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Am J Physiol Endocrinol Metab 286: E136-E143, 2004. First published September 16, 2003; doi:10.1152/ajpendo.00352.2003
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Protein turnover and amino acid transport kinetics in end-stage renal disease

Dominic S. C. Raj,1 Philip Zager,1 Vallbh O. Shah,1 Elizabeth A. Dominic,2 Oladipo Adeniyi,1 Pedro Blandon,1 Robert Wolfe,3 and Arny Ferrando3

1Division of Nephrology, University of New Mexico Health Sciences Center, Albuquerque 87131 and 2Albuquerque Academy, Albuquerque, New Mexico 87109; and 3Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77550

Submitted 1 August 2003 ; accepted in final form 13 September 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein and amino acid metabolism is abnormal in end-stage renal disease (ESRD). Protein turnover is influenced by transmembrane amino acid transport. The effect of ESRD and hemodialysis (HD) on intracellular amino acid transport kinetics is unknown. We studied intracellular amino acid transport kinetics and protein turnover by use of stable isotopes of phenylalanine, leucine, lysine, alanine, and glutamine before and during HD in six ESRD patients. Data obtained from amino acid concentrations and enrichment in the artery, vein, and muscle compartments were used to calculate intracellular amino acid transport and muscle protein synthesis and catabolism. Fractional muscle protein synthesis (FSR) was estimated by the precursor product approach. Despite a significant decrease in the plasma concentrations of amino acids in the artery and vein during HD, the intracellular concentrations remained stable. Outward transport of the amino acids was significantly higher than the inward transport during HD. FSR increased during HD (0.0521 ± 0.0043 vs. 0.0772 ± 0.0055%/h, P < 0.01). Results derived from compartmental modeling indicated that both protein synthesis (118.3 ± 20.6 vs. 146.5 ± 20.6 nmol·min-1·100 ml leg-1, P < 0.01) and catabolism (119.8 ± 18.0 vs. 174.0 ± 14.2 nmol·min-1·100 ml leg-1, P < 0.01) increased during HD. However, the intradialytic increase in catabolism exceeded that of synthesis (57.8 ± 13.8 vs. 28.0 ± 8.5%, P < 0.05). Thus HD alters amino acid transport kinetics and increases protein turnover, with net increase in protein catabolism.

protein synthesis; protein catabolism; amino acid metabolism; hemodialysis

LOSS OF LEAN BODY MASS is common in patients with end-stage renal disease (ESRD) (47). The etiology of uremic cachexia remains elusive and controversial. However, malnutrition is one of the most important predictors of increased mortality and morbidity. Accumulation of uremic toxins, anorexia, metabolic acidosis, loss of metabolizing renal tissue, perturbations in the production of or responsiveness to catabolic and anabolic hormones, and activation of cytokines act in concert to contribute to the muscle wasting in uremia (4, 5). A healthy adult synthesizes and degrades ~200 g of tissue protein every day; consequently, even a small but sustained change in protein balance will have a major impact on lean body mass (44). Augmented protein catabolism and/or decreased protein synthesis in ESRD could be the cause of uremic cachexia. A number of investigators have observed that protein turnover is abnormal in ESRD. Metabolic acidosis that accompanies chronic renal failure (CRF) is known to promote protein catabolism (3, 39). However, the effect of uremia per se on protein turnover independent of metabolic acidosis has not been rigorously studied. Also, the effect of hemodialysis on protein turnover remains controversial (22, 25).

Concentrations of amino acids in the blood and muscle free pool are determined by protein turnover, interorgan amino acid flux, and intracellular recycling of amino acids (1, 10, 27). In catabolic states and starvation, amino acids are released from the muscle into the blood to be utilized in other organs (1). On the other hand, amino acids can be actively taken up by muscle during anabolism. Patients with ESRD have unique intracellular and plasma amino acid profiles (2): plasma concentrations of essential amino acids are decreased, whereas those of nonessential amino acids are normal or increased (24, 37). It is well known that transmembrane transport of amino acids varies widely in health (10). However, the effects of ESRD and hemodialysis (HD) on the transmembrane transport and intracellular transport kinetics of amino acids are unknown.

The present study was designed to determine the intracellular transport kinetics and protein turnover in patients with ESRD before and during HD. We estimated the kinetics of three essential and two nonessential amino acids in the leg muscles of six ESRD patients with a three-compartmental model (artery, vein, and muscle). Protein synthesis and catabolism were determined by compartmental modeling and the precursor product approach.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Six stable ESRD patients on maintenance HD were recruited from the outpatient dialysis unit of the New Mexico Health Sciences Center. Only patients who were on maintenance HD for >=90 days without residual renal function and who were between 18 and 65 yr were included. Patients with diabetes, infection, chronic inflammation, pregnancy, hematocrit <30%, bleeding diathesis, preexisting cardiac condition, and 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.

Methods. Participants were placed on a diet containing 35 kcal/kg, 1.2 g protein·kg-1·day-1 (>=50% ideal protein), salt 4-5 g/day, and phosphorous 14 mg·kg-1·day-1. Patients consumed the recommended diet at home for a minimum of 14 days before the study. Dietary intake was confirmed by a 3-day dietary history before the experiment. Predialysis plasma bicarbonate (HCO3) was checked 3 wk before the experiment. If plasma HCO3 was <22 meq/l, patients were initiated on oral NaHCO3 supplementation, and the dose was adjusted to achieve a target plasma HCO3 level of >=22 meq/l. The experiments were performed only when the patient's plasma HCO3 was >22 meq/l over a period of >=2 wk.

Subjects were admitted to the General Clinical Research Center (GCRC) at the University of New Mexico Health Sciences Center 1 day before the experiment. Experiments were performed 72 h after dialysis. This timing was selected to study the protein dynamics at the peak of metabolic abnormality. Anthropometry was performed, and leg volume was estimated as previously described (10, 17). The study was performed in a postabsorptive state after an 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 indocyanine 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 amino acids was initiated at the rates given in Table 1. Infusion of all of the tracers was started at 0 h and continued throughout the experiment (Fig. 1). Blood flow to the lower extremity was measured by a dye dilution technique (23). Briefly, a continuous infusion of ICG dye into the femoral artery was initiated at a rate of 1 ml/min ~30 min before the second and third biopsies. Samples were obtained every 10 min from the femoral and arterialized wrist veins to measure plasma ICG concentrations. Dye concentration was measured spectroscopically at {lambda} 805 nm. Leg plasma flow was calculated from steady-state ICG concentrations in the femoral artery and arterialized wrist vein.


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  		Table 1 Tracer infusion rates

 


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  		Fig. 1. Study design. Six end-stage renal disease (ESRD) patients on maintenance hemodialysis (HD) were studied. The study was divided into a basal predialysis phase (postabsorptive phase, 0-300 min) and an HD phase (300-540 min). After blood samples had been taken for background at 0 min, a primed continuous infusion of L-[5-15N]glutamine, L-[ring-13C6]phenylalanine, L-[1-13C]leucine, L-[2-15N]lysine, and L-[1-13C]alanine was started and continued throughout the experiment. From 240 to 300 min and from 480 to 540 min, blood samples were obtained from artery and vein to estimate enrichment and arteriovenous balance and to measure protein kinetics. Muscle biopsies were taken at 120, 300, and 540 min. A continuous infusion of indocyanine green (ICG) was administered to the femoral artery from 240 to 270 min and from 480 to 510 min to estimate rate of blood flow to the leg. Blood samples were taken from femoral and wrist veins every 10 min from 240 to 270 min and from 480 to 510 min to estimate ICG concentration.

 

HD was initiated at 300 min and continued for 4 h. Patients were dialyzed with their usual blood and dialysate flow rates. Dialysate composition was as follows: sodium (Na+), 139 meq/l; bicarbonate (HCO3), 35 meq/l; calcium (Ca2+), 2.5 meq/l; magnesium (Mg2+), 1 meq/l; dextrose 200 mg/dl; and potassium (K+), per patient's need. To eliminate the impact of bioincompatibility on protein turnover, a new polysulfone membrane (F70, Fresenius, Hemoflow) was used. To minimize the risk of bleeding, anticoagulation was not used during dialysis.

Muscle biopsies were performed at the 2nd h to measure isotopic carbon enrichment of bound and free phenylalanine in the muscle. The second biopsy was obtained at the 5th h to estimate the fractional synthesis rate (FSR) of protein before HD. The third biopsy was obtained at the 4th h of HD. Biopsies were taken from the lateral portion of the vastus lateralis ~20 cm above the knee by use of a Bergstrom biopsy needle. Fat and connective tissue were removed, and the samples were frozen in liquid nitrogen and stored at -80°C for future analysis.

Blood urea nitrogen, creatinine, electrolyte, and hemoglobin concentrations were measured before and at the end of HD. Cytokines (IL-1, IL-6, IL-10, and TNF-{alpha}) were measured in plasma samples obtained from the participants before and at the end of HD with commercially available ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's directions. Assays were performed in duplicate, and the mean of the two measurements was used. The interassay and intra-assay coefficients of variation (CVs, in %) for each of the cytokines were <6%. Serum albumin was measured by the bromcresol green method; prealbumin by nephelometry; insulin-like growth factor, glucagon, and catecholamines by RIA; and TSH, insulin, and cortisol by immulite chemiluminescence.

Analytical procedures. Blood samples for the measurement of amino acid concentrations and enrichment were collected as previously described (10). A known amount of an internal standard mixture (100 µl/ml of blood) was added for measurement of amino acid concentrations. The composition of the internal standard was as follows: 1.13 mmol/l of [U-13C9-15N]phenylalanine, 9.27 mmol/l of [U-13C6]leucine, 4.89 mmol/l of L-[2H4]lysine, 21.55 mmol/l of [2H4]alanine, and 10.00 mmol/l of [U-13C5]glutamine. Blood amino acids were separated using cation exchange chromatography (46). The enrichment of free amino acids in the arterial and venous samples was determined by gas chromatography-mass spectrometry (GC-MS; GC HP-5890, MSD HP-5989; Hewlett-Packard, Palo Alto, CA) by selected ion (mass-to-charge ratio, m/z) monitoring. Chemical ionization was used for nitrogen-acetyl-n-propyl ester derivatives of leucine (m/z ratios 302, 303, and 308), lysine (m/z ratios 431, 432, and 235), phenylalanine (m/z ratios 336, 342, and 346), and alanine (m/z ratios 260, 261, and 264). A separate aliquot of the sulfosalicylic acid extract was processed to obtain the t-butyldimethylsilyl (t-BDMS) derivative of glutamine (46). Electron impact ionization was used for the t-BDMS derivative of glutamine (m/z ratios 431, 432, and 436).

Muscle samples were weighed and the proteins precipitated with 450 µl of 14% perchloric acid. An internal standard solution (2 µl/mg of muscle tissue) was added to measure the intracellular concentrations of the traced amino acids. The internal standard solution contained 3.00 µmol/l of [U-13C9-15N]phenylalanine, 9.64 µmol/l of [U-13C6]leucine, 6.38 µmol/l of L-[2H4]lysine, 92.3 µmol/l of [2H4]alanine, and 775.0 µmol/l of [U-13C5]glutamine. The tissue was homogenized and centrifuged, and the supernatant was collected. The enrichment and concentration of amino acids were determined as described previously (10). The tissue pellet was further washed and dried. The precipitated protein was hydrolyzed at 110°C for 24 h with 6 N constantly boiling HCl. The protein hydrolysate was then processed as blood samples, and phenylalanine enrichment was measured by GC-MS (GC 8000 series, MD 800; Fisons Instruments, Manchester, UK) with chemical ionization and the standard curve approach (14).

Compartmental model. The kinetics of intracellular amino acids were determined using a three-compartmental model (10, 23). Amino acids entering the leg through the femoral artery (Fin) and leaving the leg via the femoral vein (Fout), rates of inward (Fma) and outward (Fvm) transmembrane transport, and the intracellular appearance of amino acids (Fmo) and amino acid utilization (Fom) were computed (Fig. 2). Fmo of phenylalanine, leucine, and lysine represents the protein breakdown. Fom derived from alanine and glutamine kinetics is a measure of protein breakdown and de novo synthesis. Fom values of phenylalanine and lysine are indexes of protein synthesis, but Fom of leucine is a measure of synthesis and oxidation

where Ca and Cv are the amino acid concentrations in the artery and vein, and BF is plasma flow to the leg.



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  		Fig. 2. Three-compartmental model with unidirectional flow of amino acids (AA) between artery (A), vein (V), and muscle (M) compartments. AA enter leg through femoral artery (Fin) and exit through femoral vein (Fout). Fva is the shunt between artery and vein. Fma is inward transport of AA from artery; Fvm is outward transport of AA from muscle to vein. Fmo is rate of appearance of AA in a intracellular compartment. It represents protein breakdown for Phe, Leu, and Lys and proteolysis and de novo synthesis for Ala. Fom is the rate of disappearance of AA in the intracellular compartment. It represents protein synthesis for Phe and Lys and protein synthesis and other metabolic pathways for Ala and Leu.

 

Net amino acid balance (NB) in the leg can be calculated as

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

The sum of inward transport of amino acids and intracellular appearance from de novo sources (Ram) can be calculated as

The intracellular amino acid appearance from protein breakdown and or de novo synthesis (Fmo) is calculated as

Because NB is the difference between Fma and Fmo, by integrating Eq. 8, the rate of intracellular amino acid utilization (Fom) can be estimated as

FSR will be calculated as

where Em(1) and Em(2) represent the L-[ring-13C6]phenylalanine enrichments in the free muscle pool in the consecutive biopsies. T is the time interval between the first and second biopsies.

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. Linear regression analysis was used to identify the relationship between variables.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Patient characteristics and biochemical profiles are described in Table 2. Etiology of renal failure was hypertension in two, glomerulonephritis in one, tubulointerstitial nephropathy in one, and unknown in two patients. Protein and calorie intake amounts were 1.46 ± 0.07 g·kg-1·day-1 and 32.9 ± 3.1 kcal·kg-1·day-1, respectively. Predialysis plasma bicarbonate was 24.3 ± 0.7 meq/l. The total ultrafiltration during HD was 2.9 ± 0.3 liters. Mean blood and dialysate flow rates during HD were 376.2 ± 12.8 and 800 ml/min, respectively. Blood flow to the leg did not change significantly during HD compared with predialysis flow rates (4.26 ± 0.25 vs. 4.25 ± 0.17 ml·min-1·100 ml leg-1). Plasma levels of cytokines (pg/dl) IL-1 (11.8 ± 2.4 vs. 26.9 ± 4.3, P < 0.03), IL-6 (1.2 ± 0.04 vs. 1.5 ± 0.09, P < 0.02), and IL-10 (0.9 ± 0.3 vs. 2.2 ± 0.5, P < 0.01) increased during HD from baseline. In contrast, TNF-{alpha} (0.8 ± 0.1 vs. 0.9 ± 0.4 pg/dl, P = NS) levels did not change during HD.


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  		Table 2. Demography and biochemistry

 

Plasma concentrations of amino acids in the artery and vein decreased significantly during HD (Table 3). Amino acid concentration in the vein was higher than that in the artery during HD, indicating a net release of amino acids from the muscle. Concentrations of the amino acids in the muscle free pool was higher than that in arterial and venous blood, but the tissue-to-artery gradient varied widely for different amino acids. The concentrations of the traced amino acids in the muscle did not change significantly during HD despite a decrease in arterial and venous concentrations, resulting in an increased tissue-to-artery gradient during HD for all of the amino acids studied (P < 0.05). The intracellular enrichments in the muscle were lower than those in artery and vein because of intracellular tracer dilution from unlabeled amino acids originating from de novo synthesis and protein breakdown (Table 4).


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  		Table 3. Free amino acid concentrations in plasma samples of femoral artery and vein, skeletal muscle, and dialysate

 

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  		Table 4. Free amino acid enrichment in femoral artery, vein, and muscle

 

The absolute rates of transmembrane transport of the traced amino acids varied widely (Table 5). To eliminate the impact of differences in the arterial concentrations of amino acids on the inward transport, the absolute inward transport rate (Fma) was normalized to the prevailing amino acid concentration in the artery (Ca). The normalized inward transport values (Fma/Ca) tended to increase during HD compared with pre-HD values: phenylalanine (1.99 ± 0.31 vs. 2.47 ± 0.38), leucine (1.21 ± 0.12 vs. 2.0 ± 0.38), alanine (1.75 ± 0.40 vs. 2.30 ± 0.43), and glutamine (1.10 ± 0.10 vs. 1.24 ± 0.15); however, they reached statistical significance only for lysine (1.29 ± 0.14 vs. 2.03 ± 0.34, P < 0.01). The outward transport (Fvm) of leucine and lysine increased during HD from baseline (P < 0.05). Fvm was significantly higher than Fma for all traced amino acids during HD. The outward transport values, as a function of intracellular amino acid concentrations (Fvm/Cm) before and during HD, were 1.74 ± 0.22 vs. 2.21 ± 0.32 for phenylalanine, 1.57 ± 0.13 vs. 1.92 ± 0.17 for leucine (P < 0.01), 0.32 ± 0.06 vs. 0.47 ± 0.09 for lysine (P < 0.01), 0.42 ± 0.13 vs. 0.51 ± 0.15 for alanine, and 0.05 ± 0.01 vs. 0.06 ± 0.01 for glutamine.


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  		Table 5. Amino acid transport kinetics before and during HD

 

Rates of utilization of intracellular amino acids (Fom) increased during HD for all traced amino acids except glutamine (Fig. 3). However, the increase was statistically significant only for phenylalanine and lysine. Intracellular amino acid appearance (Fmo) increased intradialysis for all essential amino acids, indicating net protein breakdown (P < 0.01). Ram, the total intracellular amino acid appearance (sum of inward transport, protein breakdown, and de novo synthesis), increased significantly for phenylalanine, leucine, and alanine during HD. The ratio of Fom to Fmo was higher during pre-HD compared with HD for phenylalanine (1.02 ± 0.03 vs. 0.84 ± 0.06, P < 0.05) and leucine (0.96 ± 0.04 vs. 0.76 ± 0.09, P < 0.05), lysine (0.90 ± 0.09 vs. 0.75 ± 0.07), alanine (0.84 ± 0.09 vs. 0.83 ± 0.08), and glutamine (0.97 ± 0.03 vs. 0.88 ± 0.03). Fom and Fmo were correlated with each other before (r2 = 0.95, P < 0.001) and during HD (r2 = 0.68, P < 0.05).



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  		Fig. 3. Protein synthesis (Fom) and catabolism (Fmo) estimated from compartmental modeling. Data are expressed as nmol·min-1·100 ml leg-1. Both protein synthesis (28.0 ± 8.5%) and catabolism (57.8 ± 13.8%) increased during HD (P < 0.01). Protein synthesis was comparable to breakdown before dialysis (pre-HD). However, protein catabolism was higher than synthesis during HD (P < 0.05).

 

The increment {Delta}Ep of the enrichment of muscle protein-bound phenylalanine increased during HD (6.40·10-5 ± 0.27·10-5 vs. 14.5·10-5 ± 0.51·10-5, P < 0.05), and the intracellular phenylalanine enrichment decreased (0.0412 ± 0.0132 vs. 0.0308 ± 0.00953, P < 0.05). The net result was a significant increase in FSR of muscle protein during HD compared with pre-HD (0.0521 ± 0.0043 vs. 0.0772 ± 0.0055%/h, P < 0.01; Fig. 4). Protein synthesis estimated from the precursor product approach (FSR) and that derived from compartmental modeling (Fom) were correlated (r2 = 0.59, P < 0.03). There was no significant correlation between Fma and Fom.



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  		Fig. 4. Muscle protein synthesis was measured before (pre-HD) and during HD by the precursor product approach in 6 ESRD patients. Fractional protein synthesis rate increased by 50.3 ± 9.7% during dialysis compared with predialysis (P < 0.01).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We estimated the protein synthesis and catabolism in ESRD patients who were without metabolic acidosis and who consumed an adequate protein diet, and we demonstrated that the balance between synthesis and catabolism is maintained predialysis. However, HD increases net protein catabolism. We also estimated the transmembrane transport of amino acids by using multiple tracers. We found that the net outward transport (Fvm) of amino acids increases during HD, with no significant change in inward transport (Fma). The complex physiological system of amino acids and protein kinetics can be studied by suitable compartmental modeling. The advantage of compartmental modeling is that it enables the calculation of kinetic factors occurring in nonsampled, intracellular compartments. Cobelli et al. (15) proposed a ten-compartmental model to estimate protein synthesis, catabolism, and leucine oxidation. In this study, we have used a robust three-compartmental model developed in our laboratory (10).

Borah et al. (13) noted that protein balance is either more negative or less positive on the day of dialysis compared with interdialysis days. Garibotto et al. (18), using phenylalanine kinetics, observed a balanced increase in protein synthesis and catabolism with no net increase in catabolism in patients with CRF. Our study also shows that ESRD patients are in a state of metabolic equilibrium despite the uremia before dialysis. The possible mechanisms for protein conservation in CRF are downregulation of genes promoting protein catabolism (38), correction of metabolic acidosis, and adequate protein and calorie intake by the participants. Berkelhammer et al. (6), using an L-[1-13C]leucine tracer, found that protein breakdown in ESRD is not different from that in normal subjects. However, they reported increased leucine oxidation in ESRD patients compared with control subjects (6). Conley et al. (16) reported that, in children with ESRD, protein flux is increased after initiation of dialysis. Lim et al. (26) measured longitudinally whole body leucine flux in patients with CRF before and after initiation of dialysis. Using mass balance calculations, they found that the protein synthesis increase was more than the degradation after initiation of HD. However, very few studies have examined the effect of HD on protein turnover. Lim et al. (25) reported normal basal leucine flux, a transient decrease in protein synthesis, and a negative protein balance during HD. Ikizler et al. (22), using L-[1-13C]leucine and L-[ring-2H5]phenylalanine, observed that protein catabolism is increased during HD, with no significant change in protein synthesis. The net result was an increase in whole body and muscle proteolysis. We estimated protein synthesis and catabolism by the precursor product approach and compartmental modeling, using multiple tracers, and observed a significant increase in both protein synthesis and catabolism during HD. However, the intradialytic increase in catabolism exceeded that of synthesis (57.8 ± 13.8 vs. 28.0 ± 8.5%, P < 0.05).

About 80% of amino acids utilized for protein synthesis is derived from recycling of amino acids from protein breakdown, mostly in skeletal muscle (44). The correlation between Fmo and Fom was stronger in the predialysis phase than during HD, indicating that reutilization of amino acids derived from catabolism is more effective during the pre-HD phase than during HD. Also, this observation is supported by the fact that the ratio of Fom to Fmo decreased, and net balance (NB) became more negative during HD compared with pre-HD. Negative nitrogen balance during HD was associated with a large efflux of nitrogen on the two major carriers, alanine and glutamine. However, the contribution of glutamine to negative nitrogen balance is far greater than that of alanine, because glutamine carries both amino and amide nitrogens. Similar to HD, glutamine synthesis is decreased (8) and the Fvm of glutamine is increased (33) in other catabolic conditions. Considering the prolonged nature of the study, the possibility of recycling of tracer from the body proteins is possible. We previously examined the recycling of L-[5-15N]glutamine by estimating the M+1 enrichment of the t-BDMS derivative of alanine in the blood samples and found no [15N]alanine enrichment above the background after 5 h of tracer infusion (10). These results indicate that nitrogen derived from the deamidation of glutamine to glutamate is diluted in the large ammonia pool before incorporation into other amino acids (35, 45).

The amino acids traced were chosen for their unique metabolic pathways and transport kinetics. The transport systems of these amino acids are also different: phenylalanine and leucine are transported through the L system, lysine by the y+ system, alanine by the A, ASC, and L system, and glutamine is carried by the N, A, ASC, and L systems (20). Phenylalanine and lysine are not synthesized or oxidized in skeletal muscle. Also, phenylalanine is not hydroxylated to tyrosine in the muscle. Leucine is catabolized in the muscle (34). Skeletal muscle is the main site of synthesis of alanine and glutamine. In catabolic states, alanine release from muscle is accelerated, but glutamine release is either increased or unchanged (12). Biolo et al. (9) observed that the outward transport increased and the absolute rates of inward transport decreased in hypercatabolic patients. During HD, both inward (Fma/Ca) and outward transport (Fvm/Cm) of all of the traced amino acids showed a tendency to increase. The magnitude of increase in outward transport was significantly higher than that in inward transport for all of the amino acids, confirming net release of amino acids from the muscle during HD.

Amino acid concentrations, protein synthesis, and catabolism are interdependent and regulatory. We previously demonstrated that hyperaminoacidemia stimulates protein synthesis by augmenting amino acid transport into the cell (11, 43). The lack of correlation between inward transport (Fma) and rate of utilization (Fom) of the amino acid in the present study suggests that the increased protein synthesis during HD is not due to augmented inward transport. Despite the loss of amino acids in the dialysate and the resultant decrease in plasma amino acid concentrations, the intracellular amino acid levels did not change significantly during HD. The intracellular amino acid levels are probably maintained by accelerated proteolysis. The intracellular hyperaminoacidemia in turn promotes protein synthesis. However, it is also possible that increased protein synthesis is due to removal of inhibitors of protein synthesis or a compensatory mechanism to prevent excessive protein catabolism (29). Because the increase in inward transport was less than that of the outward transport, augmented inward transport alone could not have contributed to the maintenance of the intracellular concentration of amino acids. During HD, the plasma concentrations of amino acids decreased, indicating that increased catabolism and outward transport are not able to compensate for the dialysate loss of amino acids.

Perturbation in protein and amino acid metabolism in ESRD could be due to hormonal dysregulation. We found a significant increase in plasma cortisol and a modest decrease in insulin during HD. Insulin and IGF-I induce protein anabolism. Metabolic acidosis and cytokine activation mediate muscle catabolism through the glucocortoid-dependent ubiquitin-proteasome pathway (31, 42). We observed a significant increase in cytokine levels during HD. IL-6 plays a major role in regulating protein breakdown in inflammatory states (41). We found a significant correlation between plasma IL-6 levels and caspase-3, ubiquitin, and branched-chain keto acid dehydrogenase-E2 gene expression in the skeletal muscle of the patients (38). Thus cytokines may also play a role in mediating the increased protein catabolism during HD through activation of the ubiquitin-proteasome pathway (32).

Luzi et al. (28) demonstrated that the abnormal protein metabolism in diabetic ESRD patients improves after kidney transplantation. However, combined kidney-pancreas transplantation leads to near normalization of the metabolic abnormalities. Any dialysis therapy can at best be viewed only as an imperfect substitute for the kidney. Although HD may replace the excretory function of the kidney, other components of the uremic syndrome remain uncorrected and continue unabated. Also, inflammation in uremia is further augmented by HD. Muscle protein catabolism and release of amino acids are essential to maintain plasma amino acid concentrations and for synthesis of acute phase response proteins (30). However, prolonged and recurrent episodes of protein catabolism will result in loss of lean body mass. Decreased lean body mass is a strong predictor of outcome in a variety of acute and chronic illnesses (21, 40). Intradialytic protein breakdown can be attenuated by replacement of amino acids lost in the dialysate (19, 36) and by blocking the activation of cytokines (7).

To summarize, a balance between protein synthesis and catabolism is maintained in stable ESRD without metabolic acidosis. However, HD induces an unbalanced increase in synthesis and catabolism, with net increase in protein catabolism. The net outward transmembrane transport of amino acids is increased compared with inward transport during dialysis. The intracellular concentrations of amino acids are maintained in the face of dialysate loss of amino acids and declining plasma amino acid levels in the artery and vein, probably through augmented protein catabolism.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a Paul Teschan Research Grant, a Young Investigator Award from the National Kidney Foundation, and a Research Allocation Committee Grant from the University of New Mexico (all to D. Raj), and National Institutes of Health National Center for Research Resources GCRC Grant 5 MO1 RR-OO997.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent nursing care provided by Rachael Bonner, Sandra McClelland, and Lorraine Martinez; pharmacy support by Nancy Morgan; technical support by Gaurng Jariwala, Kripali Patel, and William McCurdy; and cooperative help from research dietician Rosemary Wold, Rachael Cunnick, Mary Martinez, and the dialysis unit staff at the University of New Mexico.


   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
 

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