The physiologic concentration of ammonia is 10- to 20-fold higher in portal blood than in systemic blood and reaches levels of approximately 300 to 600 µmol/L. In systemic blood, these levels would be toxic to the brain [9]. As blood passes through the hepatic lobule, ammonia is converted to urea by the urea cycle, which occurs mainly in periportal hepatocytes (Figure 1). Most of the ammonia that escapes the urea cycle is incorporated into glutamine by glutamine synthetase, which is located in pericentral hepatocytes [10, 11] (Figure 1).

View larger version (40K): [in this window] [in a new window]   Figure 1. Nonhomogeneous distribution of enzymes in the hepatocytes of an acinar sinusoid as they are linearly distributed from the protal triad to the region of the central vein or terminal hepatic venule. The specific enzymatic reactions shown are for an individual periportal and perivenous hepatocyte. The glutamine synthetase (GS) and ornithine aminotransferase (OAT) enzyme activities are expressed exclusively in the first three cell layers that surround the central vein (that is, the region of zone 3 of the liver lobule), whereas the urea cycle enzymes are concentrated within the periportal hepatocytes. However, the urea cycle enzyme activities are higher in zone 1 (immediately surrounding the portal triad) than in zone 2. Squares represent hepatocytes, circles represent hepatic mitochondria, and the interrupted line on either side of the linear array of hepatocytes represents the lining of the space of Disse. -KG = -ketoglutarate; Arg = arginine; ASA = argininosuccinate; Asp = aspartate; ATP = adenosine triphosphate; Cit = citrulline; CO2 = carbon dioxide or bicarbonate; CP = carbamoyl phosphate; CPS I = carbamoyl phosphate synthetase I; Gln = glutamine; Glu = glutamate; NH3 = ammonia; Orn = ornithine; P5C = delta1 -pyrroline 5-carboxylate; P5CDH = 1-pyrroline-5-carboxylate dehydrogenase.

Attempts to elucidate the cause of hyperammonemia after organ transplantation have been unsuccessful, and no defect in the function of the urea cycle has been found to explain this phenomenon. The paradox that led to our study was the normal or slightly low-normal levels of glutamine in the blood of most reported patients with post-transplantation hyperammonemia [3]. This finding contrasts with that seen in symptomatic patients with urea cycle disorders, in whom plasma levels of glutamine are invariably markedly elevated before treatment [12]. We postulated that dysfunctional or damaged pericentral hepatocytes or perturbed glutamine synthetase activity within these hepatocytes may cause hyperammonemia in the presence of normal function of the urea cycle.

Case Reports

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Patient 1

A more detailed clinical case report of this patient was published elsewhere [13]. A 51-year-old woman had single orthotopic lung transplantation for treatment of primary pulmonary hypertension. After surgery, she received cyclosporine, azathioprine, and methylprednisolone. Three days after surgery, she had acute rejection that was treated with high-dose methylprednisolone. On the fourth day after surgery, while the rejection was resolving, she became agitated and her mental state rapidly deteriorated. She had tonic-clonic seizures and was treated with phenytoin and phenobarbital, but the convulsions continued, requiring induction of pentobarbital coma. On the fifth day, the plasma level of ammonium was found to be 1642 µmol/L (normal level < 35 µmol/L). The serum level of alanine aminotransferase was 75 U/L (normal level < 40 U/L); serum levels of aspartate aminotransferase, alkaline phosphate, and -glutamyltransferase were normal. The serum bilirubin level was 1.2 mg/dL (normal level < 1.2 mg/dL). Quantitation of plasma amino acid revealed normal levels of citrulline, arginine, alanine, and asparagine and a mildly elevated glutamine level (1076 µmol/L; normal level, 285 to 832 µmol/L). Orotic acid in urine was not detectable. The level of glutamine in cerebrospinal fluid was markedly elevated (31 070 µmol/L; normal level, 339 to 865 µmol/L). Pulmonary function was normal, and the patient did not have fever or hypotension. She was given arteriovenous hemodialysis and oral and rectal lactulose; nonetheless, the ammonium level continued to increase, reaching 3207 µmol/L 7 days after surgery. Results of a percutaneous liver biopsy showed severe microvesicular steatosis without necrosis or hepatitis. Lipofuscin was present in zone 3 hepatocytes. Periodic acid-Schiff staining with diastase digestion showed an increased number of Kupffer cells. Electron microscopy revealed bizarre giant mitochondria that contained crystalline structures and had loss of cristae.

Because the patient had no electrical activity in the brain, life support was withdrawn. On autopsy, analysis of the liver done by using light and electron microscopy showed findings similar to those reported in the Reye syndrome.

Patient 2

A 58-year-old woman underwent orthotopic lung transplantation for severe chronic obstructive lung disease. After successful transplantation, she was treated with cyclosporine, azathioprine, and methylprednisolone and was given total parenteral nutrition. Because the transplanted lung contained bacteria, the patient was given ceftazidine and vancomycin. On the seventh day after surgery, she became lethargic. The plasma level of ammonium was 118 µmol/L, and the patient received lactulose and neomycin. The plasma level of ammonium fluctuated between 89 and 179 µmol/L until day 14 after surgery, when it increased to 440 µmol/L. The patient was then treated with intravenous sodium benzoate and underwent hemodialysis; this treatment reduced the ammonium level to between 42 and 100 µmol/L. Plasma levels of glutamine ranged from 501 to 593 µmol/L (normal level, 285 to 832 µmol/L). The level of orotic acid in urine was normal. On day 18, the patient developed an acute abdomen after cecal perforation, which was repaired surgically. Her condition deteriorated, and, 32 days after transplantation, she became oliguric and unresponsive. By day 35, the plasma level of ammonium was 535 µmol/L. The serum level of bilirubin was 1.8 mg/dL (normal level < 1.2 mg/dL), the alanine aminotransferase level was 22 U/L (normal level, 0 to 40 U/L), and the alkaline phosphatase level was 165 U/L (normal level, 35 to 125 U/L). Life support was withdrawn, and the patient died 35 days after transplantation. On autopsy, histologic examination of the liver showed cholestasis but no evidence of hepatocellular damage.

Methods

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Liver tissue was obtained from patient 1 before death and from patient 2 one hour after death; both patients' family members had given informed consent. Sections were fixed for histologic analysis, and a portion of tissue was rapidly frozen in liquid nitrogen and stored at –80°C for later analysis by enzyme assays. Controls were specimens of frozen normal human liver tissue from five adults obtained through the University of Minnesota's liver tissue procurement and distribution system, by contract from the National Institutes of Health. The five livers from which the control specimens were taken were structurally normal and had no dysfunction before harvest. They were frozen in liquid nitrogen immediately after harvesting and were stored at –80°C until analysis was done. Assays were performed to measure the catalytic activities of carbamoyl phosphate synthetase I and ornithine carbamoyltransferase in liver homogenates, as described elsewhere [14]. The same homogenates were used in a spectrophotometric assay of glutamine synthetase catalytic activity [14], which was performed in the forward reaction as well as in the reverse transferase reaction [15]. Western blot assays of the liver homogenates for glutamine synthetase were performed [16, 17] by using monoclonal mouse antisheep glutamine synthetase antibody (Chemicon International, Inc., Temecula, California). This antibody strongly cross-reacts with rat and human glutamine synthetase. We did this analysis to correlate the enzyme activity with the amount of enzyme present in the tissue.

Results

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The Table 1 shows the results of enzymatic studies performed on liver tissue from the two patients and the five controls. Hepatic glutamine synthetase activity in the patients was 12% and 28% of that in the controls, whereas activities of carbamoyl phosphate synthetase I and ornithine carbamoyltransferase were similar to those of the controls. The results of analysis of glutamine synthetase done by the transferase method are not shown in the (Table 1). On the transferase (reverse) assay, liver specimens from controls 2 and 3 had glutamine synthetase activities of 97.6 to 318.6 µmol/g of weight (0.54 to 1.4 µmol/mg of protein), whereas the livers of patients 1 and 2 had activities of 44.2 µmol/g of weight (0.24 µmol/mg of protein) and 8.3 µmol/g of weight (0.053 µmol/mg of protein), respectively. The deficiency of glutamine synthetase in the patients resulted from markedly reduced amounts of enzyme protein in their livers, based on Western blot assay (data not shown).

Discussion

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We studied hepatic glutamine synthetase deficiency in two patients who had fatal hyperammonemia after lung transplantation. The paradoxical finding of normal plasma levels of glutamine in the presence of massive hyperammonemia may be explained by deficient glutamine synthetase activity; this may have been partially or completely responsible for the hyperammonemia.

Patients with severe multiorgan disease have negative nitrogen balance and enhanced degradation of proteins. However, a catabolic condition alone is unlikely to cause hyperammonemia because this complication is not usually found in very ill patients in intensive care units. Alternatively, elevated levels of ammonia in the absence of proportionally elevated plasma levels of glutamine may be caused by hypoperfusion of the liver by portal circulation or massive production of ammonia in tissues other than those drained by the portal vein. In these hypothetical situations, blood ammonia would not be completely available to be metabolized by liver enzymes and would subsequently enter the brain through the systemic circulation. Large amounts of glutamine would then be produced by brain glutamine synthetase; this would be shown by massively elevated glutamine levels in cerebrospinal fluid. Autopsy revealed no evidence of hepatic circulation problems in the two patients, but the possibility of enhanced ammonia production by tissues other than the intestine must be investigated in future patients.

The cause for reduced glutamine synthetase protein activity in the livers of the patients remains obscure. Glutamine synthetase seems highly susceptible to oxidation by various compounds [18] and seems to be a target of acetaminophen toxicity [19]. Destruction of rat perivenous hepatocytes with carbon tetrachloride results in diminished ammonia extraction from portal blood [20]. When cirrhosis was induced in rats by using carbon tetrachloride, serum levels of ammonia showed an inverse correlation with the specific activity of glutamine synthetase, while urea synthesis did not diminish. This and other studies show the importance of hepatic glutamine synthetase for ammonia detoxification and suggest that deficiency of glutamine synthetase may contribute to hyperammonemia in liver disease.

Essentially, all patients undergoing transplantation procedures are treated with multiple agents, including immunosuppressive medications, steroids, antibiotics, and analgesics. Some persons may be predisposed to glutamine synthetase protein denaturation or degeneration of pericentral hepatocytes as a result of the unknown toxic effects of drugs given after transplantation or as a result of unrecognized humoral or immune factors.

We studied only two patients with serious multisystem disease that could have affected glutamine synthetase in a nonspecific manner. However, glutamine synthetase is an ammonia scavenger, and a deficiency of this enzyme may cause hyperammonemia under certain conditions. The findings in these two patients may provide a clue to the cause of hyperammonemia after organ transplantation.

Dr. Lichtenstein: Department of Medicine, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104-4283.

Dr. Furth: Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104.

Dr. Bavaria: Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104.

Drs. Kaplan and Berry: Department of Metabolism and Genetics, The Children's Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104-4399.

Dr. Gibson: Arkansas Children's Hospital, 800 Marshall Street, Little Rock, AR 72202-3591.