Short-term metabolic fate of [13N]ammonia in rat liver in vivo

The short-term metabolic fate of [13N]ammonia in the livers of adult male, anesthetized rats was determined. Following a bolus injection of tracer quantities of [13N]ammonia into the portal vein, the single pass extraction was approximately 93%, in good agreement with the portal-hepatic vein difference of approximately 90%. High performance liquid chromatographic analysis of deproteinized liver samples indicated that labeled nitrogen is exchanged rapidly among components of: mitochondrial aspartate aminotransferase and glutamate dehydrogenase reactions and cytoplasmic aspartate aminotransferase and alanine aminotransferase reactions (t1/2 for the exchange of label toward equilibrium is on the order of seconds). Comparison of specific activities of glutamate and ammonia suggests that at 5 s most labeled glutamate was mitochondrial, whereas at 60 s approximately 93% was cytosolic; this change is presumably brought about by the combined action of the mitochondrial and cytosolic aspartate aminotransferases and the aspartate carrier of the malate-aspartate shuttle. Specific activity measurements of glutamate, alanine, and aspartate are in accord with the proposal by Williamson et al. (Williamson, D.H., Lopes-Vieira, O., and Walker, B. (1967) Biochem. J. 104, 497-502) that the components of the aspartate aminotransferase reaction are in thermodynamic equilibrium, whereas the components of the alanine aminotransferase reaction are in equilibrium but compartmented in the rat liver. Despite considerable label in citrulline at early time points, no radioactivity (less than or equal to 0.25% of the total) was detected in carbamyl phosphate, suggesting very efficient conversion to citrulline with little free carbamyl phosphate accumulating in the mitochondria. Our data also show that some portal vein-derived ammonia is metabolized to glutamine in the rat liver, but the amount is small (approximately 7% of that metabolized to urea) in part because liver glutamine synthetase is located in a small population of perivenous cells "downstream" from the urea cycle-containing periportal cells. Finally, no tracer evidence could be found for the participation of the purine nucleotide cycle in ammonia production from aspartate. The present work continues to emphasize the usefulness of [13N]ammonia for short-term metabolic studies under truly tracer conditions, particularly when turnover times are on the order of seconds.     

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.     

Chronic metabolic effects of ammonia in mouse brain.

Chronic ammonia toxicity in experimental mice was induced by exposing them for 2 and 5 days to 5 % (v/v) ammonia solution. The enzymes concerned with glutamate metabolism (aspartate-, alanine- and tyrosine aminotransferases, glutamate dehydrogenase and glutamine synthetase) and (Na+ + K+)-ATPase were estimated in the three regions of brain (cerebellum, cerebral cortex and brain stem) and in liver. Glutamate, aspartate, alanine, glutamine and GABA, RNA and protein were also estimated in the three regions of brain and liver. A significant rise in the activity of (Na+ + K+)-ATPase in all the three regions of brain along with a fall in the activity of alanine aminotransferase was noticed. Changes in the activities of other enzymes were also observed. A significant increase in alanine and a decrease in glutamic acid was observed while no change was observed in the content of other amino acids belonging to the glutamate family. As a result of this, changes in the ratios of glutamate/glutamine and glutamate + aspartate/GABA was observed. The results indicated that the brain was in a state of more depression and less of excitation. Under these conditions the liver tissue was showing a profound rise in the activity of the enzymes of glutamate metabolism. The results are further discussed.     

The metabolic fate of intravenously injected peptide-bound chondroitin sulphate in the rat.

The degradation of intravenously administered chondroitin sulphate-peptide, obtained by trypsin digestion of rat cartilage preparations labelled in vitro with 35S (and, in some cases, with 3H), was studied in rats. As with free chains of chondroitin sulphate, the major site of accumulation and degradation in the body was the liver, although peptide-linked chains were taken up more rapidly than free chains. In the first 2h after intravenous injection of a chondroitin sulphate-peptide fraction, labelled macromolecular components were excreted in the urine. These were shown to be chondroitin sulphate-peptide of the same degree of sulphation but of smaller average size than the injected material. A similar observation was made when free chains of chondroitin sulphate from the same source were administered intravenously. An isolated perfused rat kidney failed to de-sulphate circulating chondroitin sulphate-peptide, but a component of lower average molecular weight was excreted in the urine. When a chondroitin sulphate-peptide fraction of relatively larger hydrodynamic volume was administered, very little chondroitin sulphate appeared in the urine in the first 2h. It was concluded that, depending on size and/or peptide content, the chondroitin sulphate-peptide released from connective tissues into the circulation would probably be subjected to one of two alternative fates. The smaller fragments are more likely to be excreted in the urine, whereas the larger ones are taken up by the liver and there degraded to inorganic sulphate and undefined carbohydrate components.     

Acute metabolic effects of ammonia in mouse brain

Acute effects of intraperitoneal administration of ammonium chloride (200 mg/kg) on Na⁺,K⁺-ATPase and amino acid content of the glutamate family (glutamate, aspartate, alanine, glutamine, and GABA), as well as the enzymes involved in the metabolism of these amino acids, have been studied in the different regions of brain and liver in mice. A significant increase in the activity of Na⁺,K⁺-ATPase was observed in the cerebellum, cerebral cortex, and brain stem. A similar increase in the activity of glutamate dehydrogenase was observed in the brain stem, while a moderate increase in the activity of this enzyme was observed in the cerebral cortex and liver in the mice treated with ammonium chloride. In all three regions of brain, a 50% decrease was observed in the activity of alanine aminotransferase, while the activity of aspartate aminotransferase significantly rose in the brain stem. The activity of glutamine synthetase did not change much in the three regions of brain, and a significant fall was registered in the liver. The activity of tyrosine aminotransferase showed a rise in the cerebellum, brain stem, and in liver. Not much change was observed in the protein content in either brain or liver, whereas there was a 1.5-fold increase in the total RNA content in the liver of the animals treated with ammonium chloride. Under the experimental conditions, there was an increase only in the content of glutamine, of all the amino acids tested, in the cerebral cortex and liver. Similar results were obtained with homogenates of tissues enriched with ammonium chloride (in vitro) for the enzyme systems studied. These results are discussed, and the probable metabolic and functional significance of ammonia in brain is indicated.     

Observations on the fate of cystathionine in rat brain.

Direct intracerebral administration of ³⁵S-cystathionine has been shown to be capable of labelling the normal pools of cystathionine in rat brain tissue and thus capable of yielding useful information about the metabolic fate of the amino acid. Cystathionine was shown to be metabolised slowly to yield inorganic SO₄^2?, taurine and cystine. Sub-cellular fractionation studies concerned with the distribution of ³⁵S-cystathionine coupled with similar investigations involving enzymes of the transsulphuration pathway indicate that cystathionine fulfill some of the requirements of a neurotransmitter substance.     

Prevention of ammonia toxicity byl-carnitine: metabolic changes in brain

l-Carnitine when injected in mice 30 min before an LD₁₀₀ of ammonium acetate (12 mmol/kg body weight, intraperitoneal) reduced mortality (100% survival with 16 mmoll-carnitine/kg) and prevented the appearance of symptoms of ammonia toxicity. Brain ammonia decreased in the animals givenl-carnitine. Ammonia decreased the levels of glutamate in brain; they were partially restored byl-carnitine, which also reduced the increase in brain glutamine in animals given only ammonia. The redox state of the brain was altered following ammonia intoxication. The ratio of lactate to pyruvate in the cytosol increased while that of glutamate to -ketoglutarate in the mitochondria decreased. These ratios were partially restored byl-carnitine. The implications of these findings are discussed relative to the mechanism of ammonia toxicity.     

The acute action of ammonia on rat brain metabolism in vivo

1. Acute NH(4) (+) toxicity was studied by using a new apparatus that removes and freezes the brains of conscious rats within 1s. 2. Brains were removed and frozen 5min after intraperitoneal injection of ammonium acetate (2-3min before the onset of convulsions). Arterial [NH(4) (+)] rose from less than 0.01 to 1.74mm at 4-5min. The concentrations of all glycolytic intermediates measured, except glucose 6-phosphate, were increased by the indicated percentage above the control value as follows: glucose (by 41%), fructose 1,6-diphosphate (by 133%), dihydroxyacetone phosphate (by 164%), alpha-glycerophosphate (by 45%), phosphoenolpyruvate (by 67%) and pyruvate (by 26%). 4. Citrate and alpha-oxoglutarate concentrations were unchanged and that of malate was increased (by 17%). 5. Adenine nucleotides and P(i) concentrations were unchanged but the concentration of creatine phosphate decreased slightly (by 6%). 6. Brain [NH(4) (+)] increased from 0.2 to 1.53mm. Net glutamine synthesis occurred at an average rate of 0.33mumol/min per g. 7. The rate of brain glucose utilization was measured in vivo as 0.62mumol/min per g in controls and 0.81mumol/min per g after NH(4) (+) injection. 8. The arteriovenous difference of glucose and O(2) increased by 35%. 9. No significant arteriovenous differences of glutamate or glutamine were detected. Thus, although much NH(4) (+) was incorporated into glutamine the latter was not rapidly released from the brain to the circulation. 10. Plasma [K(+)] increased from 3.3 to 5.4mm. 11. The results indicate that NH(4) (+) stimulates oxidative metabolism but does not interfere with brain energy balance. The increased rate of oxidative metabolism could not be accounted for only on the basis of glutamine synthesis. We suggest that increased extracellular [NH(4) (+)] and [K(+)] decreased the resting transmembrane potential and stimulated Na(+),K(+)-stimulated adenosine triphosphatase activity thus accounting for the increased metabolic rate.     

The metabolic fate of chlormadinone acetate in the baboon.

The metabolic fate of chlormadinone acetate (17alpha-acetoxy-6-chloro-4, 6-pregnadiene-3, 20-dione; CAP) was studied in intact and biliary fistula baboons. The steroid was labeled with 3H at position 1 and with 14C at the carboxyl moiety of the 17alpha-acetate, thus affording the opportunity to ascertain the loss of the 17alpha-acetoxy group and the fate of both labels. The averages of the radioactivity excreted, given as percentages of the amounts injected, and the standard deviations were as follows: In the urine of intact animals after 6 hours, 5.7 +/- 0.2% and 5.5 +/- 0.7% of the 3H and 14C were recovered, respectively. After 6 days, there was 17.5% of the 3H and 16.2% of the 14C in the urine plus 15.3% of the 3H and 16.4% of the 14C in the feces. In baboons with biliary fistulas, the total radioactivity excreted was 7.8 +/- 0.7% of the 3H and 11.6% of the 14C in the urine, and 30.9 +/- 4.4% of the 3H and 30.7% of the 14C in the bile after 6 hours. Glucosiduronates were the predominant conjugates in the urine and bile. The similarity in the urinary excretion of radioactivity in the first 6 hours in intact and biliary fistula animals, the relatively low excretion of radioactivity in the bile and after 6 days in the urine, and the low fecal excretion suggest that the metabolites of CAP are not involved in an extensive enterohepatic circulation in the baboon. Deacetylation of the 17alpha-acetate in CAP was detected in the early collection periods of the urine and bile and constituted a very small percentage of the injected compound. No significant oxygenation of CAP at position 1 was detected. The metabolism of CAP is discussed and compared to our previously reported data on the metabolism of progesterone, ethynodiol diacetate and medroxyprogesterone acetate and the data on other progestogens reported in the literature. It appears that the excretion of CAP is significantly slower in the baboon than that of the other progestogens. The amounts of glucosiduronates of CAP and/or its metabolites formed in vivo are less than those formed with the other progestogens. Also, the extent of deacetylation of the 17alpha-acetate of CAP is much less than that of the 3beta-acetate of ethynodiol diacetate.     

The metabolic fate of medroxyprogesterone acetate in the baboon.

The metabolic fate of medroxyprogesterone acetate (6alpha-methyl-17a lpha-acetoxyprogesterone; MAP)was studied in intact baboons and in those with bile fistulas. The steroid moiety was labeled with tritiated hydrogen at positions 1 and 2 and the 17alpha-acetate with carbon-14, thus affording the opportunity to ascertain the loss of the 17-acetoxy group and the fate of both labels. Following the iv administration of labeled MAP only a small percentage (less than 15%) of the administered dose was recovered in the urine in 7 hours in intact baboons, as well as in the urine of baboons with biliary fistulas. Higher amounts of radioa ctivity were excreted in the bile (approximately 25%), amounting to almost double the percentage excreted into the urine. The similarity in the urinary excretion of radioactivity in intact and biliary fistula animals indicates that, even though a substantial biliary excretion of the labeled MAP occurred, the amount involved in an enterohepatic circulation is probably small. Glucosiduronates were the predominant conjugates, both in the urine and bile. The loss of the 17alpha-acetate group appeared to be rather extensive, ranging 30-70% among different co njugated and unconjugated metabolities of MAP. The degree of in vivo hydrolysis of the axial 17alpha-acetate of MAP, though extensive appeared to be of a significantly lesser magnitude than that exhibited toward the equatorial 3beta and 17beta acetate groups of labeled ethynodiol diacetate injected into baboons. The deacetylation of the 17alpha-acetate in MAP was similar to that observed in humans given the drug. Oxygenation of MAP at positions 1 and/or 2 appeared to be rather minimal (less than 5%).     

Metabolism of [13N]ammonia in rat lung

Bolus injection of [13N]ammonia into the femoral vein of pentobarbital-anesthetized rats was followed by rapid clearance from the blood and first-pass extraction of nearly 30% by the lungs. Of the label present in the lungs at 6 s after injection (about 27% of the dose), more than 20% was in metabolized form. Of the label present in the lungs at 2 min after injection (about 10% of the dose), 18-25% was in ammonia, about 75% was in glutamine (amide) and less than 1% was in glutamate and aspartate. Thus, despite the presence of significant amounts of glutamate dehydrogenase, the overwhelming route for metabolism of ammonia entering the rat lung in vivo was the glutamine synthetase reaction. Lung tissue that was removed 6 s after intravenous injection of [13N]ammonia and incubated in Krebs-Ringer glucose medium at 37 degrees C for 20 min, showed a significant increase (more than one-third), compared to unincubated lung tissue in the quantity of label in glutamine. Between 6s and 2 min after injection, during which time the total 13N content of the lungs decreased by more than 60%, the maintenance of a quasi-steady state in the concentration of labeled glutamine suggested a short-term balance between formation from extracted ammonia and loss of glutamine into the circulation. Our data support the concept that the lungs are a source of circulating glutamine in the rat. Despite the large fractional extraction of blood-borne [13N]ammonia by the lungs, only minute amounts of tracer (0.2-0.6 ppm of the injected dose) were detected in the expired air within the first 5 min after administration of [13N]ammonia to anesthetized rats, so that pulmonary excretion was not a significant pathway of ammonia elimination. The present findings emphasize the importance of the lungs in the maintenance of whole-body nitrogen homeostasis and suggest the use of [13N]ammonia and 13N-labeled amino acids as non-invasive probes in the study of normal and diseased lung metabolism.     

The fate of extracellular glutathione in the rat.

When intravenously administered to rats, [U-14C]glycine-labelled GSSG, GSH and its analogue ophthalmic acid were rapidly removed from the blood. In perfusion studies with isolated liver, however, the compounds did not enter the liver tissue. Thus, uptake by this tissue is obviously not responsible for the removal of gamma-glutamyl tripeptides from the blood. Instead, rapid hydrolysis of the tripeptides was observed. The undegraded tripeptides were only detected in the blood immediately after administration. Within tissue the degradation product glycine accounted for all the radioactivity. After intravenous injection of the labelled tripeptides the radioactivity accumulated first in the kidney, as shown by autoradiographic studies and chemical analysis of different tissues. The hydrolysis of the gamma-glutamyl tripeptides decreased markedly after the renal arteries were clamped. These observations strongly suggest that renal tissue is the principal site of the degradation of the tripeptides. Inhibition studies and experiments with isolated renal tubules revealed that gamma-glutamyl transpeptidase catalyses the fast hydrolysis of the extracellular peptides. The results indicate that, when entering the extracellular space, glutathione and its analogues are completely hydrolysed and must be resynthesized after reuptake of the constituent amino acids. It is concluded that the degradation occurs mainly on the luminal surface of the renal brush-border membrane and that gamma-glutamyl transpeptidase is a glutathionase acting on extracellular glutathione.     

The metabolic fate of dietary polyphenols in humans

Dietary polyphenols are widely considered to contribute to health benefits in humans. However, little is yet known concerning their bioactive forms in vivo and the mechanisms by which they may contribute toward disease prevention. Although many studies are focusing on the bioavailability of polyphenols through studying their uptake and the excretion of their conjugated forms, few are emphasizing the occurrence of metabolites in vivo formed via degradation by the enzymes of colonic bacteria and subsequent absorption. The purpose of this research was to investigate the relationship between biomarkers of the colonic biotransformation of ingested dietary polyphenols and the absorbed conjugated polyphenols. The results show that the majority of the in vivo forms derive from cleavage products of the action of colonic bacterial enzymes and subsequent metabolism in the liver. Those include the glucuronides of 3-hydroxyphenylacetic, homovanillic, vanillic and isoferulic acid as well as 3-(3-methoxy-4-hydroxyphenyl)-propionic, 3-(3-hydroxyphenyl)-propionic acid, and 3-hydroxyhippuric acid. In contrast, intact conjugated polyphenols themselves, such as the glucuronides of quercetin, naringenin and ferulic, p-coumaric, and sinapic acid were detected at much lower levels. The results suggest that consideration should be given to the cleavage products as having a putative role as physiologically relevant bioactive components in vivo.