidney/min. This rate is 3-4 times larger than in liver and is in agreement with published rates of the purine nucleotide cycle in kidney. The addition of 0.1 mM fluorocitrate to glutamate perfusions stimulated ammonia formation 3 1/2-fold. The turnover of the 6-amino group of adenine nucleotides increased during the first 5 min after adding fluorocitrate to form ammonia predominately from tissue glutamate and aspartate. This turnover correlates with a 3 1/2-fold increase in kidney tissue IMP levels. As the ATP/ADP ratio fell the purine nucleotide cycle was inhibited and glutamate dehydrogenase was stimulated to form ammonia stoichiometric with glutamate taken up from the perfusate. Ammonia formation via glutamate dehydrogenase occurred at a rate of 1.0 mumol/g of kidney/min. Fluorocitrate completely blocked ammonia formation from aspartate in perfusions. The perfused kidney formed ammonia from aspartate via the purine nucleotide cycle at a rate of 1.0 mumol/g of kidney/min. The results indicate a discrete role for aspartate in renal metabolism. Ammonia formation via the purine nucleotide cycle can occur at significant rates and equal to the rate of ammonia formation from glutamate via glutamate dehydrogenase.