Metabolic Abnormalities in Renal Failure Guarnieri G, Panetta G, Toigo G (eds): Metabolic and Nutritional Abnormalities in Kidney Disease. Contrib Nephrol. Basel, Karger, 1992, vol 98, pp 1-10
Muscle Amino Acid and Protein Metabolism in Chronic Renal Failure Giacomo Garibotto, Rodolfo Russo, Cristina Robaudo, Stefano Saffioti, Alberto Magnasco, Giacomo Deferrari, Alberto Tizianello Department of Internal Medicine, Divisions of Nephrology and Clinical Methodology, University of Genova, Italy
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Information on protein turnover in patients with chronic renal failure (CRF) is still poor. Uremic patients, especially those undergoing maintenance dialysis, show a high prevalence of malnutrition [1], attributed mainly to inadequate intake of nutrients and/or superimposed illnesses. A major question is whether or not CRF per se affects protein turnover. The occurrence of alterations in levels of circulating amino acids (AA) and in AA exchange across the organs in well-nourished patients with moderate renal failure suggests that CRF per se, independently of exogenous malnutrition, can play a role in abnormalities in AA metabolism [2]. In addition, a number of alterations in muscle exchange of ΑΑ have been observed in patients studied in the postabsorptive and protein-fed state [2-4]. These abnormalities may be a telltale sign of an altered protein metabolism in muscle. Results reported here have been obtained from studies carried out in patients with moderate CRF (creatinine clearance 18 ± 3 ml/min • 1.73 m2, range 10-35) who were on a diet which provided 30-35 kcal/day and 0.80.9 g protein/kg/day and were actively employed. Muscle AA and protein metabolism was evaluated by the arteriovenous difference technique across the leg and forearm [2-6].
Garibotto/Russo/Robaudo/Saffioti/M agnasco/Deferrari/Tizi anello
a 140 c
~
Εi
120 75 -
50
Ø
-
J 0
Total AA
Irι Ala
Gin
ΣPhe+Tyr+Lys
Ii
Val Leu
2
Ι
ile
Fig. 1. Net release of total AA, Ala, Gin and the sum of Phe, Tyr and Lys (Σ) (a) and BCAA (b) from the leg in patients with CRF (0) and in subjects with normal renal function (c) in the postabsorptive state [from 2].
Abnormalities in Muscle AA Metabolism in the Postabsorptive State in Patients with CRF
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Every day in a healthy 70-kg man about 300 g of protein are degraded and 80% of released AA reutilized for new protein synthesis. Peripheral tissues play an important role in endogenous protein turnover inasmuch as these tissues, chiefly muscle, are the largest reservoir of body protein. Accordingly, evaluation of AA exchange across peripheral tissues, namely leg and forearm, can provide an indirect approach to an understanding of muscle AA and protein metabolism. In postabsorptive patients with CRF, peripheral tissues release most individual AA, including glutamine and alanine, as well as total AA, in amounts similar to controls (fig. 1) [2]. The same is true for phenylalanine, tyrosine and lysine, which are neither catabolized nor synthesized by muscle and therefore express net proteolysis. These findings confirm previous studies in which leg blood flow was not measured [3, 4]. All the data suggest that in renal patients before the uremic stage, net proteolysis is not increased. Different results are obtained in uremic rats, in which an augmented release of AA was generally observed [2]. It has to be pointed out that the arteriovenous difference technique reveals only the net bal-
Muscle Amino Acid and Protein Metabolism in Renal Failure
Controls: ΕΑΑ CRF: ΕΑΑ
a 0.9
.-. •
-•
ΝΕΑΑ o—o ΝΕΑΑ -.ο
b
Oral ΑΑ
ΤΡ ''1`T
,
0.7
c
',
,,
ι
ν
120 -
-λ
a
80 -
0.5
o
w m
C
~ Ε ν
0.3
40w
ν
U
C
0.1
20o
15
30
45
Time, min
60 70
(J Controls CRF
~ 100 -
ι
~
η
3
1
I
ΕΑΑ ΝΕΑΑ Total ΑΑ
ance of AA and net proteolysis. No information on the absolute rates of tissue protein breakdown and synthesis can therefore be deduced by this method and the occurrence of counterbalanced alterations in protein synthesis and/or breakdown cannot be ruled out. It is noteworthy that even small changes in protein turnover may be crucial for maintaining body mass, owing to the large amount of protein degraded and synthesized every day [7]. The above reported studies [2-5] have also shown the existence of selective disturbances in leg metabolism of branched-chain AA (BCAA) in postabsorptive patients with CRF. In patients, the release of valine and leucine from the leg is lower than in controls (fig. 1). In addition, no significant release of any individual branched-chain keto-acid (BCKA) has been observed, whereas, in controls, small amounts of keto-leucine and keto-isoleucine are released by the leg [2, 8]. It has also been demonstrated
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Fig. 2. Increments above basal values in arterial levels of ΕΑΑ and ΝΕΑΑ (a) and their respective arterial areas (b) after AA ingestion in patients with CRF and in subjects with normal renal function. Significance of difference from the corresponding value in normal subjects: a p < 0.025; b p < 0.01; c p < 0.001 [from 17, with permission].
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
4
that the low muscle release of BCAA is responsible for their reduced circulating levels [3, 4, 9]. The reason why in CRF the release of BCAA from peripheral tissues is low is not clear, but an increased degradation of BCAA can be suspected. The low utilization of glucose by muscle cells caused by insulin resistance [ 10, 11 ] may increase BCAA degradation in order to meet energy fuel demand. Α potential role of metabolic acidosis in promoting BCAA degradation is suggested by recent data obtained in rats [ 12]. Such a possibility is supported by the observation that in chronic hemodialysis patients valine levels in muscle cells are reduced and directly correlated with extracellular ΗCO3 concentration [ 13]. Abnormalities in muscle BCAA metabolism in renal patients might affect muscle protein turnover, taking into consideration the regulatory role played by these AA, mainly leucine, in muscle protein turnover. The altered BCAA metabolism may exert a catabdlic effect, as it has been shown in rats that leucine stimulates muscle protein synthesis, while keto-leucine inhibits protein breakdown [ 12]. Furthermore, if, as commonly believed, protein synthesis requires the supply of a high quality AA pattern [14, 15], in CRF the availability of BCAA, mainly valine, in muscle may be insufficient. Accordingly, in patients the reduced muscle intracellular valine may become rate limiting for protein synthesis.
Despite a large number of studies dealing with nutritional treatment of patients with CRF, data on muscle metabolism of AA in the protein-fed state are scanty. Evidence has recently been provided that in patients with CRF after protein and/or AA ingestion, arterial AA profile is markedly altered and characterized by exaggerated increments in total AA mainly owing to nonessential AA (ΝΕΑΑ) levels (fig. 2) [ 16, 17]. Major abnormalities regard glutamine, proline, glutamate, serine, glycine and alanine [ 16, 17]. The altered postprandial AA profile depends chiefly on an abnormally increased escape of ΝΕΑΑ from splanchnic organs [ 17]. Effects of the abnormal AA supply on the AA exchange across peripheral tissues have been recently investigated [2, 5]. After AA ingestion in patients, as well as in controls, the release of most AA, observed in the basal state, is promptly reversed into a marked uptake (fig. 3). While the uptake of essential AA (ΕΑΑ) is similar in both groups, in patients the leg extracts ΝΕΑΑ in by far
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Abnormalities in Muscle AΑ Metabolism in the Protein-Fed State in Patients with CRF
5
Muscle Amino Acid and Protein Metabolism in Renal Failure
Controls: ΕΑΑ •---• ΝΕΑΑ 0-o CRE: ΕEE •-+ ΝΕΑΑ o-o α
ι
ι
0 15 30 45 60 Time, min
75
ΕΑΑ
ΝΕΑΑ
Total ΑΑ
greater amounts than in controls, paralleling changes in circulating ΝΕΑΑ levels. It follows that also the uptake of total ΑΑ by peripheral tissues is increased. At the same time in patients BCAA uptake by the leg is similar to controls [5, 9]. However, as a consequence of the increased uptake of total AA, the uptake of BCAA by the leg is only one-third of that of total AA, whereas it is about one-half ín controls [5, 9]. So, in renal failure, after AA ingestion, peripheral tissues cope with the altered supply of AA by an increased and unbalanced uptake of them. AA are removed from arterial blood far in excess of their relative frequencies in muscle protein and, consequently, only a fraction of them can be utilized for protein synthesis. Likely, ΝΕΑΑ are degraded to a greater extent. Taken together, these data demonstrate that in patients with CRF muscle metabolism of AA is abnormal both in the postabsorptive and protein-fed state. Repercussions, if any, on protein turnover are unknown.
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Fig. 3a, b. Leg exchange of EAA, NEØ and total AA after AA ingestion in patients with CRF and in subjects with normal renal function. * Statistically different from controls [from 2, 5].
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
6
Data on protein turnover in CRF are fragmentary; most studies have been carried out in rats and sometimes arbitrarily extrapolated to man. Uremic rats show an enhanced net proteolysis during fasting [ 18, 19]. Starvation reduces muscle protein synthesis and increases protein muscle breakdown more severely in uremic rats than in controls [ 18, 19]. In fed uremic rats, muscle protein synthesis is reported to be decreased [20, 21] or, conversely, normal [ 18, 19]. Insulin resistance and abnormal energy metabolism in muscle [12] have been proposed as major potential catabolic factors in uremic rats. Recently, evidence has been provided that metabolic acidosis is responsible for the increased muscle protein breakdown in uremic rats [ 12]. Data dealing with muscle protein metabolism in humans with CRF are sparse and somewhat contradictory. In patients with advanced uremia and/or under dialytic treatment, loss of lean body mass occurs frequently [1]. Furthermore, muscle RNA/DNA ratio is decreased in advanced uremia or under protein-restricted diets [22]. Protein turnover in patients with CRF has so far been investigated by the whole-body isotope dilution technique. A reduced whole-body protein turnover has been reported in children in hemodialysís [23]; many of these patients were malnourished and no control group was studied. Conversely, a reduced protein synthesis in the presence of a normal protein breakdown has been reported in apparently well-nourished adults in hemodialysís [24]. On the other hand, a normal protein degradation, but an enhanced proteolytic response to thyroid hormones have been reported in adult hemodialysis patients [25]. Recently, the adaptation of patients with CRF to low protein diets has been studied [26]; in these patients, AA oxidation and protein degradation decreased as in controls. Advanced uremia, dialysis therapy and different calorie and/or protein intake can partially explain differences among these studies. Recently, a technique combining the method of the arteriovenous difference across the forearm with the systemic infusion of 3H-phenylalanine has been proposed in order to estimate the individual rates of protein synthesis and breakdown in muscle [27, 28]. The method is based on the steady-state kinetics of phenylalanine in muscle. Since phenylalanine is not metabolized in muscle, the rates of phenylalanine disposal across the forearm reflect its rates of incorporation into protein, while rates of appearance of phenylalanine from muscle reflect its release from muscle protein
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Muscle Protein Metabolism in CRF
Muscle Amino Acid and Protein Metabolism in Renal Failure
7
breakdown. Net balance of phenylalanine across the forearm expresses net proteolysis. This procedure has been used in order to evaluate muscle protein turnover in well-nourished patients with CRF in the postabsorptive state [6]. In controls, data on forearm protein turnover are fairly comparable with those previously reported in normal postabsorptive subjects [27, 28]. In patients, phenylalanine appearance from the forearm is 30% increased (fig. 4) indicating an increased protein breakdown. Also, phenylalanine disposal is augmented, outlining an increased protein synthesis. Net balance of phenylalanine, that is net proteolysis, is not changed. The release of tyrosine and lysine and total AA is similar to controls and confirms results obtained when AA exchange across the leg was evaluated [2, 4]. It is interesting to note that most patients had metabolic acidosis. Moreover, both in patients and controls protein breakdown was inversely
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Fig. 4. Rates of release (protein breakdown), rates of disposal (protein synthesis) and net balance of phenylalanine (net proteolysis) across the forearm in patients with CRF (0) and in subjects with normal renal function (σ) in the postabsorptive state [from 6, with permission].
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
8
related to arterial HCO3-concentration [6]. These data are in accordance with results obtained in uremic rats [ 12] and support the role of metabolic acidosis in increasing muscle protein degradation in humans with CRF. In conclusion, in patients with CRF, muscle ΑΑ metabolism is altered rather early both in the postabsorptive and protein-fed state. In the postabsorptive state, major ΑΑ alterations regard BCAA, which are released from peripheral tissues into the circulation in reduced amounts. In the proteinfed state, muscle uptake of NEEA is increased, counterbalancing alterations in arterial NEAE levels. Possible consequences of the altered muscle ΑΑ metabolism on protein turnover have to be addressed. However, preliminary data indicate that in postabsorptive patients with CRF, muscle protein breakdown is increased and correlated with the degree of metabolic acidosis. Protein synthesis tends to counterbalance protein breakdown; accordingly, net proteolysis is not augmented.
Acknowledgements This study was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Assegnazione per la ricerca scientifica 40 % e 60 %) and by grant No. 91.00255.41 from The National Research Council ((CNR)-Targeted Project — Prevention and Control Disease Factors); Subproject Spl Alimentazione. The authors thank Mr. M. Bruzzone and M. Marchelli for their technical assistance and Mrs. F. Tincani for preparation of the manuscript and figures.
References
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
1 Kopple JD: Causes of catabolism and wasting in acute or chronic renal failure; in Robinson RR (ed): Nephrology. New York, Springer, 1984, pp 1498-1515. 2 Tizianello A, Deferrari G, Garibotto G, Robaudo C, Saffioti S, Paoletti E: Abnormalities of amino acids and ketoacid metabolism in chronic renal failure; in Davison AM (ed): Nephrology. London, Baillière Tindall, 1988, pp 1011-1025. 3 Tizianello A, Deferrari G, Garibotto G, Robaudo C, Lutman M, Passerone GC, Bruzzone M: Branched-chain amino acid metabolism in chronic renal failure. Kidney Int 1983;24:S17—S22. 4 Deferrari G, Garibotto G, Robaudo C, Canepa A, Bagnasco S, Tizianello A: Leg metabolism of amino acids and ammonia in patients with chronic renal failure. Clin Sci 1985;69:143-151. 5 Garibotto G, Deferrari G, Robaudo C, Saffioti S, Paoletti E, Tizianello A: Leg metabolism of amino acids after amino acid ingestion in chronic renal failure. Abstr Xth Int Congr Nephrology, London 1987, p 501.
9
6 Garibotto G, Russo R, Sala MR, Ancarani P, Robaudo C, Sofia A, Deferrari G, Tizianello A: Muscle protein turnover and amino acid metabolism in patients with chronic renal failure. Miner Electrolyte Metab, in press. 7 Young VR: Some metabolic and nutritional considerations of dietary protein restriction; in Mitch WE (ed): The Progressive Nature of Renal Disease. New York, Churchill Livingstone, 1986, pp 263-283. 8 Garibotto G, Ancarani P, Russo R, Sala MR, Fiorini F, Paoletti E: Reversed-phase HPLC analysis of branched-chain keto acids hydrazine derivatives; optimization of techniques and application to BCKA balance studies across the forearm. J Chromatogr Biomed App1 1991;572:11-23. 9 Tizianello A, Deferrari G, Garibotto G, Robaudo C, Saffioti S, Pontremoli R, Sala MR: Abnormalities in amino acid metabolism in chronic renal failure; in Albertazzi A, Cappelli P, Del Rosso G, Di Paolo B, Evangelista M, Palmieri PF (eds): Nutritional and Pharmacological Strategies in Chronic Renal Failure. Contrib Nephrol. Basel, Karger, 1990, vol 81, pp 169-180. 10 De Fronzo RA, Alvestrand A, Smith D, Hendler R, Hendler E, Wharen J: Insulin resistance in uremia. J Clin Invest 1981;67:563-568. 11 Deferrari G, Garibotto G, Robaudo C, Lutman M, Viviani G, Sala MR: Glucose interorgan exchange in chronic renal failure. Kidney Int 1983;24:S115-5120. 12 Mitch WE: Uremia and the control of protein metabolism. Nephron 1988;49:8993. 13 Bergstrom J, Alvestrand A, Furst P: Plasma and muscle free amino acids in maintenance hemodialysis patients without protein malnutrition. Kidney Int 1990;38: 108-114. 14 Munro ΗΝ: Free amino acid pools and their role in regulation; in Munro ΗΝ (ed): Mammalian Protein Metabolism. New York, Academic Press, 1970, vol 4, pp 299386. 15 Austin SA, Clemens MJ: The regulation of protein synthesis in mammalian cells by amino acid supply. Biosci Rep 1981;1:35-47. 16 Garibotto G, Deferrari G, Robaudo C, Saf ιοti S, Salvidio G, Paoletti E, Tizianello A: Effect of amino acid ingestion on blood amino acid profile in patients with chronic renal failure. Am J Clin Nutr 1987;46:949-954. 17 Deferrari G, Garibotto G, Robaudo C, Sala MR, Tizianello A: Splanchnic exchange of amino acids after amino acid ingestion in patients with chronic renal insufficiency. Am J Clin Nutr 1988;48:72-83. 18 Holliday MA, Chantler CA, MacDonnell R, Keitges J: Effect of uremia on nutritionally-induced variations in protein metabolism. Kidney Int 1977;11:236. 19 Li JB, Wassner SJ: Protein synthesis and degradation in skeletal muscle of chronically uremic rats. Kidney Int 1986;29:1 136-1143. 20 Garber AJ: Skeletal muscle protein and amino acid metabolism in experimental chronic uremia in the rat. J Clin Invest 1978;62:623-632. 21 Harter Η, Birge SJ, Martin KJ, Klahr S, Karl IE: Effects of vitamin D metabolites on protein catabolism of muscle from uremic rats. Kidney Int 1983;23:465-472. 22 Guarnieri G, Toigo G, Situlin M, Carraro M, Tamaro G: The assessment of nutritional status in chronically uremic patients; in Gretz N, Strauch M, Giovannetti S (eds): Low-Protein Diets in Renal Patients: Composition and Absorption. Contrib Nephrol. Basel, Karger, vol 72, pp 73-103.
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Muscle Amino Acid and Protein Metabolism in Renal Failure
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
10
Giacomo Garibotto, MD, Dipartimento di Medicina Interna, Divisione di Nefrologia, Viale Benedetto XV, 6, I-16132 Genova (Italy)
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
23 Conley SB, Rose GM, Robson ΑΜ, Bier DM: Effects of dietary intake and hemodíalysis on protein turnover in uremic children. Kidney Int 1980;17:837-846. 24 Berkelhammer CH, Baker JP, Leiter LA, Uldall PR, Whittall R, Slater A, Wolman SL: Whole-body protein turnover in adult hemodialysis in patients as measured by 13C-leucine. Am J Clin Nutr 1987;46:778-783. 25 Lim VS, Tsalikian E, Flanigan MS: Augmentation of protein degradation by Ltriiodothyronine in uremia. Metabolism 1989;38:1210-1215. 26 Goodship THJ, Mitch WE, Hoerr RA, Wagner DA, Steinman TI, Young VR: Adaptation to low-protein diets in renal failure: leucine turnover and nitrogen balance. J Am Soc Nephrol 1990;1:66-75. 27 Gelfand RA, Barrett EJ: Effect of physiological hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest 1987;80:1-6. 28 Fryburg DA, Barrett EJ, Louard RJ, Gelfand RA: Effect of starvation on human muscle protein metabolism and its response to insulin. Am J Physiol 1990;259: Ε477—Ε482.
Muscle Amino Acid and Protein Metabolism in Chronic Renal Failure Giacomo Garibotto, Rodolfo Russo, Cristina Robaudo, Stefano Saffioti, Alberto Magnasco, Giacomo Deferrari, Alberto Tizianello Department of Internal Medicine, Divisions of Nephrology and Clinical Methodology, University of Genova, Italy
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Information on protein turnover in patients with chronic renal failure (CRF) is still poor. Uremic patients, especially those undergoing maintenance dialysis, show a high prevalence of malnutrition [1], attributed mainly to inadequate intake of nutrients and/or superimposed illnesses. A major question is whether or not CRF per se affects protein turnover. The occurrence of alterations in levels of circulating amino acids (AA) and in AA exchange across the organs in well-nourished patients with moderate renal failure suggests that CRF per se, independently of exogenous malnutrition, can play a role in abnormalities in AA metabolism [2]. In addition, a number of alterations in muscle exchange of ΑΑ have been observed in patients studied in the postabsorptive and protein-fed state [2-4]. These abnormalities may be a telltale sign of an altered protein metabolism in muscle. Results reported here have been obtained from studies carried out in patients with moderate CRF (creatinine clearance 18 ± 3 ml/min • 1.73 m2, range 10-35) who were on a diet which provided 30-35 kcal/day and 0.80.9 g protein/kg/day and were actively employed. Muscle AA and protein metabolism was evaluated by the arteriovenous difference technique across the leg and forearm [2-6].
Garibotto/Russo/Robaudo/Saffioti/M agnasco/Deferrari/Tizi anello
a 140 c
~
Εi
120 75 -
50
Ø
-
J 0
Total AA
Irι Ala
Gin
ΣPhe+Tyr+Lys
Ii
Val Leu
2
Ι
ile
Fig. 1. Net release of total AA, Ala, Gin and the sum of Phe, Tyr and Lys (Σ) (a) and BCAA (b) from the leg in patients with CRF (0) and in subjects with normal renal function (c) in the postabsorptive state [from 2].
Abnormalities in Muscle AA Metabolism in the Postabsorptive State in Patients with CRF
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Every day in a healthy 70-kg man about 300 g of protein are degraded and 80% of released AA reutilized for new protein synthesis. Peripheral tissues play an important role in endogenous protein turnover inasmuch as these tissues, chiefly muscle, are the largest reservoir of body protein. Accordingly, evaluation of AA exchange across peripheral tissues, namely leg and forearm, can provide an indirect approach to an understanding of muscle AA and protein metabolism. In postabsorptive patients with CRF, peripheral tissues release most individual AA, including glutamine and alanine, as well as total AA, in amounts similar to controls (fig. 1) [2]. The same is true for phenylalanine, tyrosine and lysine, which are neither catabolized nor synthesized by muscle and therefore express net proteolysis. These findings confirm previous studies in which leg blood flow was not measured [3, 4]. All the data suggest that in renal patients before the uremic stage, net proteolysis is not increased. Different results are obtained in uremic rats, in which an augmented release of AA was generally observed [2]. It has to be pointed out that the arteriovenous difference technique reveals only the net bal-
Muscle Amino Acid and Protein Metabolism in Renal Failure
Controls: ΕΑΑ CRF: ΕΑΑ
a 0.9
.-. •
-•
ΝΕΑΑ o—o ΝΕΑΑ -.ο
b
Oral ΑΑ
ΤΡ ''1`T
,
0.7
c
',
,,
ι
ν
120 -
-λ
a
80 -
0.5
o
w m
C
~ Ε ν
0.3
40w
ν
U
C
0.1
20o
15
30
45
Time, min
60 70
(J Controls CRF
~ 100 -
ι
~
η
3
1
I
ΕΑΑ ΝΕΑΑ Total ΑΑ
ance of AA and net proteolysis. No information on the absolute rates of tissue protein breakdown and synthesis can therefore be deduced by this method and the occurrence of counterbalanced alterations in protein synthesis and/or breakdown cannot be ruled out. It is noteworthy that even small changes in protein turnover may be crucial for maintaining body mass, owing to the large amount of protein degraded and synthesized every day [7]. The above reported studies [2-5] have also shown the existence of selective disturbances in leg metabolism of branched-chain AA (BCAA) in postabsorptive patients with CRF. In patients, the release of valine and leucine from the leg is lower than in controls (fig. 1). In addition, no significant release of any individual branched-chain keto-acid (BCKA) has been observed, whereas, in controls, small amounts of keto-leucine and keto-isoleucine are released by the leg [2, 8]. It has also been demonstrated
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Fig. 2. Increments above basal values in arterial levels of ΕΑΑ and ΝΕΑΑ (a) and their respective arterial areas (b) after AA ingestion in patients with CRF and in subjects with normal renal function. Significance of difference from the corresponding value in normal subjects: a p < 0.025; b p < 0.01; c p < 0.001 [from 17, with permission].
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
4
that the low muscle release of BCAA is responsible for their reduced circulating levels [3, 4, 9]. The reason why in CRF the release of BCAA from peripheral tissues is low is not clear, but an increased degradation of BCAA can be suspected. The low utilization of glucose by muscle cells caused by insulin resistance [ 10, 11 ] may increase BCAA degradation in order to meet energy fuel demand. Α potential role of metabolic acidosis in promoting BCAA degradation is suggested by recent data obtained in rats [ 12]. Such a possibility is supported by the observation that in chronic hemodialysis patients valine levels in muscle cells are reduced and directly correlated with extracellular ΗCO3 concentration [ 13]. Abnormalities in muscle BCAA metabolism in renal patients might affect muscle protein turnover, taking into consideration the regulatory role played by these AA, mainly leucine, in muscle protein turnover. The altered BCAA metabolism may exert a catabdlic effect, as it has been shown in rats that leucine stimulates muscle protein synthesis, while keto-leucine inhibits protein breakdown [ 12]. Furthermore, if, as commonly believed, protein synthesis requires the supply of a high quality AA pattern [14, 15], in CRF the availability of BCAA, mainly valine, in muscle may be insufficient. Accordingly, in patients the reduced muscle intracellular valine may become rate limiting for protein synthesis.
Despite a large number of studies dealing with nutritional treatment of patients with CRF, data on muscle metabolism of AA in the protein-fed state are scanty. Evidence has recently been provided that in patients with CRF after protein and/or AA ingestion, arterial AA profile is markedly altered and characterized by exaggerated increments in total AA mainly owing to nonessential AA (ΝΕΑΑ) levels (fig. 2) [ 16, 17]. Major abnormalities regard glutamine, proline, glutamate, serine, glycine and alanine [ 16, 17]. The altered postprandial AA profile depends chiefly on an abnormally increased escape of ΝΕΑΑ from splanchnic organs [ 17]. Effects of the abnormal AA supply on the AA exchange across peripheral tissues have been recently investigated [2, 5]. After AA ingestion in patients, as well as in controls, the release of most AA, observed in the basal state, is promptly reversed into a marked uptake (fig. 3). While the uptake of essential AA (ΕΑΑ) is similar in both groups, in patients the leg extracts ΝΕΑΑ in by far
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Abnormalities in Muscle AΑ Metabolism in the Protein-Fed State in Patients with CRF
5
Muscle Amino Acid and Protein Metabolism in Renal Failure
Controls: ΕΑΑ •---• ΝΕΑΑ 0-o CRE: ΕEE •-+ ΝΕΑΑ o-o α
ι
ι
0 15 30 45 60 Time, min
75
ΕΑΑ
ΝΕΑΑ
Total ΑΑ
greater amounts than in controls, paralleling changes in circulating ΝΕΑΑ levels. It follows that also the uptake of total ΑΑ by peripheral tissues is increased. At the same time in patients BCAA uptake by the leg is similar to controls [5, 9]. However, as a consequence of the increased uptake of total AA, the uptake of BCAA by the leg is only one-third of that of total AA, whereas it is about one-half ín controls [5, 9]. So, in renal failure, after AA ingestion, peripheral tissues cope with the altered supply of AA by an increased and unbalanced uptake of them. AA are removed from arterial blood far in excess of their relative frequencies in muscle protein and, consequently, only a fraction of them can be utilized for protein synthesis. Likely, ΝΕΑΑ are degraded to a greater extent. Taken together, these data demonstrate that in patients with CRF muscle metabolism of AA is abnormal both in the postabsorptive and protein-fed state. Repercussions, if any, on protein turnover are unknown.
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Fig. 3a, b. Leg exchange of EAA, NEØ and total AA after AA ingestion in patients with CRF and in subjects with normal renal function. * Statistically different from controls [from 2, 5].
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
6
Data on protein turnover in CRF are fragmentary; most studies have been carried out in rats and sometimes arbitrarily extrapolated to man. Uremic rats show an enhanced net proteolysis during fasting [ 18, 19]. Starvation reduces muscle protein synthesis and increases protein muscle breakdown more severely in uremic rats than in controls [ 18, 19]. In fed uremic rats, muscle protein synthesis is reported to be decreased [20, 21] or, conversely, normal [ 18, 19]. Insulin resistance and abnormal energy metabolism in muscle [12] have been proposed as major potential catabolic factors in uremic rats. Recently, evidence has been provided that metabolic acidosis is responsible for the increased muscle protein breakdown in uremic rats [ 12]. Data dealing with muscle protein metabolism in humans with CRF are sparse and somewhat contradictory. In patients with advanced uremia and/or under dialytic treatment, loss of lean body mass occurs frequently [1]. Furthermore, muscle RNA/DNA ratio is decreased in advanced uremia or under protein-restricted diets [22]. Protein turnover in patients with CRF has so far been investigated by the whole-body isotope dilution technique. A reduced whole-body protein turnover has been reported in children in hemodialysís [23]; many of these patients were malnourished and no control group was studied. Conversely, a reduced protein synthesis in the presence of a normal protein breakdown has been reported in apparently well-nourished adults in hemodialysís [24]. On the other hand, a normal protein degradation, but an enhanced proteolytic response to thyroid hormones have been reported in adult hemodialysis patients [25]. Recently, the adaptation of patients with CRF to low protein diets has been studied [26]; in these patients, AA oxidation and protein degradation decreased as in controls. Advanced uremia, dialysis therapy and different calorie and/or protein intake can partially explain differences among these studies. Recently, a technique combining the method of the arteriovenous difference across the forearm with the systemic infusion of 3H-phenylalanine has been proposed in order to estimate the individual rates of protein synthesis and breakdown in muscle [27, 28]. The method is based on the steady-state kinetics of phenylalanine in muscle. Since phenylalanine is not metabolized in muscle, the rates of phenylalanine disposal across the forearm reflect its rates of incorporation into protein, while rates of appearance of phenylalanine from muscle reflect its release from muscle protein
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Muscle Protein Metabolism in CRF
Muscle Amino Acid and Protein Metabolism in Renal Failure
7
breakdown. Net balance of phenylalanine across the forearm expresses net proteolysis. This procedure has been used in order to evaluate muscle protein turnover in well-nourished patients with CRF in the postabsorptive state [6]. In controls, data on forearm protein turnover are fairly comparable with those previously reported in normal postabsorptive subjects [27, 28]. In patients, phenylalanine appearance from the forearm is 30% increased (fig. 4) indicating an increased protein breakdown. Also, phenylalanine disposal is augmented, outlining an increased protein synthesis. Net balance of phenylalanine, that is net proteolysis, is not changed. The release of tyrosine and lysine and total AA is similar to controls and confirms results obtained when AA exchange across the leg was evaluated [2, 4]. It is interesting to note that most patients had metabolic acidosis. Moreover, both in patients and controls protein breakdown was inversely
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Fig. 4. Rates of release (protein breakdown), rates of disposal (protein synthesis) and net balance of phenylalanine (net proteolysis) across the forearm in patients with CRF (0) and in subjects with normal renal function (σ) in the postabsorptive state [from 6, with permission].
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
8
related to arterial HCO3-concentration [6]. These data are in accordance with results obtained in uremic rats [ 12] and support the role of metabolic acidosis in increasing muscle protein degradation in humans with CRF. In conclusion, in patients with CRF, muscle ΑΑ metabolism is altered rather early both in the postabsorptive and protein-fed state. In the postabsorptive state, major ΑΑ alterations regard BCAA, which are released from peripheral tissues into the circulation in reduced amounts. In the proteinfed state, muscle uptake of NEEA is increased, counterbalancing alterations in arterial NEAE levels. Possible consequences of the altered muscle ΑΑ metabolism on protein turnover have to be addressed. However, preliminary data indicate that in postabsorptive patients with CRF, muscle protein breakdown is increased and correlated with the degree of metabolic acidosis. Protein synthesis tends to counterbalance protein breakdown; accordingly, net proteolysis is not augmented.
Acknowledgements This study was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (Assegnazione per la ricerca scientifica 40 % e 60 %) and by grant No. 91.00255.41 from The National Research Council ((CNR)-Targeted Project — Prevention and Control Disease Factors); Subproject Spl Alimentazione. The authors thank Mr. M. Bruzzone and M. Marchelli for their technical assistance and Mrs. F. Tincani for preparation of the manuscript and figures.
References
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
1 Kopple JD: Causes of catabolism and wasting in acute or chronic renal failure; in Robinson RR (ed): Nephrology. New York, Springer, 1984, pp 1498-1515. 2 Tizianello A, Deferrari G, Garibotto G, Robaudo C, Saffioti S, Paoletti E: Abnormalities of amino acids and ketoacid metabolism in chronic renal failure; in Davison AM (ed): Nephrology. London, Baillière Tindall, 1988, pp 1011-1025. 3 Tizianello A, Deferrari G, Garibotto G, Robaudo C, Lutman M, Passerone GC, Bruzzone M: Branched-chain amino acid metabolism in chronic renal failure. Kidney Int 1983;24:S17—S22. 4 Deferrari G, Garibotto G, Robaudo C, Canepa A, Bagnasco S, Tizianello A: Leg metabolism of amino acids and ammonia in patients with chronic renal failure. Clin Sci 1985;69:143-151. 5 Garibotto G, Deferrari G, Robaudo C, Saffioti S, Paoletti E, Tizianello A: Leg metabolism of amino acids after amino acid ingestion in chronic renal failure. Abstr Xth Int Congr Nephrology, London 1987, p 501.
9
6 Garibotto G, Russo R, Sala MR, Ancarani P, Robaudo C, Sofia A, Deferrari G, Tizianello A: Muscle protein turnover and amino acid metabolism in patients with chronic renal failure. Miner Electrolyte Metab, in press. 7 Young VR: Some metabolic and nutritional considerations of dietary protein restriction; in Mitch WE (ed): The Progressive Nature of Renal Disease. New York, Churchill Livingstone, 1986, pp 263-283. 8 Garibotto G, Ancarani P, Russo R, Sala MR, Fiorini F, Paoletti E: Reversed-phase HPLC analysis of branched-chain keto acids hydrazine derivatives; optimization of techniques and application to BCKA balance studies across the forearm. J Chromatogr Biomed App1 1991;572:11-23. 9 Tizianello A, Deferrari G, Garibotto G, Robaudo C, Saffioti S, Pontremoli R, Sala MR: Abnormalities in amino acid metabolism in chronic renal failure; in Albertazzi A, Cappelli P, Del Rosso G, Di Paolo B, Evangelista M, Palmieri PF (eds): Nutritional and Pharmacological Strategies in Chronic Renal Failure. Contrib Nephrol. Basel, Karger, 1990, vol 81, pp 169-180. 10 De Fronzo RA, Alvestrand A, Smith D, Hendler R, Hendler E, Wharen J: Insulin resistance in uremia. J Clin Invest 1981;67:563-568. 11 Deferrari G, Garibotto G, Robaudo C, Lutman M, Viviani G, Sala MR: Glucose interorgan exchange in chronic renal failure. Kidney Int 1983;24:S115-5120. 12 Mitch WE: Uremia and the control of protein metabolism. Nephron 1988;49:8993. 13 Bergstrom J, Alvestrand A, Furst P: Plasma and muscle free amino acids in maintenance hemodialysis patients without protein malnutrition. Kidney Int 1990;38: 108-114. 14 Munro ΗΝ: Free amino acid pools and their role in regulation; in Munro ΗΝ (ed): Mammalian Protein Metabolism. New York, Academic Press, 1970, vol 4, pp 299386. 15 Austin SA, Clemens MJ: The regulation of protein synthesis in mammalian cells by amino acid supply. Biosci Rep 1981;1:35-47. 16 Garibotto G, Deferrari G, Robaudo C, Saf ιοti S, Salvidio G, Paoletti E, Tizianello A: Effect of amino acid ingestion on blood amino acid profile in patients with chronic renal failure. Am J Clin Nutr 1987;46:949-954. 17 Deferrari G, Garibotto G, Robaudo C, Sala MR, Tizianello A: Splanchnic exchange of amino acids after amino acid ingestion in patients with chronic renal insufficiency. Am J Clin Nutr 1988;48:72-83. 18 Holliday MA, Chantler CA, MacDonnell R, Keitges J: Effect of uremia on nutritionally-induced variations in protein metabolism. Kidney Int 1977;11:236. 19 Li JB, Wassner SJ: Protein synthesis and degradation in skeletal muscle of chronically uremic rats. Kidney Int 1986;29:1 136-1143. 20 Garber AJ: Skeletal muscle protein and amino acid metabolism in experimental chronic uremia in the rat. J Clin Invest 1978;62:623-632. 21 Harter Η, Birge SJ, Martin KJ, Klahr S, Karl IE: Effects of vitamin D metabolites on protein catabolism of muscle from uremic rats. Kidney Int 1983;23:465-472. 22 Guarnieri G, Toigo G, Situlin M, Carraro M, Tamaro G: The assessment of nutritional status in chronically uremic patients; in Gretz N, Strauch M, Giovannetti S (eds): Low-Protein Diets in Renal Patients: Composition and Absorption. Contrib Nephrol. Basel, Karger, vol 72, pp 73-103.
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
Muscle Amino Acid and Protein Metabolism in Renal Failure
Garibotto/Russo/Robaudo/Saffioti/Magnasco/Deferrari/Tizianello
10
Giacomo Garibotto, MD, Dipartimento di Medicina Interna, Divisione di Nefrologia, Viale Benedetto XV, 6, I-16132 Genova (Italy)
Downloaded by: Université de Paris 193.51.85.197 - 2/18/2020 2:41:31 PM
23 Conley SB, Rose GM, Robson ΑΜ, Bier DM: Effects of dietary intake and hemodíalysis on protein turnover in uremic children. Kidney Int 1980;17:837-846. 24 Berkelhammer CH, Baker JP, Leiter LA, Uldall PR, Whittall R, Slater A, Wolman SL: Whole-body protein turnover in adult hemodialysis in patients as measured by 13C-leucine. Am J Clin Nutr 1987;46:778-783. 25 Lim VS, Tsalikian E, Flanigan MS: Augmentation of protein degradation by Ltriiodothyronine in uremia. Metabolism 1989;38:1210-1215. 26 Goodship THJ, Mitch WE, Hoerr RA, Wagner DA, Steinman TI, Young VR: Adaptation to low-protein diets in renal failure: leucine turnover and nitrogen balance. J Am Soc Nephrol 1990;1:66-75. 27 Gelfand RA, Barrett EJ: Effect of physiological hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest 1987;80:1-6. 28 Fryburg DA, Barrett EJ, Louard RJ, Gelfand RA: Effect of starvation on human muscle protein metabolism and its response to insulin. Am J Physiol 1990;259: Ε477—Ε482.
