Alan S. Gelbard

The dynamics of ammonia metabolism in man. Effects of liver disease and hyperammonemia.

The cyclotron-produced radionuclide, 13N, was used to label ammonia and to study its metabolism in a group of 5 normal subjects and 17 patients with liver disease, including 5 with portacaval shunts and 11 with encephalopathy. Arterial ammonia levels were 52-264 micron. The rate of ammonia clearance from the vascular compartment (metabolism) was a linear function of its arterial concentration: mumol/min = 4.71 [NH3]a + 3.76, r = +0.85, P less than 0.005. Quantitative body scans showed that 7.4 +/- 0.3% of the isotope was metabolized by the brain. The brain ammonia utilization rate, calculated from brain and blood activities, was a function of the arterial ammonia concentration: mumol/min per whole brain = 0.375 [NH3]a - 3.6, r = +0.93, P less than 0.005. Assuming that cerebral blood flow and brain weights were normal, 47 +/- 3% of the ammonia was extracted from arterial blood during a single pass through the normal brains. Ammonia uptake was greatest in gray matter. The ammonia utilization reaction(s) appears to take place in a compartment, perhaps in astrocytes, that includes less than 20% of all brain ammonia. In the 11 nonencephalopathic subjects the [NH3]a was 100 +/- 8 micron and the brain ammonia utilization rate was 32 +/- 3 mumol/min per whole brain; in the 11 encephalopathic subjects these were respectively elevated to 149 +/- 18 micron (P less than 0.01), and 53 +/- 7 mumol/min per whole brain (P less than 0.01). In normal subjects, approximately equal to 50% of the arterial ammonia was metabolized by skeletal muscle. In patients with portal-systemic shunting, muscle may become the most important organ for ammonia detoxification. Muscle atrophy may thereby contribute to the development of hyperammonemic encephalopathy with an associated increase in the brain ammonia utilization rate.

Cerebral ammonia metabolism in hyperammonemic rats.

The short‐term metabolic fate of blood‐borne [13N]ammonia was determined in the brains of chronically (8‐ or 14‐week portacaval‐shunted rats) or acutely (urease‐treated) hyperammonemic rats. Using a “freezeblowing” technique it was shown that the overwhelming route for metabolism of blood‐borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L‐[amide‐13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14‐week portacaval‐shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight‐matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.

Perfusion of colorectal hepatic metastases. Relative distribution of flow from the hepatic artery and portal vein

The importance of portal circulation in the delivery of drugs and nutrients to colorectal hepatic metastases is controversial. Using 13N (nitrogen 13) amino acids and ammonia with dynamic gamma camera imaging, we demonstrate, for the first time in human beings, a quantitative advantage of hepatic artery compared with portal vein infusion. Eleven patients were studied by hepatic artery injection, five patients were studied by portal vein injection, and two patients had injections through both routes. Data collected from the liver for 10 minutes after rapid bolus injection of 13N L‐glutamate, L‐glutamine, or ammonia were compared with 99mTc (technetium) macroaggregated albumin (MAA) images produced after injection through the hepatic artery or portal vein at the same session. Tumor regions defined from 99mTc sulfur colloid scans were compared with nearby liver areas of similar thickness. For the 13N compounds, the area‐normalized count rate at first pass maximum (Qmax) and the tissue extraction efficiency were computed. The tumor/liver Qmax ratios for MAA and 13N compounds were highly correlated. Both tumor and liver extracted more than 70% of the nitrogenous compounds. The tumor/liver Qmax ratios reflect the relative delivery of injected tracer per unit volume of tissue. After hepatic artery injection the Qmax ratio was 1.03 ± 0.33 (mean ± SD), significantly exceeding the Qmax ratio of 0.50 ± 0.34 after portal vein injection (P < 0.003). Therefore, (1) more than twice as much of a nutrient substrate is delivered per volume of tumor relative to liver by the hepatic artery as by the portal vein; (2) the high extraction efficiency demonstrates that the hepatic artery flow is nutritive; and (3) the delivery of substance in solution (such as nutrients or drugs) to tumor and liver tissue correlates with the distribution of colloids such as macroaggregated albumin after hepatic arterial and portal venous injection.

Enzymatic Synthesis and Organ Distribution Studies with 13N-Labeled L-Glutamine and L-Glutamic Acid1

A method was developed for producing several hundred mCi of 13NH3 with subsequent enzymatic synthesis of large amounts of 13N-labeled glutamine and glutamic acid. Dynamic measurements and quantitative whole body scans demonstrated greater glutamine uptake in the liver region of mongrel dogs than uptake of glutamic acid or NH3, although all concentrated in the liver to a significant degree. Myocardial uptake of glutamine and glutamic acid was low, while NH3 was incorporated into the heart, brain, bladder, and kidneys at a greater rate than the amino acids.

The use of immobilized glutamate dehydrogenase to synthesize 13N-labeled l-amino acids☆

Abstract By utilizing glutamate dehydrogenase immobilized onto CNBr-activated Sepharose it is possible to synthesize six l -13N-amino acids in high radiochemical yield (5–140 mCi) and in high (>99%) radiochemical purity. These 13N-amino acid solutions are potentially suitable for whole body and organ imaging in large animals and man.

Glutamine biosynthesis and the utilization of succinate and glutamine by Rhizobium etli and Sinorhizobium meliloti.

Sinorhizobium meliloti 1021 and Rhizobium etli CE3 turn over nitrogen and carbon from glutamine to ammonium and CO2, respectively. Some of the ammonium released is assimilated back into glutamine, indicating that a glutamine cycle similar to that in Neurospora operates in Rhizobium. In addition, a previously unrecognized metabolic pathway in Rhizobium was discovered--namely, conversion of glutamine-carbon to gamma-hydroxybutyric acid and beta-hydroxybutyric acid. Additionally, some of the 2-oxoglutarate derived from glutamine catabolism in Rhizobium is converted to succinate in glutamine-containing medium. Both S. meliloti 1021 and R. etli CE3 oxidize succinate preferentially over glutamine when provided with both carbon sources. In contrast to Sinorhizobium meliloti 1021 and Rhizobium etli CE3, an S. meliloti double mutant that lacks both glutamine synthetase (GS) I and II preferentially oxidizes glutamine over succinate when supplied with both substrates. GSII activity is induced in wild-type S. meliloti 1021 and R. etli CE3 grown in succinate-glutamine medium, and this enzyme participates in the cycling of glutamine-carbon and -nitrogen. On the other hand, GSII activity is repressed in both micro-organisms when glutamine is the only carbon source. These findings show that, in medium containing both glutamine and succinate, glutamine synthesis helps drive the utilization of succinate. When glutamine is in excess as an energy-providing substrate its synthesis is restricted, allowing for more effective utilization of glutamine as an energy source.

High Activities of Glutamine Transaminase K (Dichlorovinylcysteine β‐Lyase) and ω‐Amidase in the Choroid Plexus of Rat Brain

Abstract: Certain halogenated hydrocarbons, e.g., dichlo‐roacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine‐S‐conjugate β‐lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine‐S‐conjugate β‐lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and ω‐amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and α‐keto acid substrates. The ω‐amidase also has a broad specificity; presumably, however, the natural substrates are α‐ketoglutaramate and α‐ketosuccinamate, the α‐keto acid analogues of glutamine and aspara‐gine, respectively. The high activities of both glutamine transaminase K and ω‐amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., l‐glutamine and α‐keto acid α‐ketoglutarate and l‐amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transaminase K and its presence in the choroid plexus.

Quotient imaging with N‐ 13 L‐glutamate in osteogenic sarcoma: Correlation with tumor viability

An investigation was performed to correlate the regional uptake of N‐13 L‐glutamate with histologic changes in tumor tissue in patients undergoing adjuvant chemotherapy for osteogenic sarcoma. A parametric image was produced by calculating the ratio of N‐13 uptake in the tumor in a pixel‐by‐pixel fashion, using the presurgical scan as the numerator and the pretherapy scan as the denominator. The change in N‐13 uptake in 2 × 2‐cm regions of the tumor was compared with residual cell viability as determined by microscopic examination of multiple thin sections obtained from the surgical specimens. Regions that showed decreases in N‐13 uptake of more than 30% were frequently associated with areas of highly necrotic tumor, and regions that showed increasing uptake were associated with high residual cell viability and incomplete response to chemotherapy.

High-performance liquid chromatographic on-line flow-through radioactivity detector system for analyzing amino acids and metabolites labeled with nitrogen-13

A flow-through radioactivity detector was used for the high-performance liquid chromatographic determination of amino acids and other nitrogenous substances labeled with 13N, a short-lived (t1/2 9.96 min) positron-emitting radionuclide. 13N-Labeled compounds were analyzed using cation, anion and amino columns, or as the o-phthaldialdehyde derivative on an ODS column. Use of column-switching valves and a high-performance liquid chromatographic system with a quaternary eluting capability permits two to three 20-min analyses of labeled samples from a single 13N experiment to be carried out on different columns using a binary or a single mobile phase. Radioactivity in liver metabolites was quantified using an on-line flow-through monitor with data processing capability for integrating peaks and correcting for radioactivity decay. As an example, 1 min following an L-[13N]glutamate injection via the hepatic portal vein, 77% of the label in the liver was in a metabolized form; at least ten labeled products were formed.

Imaging of tumors involving bone with 13N-glutamic acid.

Nitrogen-13 labeled L-glutamic acid was evaluated as an imaging agent for tumors involving bone. The enzymatically prepared labeled compound was administered intravenously to dogs with spontaneous tumors, and tumor uptake was determined with a gamma camera and rectilinear scanner. These tumors were well visualized with 13N-glutamic acid, and the results compared favorably with uptake studies performed on the same animals with 99mTc-diphosphonate.