Hemodialysis stimulates muscle and whole body protein loss and alters substrate oxidation

T. Alp Ikizler1, Lara B. Pupim1, John R. Brouillette1, Deanna K. Levenhagen2, Kali Farmer2, Raymond M. Hakim1, and Paul J. Flakoll2,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The hemodialysis (HD) procedure has been implicated as a potential catabolic factor predisposing the chronic HD (CHD) patients to protein calorie malnutrition. To assess the potential effects of HD on protein and energy metabolism, we studied 11 CHD patients 2 h before, during, and 2 h after HD by use of primed constant infusion of L-[1-13C]leucine and L-[ring-2H5]phenylalanine. Our results showed that HD led to increased whole body (10%) and muscle protein (133%) proteolysis. Simultaneously, whole body protein synthesis did not change, and forearm synthesis increased (120%). The net result was increased net whole body protein loss (96%) and net forearm protein loss (164%). During the 2-h post-HD period, the muscle protein breakdown trended toward baseline, whereas whole body protein breakdown increased further. Substrate oxidation during the post-HD was significantly altered, with diminished carbohydrate and accelerated lipid and amino acid oxidation. These data demonstrate that hemodialysis is an overall catabolic event, decreasing the circulating amino acids, accelerating rates of whole body and muscle proteolysis, stimulating muscle release of amino acids, and elevating net whole body and muscle protein loss.

catabolism; malnutrition; inflammation; energy expenditure; metabolism


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MORTALITY RATE of treated end-stage renal disease (ESRD) patients remains high (37). In the United States, the life expectancy of ESRD patients is 20-25 yr less than that of the normal age-, sex-, and race-matched US population over the age of 45 yr. Among many factors, including treatment characteristics and comorbid conditions, protein and calorie malnutrition has been shown to be a major risk factor for increased mortality in the ESRD patient population (4, 17). Multiple factors predispose ESRD patients to protein calorie malnutrition. These include decreased dietary nutrient intake, higher resting energy expenditure, hormonal and metabolic derangements due to loss of kidney function, and comorbid conditions such as presence of diabetes mellitus (4, 17).

An ongoing debate is whether the treatment modalities for ESRD contribute to the poor nutritional status of the ESRD patients (24). Specifically, chronic intermittent hemodialysis has been implicated as a catabolic process that worsens the nutritional status of the ESRD patients treated with this modality. Indeed, we have shown that chronic hemodialysis causes negative protein and calorie balance by the inevitable losses of amino acids and increased energy expenditure during hemodialysis (16, 19). Another protein catabolic effect of hemodialysis treatment is observed when dialyzer membranes that activate the complement system are utilized (13, 33). However, the catabolic impact is less when dialyzers with synthetic noncomplement-activating membranes are used.

Studies examining the metabolic and hormonal effects of the hemodialysis treatment per se are limited. This information is critically important, because chronic intermittent hemodialysis treatment is the primary mode of renal replacement therapy in ESRD patients. In this prospective study, we evaluated the effects of the hemodialysis procedure on protein, carbohydrate, and lipid metabolism. Specifically, we studied 11 chronic hemodialysis (CHD) patients before, during, and after a single hemodialysis session using a combination of stable isotopic techniques, arteriovenous balance measurements, and indirect calorimetry to assess 1) the dynamics of whole body as well as forearm muscle protein metabolism, 2) forearm amino acid and glucose balance, 3) energy expenditure, and 4) oxidation of amino acids, carbohydrate, and lipids.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Patients. Patients were recruited from the Vanderbilt University Outpatient Dialysis Unit. All study patients were clinically stable, without any overt active inflammatory disease and were on chronic hemodialysis treatment for a minimum of 6 mo. The Institutional Review Board of Vanderbilt University approved the study protocol procedures performed at the Vanderbilt University General Clinical Research Center (GCRC). After an explanation of the procedures, written informed consent was obtained from all study patients eligible for participation.

Study design. The study was a prospective design. Within 1 wk before each study, the following procedures were performed. Dual-energy X-ray absorptiometry (DEXA) was performed to estimate lean body mass and body fat mass. Resting metabolic rate was measured via indirect calorimetry to establish energy requirements before experimentation. Infusion of stable isotopes and blood sampling were done using the arteriovenous (av) shunt created for hemodialysis. The av shunt is the commonly used term for a fistula created in the upper extremity by connecting an artery to a nearby vein either by direct surgical anastomosis of the native vessels or with an artificial synthetic vascular graft. In our study subjects, five patients had the native fistula, and six patients had the artificial graft. The only circumstance that would affect the purity of the arterial or venous samples would be if there were stenoses in the fistula causing the venous blood to mix with arterial blood (recirculation). Therefore, recirculation of the av shunt as well as total vascular access blood flow to assess for stenosis in the av shunt was measured in every patient before the study by use of ultrasound dilution technique (Transonics, Ithaca, NY). Patients with access blood flow <750 ml/min and/or with any recirculation in the access were not included in the study.

A schematic diagram of the study design is depicted in Fig. 1. The subjects were admitted to the GCRC the day before the study at ~7:00 PM and received a meal from the dietary kitchen of the GCRC upon admission. The meal consisted of 18% protein and 30% fat, and energy intake was kept at maintenance levels based upon the Harris-Benedict equation and each subject's gender, height, weight, and activity level. The subjects remained fasting after 8:00 PM, such that the last meal before the study was >= 10 h before the initiation of the study (water consumption was allowed). During the study, patients were monitored closely for any signs or symptoms of hypoglycemia or hyperglycemia, and blood glucose levels were checked as needed.


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Fig. 1.   Experimental design. Each study consisted of a 120-min equilibration period (-150 to -30 min), a 30-min basal period, a 240-min hemodialysis period (0-240 min), and a 120-min postdialysis period. Blood sampling time points are indicated by the arrows. A continuous infusion of leucine and phenylalanine isotopes was maintained throughout the study.

Each study consisted of a 2-h equilibration period, a 0.5-h basal sampling period, the usual 4-h dialysis period, and a 2-h postdialysis period. The subjects were initiated on isotope infusion at 6:00 AM via a dialysis needle (15-gauge) placed on the venous side of the av shunt. A needle draining from the arterial side of the av shunt was used for arterial blood sampling. Because there was no recirculation in the av shunt, blood from venous and arterial sides of the shunt was not mixed, providing appropriate routes for infusion of tracers and blood sampling, respectively. Another catheter was placed on the contralateral (nonaccess) forearm for sampling of venous blood draining the forearm. After collection of blood and breath samples to determine isotopic backgrounds (-150 min), a bolus infusion of [13C]NaHCO3 (0.12 mg/kg), L-[1-13C]leucine (7.2 µmol/kg), and L-[ring-2H5]phenylalanine (7.2 µmol/kg) was given to prime the carbon dioxide, leucine, and phenylalanine pools, respectively. Subsequently, a continuous infusion of L-[1-13C]leucine (0.12 µmol · kg-1 · min-1) and L-[ring-2H5]phenylalanine (0.12 µmol · kg-1 · min-1) was initiated and continued throughout the remainder of the study.

At the conclusion of the basal period, the patients were initiated on hemodialysis. Patients were dialyzed for 4 h, with blood flow of 400 ml/min and dialysate flow of 800 ml/min. All patients were dialyzed with new biocompatible membranes (Fresenius F-80B, Walnut Creek, CA). Ultrafiltration rates were determined by the patients' previously established "estimated dry weight." The composition of the dialysate used during the study was identical for all treatments and consisted of 139 meq/l sodium, 2 meq/l potassium, 2.5 meq/l calcium, 200 mg/dl glucose, and 39 meq/l bicarbonate. Hemodialysis treatment was performed under the careful supervision of a hemodialysis nurse. After hemodialysis was finished and patients were taken off the machine, a 2-h postdialysis period ensued. At the conclusion of the study, peripheral catheters were removed, and the subjects were fed a meal and discharged from the GCRC. Patients continued their subsequent dialysis treatments at the outpatient unit as scheduled.

Arterial and venous blood samples were taken every 15 min during the basal period and every 30 min during the dialysis and postdialysis periods. Simultaneously with the blood samples, breath samples were collected for 1 min from each subject in a Douglas bag, and duplicate 20-ml samples were placed into nonsiliconized evacuated glass tubes for the determination of breath 13CO2 enrichment. Forearm blood flow measurements were determined by plethysmography (model 2560 with URI/CP software version 3.0; UFI, Morro Bay, CA) (32). Finally, carbon dioxide production and oxygen consumption were determined throughout each period by indirect calorimetry (Sensormedics 2900 metabolic cart, Palo Alto, CA) to assess energy expenditure, respiratory quotient, and substrate oxidation.

Analytical procedures. Blood samples were collected into Venoject tubes containing 15 mg Na2EDTA (Terumo Medical, Elkton, MD). A 3-ml aliquot of blood was transferred to a tube containing EDTA, and reduced glutathione with the plasma was stored at -80°C for later measurement of plasma epinephrine and norepinephrine concentrations by HPLC. 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 (model II Glucose Analyzer, Beckman Instruments).

Immunoreactive insulin was determined in plasma with a double-antibody system. Plasma aliquots for glucagon determination were placed in tubes containing 25 kallikrein-inhibitor units of aprotinin (Trasylol; 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 (30) 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 (Travenol-GenTech, Cambridge, MA) was used to measure plasma cortisol concentrations. Plasma insulin-like growth factor I concentrations were determined by a radioimmunoassay acid-extraction procedure (Nichols Institute Diagnostics, San Juan Capistrano, CA). Plasma amino acid concentrations were determined by reverse-phase HPLC after derivatization with phenylisothiocyanate (15). 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, phenylalanine, threonine, 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 L-[ring-2H5]phenylalanine were determined using gas chromatography-mass spectrometry (GC-MS) (Hewlett-Packard 5890a GC and 5970 MS, 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 (31). After drying under nitrogen gas, both the keto and amino acid fractions were derivatized (35) with N-methyl-N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide containing 1% t-butyldimethylchlorosilane (Regis Technologies, Morton Grove, IL). The derivatized samples were then analyzed with GC-MS for plasma leucine and KIC enrichments. Breath 13CO2 enrichment was measured by isotope ratio mass spectrometry (Metabolic Solutions, Nashua, NH) (36).

Calculations. Net skeletal muscle protein balance (synthesis-breakdown) was determined by dilution and enrichment of phenylalanine across the hindlimb, as described by Gelfand and Barrett (12). Because phenylalanine is neither synthesized nor metabolized by skeletal muscle, rate of appearance (Ra) of unlabeled phenylalanine reflects muscle protein breakdown, whereas the rate of disappearance (Rd) of labeled phenylalanine estimates muscle protein synthesis (12). 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 arm plasma flow (12, 38). Net phenylalanine Ra was calculated by subtracting the net av balance of phenylalanine across the upper limb from the phenylalanine Rd (12, 38). Rates of skeletal muscle protein breakdown and net synthesis were determined from the phenylalanine Rd and Ra, on the assumption that 3.8% of skeletal muscle protein is comprised of phenylalanine.

The steady-state whole body leucine Ra (an estimate of whole body protein breakdown) was calculated by dividing the [13C]leucine infusion rate by the plasma [13C]KIC enrichment (38). Plasma KIC provides a better estimate of intracellular leucine enrichment than does plasma leucine enrichment, because KIC is derived from intracellular leucine metabolism (38). Breath 13CO2 production was determined by multiplying the total CO2 production rate by the breath 13CO2 enrichment (38). 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 period 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, on the assumption that 7.8% of whole body protein is comprised of leucine (11).

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 due to 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 respiratory quotient (20). The assumptions and limitations of calculating net substrate oxidation on the basis of indirect calorimetry measurements have been reviewed previously (20).

Statistical analysis. For each protocol, mean variables for each period (basal, dialysis, and postdialysis periods) were calculated. Values presented in the text and figures are means ± SE for each period. Differences between the mean values were assessed using a repeated-measures analysis of variance and paired t-tests (Statistical Analysis System for Windows, 1996, release 6.12, SAS Institute, Cary, NC). A P value of <0.05 was required to reject the null hypothesis of no difference between the means.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Patient characteristics. The patient characteristics are depicted in Table 1. There were 11 patients, six male and five female. The mean weight was 82.6 ± 5.2 kg. Mean body mass index was 28.3 ± 1.9 kg/m2. Mean fat mass was 29 ± 2.1% by DEXA.

                              
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Table 1.   Demographic and nutritional characteristics of the study population

Blood chemistries. Measurement of pre- and posthemodialysis blood chemistries including blood urea nitrogen, serum creatinine, sodium, potassium, chloride, and total bicarbonate showed expected changes after hemodialysis treatment. The mean urea reduction ratio was 70% for the hemodialysis procedures performed during the study. Table 1 depicts the results of predialysis biochemical parameters for the study patients.

Metabolic hormones. Table 2 shows results for the plasma concentrations of several metabolic hormones during the three phases of the study. Plasma insulin concentration increased 60% during the hemodialysis procedure and returned to baseline values during the postdialysis period. Consistent with these results, plasma glucagon concentrations decreased 40% during hemodialysis. Although there was a trend toward normalization, mean glucagon concentration during postdialysis was still 24% less than during predialysis. Circulating growth hormone concentrations were low before dialysis and decreased further during hemodialysis. However, postdialysis growth hormone levels were not statistically significantly different from basal levels. Serum insulin-like growth factor I concentrations were not significantly different between periods. Circulating cortisol concentrations were elevated throughout the study but were not different between periods. Serum norepinephrine concentrations also were not altered due to dialysis. Epinephrine values were decreased by 47% during dialysis but returned to near basal during the postdialysis period.

                              
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Table 2.   Plasma metabolic hormone concentrations

Hematocrit, forearm blood flow, and glucose metabolism. Hematocrit values were increased during each subsequent period (Table 3). Hematocrit values during dialysis and postdialysis periods were increased 4 and 6%, respectively, compared with basal values. The observed increase in forearm plasma flow during hemodialysis was not statistically significant (Table 3). However, plasma flow was significantly different between dialysis and postdialysis periods. Compared with basal levels, plasma glucose concentrations increased 32% during hemodialysis (Table 3). Glucose concentrations returned to baseline levels after completion of hemodialysis. Glucose uptake by the forearm was more than threefold greater during hemodialysis compared with baseline measurements. Interestingly, as great as this increase was during the dialysis period, forearm glucose uptake during the postdialysis period returned to near basal levels.

                              
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Table 3.   Glucose concentration and uptake by the forearm

Amino acid profiles and net forearm balance. The individual as well as total essential and nonessential arterial plasma amino acid concentrations during the three phases of the study are included in Table 4. Each of the individual plasma amino acids displayed a decrease during the hemodialysis period compared with predialysis concentrations. As a group, BCAA decreased 31%, EAA decreased 33%, NEAA decreased 38%, and TAA decreased 36%. During the postdialysis phase, each of the individual plasma amino acids moved back toward baseline values. Only 3-methylhistidine, aspartate, glutamate, and hydroxyproline were not significantly different for postdialysis vs. dialysis periods. However, all of the amino acids, with the exception of arginine, glutamate, leucine, phenylalanine, taurine, tryptophan, and tyrosine, were still significantly low when compared with baseline values. As a group, BCAA decreased 15%, EAA decreased 15%, NEAA decreased 23%, and TAA decreased 20% for the postdialysis period vs. basal period values.

                              
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Table 4.   Arterial plasma amino acids

Net forearm amino acid balances during the three phases of the study are included in Table 5. During the basal period, there was a net uptake of BCAA, a net release of NEAA, and EAA were in a state of net equilibrium. Overall, there was a net release of TAA. During hemodialysis, there was either a decreased uptake or increased release of most individual amino acids. This was statistically significant for 1-methylhistidine, 3-methylhistidine, isoleucine, phenylalanine, proline, valine, and BCAA as a group. Net balance of EAA switched from a state of equilibrium to a net release, with the difference reaching a statistical trend (P < 0.05). After the completion of hemodialysis, there was either an increased uptake or decreased release of most individual amino acids compared with the dialysis period. This was statistically significant for 1-methylhistidine, 3-methylhistidine, alanine, glycine, hydroxyproline, leucine, methionine, proline, threonine, tryptophan, valine, BCAA, gluconeogenic amino acids, EAA, NEAA, and TAA. Interestingly, forearm fractional extraction of phenylalanine determined using the stable isotopic tracer of phenylalanine was not different between basal and dialysis periods (10 ± 2 and 15 ± 2%, respectively). However, fractional extraction of phenylalanine was significantly increased between basal and postdialysis (16 ± 3%) periods.

                              
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Table 5.   Net forearm balance of amino acids

Dynamics of whole body and forearm protein metabolism. Measurements of whole body protein dynamics during the three phases of the study are included in Table 6. During the hemodialysis treatment, whole body proteolysis was increased by 10% or 0.32 mg · fat-free mass (FFM)-1 · min-1 (Fig. 2). Based on the loss of leucine into the dialysate (47.2 ± 1.3 µg · FFM-1 · min-1), the amino acid loss into the dialysate was calculated to be 0.61 ± 0.02 mg · FFM-1 · min-1. This was 16% of the total rate of amino acid disappearance. The rate of whole body protein synthesis did not increase simultaneously during the hemodialysis period, but protein synthesis was reduced from 84% of the total rate of amino acid disappearance at baseline to 72% of the total during hemodialysis period. Therefore, the overall net whole body protein loss, which is the difference between protein breakdown and protein synthesis, was increased by 0.51 mg · FFM-1 · min-1, or approximately twofold, during hemodialysis.

                              
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Table 6.   Dynamic components of whole body and forearm protein metabolism



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Fig. 2.   Whole body protein metabolism. Values reported are means ± SE of each period. FFM, fat-free mass. *Significant difference from the basal period (P < 0.05); §significant difference between the dialysis and postdialysis periods (P < 0.05).

Whole body protein breakdown continued to be elevated by 11% during the 2-h period after dialysis compared with the mean baseline values. Whole body protein synthesis during the postdialysis period was also statistically significantly higher compared with baseline and dialysis periods. Overall, net whole body protein loss during the postdialysis period was significantly decreased compared with the dialysis period but was still 21% greater than during the baseline period.

Table 6 also contains the results for the dynamics of forearm protein metabolism. Similar to the measurements of whole body protein metabolism, muscle protein breakdown was increased more than twofold during the hemodialysis period (Fig. 3). However, forearm protein synthesis also was increased, but by a slightly smaller magnitude. Overall, net forearm loss of protein was increased nearly threefold during hemodialysis. During the postdialysis period, forearm protein breakdown was significantly decreased from the dialysis period, but there was still a trend for protein breakdown to be 84% greater than during the basal period. Similarly, forearm protein synthesis was significantly decreased from the dialysis to the postdialysis period, but there was still a trend for protein synthesis to be 75% greater than during the basal period. Ultimately, net forearm protein loss was similar between basal and postdialysis periods.


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Fig. 3.   Net forearm muscle protein loss comparing predialysis and dialysis periods for individual data points. Each character represents a single patient.

Energy expenditure, respiratory quotient, and substrate oxidation. Energy expenditure adjusted for FFM increased by 7% during the hemodialysis phase (Table 7). Energy expenditure continued to increase during the postdialysis period (12% greater than basal), such that postdialysis energy expenditure was significantly greater than that during basal and dialysis periods. Respiratory quotient values were not altered by hemodialysis but decreased by ~10% during the postdialysis period compared with baseline. The rates and proportions of amino acid, carbohydrate, and lipid oxidation were not different during hemodialysis. Amino acid oxidation rates were slightly but significantly different between basal and postdialysis periods. On the other hand, postdialysis rates of carbohydrate oxidation were decreased 52%, and rates of lipid oxidation were increased by 65% compared with baseline. Therefore, significantly more energy was derived from lipid oxidation, and less was derived from carbohydrates in the 2-h period after dialysis.

                              
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Table 7.   Components of energy expenditure


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protein calorie malnutrition (PCM) is highly prevalent in CHD patients and has a significant impact on the morbidity, mortality, and costs associated with this disease (17). The process of PCM is multifactorial, including several non-dialysis- and dialysis-related causes. The hemodialysis procedure itself has been considered another potential catabolic factor predisposing the patient to PCM (24). In the present study, the acute metabolic effects of hemodialysis treatment were evaluated in 11 stable CHD patients in a systematic approach. Our results indicate that the procedure of hemodialysis treatment significantly impacts protein, fat, and carbohydrate metabolism. Specifically, the hemodialysis procedure leads to catabolism of whole body and muscle protein and a net loss of protein stores. There is a lack of adequate compensatory protein anabolism during this process, with a net result of increased protein catabolism during hemodialysis. Simultaneously, there is an increase in resting energy expenditure. Although muscle protein breakdown tends to lessen during the 2-h posthemodialysis period, whole body protein breakdown increases further, resulting in persistently elevated net protein catabolism during this period. Furthermore, substrate oxidation during the 2 h after completion of hemodialysis is significantly altered due to diminished carbohydrate oxidation.

We studied the impact of the hemodialysis procedure on the components of whole body and muscle protein dynamics in our study population. Although the results showed that rates of both whole body and muscle proteolysis were significantly increased during hemodialysis, the magnitude of this increase was much greater for muscle than for whole body measurements, suggesting that most of the increased proteolysis was due to alterations in skeletal muscle proteolysis. Measurements of forearm amino acid balance are consistent with the measurements of muscle protein breakdown, as during the hemodialysis period there was either a decreased uptake or increased release of most individual amino acids. Net balance of essential amino acids as a group switched from a state of equilibrium to a net release. This is consistent with the studies of Gutierrez et al. (14), who reported an increase in the release of amino acids from the leg due to hemodialysis. Despite this increase in amino acid release from muscle, plasma amino acid concentrations fell dramatically during hemodialysis, suggesting that the release of amino acids cannot keep up with the transient, but large, losses occurring during hemodialysis. This finding confirms previously published data from our laboratory (16).

The results of stable isotope dilution and enrichment were also consistent with the aforementioned findings, suggesting that loss of amino acids into the dialysate was the major factor resulting in a decrease in plasma amino acid concentrations. This is due to the fact that there are three likely sources of amino acid loss from the plasma, including amino acid oxidation, amino acid loss into the dialysate, and amino acid conversion to protein. Although rates of amino acid oxidation and whole body protein synthesis were not increased by hemodialysis, as shown by L-[1-13C]leucine kinetics, rates of both muscle protein synthesis and amino acid loss into the dialysate were increased during the hemodialysis period. Although whole body protein synthesis decreased from 84% of total amino acid utilization during the basal period to 72% during the hemodialysis period, the rate of muscle protein synthesis increased nearly twofold, suggesting one potential source of amino acid disappearance from the plasma pool. Indeed, there was a trend for increased fractional extraction of amino acids (from 10% basally to 15% during hemodialysis). This suggests that muscle tissue removed a greater portion of the amino acids provided to it during hemodialysis compared with that during the predialysis period. In addition, this demonstrates that alterations in muscle protein homeostasis during dialysis are most likely not due to derangements in muscle uptake of amino acids. With the assumption that 30% of the body mass in these subjects is muscle mass, the increased use of amino acids for protein synthesis would be ~4 g during the 4-h dialysis procedure. In comparison, we measured a loss of ~8.5 g of amino acids into the dialysate during the 4-h dialysis procedure. Thus removal of amino acids by dialysis was the primary cause, accounting for ~66%, for the decline in plasma amino acid concentrations. Interestingly, despite a simultaneous threefold increase in muscle protein catabolism, the plasma amino acid concentrations still decreased during hemodialysis.

The most important variable of protein dynamics is net protein accretion or loss, which is the difference between protein synthesis and protein breakdown. Because of an increase in the ratio between protein breakdown to protein synthesis during hemodialysis, net whole body protein loss was increased twofold and net muscle protein loss was increased threefold. The whole body loss during dialysis alone translates to an increased loss of ~7 g of protein per dialysis period. This is similar to the mass of amino acids lost in the dialysate. For a patient with a dialysis schedule of three times weekly, this protein loss would translate to a loss of ~2 kg of lean mass in a span of 1 yr. This is similar to what is often observed clinically.

We also evaluated the components of protein metabolism during a 2-h postdialysis period. The whole body protein breakdown as well as forearm muscle protein breakdown remained elevated compared with the baseline period for 2 h after dialysis. However, there was a significant decrease in muscle protein breakdown during the postdialysis period compared with the hemodialysis period. Plasma amino acid concentrations increased, trending toward baseline period, and amino acid uptake by muscle, fractional extraction of amino acids, and whole body protein synthesis increased during the 2-h period after dialysis. However, the persistence of increased net protein loss during this period suggests that there are other potential mechanisms in addition to amino acid losses inducing protein catabolism that are directly related to the hemodialysis procedure.

Another potential mechanism for hemodialysis-associated catabolism is the initiation of an inflammatory process that triggers enhanced proteolysis. Earlier studies by Gutierrez et al. (13) suggested that the structure of the hemodialysis membrane, in particular its bioincompatibility, plays a role in this process. However, in the present study, we exclusively used synthetic membranes that minimally activate the complement system, albeit some complement activation also occurs with this membrane. Other mechanisms by which inflammation can be triggered during hemodialysis include back-filtration of dialysate. Furthermore, other markers of stress and inflammation, such as interleukin-1, tumor necrosis factor-alpha , and C-reactive protein, have been shown to be elevated in the dialysis patients (18, 21). These mediators are also thought to increase muscle protein degradation and muscle amino acid release (10, 25). Of interest is that the peak concentration for these cytokines appears to be ~6 h after the initiation of dialysis (13). The finding that protein catabolism is still elevated for an additional 2 h after completion of a 4-h session suggests that an inflammatory process may still be in effect in these patients. Nevertheless, the relationship between increased protein breakdown and hemodialysis-initiated inflammatory response needs further study.

Previous studies have demonstrated a role for metabolic acidosis in increased protein catabolism, reduced albumin synthesis, and negative nitrogen balance (3, 26). Subsequent observations by Mitch et al. (29) demonstrated that an ATP-dependent pathway involving ubiquitin and proteasomes during metabolic acidosis stimulates muscle proteolysis. Total carbon dioxide levels, a measure of acidosis in our study patients, were slightly decreased, which is a common observation in predialysis blood analysis. The level of acidosis observed in this study is similar to levels in the previous studies, suggesting that acidosis may very well be a reasonable explanation for the observed findings (2). Moreover, diabetes in the presence of a higher level of glucocorticoids also activates the ubiquitin-proteasome system, potentially contributing to worsened proteolysis (27, 34). Nevertheless, whether this mild depression observed in total carbon dioxide levels and increased protein catabolism is related to the observations of Mitch et al. will require further investigation.

Hemodialysis-related proteolysis may also be related to hormonal derangements. For example, hypercortisolemia has been linked to increased breakdown of protein (28). In our study, however, cortisol concentrations were elevated basally, but they did not increase further during the hemodialysis procedure. It is possible that this chronic elevation in cortisol may provide a setting that facilitates other mediators during hemodialysis. Another counterregulatory hormone, glucagon, which has its major interaction with amino acid metabolism by increasing hepatic uptake of amino acids for gluconeogenesis, also does not appear to play a major role, because it falls significantly during hemodialysis. The actions of growth hormone, which has been shown to have positive effects on muscle protein synthesis, have been associated with a significant resistance in uremia (7). Growth hormone concentrations were significantly reduced during dialysis in this study. However, this change was small, and the physiological significance of this change is unclear. Certainly, insulin resistance could play a role in these observations. Insulin levels were increased during hemodialysis, and it would be expected that, if anything, this would reflect a decrease in protein breakdown (8). However, protein breakdown was increased, suggesting impairment in insulin-mediated protein metabolism.

In an earlier study, Lim et al. (23) studied the effects of hemodialysis on protein metabolism. They concluded that there were not any significant alterations in whole body protein breakdown during hemodialysis; rather, they reported a decrease in amino acid oxidation, a nonsignificant decrease in protein synthesis, and a significant increase in net protein loss. Although the reasons for differences between the present study and this previous report are not clear, differences in experimental conditions and hemodialysis parameters may contribute. For example, Lim et al. used a 3-h dialysis period, and plasma leucine concentrations did not decrease in their studies. Nevertheless, the comparison of the magnitude of net protein loss in two studies is comparable (65% in Lim et al. and 96% in our study).

Another aim of the present study was to evaluate the energy expenditure and substrate oxidation during the hemodialysis procedure. Similar to the magnitude demonstrated in an earlier study (19), the hemodialysis procedure was associated with a significant increase in energy expenditure. Energy expenditure was found to further increase during the 2-h period after dialysis. Because protein synthesis and breakdown are processes that require energy, part of this energy increase may be due to the increased protein turnover. Each peptide bond formed requires approximately five ATP of energy. This would translate to 4.5 kJ or 1.076 kcal/g of protein. Therefore, the additional energy cost of synthesizing the protein lost during the dialysis and postdialysis periods would be 0.022 kcal energy ·FFM-1 · h-1. This accounts for approximately one-fourth of the total increase in energy expenditure observed, leaving a significant proportion of the increase in energy expenditure that is unexplained. Although the actual cause of the increased energy expenditure is not clear, it represents an additional catabolic response to hemodialysis procedure and possibly uremia. Another interesting observation in our study was the changes that occurred in substrate oxidation. Specifically, during the 2-h postdialysis period, carbohydrate oxidation decreased substantially, and there were significant increases in lipid and amino acid oxidation. Determining the mechanisms for these changes in substrate oxidation will require further study.

Despite the intriguing results presented in this paper, there are several potential caveats that should be considered. Although the techniques used in this study are associated with a margin of error, they are well-accepted and validated methods. In addition, we have used these techniques in several study populations and have found them to be reliable and reproducible (5, 6, 9, 22). The patients in this study were well nourished. Because of this and the acute nature of the studies, any conclusions with regard to potential effects of malnutrition and inflammation in CHD patients are clearly speculative. It is possible that malnourished and/or inflamed patients might have a different metabolic profile. Clearly, those patients should be studied and compared with the population presented in this study. Furthermore, we report only short-term effects of the hemodialysis procedure and cannot speculate on the repetitive nature of this treatment modality on protein and energy metabolism over a longer period of time. Our study population includes only two diabetic patients. Although there are not any numerical or statistical differences in our results whether the diabetic patients are included or excluded (data not shown), we recognize the possibility that, if a larger subset of diabetic patients had been included, our results might have been altered. Finally, the present study does not provide complete insight into the mechanism(s) involved in induction of hemodialysis-associated catabolism. Except for amino acid losses into the dialysate, the other explanations are speculative and need further study.

In conclusion, this study demonstrates that hemodialysis is, overall, a catabolic event, decreasing circulating amino acids, accelerating rates of whole body and muscle proteolysis, stimulating muscle release of amino acids, elevating net whole body and muscle protein loss, and increasing energy expenditure. Several of these variables continue to be altered for at least 2 h after hemodialysis. These changes provide a mechanistic basis for alterations in nutrient stores, increasing the potential for the development of malnourished patients. Furthermore, these findings emphasize that there is a need to design nutrient therapies to combat the degenerative process associated with hemodialysis.


    ACKNOWLEDGEMENTS

We thank the patients and staff of the Vanderbilt University Medical Center Outpatient Dialysis Unit for their participation in the study. The excellent technical assistance of Jennifer Gresham, Suzan Vaughan, Wanda Snead, Janice Harvell, and the nursing staff on the GCRC is also appreciated.


    FOOTNOTES

This study is supported in part by National Institutes of Health Grant RO1-45604, Federal Drug Administration Grant 000903, Clinical Nutrition Research Unit Grant DK-26657, General Clinical Research Center Grant RR-00095, and Diabetes Research and Training Center Grant DK-20593.

Address for reprint requests and other correspondence: T. A. Ikizler, Vanderbilt University Medical Center, 1161 21st Ave. South & Garland, Div. of Nephrology, S-3223 MCN, Nashville, TN 37232-2372 (E-mail:alp.ikizler{at}mcmail.vanderbilt.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.

Received 1 May 2001; accepted in final form 27 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 282(1):E107-E116
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