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Peptides in clinical nutrition
Clinical Nutrition (1991) 10, SUPP: 19-24 Q Longman GroupUK Ltd 1991
SESSION 2: CHAIRMAN’S INTRODUCTION
Peptides in clinical nutrition P. FijRST Institute for Biological Chemistry and Nutrition, Stuttgart 70, Germany
University of Hohenheim,
Short chain peptides -past and present questions
Six years ago, in 1984, many of us had the privilege of attending the first ESPEN-AJINOMOTO Research Symposium here in Hotel Atlantic. On that occasion I was asked to introduce the session devoted to a novel topic - peptides. I recall the commencement of my presentation: ‘. . . Peptides might be considered as brand new candidates for parenteral nutrition. Their potential use is based on the assumption that ‘tailored’ amino-acid solutions will increase the benefits of intravenous nutrition for specific patient groups. Undoubtedly this new approach has introduced a new dimension, though the explosion of new information about peptide assimilation is only a prelude to intravenous use of peptides in common clinical practice . .’ (1). I closed my lecture with the statement: ‘. . . in the light of the many important unresolved questions, some of which have been emphasised in this presentation, it must be stressed that studies in healthy and diseased man are urgently required before the use of peptides in parenteral nutrition can be established. Thus, it now appears that this novel form of therapy must await increased fundamental knowledge . . .’ (1). Indeed, during the last 6 years, the great interest directed towards peptide research is mirrored by the numerous publications on peptide synthesis and characterisation as well as relative to their in vivo uptake and subsequent utilisation. Considering the present and past problems I suggest three important topics of interest. The first is the availability of suitable peptides. They should be highly soluble and stable during sterilisation procedures. In addition, they must be sufficiently pure to be included in parenteral solutions. The second relates to human studies in healthy volunteers and stressed or malnourished patients. Thirdly, in vivo peptide handling might be of great interest when examining the capacity for and
Garbenstrape
30, D-7000
efficacy of utilisation as well as the affinity for hydrolysis of the individual peptides. The availability of suitable peptides
One of the major problems is the availability of dipeptides for experimental and therapeutical purposes. Availability depends upon the development of suitable methods for production to reduce the costs of synthesis by current techniques. The commonly used methods for synthesis of shortchain peptides are expensive and laborious. Enzyme-catalysed synthesis of dipeptides, applying proteases as biocatalyst, might be one economical and easily manageable approach. Various proteases of animal, plant, and microbial origin have been advocated for their applicability in catalysing the sequential synthesis of peptides and for the condensation of peptide fragments (2). Clear advantages of enzymatic peptide synthesis compared with chemical methods are (stereo)selectivity of the reaction and minimum protection of functional groups. The possibility of using free amino-acids as amino components and the application of ester substrates with selectivity cleavable N-protecting groups facilitate an easy release of the unprotected peptides. Recently, we suggested a novel approach employing papain or ficin as biocatalysts and using free amino-acids as nucleophiles. In a series of experiments, the optimum synthetic conditions and ester concentration, (PH, nucleophile reaction time) have been established for the model reaction between Z-Ala-OMe and free alanine. By using these optimised conditions. various glycine-, glutamine-, and cysteine-containing dipeptides could be synthesised in high yields (3, 4, 5). Indeed, a prerequisite for a commercial realisation of this biotechnological peptide synthesis is the development of a continuous synthetic procedure on an industrial scale. In preliminary studies, we
20
PEPTIDES IN CLINICAL NUTRITION
successfully used immobilised papain or ficin as a biocatalyst for the synthesis of dipeptides employing free amino-acids as the nucleophilic component. This approach offers the possibility of developing a suitable industrial reactor enabling biotechnological dipeptide synthesis in higher quantities (up to g or kg scale) (3, 4). Studies in healthy and diseased man
Animal studies with various synthetic dipeptides provide convincing evidence that peptides are easily available and their constituent amino-acids are instantly incorporated in various tissue proteins (6-10). Under conditions of continuous TPN or following a bolus injection, parenterally administered glutamine, tyrosine, and cystine containing dipeptides provide these amino-acids in free form for maintenance of their intra- and/or extra-cellular pools (11, 12). Interestingly, parenteral provision of L-alanyl-L-glutamine apparently reduces muscle loss of glutamine during stress (13, 14). In December 1985, one and a half years after the first ESPEN-AJINOMOTO Research Symposium we performed the first studies in healthy human volunteers in co-operation with Drs Vinnars and Westerman in Stockholm. In kinetic studies and/or following continuous infusion, it was demonstrated that the dipeptides L-alanyl-L-glutamine and glycyl-L-tyrosine injected or infused are readily hydrolysed. Peptide disappearance was accompanied by a prompt and considerable increase in the concentration of the constituting amino-acids. We observed no side-effects and no complications were reported (15,16). Very similar results were observed in subsequent studies by Brand1 et al (17) and also by Drs Roth and Lochs in Vienna (18, 19). These studies in healthy man provide firm evidence that synthetic dipeptides can be used as a safe and efficient source of free glutamine and tyrosine. In vivo utilisation of highly stable cystine containing peptides has, as yet, only been shown in animal experiments. Following intravenous injection of L-alanyl-L-cystine and glycyl-L-cystine the peptides are rapidly hydrolysed (12). This may serve as first evidence that short-chain peptides with C-terminal cystine residue may represent efficient sources of free cystine in parenteral nutrition and shows that human studies are warranted. As repeatedly emphasised, profound intracellular glutamine depletion is characteristic of injury
and other hypercatabolic conditions (20, 21). If maintenance of the intracellular glutamine pool promotes conservation of muscle protein (22, 23, 24), there is an obvious indication for glutamine supplements in the parenteral nutrition of patients with injury and infection, although instability prevents its addition to existing preparations (25). This can be overcome by the use of synthetic glutamine-containing dipeptides. Accordingly, with the use of a L-alanyl-L-glutamine-containing solution (- 20g peptide, corresponding to 12g glutamine) we showed that intramuscular glutamine concentration could be maintained in postoperative patients, whereas with an isonitrogenous-isoenergetic regimen but without the peptide it decreased significantly. Maintenance of the intracellular glutamine pool was associated with significantly better N-balance on each day of the study (26). Drs Vinnars and Wernerman showed that administration of both free glutamine (27) and glutamine containing dipeptides (28, 29) to postoperative patients was accompanied by improved N-balance and an improved intracellular glutamine pool. In severe injury, however, the daily provision of 20g peptide (12g glutamine) did not affect intracellular glutamine concentrations, while the cumulative nitrogen balance was significantly better than in an isonitrogenous, isoenergetic control group (14, 25). This positive effect was entirely due to initial improvements in this balance, as also found by Dr Wilmore et al (30). In critically ill patients, little influence of dipeptide administration was observed by Dr Roth et al (31, 32). Since, in these patients, high amounts of dipeptides were administered, the question must be raised whether the ability to utilise substrate is seriously limited in these patients. It is well known that substrate utilisation is ultimately dependent on the cellular function, and thus disturbed or impaired cellular activity may hamper substrate utilisation (33). Cellular viability and therefore the ability to utilise substrates are ultimately dependent on the rate of intracellular energy expenditure and are limited at low level of free energy (33). These lines of reasoning may lead to the conclusion that delivery of very large amounts of depeptide or glutamine is of questionable value. Nevertheless, a cautious interpretation of these results demands a consideration of pertinent recent knowledge about glutamine uptake and utilisation. Recent animal studies have demonstrated that glutamine consumption by the intestinal tract is markedly increased during catabolism (34, 35); the enhanced uptake is certainly due to
CLINICAL
the very favourable utilisation of glutamine in mucosal cells as described by Windmtiller and Spaeth (36). Furthermore, Newsholme emphasises that rapidly proliferating cells use glutamine preferentially as metabolic fuel (37, 38). According to the original hypothesis, the fate of intramuscular glutamine was to supply hepatic processes with carbon and nitrogen. Any deficit in glutamine could limit these processes during catabolic stress (20, 39, 40). However, in the light of our present knowledge, a revision of this hypothesis may be required. Thus, glutamine may serve primarily as an obligatory nutrient necessary for normal maintenance of the intestinal mucosa and, presumably, of proliferating cells. The reported increased intestinal requirement for metabolic fuel during catabolic stress might be matched by an enhanced demand for muscle glutamine and lead to intracellular glutamine depletion. Consequently, the delivery of adequate amounts of glutamine is essential to maintain the integrity of the mucosa (41,42,43) and of rapidly proliferating cells (37, 38), to preserve the muscle glutamine pool (14,26,30), and to improve overall nitrogen economy during conditions of stress. In post-operative patients, I suggest that the increased intestinal requirement and the cellular demand for metabolic fuel could be met by a daily provision of 12-13g of glutamine per day (26). In contrast, after severe accidental injury the same amount of the peptide (20g/day) did not influence the muscle free intracellular glutamine pool (14. 31, 32), although the nitrogen economy was improved in the immediate post-injury phase. Thus, the increased intestinal requirement and the cellular demand for metabolic fuel are partly compensated, but not met, in these patients. Interestingly, Wilmore et al obtained very similar results by supplementing, with 20g of glutamine per day, the nutrition of patients undergoing bone marrow transplantation and total body irradiation. Improvement in nitrogen balance with this amount was restricted to the first few days of the study and was followed by successive deterioration of the retention. Administration of 40g of glutamine per day. however, resulted in a prolongation of the beneficial effect throughout the study (30, 44) (personal communication). In vivo peptide handling The site of hydrolysis. The occurrence of equimolar increments of alanine, glutamine, glycine, and tyrosine, together with the prompt liberation of these amino-acids (15,16) suggests extracellular
NUTRITION
21
hydrolysis of the infused dipeptides. Intracellular cleavage of dipeptides has been repeatedly demonstrated after the enteral administration of the peptides, preferentially at the site of the renal or intestinal brush-border (45). This interpretation of intracellular hydrolysis after intravenous provision of the peptides would require an equimolar efflux/reabsorption from tissues or organs of the liberated amino-acids, a difficult proposition to accept, given the heterogenity of the various transport systems and intermediary metabolism of the constituent amino-acids. Considerable hydrolase activity in plasma as well as the recent report describing hepatic assimilation of dipeptides by enzymes located on the liver sinusoidal plasma membranes also suggest extracellular hydrolysis (46). Accordingly, Hundal and Rennie were able to identify a dipeptide hydrolase from the plasma membrane in skeletal muscle (47). Current studies, employing a newly developed in vitro assay for plasma hydrolase activity, demonstrate that glutamine-, tyrosine-, and cystine-containing peptides are substrates for human plasma free peptidases, the rate of hydrolysis clearly depending on the amino-acid composition of the peptides (48). Calculation of the total plasma hydrolase capacity against Ala-Gln and Gly-Tyr revealed 149 FmoYmin and 104 FmoY min, respectively. These high capacities may explain in part the extreme short plasma half-lives of these peptides observed in previous studies in vivo. Prolonged or rapid elimination of dipeptides. A benefit or disadvantage. Glycyl dipeptides are
claimed to be superior to other dipeptides since they exhibit a more prolonged half-life than that of the dipeptides with alternative N-terminal aminoacid residues (49, 50). This belief rests on the assumption that, due to their longer half-life, a greater fraction of the infused glycyl dipeptides would reach the tissues in an intact form, but no peptide could ever be detected in any tissues, even when infused in a large excess. Furthermore, as emphasised above, a growing body of evidence supports extracellular hydrolysis of parenterally administered dipeptides. Rapid elimination of the peptides offers the advantage that substantial amounts can be administered without their accumulation in body fluids, perhaps thus avoiding the possible risk of undesirable pharmacological and/or physiological sideeffects (51). This prediction is verified by the fact that, despite its presence as a peptide load. the
22
PEPTIDES IN CLINICAL NUTRITION
plasma concentration of the peptide given remained constant at trace levels throughout the infusion, accompanied by apparent steady state concentrations of the constituent free amino-acids (16). This stable amino-acid/peptide ratio allows an estimation of the plasma clearance rates of the dipeptides. The very similar metabolic clearance rate for L-alanyl-L-glutamine and glycyl-l-tyrosine are in excellent agreement with the half-lives of these dipeptides determined from bolus kinetics (15). These data, obtained from experiments with different protocols and underlying assumptions strongly indicate, by their agreement, that these dipeptides are handled similarly when infused intravenously. In general, we believe that speculations concerning the correlation between dipeptide structure and affinity for hydrolysis (49, 50) are unhelpful. An appropriate evaluation of the influence of structure on hydrolytic behaviour would require consideration of the effects of variation in about 400 possible structures. Indeed, given the intention to improve clinical nutrition using intravenously administered dipeptides, marginal differences in half-lives are of little importance. We suggest that the demonstration of specific or selective organ utilisation of a given dipeptide or amino-acidldipeptide combination is of much greater value. Future role for peptides in parenteral which of them - whither the future?
nutrition:
The potential use of short chain peptides as additional or alternative substrates for aminoacids in the frame of parenteral nutrition has recently been reviewed. As a main advantage, Adibi cites the low osmolarity of peptide-based parenteral solutions, thus enabling them to meet the nitrogen requirement of patients with severe fluid restriction (50). Another approach is based upon the premise that improvement in the quality of available amino-acid solutions, currently lacking glutamine, and also tyrosine and cystine is a major step in resolving the unsolved problem of how to formulate and prepare a ‘complete, wellbalanced’ amino-acid solution (52). The aim of this review was to consider the nutritional potential of short chain peptides and the need for glutamine peptides, especially in catabolic conditions. It is certainly too early to give an overall recommendation concerning the nature and specification of the most favourable peptide or peptide groups. The potential utilisation of a peptide by target tissues will in all probability vary
according to its structure and biological effects (13, 19, 48, 53, 54). Specific diseases may lead to certain amino-acid deficiences (20, 21), antagonisms or imbalances in various tissues; these conditions might cause a nutritional requirement for one or more specific peptides which are appropriate for use only in that specific condition to support the depleted tissue.
References 1. Ftirst P 1985 Peptides in clinical nutrition. Clinical Nutrition 4 (spec. suppl.): 105-115 2. Jakubke H-D. Kuhl P. Konnecke A 1985 Grundprinzipien der proteasekatalysierten Kniipfung der Peptidbindung. Angewandte Chemie 97: 79-87 3. Groeger U, Stehle P, Fiirst P, Leuchtenberger W, Drauz K 1989 Papain-catalysed synthesis of dipeptides. Food Biotechnology 2: 187-198 4. Stehle P, Bahsitta H-P, Monter B, Fiirst P 1990 Papain. catalysed synthesis of dipeptides. A novel approach using free amino-acids as nucleophiles. Enzyme and Microbial Technology 12: 56-60 5. Monter B, Herzog B, Stehle P, Fiirst P 1991 Kinetically controlled synthesis of dipeptides using ficin as biocatalyst. Applied Biotechnology and Microbiology (in press). 6. Daabees T T, Stegink L D 1978 L-Alanyl-L-tyrosine as a tyrosine source during intravenous nutrition of the rat. Journal of Nutrition 108: 1104-1113 7. Krzysik B A, Adibi S A 1979 Comparison of metabolism of glycine injected intravenously in free and dipeptide forms. Metabolism 28: 1211-1217 8. Adibi S A, Morse E L 1982 Enrichment of glycine pool in plasma and tissues by glycine, di-. tri-. and tetraglycine. American Journal of Physiology 243: E413E417 9. Amberger I, Fiirst P, Godel H, Graser Th, Stehle P, Pfaender P 1983 The potential parenteral application of the peptide L-alanyl-L-glutamine as a nitrogen source in severe catabolic states. Hoppe-Seyler’s Zeitschrift fiier Physiologische Chemie 364: 1253-1254 10. Stehle P, Ratz I, Ftirst P 1989 In vivo utilisation of intravenously supplied L-alanyl-L-glutamine in various tissues of the rat. Nutrition 5: 411-415 11. Albers S, Abele R, Amberger I, Mangold J, Pfaender P, Fiirst P 1984 Komplette parenterale Ernahrung mit und ohne einem synthetischen Dipeptid (L-Alanyl-Lglutamin) bei Ratten mit Verbrennungen. Aktuelle Ernaehrungsmedizin 9: 147-149 12. Stehle P, Albers S, Pollack L, Fiirst P 1988 In vivo utilisation of cystine-containing synthetic short chain peptides after intravenous bolus injection in the rat. Journal of Nutrition 118: 1470-1474 13. Roth E, Karner J, Ollenschlager G. Simmel A, Ftirst P. Funovics J 1988 Alanylglutamine reduces muscle loss of alanine and glutamine in post-operative anaesthetised dogs. Clinical Science 75: 641-648 14. Karner J, Roth E, Stehle P, Albers S, Fiirst P 1989 Influence of glutamine-containing dipeptides on muscle amino-acid metabolism. In: Nutrition in Clinical Practice. Hartig W, Dietze G, Weiner R. Ftirst P (eds). Karger, Basel, ~~56-70
CLINICAL NUTRITION 15. Albers S, Wernerman J, Stehle P, Vinnars E, Filrst P 1988 Availability of amino-acids supplied intravenously in healthy man as synthetic dipeptides: kinetic evaluation of L-alanyl-L-glutamine and glycyl-L-tyrosine. Clinical Science 75: 463-468 16. Albers S, Wernerman J, Stehle P, Vinnars E, Furst P 1989 Availability of amino-acids supplied by constant intravenous infusion of synthetic dipeptides in healthy man. Clinical Science 76: 643-648 17. Brand1 M, Sailer D, Langer K, Engelhardt A, Kleinhenz H, Adibi S A, Fekl W 1987 Parenteral nutrition with an amino-acid solution containing a mixture of dipeptides in man. Contributions to Infusion Therapy and Clinical Nutrition 17: 103-115 18. Roth E (1986) Veraanderungen im Aminosauren- und Proteinstoffwechsel bei chirurgischen Patienten. W. Zuckschwerdt, Muenchen, Bern, Wien. San Francisco. ppl-117 19. Hiibl W, Drum1 W, Langer K, Lochs H 1989 Influence of molecular structure and plasma hydrolysis on the metabolism of glutamine-containing dipeptides in humans. Metabolism 38 (suppl. 1): 5962 20. Furst P 1983 Intracellular muscle free amino-acids their measurement and function. Proceedings of the Nutrition Society 42: 451-462 21. Fiirst P 1985 Regulation of intracellular metabolism of amino-acids. In: Nutrition in Cancer and Trauma Sepsis. Bozzetti F, Dionigi R (eds). Karger, Base], ~~21-53 22. Jepson M M, Bates P C, Broadbent P, Pell J M, Millward D J 1988 Relationship between glutamine concentration and protein synthesis in rat skeletal muscle. American Journal of Physiology 255: E166E172 23. MacLennan P A, Brown R A, Rennie M J 1987 A positive relationship between protein synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle. FEBS Letters 215: 187-191 24. MacLennan P. Smith K, Weryk B, Watt P W, Rennie M J 1988 Inhibition of protein breakdown by glutamine in perfused rat skeletal muscle. FEBS Letters 237: 133-136 25. Fiirst P, Albers S, Stehle P 1990 Glutamine-containing dipeptides in parenteral nutrition. Journal of Parenteral and Enteral Nutrition 14: 118S124S 26. Stehle P, Zander J, Mertes N, Albers S. Puchstein Ch. Lawin P, Furst P 1989 Effect of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance after major surgery. The Lancet i: 231-233 27. Hammarqvist F, Wernerman J, Ali R. Von der Decken A, Vinnars E 1989 Addition of glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the fall in muscle protein synthesis. and improves nitrogen balance. Annals of Surgery 209: 455-461 28. Vinnars E, Hammarqvist F, Von der Decken A. Wernerman J 1990 Role of glutamine and its analogs in post-traumatic muscle amino-acid metabolism. Journal of Parenteral and Enteral Nutrition 14: 125S-129s 29. Hammarqvist F, Wernerman J, Von der Decken A, Vinnars E 1990 Alanyl-glutamine counteracts the depletion of free glutamine and the decline in protein synthesis post-operatively in skeletal muscle. Annals of Surgery (in press) 30. Ziegler T R, Benfell K, Smith R J, Young L S, Brown E. Ferrari-Balliviera E, Lowe D K, Wilmore D W 1990 Safetv and metabolic effects of L-elutamine
31.
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47.
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administration in humans. Journal of Parenteral and Enteral Nutrition 14: 137S-146s Karner J, Roth E 1990 Alanylglutamine infusions to patients with acute pancreatitis. Clinical Nutrition 9: 43-45 Steininger R, Karner J, Roth E, Langer K 1989 Infusion of dipeptides as nutritional substrates for glutamine, tyrosine, and branched-chain amino-acids in patients with acute pancreatitis. Metabolism 38 (suppl. 1): 78-81 Kinney J M, Ftirst P, Elwyn D H, Carpentier Y A 1988 The intensive care patient. In: Nutrition and Metabolism in Patient Care. Kinney J M, Jeejeebhoy K N, Hill Cl L, Owen 0 E (eds). W.B. Saunders, Philadelphia, London, Toronto, Montreal, Sydney, Tokyo, ~~656671 Souba W W, Wilmore D W 1983 Post-operative alteration of arteriovenous exchange of amino-acids across the gastrointestinal tract. Surgery 94: 342-350 Souba W W, Smith R J, Wilmore D W 1985 Glutamine metabolism by the intestinal tract. Journal of Parenteral and Enteral Nutrition 9: 608617 Windmueller H G, Spaeth A E 1974 Uptake and metabolism of plasma glutamine by thesmall intestine. Journal of Bioloaical Chemistrv 249: 507@5079 Newsholme E AT Crabtree B. krdawi M S M 1985 The role of high rates of glycolysis and glutamine utilisation in rapidly dividing cells. Bioscience Reports 5: 393400 Newsholme E A, Newsholme P. Curi R, Challoner E, Ardawi M S M 1988 A role for muscle in the immune system and its importance in surgery. trauma. sepsis and burns. Nutrition 4: 261-268 Vinnars E. Bergstrom J. Furst P 1975 Influence of the post-operative state on the intra-cellular free amino-acids in human muscle tissue. Annals of Surgery 182: 665-671 Askanazi J, Carpentier Y A, Michelsen CB, Elwyn D H, Fiirst P, Kantrowitz L R. Gumo F E. Kinnev J M 1980 Muscle and plasma amino-acids following injury. Influence of intercurrent infection. Annals of Surgery 192: 78-85 Hwang T L. O’Dwyer S T, Smith R J. Wilmore D W 1986 Preservation of the small bowel mucosa using glutamine-enriched parenteral nutrition. Surgical Forum 37: 56-58 Fox A D. Kriple S A. De Paula J A. Berman J M. Settle R G, Rombeau J L 1988 Effect of a glutaminesupplemented enteral diet on methotrexate-induced enterocolitis. Journal of Parenteral and Enteral Nutrition 12: 325-331 Souba W W 1988 The gut as a nitrogen-processing organ in the metabolic response to critical illness. Nutritional Support Services 8: 15-22 Smith R J, Wilmore D W 1990 Glutamine nutrition and requirements. Journal of Parenteral and Enteral Nutrition 14: 94S-99s Ganapathy V. Leibach F H 1986 Carrier-mediated reabsorption of small peptides in renal proximal tubule. American Journal of Physiology 251: F945F953 Lochs H. Morse E L. Adibi S A 1986 Mechanism of hepatic assimilation of dipeptides. Transport versus hydrolysis. Journal of Biological Chemistry 261: 14976 14981 Hundal H S. Rennie M J 1988 Skeletal muscle tissue
contains extracellular aminopeptidase activity against Ala-Gin but no peptide transporter. European Journal Clinical Investigation 18: 16sA34 (Abstract) 48. Stehle P. Furst P 1990 In vitro hydrolysis of glutamine-, tyrosine- and cystine-containing short chain peptides. Clinical Nutrition 9: 37-38
of
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PEPTIDES IN CLINICAL NUTRITION
49. Adibi S A. Paleos G A, Morse E L 1986 Influence of molecular structure on half-life and hydrolysis of dipeptides in plasma: importance of glycine as Nterminal amino-acid residue. Metabolism 35: 830-836 50. Adibi S A 1987 Experimental basis for use of peptides as substrates for parenteral nutrition: a review. Metabolism 36: 1001-1011 51. McCain H W, Bilotta J, Lamster I B 1987 Endocorphinergic modulation of immune function: potent action of the dipeptide glycyl-L-glutamine. Life Sciences 41: 169-176
52. Wretlind A 1981 Parenteral nutrition. Nutrition Reviews 39: 257-265 53. Lochs H, HubI W 1990 Metabolic basis for selecting glutamine-containing substrates for parenteral nutrition. Journal of Parenteral and Enteral Nutrition 14: 114S117s 54. Abumrad N N. Morse E L. Lochs H, Williams P E, Adibi S A 1989 Possible sources of glutamine for parenteral nutrition: impact on glutamine metabolism. American Journal of Physiology 257: E228-E234
SESSION 2: CHAIRMAN’S INTRODUCTION
Peptides in clinical nutrition P. FijRST Institute for Biological Chemistry and Nutrition, Stuttgart 70, Germany
University of Hohenheim,
Short chain peptides -past and present questions
Six years ago, in 1984, many of us had the privilege of attending the first ESPEN-AJINOMOTO Research Symposium here in Hotel Atlantic. On that occasion I was asked to introduce the session devoted to a novel topic - peptides. I recall the commencement of my presentation: ‘. . . Peptides might be considered as brand new candidates for parenteral nutrition. Their potential use is based on the assumption that ‘tailored’ amino-acid solutions will increase the benefits of intravenous nutrition for specific patient groups. Undoubtedly this new approach has introduced a new dimension, though the explosion of new information about peptide assimilation is only a prelude to intravenous use of peptides in common clinical practice . .’ (1). I closed my lecture with the statement: ‘. . . in the light of the many important unresolved questions, some of which have been emphasised in this presentation, it must be stressed that studies in healthy and diseased man are urgently required before the use of peptides in parenteral nutrition can be established. Thus, it now appears that this novel form of therapy must await increased fundamental knowledge . . .’ (1). Indeed, during the last 6 years, the great interest directed towards peptide research is mirrored by the numerous publications on peptide synthesis and characterisation as well as relative to their in vivo uptake and subsequent utilisation. Considering the present and past problems I suggest three important topics of interest. The first is the availability of suitable peptides. They should be highly soluble and stable during sterilisation procedures. In addition, they must be sufficiently pure to be included in parenteral solutions. The second relates to human studies in healthy volunteers and stressed or malnourished patients. Thirdly, in vivo peptide handling might be of great interest when examining the capacity for and
Garbenstrape
30, D-7000
efficacy of utilisation as well as the affinity for hydrolysis of the individual peptides. The availability of suitable peptides
One of the major problems is the availability of dipeptides for experimental and therapeutical purposes. Availability depends upon the development of suitable methods for production to reduce the costs of synthesis by current techniques. The commonly used methods for synthesis of shortchain peptides are expensive and laborious. Enzyme-catalysed synthesis of dipeptides, applying proteases as biocatalyst, might be one economical and easily manageable approach. Various proteases of animal, plant, and microbial origin have been advocated for their applicability in catalysing the sequential synthesis of peptides and for the condensation of peptide fragments (2). Clear advantages of enzymatic peptide synthesis compared with chemical methods are (stereo)selectivity of the reaction and minimum protection of functional groups. The possibility of using free amino-acids as amino components and the application of ester substrates with selectivity cleavable N-protecting groups facilitate an easy release of the unprotected peptides. Recently, we suggested a novel approach employing papain or ficin as biocatalysts and using free amino-acids as nucleophiles. In a series of experiments, the optimum synthetic conditions and ester concentration, (PH, nucleophile reaction time) have been established for the model reaction between Z-Ala-OMe and free alanine. By using these optimised conditions. various glycine-, glutamine-, and cysteine-containing dipeptides could be synthesised in high yields (3, 4, 5). Indeed, a prerequisite for a commercial realisation of this biotechnological peptide synthesis is the development of a continuous synthetic procedure on an industrial scale. In preliminary studies, we
20
PEPTIDES IN CLINICAL NUTRITION
successfully used immobilised papain or ficin as a biocatalyst for the synthesis of dipeptides employing free amino-acids as the nucleophilic component. This approach offers the possibility of developing a suitable industrial reactor enabling biotechnological dipeptide synthesis in higher quantities (up to g or kg scale) (3, 4). Studies in healthy and diseased man
Animal studies with various synthetic dipeptides provide convincing evidence that peptides are easily available and their constituent amino-acids are instantly incorporated in various tissue proteins (6-10). Under conditions of continuous TPN or following a bolus injection, parenterally administered glutamine, tyrosine, and cystine containing dipeptides provide these amino-acids in free form for maintenance of their intra- and/or extra-cellular pools (11, 12). Interestingly, parenteral provision of L-alanyl-L-glutamine apparently reduces muscle loss of glutamine during stress (13, 14). In December 1985, one and a half years after the first ESPEN-AJINOMOTO Research Symposium we performed the first studies in healthy human volunteers in co-operation with Drs Vinnars and Westerman in Stockholm. In kinetic studies and/or following continuous infusion, it was demonstrated that the dipeptides L-alanyl-L-glutamine and glycyl-L-tyrosine injected or infused are readily hydrolysed. Peptide disappearance was accompanied by a prompt and considerable increase in the concentration of the constituting amino-acids. We observed no side-effects and no complications were reported (15,16). Very similar results were observed in subsequent studies by Brand1 et al (17) and also by Drs Roth and Lochs in Vienna (18, 19). These studies in healthy man provide firm evidence that synthetic dipeptides can be used as a safe and efficient source of free glutamine and tyrosine. In vivo utilisation of highly stable cystine containing peptides has, as yet, only been shown in animal experiments. Following intravenous injection of L-alanyl-L-cystine and glycyl-L-cystine the peptides are rapidly hydrolysed (12). This may serve as first evidence that short-chain peptides with C-terminal cystine residue may represent efficient sources of free cystine in parenteral nutrition and shows that human studies are warranted. As repeatedly emphasised, profound intracellular glutamine depletion is characteristic of injury
and other hypercatabolic conditions (20, 21). If maintenance of the intracellular glutamine pool promotes conservation of muscle protein (22, 23, 24), there is an obvious indication for glutamine supplements in the parenteral nutrition of patients with injury and infection, although instability prevents its addition to existing preparations (25). This can be overcome by the use of synthetic glutamine-containing dipeptides. Accordingly, with the use of a L-alanyl-L-glutamine-containing solution (- 20g peptide, corresponding to 12g glutamine) we showed that intramuscular glutamine concentration could be maintained in postoperative patients, whereas with an isonitrogenous-isoenergetic regimen but without the peptide it decreased significantly. Maintenance of the intracellular glutamine pool was associated with significantly better N-balance on each day of the study (26). Drs Vinnars and Wernerman showed that administration of both free glutamine (27) and glutamine containing dipeptides (28, 29) to postoperative patients was accompanied by improved N-balance and an improved intracellular glutamine pool. In severe injury, however, the daily provision of 20g peptide (12g glutamine) did not affect intracellular glutamine concentrations, while the cumulative nitrogen balance was significantly better than in an isonitrogenous, isoenergetic control group (14, 25). This positive effect was entirely due to initial improvements in this balance, as also found by Dr Wilmore et al (30). In critically ill patients, little influence of dipeptide administration was observed by Dr Roth et al (31, 32). Since, in these patients, high amounts of dipeptides were administered, the question must be raised whether the ability to utilise substrate is seriously limited in these patients. It is well known that substrate utilisation is ultimately dependent on the cellular function, and thus disturbed or impaired cellular activity may hamper substrate utilisation (33). Cellular viability and therefore the ability to utilise substrates are ultimately dependent on the rate of intracellular energy expenditure and are limited at low level of free energy (33). These lines of reasoning may lead to the conclusion that delivery of very large amounts of depeptide or glutamine is of questionable value. Nevertheless, a cautious interpretation of these results demands a consideration of pertinent recent knowledge about glutamine uptake and utilisation. Recent animal studies have demonstrated that glutamine consumption by the intestinal tract is markedly increased during catabolism (34, 35); the enhanced uptake is certainly due to
CLINICAL
the very favourable utilisation of glutamine in mucosal cells as described by Windmtiller and Spaeth (36). Furthermore, Newsholme emphasises that rapidly proliferating cells use glutamine preferentially as metabolic fuel (37, 38). According to the original hypothesis, the fate of intramuscular glutamine was to supply hepatic processes with carbon and nitrogen. Any deficit in glutamine could limit these processes during catabolic stress (20, 39, 40). However, in the light of our present knowledge, a revision of this hypothesis may be required. Thus, glutamine may serve primarily as an obligatory nutrient necessary for normal maintenance of the intestinal mucosa and, presumably, of proliferating cells. The reported increased intestinal requirement for metabolic fuel during catabolic stress might be matched by an enhanced demand for muscle glutamine and lead to intracellular glutamine depletion. Consequently, the delivery of adequate amounts of glutamine is essential to maintain the integrity of the mucosa (41,42,43) and of rapidly proliferating cells (37, 38), to preserve the muscle glutamine pool (14,26,30), and to improve overall nitrogen economy during conditions of stress. In post-operative patients, I suggest that the increased intestinal requirement and the cellular demand for metabolic fuel could be met by a daily provision of 12-13g of glutamine per day (26). In contrast, after severe accidental injury the same amount of the peptide (20g/day) did not influence the muscle free intracellular glutamine pool (14. 31, 32), although the nitrogen economy was improved in the immediate post-injury phase. Thus, the increased intestinal requirement and the cellular demand for metabolic fuel are partly compensated, but not met, in these patients. Interestingly, Wilmore et al obtained very similar results by supplementing, with 20g of glutamine per day, the nutrition of patients undergoing bone marrow transplantation and total body irradiation. Improvement in nitrogen balance with this amount was restricted to the first few days of the study and was followed by successive deterioration of the retention. Administration of 40g of glutamine per day. however, resulted in a prolongation of the beneficial effect throughout the study (30, 44) (personal communication). In vivo peptide handling The site of hydrolysis. The occurrence of equimolar increments of alanine, glutamine, glycine, and tyrosine, together with the prompt liberation of these amino-acids (15,16) suggests extracellular
NUTRITION
21
hydrolysis of the infused dipeptides. Intracellular cleavage of dipeptides has been repeatedly demonstrated after the enteral administration of the peptides, preferentially at the site of the renal or intestinal brush-border (45). This interpretation of intracellular hydrolysis after intravenous provision of the peptides would require an equimolar efflux/reabsorption from tissues or organs of the liberated amino-acids, a difficult proposition to accept, given the heterogenity of the various transport systems and intermediary metabolism of the constituent amino-acids. Considerable hydrolase activity in plasma as well as the recent report describing hepatic assimilation of dipeptides by enzymes located on the liver sinusoidal plasma membranes also suggest extracellular hydrolysis (46). Accordingly, Hundal and Rennie were able to identify a dipeptide hydrolase from the plasma membrane in skeletal muscle (47). Current studies, employing a newly developed in vitro assay for plasma hydrolase activity, demonstrate that glutamine-, tyrosine-, and cystine-containing peptides are substrates for human plasma free peptidases, the rate of hydrolysis clearly depending on the amino-acid composition of the peptides (48). Calculation of the total plasma hydrolase capacity against Ala-Gln and Gly-Tyr revealed 149 FmoYmin and 104 FmoY min, respectively. These high capacities may explain in part the extreme short plasma half-lives of these peptides observed in previous studies in vivo. Prolonged or rapid elimination of dipeptides. A benefit or disadvantage. Glycyl dipeptides are
claimed to be superior to other dipeptides since they exhibit a more prolonged half-life than that of the dipeptides with alternative N-terminal aminoacid residues (49, 50). This belief rests on the assumption that, due to their longer half-life, a greater fraction of the infused glycyl dipeptides would reach the tissues in an intact form, but no peptide could ever be detected in any tissues, even when infused in a large excess. Furthermore, as emphasised above, a growing body of evidence supports extracellular hydrolysis of parenterally administered dipeptides. Rapid elimination of the peptides offers the advantage that substantial amounts can be administered without their accumulation in body fluids, perhaps thus avoiding the possible risk of undesirable pharmacological and/or physiological sideeffects (51). This prediction is verified by the fact that, despite its presence as a peptide load. the
22
PEPTIDES IN CLINICAL NUTRITION
plasma concentration of the peptide given remained constant at trace levels throughout the infusion, accompanied by apparent steady state concentrations of the constituent free amino-acids (16). This stable amino-acid/peptide ratio allows an estimation of the plasma clearance rates of the dipeptides. The very similar metabolic clearance rate for L-alanyl-L-glutamine and glycyl-l-tyrosine are in excellent agreement with the half-lives of these dipeptides determined from bolus kinetics (15). These data, obtained from experiments with different protocols and underlying assumptions strongly indicate, by their agreement, that these dipeptides are handled similarly when infused intravenously. In general, we believe that speculations concerning the correlation between dipeptide structure and affinity for hydrolysis (49, 50) are unhelpful. An appropriate evaluation of the influence of structure on hydrolytic behaviour would require consideration of the effects of variation in about 400 possible structures. Indeed, given the intention to improve clinical nutrition using intravenously administered dipeptides, marginal differences in half-lives are of little importance. We suggest that the demonstration of specific or selective organ utilisation of a given dipeptide or amino-acidldipeptide combination is of much greater value. Future role for peptides in parenteral which of them - whither the future?
nutrition:
The potential use of short chain peptides as additional or alternative substrates for aminoacids in the frame of parenteral nutrition has recently been reviewed. As a main advantage, Adibi cites the low osmolarity of peptide-based parenteral solutions, thus enabling them to meet the nitrogen requirement of patients with severe fluid restriction (50). Another approach is based upon the premise that improvement in the quality of available amino-acid solutions, currently lacking glutamine, and also tyrosine and cystine is a major step in resolving the unsolved problem of how to formulate and prepare a ‘complete, wellbalanced’ amino-acid solution (52). The aim of this review was to consider the nutritional potential of short chain peptides and the need for glutamine peptides, especially in catabolic conditions. It is certainly too early to give an overall recommendation concerning the nature and specification of the most favourable peptide or peptide groups. The potential utilisation of a peptide by target tissues will in all probability vary
according to its structure and biological effects (13, 19, 48, 53, 54). Specific diseases may lead to certain amino-acid deficiences (20, 21), antagonisms or imbalances in various tissues; these conditions might cause a nutritional requirement for one or more specific peptides which are appropriate for use only in that specific condition to support the depleted tissue.
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