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Am J Physiol Endocrinol Metab 292: E1534-E1542, 2007. First published January 30, 2007; doi:10.1152/ajpendo.00599.2006
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Amino acid repletion does not decrease muscle protein catabolism during hemodialysis

Dominic S. C. Raj,1 Oladipo Adeniyi,1 Elizabeth A. Dominic,2 Michel A. Boivin,3 Sandra McClelland,4 Antonios H. Tzamaloukas,1 Nancy Morgan,4 Lawrence Gonzales,4 Robert Wolfe,5 and Arny Ferrando5

1Division of Nephrology, University of New Mexico Health Sciences Center, Albuquerque; 2Albuquerque Academy, Albuquerque; 3Division of Pulmonary and Critical Care, 4General Clinical Research Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico; and 5Department of Geriatrics, University of Arkansas, Little Rock, Arkansas

Submitted 9 November 2006 ; accepted in final form 29 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol·min–1·100 ml –1) was more negative during HD-O compared with pre-HD (–33.7 ± 1.5 vs. –6.0 ± 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (–16.9 ± 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

protein turnover; cytokines; end-stage renal disease; malnutrition; caspase-3; skeletal muscle

LOSS OF LEAN BODY MASS is common and is one of the most important predictors of increased mortality and morbidity in patients with end-stage renal disease (ESRD). Muscle and whole body protein turnover studies have consistently demonstrated a balanced reduction in protein synthesis and degradation in stable ESRD patients (1, 13, 21, 26). On the other hand, investigators unanimously agree that hemodialysis (HD) induces net protein catabolism (27, 31, 40). About 10–12 g of amino acids are lost in the dialysate per HD session. Animal (48) and human (8) studies have indicated that availability of precursor amino acids is a potent stimulus for protein synthesis. Our group reported that amino acid depletion by HD promotes protein catabolism to maintain the plasma and intracellular amino acid concentrations (39). Our group also has shown that abnormal protein turnover in ESRD could be due to altered amino acid transport kinetics (40) and that interleukin-6 (IL-6) plays a key role in HD-induced muscle protein catabolism (42, 43). Amino acid utilization for protein synthesis is impaired in patients with chronic renal failure (11). Anabolic response to amino acid infusion, however, was restored by concomitant administration of insulin (10, 32), possibly through augmentation of intracellular amino acid transport (3).

The effect of intradialytic amino acid infusion on the transmembrane amino acid transport kinetics and protein turnover is unknown. A defect in amino acid transport could impair the ability of the muscle to utilize amino acids for protein synthesis. We hypothesized that amino acid repletion during HD would increase muscle protein synthesis but would not decrease protein breakdown. Thus the principal aim of this study was to examine the effect of intradialytic amino acid infusion on amino acid transport kinetics and muscle protein turnover in ESRD patients. We determined the protein and amino acid kinetics at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA) using stable isotopes of phenylalanine, leucine, and lysine. Our results indicate that protein balance in the muscle is switched from a state protein balance pre-HD to a net protein loss during HD-O, which is partially corrected by amino acid replacement.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Eight stable ESRD patients on maintenance HD for >90 days were studied. Patients with diabetes mellitus, infection, chronic inflammation, pregnancy, hematocrit <30%, bleeding diathesis, preexisting cardiac condition, catabolic illnesses, or unexplained weight loss were excluded from the study. The study was approved by the Human Research Review Committee at the University of New Mexico Health Sciences Center (UNMHSC).

Methods. Participants were placed on a diet containing 35 kcal/kg, 1.2 g protein·kg–1·day–1, for a minimum of 14 days before the study. Dietary intake was confirmed by a 3-day dietary history preceding the experiment. Subjects were admitted to the General Clinical Research Center (GCRC) at the UNMHSC 1 day before the experiment. Leg volume was estimated as described previously (6). The study was performed in a postabsorptive state after overnight fast. Polyethylene catheters were inserted in the femoral artery and vein on the same side. The femoral arterial catheter was used for infusion of indocianin green (ICG). Catheters were also placed in the nonaccess forearm veins for infusion of labeled amino acids and in the right wrist vein for arterialized blood sampling.

After a blood sample was obtained for background amino acid enrichment, a primed continuous infusion of L-[ring-13C6]phenylalanine (prime, 2.0 µmol/kg; infusion, 0.05 µmol·kg–1·min–1), L-[1-13C] leucine (prime, 4.8 µmol/kg; infusion, 0.08 µmol·kg–1·min–1), and L-[2-15N]lysine (prime, 7.2 µmol/kg; infusion, 0.08 µmol·kg–1·min–1) was maintained throughout the experiment (Fig. 1). Tracer infusion rates were increased by 10% during HD+AA to maintain stable enrichment. Blood flow to the lower extremity was measured by dye dilution technique as described by our group previously (40).


Figure 1
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  		Fig. 1. Study design. End-stage renal disease (ESRD) patients were studied before hemodialysis (pre-HD; postabsorptive phase, 0 to 300 min) and during HD (300 to 540 min). The HD phase was further divided into an initial 2 h of HD without amino acid infusion (HD-O; 300 to 420 min) and a subsequent 2 h during which the patients received a primed constant infusion of unlabeled amino acid mixture (HD+AA; 420 to 540 min). After blood samples were obtained for background enrichment at 0 min, a primed continuous infusion of L-[ring-13C6]phenylalanine, L-[1-13C]leucine, and L-[2-15N]lysine was started and continued throughout the experiment. From 240 to 300 min, 360 to 420 min, and 480 to 540 min, blood samples were obtained from artery and vein to estimate enrichment and arteriovenous (A-V) balance and to measure protein kinetics. Muscle biopsies were obtained at baseline and 300, 420, and 540 min. A continuous infusion of indocianin green (ICG) was administered into the femoral artery between 240 and 270 min, 360 and 390 min, and 480 and 510 min to estimate the blood flow rate to the leg. Blood samples were taken every 10 min from the femoral vein and wrist vein during that period to estimate ICG concentration.

 
HD was initiated at 300 min and continued for 4 h. Patients were dialyzed using their usual blood and dialysate flow rates. Dialysate composition was as follows: sodium, 139 meq/l; bicarbonate, 35 meq/l; calcium, 2.5 meq/l; magnesium, 1 meq/l; dextrose, 200 mg/dl; and potassium, per patient's need. A new polysulfone membrane (F70, Fresenius, Hemoflow) was used for dialysis. Anticoagulation was not used to minimize the risk of bleeding. During the last 2 h of dialysis, a primed (50 ml) constant infusion (125 ml/h) of unlabeled amino acid mixture was maintained through the venous port of the dialysis circuit. The amino acid mixture was prepared from commercial amino acid preparation (10% TrophAmine; Braun Medical, Irvine, CA) to which freshly prepared glutamine was added. The final concentrations of amino acids in the infusate were as follows: alanine, 2.51 g/l; arginine, 5.64 g/l; aspartic acid, 1.48 g/l; cysteine, 0.11 g/l; glutamine, 6.68 g/l; glycine, 1.69 g/l; glutamate, 1.96 g/l; histidine, 2.25 g/l; isoleucine, 3.82 g/l; leucine, 6.54 g/l; lysine, 3.82 g/l; methionine, 1.58 g/l; phenylalanine, 2.25 g/l; proline, 3.18 g/l; serine, 1.78 g/l; taurine, 0.12 g/l; threonine, 1.95 g/l; tryptophan, 0.94 g/l; tyrosine, 1.22 g/l; and valine, 3.65 g/l.

Representative dialysate samples were collected every 10 min in a sterile container. The samples were collected separately during HD-O and HD+AA. The spent dialysate sample was mixed thoroughly, and a sample was taken and stored at –80°C for future analysis.

Muscle biopsies were performed at the second hour to measure isotopic carbon enrichment of bound and free phenylalanine in the muscle. Subsequent biopsies were obtained at the fifth, seventh, and ninth hours of the experiment to determine the intracellular enrichments and to calculate the fractional synthesis rate of protein during pre-HD, HD-O, and HD+AA, respectively. Biopsies were taken from lateral portion of vastus lateralis ~20 cm above the knee with a Bergstrom biopsy needle. Considering the possibility that repeated muscle biopsies could activate local inflammatory response, the first and second biopsies were performed through the same incision, but the needle was directed away from the previously biopsied site. The third and fourth biopsies were taken from an adjacent but different incision. Fat and connective tissues were removed, and the samples were frozen in liquid nitrogen and stored at –80°C for future analysis.

Blood samples were obtained at baseline and during the last 10 min of HD-O and HD+AA for blood urea nitrogen (BUN), creatinine, electrolytes, albumin, and fibrinogen measurement. Albumin was measured by using the bromcresol green method, glucagon by radioimmunoassay (RIA), and insulin, cortisol, and C-reactive protein (CRP) by immulite chemiluminescence. Plasma IL-6 was measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's directions.

Analytical procedures. Blood samples for the measurement of amino acid concentrations and enrichment were collected as previously described (6). Muscle samples were weighed, and the proteins were precipitated with 450 µl of 14% perchloric acid. An internal standard solution was added to measure the intracellular concentrations of the traced amino acids (6). This method measures muscle free water enrichment. The tissue pellet was further washed and dried. The precipitated protein was hydrolyzed at 110°C for 24 h with 6 N constant boiling HCl. The protein hydrolysate was then processed, while blood samples and phenylalanine enrichment were measured by GC-MS (GC 8000 series, MD 800; Fisons Instruments, Manchester, UK) using chemical ionization and the standard curve approach.

Compartmental model. The kinetics of intracellular amino acids were determined using a three-compartment model (6). Amino acids entering the leg through the femoral artery (Fin) and leaving the leg via femoral vein (Fout), rates of inward (Fma) and outward transmembrane transport (Fvm), and the intracellular appearance of amino acids (Fmo) and amino acid utilization (Fom) were computed (Fig. 2).

Formula

Formula
where Ca and Cv are the amino acid concentrations in the artery and vein, and BF is the plasma flow to the leg. Net amino acid balance (NB) in the leg was calculated as

Formula

Formula
where Ea, Ev, and Em are amino acid enrichments in the femoral artery, vein, and muscle, respectively.

Formula


Figure 2
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  		Fig. 2. Three-pool model for calculation of amino acid transport kinetics. A 3-compartment model with unidirectional flow of amino acids among artery (A), vein (V), and muscle (M) compartments is shown. Amino acids enter the leg through the femoral artery (Fin) and exit through the femoral vein (Fout). Fva is the shunt to the vein from the artery. Fma is the inward transport of amino acid to the muscle from the artery, and Fvm is the outward transport of amino acids to the vein from the muscle intracellular free pool. Fmo is the rate of intracellular appearance and Fom is the rate of utilization of amino acids in the intracellular compartment, respectively.

 
The intracellular amino acid appearance (Fmo) was calculated as

Formula
Fmo represents the protein breakdown.

The rate of intracellular amino acid utilization (Fom) was calculated as

Formula
The Fom of phenylalanine and lysine is an index of protein synthesis, but Fom of leucine is a measure of synthesis and oxidation (12).

Protein synthesis efficiency, an estimate of the fraction of amino acid that appears in the intracellular pool that is directed to protein synthesis, was computed as

Formula
Fractional synthesis rate (FSR) was calculated by precursor product approach as described earlier (42).

The rates of release of branched chain amino acid (BCAA) from protein catabolism were calculated from the rate of phenylalanine release, corrected for the molar ratios of leucine to phenylalanine, isoleucine to phenylalanine, and valine to phenylalanine (7):

Formula
The calculated rate of BCAA catabolism (i.e., nonprotein BCAA disposal) is not equivalent to the actual rate of BCAA oxidation. However, the difference between the rates of BCAA catabolism and oxidation is likely to be very small (45).

Real-time PCR for IL-6. Real-time PCR was performed as described by our group previously (41). Briefly, muscle biopsy was homogenized in Qiagen's RNeasy lysis buffer containing 2-mercaptoethanol at room temperature, and RNA was isolated. PCR was performed on the MJ Research Opticon 2 instrument. Negative controls for PCR included reactions containing no template. The amount of target gene in ESRD patients was normalized to an endogenous control (GAPDH) and to the mRNA level in the controls (9). Quantifying the relative changes in gene expression using real time was performed using the 2{{uparrow}–{Delta}{Delta}CT} method (33).

Western blotting for carboxy terminus of 14-kDa actin fragments. Western blotting was performed in the muscle biopsy samples obtained during pre-HD, HD-O, and HD+AA phases of the study from one representative patient. Skeletal muscle samples were dissected free of connective tissue and placed in 350 µl of hypotonic muscle lysis buffer [5 mM Tris·HCl (pH 8.0), 1 mM EDTA, 1 mM EGTA, 1 mM beta-mercaptoethanol, 1% glycerol, and 40 µl/ml protease inhibitor concentrate (no. P2714; Sigma, St. Louis, MO)] according to the description by Du et al. (14). The tissue was then homogenized for 30 s at level 6 with a Polytronic PT 1200C tissue homogenizer (Kinematica, Lucerne, Switzerland). The samples were centrifuged at 5,000 g for 10 min. The pellets were considered the "insoluble fraction," and the supernatant was saved as the "soluble fraction." The insoluble fractions were resuspended in a 1 mg-to-10 µl ratio of a 1:1 mixture of 2x Laemmli buffer (with 50 µl/ml beta-mercaptoethanol) and PBS with protease inhibitor concentrate. The soluble samples were assayed for protein content (Lowry method), and equal protein loading was used. The soluble samples were mixed 1:1 with 2x Laemmli buffer and boiled for 7 min. They were loaded on 4–20% SDS-PAGE gradient gels and run for 90 min at 130 V. They were transferred to 0.22 µM polyvinylidene difluoride membranes overnight at 20 V and blocked for 1 h with Zymed membrane blocking solution (Invitrogen, Carlsbad, CA). Following blocking, membranes were incubated with the combined primary-secondary antibody (anticarboxy-terminal actin conjugated with horseradish peroxidase antibody, SC-1615HRP; Santa Cruz Biotechnologies, Santa Cruz, CA) in Zymed blocking solution at a dilution of 1:1,000. After 1 h, the membranes were washed four times with Tris-buffered saline plus Tween, saturated with luminal reagent (Santa Cruz Biotech), and exposed to Kodak film.

Statistical methods. Data are given as means ± SE; {alpha} was set at 0.05. Student's paired and unpaired t-tests were used as appropriate. The Wilcoxon signed rank test was used when the results were not normally distributed. A repeated-measures ANOVA with a post hoc Tukey's test was used when comparing more than two variables. Linear regression analysis was used to identify relationship between variables.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The clinical and biochemical characteristics of the subjects are shown in Table 1. Mean age of the patients was 42.4 ± 4.7 yr, and there was one female participant. The etiology of renal failure was hypertension in three, glomerulonephritis in two, tubulointerstitial nephropathy in one, and unknown in two patients. Protein (1.51 ± 0.24 vs. 1.47 ± 0.09 g·kg–1·day–1) and calorie (29.8 ± 7.1 vs. 31.2 ± 3.9 kcal·kg–1·day–1) intakes estimated at baseline and during the 2-wk run-in period before the experiment were not significantly different. The total amount of amino acid infused during HD+AA was 16.1 ± 2.7 g. Amino acid loss during HD+AA was more than the loss during HD-O (7.6 ± 1.2 vs. 4.3 ± 0.8 g, P < 0.01). About 31.7, 44.4, and 57.6% of the infused leucine, lysine, and phenylalanine were lost in the dialysate, respectively. Glucose, insulin, and cortisol levels increased significantly during HD+AA. Blood flow to the leg did not change significantly during HD-O (4.23 ± 0.29 ml·min–1·100 ml leg–1) or HD+AA (4.47 ± 0.26 ml·min–1·100 ml leg–1) compared with predialysis (4.12 ± 0.25 ml·min–1·100 ml leg–1). Urea reduction ratio was 56.3 ± 2.7%. Plasma IL-6 and CRP levels increased significantly during HD.


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  		Table 1. Clinical and biochemical profile of participants

 
The intracellular enrichment in the muscle was lower compared with those in artery and vein (P < 0.001) (Table 2). Enrichment in all the three compartments decreased significantly during HD+AA compared with pre-HD and HD-O. Enrichment during each phase of the study (pre-HD, HD-O, and HD+AA), however, remained stable, thus maintaining a steady state. Plasma levels of amino acids in the artery declined during HD-O but increased with amino acid infusion in all three compartments (P < 0.001) (Tables 3 and 4). Concentrations of amino acids in the vein were higher than in the artery during HD-O (P < 0.001). Intracellular amino acid concentrations were significantly higher than in arterial and venous compartments at all time points. The tissue/artery gradient varied for different amino acids and showed a tendency to decrease during HD+AA. Dialysate concentration of amino acids increased during HD+AA compared with HD-O (P < 0.05).


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  		Table 2. Enrichment of free amino acids in the artery, vein, and muscle compartments

 

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  		Table 3. Changes in concentrations of free amino acids in the femoral artery, femoral vein, and muscle compartments

 

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  		Table 4. Arteriovenous balance of amino acids during the different phases of the study

 
The model-derived leg amino acid kinetic parameters are shown in Table 5. Amino acid infusion increased the delivery of amino acids (Fin) to the leg and also the efflux from the leg (Fout) (P < 0.001). Net balance became profoundly negative during HD-O, and amino acid infusion during HD did not shift the balance from net release to net uptake. The net balance of 3-methyl histidine became more negative during HD-O (–17.1 ± 7.5) and HD+AA (–21.3 ± 6.7 nmol·min–1·100 ml leg–1) compared with pre-HD (–0.07 ± 6.8 nmol·min–1·100 ml leg–1) (P < 0.04). Change in net balance of 3-methyl histidine from baseline correlated with that of phenylalanine (r2 = 0.28, P < 0.04). The absolute rates of inward transport of phenylalanine, leucine, and lysine increased during HD-O compared with pre-HD (P < 0.01) and were further augmented during HD+AA (P < 0.001). The outward transport for all of the traced amino acid also increased significantly during HD-O and HD+AA compared with pre-HD. The outward transport was faster than inward transport during HD-O for leucine and lysine (P < 0.01).


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  		Table 5. Amino acid transport kinetics during hemodialysis with and without amino acid infusion

 
The increment {Delta}Ep of the enrichment of the muscle protein-bound phenylalanine increased during HD+AA (4.86 ± 0.18 x 10–5) compared with pre-HD (3.28 ± 0.01 x 10–5) and HD-O (3.53 ± 0.11 x 10–5) (P < 0.001). The FSR of muscle protein increased during HD-O and HD+AA (P < 0.001) (Fig. 3). Intracellular utilization of all the traced amino acids increased during HD+AA compared with pre-HD and HD-O (P < 0.001). Intracellular appearance of amino acids from muscle protein breakdown increased during HD-O and increased further during HD+AA (P < 0.001). Protein synthesis efficiency decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). BCAA catabolism was significantly higher during HD-O and HD+AA compared with pre-HD (Fig. 4). Nonprotein leucine disposal increased from –14.2 ± 9.5 nmol·min–1·100 ml leg–1 pre-HD to 42.5 ± 16.7 and 34.7 ± 12.8 nmol·min–1·100 ml leg–1, respectively, during HD-O and HD+AA (pre-HD vs. HD-O and HD+AA, P < 0.02). IL-6 gene expression progressively increased during HD-O and HD+AA from baseline (P < 0.001) (Fig. 5). Western blotting demonstrated a progressive increase in 14-kDa actin fragments in the skeletal muscle during HD-O and HD+AA (Fig. 6).


Figure 3
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  		Fig. 3. Fractional muscle protein synthesis rate (FSR; %h) derived from precursor product approach. FSR increased by 31.9 and 88.7% from baseline (pre-HD) during HD-O and HD+AA, respectively. *P < 0.001, HD-O vs. pre-HD. {dagger}P < 0.001, HD+AA vs. HD-O and pre-HD.

 

Figure 4
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  		Fig. 4. Branched chain amino acid (BCAA) catabolism increased from baseline (pre-HD) during HD-O and did not change significantly during HD+AA. *P < 0.01, HD-O and HD+AA vs. pre-HD.

 

Figure 5
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  		Fig. 5. Interleukin-6 (IL-6) gene expression in the skeletal muscle was determined using real-time PCR. The mRNA level of IL-6 progressively increased from baseline (pre-HD) during the initial 2 h of HD-O and increased further during 2 h of HD+AA. *P < 0.001, HD-O vs. pre-HD. {dagger}P < 0.001, HD+AA vs. pre-HD and HD-O.

 

Figure 6
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  		Fig. 6. Representative Western blot of the COOH-terminal 14-kDa actin fragment in muscle biopsies obtained pre-HD, during HD-O, and during HD+AA. There was a progressive increase in 14-kDa actin fragments during HD-O and HD+AA, indicating augmented protein catabolism despite amino acid repletion during hemodialysis.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrates that amino acid depletion during HD induces net muscle protein breakdown. Repletion of amino acids in plasma and intracellular pools by amino acid infusion during HD increased protein turnover in the muscle with a balanced increase in both synthesis and catabolism. This could be due to the opposing effects of increased availability of precursor amino acids promoting protein synthesis and protein catabolic signals from concomitant increase in plasma cortisol (17), and activation of proinflammatory cytokines (42) during HD. Insulin has been shown to facilitate utilization of amino acids for protein synthesis (3). Despite increase in plasma insulin level during HD+AA, protein synthesis efficiency did not improve. Net balance became less negative with amino acid repletion during HD+AA but did not approach the pre-HD state of protein balance. Thus amino acid infusion during HD does not decrease muscle protein breakdown and had only a modest net anabolic effect.

In an animal model, using a similar study design, Kobayashi et al. (28) demonstrated that amino acid depletion by HD decreased muscle protein synthesis and increased protein breakdown between 30 and 60 min after the start of HD. Muscle protein synthesis was stimulated when amino acids were replaced during HD; protein catabolism, however, was unchanged. In the present study, a disproportionate increase in muscle protein breakdown was noted during HD-O, but amino acid replacement induced a proportional increase in both synthesis and catabolism. Sham HD has been shown to induce muscle protein catabolism even in the absence of amino acid loss (23). Augmented protein catabolism that is uncorrected by amino acid infusion could be due to concomitant increase in plasma cortisol (37) and cytokine activation (19, 44); both are known to induce protein catabolism. Similar changes in protein kinetics are observed in other stress states (4, 16). Not all investigators have demonstrated that exogenous amino acids ameliorates muscle protein catabolism (25, 47). A close link exists between rates of muscle protein synthesis and breakdown (50). In catabolic (25) or anabolic (8) states, the extent of changes in muscle protein synthesis and breakdown differs with respect to each other, but the two factors generally change in the same direction (28). In contrast to findings of the present study, Pupim et al. (38) demonstrated that intradialytic parenteral nutrition reversed the net forearm protein balance from a negative to a positive state. The difference in these results could be due to differences in the amino acid replacement used and in the duration of infusion. Furthermore, investigators have demonstrated that providing calories together with amino acids attenuates protein breakdown in chronic renal failure (29) and other catabolic states (34). A more robust increase in insulin secretion with calorie supplementation could potentially facilitate amino acid utilization for protein synthesis.

Consistent with our previous study (40), concentrations of amino acids in the artery and vein decreased during HD-O, but intracellular concentration remained stable. Our group previously reported (39) that intracellular amino acid concentration was maintained during dialysis by augmented muscle protein catabolism. Exogenous amino acid infusion during HD+AA increased the concentrations of phenylalanine, leucine, and lysine in all three compartments. Arteriovenous balance studies have shown that there is a net efflux of amino acids from the muscle into the vein during HD-O but not pre-HD. The net balance of amino acids switched from a state of equilibrium pre-HD to a net release during HD-O, and the balance was partially restored by amino acid infusion during HD+AA.

The amino acids traced were chosen for their unique metabolic pathways and transport kinetics. Phenylalanine and lysine are not synthesized or oxidized in skeletal muscle. Leucine, on the other hand, is oxidized in the muscle (36). The transport systems of these amino acids are also different: phenylalanine and leucine are transported through the L-system, lysine by the y+ system (22). Both inward and outward transport of all the traced amino acids increased during HD-O and increased further during HD+AA. Outward transport of the traced amino acids were faster than inward transport during HD-O, probably to maintain plasma levels of amino acids. Augmented transport of the amino acids is substantiated by the increase in intracellular concentration of these amino acids.

The rate of intracellular amino acid utilization (Fom) of phenylalanine and lysine is an index of muscle protein synthesis, but for leucine it is a measure of muscle protein synthesis and amino acid oxidation. The Fmo of phenylalanine, leucine, and lysine represents muscle protein breakdown. There was an unbalanced increase in muscle protein catabolism (Fmo) compared with synthesis (Fom) during HD-O for phenylalanine (16.7 vs. 48.9%), leucine (17.6 vs. 43.0%), and lysine (18.0 vs. 41%). The augmented protein catabolism during HD-O could be due to depletion of nutrients by the dialysis procedure, especially in the postabsorptive state. During HD+AA, however, increase in Fom and Fmo from baseline for phenylalanine (70.2 vs. 77.9%), leucine (90.7 vs. 90.0%), and lysine (101.2 vs. 104.0%) were comparable.

We further substantiated increased protein breakdown during HD+AA by demonstrating an increase in 14-kDa carboxy-terminal actin in the muscle. Muscle protein catabolism involves activation of the ubiquitin-proteasome system (43). The initial rate-limiting step in muscle protein breakdown is activation of caspase-3, which cleaves actomyosin to create a substrate that is degraded by the ubiquitin-proteasome system (14). The caspase-3-mediated protein cleavage leads to a characteristic "footprint" remaining in the myofibril fraction of the muscle, a 14-kDa actin fragment. In an elegant study, Workeneh et al. (51) demonstrated that the abundance of 14-kDa actin fragments in the muscle has been shown to correlate with muscle protein catabolic rate. The lack of decrease in muscle protein catabolism during HD+AA could be due to catabolic signals initiated during the first 2 h that are being carried over to the second phase when amino acids were infused.

Fractional muscle protein synthesis, determined by precursor product approach, increased by 31.9 and 88.7% from baseline during HD-O and HD+AA, respectively. Lack of a decrease in protein catabolism, despite an increase in plasma insulin and abundant amino acid supply during HD+AA, could be due to a concomitant increase in plasma cortisol (37) and proinflammatory cytokine activation (19). Preliminary evidence from our laboratory has shown that IL-6 is released from skeletal muscle (41) and may mediate muscle protein catabolism (42, 43). Biolo et al. (2) demonstrated that pentoxifylline decreases whole body proteolysis in patients with chronic renal failure, possibly through inhibition of tumor necrosis factor-{alpha}. Tsujinaka et al. (46) observed muscle atrophy in transgenic mice that overexpressed IL-6. Haddad et al. (24) reported that local infusion of IL-6 increased myofibrillar protein loss. IL-6 has been shown to increase the mRNA levels of proteolytic enzymes (46) and decrease the phosphorylation of ribosomal protein S6 kinase 1 (S6K-1) (24). Both insulin and IGF-1 stimulate protein translation in mature muscle cells by activating Akt and mammalian target of rapamycin (mTOR) and by phosphorylation of S6K-1, which may be blocked by proinflammatory cytokines (15, 30). Some investigators, on the other hand, have demonstrated that muscle protein catabolism could progress independently of IL-6 (20, 49). The progressive increase in plasma IL-6 and the mRNA level in the skeletal muscle during HD-O and the further increase during HD+AA are due to longer exposure to HD procedure. We cannot, however, completely exclude the role of local trauma inducing an increase in the inflammatory response and cytokine gene expression.

Release of 3-methyl histidine from the muscle is an index of muscle proteolysis (35). Arteriovenous balance studies showed that the net efflux of 3-methyl histidine from the muscle increased during HD-O and did not decrease despite amino acid repletion. BCAA have a regulatory effect on protein metabolism and are the main donor for synthesis of alanine and glutamine in the skeletal muscle. BCAA catabolism is increased during HD-O, possibly to sustain glutamine synthesis (39). However, BCAA catabolism did not decrease significantly during HD+AA despite inclusion of glutamine in the infusate. This could be because the glutamine-alanine-BCAA interaction is further modulated by coexisting inflammation and cytokine activation observed during HD (5, 18).

To summarize, protein balance is maintained in ESRD patients pre-HD. Amino acid depletion by HD facilitates net protein loss. Amino acid replacement during HD increased muscle protein synthesis but did not decrease breakdown. Net balance became less negative during HD+AA but did not become positive, indicating that amino acid infusion has only negligible net anabolic effect. Furthermore, repletion of amino acids in the blood and muscle compartments during HD+AA did not improve protein synthesis efficiency or decrease BCAA catabolism. The role of increased plasma cortisol and activation of proinflammatory cytokines during HD in counterbalancing the anabolic response to amino acid repletion needs further investigation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grant AG-21560, a Norman Coplan Satellite Research Grant, a Young Investigator Award from the National Kidney Foundation, a Paul Teschan Research Grant, and NIH GCRC Grant 5 M01 RR-00997.


   ACKNOWLEDGMENTS
 
We express sincere gratitude to the brave participants, GCRC nurses, and acute dialysis unit nurses Renita Truujillo and Louis Ashly.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. S. C. Raj, Division of Nephrology, 5th Floor ACC, 2211 Lomas Blvd. NE, Albuquerque, NM 87131-5271 (e-mail: draj{at}salud.unm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Berkelhammer CH, Baker JP, Leiter LA, Uldall PR, Whittall R, Slater A, Wolman SL. Whole-body protein turnover in adult hemodialysis patients as measured by 13C-leucine. Am J Clin Nutr 46: 778–783, 1987.[Abstract/Free Full Text]
  2. Biolo G, Ciocchi B, Bosutti A, Situlin R, Toigo G, Guarnieri G. Pentoxifylline acutely reduces protein catabolism in chronically uremic patients. Am J Kidney Dis 40: 1162–1172, 2002.[CrossRef][ISI][Medline]
  3. Biolo G, Declan Fleming RY, Wolfe RR. Physiologic hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle. J Clin Invest 95: 811–819, 1995.[ISI][Medline]
  4. Biolo G, Fleming RY, Maggi SP, Nguyen TT, Herndon DN, Wolfe RR. - Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients. J Clin Endocrinol Metab 87: 3378–3384, 2000.[CrossRef]
  5. Biolo G, Fleming RY, Maggi SP, Nguyen TT, Herndon DN, Wolfe RR. Inhibition of muscle glutamine formation in hypercatabolic patients. Clin Sci (Lond) 99: 189–194, 2000.[Medline]
  6. Biolo G, Fleming RY, Maggi SP, Wolfe RR. Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle. Am J Physiol Endocrinol Metab 268: E75–E84, 1995.[Abstract/Free Full Text]
  7. Biolo G, Iscra F, Bosutti A, Toigo G, Ciocchi B, Geatti O, Gullo A, Guarnieri G. Growth hormone decreases muscle glutamine production and stimulates protein synthesis in hypercatabolic patients. Am J Physiol Endocrinol Metab 279: E323–E332, 2000.[Abstract/Free Full Text]
  8. Biolo G, Tipton KD, Klein S, Wolfe RR. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am J Physiol Endocrinol Metab 273: E122–E129, 1997.[Abstract/Free Full Text]
  9. Bruce CR, Carey AL, Hawley JA, Febbraio MA. Intramuscular heat shock protein 72 and heme oxygenase-1 mRNA are reduced in patients with type 2 diabetes: evidence that insulin resistance is associated with a disturbed antioxidant defense mechanism. Diabetes 52: 2338–2345, 2003.[Abstract/Free Full Text]
  10. Castellino P, Luzi L, Giordano M, DeFronzo RA. Effects of insulin and amino acids on glucose and leucine metabolism in CAPD patients. J Am Soc Nephrol 10: 1050–1058, 1999.[Abstract/Free Full Text]
  11. Castellino P, Solini A, Luzi L, Barr JG, Smith DJ, Petrides A, Giordano M, Carroll C, De Fronzo RA. Glucose and amino acid metabolism in chronic renal failure: effect of insulin and amino acids. Am J Physiol Renal Fluid Electrolyte Physiol 262: F168–F176, 1992.[Abstract/Free Full Text]
  12. Cobelli C, Saccomani MP, Tessari P, Biolo G, Luzi L, Matthews DE. Compartmental model of leucine kinetics in humans. Am J Physiol Endocrinol Metab 261: E539–E550, 1991.[Abstract/Free Full Text]
  13. Conley SB, Rose GM, Robson AM, Bier DM. Effects of dietary intake and hemodialysis on protein turnover in uremic children. Kidney Int 17: 837–846, 1980.[ISI][Medline]
  14. Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price SR, Mitch WE. Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. J Clin Invest 113: 115–123, 2004.[CrossRef][ISI][Medline]
  15. Fan J, Char D, Bagby GJ, Gelato MC, Lang CH. Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding proteins by tumor necrosis factor. Am J Physiol Regul Integr Comp Physiol 269: R1204–R1212, 1995.[Abstract/Free Full Text]
  16. Ferrando AA, Raj DSC, Wolfe RR. Amino acid control of muscle protein turnover in renal disease. J Ren Nutr 15: 34–36, 2005.[CrossRef][ISI][Medline]
  17. Ferrando AA, Stuart CA, Sheffield-Moore M, Wolfe RR. Inactivity amplifies the catabolic response of skeletal muscle to cortisol. J Clin Endocrinol Metab 84: 3515–3521, 1999.[Abstract/Free Full Text]
  18. Fischer CP, Bode BP, Abcouwer SF, Lukaszewicz GC, Souba WW. Hepatic uptake of glutamine and other amino acids during infection and inflammation. Shock 3: 315–322, 1995.[ISI][Medline]
  19. Fujita J, Tsujinaka T, Yano M, Ebisui C, Saito H, Katsume A, Akamatsu K, Ohsugi Y, Shiozaki H, Monden M. Anti-interleukin-6 receptor antibody prevents muscle atrophy in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-dependent proteolytic pathways. Int J Cancer 68: 637–643, 1996.[CrossRef][ISI][Medline]
  20. Garcia-Martinez C, Lopez-Soriano FJ, Argiles JM. Interleukin-6 does not activate protein breakdown in rat skeletal muscle. Cancer Lett 76: 1–4, 1994.[CrossRef][ISI][Medline]
  21. Goodship TH, Lloyd S, Clague MB, Bartlett K, Ward MK, Wilkinson R. Whole body leucine turnover and nutritional status in continuous ambulatory peritoneal dialysis. Clin Sci (Lond) 73: 463–469, 1987.[Medline]
  22. Guidotti GG, Gazzola GC. Amino acid transporters: systematic approach and principles of controls. In: Mammalian Amino Acid Transport, edited by Kilberg MS and Haussinger D. New York: Plenum, 1992, p. 9–29.
  23. Gutierrez A, Alvestrand A, Wahren J, Bergstrom J. Effect of in vivo contact between blood and dialysis membranes on protein catabolism in humans. Kidney Int 38: 487–494, 1990.[ISI][Medline]
  24. Haddad F, Zaldivar F, Cooper DM, Adams GR. IL-6-induced skeletal muscle atrophy. J Appl Physiol 98: 911–917, 2005.[Abstract/Free Full Text]
  25. Hart DW, Wolf SE, Chinkes DL, Gore DC, Mlcak RP, Beauford RB, Obeng MK, Lal S, Gold WF, Wolfe RR, Herndon DN. Determinants of skeletal muscle catabolism after severe burn. Ann Surg 232: 455–465, 2000.[CrossRef][ISI][Medline]
  26. Hou JC, Zhou JN, Zhu HW, Wu JZ, Wu JC, Zhang MW. Dynamic aspects of whole-body nitrogen metabolism in uremic patients on dietary therapy. Nephron 44: 288–294, 1986.[ISI][Medline]
  27. Ikizler TA, Pupim LB, Brouillette JR, Levenhagen DK, Farmer K, Hakim RM, Flakoll PJ. Hemodialysis stimulates muscle and whole body protein loss and alters substrate oxidation. Am J Physiol Endocrinol Metab 282: E107–E116, 2002.[Abstract/Free Full Text]
  28. Kobayashi H, Borsheim E, Anthony TG, Traber DL, Badalamenti J, Kimball SR, Jefferson LS, Wolfe RR. Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B. Am J Physiol Endocrinol Metab 284: E488–E498, 2003.[Abstract/Free Full Text]
  29. Kopple JD, Monteon FJ, Shaib JK. Effect of energy intake on nitrogen metabolism in nondialyzed patients with chronic renal failure. Kidney Int 29: 734–742, 1986.[ISI][Medline]
  30. Lang CH, Hong-Brown L, Frost RA. Cytokine inhibition of JAK-STAT signaling: a new mechanism of growth hormone resistance. Pediatr Nephrol 20: 306–312, 2005.[CrossRef][ISI][Medline]
  31. Lim VS, Bier DM, Flanigan MJ, Sum-Ping ST. The effect of hemodialysis on protein metabolism. A leucine kinetic study. J Clin Invest 91: 2429–2436, 1993.[ISI][Medline]
  32. Lim VS, Yarasheski KE, Crowley JR, Fangman J, Flanigan M. Insulin is protein-anabolic in chronic renal failure patients. J Am Soc Nephrol 14: 2297–2304, 2003.[Abstract/Free Full Text]
  33. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2{{uparrow}–{Delta}{Delta}CT} method. Methods 25: 402–408, 2001.[CrossRef][ISI][Medline]
  34. Loder PB, Smith RC, Kee AJ, Kohlhardt SR, Fisher MM, Jones M, Reeve TS. What rate of infusion of intravenous nutrition solution is required to stimulate uptake of amino acids by peripheral tissues in depleted patients? Ann Surg 211: 360–368, 1990.[ISI][Medline]
  35. Lowell BB, Ruderman NB, Goodman MN. Regulation of myofibrillar protein degradation in rat skeletal muscle during brief and prolonged starvation. Metabolism 35: 1121–1127, 1986.[CrossRef][ISI][Medline]
  36. Odessey R, Goldberg AL. Oxidation of leucine by rat skeletal muscle. Am J Physiol 223: 1376–1383, 1972.[Free Full Text]
  37. Paddon-Jones D, Sheffield-Moore M, Creson DL, Sanford AP, Wolf SE, Wolfe RR, Ferrando AA, Ferrando AA. Hypercortisolemia alters muscle protein anabolism following ingestion of essential amino acids. Am J Physiol Endocrinol Metab 284: E946–E953, 2003.[Abstract/Free Full Text]
  38. Pupim LB, Flakoll PJ, Brouillette JR, Levenhagen DK, Hakim RM, Ikizler TA. Intradialytic parenteral nutrition improves protein and energy homeostasis in chronic hemodialysis patients. J Clin Invest 110: 483–492, 2002.[CrossRef][ISI][Medline]
  39. Raj DS, Welbourne T, Dominic EA, Waters D, Wolfe R, Ferrando A. Glutamine kinetics and protein turnover in end-stage renal disease. Am J Physiol Endocrinol Metab 288: E37–E46, 2005.[Abstract/Free Full Text]
  40. Raj DS, Zager P, Shah VO, Dominic EA, Adeniyi O, Blandon P, Wolfe R, Ferrando A. Protein turnover and amino acid transport kinetics in end-stage renal disease. Am J Physiol Endocrinol Metab 286: E136–E143, 2004.[Abstract/Free Full Text]
  41. Raj DSC, Dominic EA, Pai A, Osman F, Morgan M, Pickett G, Shah VO, Ferrando A, Moseley P. Skeletal muscle, cytokines and oxidative stress in End-stage renal disease. Kidney Int 68: 2338–2344, 2005.[CrossRef][ISI][Medline]
  42. Raj DSC, Dominic EA, Wolfe RA, Shah VO, Bankhurst A, Zager P, Ferrando A. Co-ordinated increase in albumin, fibrinogen and muscle protein synthesis during hemodialysis: role of cytokines. Am J Physiol Endocrinol Metab 286: E658–E664, 2004.[Abstract/Free Full Text]
  43. Raj DSC, Shah H, Shah V, Ferrando A, Bankhurst A, Wolfe R, Zager P. Markers of inflammation, proteolysis and apoptosis in ESRD. Am J Kidney Dis 42: 1212–1220, 2003.[CrossRef][ISI][Medline]
  44. Sakurai Y, Zhang XJ, Wolfe RR. Effect of tumor necrosis factor on substrate and amino acid kinetics in conscious dogs. Am J Physiol Endocrinol Metab 266: E936–E945, 1994.[Abstract/Free Full Text]
  45. Tessari P, Garibotto G, Inchiostro S, Robaudo C, Saffioti S, Vettore M, Zanetti M, Russo R, Deferrari G. Kidney, splanchnic, and leg protein turnover in humans. Insight from leucine and phenylalanine kinetics. J Clin Invest 98: 1481–1492, 1996.[ISI][Medline]
  46. Tsujinaka T, Fujita J, Ebisui C, Yano M, Kominami E, Suzuki K, Tanaka K, Katsume A, Ohsugi Y, Shiozaki H, Monden M. Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice. J Clin Invest 97: 244–249, 1996.[ISI][Medline]
  47. Volpi E, Ferrando AA, Yeckel CW, Tipton KD, Wolfe RR. Exogenous amino acids stimulate net muscle protein synthesis in the elderly. J Clin Invest 101: 2000–2007, 1998.[ISI][Medline]
  48. Watt PW, Corbett ME, Rennie MJ. Stimulation of protein synthesis in pig skeletal muscle by infusion of amino acids during constant insulin availability. Am J Physiol Endocrinol Metab 263: E453–E460, 1992.[Abstract/Free Full Text]
  49. Williams A, Wang JJ, Wang L, Sun X, Fischer JE, Hasselgren PO. Sepsis in mice stimulates muscle proteolysis in the absence of IL-6. Am J Physiol Regul Integr Comp Physiol 275: R1983–R1991, 1998.[Abstract/Free Full Text]
  50. Wolfe RR, Miller SL. Amino acid availability controls muscle protein metabolism. Diabetes Nutr Metab 12: 322–328, 1999.[ISI][Medline]
  51. Workeneh BT, Rondon-Berrios H, Zhang L, Hu Z, Ayehu G, Ferrando A, Kopple JD, Wang H, Storer T, Fournier M, Lee SW, Du J, Mitch WE. Development of a diagnostic method for detecting increased muscle protein degradation in patients with catabolic conditions. J Am Soc Nephrol 17: 3233–3239, 2006.[Abstract/Free Full Text]




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