PHYSIOLOGICAL REVIEWS Vol. ‘70, No. 3, July 1990

Printed

in U.S.A.

Nitrogen ALFRED

Metabolism

J. MEIJER,

WOUTER

and Ornithine H. LAMERS,

AND

ROBERT

Cycle Function A. F. M. CHAMULEAU

E. C. Slater Institute jbr Biochemical Research, Department of Anatomy and Embryology, Experimental Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam,

and Laboratory of The Netherlands

I. Overview ............................................................................................. II. Control of Intracellular Protein Degradation ...................................................... A. Mechanisms of protein degradation ............................................................. B. Control of hepatic (autophagic) proteolysis ..................................................... C. Control of protein degradation in muscle ....................................................... D. Degradation of nonfibrillar and fibrillar muscle protein ........................................ III. Interorgan Amino Acid Fluxes ...................................................................... A. Feeding ........................................................................................... B. Fasting ........................................................................................... C. Diabetes and trauma ............................................................................ IV. Amino Acid Transport in Hepatocytes ............................................................. A. Amino acid transport systems ................................................................... B. Control of amino acid metabolism by amino acid transport across plasma membrane ........ V. Ornithine Cycle ...................................................................................... A. Introduction ...................................................................................... B. Substrates for urea synthesis .................................................................... C. Properties of individual enzymes involved in urea synthesis ................................... D. Comparison of kinetic parameters of ornithine cycle enzymes with concentrations of key metabolites of urea synthesis in vivo ....................................................... E. Control of ornithine cycle activity ............................................................... F. Urea synthesis and hepatic glutamine metabolism ............................................. VI. Amino Acid Metabolism and pH Homeostasis ..................................................... A. Urea synthesis and pH homeostasis ............................................................. B. Mechanisms responsible for inhibition of urea synthesis at low pH ........................... C. Interorgan glutamine flow in acidosis ........................................................... VII. Urea Synthesis and Development ................................................................... A. First appearance of ornithine cycle enzymes .................................................... B. Prenatal function of ornithine cycle ............................................................. C. Neonatal function of ornithine cycle ............................................................ VIII. Topographic Distribution of Ornithine Cycle Enzymes in Liver ................................... IX. Pathophysiology of Hyperammonemia ............................................................. A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Inborn errors of urea synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Secondary hyperammonemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Neurotoxicity of ammonia . . . ..*.................................................................

I. OVERVIEW

. During catabolism OIFamino aclas, large amounts OI NH,+ and HCO, (in addition to CO,) are formed: in humans, -1 mol of each per day is formed when the daily protein consumption is 100 g (14, 18). In mammals the major metabolic pathway responsible for the removal of these two products is the synthesis of nontoxic urea in the liver (Fig. 1). The activity of the ornithine cycle, which in this review is defined as the magnitude of the flux through l

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this pathway at a given constant intrahepatic ammonia’ concentration, must be carefully controlled for several reasons. Entry of ammonia into the systemic circulation must be prevented, because at plasma concentrations 140 PM the compound is toxic to the central nervous system. On the other hand, it is important that ammonia is not completely converted into urea, because ammo’ In this review we use the term ammonia to indicate the sum of NH, and NH& Whenever either NH, or NH: is meant, it is specifically mentioned.

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MEIJER,

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AND CHAMULEAU

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Mitochondrion H+?*--

Glutamate-

f

l AC’

Ornithine+

[Fumarate--1

2 - oxoglutarateAmino @WI2

N -Acdyl9lutamate”



b?

.I>

Orni!hine+

1

,,*

FIG. 1. The ornith ine cycle. [From H%u ssinger et al. (230). 1

PPi AMP

ATP

- 0

-.. Glutainate-

> GWimare-

I &id3 1 2-Oxoacids)

nia is also an essential metabolite in a number of vital metabolic processes, such as the synthesis of nonessential amino acids. Through some of these amino acids, ammonia is used for the synthesis of pyrimidines and purines. Thus it is important that the intrahepatic concentration of ammonia is kept within certain limits. The concentration of HCO, must also be kept constant: although a small amount of HCO; (2.5-5s of the total amount produced by amino acid catabolism) can leave the body via the urine, the remainder can only be eliminated via synthesis of urea (14, 18). As will be seen, urea synthesis (periportal) is essentially part of a device, which also includes hepatic glutamine synthetase (pericentral), that enables the liver to fulfill both criteria, i.e., to buffer the concentration of both ammonia and of HCO, and, connected with the latter anion, of H+. Switching disposal of nitrogen from urea to urinary NH,+ excretion, with glutamine as the nontoxic nitrogen carrier between extrarenal tissues and the kidneys, allows the body to retain HCO; in order to neutralize the excess plasma protons in acidosis (14, 18, 227, 461). To set the stage for a discussion of the various aspects of control of urea synthesis, this metabolic pathway must first be defined under in vivo conditions. Following Kacser and Burns (281), it is important to stress that control of flux through a metabolic pathway can only be studied in an appropriate way if the pathway is located between an initial substrate (or substrates) and a final product and if the concentrations of these are either constant or vary minimally such that pathway flux is unaffected. In vivo, the concentration of the final product, urea, is rather constant under normal conditions. Even when its concentration varies, little effect on the flux through the ornithine cycle is expected

‘*&Yl&ie

because of lack of feedback inhibition by urea. At very high concentrations only, such as found in uremia, there is some inhibition of argininosuccinate lyase (427). The substrate side of urea synthesis is less clear. Obviously, ammonia, bicarbonate, and aspartate are the direct substrates for the ornithine cycle. However, in vivo these compounds are not the beginning of the pathway of urea synthesis, since they are derived from the catabolism of amino acids that, in turn, are derived from breakdown of either body or dietary protein. Ultimately the pathway of urea synthesis starts with proteolysis and ends with urea as the final product. Although this notion seems trivial, its consequences are extremely important. As long as the ornithine cycle does not operate at full capacity (which is the case in vivo), moderate changes in activity of one of the enzymes involved in the synthesis of urea from ammonia, by changing its maximum rate ( Vmax)or its Michaelis constant (K,), will not affect the urea production rate unless such a change in the concentration of hepatic ammonia feeds back to hepatic amino acid degradation and to protein breakdown either in the liver or elsewhere in the body. In the liver, amino acid catabolism and proteolysis (in starvation) are rather insensitive to product inhibition by ammonia, at least in the physiological concentration range of 0.2-l mM in the liver (312,417,421). Only high concentrations of ammonia, >2 mM, inhibit hepatic proteolysis, because they increase the intralysosomal pH (562, 563). A moderate change in hepatic ammonia is also unlikely to affect muscle proteolysis directly, since normally ammonia does not escape the liver and blood ammonia concentrations remain very low. On the other hand, moderate changes in intrahepatic ammonia could result in changes in intrahepatic glutamate and aspartate, which affect hepatic autophagic proteolysis (see

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sect. II@. Likewise, a moderate change in hepatic ammonia, due to a relatively small change in activity of one of the ornithine cycle enzymes, could be transmitted to the muscle via changes in the plasma concentration of glutamine (after a change in hepatic glutamine synthesis), which may affect muscle proteolysis (see sect. I@). As yet, however, such a coupling between ornithine cycle activity and both liver and muscle proteolysis remains to be demonstrated (4a). In the fed state, when amino acids for urea synthesis are mainly derived from the diet, a relatively small change in ornithine cycle activity will not affect amino acid degradation because of the lack of feedback by ammonia. In the absence of feedback by ammonia, control of the ornithine cycle is essentially control of substrate (i.e., ammonia) concentration rather than control of flux (421, 670). Interestingly, in patients with a partial deficiency in one of the ornithine cycle enzymes the concentration of urea in plasma is usually quite normal (although urinary output may be less than normal) in contrast to that of ammonia, which is greatly enhanced (570). Only in experiments in vitro where the ammonia concentration can be kept constant, such as in the isolated perfused liver or in isolated hepatocytes, will activation or inhibition of one of the ornithine cycle enzymes be able to affect flux through the ornithine cycle (421). Having defined the metabolic pathway of urea synthesis, we now discuss its individual components: proteolysis, interorgan amino acid transport, transport of amino acids across the plasma membrane of the hepatocyte, and the ornithine cycle itself. We focus on more recent developments in these fields.

II.

CONTROL

OF INTRACELLULAR

PROTEIN

DEGRADATION

A. Mechanisms

of Protein

Degradation

Body proteins are mobilized in order to supply amino acids for various purposes: the synthesis of essential proteins and other nitrogen-containing compounds (purines, pyrimidines, creatine, porphyrins), precursors for the synthesis of glucose and ketone bodies, or oxidizable substrates for ATP production. Quantitatively, liver and muscle are the most important tissues in this respect. Nutrition and growth-induced changes in liver mass are primarily caused by changes in the rate of protein degradation rather than of protein synthesis (444, 558). Changes in skeletal muscle mass are caused by changes in’ both the rate of protein synthesis and degradation, although these do not always occur in opposite directions (192,432,433). The parallel increase in rates of protein synthesis and degradation in muscle during growth and during recovery after exercise has

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been ascribed to the need for extensive remodeling of this highly organized tissue (556, 650). Nutritional control of muscle protein turnover, in addition to that by certain amino acids (see sect. IIC), is mediated by hormones, such as insulin, corticosteroids, and thyroid hormone. Insulin increases protein synthesis and decreases protein degradation, corticosteroids have the opposite effect, and thyroid hormone stimulates both protein synthesis and degradation, with a relatively greater effect on the former than on the latter (432). Nutritional control of liver protein turnover, in addition to that by amino acids, mainly occurs through the actions of insulin and glucagon (444). Both intra- and extralysosomal pathways are responsible for the degradation of intracellular protein. The extralysosomal pathway is, at least in part, responsible for the turnover of short-lived and of abnormal proteins. It is ATP dependent and is not affected by the nutritional status (84,444). Various proteolytic systems, including the ubiquitin pathway, are responsible for extralysosomal protein breakdown (50). The intralysosomal pathway of intracellular protein degradation, the autophagic pathway, is mainly responsible for the degradation of long-lived protein. Two classes of autophagy are involved in the breakdown of these stable proteins. The first class is macroautophagy, a process that is responsible for the massive breakdown of intracellular protein when amino acid concentrations fall. For example, in rats and mice, ZO-30% loss of hepatic protein occurs after 24 h of starvation (261,593). In this process, portions of cytoplasm, which may even include whole organelles, are surrounded by a membrane that appears to originate from the endoplasmic reticulum (454). The autophagosome formed in this way then fuses with an existing lysosome and proteolysis proceeds. Flux through this pathway is also ATP dependent (256, 478) but in addition is acutely controlled by changes in concentration of a number of factors, including amino acids and hormones (see next section). In microautophagy, or basal autophagy, part of the cytosol is directly sequestered by the lysosomes by invagination of their membranes (382). This process is subject only to long-term regulation and decreases after l-2 days of starvation (443). Although it was thought that autophagic protein degradation is nonselective (240, 310), recent evidence suggests strongly that proteins can be specifically targeted to the lysosomes for degradation (83,335). The relative contribution to total proteolysis of the intra- and extralysosomal pathways of protein degradation can be estimated with inhibitors that specifically interfere with the lysosomal function, such as weak bases that cause increases in intralysosomal pH. In rat hepatocytes, for example, autophagy accounts for -70% of total proteolysis under amino acid-deprived conditions (562). This is in agreement with calculations showing that in vivo autophagic proteolysis contributes between 60 and 90% to overall proteolysis, depending on the nutritional conditions (239,298). In isolated skeletal muscle, the lysosomal pathway contributes 50% to over-

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all protein breakdown under amino acid-deprived conditions (164). It must be stressed that inhibitors of thiol proteases, such as leupeptin, chymostatin, and E-64, cannot be used to distinguish between intra- and extralysosomal pathways. Originally it was believed that these inhibitors only acted on lysosomal proteases, but this view is incorrect, since cytosolic thiol proteases are also inhibited (for discussion see Refs. 164, 687). It has been known for a long time that amino acids and insulin are able to suppress autophagic proteolysis both in liver and muscle, whereas glucagon accelerates this process in liver, and that the glucagon-to-insulin ratio is the relevant parameter (470). However, surprisingly little is known about the mechanism of these hormonal effects (202, 444, 555). The changes in flux through the macroautophagic pathway in response to these factors can be extremely rapid, with the half-life of the autophagosomes being 8 min (444,555). It is certain that major control by amino acids and hormones is exerted at the first step in the pathway of macroautophagy, the formation of autophagosomes (555,559,560), although some additional effect on the fusion of autophagosomes and existing lysosomes cannot be excluded (559, 560). B. Control of Hepatic (Autophagic) Proteolysis

Because in liver the increased rate of protein breakdown induced by starvation can be totally accounted for by increased autophagic proteolysis, we first discuss control of this process by amino acids. Despite numerous attempts to get information on the nature of the amino acids interacting with the macroautophagic pathway, it is still not known which amino acids are responsible for the inhibition of this process. Several amino acids have been mentioned as being particularly effective: among these are tryptophan (201, 256, 598), phenylalanine (256), asparagine (561), leucine (482, 561), and methionine (256, 598). According to Mortimore and colleagues (444, 445, 480), there is a regulatory group of amino acids containing leucine, tyrosine, glutamine, proline, histidine, tryptophan, and methionine that, together with the synergistic coregulator alanine (which does not inhibit proteolysis on its own), is responsible for the antiproteolytic response. A problem is that in studies with isolated hepatocytes, unphysiologically high concentrations of amino acids are required for significant inhibition of autophagic proteolysis (256, 561, 598) in contrast to the flowthrough perfused liver, a system in which inhibition is obtained at physiological (portal) concentrations of amino acids (444, 480, 482, 555). Recently, however, it was shown that autophagic proteolysis in perifused hepatocytes is as sensitive to inhibition by amino acids as the perfused liver (341): low concentrations of leucine act synergistically with either alanine, asparagine, glutamine, or proline in their effect on proteolysis. These observations have been confirmed with the nerfused

AND

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liver (445). Because inhibition by alanine, but not that by proline, asparagine, or glutamine, was sensitive to the transaminase inhibitor aminooxyacetate, it was suspected that in the case of alanine its metabolism is required for its effect on proteolysis (67). Indeed, inhibition of proteolysis under steady-state conditions by such combinations of amino acids is correlated with increases in intracellular leucine (which is not catabolized in rat liver) in combination with increases in intracellular glutamate or aspartate (67, 341). Accumulation of ammonia does not seem to be involved, since the inhibitory effects were seen in the presence of ornithine, which decreases the level of ammonia (67,341). Inhibition of proteolysis is not observed in hepatocytes perifused with K+-depleted medium: intracellular glutamate and aspartate levels are strongly decreased because of efflux of these amino acids from the cells (341). It is not clear at present whether intracellular glutamate or aspartate (in combination with leucine) are directly responsible for inhibition of proteolysis or whether it is a metabolite, derived from these amino acids, that is responsible for these effects. Stimulation of hepatic protein degradation by glucagon only occurs at low amino acid concentrations (555). In the perfused liver, stimulation by glucagon can be mimicked by omission of alanine from a complete mixture of amino acids (480). It is tempting to speculate that decreases in intrahepatic glutamate under both conditions are responsible for the acceleration in protein degradation, especially because glucagon is known to decrease intramitochondrial 2-oxoglutarate by activation of 2-oxoglutarate dehydrogenase (603, 604). C. Control of Protein Degradation in Muscle

As in liver proteolysis, the intralysosomal pathway of muscle proteolysis is stimulated on amino acid deprivation and is inhibited by insulin (63,163). Inhibition of proteolysis by insulin is probably unrelated to stimulated glucose and amino acid transport, because inhibition is also observed when transport is not stimulated (163). The extralysosomal pathway of proteolysis is stimulated by Ca2+ and is responsible for increased protein breakdown in various diseased states (fever, sepsis, trauma) (for review see Ref. 149). Distinction between the two proteolytic pathways can be made using weak bases that specifically affect lysosomal function (164, 355, 687). In vitro experiments have shown that prostaglandin E, is able to stimulate the Ca2+-dependent component of the extralysosomal proteolytic pathway2 (188,521,522). This suggested that inhibitors of prostaglandin synthesis, such as aspirin and indomethacin, may be useful as therapeutic agents to suppress the excessive loss of body protein in patients with trauma, sepsis, or injury. However, the results ob-

than

2 The earlier view the extralysosomal

that Ca2+ stimulated pathway (188,521,522)

the lysosomal rather was later abandoned

(149.164).

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tained after treatment of rats with such agents do not support this view. Thus burn-induced muscle protein breakdown was not affected by several cyclooxygenase inhibitors (407,458) despite the fact that prostaglandin E, levels were greatly reduced (407). Similar data were reported for skeletal muscle in septic rats (219) and in infected guinea pigs (595). These apparent conflicting data may be reconciled by assuming that prostaglandin E, is important in the initiation of enhanced proteolysis but not in its continuation (149,407,458). Experiments showing that monocyte supernatants containing interleukin 1 were able to increase skeletal muscle proteolysis in vitro, a phenomenon that was accompanied by increases in prostaglandin E, and that could be prevented in part by indomethacin, led to the suggestion that interleukin 1 was responsible for the stimulation of muscle protein breakdown (28,127). However, recent studies with recombinant interleukin 1 failed to reproduce these results (149, 437), indicating that a contaminant in the original preparation of interleukin 1, but not interleukin 1 itself, must have been responsible for these effects. On the same grounds, tumor necrosis factor (45), another product of mononuclear phagocytes, has been eliminated as a possible candidate directly responsible for enhanced muscle proteolysis in trauma and inflammation (437). For a more detailed discussion of these issues, the reader is referred to a recent review of Hasselgren et al. (218). Among the various amino acids it is only the branched-chain amino acids, particularly leucine, that are effective inhibitors of muscle proteolysis, both in skeletal muscle (63, 163, 343) and in heart (87). Unlike liver, transamination of leucine to cw-ketoisocaproate is required for this effect in muscle (434, 624). Leucine is also known to stimulate protein synthesis in vitro (63). However, because in many conditions with increased muscle proteolysis intramuscular leutine is increased rather than decreased (l&429,649), it is unlikely that, in vivo, leucine alone is involved in the control of muscle protein turnover. Indeed, branchedchain amino acids infused into postabsorptive rats failed to stimulate muscle protein synthesis on their own (412). However, in their presence the sensitivity of protein synthesis to stimulation by low concentrations of insulin is greatly increased (173). Because other oxidizable substrates in addition to leucine also inhibit proteolysis (87, 466, 518, 582), it is possible that the cytosolic NAD redox state may be involved in the control of muscle proteolysis. A low NADH/NAD+ ratio is associated with a high proteolytic rate (8, 196, 623, 625, 626). Also, it has been suggested that glutathione disulfide links act as a signal for initiation of proteolysis (626). Although attractive as a unifying hypothesis, it is now clear that this correlation certainly does not hold under all conditions (150,434). This may be due to other factors also influencing muscle proteolysis (151). Another hypothesis concerning the control of muscle protein turnover has been proposed by Rennie et al. (515). According to them, muscle protein synthesis and

presumably proteolysis (411, 583) are under the control of the size of the intramuscular glutamine pool. Under a variety of conditions, both in vitro and in vivo, there is a nearly linear relationship between the magnitude of the intramuscular glutamine concentration and the protein synthetic rate (268, 410, 515). In their view, glutamine transport out of the muscle cells plays a crucial role. Muscle glutamine efflux is mediated by a Na+-dependent carrier, which closely resembles system N present in hepatocytes (259; see sect. IvA). Conditions leading to increases in intracellular Na+, such as injury, sepsis, and chronic disease, lead to a rapid loss of muscle glutamine and hence to a net increase in muscle protein degradation (see Ref. 515). Leucine may interfere with muscle proteolysis, because it noncompetitively inhibits glutamine efflux (515). Consistent with the hypothesis of Rennie and colleagues is a recent report showing that parenteral administration of the dipeptide alanylglutamine in surgical patients partly suppressed the negative nitrogen balance and prevented loss of muscular glutamine (607). Although the correlation between intracellular glutamine and muscle protein turnover holds under many conditions, in starvation intramuscular glutamine tends to increase rather than decrease (627). D. Degradation of Nonfibrillar hhmle Protein

and Fibrillar

It must be pointed out that until recently no distinction has been made with regard to degradation of nonfibrillar and myofibrillar protein in muscle. Turnover of myofibrillar proteins, which comprise -60% of total skeletal muscle protein, is much slower than that of nonmyofibrillar proteins (31). It has now been shown that degradation of myofibrillar proteins, which can be followed by the production of 3=methylhistidine, occurs by an extralysosomal pathway (355) and is not responsive to regulation by amino acids or insulin (355, 411) nor is it responsive to changes in the intracellular concentration of Ca2+ (195). Because of the fact that turnover of myofibrillar proteins is much slower than that of nonfibrillar proteins, changes in total cell proteolysis, usually measured as tyrosine release, are unlikely to reflect myofibrillar proteolysis. Likewise, changes in myofibrillar proteolysis can occur in situations where total proteolysis does not reveal a major change (195). III.

INTERORGAN

AMINO

ACID

FLUXES

Excellent reviews on the flow of amino acids between the various tissues have appeared in the past (66, 85,155,238,365, 668). Because it is not feasible to cover this immense field of research in full in this review, we only summarize the major features of the traffic of amino acids among the various tissues. A. Feeding

After the consumption of a protein-containing meal, free amino acids produced by the actions of pro-

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teases in the alimentary tract and peptidases in the intestinal mucosal cells (384) enter the circulation. In the mucosal cells conversion of glutamine, glutamate, and aspartate to alanine, citrulline, ornithine, proline, and ammonia occurs (265, 677-680). There is net uptake by the liver of significant fractions of nearly all amino acids except for the branchedchain amino acids (155,265,519), with the highest fractional uptake, ~40-50 % , for alanine, asparagine, glytine, serine, and histidine (513). In this way the liver contributes to buffering the effect of protein ingestion on the systemic concentration of most amino acids. The branched-chain amino acids, which undergo little degradation in the liver because of low transaminase activity (262), are transported to the periphery so that after a meal these tissues are confronted with an amino acid mixture that is enriched in leucine, isoleucine, and valine. In muscle, which is the major site in the body where they are metabolized, the branched-chain amino acids are used for protein synthesis or they are oxidized (2, 155, 349). During oxidation in rat muscle (349), but not in human muscle (141), part of the branched-chain keto acids formed may leave the muscle and be further oxidized in liver. The branched-chain amino acids are also oxidized in adipose tissue (189) to the extent of about one-sixth of the total amount oxidized in skeletal muscle (238). Brain may be another consumer of branchedchain amino acids (155), especially in diabetes (58). The main end products of muscle amino acid degradation are alanine and glutamine (155). Alanine produced by muscle is consumed by the liver, whereas glutamine derived from muscle is used by the intestine, by the liver, and, especially in acidosis, by the kidney (553, 554). In addition, glutamine is an important respiratory substrate for cells of the immune system (452).

B. Fasting

In the postabsorptive state, proteolysis causes a release of most amino acids from the muscle, including branched-chain amino acids; however, as in the fed state, alanine and glutamine still account for >50% of the total a-amino nitrogen released by the muscle (155, 238, 265). This percentage is completely out of proportion to their abundance in muscle protein. There has been controversy with regard to the origin of the carbon skeleton of alanine. Although it has been claimed that the carbon skeleton is derived from glucose (75), it is evident that as long as there is a net contribution of alanine to body glucose (to be utilized by brain) through hepatic gluconeogenesis, its carbon is derived from other amino acids produced by muscle proteolysis and in particular from valine and isoleucine (172,585). However, when alanine is part of the glucosealanine cycle (155), its carbon must be ultimately derived from glucose (76,172,585). Thus, in exercise, high glycolytic flux is accompanied by a greatly increased output of alanine from the muscle (155).

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With regard to the source of the carbon for glutamine synthesis in muscle, it is likely that sufficient glutamate can be formed during intracellular amino acid catabolism (76,172,529). Plasma glutamate also contributes (377). It has been proposed that in starvation increases in plasma concentrations of branched-chain keto acids promote hepatic glutamate efflux (224). If this mechanism is correct, it implies direct cooperation of muscle and liver in the synthesis of muscle glutamine. Also of interest is that the rate of transport of glutamate across the plasma membrane of the muscle cell increases with decreasing pH (516): this effect may contribute to the increase in muscle glutamine production in acidosis (554; see sect. VIC) where glutamate is also derived, at least in part, from liver (662). Free ammonia required for conversion of glutamate to glutamine may be derived from the following sources: from glutamate dehydrogenase, although activity of this enzyme is low in muscle (667); via the purine nucleotide cycle, which catalyzes net conversion of aspartate to ammonia and fumarate (356); by direct production from certain amino acids, e.g., glycine (238, 529); and from the circulation when plasma ammonia concentrations are increased (132,246, 552). The major sites of glutamine uptake are kidney and intestine but not the liver from which there is a net output of glutamine in fasting (265). In kidney, glutamine may be used for ammoniagenesis (614, 683), whereas the intestine releases glutamine nitrogen in the form of alanine, ammonia, ornithine, citrulline, and proline (679). In the postabsorptive state, alanine accounts for ~50% of total hepatic amino acid uptake (155, 265). Other gluconeogenic amino acids are also consumed by the liver, with major contributions from glycine and serine (155,265): glycine being derived from serine in muscle and intestine, whereas the kidney converts some glycine to serine (58, 155, 265). Production of citrulline by the intestinal mucosa is important for anapleurosis of the ornithine cycle in liver under fasting conditions. However, citrulline cannot be used by the liver directly, because the plasma membrane of the hepatocyte is not readily permeable for plasma citrulline (681). Instead, citrulline is converted to arginine by the kidney, and part of the arginine formed is taken up by the liver (523, 681). In cats, intestinal conversion of glutamine to citrulline does not occur, and arginine becomes an essential amino acid: adminstration after an overnight fast of a single meal containing all amino acids except arginine leads within a few hours to hyperammonemia and to all the clinical symptoms of ammonia toxicity (442). In short-term starvation, as in the postabsorptive state, alanine is also the major gluconeogenic substrate; under these conditions, plasma concentrations of branched-chain amino acids are increased, presumably due to the fall in insulin (155). This is also observed in diabetes (see next section). In long-term starvation, gluconeogenesis (and also ureogenesis) decreases because of a decrease in muscle proteolssis that mav be related

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tially purified (237) and has been reconstituted in liposomes (485). System ASC, which is also Na+ dependent, also mediates transport of alanine, serine, glycine, and threonine but, in contrast to system A, mediates cysteine transport as well. Discrimination between the two systems can be achieved by the use of a)-aminoisobutyric acid (AIB) and cu-(methylamino)isobutyric acid (MeAIB); these nonmetabolizable alanine analogues interfere with alanine transport via system A but not with that via system ASC. A separate system (Gly) exists for exclusive transport of glycine and sarcosine (86,546). Glutamine, histidine, and asparagine are transported via a separate Na+-dependent transport system (called N). Because this system is not inhibited by AIB and MeAIB and because glutamine, histidine, and asparagine do not interfere with cysteine transport (304), it can be distinguished from systems A and ASC. Efflux of glutamine from pericentral hepatocytes, after its synthesis by glutamine synthetase, presumably occurs via a Na+-independent mechanism (176). Transport of the branched-chain and aromatic amino acids occurs via system L, which does not require Na+. This system is not concentrative but allows equilibration of the concentrations of these amino acids on both sides of the plasma membrane (399, 542, 564) and can be inhibited by Z-aminobicyclo-2,&l-heptane-Zcarboxylic acid (BCH). In Chang liver cells, intracellular pH changes monitored by pH indicators after plasma membrane leucine transport indicate that transport of leucine is coupled to H+ cotransport (435). On the other hand, it has been observed that transport of leucine in rat hepatocytes is not very much affected by changes in the extracellular pH (304). Transport of the acidic amino acids, glutamate and aspartate, occurs via a Na+-dependent, high-affinity, low-capacity system (Xio) (175,178,372,578). This system is largely confined to the small population of pericentral hepatocytes containing glutamine synthetase;

to the rise in plasma ketone bodies under these conditions (155, 196, 582, 585). C Diabetes and Trauma

The situation in diabetes (58, 155) and in trauma (365,668) strongly resembles that in short-term starvation. In both conditions there is an increased output of alanine and glutamine from the muscle, and arterial plasma concentrations of branched-chain amino acids rise while plasma alanine concentrations fall because of accelerated gluconeogenesis. In diabetes, plasma branched-chain amino acid concentrations rise. The low level of insulin diminishes uptake of these amino acids by the muscle, and net muscle protein breakdown is increased. Likewise, increased liver proteolysis may also contribute to the rise in plasma branched-chain amino acids (48). In trauma, even though insulin is high, there is insulin resistance because of the high concentrations of glucagon, catecholamines, and cortisol so that protein catabolism is greatly increased (180, 365, 668). IV.

AMINO

ACID

TRANSPORT

IN HEPATOCYTES

A. Amino Acid Transport Systems

Transport of amino acids across the plasma membrane of the hepatocyte is mediated by various transport systems with overlapping specificities. Some of these systems are Na+ dependent and allow transport of their amino acid substrates against a concentration gradient. The following systems, most of which also occur in other mammalian cells (85,99,209,300-302,394, 572), have been characterized in hepatocytes (Table 1). System A mediates transport of alanine, glycine, serine, proline, and, to a lesser extent, that of asparagine and methionine (301); it is Na+ dependent and electrogenic (316,580,637). The carrier system has been par-

TABLE

707

UREA SYNTHESIS

1990

1. Amino acid transport in hepatocytes Transport A

Substrate

Ala GlY

Na+ dependence Effect of H+ Hormonal regulation Adaptive regulation

Ser Yro Met AIB MeAIB (Thr?) (A@ (His) Yes Inhibits Yes Yes

ASC

Ala Ser

GUY GlY

Sarc

CYS GUY

Systems N

Gln His Asn

Leu Ileu Val

xi,

Glu

Y+

ASP

A% Lys? Orn?

Yes Inhibits Yes ?

No ? ? ?

TYr

Thr (Met?)

Yes None No No

L

Phe TrP

Yes None ? No?

Yes Inhibits Yes Yes

No None ? ?

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708

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LAMERS,

after induction of pericentral liver cell necrosis by Ccl,, the glutamate transport system is lost (222, 619). Kinetic data on glutamate transport in hepatocyte cultures enriched with either periportal or pericentral hepatocytes have supported this conclusion (176). It has been proposed that this system is involved in the delivery of glutamate for synthesis of glutamine (110, 222, 619). Inconsistent with this proposal is the fact that activity of the transport system decreases with decreasing pH (Vi%), whereas hepatic synthesis of glutamine sometimes increases in acidosis (see sect. VIBS?). Moreover the capacity of the glutamate transport system is at least one order of magnitude below the Vmaxof glutamine synthetase. It is doubtful, therefore, that synthesis of glutamine in pericentral cells can occur at the expense of extracellular glutamate only except when flux through glutamine synthetase is low (see sect. vF). During amino acid catabolism in periportal hepatocytes, glutamate efflux can occur in a Na+-independent manner; this process is stimulated by certain oxo-acids, especially by those derived from leucine, isoleucine, and methionine (224). This system is presumably identical to that described by Gebhardt and Mecke (178) and by Ballatori et al. (25). The same system is possibly also involved in the export of glutamate derived from glutamine (662). Transport of cationic amino acids [reviewed by White (671)] is mediated by system Y+, which is not dependent on Na+. The system has low activity in hepatocytes but is more active in hepatoma cell lines (672).

B. Control of Amino Acid Metabolism by Amino Acid Transport Across Plasma Membrane

I. Short-term regulation

There is now considerable evidence that amino acid transport systems can exert significant control on the rate at which amino acids are metabolized. Table 2 gives a compilation of kinetic data on the transport of naturally occurring amino acids. Data on alanine and glutamine transport are most abundant, because these amino acids are physiologically important in hepatic amino acid metabolism. In general, there is a remarkable agreement in the results among various laboratories. Kinetic properties of the various transport systems (Table 2) d emonstrate that at physiological portal vein amino acid concentrations [-1 mM or lower (265,513)], the systems operate at or below the K, for their substrates. Therefore amino acid fluxes across the plasma membrane of hepatocytes are very sensitive to changes in the amino acid concentration in the portal vein. Furthermore, from the kinetic properties it can be calculated that under many conditions in vivo the rate of amino acid transport controls hepatic amino acid metab-

AND CHAMULEAU

Volume 70

2. Some kinetic properties of amino acid transport in rat hepatocytes

TABLE

Amino

Acid

Alanine

Serine Glutamine

Leucine Valine Tryptophan Phenylalanine Tyrosine Glutamate

K mmh;

4.5 2.5 4 4.3 3.2 4 3.7 1.1 1.25 1 4.5 3.8 5.5 43 1.65 1.60 1.95 0.29 0.005

V max9 pm01 min-’ g dry wt-’ l

Nutritional Condition

l

16 27 19 13 29 15 13 22 5 6 17 16 11 15 3 3 2.5 0.7

Starved Starved Starved Fed Starved Starved Starved Fed? Fed Fed Starved Starved Fed Starved Fed Fed Fed Starved Fed

Reference

276 138 579 317 317 618 276 304 145 177 276 618 399 564 541 541 541 619 578

Vmaxvalues are obtained at 37°C. Because Vmaxvalues reported by various groups are not always expressed in the same units, all values were calculated back to pmol min-’ g dry wt-l for direct comparison. In these calculations the following conversion factors were used: I g dry wt = 3.7 g wet wt = 2.7 ml intracellular H,O = 0.7 g protein. In many cases (except for tryptophan, tyrosine, and phenylalanine), no indication was given as to which amino acid transport system was responsible for the reported kinetic properties. In the case of alanine, for example, transport kinetics are presumably a mixture of properties of both systems A and ASC. For detailed information concerning kinetic constants of Na+-dependent and Na+-independent transport of glutamate and glutamine in hepatocyte cultures enriched with either periportal or pericentral hepatocytes, see Ref. 176. l

l

olism

(147). This is supported by a comparison of the Vmaxvalues of the transport of alanine, glutamine, and

serine (which are major gluconeogenic substrates) with those for activity of argininosuccinate synthase, argininosuccinate lyase, and carbamoyl-phosphate synthase (which are enzymes of the ornithine cycle with the lowest activities). Both transport and enzyme Vmaxvalues are of the same order of magnitude (see Table 3; see sect. vD). Transport of alanine is of particular interest, because, among the amino acids, it contributes most to gluconeogenesis and ureogenesis (see sect. III). Essential for our understanding of the control of hepatic alanine catabolism is the fact that under steady-state conditions the flux of alanine across the plasma membrane of the hepatocyte does not respond to changes in the intracellular concentration of alanine (205,317). This finding appears to be in contradiction to the phenomenon of transinhibition (inhibition of MeAIB influx by intracellular MeAIB in this case) that has been reported for transport of MeAIB, the nonmetabolizable substrate of transport system A in hepatoma cells (673). The reason for this discrepancs has never been exnlained but ner-

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July l&w

UREA

SYNTHESIS

haps resides in the fact that transport of alanine not only proceeds via system A but also via system AX (618); the latter system may be subject to transstimulation (accelerative countertransport) (572). This insensitivity of alanine transport to intracellular alanine implies, by definition (281), that at a particular extracellular concentration of alanine, alanine catabolism is fully controlled by its rate of transport across the plasma membrane (i.e., has a flux control coefficient of I) (205, 206). Any change in the rate of intracellular alanine metabolism will only result in a change in the intracellular concentration of alanine; this, however, does not feed back to influence the flux of alanine across the plasma membrane. In hepatocytes incubated under steady-state conditions, either with ala-= nine alone or with a complete mixture of all amino acids, intracellular alanine is below the extracellular alanine concentration unless intracellular alanine metabolism is inhibited (51,205). In the latter case, the intracellular concentration of alanine greatly exceeds its extracellular concentration. These observations indicate that alanine transport is relatively slow in comparison with the rate of its intracellular metabolism. Other evidence in support of the view that alanine transport controls hepatic alanine metabolism has also been given (147, 317,404). Transport of glutamate, as measured in the entire hepatocyte population, is at least one order of magnitude below the Vmaxof glutamine synthetase in pericentral cells (578) so that there is no doubt that flux through glutamine synthetase at the expense of extracellular glutamate is controlled by the rate of glutamate transport. Transport of aromatic amino acids also exerts considerable control on their intracellular metabolism, especially when the initial enzymes involved in the degradation of phenylalanine, tyrosine, and tryptophan are maximally induced by dexamethasone (542). Short-term regulation of amino acid transport may be exerted by changes in the pH (systems A and N), by competition with amino acids for the same carrier system, or by hormones such as glucagon. Short-term stimulation of transport of alanine by glucagon (137,373) is likely to be due to membrane hyperpolarization by the hormone (446,447). Transport of alanine is inhibited at low pH (51,304, 336, 566, 577, 580), which is important in acidosis when urea synthesis is inhibited (see sect. VIBI). A similar inhibition by low pH has been reported for transport of glutamine via system N (145,304,340). An example of regulation by substrate competition comes from studies with the isolated perfused rat liver. In these studies, physiological concentrations of histidine (an amino acid that is also a substrate for system N) not only inhibits glutamine catabolism in periportal hepatocytes by competing for transport but also causes accumulation of glutamine in glutamine synthetasecontaining pericentral hepatocytes by inhibiting glutamine efflux from these cells (233).

709

2. Hormonal control and long-term regulation of plasma membrane amino acid transport

In addition to short-term regulation, there is also long-term regulation of the activity of amino acid transport systems. This involves a change in the number of carrier molecules in response to the nutritional or hormonal status of the animal (486, 526, 543, 546). In general, the effects on system A (alanine transport) are much larger than those on system N (glutamine transport). In starvation, activity of system A is increased in comparison to the fed state (154,236,303,486), but this is not the case for system N (236). Hepatic transport of alanine is increased in streptozotocin-diabetic animals (544) and in rats fed a high-protein diet (146,147). Glytine transport also adapts to high-protein feeding. Three hours after feeding a high-protein diet to rats, hepatic extraction of glycine was increased lo-fold, yet the concentration of glycine in the portal vein was similar to that after feeding a protein-free diet (265). Several hormones, including glucagon, insulin, glucocorticoids, and thyroid hormone, have been shown to increase the activity of transport system A (153, 209, 301, 304, 572). Although it was originally thought that system N was insensitive to hormones (304), later studies showed that this was incorrect. Thus, in cultured hepatocytes, both insulin and glucagon are able to stimulate glutamine transport, an effect that is synergistic to that exerted by glucocorticoids (177). Also, in diabetic rats, activity of system N is increased (30). Another system that is hormonally controlled is the Na+-dependent transport of glutamate, the capacity of which is increased by glucocorticoids (178). This may account for the fact that hepatic glutamate uptake is increased by high-protein diets (513). Activity of systems A and N can also be controlled by a phenomenon that is known as adaptive regulation: when hepatocytes are cultured in a medium devoid of amino acids, there is an increase in the activity of the two transport systems (295) that is dependent on the synthesis of a glycoprotein in the plasma membrane of the hepatocyte (29,301,304). However, adaptive stimulation of system A is much stronger than that of system N (304). Activity of system A, after being induced either by hormones or by adaptive regulation, can be decreased back to the unstimulated activity by repression. When hepatocytes with elevated system A activity are cultured in the presence of amino acids, system A activity falls back to basal values in the course of several hours, with a half-life of 1.5 h. No such decay is observed in the absence of added amino acids (148,214). Addition of single amino acids, but in particular’of those that are substrates for system A, is sufficient to cause repression of system A (52, 148, 301). Because this amino aciddependent repression is prevented by inhibitors of RNA and protein synthesis, it has been proposed that the syn-

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thesis of a transport-inactivating protein is responsible for the decline in activity of system A (214). V.

ORNITHINE

CYCLE

A. Introduction

Since the discovery of the ornithine cycle by Krebs and Henseleit (314) in 1932, many studies on the regulation of this complex pathway have been performed. Severa1 reviews on this topic have appeared in the past (57, 6la, 96, 203, 230, 312, 420, 484, 505, 590, 597, 653, 654). Historical surveys of the discovery of the ornithine cycle can be found in References 158 and 311. The liver is the only organ in which urea formation occurs from ammonia. Although some of the ornithine cycle enzymes also occur in extrahepatic tissues, none of these tissues is capable of synthesizing urea from ammonia to a significant extent (505). The presence of N-acetylglutamate synthase (636), carbamoyl-phosphate synthase, and ornithine carbamoyltransferase in the intestinal mucosa allows this tissue to produce citrulline from glutamine (681; see sect. IIIA). The presence of argininosuccinate synthase and argininosuccinate lyase in the kidney allows production of arginine from citrulline (see sect. IIIB). The function of arginase, which is present in many extrahepatic tissues (505), is presumably to produce ornithine for polyamine synthesis (610). A single gene codes for each of the ornithine cycle enzymes in the various tissues, with the exception of arginase in which more than one locus may be involved (266). B. Substrates

for Urea Synthesis

As was seen in section III, the form in which nitrogen enters the liver is dependent on the nutritional and hormonal state of the animal. In the fed state, most amino acids reaching the liver, except for the branched-chain amino acids, can serve as precursors for ureogenesis. Free ammonia, derived from metabolism of glutamine in the intestinal mucosa and hepatocytes and from bacterial urease in the gut, also contributes to urea synthesis. About 25% of hepatic urea synthesis may be recycled via bacterial urease (653,684). On the basis of isotopic measurements in the adult anesthetized rat, the following estimates for the contribution of the various sources of ammonia to urea synthesis in the fed state have been made: portal vein ammonia, 33% [about the same as in humans (653)]; portal vein glutamine (amide), 6-13%; mitochondrial glutamate via glutamate dehydrogenase (glutamate derived from, e.g., alanine, glutamine, proline, histidine), 20% ; and other sources, including hepatic artery ammonia and glutamine and many enzymes in liver capable of generating ammonia directly (e.g., from asparagine, threonine, glycine, serine), 33-40% (109). According to

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these data, flux through glutamate dehydrogenase is relatively small, and activity of glutamate dehydrogenase is high enough to maintain near-equilibrium of the reactants (109,675); net production of ammonia via glutamate dehydrogenase requires continuous removal of 2-oxoglutarate from the equilibrium, because this metabolite strongly inhibits glutamate dehydrogenase in the direction of ammonia production (334, 657). Although alanine is important as substrate for urea synthesis (see sect. III), in vivo little alanine is deaminated; instead, most alanine nitrogen is metabolized via aspartate (see Ref. 676). The role of glutamate dehydrogenase in the production of ammonia for urea synthesis (54) has been questioned by McGivan and Chappell (401): they proposed that ammonia is produced from aspartate in the purine nucleotide cycle and that the main function of glutamate dehydrogenase is to synthesize glutamate. However, kinetic data obtained with [15N]alanine in hepatocytes revealed that the rate of incorporation of 15N into urea was B-fold higher than its rate of incorporation in the six-amino group of adenine nucleotides (313). Other data have supported this conclusion (108, 524). In catabolic states such as starvation, diabetes, and trauma, alanine is the major substrate for urea synthesis (sect. III, B and c). However, because of glutamine cycling, glutamine is also an important substrate for urea synthesis under these conditions, even though there may be no net uptake of glutamine by the liver (220; see sect. VF).

C. Properties of Individual in Urea Synthesis

Enzymes

Involved

The kinetic properties of the individual enzymes involved in urea synthesis have been extensively reviewed elsewhere (266,420,505,654). We restrict ourselves to a brief summary. Table 3 lists the K, and Vmax values for the enzymes of rat liver, since in vivo concentrations of ornithine cycle intermediates, to which the kinetic parameters must be related, are only available for this organ. In vivo concentrations are also given in Table 3. Kinetic data for the enzymes of human liver are, in general, quite similar to those of the rat (420, 654). The K, values in Table 3 were determined in situ or in experiments carried out with permeabilized mitochondria, intact mitochondria, or hepatocytes. These methods are described by Htiussingerto Table et al. (230) and in the references cited in the legend 3 .

1. Carbamoyl-phosphate synthase (ammonia)

Production of car bamoyl phosphate from ammonia and bicarbonate, the first step in urea synth esis from ammonia, is catalyzed by carbamoyl-phosphate synthase in the mitochondria

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July

Itwo

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711

3. Kinetic properties of the enzymes of the ornithine cycle in rat liver and concentration of ornithine cycle intermediates in rat liver in vivo

TABLE

Enzyme

Carbamoyl-phosphate synthase (ammonia)

Ornithine carbamoyltransferase Argininosuccinate synthase Argininosuccinate lyase Arginase N-acetylglutamate synthase

Reactant

or Activator

NH,+ a HCO, MgATP me+ N-acetylglutamate (free)b N-acetylglutamate (free + bound)” Ornithine Carbamoyl phosphate Citrulline Aspartate MgATP Argininosuccinate Arginine Glutamate Ace tyl CoA Arginine

Km,

1-2 4-5 0.5-3 0.17-Z 0.04-0.1

mM

(0.6) (2) (1.2) (0.15) 0.04 (>0.2) 0.02 0.15 0.04-0.13 (>0.03) (bovine liver) 3.5 (pH 7.5) (>0.06) 3 0.7 0.01 (0.05)

pmol

l

min-l

V maxf g dry l

21

wt-l

Concentration Metabolite,

of mM

0.2-0.5d 25” lo-13 (mitochondrial) 0.4-Z (mitochondrial)

799

7.4 13.3 5,143 0.22

0.4-l .2 lO PM (290) so that variations in mitochondrial arginine do lead to changes in enzyme activity. Furthermore, the extent of activation of N-acetylglutamate synthase by saturating concentrations of arginine increases with the time after feeding, reaching a six- to ninefold increase in Vmaxafter 9 h (289, 290). D. Comparison of Kinetic Parameters of Ornithine Cycle Enzymes With Concentrations of Key Metabolites of Urea Synthesis In Vivo

In the presence of excess substrate, maximal rates of urea synthesis are limited by the enzymes with the

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lowest Vmaxvalues, namely argininosuccinate synthase and argininosuccinate lyase (Table 3). Thus, in perfused liver or isolated hepatocytes exposed to saturating concentrations of ammonia, ornithine, and lactate (to supply oxaloacetate for aspartate formation), citrulline and argininosuccinate will accumulate (56, 419, 421, 533). This can also occur in vivo under extreme conditions: substantial intrahepatic accumulation of citrulline and aspartate were observed in rats subjected to a sudden transition from a low- to a high-protein diet (532) and in rats injected with large doses of a mixture of 20 amino acids (608). From the data of Table 3 it can be concluded that in vivo most enzymes involved in urea synthesis operate at or below the K, value for their substrates. This is in good agreement with other calculations showing that in vivo flux through the ornithine cycle is 2050% of its maximal capacity (108, 204). Apparently, the overcapacity of the ornithine cycle is not very high. In response to a high-protein diet, however, the liver can increase its ureogenic capacity by extra synthesis of the ornithine cycle enzymes, although the adaptation requires time (see sect. vEI). Exceptions to the rule that ureogenic enzymes work at or below the K, for their substrates are the saturation of carbamoyl-phosphate synthase and of argininosuccinate synthase with ATP and the saturation of argininosuccinate synthase with aspartate. Saturation with ATP suggests that urea synthesis is not controlled by ATP production and that competition with other ATP-utilizing processes presumably does not occur. However, because ADP inhibits carbamoyl-phosphate synthase, definite conclusions cannot be drawn (see Ref. 420). Production of aspartate for urea synthesis mainly occurs in the mitochondria. Aspartate leaves the mitochondria in exchange for glutamate via the glutamateaspartate translocator (419; Fig. 1). Stoichiometric formation of carbamoyl phosphate and aspartate for urea synthesis is achieved by the fact that aspartate aminotransferase is close to equilibrium (108, 675) so that removal of aspartate by its interaction with citrulline to form argininosuccinate is automatically followed by its replacement (312). The question of whether or not carbamoyl-phosphate synthase is controlled by changes in the concentrations of N-acetylglutamate and HCO, is discussed in sections vE3 and v1B4, respectively.

E. Control of Ornithine Cycle Activity

Control of metabolic pathways can either term, involving the synthesis and degradation zyme molecules (time range of hours or days), term, via activation or inhibition of existing molecules (time range of seconds or minutes). nithine cycle is no exception to this rule.

be long of enor short enzyme The or-

AND CHAMULEAU 1. Long-term control of ornithine

Volume

70

cycle

Whenever the liver faces a long-term change in the rate of amino acid catabolism, it responds by changing the concentrations of ornithine cycle enzymes. Thus increases in enzyme concentrations are observed after feeding a protein-rich diet (161,549), in starvation (550), in diabetes (62, 161, 275, 409), and during development (260,322,498) (see sect. VII). Presumably many of these changes in enzyme concentrations are caused by alterations in plasma concentrations of relevant hormones, such as corticosteroids (161,327,345,408,551), glucagon (345,409,591), thyroid hormones (596), and growth hormones (408, 468). N-acetylglutamate synthase also increases with increasing protein content of the diet (27) or in diabetes (275). Together with the short-term control mechanisms of ornithine cycle activity (discussed next), these longterm adaptations allow a large change in flux through the ornithine cycle at a relatively constant ammonia concentration. 2. Short-term control of ornithine cycle

Essential for understanding the short-term control of ornithine cycle activity is that carbamoyl-phosphate synthase is hardly sensitive to inhibition by its product, carbamoyl phosphate. This is not only true for the isolated enzyme (143) but also in isolated mitochondria (658) and in hepatocytes (421). This property of carbamoyl-phosphate synthase, in combination with the fact that in vivo the other ornithine cycle enzymes are highly responsive to changes in the concentration of ornithine cycle intermediates (because they operate far below saturation), makes the enzyme, almost by definition (206, Zsl), the major rate-controlling step in the ornithine cycle (provided that the supply of ammonia and bicarbonate is constant). Its flux control coefficient is close to 1 in today’s terminology. If this were not the case and if any enzyme involved in urea synthesis beyond carbamoyl-phosphate synthase were to have a lower activity than carbamoyl-phosphate synthase, then intramitochondrial carbamoyl phosphate would be expected to rise to high concentrations. However, this is not observed under normal conditions in vivo (see sect. vCZ). Only when there is a deficiency in ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, arginase, or in the the ornithine-citrulline transporter will carbamoyl phosphate accumulate. If this accumulation is extensive, carbamoyl phosphate can escape the mitochondria despite the low permeability of the mitochondrial inner membrane for this compound (450) and is used for pyrimidine biosynthesis, which results in production of erotic acid (217,472,617, 633,654). It must be pointed out, however, that the absolute rate of erotic acid synthesis in such cases is very low [70%) by suckling rats (391, 431) and, more indirectly, from the effects of litter size on body growth (586) and from a correlation between the protein content of milk and the neonatal growth rate in a number of species (42, 391, 431). No such relationship could be found for any other nutrient. To achieve a more economic nitrogen balance, protein and/or amino acid degradation could be reduced. In the perinatal rat, it appears that both options occur, resulting in the very high protein accretion rates [40-55%/day during the fetal period and -lZ%/day during the suckling period (682)]. 2. Protein turnover

Whole body protein turnover in the perinatal period is high to allow for structural remodeling of rapidly growing organs. During this period in rats, the fractional rate of protein synthesis (i.e., the percentage of protein mass synthesized per day) of the whole body and several large organs is two- to threefold higher than the fractional rate of protein degradation (135,190,191,193, 269, 342, 389). The fractional rate of protein synthesis decreases ~50% between the fetal and the weaning period when adult levels are approached. Transient peaks in whole body protein synthesis may be found in the middle of the 1st and 3rd postnatal wk, respectively (431). As weaning approaches, the ratio of the rate of protein synthesis and degradation slowly approaches unity. During human (556,686) and sheep (297,413,641) development, the rate of total body protein synthesis and degradation decreases with a similar time constant; the ratio between the rate of protein synthesis and degradation in the perinatal period is only 1.2-1.5 in humans rather than 2-3 as in rats (72, 120, 413, 475). Because of this difference, only 15-20% of protein synthesized is retained by human neonates, representing net protein gain, whereas this is 60-70% in rat neonates. As a result the metabolic cost of protein accretion is much higher in humans than in neonatal rats (428). In whole body growth and in growth of most organs, changes in overall growth rate reflect changes in the rate of protein synthesis. The liver, however, provides a well-documented example where a lower rate of protein breakdown leads to accumulation of protein during growth (102, 103, 556, 557). Thus neonatal mouse liver has a 40% higher fractional rate of stable, nonsecreted protein synthesis and a 45% lower fractional rate of stable protein degradation compared with adult mouse liver (103). In regenerating livers (557) and in livers of protein-starved, refed animals (102), protein degradation is depressed to an even larger extent. A decrease in the rate of protein breakdown is a much quicker and more efficient way to accumulate tissue protein than an increased rate of synthesis (471). The mechanism underlying the decrease in proteolysis is not yet known. However, agents like CAMP that have pronounced catabolic effects in the adult do not have such effects in neonatal liver (306). The developmental change toward

725

the adult catabolic effects of CAMP follows those of amino acid-catabolizing enzymes (see Refs. 586, 588) and those of the ornithine cycle enzymes (see Refs. 174, 327,430,494,501).

3. Amino acid catabolism

In rats, amino acid concentrations in plasma and liver, which are elevated in the fetus, abruptly decrease to adult levels at birth (115, 393, 512). The reduced excretion of urea that is observed in suckling rats (263) is probably the result of a limited capacity for amino acid catabolism and not a result of limited capacity of the ornithine cycle (499) or a decreased pool size of amino acids as a result of the postnatally reduced rate of protein degradation in the liver (103). This hypothesis is supported by the observation that the in vivo oxidation of alanine, leucine, glutamate, and phenylalanine to CO, is substantially lower at 3 days than at 3 wk after birth (115, 674), whereas their incorporation into protein strongly decreases in that interval (115). Furthermore, no significant incorporation of labeled alanine into glycogen was observed when 3-day-old animals were starved, but at 3 wk after birth a IO-fold increase was seen (674). Recent results indicate that in suckling rats glutamine rather than alanine is the major carrier of nitrogen to the liver (68, 585). The intrahepatic fate of this glutamine is not yet clear, but its increased availability apparently does not ensure an enhanced rate of urea biosynthesis. Gluconeogenesis from nonnitrogen carbon sources increases rapidly in the liver of newborn animals and has, during the suckling period, a much higher capacity than gluconeogenesis from a saturating mixture of amino acids (184, 586, 647). This indicates that the flux through several amino acid-degrading enzymes must be limiting. In agreement with this conclusion are the low enzymatic activities of many aminotransferases, oxidases, and dehydratases in the liver of suckling rats and their developmental increase to adult values at weaning (for review see Ref. 588). In addition, the developmental changes in periportally localized glutamate dehydrogenase activity parallel those of ornithine cycle enzymes and urea excretion (323). Periportally, but not pericentrally, localized glutamate dehydrogenase is thought to be implicated in gluconeogenesis and ureagenesis from extrahepatic amino acids (323). Only gluconeogenesis from amino acids entering this pathway via hydroxypyruvate was appreciable during the suckling period (for review see Ref. 586). The potential hydroxypyruvate precursors hydroxyproline, glytine, and serine are all present at elevated levels in the circulating blood of suckling animals (68, 512), and the required mitochondrial enzymes are elevated above adult levels throughout the suckling period (587, 588). 4. Summary

Whole body protein turnover in the perinatal period is high to allow for structural remodeling of rapidly

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growing organs. Such rapid growth can therefore only be achieved when rates of protein synthesis exceed those of protein degradation. Rodents appear to have evolved special adaptations to support their very high postnatal growth rates. In rodent liver, rapid growth is made possible by a combination of a high rate of protein synthesis and a low rate of protein degradation. The effects of a limited supply of amino acids in the milk on the availability of amino acids for protein synthesis is offset by very low rates of hepatic amino acid degradation and hence a low rate of urea biosynthesis. A better understanding of these phenomena in rat and mouse neonates may prove to be very useful for the feeding of human preterm neonates who apparently cannot economize their amino acids as effectively (428). VIII.

TOPOGRAPHIC CYCLE

ENZYMES

DISTRIBUTION

OF ORNITHINE

IN LIVER

In adult rat liver, ornithine cycle enzymes are localized in a wide periportal zone that comprises >90% of all hepatocytes (166; R. Charles, P. G. Mooren, A. F. M. Moorman, and W. H. Lamers, unpublished observations). The remaining 10% of hepatocytes, localized as a one- to two-cell-wide zone around the central vein, do not normally contain ornithine cycle enzymes but contain a high concentration of glutamine synthetase (165, 179). The functional significance of this complementary distribution pattern is discussed in section ~8’. This mutually exclusive distribution pattern of ornithine cycle enzymes and glutamine synthetase is unique, as the distribution patterns of enzymes belonging to other complementary metabolic pathways with either a periportal or a pericentral localization usually show a wide overlap (280). The similarity of the developmental changes in ornithine cycle enzymes (327,328,430,494,501) and glutamine synthetase (165,309,685) suggests that those factors that determine the rate of expression of both genes under normal circumstances are not responsible for the intrahepatic pattern of expression of both genes. In rats, the distribution pattern of ornithine cycle enzymes arises gradually during development. At embryonic day 20, the pericentral hepatocytes contain markedly less carbamoyl-phosphate synthase and arginase than the periportal hepatocytes (170). At and shortly after birth, when hepatic enzyme concentrations have increased severalfold, all hepatocytes stain equally densely (167, 324). During the threefold increase in carbamoyl-phosphate synthase activity in the preweaning period, carbamoyl-phosphate synthase expression becomes confined to the periportal hepatocytes. Similarly, glutamine synthetase expression becomes gradually confined to the pericentral hepatocytes in perinatal rat (165,324) and mouse (39, 319) liver development, despite a large increase in hepatic glutamine synthetase concentration. The observation that a severalfold increase in enzyme concentration leads to a homogeneous distribution in the perinatal period, but no longer in the weaning

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period, has led to the hypothesis that the expression of these enzymes becomes gradually confined to a compartment (324). A compartment is a defined zone of liver parenchyma to which expression of genes is confined irrespective of the concentration of modulatory factors. The development of these compartments was shown to be related to the development of the architecture of the liver (324). A temporal correlation between the development of hepatic noradrenergic innervation and the appearance of a heterogeneous distribution of carbamoylphosphate synthase was observed (326). However, the effects of chemical sympathectomy with 6=hydroxydopamine showed that the development of noradrenergic innervation is not causally related to the development of a heterogeneous gene expression pattern but that it can serve as an additional parameter to follow the development of the hepatic architecture (326). Most likely, therefore, the determinants of the compartments of gene expression will have to be found in cell-cell interactions and/or in the extracellular matrix. In postnatal rats, no conditions have been identified where glutamine synthetase expression can be induced in periportal hepatocytes and only two rather extreme conditions where carbamoyl-phosphate synthase and glutamine synthetase are coexpressed in pericentral hepatocytes. Such a coexpression is found in the perinatal period (165,324) and in diabetic rats that are treated with high doses of glucocorticoids (122). Both conditions are characterized by the simultaneous presence of high circulating concentrations of glucocorticoids and high intrahepatic concentrations of CAMP (89, 126, 380). Glucocorticoids or CAMP alone has no such effect (122). In the perinatal rat, changes in the ratio of insulin to glucagon, which is directly related to intrahepatic CAMP concentration (565), are very pronounced compared with other species (331). The distribution pattern of carbamoyl-phosphate synthase and glutamine synthetase in human liver is comparable to that in rat liver (439). Carbamoyl-phosphate synthase is present in a periportal zone that comprises only 25-35% of the portocentral distance. Even though the human liver lobule, and hence the portocentral distance, is approximately threefold larger than that of rats, the same number of carbamoyl-phosphate synthase-positive cells line a sinusoid between a terminal branch of the portal vein and the central vein as in rats (439). Because the number of glutamine synthetase-positive cells along a sinusoid in humans and in rats is the same, an “empty” zone exists between the carbamoyl-phosphate synthase and glutamine synthetase expression area (439). In contrast to the mitochondrial ornithine cycle enzymes, the cytosolic enzymes argininosuccinate synthase and arginase appear to be homogeneously distributed in human liver (448, 539) rather than being present only in periportal cells similar to that of rat liver. The functional implications of the different distribution pattern of the mitochondrial and cytosolic ornithine cycle enzymes for the distribution and control of metabolite fluxes are not yet clear. In human liver the periportal zone of hepatocytes,

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as defined by the carbamoyl-phosphate synthase-positive phenotype, is relatively small compared with that in rat liver (325). A reconstruction of the spatial distribution of carbamoyl-phosphate synthase-positive cells shows that the periportal zone is present as a three-dimensional network within the liver. A reconstruction of the spatial distribution of the glutamine synthetase-positive cells (and the empty zone) shows that the pericentral zone follows the branching distribution pattern of the central veins (325). On sections, this pattern resembles that of the concept of the classic liver lobulus of Kiernan (299; Fig. 2A) rather than the present widely accepted concept of the liver acinus of Rappaport (504; Fig. 2B). The lobular concept of the liver architecture is found in both humans and rats and is furthermore supported by the distribution pattern of other enzymes as well as by the blood perfusion and oxygenation pattern (325) of the liver. However, under certain pathophysiological conditions, such as a low net perfusion pressure (shock), a high viscosity of the blood (or perfusate), or a high extraction of 0, by hepatocytes from the blood (e.g., on exposure to ethanol), the composition of the blood in the peripheral parts of the periportal zone, in particular near the ends of the distributing portal veins, changes toward a composition that is typical of the pericentral zone. As a consequence, the lobular pattern of the hepatic functional architecture reverses to an acinar pattern (325). IX.

PATHOPHYSIOLOGY

727

B

OF HYPERAMMONEMIA

A. Introduction

In this section we discuss inborn errors in urea synthesis, ical mechanisms underlying syndromes, hyperammonemia ease, and the neurotoxicity of B. Inborn I. Ornithine

Errors

some general aspects of possible pathophysiologother hyperammonemic in relation to liver disammonia (Table 4).

of Urea Synthesis

cycle enzymes

A large number of in-depth reviews on inborn errors of urea synthesis has appeared in recent years (61, 61a, 100, 266, 357, 517, 534, 570, 654). A brief summary of various aspects is presented. Enzymatic defects can occur at all steps of the urea cycle (Table 4). All are inherited as an autosomal recessive trait except for ornithine carbamoyltransferase deficiency, which is an X-linked trait. In all cases the common denominator is hyperammonemia, which may be observed in the fasting state but which is always induced by a protein load. Early in the course of hyperammonemia major symptoms are lethargy and irritability, frequently ac-

FIG. 2. Distribution pattern of enzymes on liver sections as predicted by a lobular (A) or an acinar (B) concept of liver architecture. Idealized sections are cut in plane of distributing portal veins and perpendicular to central veins. Spatially, the lobular concept stresses circular layers of periportal and pericentral cells around perpendicularly oriented afferent and efferent vessels, respectively, whereas acinar concept primarily stresses a rotationally symmetrical distribution of periportal hepatocytes. Hence adjacent lobuli are characterized by contiguous periportal zones and discrete pericentral zones, whereas adjacent acini are characterized by discrete periportal zones and contiguous pericentral zones. Certain pathophysiological conditions (see text) may entail a blood composition typical for pericentral zone at periphery of periportal zones and may thus change distribution pattern of hepatic metabolism from lobular to acinar. Hatched areas, periportal zone; stippled areas, pericentral zone; triangles, portal tracts with conducting part of portal vein, hepatic artery, and bile duct; dashed lines, distributing part of the portal vein; circles, central (terminal hepatic) vein. [A: modified from Lamers et al. (325). B: modified from Rappaport (504).]

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728 TABLE 4. Diseases and syndromes with hyperammonemia

MEIJER,LAMERS, associated

Inborn errors of urea metabolism Carbamoyl-phosphate synthase deficiency Ornithine carbamoyltransferase deficiency Citrullinemia Argininosuccinic aciduria Argininemia N-acetylglutamate synthase deficiency Hyperornithinemia, hyperammonemia, and homocitrullinemia syndrome Secondary hyperammonemias Disorders leading to CoA sequestration Organic acidurias Reye’s syndrome Environmental factors (valproic acid, hypoglycin) Familial protein intolerance Periodic hyperlysinemia with hyperammonemia Transient hyperammonemia of the newborn Liver disease

companied by vomiting. Persistence of hyperammonemia or further deterioration leads to confusion, stupor, and coma. In the terminal phase there is an increase in intracranial pressure and brain edema occurs. Patients with residual activity of ornithine cycleassociated enzymes ~5% of normal show an acute clinical course that, without intervention, leads to death. In patients with a higher residual activity (5-15s of normal), episodic hyperammonemia occurs in the first few years of life. Hyperammonemia is most severe in patients with deficiencies of carbamoyl-phosphate synthase, ornithine carbamoyltransferase, argininosuccinate synthase, and argininosuccinate lyase and is of minor importance in patients deficient in arginase (61). This appears to be true because arginase has by far the highest capacity of all ornithine cycle enzymes (see sect. vCS). Moreover, a deficiency in arginase leads to a higher steady-state concentration of arginine in the liver, which may promote synthesis of N-acetylglutamate and may thereby activate carbamoyl-phosphate synthase (see sect. vCS). From a diagnostic point of view it is important to stress that defects distal to production of carbamoyl phosphate are always accompanied by overproduction of erotic acid, because mitochondrial carbamoyl phosphate escapes to the cytosol under such pathological conditions and is used for pyrimidine biosynthesis (see sect. ~$2). In contrast, errors in production of carbamoyl phosphate do not result in erotic acid production. In recent years, application of recombinant DNA techniques has led to the cloning and characterization of the genes for the enzymes involved in urea synthesis. Progress in this area now makes it possible to study gene regulation and the molecular basis of the enzyme defects involved (266, 348, 460, 534). 2. Hyperornithinemia, h yperammonemia, homocitrullinemia syndrome

and

The HHH syndrome is characterized by hyperornithinemia, hyperammonemia, and homocitrullinemia,

AND CHAMULEAU

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which lead to a variable degree of mental retardation. It is probably caused by a defect in the transport of ornithine from the cytosol to the mitochondrion (254, 264). Because of the low intramitochondrial ornithine concentration, flux through both ornithine carbamoyltransferase and ornithine aminotransferase is low; this results in accumulation of ornithine in the cytosol and a rise in the level of ammonia. Under these conditions, lysine can replace ornithine as substrate for ornithine carbamoyltransferase (see sect. vCZ) and homocitrulline is formed. Protein restriction can control hyperammonemia, and arginine treatment can improve the clinical conditon (570). The synthesis of creatine is reduced in HHH syndrome because of product inhibition of arginine:glycine amidinotransferase by the high concentrations of ornithine (651). This is also observed in patients with ornithine aminotransferase deficiency, who also have high plasma ornithine concentrations but do not suffer from hyperammonemia (576). 3. Inborn errors of urea synthesis and pH homeostasis

Because the function of urea synthesis is to eliminate both ammonia and bicarbonate, a defect in urea synthesis can be expected to result in accumulation not only of ammonia but also of bicarbonate and hence in metabolic alkalosis. To study this, Bachmann and Colombo (20) have analyzed, before treatment, the acid-base status of 51 patients with various defects in urea synthesis. Eighteen patients had a normal blood pH, whereas in the other patients, both alkalosis (n = 15) and acidosis (n = 18) were found. They concluded that apparently the role of urea synthesis in pH regulation is minor. However, as pointed out by Atkinson and Bourke (15), it is not always valid to infer from observations of pathological conditions how control is normally exerted, since secondary interactions and compensatory effects of many kinds may occur. Hgussinger et al. (230) have drawn attention to the fact that a blockade in the ornithine cycle need not necessarily lead to hyperbicarbonatemia. For example, in patients with citrullinemia treated with ornithine supplementation in order to maintain excretion of waste nitrogen as citrulline, more bicarbonate is consumed than ammonia is eliminated (see also Fig. 1). C. Secondary Hyperammonemias I. Disorders leading to CoA sequestration

Whenever N-acetylglutamate synthesis is impaired, urea synthesis decreases and hyperammonemia develops. Factors leading to this condition may be congenital (100; Table 4), environmental, or a combination of both. In addition to a direct deficiency of N-acetylglutamate synthase, other defects can also be involved. For example, when a deficiency of enzymes involved in the

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SYNTHESIS

metabolism of certain fatty acyl-CoA compounds occurs, intramitochondrial CoASH will be trapped. Synthesis of N-acetylglutamate will decrease because of a lack of acetyl-CoA and because accumulating fatty acyl-CoA itself directly inhibits N-acetylglutamate synthase. Because mitochondrial acetyl-CoA is also required as an essential activator of pyruvate carboxylase, these conditions concomitantly lead to hypoglycemia. Hyperammonemia associated with propionic and methylmalonic aciduria (187) has been shown to be due to decreased synthesis of N-acetylglutamate (114, 497, 609). The same mechanism is probably responsible for hyperammonemia in other cases, such as in patients with a deficiency in medium-chain CoA-dehydrogenase [dicarboxylic aciduria (606)]. In propionic aciduria, inhibition of mitochondrial ATP production by propionate may also be involved (69, 187). This is particularly important in the newborn where hyperammonemia in propionic aciduria is most severe and the capacity to synthesize ATP via oxidative phosphorylation is still low (69). Depletion of N-acetylglutamate is also the underlying mechanism of hyperammonemia after therapeutic administration of the antiepileptic drug valproate (36, 181) and after intoxication with hypoglycin, a shortchain fatty acid that is the toxic compound involved in Jamaican vomiting sickness (9). These compounds also interfere with CoA metabolism. After valproate administration, the hyperammonemia may also be of renal origin. This is because valproate interferes with renal fatty acid oxidation and renal glutamine utilization increases with release of ammonia in the renal vein (251). Short-chain fatty acids may also be responsible, at least in part, for the hyperammonemia in Reye’s syndrome, which is characterized by acute encephalopathy and fatty infiltration of many organs (632). A possibility, albeit speculative, is that in Reye’s syndrome hyperammonemia is precipitated by a partial deficiency in the oxidation of these fatty acids in combination with environmental factors, such as viral infection (Varicella, influenza B) and the use of aspirin, which is metabolized by the liver after conversion to its CoA derivative (162, 632). Short-chain fatty acids not only sequester CoA, which results in a reduced intramitochondrial concentration of N-acetylglutamate, but also directly reduce the activity of carbamoyl-phosphate synthase and ornithine carbamoyltransferase (59,575,613). 2. Familial protein intolerance with de$cient transport of basic amino acids (lysinuric protein intolerance)

Patients with lysinuric protein intolerance have a defect in the renal and intestinal transport of the basic amino acids lysine, arginine, and ornithine, resulting in enhanced urinary excretion and low plasma concentrations of these amino acids. In addition, transport of these amino acids across the plasma membrane of the hepatocyte is probably decreased (574). Hyperammonemia in the patients is therefore likely to be the result of the reduced availability of ornithine cycle interme-

diates. Indeed, administration of ornithine or arginine to the patient is able to prevent hyperammonemia after an intravenous load of alanine (654).

3. Periodic hyperlysinemia

with hyperammonemia

A defect in the degradation of lysine leads to high plasma concentrations of this amino acid (182). Hyperammonemia in these patients may be the result of direct inhibition of ornithine carbamoyltransferase (287), argininosuccinate synthase (sect. vCZ), arginase, and the mitochondrial transport of ornithine by lysine (287).

4. Transient hyperammonemia of newborns

This was discussed in section

VIIC~.

5. Liver disease

Both chronic and acute liver insufficiencies are associated with increased blood ammonia levels. Hyperammonemia in cirrhosis is the result of a diminished capacity of the liver to synthesize urea (272, 371, 449, 530), to portosystemic shunting, and to a decrease in glutamine synthetase (282). Maintenance of a normal flux through the urea cycle despite a 80% decrease of its capacity is only possible at an elevated intramitochondrial ammonia concentration. The fourfold increased flux through glutaminase in cirrhosis, as shown by Kaiser et al. (282), is in agreement with this assumption. However, because glutamine synthetase flux in cirrhotic liver is also decreased by ~80%, hyperammonemia is the result (282). Diseases that cause perivenous liver cell necrosis (e.g., poisoning with paracetamol or Ccl,, right-sided backward failure of the heart), are associated with hyperammonemia because of impairment of glutamine synthetase, the high-affinity system for ammonia detoxification in the liver, whereas flux through the ornithine cycle is relatively normal. Other factors that contribute to the hyperammonemia in chronic liver insufficiency are impaired muscular glutamine synthesis (352,464) and increased renal vein ammonia output (171). The latter may be caused by hypokalemia and alkalosis induced by diuretics (22,463). It should be stressed that inhibition of liver carbonic anhydrase by diuretics can also contribute to the development of hyperammonemia in cirrhotic patients (229). As discussed by Haussinger et al. (230), many patients with chronic liver disease may develop metabolic alkalosis, which is believed to be the result of, at least in part, decreased hepatic bicarbonate disposal that in turn results from impaired urea synthesis. However, other factors, such as vomiting, therapy with diuretics, and hypokalemia, may also contribute to the alkalosis (230).

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730

MEIJER,LAMERS,

D. Neurotoxicity

of Ammonia

I. Ammonia concentration in brain

At elevated concentrations ammonia is toxic to the central nervous system (I). Both chronic and acute liver insufficiency are associated with increased blood ammonia concentrations, although the blood ammonia concentration does not necessarily correlate with the degree of hepatic encephalopathy (HE) (605). Nevertheless, according to many investigators, ammonia plays a key role in the pathogenesis of HE (547). The normal value for fasting arterial blood ammonia in humans is (50 pmol/l (111, 258). The brain-toblood ammonia concentration ratio is in the range of 1.5:1 to 3:l. This ratio is maintained by a complex interaction of blood flow, the pH difference across the blood-brain barrier, and a balance between enzymatic removal and synthesis (111). The brain itself normally detoxifies ammonia by one major enzymic reaction, glutamine synthetase (111, 136). Although the K, value of brain glutamine synthetase for ammonia is 180 PM (469), the K, of brain glutamate dehydrogenase for ammonia is much higher [lo mM (SO)]. Thus in moderate hyperammonemia (plasma concentrations of 100-200 PM), brain glutamine synthetase begins to be less responsive to further increases in ammonia while flux through glutamate dehydrogenase is still very low. This has been directly demonstrated by tracer studies with [13N]ammonia (106,107). Because of the limited capacity of glutamine synthetase and the relative inability of brain to use other pathways of ammonia detoxification, acute ammonia loading superimposed on chronic hyperammonemia is highly toxic to cerebral function (139, 185,451,493). This phenomenon is well known in experimental research. An example is the poor tolerance to acute ammonia loading in animals with a portacaval shunt (PCS). An example in a clinical setting is the provocation of HE by gastrointestinal bleeding in a cirrhotic patient (134). 2. Possible mechanisms of ammonia toxicity

Although there is strong evidence that a major neurotoxin (38,185), its mechanism still a matter of debate. Possible mechanisms nia neurotoxicity are reviewed by Cooper (Ill) and are summarized in Table 5.

ammonia is of action is for ammoand Plum

TABLE 5. Possible mechanisms of ammonia neurotoxicity l

l l l l

Structural and morphological astrocytes, and neurons Pathophysiological changes of Effects on electrophysiological Interference with neurotransmitter Interference with biochemical Modified

from

Cooper

changes

in blood-brain

cerebral blood flow properties of central function pathways

and Plum

(111).

barrier,

nervous

system

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I) STRUCTURAL CHANGES. Because, in clinical practice, the symptoms in chronic HE are completely reversible, any postulated mechanism of neurotoxicity of ammonia must take this reversibility into account. Thus neurotoxicity of ammonia in chronic HE is unlikely to be due to ammonia-induced structural and/or morphological lesions of the central nervous system (e.g., enlarged protoplasmic astrocytes Alzheimer type II cells), since the appearance of these lesions in cerebra during chronic HE does not correlate with the severity of the hepatic lesion (105). However, the terminal phase of acute HE, as is observed in acute fulminant hepatitis, is irreversible and is associated with brain edema (112). II)CEREBRAL BLOOD FLOW. In the pathogenesis of chronic HE it is unlikely that ammonia has important effects on the physiology of cerebral blood vessels and cerebral blood flow (CBF) (Ill). The effects of acute hyperammonemia on cerebral circulation in the whole animal are inconsistent, since they vary among species and show regional differences. Chronic hyperammonemia does appear to be associated with an increased CBF in experimental animals (185). In humans few unequivocal results exist. An increase in CBF has been observed after PCS (46), but a decline in CBF during the development of HE (479) occurs. Neither observation was significantly correlated with plasma ammonia concentrations. III)ELECTROPHYSIOLOGY. Neurophysiologicalstudies indicate that ammonia has major effects both on excitatory and inhibitory synaptic transmission. Ammonia inhibits the extrusion of Cl- from neurons by an unknown mechanism and inhibits in a dose-dependent manner the generation of the hyperpolarizing potential, and thus no inhibitory postsynaptic potential (IPSP) occurs (350,369,370,492). This effect is probably related to increased slow wave activity in the electroencephalogram, as observed in hyperammonemia in intact animals (74,459) and humans (639). In brain slices, ammonia partially suppresses the frequencies of spontaneous action potentials. Alger and Nicoll(4) and Raabe (490) also observed a decrease of the amplitude of the excitatory postsynaptic potential (EPSP) during ammonia intoxication. If the amplitude of the EPSP is sufficiently decreased, the EPSP becomes too small to generate an action potential (621, 622). The concentrations at which ammonia becomes neurotoxic appear to be rather variable. In encephalopathy resulting from acute ammonia intoxication, the earliest behavioral neurological symptom, lethargy, occurs when brain ammonia concentration increases to -750 pmol/kg (139). An advanced stage of HE is associated with brain ammonia concentrations of l,ZOO-1,800 pmol/kg (139, 248, 548). These observations may be related to results of Raabe and Lin (491, 492), which show that I mM ammonia produces a maximal effect on inhibitory transmission, with only a very small effect on excitatory synaptic transmission; for effects on the latter process, higher concentrations of ammonia are required. Therefore it is suggested that in the early stage of acute HE, the IPSP is decreased and that both

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1990

Pre-

synaptic

neuron

Astrocyte

\I 1

Glutamine

FIG. 3. COmpartmentatiOn Of ammonia metabolism in brain. GABA, y-aminobutyric acid; GAD, glutamic acid decarboxylase; GDH, glutamate dehydrogenase.

Nitrogen metabolism and ornithine cycle function.

PHYSIOLOGICAL REVIEWS Vol. ‘70, No. 3, July 1990 Printed in U.S.A. Nitrogen ALFRED Metabolism J. MEIJER, WOUTER and Ornithine H. LAMERS, AND...
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