Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Fatty acid metabolism wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Microbial metabolism wikipedia , lookup
Citric acid cycle wikipedia , lookup
Gaseous signaling molecules wikipedia , lookup
Biosynthesis wikipedia , lookup
Biochemistry wikipedia , lookup
Nitrogen cycle wikipedia , lookup
Clinical Application of Blood Ammonia Determinations C/E Update: Chemistry III by Allen M. Glasgow, M.D. Category A-l continuing education credit is available to anyone who studies a C/E Update series and completes a written exam. Exams can be ordered from the ASCP and will be sent to participants following the appearance of the final article of each series in LABORATORY MEDICINE. After receipt of a completed answer sheet at ASCP prior to the deadline stated on the exam, a certificate of credit will be awarded to each participant. An exam order form appears on page 175 in this issue. Introduction This article briefly reviews ammonia toxicity and ammonia metabolism. Disorders of ammonia metabolism are described. Methods for measuring plasma ammonia are then described, followed by a discussion of the technical difficulties of ammonia assays. The article concludes with a discussion of alternate methods for assessing ammonia metabolism. Teleologic Problem Ammonia is a waste product of protein and amino acid metabolism. In aquatic animals, ammonia is easily disposed of by simple diffusion into the water. As life evolved onto land, it became necessary to From the Department of Endocrinology and Metabolism, Children's Hospital National Medical Center, Washington, D. C. develop other means of eliminating ammonia. Birds excrete waste nitrogen as uric acid, which can be highly concentrated, thus enabling them to conserve water and keep their weight low. Excess nitrogen in mammals is eliminated as urea. Ammonia as a Toxin A l t h o u g h elevated a m m o n i a levels affect the metabolism of a variety of tissues, the toxic effects on the central nervous system dominate the clinical picture in hyperammonemia. The clearest clinical example of this toxicity is seen in the inherited disorders of the urea cycle. The mechanism of ammonia neurotoxicity is not well understood. An elevated neuronal ammonia may shift the equilibrium of the reaction sequence: Alpha-ketoglutarate + NH 4 + + NADH -» Clutamate + NAD + + H 2 0 Glutamate + NH 4 + —> Glutamine It has been postulated that this shift may decrease the concentration of alpha-ketoglutarate and thus impair the activity of the cerebral citric acid cycle. 1 - 3 Alternately, the elevated glutamine may be converted, in part, to alphaketoglutaramate, which is neurotoxic. 4 Another possibility is that the neurotoxicity of ammonia may result from an alteration of neuronal ionic equilibrium or altered neurotransmitter function. 5 Hyperammonemia causes both severe reversible cerebral dysfuction and chronic irreversible cerebral impairment. Severe, even p r o f o u n d , cerebral dysfunction may be reversible if acute hyperammonemia can be treated successfully. O n the other hand, chronic mild hyperammonemia without immediate symptoms may cause irreversible cerebral damage.7 Acute hyperammonemia leads to lethargy, vomiting, agitation, seizures, coma and eventual loss of brainstem functions. Hyperventilation leading to a respiratory alkalosis is c o m m o n in hyperammonemic states, presumably due to a stimulation of the central respiratory center. The major effect of chronic hyperammonemia is impairment of intellectual function. Ammonia Metabolism Production When considering ammonia p r o d u c t i o n , it is better to think of the " a m m o n i a " or nitrogen load rather than free ammonia product i o n . Some " a m m o n i a " is transported to the liver as alanine, glutamine and other amino acids. While this " a m m o n i a " may never appear in the plasma as free ammonia, it is in equilibrium with free ammonia and constitutes part of the total ammonia load. Ultimately, ammonia comes from two sources: 1) ingested nitrogen (primarily protein); and 2) hydrolysis of urea in the gastrointestinal tract. The latter source 0007-5027/81/0300/151 $00.85 © American Society of Clinical Pathologists 151 contributes to the ammonia load because the ammonia must be reconverted to urea. Since 30% to 40% of the urea produced in the liver is hydrolysed to ammonia in the intestine, 8 the total ammonia load is about 30% to 40% greater than ingested nitrogen, minus small amounts of nitrogen eliminated by mechanisms other than the urea cycle. These mechanisms include net protein synthesis in growing children, skin loss, ammonia in expired air, stool nitrogen, and nitrogen excreted as creatinine, uric acid and urinary ammonia. In general, the nitrogen eliminated by these mechanisms is small compared with the ammonia load. In addition, the amount is relatively constant and not influenced by nitrogen intake. GLUTAMINE + C O , Carbamyl phosphate is also produced in the cytoplasm by another carbamyl phosphate synthetase 152 GLUTAMATE I OROTIC A C I D ORNITHINE CITRULLINE PYRIMIDINES \9 CYTOPLASM /So MITOCHONDRIA f ORNITHINE CITRULLINE ASPARTATE UREA ARGININOSUCCINATE The Urea Cycle The complete urea cycle (Fig. 1) is present only in the liver. The first step of the urea cycle is catalyzed by mitochondrial carbamyl phosphate synthetase. A m monia, C 0 2 and t w o ATPs are converted to carbamyl phosphate, phosphate and two ADPs. Mitochondrial carbamyl phosphate synthetase is inactive in the absence of N-acetyl glutamate. Nacetyl glutamate is f o r m e d f r o m acetyl CoA and glutamate by glutamate N-acetylase, which is activated by arginine. The activity of this enzyme is also increased in animals that are fed a high-protein diet, suggesting that increased protein intake is one way in which the urea cycle is regulated. 9 An inheritable deficiency of glutamate N-acetylase has never been described, but it is likely that such a deficiency w o u l d produce a disorder similar to the urea cycle enzyme deficiencies discussed below. ACETYL COA CAR BAM YL PHOSPHATE " ^ t . ARGININE FUMARATE Fig. 7. The urea cycle and related reactions (see text). Key to enzymes and transport processes: 7 = carbamyl phosphate synthetase (mitochondrial); 2 = ornithine transcarbamylase; 3 = argininosuccinate synthetase; 4 = argininosuccinate lyase; 5 = argininase; 6 = glutamate Nacetylase; 7 = ornithine aminotransferase; 8 = carbamyl phosphate synthetase (cytoplasmic); 9 = ornithine transport process; 10 = citrulline transport process. which utilizes glutamine rather than ammonia as a preferred substrate and does not require Nacetyl glutamate as an activator. Cytoplasmic carbamyl phosphate is part of the pyrimidine synthetic pathway. Normally, the mitochondrial (urea cycle) pool of carbamyl phosphate seems to be separate from the cytoplasmic (pyrimidine) pool. However, when mitochondrial carbamyl phosphate accumulates (as in ornithine transcarbamylase deficiency), carbamyl phosphate diffuses into the cytoplasm, leading to an overproduction of orotic acid and other pyrimidines. LABORATORY MEDICINE • VOL. 12, NO. 3, MARCH 1981 The second urea cycle enzyme, ornithine transcarbamylase, combines carbamyl phosphate and o r n i t h i n e , forming citrulline and releasing phosphate. This reaction occurs in the mitochondrial matrix. Ornithine enters the mitochondria by a specific transport process. 10 There is also a specific transport process for moving citrulline out of the mitochondria. 1 0 The remainder of the urea cycle enzymes, which regenerate ornithine f r o m citrulline and release urea, are cytoplasmic. The first cytoplasmic step catalysed by argininosuccinate synthetase converts citrulline and asparatate t o argininosuccinate, while consuming the energy of one high-energy phosphate bond of ATP. Argininosuccinate lyase then catalyzes the formation of arginine and fumarate from argininosuccinate. Ornithine is regenerated from arginine by argininase, releasing urea. The net effect of the sequence of reactions is that ammonia, aspartate, C 0 2 and three ATPs are converted t o urea, fumarate and three ADPs. centration is normally about 300 /xmol/L, whereas the plasma ammonia concentration is normally about 30 /umol/L. Thus, small rises in alanine and giutamine can " b u f f e r " relatively large rises in ammonia. While such reactions limit the rise in blood ammonia, they do not result in the elimination of ammonia. The proper functioning of the urea cycle requires adequate levels of ornithine. If dietary arginine is low, ornithine must be produced. If dietary arginine and ornithine are in excess, they can be converted to glutamate. Production and degradation of ornithine occurs by a two-step process catalyzed by ornithine aminotransferase and glutamate semialdehyde dehydrogenase. These t w o enzymes, rather than ornithine transcarbamylase, are probably the major determinants of the hepatic ornithine level. In man, dietary arginine appears to be in excess normally, and the physiologic direction of this sequence is toward glutamate. Hyperornithinemia with gyrate atrophy of the choroid and retina is the only known disorder associated with hypoammonemia." The hyperornithinemia is due to a deficiency of ornithine aminotransaminase activity. Amination Reactions An acute change in ammonia level may be temporarily "buffered" by incorporating ammonia into amino acids. This buffering may be important in limiting fluctuations in tissue ammonia levels. Incorporation of ammonia into alanine and giutamine seems to be most important physiologically. Although it has been proposed that the formation of giutamine from alpha-ketoglutarate may contribute to ammonia toxicity, it is equally likely that the formation of giutamine and alanine is protective. The serum alanine concentration is normally about 200 /umol/L and the serum giutamine con- Disorders of Ammonia Metabolism Hypoammonemia Hyperammonemia Because of the body's large reserve capacity for urea synthesis, there are few examples of hyperammonemia due to increased production of ammonia. Infection of an obstructed urinary tract with Proteus, a urea-splitting organism, has been reported to cause hyperammonemia. 1 2 The hyperammonemia in Reye's syndrome may be due in part to increased ammonia production. 1 3 Generalized Liver Disease Hyperammonemia may occur in any patient with severe liver disease including acute hepatitis, cirrhosis or Reye's syndrome. Hyperammonemia u n d o u b t e d l y contributes to the encephalopathy in these disorders; however, other metabolic abnormalities due to the hepatic dysfunction also play a role. Hyperammonemia is relatively less important in acute fulminant hepatic failure than in chronic hepatic encephalopathy and Reye's syndrome. The e n cephalopathy of fulminant hepatic failure will often respond tem- porarily to L-dopa, suggesting that the encephalopathy is partly due to false neurotransmitters. 1 4 In patients with chronic liver disease, the encephalopathy is usually made worse by measures that increase blood ammonia and improved by measures that decrease blood ammonia. 16,17 O n the other hand, the correlation between the severity of the encephalopathy and the blood ammonia level is not strong. Increased levels of plasma aromatic amino acids and decreased levels of plasma branched-chain amino acids may also contribute to the encephalopathy of chronic liver disease.18 Increased aromatic amino acids in the brain may increase the formation of serotonin and other neurotransmitters. In Reye's syndrome, there is a reasonably good correlation between the blood ammonia levels at admission and the severity of the encephalopathy. 1 9 However, hepatic function invariably improves and the ammonia level falls, even in patients w h o eventually die from this disorder. Patients with Reye's syndrome have a marked increase in ammonia production and probably also have impaired hepatic ureagenesis. 13 Although the cause of the i m paired ureagenesis is not certain, there is a transient decrease in hepatic carbamyl phosphate synthetase and ornithine transcarbamylase activity. Urea Cycle Disorders Carbamyl phosphate synthetase (CPS) deficiency has been reported in only a few patients. O n e patient with complete CPS deficiency died from overwhelming hyperammonemia shortly after birth. 2 0 Children with partial CPS deficiency may have severe mental retardation, and may experience episodic vomiting and lethargy LABORATORY MEDICINE • VOL 12, NO. 3, MARCH 1981 1 5 3 following ingestion of high-protein meals. 21 Ornithine transcarbamylase (OTC) deficiency is the most c o m m o n inheritable urea cycle disorder. The gene for OTC is on the X chromosome. 2 2 Males with OTC deficiency have only the abnormal gene, while females with OTC deficiency have t w o genes, one normal and one abnormal. The most common mutation(s) of this enzyme results in total loss of activity. Affected males have no OTC activity and usually die within two to three days of birth. 2 3 Symptoms in females vary widely, depending on the balance between the inactivation of the normal and abnormal genes 24,25 : some have severe mental retardation and recurrent episodes of acute encephalopathy; some have only protein intolerance; still others are asymptomatic. In rare cases, male patients may have an OTC mutation in which the enzyme retains partial activity; these patients have symptoms similar t o those of females. Patients with OTC deficiency excrete excessive amounts of urinary orotic acid. The best way to detect asymptomatic females with OTC deficiency is to measure the urinary orotic acid response t o a protein load. 26 Citrullinemia is caused by a deficiency of argininosuccinate synthetase. Complete deficiency of this enzyme causes severe, usually lethal, hyperammonemia in infancy. Variants with partial activity cause mental retardation and episodic encephalopathy. 2 7 Plasma and urinary citrulline are markedly elevated. It is not clear if the hyperammonemia is due to a "back u p " inhibition of OTC by citrulline or a relative ornithine deficiency. Argininosuccinic aciduria causes mental retardation and episodic 154 encephalopathy resembling the milder forms of the disorders described above. Some patients with this disorder have short, brittle hair due t o arginine deficiency. The hyperammonemia in this disorder is caused by the markedly increased excretion of argininosuccinic acid, which leads to an arginine and ornithine deficiency. 28 Argininemia is often associated with modest elevations of blood ammonia. Elevated arginine may be more important than hyperammonemia in causing the athetosis and spasticity seen in patients with this disorder. Disorders of Ornithine Metabolism Lysinuric protein intolerance results from a defect in the transport of dibasic amino acids in the intestine, kidney and other tissues. 31,32 Poor a b s o r p t i o n , i n creased excretion and poor cellular uptake of ornithine and arginine lead to hyperammonemia because of a deficiency of hepatic ornithine. This disorder, which is relatively common in Finland, usually presents in infancy. The symptoms of lysinuric protein intolerance are failure to thrive, vomiting, diarrhea, hepatomegaly, osteoporosis and hyperammonemia. Urinary dibasic amino acids are increased, and plasma levels tend to be low. Arginine supplementation may improve the hyperammonemia. A disorder characterized by ornithinemia, hyperammonemia has been and homocitrullinuria postulated to be due to a defect in mitochondrial ornithine transport. 33 The few patients reported with this disorder have been severely retarded. The hyperammonemia may respond to ornithine supplementation. The h o m o c i trulline is presumably formed by the replacement of ornithine with lysine in the ornithine transcarbamylase step. LABORATORY MEDICINE • VOL 12, NO. 3, MARCH 1981 may cause Hyperalimentation hyperammonemia if the administered amino acids contain relatively low levels of arginine. Other Causes of Hyperammonemia Most premature infants have slightly higher blood ammonia levels than full-term infants, although it is not known if this increased level is deleterious. A few preterm infants have marked hyperammonemia that lasts t w o to three days and is associated w i t h severe neurologic symptoms. 3 3 The cause of this condition is u n k n o w n . If the infant can be supported, the prognosis is g o o d . After recovery, ammonia metabolism appears to be normal. Patients with a number of organic acidemias including propionic acidemia, methylmalonic acidemia, isovaleric acidemia and glutaric acidemia may have hyperammonemia, especially during acute episodes of acidosis. The hyperammonemia may be due to inhibition of the urea cycle by the accumulating organic acid or the coenzyme A derivative. Valproic acid therapy may also cause mild hyperammonemia. Hyperammonemia is also seen in nonketotic hyperglycinemia, and mild hyperammonemia may occur in systemic carnitine deficiency. Measurement of Blood Ammonia There are three satisfactory ways of measuring blood ammonia: 1) resin absorption methods, 2) enzymatic methods, and 3) methods using ammonia electrodes. Diffusion methods, still in use in some laboratories, generally yield high values and therefore are not recommended. Resin absorption methods involve the separation of ammonia from plasma by a cation-exchange resin. 36,37 Ammonia is then eluted from the resin w i t h NaCI and measured with the phenol-hypochlorite colorimetric reaction. The resin step is necessary to separate ammonia from plasma proteins and other compounds that react with phenol-hypochlorite. A commercial kit offered by Hyland Diagnostics (Costa Mesa, CA) yields satisfactory results, and the method can be scaled d o w n to require only 0.25 ml of plasma. Enzymatic methods use glutamate dehydrogenase, 38 an enzyme that converts alpha-ketoglutarate and ammonia to glutamate while converting NADH to N A D + . The decrease in absorption of NADH at 340 nm is usually measured; however, the change in NADH can also be measured fluorometrically, 39 or 1-C H alpha-ketoglutarate conversion to glutamate can be measured. 40 Those methods that measure changes in NADH must avoid any changes in NADH due to endogenous reactions—such as conversion of pyruvate to lactate by lactate d e h y d r o g e n a s e — b y precipitating proteins with perchloric acid or by preincubation of plasma until endogenous reactions have gone to c o m p l e t i o n . A recent report indicates that this problem can also be overcome by using NADPH instead of NADH. 4 1 4 2 Ammonia electrode methods measure the change in the electrical potential as ammonia gas (NH 3 ) diffuses across a semipermeable membrane and comes into equilibrum with ammonium ions (NH 4 + ). 4 3 The ammonia must be measured at a pH such that most of it is in a gaseous state. If the pH is not carefully controlled, labile amines in plasma will break d o w n , increasing the measured ammonia. Diffusion techniques require alkalinization of plasma so that ammonia gas will diffuse into an acidic trap. The trapped ammonia is measured by titration or colorimetrically with phenol-hypochlorite. All diffusion methods yield high values because the high pH liberates ammonia f r o m labile amines. PhysioJogic Factors Affecting Ammonia Levels Arterial vs. venous blood. Some laboratories always measure arterial blood ammonia levels; however, there is normally very little difference between arterial and venous blood ammonia levels. 44 Measurement of venous blood ammonia is satisfactory for clinical purposes. Fasting vs. fed patients. Even after a high-protein meal, blood ammonia levels increase very little in normal individuals. However, substantial increases may occur in patients with impaired ammonia metabolism. Plasma vs. whole blood. Red cell ammonia levels are from 50% to 100% higher than plasma ammonia levels. Exercise. Moderate exercise has little effect on blood ammonia, 4 4 but extreme exercise can cause a marked elevation. Technical Problems Affecting Ammonia Determinations Measurement of ammonia levels in plasma is very difficult. The c o n c e n t r a t i o n of a m m o n i a in plasma is very low compared with the potential ammonia that may be released from labile compounds in plasma and to the amount of ammonia in the laboratory environment. While many modern assays measure analytes present in much lower concentrations, few of these analytes are as ubiquitous as ammonia. It is difficult to identify exactly which precautions are necessary to avoid contamination of specimens with ammonia. For example, several investigators report success in storing samples for short periods. However, we have had difficulty in storing specimens w i t h o u t en- countering an increase in the ammonia level. The f o l l o w i n g precautions taken from our experience and the experience of others 45 indicate the care that must be taken in order to measure accurately plasma ammonia levels. Patients should relax before the test and should not smoke. Blood should be drawn by accurate swift venipuncture w i t h minimal turbulence and with minimal use of a tourniquet (brief use of a tourniquet for ten to 15 seconds is acceptable). The blood should be drawn directly into a c o o l e d , heparinized, evacuated tube or it should be immediately transferred from a syringe into an o p e n , c o l d , heparinized tube w i t h o u t using a needle. The tube should be immersed immediately in ice and rotated to facilitate mixing and cooling. The specimen should be centrifuged rapidly at 4°C for about five minutes. The plasma should be separated as soon as possible. An experienced technologist should be ready to perform the assay immediately. The laboratory should be kept as free of ammonia as possible. Tobacco smoke, urine, N H 4 O H and other potential ammonia contaminates should be excluded from the laboratory at all times. Water and reagents must be checked frequently to make sure that they are free of ammonia and formaldehyde; formaldehyde interferes w i t h the phenol-hypochlorite ammonia reaction. Glassware must be specially cleaned and stored in very dilute sodium hydroxide or sodium hypochlorite and washed w i t h ammonia-free water just before use. Assay tubes should be kept covered at all times. A procedure that avoids acidifying or alkalinizing the sample should be used. If values obtained in a normal individual are below 40 jumol/L, LABORATORY MEDICINE • VOL 12, NO. 3, MARCH 1981 1 5 5 this indicates that precautions are adequate. Clinical Indications for Measurement of Plasma Ammonia Because of the increasing recognition of Reye's syndrome, it has become standard practice to measure plasma ammonia in any child with an unexplained encephalopathy. Such testing has contributed to the increased identification of patients with hyperammonemia due to other causes. In adults, the measurement of plasma ammonia is often l i m i t e d , perhaps erroneously, to patients w i t h other evidence of liver disease. Measurement of plasma ammonia in encephalopathic patients w i t h o u t obvious liver disease is w o r t h w h i l e because it may lead to an unsuspected diagnosis. The value of plasma ammonia measurements in patients with severe generalized liver disease is more limited, because there is relatively little correlation between plasma ammonia levels and encephalopathy. In selected patients with mental retardation, measurement of ammonia may be indicated to screen for urea cycle disorders. Such screening can probably be limited to patients with episodic vomiting or encephalopathy and to those with a history of avoidance of highprotein foods. It should be kept in mind that patients with mild urea cycle disorders are not consistently hyperammonemic. In patients with hyperammonemia w h o d o not have generalized liver disease, the level of plasma ammonia is the prime determinant of the results of treatment. Thus, repeated measurements may be very important. O n the other hand, in patients with generalized liver disease, including patients with Reye's syndrome, following 156 the plasma ammonia level is less important in evaluating therapy. Alternate Tests for Assessing Ammonia Metabolism Because of the technical difficulties encountered in measuring plasma ammonia, other methods of assessing ammonia metabolism have been considered. Spinal fluid glutamine is elevated in almost all patients w i t h hyperammonemia, 46 ' 47 and it is stable and relatively easy to assay. Most patients w i t h e n c e p h a l o p a t h y must undergo a spinal tap for other reasons; therefore, obtaining spinal fluid is not a p r o b l e m . However, the need for spinal fluid limits the use of this test in patients w h o do not otherwise need a lumbar puncture. O u r institution offers plasma ammonia determinations during the day but only spinal fluid glutamine determinations at night. Plasma glutamine and alanine are also usually elevated in patients w i t h h y p e r a m m o n e m i a . Measurement of these amino acids may be a useful adjunct in some situations; however, these amino acid levels do not parallel plasma ammonia levels closely enough to be a substitute for plasma ammonia measurement. Plasma alpha-ketoglutarate is inversely correlated with blood ammonia. A recent report 48 indicates that alpha-ketoglutarate may be sensitive to changes in ammonia metabolism; however, there has not been enough experience w i t h measuring this c o m p o u n d to assess fully its role in evaluating hyperammonemia. Urinary orotic acid is elevated in OTC deficiency. Our experience with measurement of urinary orotic acid indicates that it may be more sensitive than measurement of plasma ammonia in patients with this disorder (unpublished observation). LABORATORY MEDICINE • VOL. 12, NO. 3, MARCH 1981 Summary Hyperammonemia may occur in a number of disorders. In patients with severe generalized liver disease, hyperammonemia is only a part of the complex metabolic abnormalities. In other patients, such as those with urea cycle disorders, the hyperammonemia may be the sole cause of the symptoms. Several satisfactory methods are available for measuring plasma ammonia, but special care is necessary to avoid specimen contamination from labile amines in plasma and environmental ammonia. References 1. Bessman, S.P., and Bessman, A.N., 1955. The cerebral and peripheral uptake of ammonia in liver disease with a hypothesis for the mechanism of hepatic coma. J. Clin. Invest. 34:622-628. 2. Shorey, J., McCandless, D.W., and Schenker, S., 1967. Cerebral alpha ketoglutarate in ammonia intoxication. Gastroenterology 53:706711. 3. Schenker, S., et al., 1967. Studies on the intracerebral toxicity of ammonia. J. Clin. Invest. 46:838-848. 4. Vergara, F, Plum, F., and Duffy, T. E., 1974. Alpha ketoglutarate: Increased concentrations in cerebrospinal fluid of patients in hepatic coma. Science 183:81-83. 5. Benzi, G., et al., 1977. Metabolism and cerebral energy state: Effect of acute hyperammonemia in beagle dog. Biochem. Pharmacol. 26:2397-2404. 6. Chan, H., et al., 1977. Prolonged coma and isoelectric electroencephalogram in child with lysinuric protein intolerance. J. Pediatr. 91:79-81. 7. Batshaw, ML., et al., 1980. Cerebral dysfunction in asymptomatic carriers of ornithine transcarbamylase deficiency. N. Engl. J. Med. 302:482-485 8. Walser, M., and Bodenlos, L.J., 1959. Urea metabolism in man. J. Clin. Invest. 38:16171626. 9. Saheki.T., Katsunuma, T, andSase, M., 1977. Regulation of urea synthesis in rat liver. J. Biochem. 82:551-558. 10. Gamble, J.G., and Lehninger, A.L, 1973. Transport of ornithine and citrulline across the mitochondrial membrane. J. Biol. Chem. 248:610-618. 11. Valle, D., et al., 1979. Gyrate atrophy of choroid and retina: Correction of hyperornithinemia by diet. Pediatr. Res. 13:483. 12. Samtoy, B., and Debeukelaer, M.M., 1980. Ammonia encephalopathy secondary to urinary tract infection with Proteus mirabilis. Pediatrics 65:294-296. 13. Snodgrass, P.J., and Delong, G.R., 1976. Urea-cycle enzyme deficiencies and an increased nitrogen load producing hyperam- 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. monemia in Reye's syndrome. N. Engl. J. Med. 294:855. Parkes, J.D., Sharpstone, P., and Williams, R., 1970. Levodopa in hepatic coma. Lancet ii: 1341-1343. Fischer, J.E., 1976. L-Dopa in hepatic coma. Ann. Surg. 183:386-391. Phillips, G.B., et al., 1952. The syndrome of impending hepatic coma in patients with cirrhosis of the liver given certain nitrogenous substances. N. Engl. J. Med. 247:238-246. Gabuzda, G.J., Phillips, G.B., and Davidson, C.S., 1952. Reversible toxic manifestations in patients with cirrhosis of the liver given cationexchange resins. N. Engl. J. Med. 246:124130. James, J.H., et al., 1979. Hyperammonemia, plasma amino acid imbalance and blood-brain amino acid transport: A unified theory of portalsystemic encephalopathy. Lancet ii:772-775. Glasgow, A.M., Cotton, R.B., and Dhiensiri, K. 1972. Reyes syndrome: Blood ammonia and consideration ofthe non-histologic diagnosis. Am. J. Dis. Child. 124:827-836. Gelehrter, T.D., and Snodgrass, P.H., 1974. Lethal neonatal deficiency of carbamyl phosphate synthetase. N. Engl. J. Med. 290:430433. Batshaw, M., Brusilow, S., and Walser, M., 1975. Treatment of carbamyl phosphate synthetase deficiency with keto analogues of essential amino acids. N. Engl. J. Med. 292: 1085. Short, LM., etal., 1973. Evidence for X-linked dominant inheritance of ornithine transcarbamylase deficiency. N. Engl. J. Med. 288:7-12. Campbell, A.G.M., et al., 1973. Ornithine transcarbamylase deficiency: A cause of lethal neonatal hyperammonemia in males. N. Engl. J. Med. 288:1-6. Levin, B., Oberholzer, V.G., and Sinclair, L, 1969. Biochemical investigations of hyperammonemia. Lancet ii: 170—174. Glasgow, A.M., Kraegel, J.H., and Schulman, J.D., 1978. Studies of the cause and treatment of hyperammonemia in females with ornithine transcarbamylase deficiency. Pediatrics 62:30-37. Goldstein, A.S., et al., 1974. Metabolic and genetic studies of a family with ornithine transcarbamylase deficiency. Pediatr. Res. 8: 5-12. Whelan, D.T., Brusso, T., and Spate, M., 1976. Citrullinemia: Phenotype variations. Pediatrics 57:935-941. Brusilow, S.W., and Batshaw, ML., 1979. Arginine therapy of argininosuccinase deficiency. Lancet ii:124-125. Synderman, S.E., et al., 1977. Argininemia. J. Pediatr. 90:563-568. Terheggen, H.G., etal., 1975. Familial hyperargininaemia. Arch. Dis. Child. 50:57-62. Simell, O., et al., 1975. Lysinuric protein intolerance. Am. Med. 59:229-240. Simell, O., 1975. Diamino acid transport into granulocytes and liver slices of patients with lysinuric protein intolerance. Pediatr. Res. 9: 504-508. 33. Fell, V., 1974. Ornithinemia, hyperammonemia, and homocitrullinuria. Am. J. Dis. Child. 127: 752-756. 34. Batshaw, M.L., and Brusilow, S.W., 1978. Asymptomatic hyperammonemia in low birthweight infants. Pediatr. Res. 12:221-224. 35. Ballard, R.A., et al., 1978. Transient hyperammonemia of the preterm infant. N. Engl. J. Med. 299:920-925. 36. Oberholzer, V.G., et al., 1976. Microscale modification of a cation-exchange column procedure of plasma ammonia. Clin. Chem. 22:1976-1981. 37. Wu, J., Ash, K.O., and Mao, E., 1978. Modified micro-scale enzymatic method for plasma ammonia in newborn and pediatric patients: Comparison with a modified cation-exchange procedure. Clin. Chem. 24:2172-2175. 38. Doumas, B.T., 1979. Performance of the Du Pont aca ammonia method. Clin. Chem. 25: 175-178. 39. Nazar, B.L, and Schoolwerth, A.C., 1979. An improved microfluorometric enzymatic assay for the determination of ammonia. Anal. Biochem. 95:507-511. 40. Kalb, V.F., Jr., etal., 1978. A new and specific assay for ammonia and glutamine sensitive to 100 pmol. Anal. Biochem. 90:47-57. 41. Kli, P., and Shull, B.C., 1979. Fixed-time kinetic assay of plasma ammonia, with NADPH as factor, with a centrifugal analyzer. Clin. Chem. 25:611-613. 42. Humphries, B.A., et al., 1979. Automated enzymatic assay for plasma ammonia. Clin. Chem. 25:26-30. 43. Attili, A.F., Autizi, D., and Capocaccia, L, 1975. Rapid determination of plasma ammonia using an ion-specific electrode. Biochem. Med. 14:109-116. 44. Dawson, A.M., 1978. Regulation of blood ammonia. Gut 19:504-509. 45. Gerron, G.G., et al., 1976. Technical pitfalls in measurement of venous plasma NH3 concentration. Clin. Chem. 22:663-666. 46. Hourani, B.T., Hamlin, E.M., and Reynolds, T.B., 1971. Cerebrospinal fluid glutamine as a measure of hepatic encephalopathy. Arch. Intern. Med. 127:1033-1036. 47. Glasgow, A.M., and Dhiensiri, K., 1974. Improved assay for spinal fluid glutamine and values of children with Reye's syndrome. Clin. Chem. 20:642-644. 48. Batshaw, ML., and Brusilow, S.W., 1980. Alpha ketoglutarate as a harbinger of hyperammonemia in urea cycle enzymopathies. Pediatr. Res. 14:519. • Review Questions Chemistry III 1. It is postulated that the underlying mechanism of ammonia neurotoxicity is a shift in reaction equilibrium caused by an elevated neuronal ammonia. What compounds are involved in this reaction and what is the physiologic effect? 2. An ammonium ion is presented to a hepatocyte. Trace the possible fate of this ion and the end products that may be formed. 3. A patient's plasma ammonia level is noted to rise during hyperalimentation. What is a possible explanation? 4. Male infants with ornithine transcarbamylase (OTC) deficiency usually die; females usually live, but with varying degrees of mental retardation. Explain why. 5. A physician indicates that an elevated plasma ammonia level reported by the laboratory is too high to be consistent with the patient's clinical condition. What sources of laboratory error should be considered? 6. Give three clinical indications for measuring plasma ammonia. 7. Evaluate the following alternate laboratory determinations for the diagnosis of hyperammonemia: a. Spinal fluid glutamine b. Plasma alanine c. Urinary orotic acid Self-assessment exam order form can be found on page 175. LABORATORY M E D I C I N E • VOL. 12, NO. 3, MARCH 1981 157