Protein and amino acid metabolism after injury

zyxwvut zyxw zyxwv Protein and Amino Acid Metabolism after Injury Robert R. Wolfe,* Farook Jahoor, and Wolfgang H. Hart1 Metabolism Unit, Shriners Burns lnstitute and the Departments of Surgery and Anesthesiology, University of Texas Medical Branch, Galveston, Texas 77550 I. INTRODUCTION Trauma induces a net loss of lean body mass, which predisposes the patient to an increased risk of morbidity and mortality in the acute phase.' Furthermore, depletion of muscle mass can prolong rehabilitation to normal physiological function following recovery. Understanding the nature of the protein catabolic response to trauma is thus important from the therapeutic standpoint. Also, the response to trauma provides an excellent model with which to better understand factors regulating protein synthesis and breakdown in general. Unfortunately, the nature of changes in protein and amino acid metabolism after trauma in human subjects has not been clearly elucidated. A major problem in piecing together a coherent picture of the overall response to trauma is the difficulty of comparing studies done in different groups of patients. For example, most studies of amino acid metabolism after trauma have been done in the context of the response to elective surgery (see Ref. 2). In addition to complications arising from metabolic effects of preexisting disease, and varying body composition, other problems in data interpretation exist in this group of patients. The type of anesthesia affects the postoperative proteolysis3 as does the pre- and postoperative nutrition. Most importantly, even major surgery is not a potent stimulus for the protein catabolic response for a prolonged period of time. Amino acid kinetics may return to normal within a few days after surgery.2 Consequently, the length of time elapsing between surgery and the study day is a crucial variable. The response to traumatic injury is a much greater catabolic stimulus than is elective surgery. The extent of injury may vary greatly, however, and, although methods exist for categorizing patients according to ~ e v e r i t y ,such ~ classification systems can be imprecise. The various problems related to selection and categorization of patients to be studied can be overcome to a great extent by studying severely burned patients. The amount of injury can be easily quantified by measuring the surface area burned. Furthermore, the observed metabolic responses in patients appear to be systemic responses to injury. For example, Aulick and Wilmore5found that the rate of release of alanine from the leg was related to the extent of total body surface injury but unrelated to the size of limb burn or the rate of leg blood flow in the leg being studied. Furthermore, when patients with burns over more than 50% of the body surface are studied, differences in burn size do not markedly affect the observed responses. Finally, burn injury probably represents the most severe form of trauma, and recovery may require months. During recovery, a relatively stable physiological state exists, but accelerated protein breakdown may persist throughout. Consequently, the severely burned patient is an excellent model for understanding the response to trauma. Loss of as much as 20% of the body protein may occur in the first 2 weeks following major burn injury.6 Contrary to expectations, however, when aggressive nutritional support is provided the proteolytic response is not maximal immediately after injury but may peak as much as 1month after injury. We recently completed a longitudinal study of severely burned children burn size = 65% of body surface) designed to examine amino acid and urea dynamics in the various phases of zyxwvu zyxwvutsrq *To whom correspondence should be addressed at Shriners Burns Institute, 610 Texas Avenue, Galveston, Texas 77550. DiabetedMetabolism Reviews, Vol. 5, No. 2, 149-164 (1989) 0 1989 by John Wiley & Sons, Inc. (x CCC 0742-4221/89/020149-16$04.00 150 zyxwvutsrqpon response to i n j ~ r y We . ~ were particularly interested in defining the roles of changes in protein synthesis and breakdown in mediating the catabolic (flow phase) and then anabolic (convalescent phase) responses to injury. The absolute rate of whole-body protein breakdown was assessed from the plasma flux of the essential amino acids (EAA) leucine, valine, and lysine, as determined with stable isotopic tracers. The rate of urea production (determined with I5N2urea) was used as an index of net protein catabolism. Protein synthesis was inferred from the ratio of mean EAA flux to urea production. Patients were studied in the postabsorptive state in order to eliminate any direct dietary influence on the underlying changes in protein kinetics due to the injury. Compared with values obtained in recovered children, the plasma fluxes of all 3 EAAs were increased, indicating an increased rate of protein breakdown during the acute (2-4 days after injury), flow (2-4 weeks after injury), and convalescent (achievement of full epithelialization of all burn wounds) phases of injury. The rate of urea production, however, was only significantly increased during the flow phase, suggesting that accelerated protein breakdown was counteracted in the acute and convalescent phases by concomitant elevations in protein synthesis. The increase in net catabolism in the "flow" phase could therefore be attributed to a relative impairment in protein synthesis, as compared with the situation in either the acute or convalescent phase. The protein kinetic response did not correlate with changes in the metabolic rate, since resting energy expenditure was significantly increased above predicted levels during the acute and flow phases (40 and 50%, respectively) and returned to normal in convalescence. The dependence of our results on the time after injury at which the patients were studied may partially explain the diverse results of other investigations of the changes in whole-body protein synthesis and breakdown in response to injury. Also, variations in nutritional intake have probably contributed to apparently conflicting conclusions, since protein intake influences the rate of protein synthesis and breakdown at the whole-body level. Decreased nutrient intake decreases protein synthesis and breakdown,' and intravenous feeding, as opposed to enteral feeding, may also decrease protein turnover.' Thus, the fall in protein synthesis after surgery reported by O'Keefe et al." could as easily have been due to the fact that the patients were fed normally during their study before sur- PROTEIN AND AA METABOLISM AFTER INJURY gery, and were given only glucose and electrolytes for the 3 days before the data were collected for the turnover calculations after surgery, as to the effect of surgery per se. The failure to control protein intake similarly complicates the interpretation of the data reported by K e n et al." in burned children. In contrast to the report of O'Keefe et al.," Kien et al. used an ['5N]glycine technique and concluded that both protein synthesis and catabolism were increased after injury and that the increases were related to the size of injury. However, since their dietary regime involved giving graded doses of protein energy intake based on the size of the injury, there was also a direct relationship between protein intake and the rate of protein synthesis. It is therefore not certain which of the two variables were responsible for the increases in synthesis and breakdown. We evaluated the influence of nutritional intake on the rates of whole-body protein turnover in severely burned patients using l-'3C-leucine as tracer." With appropriate priming doses, this approach allows the calculation of rates of synthesis and breakdown in a 2-h period. This enables us to study patients in both the fed (intravenously) state as well as after 10 h of fasting. Studies were performed at the end of two 3-day dietary control periods during which they received 1.5 g protein/ kg day, or 2.5 g protein/kg d with total calories matched. When the patients were studied in the fed state, nutrient administration stimulated protein synthesis rather than inhibiting protein breakdown. When protein intake was increased, both synthesis and breakdown were stimulated correspondingly, but net balance was not affected.'* If variations in protein intake and severity of injury are accounted for, the results of previous studies still can be divided into those that indicate net protein catabolism results from a fall in protein synthesis below the breakdown whereas others suggest that it is due to an elevation in protein breakdown rate.11,14-17 The results of our longitudinal study confirm that two distinct phenomena are involved, but that they are operative during different phases of recovery after injury. More fundamental than the issue of the time after injury at which a study is performed or the rate of nutrient intake is the question of whether the concept of whole-body protein synthesis is a valid way in which to consider the complicated picture of protein metabolism in man. Furthermore, if one accepts the general concept of wholebody protein turnover, one must question if there WOLFE, JAHOOR, AND HARTL are any reliable means by which to determine the true value. The primary problem is that wholebody N flux is an entity that cannot be measured by any known method. Traditionally, investigators have utilized the flux of an essential amino acid to deduce a value for whole-body N flux, based on the assumption that the percent contribution of the essential amino acid flux to whole-body N flux is equal to its percent concentration in mixed wholebody protein.I8This assumption, however, has not been validated and seems unlikely. The rate of efflux and influx of an essential amino acid from and into a particular protein will be a function of the pool size of that protein, its percent composition of that particular essential amino acid, and the rate of turnover of the protein.” Since the wholebody is composed of numerous proteins, with different pool sizes and different turnover rates, some relatively fast (e.g., liver-soluble proteins), some very slow (e.g., skeletal muscle proteins),” and each has its own unique amino acid composition (e.g., lysine content of skeletal muscle, serum albumin, fibrinogen, and fibronectin is 5.1, 9.0, 7.5, and 3.6%, r e s p e c t i ~ e l y ) , ~it~is- ~highly ~ unlikely that taken together they will contribute equally to the whole-body flux of an essential amino acid. Consequently, although a certain amount of information can be obtained from whole-body turnover measurements, the results of these studies should be considered carefully in light of the theoretical limitations resulting from the necessity of making assumptions known to be untrue. Because of the interpretative limitations of whole-body studies, it is useful to consider the responses of individual tissues. Most attention has been focused on skeletal muscle, since clinical and biochemical evidence suggest that muscle is the major source of the N loss following severe inFurthermore, it is widely believed that increased muscle protein breakdown is in large part responsible for the net loss of skeletal muscle in trauma. This notion is supported by several studies that have reported elevated 3-methylhistidine (3-MH) excretion in elective surgical traumaz6 and septic patients29 as a reflection of muscle protein breakdown. However, since 3-MH excretion also results from the breakdown of skin and of smooth muscle, the effect of trauma on muscle protein breakdown cannot be quantified from 3-MH excretion data. This is shown by the results reported by Rennie et al.30They found that surgery caused a 40% decrease in the rate of release of 3-MH across the leg but overall a 40% increase in 151 total 3-MH excretion. Thus, the increase in 3-MH excretion after trauma may not reflect skeletal muscle protein breakdown. Rather, Rennie et al. proposed that injury causes a decreased rate of muscle protein breakdown, but that muscle protein synthesis is decreased to an even greater extent.30The concept of decreased muscle synthesis after injury is supported by estimation of protein synthesis in skeletal muscle after elective abdominal surgery using the concentration and size distribution of ribosomes. Wernerman et al.31 found that the percentage content of polyribosomes, total ribosome concentration, and the polyribosome concentrations per milligrams of tissue DNA were all decreased 1 and 3 days after abdominal surgery as compared with the values before surgery. Furthermore, total parenteral nutrition did not prevent the decrease. Isotopic uptake estimations of skeletal muscle protein synthesis studied in vivo in animals have yielded variable results. Lue3’ found mild, transient depression of mixed muscle protein synthesis following hysterectomy in rats, whereas Moldawer et al.33found muscle protein synthesis to be increased in trauma in amino acid-fed rats. Frayn and M a y ~ o c kshowed ~~ impairment of muscle protein synthesis but no effect on degradation following a 20% dorsum burn in the rat, and in the same animals they found that neither synthesis nor degradation was affected in extensor digitorum longus. Differences in animal models, nutritional status, and methods of investigation may explain some of the discrepancies in reported responses, but close inspection of each paper reveals no obvious reason for the observed differences. The study of isolated muscle taken from injured and uninjured limbs has enabled more detailed investigation of the alterations in the regulation of protein metabolism after injury. The incubated rat soleus muscle preparation taken from burned and unburned limbs has been used by both Shangraw and T ~ r i n s k yand ~ ~ Odessey and Parr.36Both authors found an increased rate of net protein breakdown in muscle taken from the injured limb but not from the uninjured limb. Consistent with the whole-body data,’ leucine oxidation was stimulated in the muscle from the burned limb. Odessey and Parr reported that insulin at a concentration of 100 pUlml was effective in stimulating protein synthesis and inhibiting net proteolysis, but leucine failed to suppress protein catabolism in burned muscle.36Both authors found that muscle from the unburned limb responded similarly in all respects to muscle from unburned zyxwvutsr 152 zyxwvutsrqpon PROTEIN AND AA METABOLISM AFTER INJURY controls, suggesting that the observed responses in the muscle from the burned limb were due to either muscle injury or other local factors resulting from the wound itself.36This finding leads to the conclusion that there is not a generalized proteolytic response to injury. However, since in this experiment only 3% of the total surface of the rat was burned, a more likely explanation is that the injury was not severe enough to elicit a systemic response. Studies assessing the response of muscle metabolism in man after injury have generally been limited to the measurement of the arterial-venous (A-V) balance across a limb. In general, these studies support the concept of accelerated muscle protein breakdown, in that net release of amino acids is accelerated from either the leg or the f ~ r e a r m . ~Alanine , ~ ~ - ~and ~ glutamine constitute more than 60% of the total amino acid efflux from the periphery. As the severity of the trauma increases, or in sepsis, the net efflux of amino acids from the periphery is even greater than after The increased amino acid uptake by the splanchnic bed serves the processes of gluconeogenesis and the synthesis of acute phase proteins. The relationship between amino acid release, gluconeogenesis, and ureagenesis will be discussed below in the context of the role of alanine and glutamine in transferring N from the periphery to the splanchnic area. The stimulation of the synthesis of acute-phase proteins, such as C-reactive protein, alpha-1-acid glycoprotein and fibrinogen, should not be confused with a generalized stimulation of all hepatic proteins. For example, albumin synthesis appears to be depressed in injured patients.& zyxwvutsr surgery.5,19,31-33,37,38,42,43 The concept of a transfer of amino acids from the periphery to the splanchnic area stems from the observation that the increased release of amino acids from the periphery is matched by increased clearance of amino acids from the slanchnic area. The rate of splanchnic clearance of amino acids reflects their peripheral rate of release, in that there is a modest increase in net uptake after elective s ~ r g e r y , and ~ , ~with ~ more severe injury (such as burns), the splanchnic uptake increases to a greater extent. Sepsis generally increases splanchnic uptake even further,38but in severely ill patients the ability of the liver to clear amino acids seems to fa11.43,46 However, it is also possible in severely ill patients that altered plasma concentrations of certain amino acids reflect altered peripheral release, rather than hepatic dysfunction. For example, plasma phenylalanine increases in severely injured patients and is particularly elevated in sepsis.47This has generally been considered to reflect hepatic insufficiency: specifically, a deficiency in hepatic phenylalanine hydroxylase, the enzyme which converts phenylalanine to tyrosine. However, this may not always be the case. It has been shown that when either nonseptic or septic burn patients, with elevated phenylalanine levels, were given an oral dose of 100 mg phenylalanine/ kg, the clearance of the absorbed phenylalanine was actually elevated in the patients, with the highest clearance rate occurring in the septic patients.47 11. SUMMARY OF RESPONSES OF PROTEIN METABOLISM TO SEVERE INJURY AND SEPSIS The general pattern of responses to injury or sepsis involves accelerated net breakdown of skeletal muscle, with an increased uptake of amino acids in the splanchnic bed. The amino acids taken up in the liver at increased rates serve as precursors for accelerated gluconeogenesis, with subsequent urea production, and for the synthesis of acute phase proteins. Additional urea production may occur as a direct consequence of the metabolism of some amino acids, particularly glutamine. The increase in urea production is the end product of the increase in net protein catabolism induced by injury. The relationships of the increased net loss of body mass to changes in protein synthesis and breakdown is not entirely clear. The results of studies have been difficult to collate coherently, due to differences in nutritional intake, severity of injury, and the time following injury at which the various studies have been performed. Our own studies suggest that accelerated protein breakdown persists from the time of injury throughout convalescence, even when this process is protracted over a period as long as several months. The phase of maximal net catabolism results from a relative impairment in protein synthesis, which is apparently refractory to nutritional support. The transition from the catabolic phase to the anabolic phase of recovery from injury is characterized by an increase in protein synthesis that is sufficient to overcome this accelerated rate of protein breakdown. The general scenario outlined above is generally consistent with other reports in the literature, but there are certainly inconsistencies. In part, zyxwvutsr zyxwvutsrqponml these inconsistencies probably stem from limitations, both conceptual and practical, in dealing with whole-body protein turnover measurements. Consequently, it is worthwhile to consider the response of the metabolism of specific amino acids, rather than relying entirely on studies focused principally on protein metabolism at the whole-body or tissue level. Below we will focus on the metabolism of the three amino acids, leucine, alanine, and glutamine, that have received the most attention with regard to their role in the response to injury or sepsis. zyxwvu 153 WOLFE, JAHOOR, AND HARTL COP Production From Leucine 3- zyxwvut zyxwvutsrqp zyxwvutsr zyxwvu e zyx (D L 2- r 0 L C 0) !! 0" 1- A. Response of Leucine Metabolism to Injury Studies in humans12 and animals33 have clearly established that leucine oxidation is increased after trauma. Since an increase in metabolic rate occurs after injury, it is possible that the increased leucine oxidation simply reflects an overall increase in energy expenditure. However, this is not the case. We performed the same experimental protocol in burn patients and normal volunteers in whom energy expenditure was increased either by cold exposure49or by modest exercise.50The results are summarized in Figure 1. In normal volunteers, increased energy expenditure caused by either exercise or cold exposure caused a decrease in the relative contribution of leucine oxidation to total C 0 2 production. This would be expected teleologically, since it would not be anticipated that an essential amino acid would be a preferred energy substrate but rather that adaptive mechanisms selectively spare essential amino acids in periods of increased energy expenditure. In contrast, after severe injury the increase in leucine oxidation is disproportionately greater than would be expected from the increase in energy expenditure. Clearly this represents a response that does not follow the normal pattern of adaptation to a state of increased energy expenditure. There are at least two possible explanations for the high rate of leucine oxidation after injury. One possibility is that there is an energy fuel deficiency stemming from an impairment of both glucose and fat oxidation in the muscle, leading to an energy-deficient state in the muscle which can only be alleviated by oxidation of branched chain amino acids. The alternative possibility is that the stimulation of muscle protein breakdown leads to an accumulation of free leucine in the muscle, and since leucine oxidation reflects its availability to at least some extent, leucine oxidation increases. Sev- Control Cold Exposure Exercise Burn Figure 1. Leucine oxidation in various hypermetabolic states. When expressed in terms of the percent contribution to overall C 0 2 production, leucine oxidation decreases in both cold exposure and exercise in normal man but is increased in burn patients. Adapted from References 12, 49, and 50. era1 lines of evidence argue against the first hypothesis and support the second possibility. First, we have shown that pyruvate oxidation is elevated, not inhibited in burn patients. Furthermore, stimulation of pyruvate oxidation with dichloroacetate does not affect leucine oxidation. Secondly, isotopic studies have generally indicated an enhanced, not impaired, ability to oxidize fat after injury and in sepsis.51 Finally, the intracellular concentration of leucine is elevated in the muscle of injured patient^.^^-^^ If leucine oxidation was increased because of an energy substrate deficit, one would expect the intracellular leucine concentration to be depleted and surely not to be increased. The proposal to infuse branched-chain amino acids (BCAA) for the nutritional therapy of critically ill patients is generally based on the thesis that an increased rate of oxidation of BCAA in muscle stems from a deficit in oxidizable energy substrates, thereby creating a state in which these essential amino acids may fall below the level necessary to optimally support protein synthesis. However, as discussed above, this proposal is groundless, since there is neither a demonstrable impairment of either glucose of fat oxidation in the zyxwv 154 zyxwvutsrqpon PROTEIN AND AA METABOLISM AFTER INJURY muscle of injured humans, nor is there a lack or decreased availability of BCAA for protein synthesis. An alternative rationale for providing BCAA to patients is that leucine can exert a specific stimulatory effect on protein synthesis. In vitro studies have shown that leucine concentration can exert a regulatory effect on the rates of both synthesis and degradation in rat skeletal musc1e.55,56 Similar findings of a regulatory effect specific to leucine in perfused rat hearts suggested that the effect was not a simple result of provision of an oxidizable substrate.57However, studies of in vivo infusions of BCAA into starving but otherwise unstressed guinea pigs5' and rats59showed protein sparing compared with starvation but not compared with glucose infusions of similar caloric value. Additional protein sparing by BCAA could not be demonstrated in a well-controlled study in dogs6' or in fasting man.61 Since the intramuscular concentration of leucine is higher after injury than before, it follows that any effect of exogenously infused leucine should be demonstrable in the normal circumstance more readily than after injury. It is therefore expected that most recent studies of the effectiveness of nutritional support regimens with enriched RCAA have failed to show a positive benefit in stressed patients (e.g., Ref. 62). zyxwvu zyxwvu IU Glucose Rate of Appearance 5 ( rnglkg min 1 0 Alanine Appearance Rate (brnolelkg rnin 1 - zyxwvuts B. Response of Alanine Metabolism to Severe Trauma and Sepsis Severe trauma and sepsis are characterized by a markedly elevated alanine flux, most of which originates in skeletal m ~ s c l e . ~ , ~ ' ,Th ~ ere ~ , " is a correspondingly marked increase in the splanchnic (liver) uptake of alanine.46,64 We have shown that this increased alanine flux is associated with an increased hepatic glucose and urea production rate,63suggesting that alanine fuels a stimulated gluconeogenic ~ a t h w a y . ~The ~ - contribution ~~ of alanine to the accelerated gluconeogenesis of trauma and sepsis is more than twice its contribution in the normal postabsorptive state.63r65 This increased gluconeogenesis, hence ureagenesis from alanine, is therefore an important component of the hyperglycemia and net loss of N characteristic of the catabolic phase of severe injury and sepsis. Although it has been suggested that the increased gluconeogenesis of severe trauma and sepsis is a direct consequence of an increased need for glucose,66we have shown (Figure 2) that infu- Gluconeogenesis 0.4 From Alanine (mglkg. rnin 1 o.2 NORMAL SEPTIC Figure 2. Glucose and alanine kinetics in normal volunteers and septic patients. In sepsis alanine is an important gluconeogenic precursor, and gluconeogenesis from alanine is not suppressed by glucose infusion at 4 mg/kg/min. Data from References 63 and 67. sions of exogenous glucose do not inhibit the rate of gluconeogenesis from alanine or urea production rate to the extent seen in normal control^.^^,^' A more likely mediator of this metabolic abnormality is the altered hormonal profile of severe trauma and sepsis. For example in severe burn injury and sepsis, although there may be hyperinsulinemia, glucagon levels are so highly elevated that the glucagonhnsulin molar ratio may be increased several-fold.68 The elevated plasma glucagon is responsible, at least in part, for the increased gluconeogenesis, hence the increased ~ r e a g e n e s i s During .~~ the simultaneous lowering of insulin and glucagon by somatostatin infusion in burned patients, we showed that glucose production decreased despite an increase in alanine flux. This response accompanied the hyperglycemia resulting from the reduced clearance of glu- zyxwv WOLFE, JAHOOR, AND HARTL cose due to the insulin deficiency created by the somatostatin infusion. These results suggested that glucose production (and gluconeogenesis) is controlled (hormonally) at the liver and not by amino acid supply.68This interpretation is further supported by our observation that the infusion of alanine at the rate of 2 mg/kg/min did not stimulate glucose prod~ction.~' Thus, although peripheral net protein breakdown and hepatic gluconeogenesis are related, gluconeogenesis is not driven by the accelerated rate of amino acid release from peripheral tissues. Hyperglycemia, caused either by somatostatin in burned patients or by exogenous glucose infusion in normal subjects (Figure 2), elicits a 25-30% increase in alanine f l ~ x . ~This ~ , ~suggests ' that the increased rate of alanine synthesis and efflux from muscle in severe trauma and sepsis is a direct consequence of an increased availability of pyruvate from an accelerated glycolysis, reflecting a higher rate of glucose production and uptake.71 That is, the rate of glycolysis is the limiting factor for alanine release from muscle in severe trauma and sepsis. This is supported by the fact that when dichloroacetate was administered to burned patients to stimulate PDH activity, hence pyruvate oxidation, there was a concomitant decrease in alanine flux, clearly suggesting that the availability of pyruvate was the major determinant of alanine synthesis and It is possible that, because of the high rate of glycolysis in trauma and sepsis, alanine synthesis acts as an alternative glycolytic end product to lactate. Diversion of pyruvate to alanine may serve the useful purpose of preventing an excessive synthesis of lactate, which can disturb the normal redox state of the cell via the lactate dehydrogenase reaction and also ultimately lead to lactic acidosis. In addition, since alanine is a major gluconeogenic precursor, it is a safe way of transporting "excess" 3-carbon ketoacids, due to a stimulated glycolysis, from the peripheral tissues to the liver for g l u c o n e ~ g e n e s i s .In ~ ~doing , ~ ~ this, however, it receives amino-N from glutamate, aspartate, and the BCAAs in peripheral tissues and transfers it to the liver where it is incorporated into urea and excreted in the urine, resulting in negative N balance. It is therefore a possibility that although the primary role of alanine is to transfer carbon from the periphery in order to avoid lactic acidosis, in so doing it leaches N from the skeletal muscle bed. This is reflected by the lower intramuscular concentrations of several amino acids but, of most significance, of glutamine, which 155 represents the largest pool of intracellular labile a m i n ~ - N . Therefore, ~ ~ - ~ ~ the depletion of intracellular glutamine in trauma and sepsis may be the result of a general lack of nonessential amino-N, and more specifically glutamate, the immediate precursor of both alanine and glutamine, which is preferentially consumed for alanine synthesis. Thus, the increase in protein breakdown in muscle may be in response to the need for amino-N to produce alanine, since only five amino acids can donate N towards alanine synthesis." If this is true, then the increased synthesis and efflux of alanine from skeletal muscle is probably the primary cause for the decrease in protein synthesis seen in sepsis and trauma, as it has been shown that there is a close relationship between intramuscular glutamine concentrations and the rate of protein synthesis.73Furthermore, it has been proposed that the reduced rate of protein synthesis is due to a lack of nonessential a m i n ~ - N . ~ ~ On the other hand it has been argued that the increased efflux of alanine and other amino acids from peripheral tissues and uptake by the central organs, is an adaptation necessary to furnish amino acids from the largest protein store in the body, skeletal muscle, to the central organs for the synthesis of vital acute phase proteins.45 zy zy C. Response of Glutamine Metabolism to Severe Trauma and Sepsis Glutamine is synthesized from glutamic acid and ammonia in a reaction catalyzed by the enzyme glutamine synthetase, which occurs in most animal tissues and organ^.^^,^^ It is a precursor of pyrimidine, the purine ring, nicotinamide adenine dinucleotide, glucosamine-6-phosphate, the imidazole ring of histidine and several other N compounds." It also plays a central role in the storage and transport of ammonia in the body, thereby acting as a detoxifying agent.7',79Although most (80-90%) N is excreted as urea, all the enzymes of the urea cycle are present in significant quantities only in hepatic cells. The glutamine synthetic pathway is therefore the mechanism responsible for maintaining the ammonia level within a nontoxic range in all other organsS7' Unlike urea, which is primarily an excretory form of N, glutamine is considered a form of N storage, since it can be transported from one part of the body to another for reutilization.'0,81Glutamine is released from peripheral tissues*','' and taken up by kidney" and splanchnic bed.80,'' The role of glutamine as a means of storing N is reflected by its 156 zyxwvutsrqpon PROTEIN AND AA METABOLISM AFTER INJURY high concentration in the body. Glutamine is presneogenesis and concomitant ureagenesis. The ent in high concentrations in all organs and tisanatomical location of the gut makes it an ideal site for glutamine metabolism, hence ammonia detoxis u e ~ . ~It~ comprises " 69% of the free amino acids of human skeletal muscles5 and about 20% of the fication, since the portal blood draining the gut plasma amino-N pool of man.78The reutilization of passes through the liver before reaching the sysN stored as glutamine is to a large part facilitated temic circulation. by the presence of two glutamine hydrolyzing Why is it necessary for the gut to adapt to the enzymes, glutaminase 1 (L-glutamine amidouse of glutamine as its primary source of fuel in hydrolyase) and glutaminase 11 (L-glutamine severe catabolic illnesses? The answer may reside transaminase W-amidase) in most organs. 78,79 in the findings of studies in which glucocorticoids Glutamine is also an important source of were administered to otherwise normal dogs.87,93 energy for the gut, which has been shown to be a After dexamethasone treatment there was a 23% major site for the uptake of circulating glutafall in intracellular amino-N in skeletal muscle, Up to 66% of dietary glutamine is glutamine accounting for 80% of this fall. There metabolized in the intestinal cells,88which have a was a marked increase in glutamine efflux from requirement for glutamine as their principal respiskeletal muscle93 and a doubling of glutamine ratory fuel in both the fed and fasted states.89 uptake by the At the same time the gut As discussed above, the response to severe switched from a net glucose uptake to net glucose trauma or sepsis is characterized by a net loss of release, suggesting that the adaptation to an inbody nitrogen, most of which originates from creased use of glutamine as a source of energy skeletal muscle pr~tein.'~,'~ There is an increased leads to the sparing and production of g l ~ c o s e . ~ ' efflux of amino acids from skeletal m ~ s c l e , ~of~ , ~ This ~ , ~may be a means of providing the extra glucose which glutamine alone accounts for as much as needed by the body to fuel the metabolism of the 50%.%This is reflected by a marked reduction in reparative tissues which have a requirement for the size of the intracellular glutamine pool of glucose as an energy source. muscle tissue. 74 Despite this increase in glutamine A sufficient supply of glutamine to the gut flux, its plasma concentration is significantly deappears to be essential for gut integrity. When rats creased, indicating that there is an increased rate are kept on TPN solutions lacking glutamine, there of removal by other organs exceeding its rate of is mucosal atrophy of the small intestine.94The release from muscle t i s ~ u e .The ~ ~organ , ~ ~ primarily addition of glutamine to standard TI" solutions responsible for this accelerated rate of glutamine diminishes the mucosal atrophy that occurs during uptake is the Postoperatively, in dogs subTI",95 and treatment with intravenous nutrition jected to a standard laparatomy, there is a 75% solution containing glutamine diminishes jejunal increase in the rate of glutamine uptake by the gut, cell loss and accelerates mucosal recovery after whereas glutamine renal uptake remains uninjury by 5-fluorouracil. In addition daily urinary changed.45The increase in gut glutamine uptake nitrogen excretion showed an improved N bala n ~ e Also . ~ ~ in rats being fed 200 kcal/kg/day may be due to glutamine's role as a respiratory fuel." In the gut about 60% of glutamine carbon is intravenously, there was a 34% reduction in 3completely oxidized to C02, and the rest appears methylhistidine excretion in a group receiving 6.57 as lactate (11%)citrate (6%) and several other mmol/day glutamine compared with another amino acids. About 38% of its nitrogen is released group receiving 6.57 mmol/day alanine instead.97 into portal circulation as ammonia, 24%as alanineSince about 50% of 3-methylhistidine is believed to N, 28% as citrulline-N, and the rest as glutamate, be of gut origin in the rat, this is further evidence proline, and ornithine.88The portal ammonia rethat glutamine plays a major role in the preservamoved by the liver is used primarily for the syntion of gut integrity. These findings suggest that thesis of glutamine and urea and the alanine for the mucosal atrophy of the small intestine in gluconeogenesis and ~reagenesis.",~~ Although severe injury could be due to a deficiency of there is also an increased release of alanine from glutamine. peripheral tissues, gut glutamine-derived alanine There is uncertainty about the underlying accounts for about 50% of total hepatic alanine factor(s) responsible for this increased net efflux uptake. Thus, in fulfilling its role as a primary and consequential intracellular depletion of glutasource of energy for the gut following trauma, mine in peripheral tissues. There is evidence to glutamine also serves as a major carrier of carbon suggest that it is mediated by the glucocorticoid and nitrogen, to fuel the increased hepatic glucohormones which elicit changes in glutamine kinet- zyxwvutsr zyxwvutsrqp WOLFE, JAHOOR, AND HARTL ics identical to those seen in severe trauma and ~ e p s i s .It~has ~,~ been ~ proposed that the increased rate of net protein breakdown in peripheral tissues is necessary to supply amino acids as a source of energy, since there is a deficit in peripheral fuel supply due to an impaired free fatty acid uptake and an inability to completely oxidize glucose.98 The increased availability of amino acids is also needed to fuel an increased rate of gluconeogenesis which is mediated by a decreased insulin/glucagon molar ratio",68 in order to satisfy a greater demand for glucose by the body.66 This would adequately explain the intracellular depletion and increased efflux of glutamine from skeletal muscle tissue^,",'^ but as mentioned above, the theory of a deficit in peripheral fuel supply has not been supported experimentally. Another proposal is that wounded tissues have a requirement for glutamine. From rat hind limb perfusion studies it was reported that the decrease in intracellular glutamine induced by wounding skeletal muscle with A-carrageenan was not due to an increased release of the amino acid, nor to a reduction in the capacity to synthesize it, but to an increase in glutamine utilization at the site of injury, most probably by macrophages and fibroblasts present in the inflammatory infiltrate.99 This would suggest that wounded tissues also have a requirement for glutamine during healing. If an imbalance in protein kinetics in peripheral tissues is responsible for the loss of muscle tissues evident in severe trauma and sepsis, one would expect an elevation in intracellular amino acid concentration and not a decrease. This is true for several amino acids, the BCAAs, the aromatics, methionine, glycine, and alanine. The concentration of lysine, histidine, arginine, and glutamine, however, are significantly decreased, resulting in a markedly lower muscle-to-plasma ratio in the case of g l ~ t a m i n eThis . ~ ~ is a clear indication that there is an enhanced transport of these amino acids out of the intracellular compartment. In muscle tissue a special transport system has been identified, in vitro, for glutamine, whose properties can explain the abnormal profile of glutamine distribution in trauma and sepsis."' The response of the glutamine transporter is such that there is an increase in the net efflux of glutamine and a fall in intramuscular concentration occurring concomitantly with an increase in intramuscular sodium, after muscle was exposed in vivo to bacteria or endotoxin, prior to in vitro study, or if plasma insulin was decreased or epinephrine and glucagon increased in vivo prior to in vitro study."' These 157 experimental conditions are comparable to the situation in severe trauma and sepsis. It is possible that the stimulation of such an amino acid transport system represents one of the underlying mechanisms responsible for the massive loss of protein from muscle following trauma or sepsis. For example, it has been proposed by several investigators that the unavailability of the essential amino acid lysine intracellularly may be the rate-limiting factor for protein synthesis.74Also, intracellular glutamine depletion could mean a lack of sufficient nonessential aminoN to provide all of the nonessential amino acids needed for protein synthesis.74 Evidence to support the latter hypothesis comes from MacLennan et al.,73who demonstrated a positive relationship between protein synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle both in the presence and absence of insulin. They further hypothesized that glutamine may have a direct stimulatory effect on protein synthesis, based on the observation that methionine sulfoximine, an inhibitor of glutamine synthetase, depressed protein synthesis but not glutamine concentration in muscle perfused without glutamine. Because the inhibitor compound and glutamine are similar in structure, it was hypothesized that methionine sulfoximine binds to the same site at which glutamine normally binds to cause a stimulation of protein synthesis." Although the exact mechanism by which an adequate intracellular glutamine concentration stimulates protein synthesis is still uncertain, it is known that maintaining normal glutamine concentrations intramuscularly after a standard laparotomy in dogs by administering adequate balanced amino acid solutions or glutamine enriched (50%) amino acid solutions leads to a reduction of amino acid efflux from hindquarters and conservation of muscle protein." Thus in severe trauma and sepsis, conditions characterized by massive loss of muscle protein, preservation of normal intramuscular glutamine concentrations may be necessary to conserve muscle protein. 111. MEDIATORS OF THE CATABOLIC RESPONSE The mechanism(s) responsible for the protein catabolic response to trauma remains undefined. Therefore, discussion of the mediators must be speculative. This discussion will focus primarily on results of studies of human subjects in order to at zyxwv zyxwvutsrqp 158 PROTEIN AND AA METABOLISM AFTER INJURY least eliminate concern about the relevance of a particular animal model to the human response. A. Counter-regulatoryHormones The so-called counter-regulatory hormones glucagon, catecholamines, and glucocorticoids are elevated (to varying extents) in stressed patients. At one time or another, all of these have been shown to stimulate the catabolism of some component of the body (i.e., glycogen, fat, protein). It is therefore logical to look to these hormones as potential mediators of the metabolic response to trauma. B. Glucagon Results from studies in which glucagon is given acutely either as a bolus or over several hours show that small increases in plasma glucagon (up to 300 pg/ml) do not affect plasma amino acid (AA) concentration."' When pancreatic insulin concentration is blocked simultaneously, a fall in the concentration of glucogenic AA can be observed, which is, however, not associated with an increase of nitrogen excretion above baseline.lo2 A more pronounced, acute rise of the plasma glucagon concentration results in a decline of almost all plasma AA concentrations. 103r104 Two different mechanisms appear to contribute to this phenomenon. Since the fall of the BCAAs is not observed in insulin-deficiency,'05f106 glucagon seems to decrease BCAA levels indirectly via simultaneous stimulation of pancreatic insulin release and subsequent (insulin-mediated) increased uptake of BCAA in peripheral tissues. On the other hand, the fall in gluconeogenic AA may mainly occur via stimulated hepatic uptake, lo7 since it is less pronounced in patients with reduced liver function.1o3 In contrast to amino acid (AA) concentration, information on AA kinetics is limited. Acute hyperglucagonemia (>600 pg/ml) stimulates hepatic gluconeogenesis from alanine"* but does not affect peripheral AA release."' However, since leucine turnover and oxidation in insulin deficiency are accelerated by high glucagon concentration^,'^^ this may indicate a net loss of splanchnic protein under these circumstances. In a few studies, effects of prolonged glucagon administration (over several days) have been examined. Thus, infusion of moderate amounts of glucagon (plasma concentration <500 pg/ml) after prolonged fasting or in the fed state decreases concentrations of gluconeogenic AA but maintains or even increases levels of B C A A . ~ ~Si~multaJ ~ ~ , ~ ~ ~ neously, urea nitrogen excretion falls, but since excretion of ammonia and other nitrogen compounds rises, total nitrogen excretion remains unchanged."," Similar effects can be seen in diabetic patient^."^ The reason for these changes is unclear. It had been speculated that exogenous glucagon infusion reduces pancreatic glucagon secretion via feedback inhibition, which would reduce hepatic glucagon supply and subsequently decrease hepatic urea production. To explain the simultaneous decline in alanine concentration together with unchanged or rising BCAA levels, a reduction in BCAA oxidation was proposed."' Chronic infusion of large amounts of glucagon (plasma concentrations > 600 pg/ml) depresses concentration of all plasma AA in the fed state.114-116 Similar changes have been observed in the fasting state."' However, in the latter condition the concentration of BCAA remains stable, which points again to a potential role of insulin in mediating the decline of BCAA during caloric support. In contrast to small increases in plasma glucagon, high concentrations of this hormone lead to a moderately increased nitrogen excretion.110~114-116 Since this is not accompanied by either a simultaneous rise of 3-methylhistidine ex~retion"~,"~ or by an elevated net release of nitrogen from peripheral it is likely that the catabolic action of glucagon involves the net loss of splanchnic protein and/or the reduction of the free AA pool. Thus, elevated glucagon resulting from glucagon infusion in normal man seems to exert a dual effect on protein metabolism. Below 400-500 pg/ml plasma concentration (as measured with conventional assays), glucagon may slightly increase renal gluconeogenesis but does not necessarily result in a net loss of nitrogen. Above this threshold, a net catabolic action becomes evident. However, the relevance to these observations to the situation in severely injured patients is speculative at best, since there may be an alteration of the effectiveness of glucagon after injury and sepsis. This is the case with regard to glucagon action on glucose metabolism. In normal man, glucagon infusion has a transient effect on glucose production. This would lead to the conclusion that a chronic elevation in glucagon concentration in patients (resulting from increased endogenous secretion would have minimal effect on glucose production). However, reduction of the glucagon/ insulin ratio toward normal by means of somato- zyxwvu WOLFE, JAHOOR, AND HARTL zyxwvutsr statin infusion markedly lowered glucose production in both severely injured patients68 and in septic dogs.'I7 This indicates that a change in responsiveness to glucagon occurs in injured or septic patients. Alternatively, the simultaneous elevation of the other counterregulatory hormones in trauma patients may have a synergistic effect on the action of glucagon on protein metabolism (see below). In either case, it will require direct investigation of glucagon effects in injured or septic patients before a conclusion can be drawn. C. Glucocorticoids 159 infusion of epinephrine decreases plasma concentrations of almost all amino a ~ i d s . ' ~ Since ~ - ' ~ the ~ rate of leucine appearance and oxidation falls simultaneously, catecholamines appear to be able to reduce proteolysis. 127~128 These effects are not caused by an increase in plasma insulin concentration (indirectly mediated by catecholamines) but involve the activation of beta receptors.'26,'28It is unclear whether beta receptors reduce protein breakdown directly or indirectly, e.g., via accelerated lipolysis, also, the major site of the action of catecholamines on protein metabolism is not known. On the other hand catecholamines also increase the rate of alanine appearance, reflecting an increased peripheral nitrogen transfer from leucine to alanine. 127~12yThis is probably a secondary effect of the stimulation of glycolysis. The increased flux of alanine could cause an increased loss of amino acid nitrogen via stimulated urea synthesis and also a possible depletion of intracellular glutamine, with a reduction in protein synthesis resulting (see above). zyxwvut zyxwvut In acute experiments, administration of high doses of glucocorticoids (GC) (>200 mg cortisol on a daily basis) increases plasma concentrations of BCAA"8,"y and accelerates leucine and alanine turnover, thereby increasing nitrogen transfer from leucine to alanine.'I8 These findings indicate that GC have an acute proteolytic effect. However, this effect can be overcome by a modest rise in plasma insulin concentration. When moderate doses of GC (<200 mg cortisol or equivalentdday) have been given over several days, neither a change in plasma AA concentration nor in total nitrogen excretion has been observed.'20-'22This could be the result of simultaneously increased plasma insulin concentrations counteracting the GC effects. In any case, these results argue against a prominent physiological role for GC as a stimulator of protein catabolism. On the other hand, administration of higher doses of cortisol or equivalents does raise daily nitrogen excretion significantly despite a simultaneous increase in plasma i n ~ u l i n . ' ~ The ~ , ' ~ob~ served rise of alanine and glutamine concentrations points to a possible induction of proteolysis in peripheral tissues. Thus, GC excess can significantly increase nitrogen loss of the body, and GC must therefore be considered as possible mediators in catabolic states. However, the major site of the catabolic action of GC in man is still unknown. Furthermore, it is generally only in the early phase of response to injury that glucocorticoids are markedly elevated.'32At the time of greatest catabolic response in burn patients, cortisol secretion may not be elevated at all. D. Catecholamines So far, few papers have addressed the effects of catecholamines on protein metabolism in man. Long term studies are not available. Thus, acute E. Hormone Interactions It may be that the response to simultaneous elevations in all three counterregulatory hormones amplifies the catabolic effect of increases in one hormone. Bessey et al.'30 showed that infusion of the three hormones together into normal volunteers for 74 h stimulated protein turnover and protein breakdown, caused an increase in metabolic rate, and increased nitrogen excretion as compared with when the same subjects were infused with saline. Gelfand et al. also found that the triple hormone infusion increases N excretion and decreases plasma AA concentrations. However, leucine flux and oxidation were unaffected. 13' Thus, the triple hormone infusion into normal volunteers produces responses comparable in some regards to the catabolic response of patients. The magnitude of the protein catabolic response, however, is far less following hormone infusion than it is in severely stressed patients. Furthermore, the failure of leucine oxidation to increase during hormone inf~sion'~' indicates that there may be fundamental differences in the nature of the responses in the two circumstances. Thus, it is possible that these hormones only play a minor role in mediating the protein catabolic response. Alternatively, it is also possible that changes in responsiveness to given levels of hormones occur in patients. Thus, there is a limitation in the extent to which results from volunteer experiments can 160 PROTEIN AND AA METABOLISM AFTER INJURY zyxwvutsr animal models were published which addressed the possible role of prostaglandins as mediators of altered protein metabolism after trauma and in sepsis. In those studies, in which the experimental approach (endotoxin or bacteria infusion, severe abdominal sepsis) lead to a rapid deterioration of the clinical course, a role of prostaglandins in meIV. TISSUE FACTORS AS MODULATORS diating the simultaneously accelerated rate of proOF PROTEIN METABOLISM AFTER INJURY tein breakdown could not be demonstrated.~ 4 - l ~ OR SEPSIS This corresponds to the finding that in the acute phase after injury or induction of sepsis, muscle A. Muscle Tissue PGE2 production does not i n ~ r e a s e ' ~ ~and - ' ~ ' sugSince none of the conventional endocrine progests that other factors are responsible for the teolytic stimuli induces protein wasting in muscle initiation of protein-wasting processes under these tissue to the extent seen in patients with trauma or circumstances. However, in studies in which the sepsis, other possible mediators have come into study period was extended to several days, PGE2 prominence. In 1983-1984 Clowes, Dinarello, and production by muscle started to rise.149,150,151 NoneG ~ l d b e r g ' ~ ~developed -'~~ the hypothesis that theless, in these studies acute inhibition of cypolypeptides from activated monocytes may be the clooxygenase did not block protein degradation, 15' trigger of the catabolic response. After stimulation which corresponds to the results of Turinsky, by bacteria or endotoxin, leukocytes release lymMcNurlan, and Palmer'52-'54obtained during feedphokines (such as interleukin 1 IL-1), which itself ing, denervation, or insulin-induced changes in or whose split products [proteolysis-inducing facprotein metabolism, respectively. Furthermore, in tor (PIF)] can induce muscular PGE2synthesis and recent studies the acceleration of protein degradasubsequent protein breakdown. In line with these tion by PGE;! could not be r e p r o d u ~ e d . 'On ~ ~the .~~~ in vitro findings was the observation that infusion other hand, continuous administration of cyclooxof biologically obtained IL-1 in vivo increased ygenase inhibitors over several days reduced the skeletal muscle protein breakdown and oxidasepsis-mediated rise in peripheral protein catabotion.137 lism significantly.149~157 Since cyclooxygenase inIt is now known that not only leukocytes hibitors may affect other second messenger sysproduce IL-1, but large amounts of leukokines can tems, such as the cyclo AMP system, the effect on also arise from wound surfaces and vascular endoprotein degradation may not be PG specific, and thelial cells, when stimulated with e n d o t o ~ i n . ' ~ ~ , ' ~the ~ exact role of PG in the regulation of protein Furthermore, recombinant human IL-1 can stimumetabolism remains to be defined. late muscular PGE2 and PGF20c production, thereby increasing the PGE2/PGF2,, ratio.'40 The B. Plasma Proteins expected antagonistic effects of these prostanoids on muscular protein metabolism would be a net It has long been recognized that the concenloss of muscle protein mass, with simultaneously tration of plasma proteins is altered after trauma accelerated rates of protein breakdown and synand during infection. The fraction of proteins that thesis.14' This corresponds well to the pattern of increases after injury (the so-called acute-phase protein metabolism observed by other authors in plasma proteins), includes C-reactive protein, altrauma and sepsis12 and is also in line with the pha-l-acid glycoprotein, fibrinogen, and others. In finding that titers of PIF correlate well with the contrast to those proteins, there are many others peripheral release of amino acids under such cirwhose concentrations fall after trauma. These inc u m s t a n c e ~ On . ~ ~ the other hand, under certain clude albumin, transferrin, prealbumin, and reticonditions the effects of IL-1 on protein metabonol-binding protein. For almost a decade it has lism may be blunted and artificial.142 Exogenous been known that substances released from actistimulation of IL-1 synthesis in healthy man to a vated leucocytes are capable of initiating acutedegree that produces the clinical symptoms of phase protein synthesis. IL-1, PIF, and another infection fails to elicit significant catabolic effects cytokine, hepatocyte-stimulating factor (HSF), are on muscle. 143 currently considered to be the main factors stimuSubsequent to the hypothesis of Clowes, Dinlating this r e s ~ o n s e . ' ~ ' The - ~ ~ same factors may arello, and Goldberg, several studies in different simultaneously decrease the synthesis of other be extrapolated to the situation in severely stressed patients. 132 Experimentation in patients designed to elucidate the role of the stress hormones is therefore necessary before it is possible to make a final conclusion. ~ z WOLFE, JAHOOR, AND HARTL zyxwvutsr export proteins, such as albumin, and further, yet unidentified, monokines with similar qualities have been described.16' Those factors may exert most of their effects on hepatocytes directly, since inhibition of cyclooxygenase generally does not alter the acute-phase response.'44,145,162 However, the increased synthetic rate of fibrinogen does not result from a direct effect of l y m p h ~ k i n e s 'but ~~ may be mediated by prostaglandins, since it is sensitive to cyclooxygenase inhibition.lU Based on in vitro evidence and on results from animal experiments, it is likely that cytokines and tissue factors are involved in chronic changes of protein metabolism after trauma or in sepsis. However, as far as muscle tissue is concerned, their exact nature and relative contribution to the observed overall changes in protein turnover is not known, nor have any results in man been reported so far. Furthermore, no study of tissue in man has been reported. The use of specific antagonist or inhibitors (e.g., anti IL-1 or anti TNF), which possess only little side effects, may be useful. Cyclooxygenaseinhibitors are probably not a practical tool for studying these problems because of possible interferences from drug- and actionrelated side effects in other organ systems. With regard to visceral tissues, evidence from human experiment^^^,'^^ makes it likely that tissue factors are indeed involved in the initiation of the acute-phase response in man. Whether they also mediate the decrease in synthetic rates of other plasma proteins is not known. 9. Sim AJW, Wolfe BM, Young VR, Sugden B, and Moore FD: Lancet i:68-72, 1979. 10. 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