Exercise augments the acute anabolic effects of intradialytic parenteral ...

Am J Physiol Endocrinol Metab 286: E589–E597, 2004. First published December 16, 2003; 10.1152/ajpendo.00384.2003.

Exercise augments the acute anabolic effects of intradialytic parenteral nutrition in chronic hemodialysis patients Lara B. Pupim,1 Paul J. Flakoll,2,3 Deanna K. Levenhagen,2 and T. Alp Ikizler1 1

Department of Medicine, Division of Nephrology, 2Department of Surgery, and 3Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Submitted 25 August 2003; accepted in final form 9 December 2003

Pupim, Lara B., Paul J. Flakoll, Deanna K. Levenhagen, and T. Alp Ikizler. Exercise augments the acute anabolic effects of intradialytic parenteral nutrition in chronic hemodialysis patients. Am J Physiol Endocrinol Metab 286: E589–E597, 2004. First published December 16, 2003; 10.1152/ajpendo.00384.2003.—Decreased dietary protein intake and hemodialysis (HD)-associated protein catabolism are among several factors that predispose chronic hemodialysis (CHD) patients to uremic malnutrition and associated muscle wasting. Intradialytic parenteral nutrition (IDPN) acutely reverses the net negative whole body and forearm muscle protein balances observed during the HD procedure. Exercise has been shown to improve muscle protein homeostasis, especially if performed with adequately available intramuscular amino acids. We hypothesized that exercise performance would provide additive anabolic effects to the beneficial effects of IDPN. We studied six CHD patients at two separate HD sessions: 1) IDPN administration only and 2) IDPN ⫹ exercise. Patients were studied 2 h before, during, and 2 h after an HD session by use of a primed constant infusion of L-[1-13C]leucine and L-[ring-2H5] phenylalanine. Exercise combined with IDPN promoted additive twofold increases in forearm muscle essential amino acid uptake (455 ⫾ 105 vs. 229 ⫾ 38 nmol䡠100 ml⫺1 䡠min⫺1, P ⬍ 0.05) and net muscle protein accretion (125 ⫾ 37 vs. 56 ⫾ 30 ␮g䡠100 ml⫺1 䡠min⫺1, P ⬍ 0.05) during HD compared with IDPN alone. Measurements of whole body protein homeostasis and energy expenditure were not altered by exercise treatment. In conclusion, exercise in the presence of adequate nutritional supplementation has potential as a therapeutic intervention to blunt the loss of muscle mass in CHD patients.

been demonstrated to facilitate muscle uptake of amino acids and muscle protein accretion in healthy subjects (25, 26, 42, 49). Exercise directs more nutrients toward muscle tissues and induces a greater sensitivity and responsiveness to metabolic events regulated by insulin compared with nutrient administration alone (45). The influence of exercise combined with nutritional supplementation on protein and energy homeostasis has not been studied in CHD patients. In the present study, we hypothesized that exercise performance followed by parenteral nutritional supplementation during a single HD treatment would augment the anabolic response in the skeletal muscle observed with nutritional supplementation alone. We studied protein homeostasis by use of stable isotope infusion techniques in six CHD patients during one HD session with IDPN alone and during another with IDPN plus exercise. Our results indicate that exercise significantly enhances the anabolic effects of IDPN administration by promoting further increases in muscle amino acid uptake and net muscle protein accretion. METHODS

and muscle wasting are highly prevalent and associated with unacceptably high rates of morbidity and mortality in chronic hemodialysis (CHD) patients (44). The causal factors for this wasting syndrome are multiple, including dialysis-related and non-dialysis-related events (12, 14, 18). Among these factors, decreased dietary intake, sedentary lifestyle, and hemodialysis (HD)-associated catabolism are important, because they can be either prevented and/or treated (15, 16, 23). We have recently shown that nutritional supplementation in the form of intradialytic parenteral nutrition (IDPN) can effectively compensate the catabolic effects of HD procedure by stimulating muscle protein synthesis, promoting a positive net skeletal muscle protein balance (17, 37). Although nutritional supplementation alone is effective in reversing uremic malnutrition to some extent, certain complementary interventions can be utilized to promote its anabolic actions. Exercise with increased amino acid availability has

Patients. Patients were recruited from the Vanderbilt University Outpatient Dialysis Unit. Inclusion criteria for the study specified patients with ⬍100 ml/day of urine output who were on CHD therapy for ⬎6 mo. The patients were receiving an adequate dose of dialysis (single pool Kt/V ⱖ1.4) on a thrice-weekly HD program with a biocompatible HD membrane (Fresenius F80, Fresenius USA, Lexington, MA). Patients were excluded from study if they had active infectious or inflammatory disease (e.g., vascular access infections and overt periodontal disease), hospitalizations within 3 mo before the study, recirculation in the vascular access, or vascular access blood flow ⬍750 ml/min detected on the arteriovenous (a-v) shunt. In addition, patients receiving steroids and/or immunosuppressive agents were excluded. The Institutional Review Board of Vanderbilt University Medical Center approved the study protocol, and written informed consent was obtained from all study patients. Patient characteristics are shown in Table 1. All study patients had sedentary baseline physical activity levels. Within a week before each study, dual-energy X-ray absorptiometry was performed to estimate lean and fat body masses, and resting metabolic rate was measured via indirect calorimetry to acclimate the subjects to this technology. In addition, patients were brought to the General Clinical Research Center (GCRC) a week before the study to estimate the workload required to achieve their maximal heart rate (46) and to test their ability to sustain exercise at 40% of this level for 15 min. Heart rate and blood pressure were monitored while the patients pedaled on a recumbent stationary cycle (Ergonomics 800, Ergolin, Bandhagen, Sweden) with incremental (⫹10 W/min)

Address for reprint requests and other correspondence: T. A. Ikizler, Vanderbilt Univ. Medical Center, 1161 21st Ave. South & Garland, Division of Nephrology, S-3223 MCN, Nashville, TN 37232-2372 (E-mail: [email protected]).

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.

malnutrition; muscle; catabolism

UREMIC MALNUTRITION

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EXERCISE AND PROTEIN METABOLISM IN CHD

Table 1. Demographic and nutritional characteristics and biochemical parameters of the study Demographics Gender (M/F) Race (Caucasian/African American) Age, yr Etiology of ESRD, %

5 (83%)/1 (17%) 1 (17%)/5 (83%) 44 ⫾ 7 3 (50%) Hypertension 2 (33%) ADPKD 1 (17%) Glomerulonephritis

Body composition Body weight, kg Body mass index, kg/cm2 Fat mass by DEXA, %

79.5 ⫾ 7.4 26.8 ⫾ 2.1 23.1 ⫾ 3.8

Baseline biochemistries Serum albumin, g/dl Serum prealbumin, mg/dl Serum transferrin, mg/dl Serum cholesterol, mg/dl Total CO2, mg/dl CRP, mg/dl Hematocrit, % n-PCR, g䡠kg⫺1 䡠day⫺1

IDPN 4.24⫾0.3 43.7⫾8.0 189⫾37 187⫾27 22.3⫾1.9 0.74⫾0.4 38.7⫾2.2 0.97⫾0.14

IDPN ⫹ Exercise 4.09⫾0.3 40.7⫾7.0 195⫾28 165⫾20 23.1⫾2.1 0.81⫾0.7 38.1⫾3.1 1.00⫾0.17

Values are means ⫾ SE of 6 subjects or absolute numbers with percentages in parentheses. Treatment protocols are intradialytic parenteral nutrition (IDPN) and IDPN ⫹ exercise. M, male; F, female; ESRD, end-stage renal disease; ADPKD, autosomal dominant polycystic kidney disease; DEXA, dual-energy X-ray absorptiometry; CRP, C-reactive protein; n-PCR, normalized protein catabolic rate.

changes in workload until the maximal heart rate was attained. ˙ O2), Subsequently, heart rate, blood pressure, oxygen consumption (V ˙ carbon dioxide production (VCO2), energy expenditure, and respiratory quotient (RQ) were monitored as the subjects cycled at 40% of the maximal heart rate for 15 min. Patients that were not able to tolerate this workload were excluded from the study (46). Design. This was a randomized crossover study. After reviewing the inclusion and exclusion criteria, eligible patients were randomly assigned to IDPN or IDPN ⫹ exercise as a first protocol by simple randomization. All patients who participated in this study were crossed over and participated in both protocols, with ⱖ4 wk between studies. Metabolic study. The patients were admitted to the GCRC on the day before the study at ⬃7 PM. They received a meal from the GCRC bionutrition services upon admission, after which they remained fasting. This meal was given ⱖ10 h before the initiation of the study for all patients and consisted of 18% protein and 30% lipids. Energy intake was kept at maintenance levels that were based on the HarrisBenedict equation and each subject’s gender, height, weight, and activity levels. A schematic diagram of the metabolic study day protocol is depicted in Fig. 1. Each study consisted of a pre-HD phase (a 2-h equilibration phase followed by a 0.5-h basal sampling phase), a 4-h HD phase, and a 2-h post-HD phase. Each metabolic study was initiated at ⬃6 AM by starting the infusion of isotopes, which continued throughout the study. The HD phase was started with the initiation of HD. During the HD phase, IDPN was started in both treatments at 30 min into the phase and continued throughout the HD phase. In the IDPN ⫹ exercise treatment protocol, a 15-min exercise was started 15 min after the initiation of HD. The post-HD phase started immediately at the conclusion of HD. A dialysis catheter was placed at the venous site of the a-v shunt of the forearm at 6 AM to collect a baseline blood sample (to assess baseline biochemical nutritional markers and isotopic backgrounds) and then to initiate the isotope infusion. An a-v shunt is commonly used for the vascular access with HD, which is created by connecting AJP-Endocrinol Metab • VOL

an artery to a nearby vein either by direct surgical anastomosis of the native vessels (fistula) or with an artificial synthetic vascular material (graft). In the present study, two patients had a native fistula and four patients had an artificial graft. The arterial side of the a-v shunt was the site of choice used for sampling arterial blood. The only occasion that would affect the arterial purity of the samples would be if there were stenoses in the a-v shunt causing the venous blood to mix with arterial blood (recirculation). Therefore, recirculation of the a-v shunt, as well as vascular access blood flow to assess stenoses in the a-v shunts, was checked in every patient before study by the ultrasound dilution technique (Transonic Systems, Ithaca, NY). Arterial vascular access obtained though the arterial side of the a-v shunt was used to perform HD and to sample arterial blood. The venous site of the a-v shunt was used to infuse the isotopes. Another catheter was placed in a superficial vein (on a retrograde insertion) of the contralateral forearm to sample blood draining the forearm muscle bed. At the start of the experiment, subjects received a bolus injection of NaH13CO3 (0.12 mg/kg), L-[1-13C]leucine (7.2 ␮mol/kg), and L-[ring-2H5]phenylalanine (7.2 ␮mol/kg) to prime the CO2, leucine, and phenylalanine pools, respectively. Subsequently, a continuous infusion of L-[1-13C]leucine (0.12 ␮mol䡠kg⫺1 䡠min⫺1) and L-[ring2 H5]phenylalanine (0.12 ␮mol䡠kg⫺1 䡠min⫺1) was started and continued throughout the remainder of the study. Patients were dialyzed for 4 h with blood flow of 400 ml/min and dialysate flow of 500 ml/min. Ultrafiltration rates were determined by the patients’ needs and “estimated dry weight” and were similar during both treatments. The composition of the dialysate used during the study was identical for all treatments and consisted of sodium (139 meq/l), potassium (2 meq/l), calcium (2.5 meq/l), glucose 200 mg/dl, and bicarbonate (39 meq/l). IDPN infusion was done via the venous port of the bubble trap on the HD tubing and continued throughout the entire HD procedure (total of 3.5 h of IDPN infusion). The IDPN treatment was based upon existing recommendations (24). The solution was given at a rate of 150 ml/h and consisted of 300 ml of an amino acid solution (15% Clinisol; Baxter Healthcare, Deerfield, IL), 150 ml of a 50% dextrose solution (Abbott Laboratories, Abbott Park, IL), and 150 ml of a lipid solution (20% IntraLipid; Kabi Pharmacia, Clayton, NC). In 100 ml of the amino acid solution, there were 1.18 g lysine, 1.04 g leucine, 1.04 g phenylalanine, 960 mg valine, 894 mg histidine, 749 mg isoleucine, 749 mg methionine, 749 mg threonine, 250 mg tryptophan, 2.17 g alanine, 1.47 g arginine, 1.04 g glycine, 894 mg proline, 749 mg glutamate, 592 mg serine, 434 mg aspartate, and 39 mg tyrosine. The total solution provided 188 kcal/h or 3.5 kcal䡠kg fat-free mass (FFM)⫺1 䡠h⫺1. The extra volume, as well as the electrolytes that IDPN provided to the patients, was accounted for and removed during HD.

Fig. 1. Schematic diagram of the metabolic study day protocol. IDPN, intradialytic parenteral nutrition. Arrowheads denote time points for blood draws, breath sample collections, and muscle plasma flow measurements. A primed constant infusion of L-[1-13C]leucine and L-[ring-2H5]phenylalanine was maintained throughout the entire study (360 min).

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In the IDPN ⫹ exercise treatment protocol, patients were placed on a recumbent stationary bicycle to begin exercise 15 min after initiation of HD. The workload during exercise performance was set at 40% of maximal heart rate, as previously explained (46). Exercise performance was continued for a period of 15 min, during which heart rate, ˙ O 2, V ˙ CO2, RQ, and energy expenditure were monitored. At the V conclusion of exercise, the patients were moved back to the dialysis chair. Simultaneous blood and breath samples were collected once before the start of the study, 3 times during the basal sampling phase, 6 times during IDPN and dialysis, and 3 times during the post-HD phase. Blood samples were obtained from arterial and forearm venous sampling sites. Breath samples were collected from the subjects via a Douglas bag with duplicate 20-ml samples placed into nonsiliconized glass vacutainer tubes for measurement of breath 13CO2 enrichment. Subjects were asked to breathe through a mask for 1 min each time blood was collected. In addition, forearm blood flow was estimated using capacitance plethysmography (Hokanson, Bellevue, WA). Simultaneous energy expenditure and RQ were determined by indirect calorimetry with a metabolic cart (Sensormedics 2900, Palo Alto, CA) ˙ CO2, and V ˙ O2. Metabolic cart assessto measure ventilation rates, V ment was also done during exercise. Once HD was finished, dialysis lines were disconnected, and the 2-h post-HD phase ensued. After the post-HD phase, all catheters were removed. The patients were given a meal and observed at the GCRC until they were stable, when they were discharged. Patients continued their chronic HD therapy at the outpatient dialysis unit as scheduled. Analytical procedures. Blood samples were collected into Venoject tubes containing 15 mg of Na2EDTA (Terumo Medical, Elkton, MD). A 3-ml aliquot of blood was transferred to a tube containing EDTA and reduced glutathione, with the plasma stored at ⫺80°C for later measurement of plasma epinephrine and norepinephrine concentrations by high-performance liquid chromatography (10). The remaining blood was spun in a refrigerated (4°C) centrifuge (Beckman Instruments, Fullerton, CA) at 3,000 rpm for 10 min, and plasma was extracted and stored at ⫺80°C for later analysis. Plasma glucose concentrations were determined by the glucose oxidase method (Beckman Instruments). Nutritional biochemical parameters were measured at a specialized end-stage renal disease clinical and special chemistry laboratory (Spectra Laboratories, San Juan, CA). Serum albumin was analyzed using the bromcresol green technique. Serum prealbumin was analyzed by an antigen-antibody complex assay, and serum transferrin was analyzed by turbidimetric reading (Boehringer Mannheim, Indianapolis, IN). C-reactive protein (CRP) was measured using nephelometric analysis at the Vanderbilt University Medical Center clinical chemistry laboratory. Immunoreactive insulin was determined in plasma with a doubleantibody system. Plasma aliquots for glucagon determination were placed in tubes containing 25 kallikrein inhibitor units of aprotinin (FBA Pharmaceutical, New York, NY) and were later measured by established radioimmunoassay with a double-antibody system modified from the method of Morgan and Lazarow (31) for insulin. Insulin and glucagon antisera and standards, as well as 125I-labeled hormones, were obtained from R. L. Gingerich (Linco Research, St. Louis, MO). The Clinical Assays Gammacoat Radioimmunoassay kit (TravenolGenTech, Cambridge, MA) was used to measure plasma cortisol concentrations. Plasma IGF-I concentrations were determined by a radioimmunoassay acid-extraction procedure (Nichols Institute Diagnostics, San Juan Capistrano, CA). Plasma amino acid concentrations were determined by reversed-phase high-performance liquid chromatography after derivatization with phenylisothiocyanate (13). Individual amino acids were also placed into groups for analysis purposes. These groups included branched-chain amino acids (BCAA: the sum of leucine, isoleucine, and valine); essential amino acids (EAA: the sum of arginine, histidine, isoleucine, leucine, lysine, methionine, AJP-Endocrinol Metab • VOL

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phenylalanine, theonine, tryptophan, and valine); total amino acids (TAA: the sum of all individual amino acids); and nonessential amino acids (NEAA: the difference between TAA and EAA). Plasma enrichments of [13C]leucine, [13C]ketoisocaproate (KIC), and [ring-2H5]phenylalanine were determined using gas chromatography-mass spectrometry (GC-MS; Hewlett-Packard, San Fernando, CA). Plasma was deproteinized with 4% perchloric acid, and the supernatant was passed over a cation exchange resin to separate the keto and amino acids. The keto acids were further extracted with methylene chloride and 0.5 M ammonium hydroxide (32). After drying under nitrogen gas, keto and amino acid fractions were derivatized (39) with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide containing 1% t-butyldimethylchlorosilane (MtBDSTFA ⫹ 1% t-BDMCS; Regis Technologies, Morton Grove, IL). The derivatized samples were then analyzed by GC-MS for plasma leucine, phenylalanine, and KIC enrichments with selected ion monitoring. The major fragments analyzed for the t-BDMCS derivative of KIC and [13C]KIC were the (M-57) ion fragment mass-to-charge ratios (m/z) 301 and 302, respectively. The enrichment was quantified in plasma as the ratio of [13C]KIC to KIC (ion abundance of 301/302 m/z). Enrichment measurements were made in duplicate, and duplicates had a coefficient of variation of ⬍3%. Breath 13CO2 was measured by isotope ratio mass spectrometry (Metabolic Solutions, Nashua, NH) (40). Calculations. Net skeletal muscle protein balance (synthesis minus breakdown) was determined by dilution and enrichment of phenylalanine across the forearm, as described by Gelfand and Barrett (9). Because phenylalanine is neither synthesized de novo nor metabolized by skeletal muscle, rate of appearance (Ra) of unlabeled phenylalanine reflects muscle protein breakdown. Accordingly, the rate of disappearance (Rd) of labeled phenylalanine estimates muscle protein synthesis (9). Phenylalanine Rd was calculated by multiplying the fractional extraction of the labeled phenylalanine (based on plasma arterial and venous phenylalanine enrichments and concentrations) by the arterial phenylalanine concentration and was normalized to forearm blood flow measured by plethysmography (expressed as 100 ml/min). Net phenylalanine Ra was calculated by subtracting the net a-v balance of phenylalanine across the extremity from the phenylalanine Rd (9, 47). Rates of skeletal muscle protein breakdown and net synthesis were determined from the phenylalanine Rd and Ra, with the assumption that 3.8% of skeletal muscle protein is comprised of phenylalanine. The steady-state rates of total whole body leucine Ra were calculated by dividing the [13C]leucine infusion rate by the plasma [13C]KIC enrichment (47). Plasma KIC provides a better estimate of intracellular leucine enrichment than does plasma leucine enrichment because KIC is derived from intracellular leucine metabolism (47). Steady-state conditions for KIC and CO2 enrichments were achieved, as evidenced by slopes within each phase not significantly different from zero (data not shown). Endogenous leucine Ra (an estimate of whole body protein breakdown) was determined by subtracting the rate of leucine infusion via the IDPN from the total Ra (expressed as mg䡠kg FFM⫺1 䡠min⫺1). Breath 13CO2 production was determined by multiplying the total CO2 production rate by the breath 13CO2 enrichment (47). The rate of whole body leucine oxidation was calculated by dividing breath 13CO2 production by 0.8 [correction factor for the retention of 13CO2 in the bicarbonate pool (1)] and by the plasma KIC enrichment. Leucine Rd during the dialysis phase was corrected for leucine loss into the ultrafiltration volume by measuring the ultrafiltration volume and the leucine concentration in the dialysate and by subtracting the leucine lost in the dialysate from the total Ra. The nonoxidative leucine Rd, an estimate of whole body protein synthesis, was determined indirectly by subtracting leucine oxidation from corrected total leucine Rd. Rates of whole body protein breakdown, amino acid oxidation, and protein synthesis were calculated from the endogenous leucine Ra, the leucine oxidation rate, and the nonoxidative leucine Rd, respectively, with the assumption that 7.8% of whole body protein is comprised of leucine (8).

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Rates of whole body amino acid, carbohydrate, and lipid oxidation were determined from indirect calorimetry in combination with the leucine oxidation data. The energy expended through amino acid oxidation was subtracted from the total energy expenditure, and the net rates of carbohydrate and lipid oxidation were calculated on the basis of the nonprotein RQ (19). The assumptions and limitations of calculating substrate oxidation on the basis of indirect calorimetry measurements have been reviewed previously (19). Statistical analysis. The null hypothesis of the present study was that exercise performance during HD would not improve protein homeostasis in the skeletal muscle in addition to the improvements already observed with IDPN alone. The primary end point was net forearm muscle protein balance (synthesis minus breakdown). We estimated that a sample size of 6 would provide 92% statistical power to detect a change of 1.5 SDs (assuming a 2-tailed ␣-level of 0.05 with a paired t-test). The change associated with IDPN alone was 56 ␮g䡠100 ml⫺1 䡠min⫺1, and the SD of the change was 40 ␮g䡠100 ml⫺1 䡠min⫺1. Therefore, the 1.5-effect size would allow us to have sufficient power to detect changes in the IDPN ⫹ exercise protocol of 116 ␮g䡠100 ml⫺1 䡠min⫺1 or greater (56 ⫹ 1.5 ⫻ 40). In fact, the observed result was 125 ␮g䡠100 ml⫺1 䡠min⫺1. For each protocol, mean variables for each phase (pre-HD, HD, and post-HD) were calculated as the averages of the time points for each phase. For comparisons between study protocols (IDPN vs. IDPN ⫹ exercise), paired t-tests were used for parametric distribution and the Wilcoxon signed-rank test for nonparametric distribution. The SPSS statistical software program (version 11.5, SPSS, Chicago, IL) was used for all analyses. All tests were two-tailed, and P values of ⬍0.05 were considered to indicate statistical significance. The results are expressed as means ⫾ SE. RESULTS

Blood chemistries. Table 1 depicts baseline biochemical nutritional markers, including serum albumin, serum prealbumin, serum transferrin, serum cholesterol, normalized protein catabolic rate, CO2, and CRP for the two study protocols: IDPN and IDPN ⫹ exercise. These measurements were not different between IDPN and IDPN ⫹ exercise protocols. As implied by the above-mentioned biochemical markers, the population studied was in an overall adequately nourished status and not inflamed during either treatment. Metabolic parameters. Table 2 shows the results for plasma metabolic hormones and glucose concentrations for the two study protocols. Plasma glucose and insulin concentrations increased during the HD phase to a similar degree in both protocols. However, during the post-HD phase, glucose re-

turned to lower levels for the IDPN only vs. the IDPN ⫹ exercise protocol. Cortisol concentrations were significantly higher during the IDPN ⫹ exercise protocol compared with IDPN only, both during the HD phase and the post-HD phase, P ⬍ 0.05 for both. During the HD phase, growth hormone levels were not statistically significantly different between the protocols. However, during the post-HD phase, growth hormone levels increased almost fourfold in the IDPN ⫹ exercise protocol, whereas levels returned to baseline in the IDPN only protocol, P ⬍ 0.05. The other metabolic hormones, epinephrine and norepinephrine, did not show statistically significant differences between protocols. Plasma AAs and forearm AA balance. The arterial plasma AA concentrations during both protocols are depicted in Fig. 2 by AA groups (TAA, NEAA, EAA, BCAA). IDPN administration resulted in statistically significant increases in the concentrations of all groups of AAs, and the addition of exercise to IDPN therapy did not have any significant impact on the plasma arterial AA levels. In the post-HD phase, plasma AA concentrations returned toward baseline values in both study protocols. Net forearm AA balances during both study protocols are depicted in Table 3 and graphed for the HD phase in Fig. 3. There was a net output of BCAA, EAA, NEAA, and TAA for both treatments during the basal phase, which switched to net uptake of these AAs during the HD phase. There was significantly greater forearm uptake of all groups of AAs during HD with IDPN ⫹ exercise compared with IDPN alone. Specifically, forearm uptake values of BCAA and EAA were nearly twofold greater, NEAA uptake was more than threefold greater, and TAA was between two- and threefold greater for IDPN ⫹ exercise vs. IDPN only (P ⬍ 0.05 for all). AA forearm balance was not different between treatments during the post-HD phase. Forearm muscle protein metabolism. Table 4 depicts the results for forearm protein metabolism components, which are graphed in Fig. 4 for the HD phase. Forearm muscle protein synthesis during the HD phase was numerically higher when patients performed exercise during IDPN administration compared with IDPN alone (300 ⫾ 70 vs. 254 ⫾ 84 ␮g䡠100 ml⫺1 䡠min⫺1, P ⫽ NS). On the other hand, forearm muscle proteolysis was numerically lower during IDPN ⫹ exercise (198 ⫾ 58 ␮g䡠100 ml⫺1 䡠min⫺1 during the IDPN protocol vs.

Table 2. Metabolic hormones and glucose concentrations Treatment

Insulin, ␮U/ml Glucagon, pg/ml Growth hormone, ng/ml Cortisol, ␮g/dl Epinephrine, pg/ml Norepinephrine, pg/ml Plasma glucose, mg/dl

IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN

⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise

Pre-HD

During HD

Post-HD

8.0⫾1.4 10.2⫾2.2 163⫾26 174⫾33 0.4⫾0.3 1.3⫾0.6 8.4⫾2.0 10.2⫾0.4 50⫾7 62⫾16 509⫾141 410⫾107 80.1⫾4.2 86.0⫾3.5

59.1⫾13.3 56.3⫾10.5 134⫾23 132⫾26 0.09⫾0.01 0.17⫾0.09 8.7⫾0.5 12.6⫾2.4* 47⫾13 53⫾11 464⫾137 394⫾124 145.0⫾10.5 145.1⫾6.4

12.1⫾1.8 12.8⫾3.7 146⫾23 162⫾32 0.6⫾0.3 2.5⫾0.9* 12.0⫾2.6 20.1⫾3.5* 179⫾21 115⫾21 451⫾103 478⫾150 69.4⫾4.8 81.7⫾4.2*

Values are means ⫾ SE of 6 subjects. HD, hemodialysis. *Significant difference between IDPN and IDPN ⫹ exercise (P ⬍ 0.05). AJP-Endocrinol Metab • VOL

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Fig. 2. Plasma amino acid (AA) concentrations over the study period, comparing IDPN and IDPN ⫹ exercise treatment protocols before (pre-HD), during, and after (post-HD) hemodialysis. Values are means ⫾ SE; units are in ␮mol/l.

175 ⫾ 48 ␮g䡠100 ml⫺1 䡠min⫺1 during the IDPN ⫹ exercise protocol, P ⫽ NS). The net result was a significant (2-fold) increase in the net forearm protein accretion during HD in the IDPN ⫹ exercise protocol (125 ⫾ 37 vs. 56 ⫾ 30 ␮g䡠100 ml⫺1 䡠min⫺1, P ⬍ 0.05). Whole body protein metabolism. Results for whole body protein metabolism components are shown in Table 4 and Fig. 5. Exercise did not significantly affect whole body protein synthesis or proteolysis. Consequently, net whole body protein balance was not statistically different between IDPN and IDPN ⫹ exercise. Energy metabolism and substrate oxidation. Table 5 shows the components of energy metabolism and substrate oxidation. Energy expenditure was numerically higher when patients performed exercise, but the difference did not reach statistical significance (1.40 ⫾ 0.03 vs. 1.32 ⫾ 0.04 kcal䡠kg FFM⫺1 䡠h⫺1, P ⬎ 0.05). There were trends for increases in AA and carbohydrate oxidation, along with a decrease in lipid oxidation, during IDPN ⫹ exercise, although none of these changes were statistically significant. Oxidation of fat increased during the post-HD phase for both treatments; however, there were no differences between these treatments. DISCUSSION

Nutritional status is an important predictor of clinical outcome in CHD patients, as uremic malnutrition is strongly associated with increased risk of death and hospitalization events (28). Accordingly, decreased muscle mass is the most

significant predictor of morbidity and mortality in CHD patients (28). It is therefore critical to implement new and effective strategies to prevent loss of muscle mass in these patients. The current study provides the first evidence that an exercise protocol combined with adequate nutritional supplementation results in net muscle protein accretion in CHD patients. An important factor that leads to loss of muscle mass in CHD patients is decreased dietary protein intake. An adequate protein and energy intake is actively fostered in CHD patients by a health care team that involves dietitians, social workers, nurses, and physicians. However, attempts to increase dietary protein intake by dietary counseling alone are inadequate to prevent or treat uremic malnutrition (12, 22). This is due to the fact that several other catabolic factors, such as hemodialysis procedure and concurrent metabolic abnormalities, further predispose CHD patients to increased protein catabolism (11, 17, 21, 27, 34). Provision of enteral and/or parenteral (intradialytic) nutritional supplementation, along with certain anabolic interventions to replenish body protein and energy stores, have been proposed to improve the nutritional status of these patients (4, 6, 37, 50, 51). In healthy individuals, exercise is accepted as an anabolic process and can alter protein and energy homeostasis in diverse ways (30, 48). Exercise alone creates an environment that enhances skeletal muscle sensitivity to insulin (45), stimulates the uptake of amino acids (2), increases intramuscular amino acid availability (2), and promotes rates of muscle protein accretion (2, 7, 35, 36, 38).

Table 3. Forearm balance of amino acids by groups, comparing IDPN and IDPN ⫹ exercise Treatment

Forearm BCAA uptake Forearm EAA uptake Forearm NEAA uptake Forearm TAA uptake

IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN

⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise

Pre-HD

During HD

Post-HD

⫺18⫾18 ⫺19⫾8 ⫺35⫾29 ⫺42⫾14 ⫺132⫾61 ⫺140⫾77 ⫺168⫾87 ⫺182⫾89

96⫾18 178⫾49* 229⫾38 455⫾105* 160⫾36 574⫾192* 390⫾71 1,029⫾296*

12⫾20 34⫾20 ⫺96⫾40 ⫺164⫾103 ⫺230⫾87 ⫺128⫾176 ⫺326⫾120 ⫺292⫾147

Values are means ⫾ SE expressed in nmol䡠100 ml⫺1 䡠min⫺1. BCAA, branched-chain amino acids; EAA, essential amino acids; NEAA, nonessential amino acids; TAA, total amino acids. *Significant difference between IDPN and IDPN ⫹ exercise (P ⬍ 0.05). AJP-Endocrinol Metab • VOL

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Fig. 3. Forearm balance of amino acids by groups during HD, comparing IDPN and IDPN ⫹ exercise treatments. Values are means ⫾ SE, with units in nmol䡠100 ml⫺1 䡠min⫺1. TAA, total amino acids; NEAA, nonessential AA; EAA, essential AA; BCAA, branched-chain AA.

Enhanced availability of essential amino acids alone also has potential to improve events that stimulate muscle protein deposition (3). The superimposition of exercise with increased amino acid availability has been demonstrated to produce a consistent additive anabolic response and increase skeletal muscle deposition across a wide variety of experimental conditions in healthy subjects (25, 26, 29, 42, 43). The specific aim of the current study was to test the hypothesis that an exercise protocol combined with adequate nutritional supplementation induces skeletal muscle protein accretion over and above what can be achieved by nutritional supplementation alone. Indeed, this is one of the first reports to demonstrate these profound anabolic effects, especially during the underpinnings of a highly catabolic state such as hemodialysis. These results can be generalized to suggest that the combination of exercise and increased availability of amino acids may be able to ameliorate the muscle wasting associated with a variety of catabolic diseases. A clinical implication of our study is its relevance to physical activity level in CHD patients. It is well known that CHD patients live a sedentary lifestyle and that their physical activity level is low. Furthermore, the level of physical activity is

Fig. 4. Forearm muscle protein metabolism during HD, comparing IDPN (open bars) and IDPN ⫹ exercise (solid bars). Units are in ␮g䡠100 ml⫺1 䡠min⫺1.

closely associated with morbidity and mortality (20, 33). This association is independent of presence or absence of uremic malnutrition and/or other comorbid condition, such as inflammation and diabetes. The decreased physical activity level in CHD patients is most likely due to muscle wasting, because physical performance depends on various muscle functions, including strength. Muscle protein synthesis and breakdown are central in determining both strength and overall function. Therefore, the broad implications of the results reported herein are potential improvements in overall physical activity level and quality of life in CHD patients. A novel aspect of our study is that the beneficial effects can be generalized to all CHD patients, not only the subgroup with additional comorbid conditions such as uremic malnutrition and inflammation. We examined an array of hormone profiles in our study. These measurements were performed to explore any mechanistic link between our observed results and changes in metabolic regulators such as insulin, cortisol, growth hormone, and catecholamines. Indeed, we were able to show significant changes in cortisol and growth hormone profiles during the IDPN ⫹ exercise protocol compared with IDPN alone. We did not observe any difference in insulin and catecholamine levels between study protocols. The difference in cortisol levels

Table 4. Components of whole body and forearm muscle protein metabolism

WB protein synthesis, mg䡠kg FFM

⫺1

䡠min

⫺1

WB proteolysis, mg䡠kg FFM⫺1 䡠min⫺1 Net WB protein balance, mg䡠kg FFM⫺1 䡠min⫺1 FM protein synthesis, ␮g䡠100 ml⫺1 䡠min⫺1 FM proteolysis, ␮g䡠100 ml

⫺1

䡠min

⫺1

Net FM protein balance, ␮g䡠100 ml⫺1 䡠min⫺1

Pre-HD

During HD

Post-HD

IDPN IDPN ⫹ Exercise IDPN IDPN ⫹ Exercise IDPN IDPN ⫹ Exercise

3.47⫾0.46 3.06⫾0.24 3.97⫾0.46 3.53⫾0.20 ⫺0.50⫾0.07 ⫺0.47⫾0.06

4.88⫾0.31 4.92⫾0.34 1.94⫾0.43 1.64⫾0.21 2.93⫾0.19 3.29⫾0.23

3.58⫾0.36 3.45⫾0.30 4.38⫾0.44 4.47⫾0.33 ⫺0.80⫾⫺0.10 ⫺1.02⫾0.31

IDPN IDPN ⫹ Exercise IDPN IDPN ⫹ Exercise IDPN IDPN ⫹ Exercise

57⫾18 43⫾13 72⫾13 57⫾12 ⫺16⫾⫺3 ⫺14⫾2

254⫾84 300⫾70 198⫾58 175⫾48 56⫾30 125⫾37*

122⫾33 99⫾66 176⫾44 164⫾53 ⫺55⫾16 ⫺65⫾20

Values are means ⫾ SE of 6 subjects. FFM, fat-free mass; WB, whole body; FM, forearm muscle. *Significant difference between IDPN and IDPN ⫹ exercise (P ⬍ 0.05). AJP-Endocrinol Metab • VOL

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Fig. 5. Whole body protein components during HD, comparing IDPN (open bars) and IDPN ⫹ exercise (solid bars). Units are in mg䡠kg fat-free mass (FFM)⫺1 䡠min⫺1.

between protocols is in the magnitude of 20% during exercise and carries over to the subsequent 2 h after hemodialysis at a magnitude of 45%. This is an important consideration regarding potential catabolic effects associated with exercise. However, our data show that there is a significant improvement in net muscle protein accretion simultaneous with these changes. This is probably due to the overwhelming anabolic actions of insulin and growth hormone. Although we did not observe a statistically significant difference in insulin levels between IDPN ⫹ exercise and IDPN-alone protocols, there was an approximately sixfold increase in insulin concentrations during both protocols (compared with the predialysis period). Furthermore, it is possible that sensitivity to insulin is potentially increased by exercise. This profound increase in insulin levels, as well as increased sensitivity to insulin, is probably sufficient to overcome any concomitant catabolic signaling associated with exercise. Overall, these results suggest that the anabolic actions of hormones such as insulin and growth hormone prevail over the catabolic effects of increased cortisol secretion during exercise with adequate nutritional supplementation. An interesting finding in our study was the fact that the observed anabolic effects were rapidly reversed during the postdialysis period when the IDPN was no longer being infused. This observation is similar for both protocols and is consistent with our previous results (37). These findings imply that IDPN provides only a transient improvement in uremia and hemodialysis-associated catabolism and cannot effectively

correct these prolonged catabolic effects. Of note, the lack of beneficial effects of nutritional supplementation in the postdialysis period was accompanied by a temporal decrease in concentrations of the anabolic hormone insulin, suggesting its critical role in these metabolic responses. In contrast to our findings, Tipton et al. (41) recently showed that the response to exercise and amino acid ingestion over 3-h and 24-h periods was not different in healthy individuals, suggesting that the acute response of net muscle protein balance reflects 24-h balance after exercise and amino acid ingestion. The discrepancy between the two reports can be explained by the two critical differences between the studies. First, the nutritional supplementation is different in these particular studies, such that parenteral nutrition vs. oral nutrition might have different bioavailability. Second, our study subjects are exposed to a highly catabolic milieu, namely the combination of underlying uremia and the hemodialysis procedure. These differences highlight the importance of future studies that examine different study protocols in patient populations with ongoing catabolic illnesses such as uremia. An additional finding in our study was a numerically, although not statistically, significant increase in energy expenditure during the IDPN ⫹ exercise treatment compared with IDPN alone. The observed difference, if extrapolated over 4 h of hemodialysis, translates into ⬃35 kcal of extra energy expenditure during the IDPN ⫹ exercise protocol compared with the energy expended during IDPN alone for a 70-kg patient. Although this difference is not enormous, one should consider adjusting the concomitant nutritional supplementation accordingly. Although the results presented herein are intriguing and promising, the current study should be considered as a pilot report providing rationale for future more comprehensive and detailed studies. The present study was designed as an initial trial to test the efficacy of a low-intensity exercise performed only once and for a short period of time. Further studies are still needed to examine the type, intensity, and duration of exercise that would result in a maximal response. In addition, the current study was performed under relatively acute conditions (1 day). It may be argued that the increases seen would only be beneficial if sustained over considerably more longterm conditions, leading to an increase in muscle mass. Castaneda et al. (5) demonstrated that resistance exercise therapy over a 12-wk period in 26 patients with moderate renal insufficiency increased muscle mass, type I and II muscle fiber cross-sectional areas, serum prealbumin levels, and muscle

Table 5. Components of energy metabolism and substrate oxidation ⫺1

Energy expenditure, kcal䡠kg FFM

䡠h

⫺1

Respiratory quotient Amino acid oxidation, mg䡠kg FFM⫺1 䡠min⫺1 Carbohydrate oxidation, mg䡠kg FFM⫺1 䡠min⫺1 Lipid oxidation, mg䡠kg FFM⫺1 䡠min⫺1

IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN IDPN

⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise ⫹ Exercise

Pre-HD

During HD

Post-HD

1.21⫾0.02 1.22⫾0.02 0.84⫾0.02 0.85⫾0.02 0.50⫾0.07 0.47⫾0.06 2.34⫾0.38 2.54⫾0.43 1.09⫾0.19 1.02⫾0.21

1.32⫾0.04 1.40⫾0.03 0.83⫾0.01 0.85⫾0.01 0.80⫾0.16 0.96⫾0.23 2.44⫾0.31 2.97⫾0.21 1.26⫾0.13 1.15⫾0.10

1.40⫾0.08 1.40⫾0.07 0.80⫾0.01 0.79⫾0.02 0.80⫾0.10 1.02⫾0.31 1.83⫾0.29 1.75⫾0.52 1.65⫾0.19 1.68⫾0.23

Values are means ⫾ SE. HD, hemodialysis; IDPN, intradialytic parenteral nutrition; FFM, fat-free mass. AJP-Endocrinol Metab • VOL

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strength while decreasing leucine oxidation. The current study did not explore the mechanistic pathways leading to the observed beneficial effects of exercise and nutritional supplementation. Clearly, future studies are warranted to explore potential metabolic steps that may be involved in this response. These future studies are also required in additional subgroups of CHD patients, such as diabetic patients, given the complex nature of response to nutritional supplementation in this particular patient population. Finally, another limitation was the fact that only one female subject was studied, and therefore no definitive conclusion can be derived from the present study regarding gender differences. In summary, we demonstrated that concomitant exercise augments the anabolic effects of nutritional supplementation provided during a single hemodialysis session. These effects include increases in muscle amino acid uptake and net muscle protein accretion. Exercise, in the setting of adequate nutritional supplementation, has the potential to blunt the loss of lean body mass in CHD patients. Further short-term and long-term studies examining different exercise protocols in more specific subgroups of CHD patients are necessary to generalize these results. ACKNOWLEDGMENTS The excellent technical assistance of Suzan Vaughan, Janice Harvell, Mu Zheng, Wanda Snead, and the nursing staff of the GCRC is appreciated. P. J. Flakoll is currently the Director of the Center for Designing Foods to Improve Nutrition at Iowa State University, Ames, IA. GRANTS This study is supported in part by National Institutes of Health Grants R01 45604 and 1K24 DK-62849, Food and Drug Administration Grant no. 000943, Satellite Health Extramural Grant Program, Clinical Nutrition Research Unit, Grant no. DK-26657, GCRC Grant no. RR-00095, and Diabetes Research Training Unit Grant no. DK-20593. L. B. Pupim is partly supported by the Marilyn Charitable Trust Young Investigator Grant of the National Kidney Foundation, the Vanderbilt University School of Medicine Clinician Scientist Award, as part of the Vanderbilt Physician Scientist Development Program, and the Master of Science in Clinical Investigation Program. REFERENCES 1. Allsop JR, Wolfe RR, and Burke JF. Tracer priming the bicarbonate pool. J Appl Physiol 45: 137–139, 1978. 2. Biolo G, Fleming RY, Maggi SP, and Wolfe RR. Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle. Am J Physiol Endocrinol Metab 268: E75–E84, 1995. 3. Bohe J, Low JF, Wolfe RR, and Rennie MJ. Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids. J Physiol 532: 575–579, 2001. 4. Cano N, Labastie-Coeyrehourq J, Lacombe P, Stroumza P, di CostanzoDufetel J, Durbec JP, Coudray-Lucas C, and Cynober L. Perdialytic parenteral nutrition with lipids and amino acids in malnourished hemodialysis patients. Am J Clin Nutr 52: 726–730, 1990. 5. Castaneda C, Gordon PL, Uhlin KL, Levey AS, Kehayias JJ, Dwyer JT, Fielding RA, Roubenoff R, and Singh MF. Resistance training to counteract the catabolism of a low-protein diet in patients with chronic renal insufficiency. A randomized, controlled trial. Ann Intern Med 135: 965–976, 2001. 6. Chertow GM, Ling J, Lew NL, Lazarus JM, and Lowrie EG. The association of intradialytic parenteral nutrition with survival in hemodialysis patients. Am J Kidney Dis 24: 912–920, 1994. 7. Ferrando AA, Tipton KD, Bamman MM, and Wolfe RR. Resistance exercise maintains skeletal muscle protein synthesis during bed rest. J Appl Physiol 82: 807–810, 1997. 8. Garlick PJ, McNurlan MA, McHardy KC, Calder AG, Milne E, Fearns LM, and Broom J. Rates of nutrient utilization in man measured by combined respiratory gas analysis and stable isotopic labeling: effect of food intake. Hum Nutr Clin Nutr 41: 177–191, 1987. AJP-Endocrinol Metab • VOL

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