1Department of Medicine, University of New Mexico Health Sciences Center, and 3Division of Rheumatology, University of New Mexico, Albuquerque 87131; 2Albuquerque Academy, Albuquerque, New Mexico 87109; and 4Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77550
Submitted 2 October 2003 ; accepted in final form 17 December 2003
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ABSTRACT |
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protein catabolism; protein turnover; inflammation; amino acid; endstage renal disease
Uremia is a state of microinflammation, with elevated cytokine and positive acute-phase protein levels (20), which is further augmented by hemodialysis (HD) (19). Acute-phase protein synthesis is transcriptionally regulated by cytokines (4). Increased production of cytokines and muscle protein breakdown have both beneficial and detrimental effects on the associated disease states. In the acute phase of illness, there is increased production of inflammatory cytokines, which accelerates the breakdown of skeletal muscle, providing the substrate for the liver to mount an acute-phase response (15). The acute-phase response is an important pathophysiological phenomenon that modulates the response to inflammation. This includes synthesis of proteins by the liver, which may positively or negatively influence the inflammatory process (15). However, continued loss of lean body mass is deleterious to the host and contributes to morbidity and mortality. Although intradialytic increases in FSR-A and FSR-F have been reported (8), their relationship to FSR-M and cytokine activation have not been rigorously studied. Arteriovenous balance studies have demonstrated net release of amino acids from the muscle during HD (18, 21, 34). We hypothesized that cytokine activation and release of amino acids from the muscle during HD may mediate the observed intradialytic increase in FSR-A and FSR-F.
This study was designed to examine the effect of hemodialysis and intradialytic activation of cytokines on albumin, fibrinogen, and muscle protein synthesis. We estimated arteriovenous balance of amino acids across the leg and fractional synthesis rates of albumin, fibrinogen, and muscle protein in nine ESRD patients and eight controls. ESRD patients were studied before (Pre-HD) and during HD.
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MATERIALS AND METHODS |
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The study population included nine ESRD patients and eight controls. ESRD patients were placed on a 35 kcal/kg and 1.2 g·kg-1·day-1 protein diet. A minimum protein intake of 1.2 g·kg-1·day-1 was recommended for healthy controls. The participants 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. Predialysis plasma bicarbonate (
) was checked 3 wk before the experiment. If plasma
was <22 meq/l, patients were initiated on oral NaHCO3 supplementation, and the dose was adjusted to achieve a target plasma
meq/l. The experiments were performed only when the patient's plasma
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 1 day before the experiment. ESRD patients were studied 72 h after dialysis treatment. This timing was selected to estimate the synthesis rates at the peak of metabolic abnormalities. All of the studies were performed in a postabsorptive state after an overnight fast. On the day of the experiment, diabetic subjects were given one-half of the usual dose of insulin or oral hypoglycemic agent. Blood sugar was closely monitored during the experiment and maintained within normal range by additional doses of regular insulin administered subcutaneously. Polyethylene catheters were inserted into the femoral artery and vein on the same side for blood sampling and in the nonaccess forearm veins for infusion of labeled phenylalanine. All of the experiments were performed at rest in a supine posture. Leg volume was estimated using an anthropometric formula as described previously (7).
L-[ring-13C6]phenylalanine (99% atom percent excess) was purchased from Cambridge Isotope Laboratories (Andover, MA). After blood samples were obtained for background amino acid enrichment, a primed (2 µmol/kg) continuous (0.1 µmol·kg-1·min-1) infusion of L-[ring-13C6]phenylalanine was initiated through the forearm vein. Tracer infusion was continued throughout the experiment. Blood samples for enrichment were collected as previously described (7) (Fig. 1). Blood samples were taken at 0 and 300 min to estimate FSR-A and FSR-F before dialysis and at 540 min to calculate FSR during dialysis. Arteriovenous balance studies were performed before and during HD. Blood flow to the lower extremity was measured by dye dilution technique (23). Briefly, a continuous infusion of indocyanine green (ICG) dye into the femoral artery was initiated at a rate of 1 ml/min 30 min before the second and third biopsies. Leg plasma flow was calculated from steady-state ICG concentrations in the femoral artery and arterialized wrist vein.
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Hemodialysis was initiated at 300 min and continued for 4 h. Patients' usual blood and dialysate flow rates were used. Dialysate composition was as follows: 139 meq/l sodium (Na+), 35 meq/l , 2.5 meq/l calcium (Ca2+), 1 meq/l magnesium (Mg2+), 200 mg/dl dextrose, and potassium (K+) per patient's need. A new polysulfone membrane (F70, Fresenius, Hemoflow) was used. Anticoagulation was not used during dialysis to minimize the risk of bleeding. Representative spent dialysate samples were collected to estimate the amino acid concentrations.
Plasma samples were obtained pre- and post-HD for blood urea nitrogen (BUN), creatinine, electrolytes, albumin, and fibrinogen. Albumin was measured by the bromcresol green method, prealbumin by nephelometry, insulin-like growth factor (IGF), catecholamines, and glucagon by radioimmunoassay (RIA), thyroid-stimulating hormone (TSH), insulin, cortisol, and C-reactive protein (CRP) by immulite chemiluminescence. Cytokines (TNF-, IL-1, IL-6, and IL-10) were measured using 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 inter- and intra-assay coefficients of variation for each of the cytokines were <6%.
Muscle biopsies were performed at the second hour to measure isotopic carbon enrichment of bound and free phenylalanine in the muscle. The second biopsy was obtained at the fifth hour to estimate the fractional synthesis rates before HD. The third biopsy was obtained at the fourth hour of dialysis. Biopsies were taken from the lateral portion of the vastus lateralis muscle 20 cm above the knee by use of a Bergstrom biopsy needle. Fat and connective tissue were removed and the samples frozen in liquid nitrogen and stored at -80°C for future analysis.
Analytical Methods
Blood. Plasma samples were stored at -80°C for future analysis of tracer enrichment and amino acid concentrations. Amino acids were separated using cation exchange chromatography and dried under vacuum using a Speed-Vac (38). The enrichment of free amino acids in the plasma sample was determined by gas chromatography-mass spectrometry (GC-MS) (GC HP 5890 and 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 (NAP) derivatives of phenylalanine (m/z 336, 342, and 346). Data are expressed as tracer-to-tracee ratios.
For determination of arteriovenous amino acid balance, 3 ml of blood from femoral artery and vein were collected into lithiumheparin tubes, centrifuged, and frozen at -80°C until analyzed. Free amino acid concentrations were determined by HPLC (Waters 2960 system; Milford, MA), precolumn derivatization, and o-phthalaldehyde and 3-mercaptopropionic acid.
Albumin and fibrinogen. Albumin was isolated by ethanol extraction from trichloroacetic acid (TCA)-precipitated plasma proteins, as described previously (39). In short, citrated plasma was precipitated with 10% TCA and centrifuged, and the supernatant was discarded. The protein pellet was dissolved in absolute alcohol. The resulting supernatant was decanted to obtain isolated plasma albumin. The pellet was dried under vacuum. Albumin was hydrolyzed at 110°C for 24 h with 6 N constant-boiling HCl. The protein hydrolysates were passed through a cation exchange column to isolate purified amino acids. NAP derivatives of the purified albumin amino acids were then separated and analyzed by GC-MS. L-[ring-13C6]phenylalanine enrichment of albumin was determined from an M+6/M+3 ratio and an isotopic dilution curve.
To determine isotopic enrichment in fibrinogen, blood samples were transferred into heparin sodium-treated tubes and centrifuged at 4°C. Fibrinogen was precipitated from 2 ml of plasma by adding 40 µl of calcium chloride (1 M) and 4 U of thrombin and incubating at 22°C for 2 h (10). The clot was washed and centrifuged. The purity of the isolate was confirmed by SDS-gel electrophoresis. The resulting pellet was analyzed for phenylalanine enrichment as described before.
Muscle. Muscle samples were weighed, and protein was precipitated with 500 µl of 14% perchloric acid. The supernatant was collected after homogenization of the issue and centrifugation. The amino acids in the pooled supernatant were separated using cation exchange chromatography. The isotopic enrichment of the intracellular amino acid was determined on their NAP derivatives in the electron impact mode. Intracellular enrichment was corrected on the basis of the chloride method (5). The tissue pellet was further washed with saline and absolute alcohol and dried at 50°C overnight. The precipitated protein was hydrolyzed at 110°C for 24 h with 6 N constant-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) using chemical ionization and the standard curve approach (9).
Calculations
Amino acids entering the leg through the femoral artery (Fin) and leaving the leg via femoral vein (Fout) were estimated as
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where Ca and Cv are concentration of amino acid in the artery and vein, respectively, and BF is plasma flow rate.
Net amino acid balance (NB) in the leg was calculated as
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The rate of disposal of arterial phenylalnine (Rd) is an index of the absolute amount of protein synthesis. Rd is calculated as
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where Ea is enrichment in the artery and Ev is enrichment in the vein.
The rate of appearance of phenylalanine (Ra) is an estimate of protein degradation. Ra was calculated as
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The whole body phenylalanine flux was calculated from the rate of tracer infusion (µmol·kg-1·min-1) divided by the arterial enrichment of phenylalanine.
FSR-M was calculated by dividing the increment in enrichment in the product (EP) by the average in the intracellular enrichment (precursor) from the first and second muscle biopsies.
EP is the difference in the enrichment of the muscle protein between the first and second muscle biopsies. The use of intracellular amino acid enrichment as a precursor for protein synthesis has been validated in in vivo studies (37).
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where Em(1) and Em(2) represent the L-[ring-13C6]phenylalanine enrichment in the free muscle pool in the consecutive biopsies. T is the time interval between first and second biopsies.
Increment in protein-bound L-[ring-13C6]phenylalanine enrichment (
EP) between first and second biopsies (
EP) is calculated as follows
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where (1) and (2) represent the isotope ratio from first and second biopsies.
FSR-A and FSR-F (%/day) between times t1 and t2 can be expressed by the following equation
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where Eb(t) is defined as the enrichment of phenylalanine bound in albumin at time t and Ef(t) the enrichment of phenylalanine free in plasma at time t2. The equations for Ef(t) will be determined by fitting the following nonlinear regression curves to the values for Ef(t1) and Ef(t2)
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These regression curves were chosen to account for the behavior of plasma phenylalanine enrichment during the primed constant infusion (2).
Subjects
Healthy volunteers were recruited from the GCRC database for healthy volunteers. ESRD patients were recruited from the outpatient dialysis unit. There were two diabetic patients in the control and ESRD arms each. Only patients on maintenance HD for 90 days and without residual renal function were included. Patients with infective or inflammatory condition, pregnancy, liver dysfunction, bleeding tendency, catabolic illness, or unexplained weight loss were excluded from the study. The study was approved by the Human Research Review Committee at the University of New Mexico Health Sciences Center.
Statistical Analysis
Data are given as means ± SE. Value was set at 0.05. Paired and unpaired t-tests were used when applicable. Repeated measures of analysis of variance (ANOVA) were used, with pre- and post-HD as the repeating factor and ESRD vs. control as the grouping factor, with a post hoc Tukey test. Linear regression analysis was used to identify relations between variables.
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RESULTS |
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Changes in plasma levels of cytokines and hormones during HD are shown in Table 2. Plasma IL-6, IL-10, and CRP increased significantly during HD, but there were no significant changes in IL-1 and TNF-. Plasma insulin and glucagon levels were higher pre-HD compared with controls and showed a tendency to decrease during HD. In contrast, plasma cortisol concentration increased during HD.
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Phenylalanine concentration in the artery decreased during HD (86.1 ± 7.7 vs. 67.6 ± 6.4 µmol/l, P < 0.01), but the venous concentration did not change significantly (86.6 ± 7.4 vs. 76.2 ± 6.8 µmol/l). Similarly, intradialytic decreases in arterial concentrations of total amino acids (TAA; 24.9 vs. 15.9%), essential amino acids (EAA; 33.4 vs. 15.8%), nonessential amino acids (NEAA; 25.4 vs. 15.8%), and branched-chain amino acids (BCAA; 20.5 vs. 9.5%) were greater than those in the vein. Amino acid inflow values into the leg (Fin) for phenylalanine, EAA, and NEAA were higher among controls compared with pre-HD and HD values (Table 3). Despite a decrease in Fin, the efflux (Fout) of the amino acids from leg increased during HD, resulting in increased negative net balance. The TAA concentration in the dialysate was 1,105 ± 490 µmol/l.
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The precursor phenylalanine reached isotopic plateau at 240 min and remained stable throughout the experiment. Enrichments of protein-bound phenylalanine in albumin, fibrinogen, and muscle protein are shown in Table 4. Both muscle protein synthesis (Rd) and catabolism increased significantly during HD (Table 5). However, the increase in muscle breakdown was higher than synthesis during HD (83.1 ± 3.6 vs. 29.1 ± 5.3 nmol·100 ml-1·min-1, P < 0.001), resulting in protein loss. However, whole body proteolysis did not change significantly during HD.
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The enrichment of albumin and fibrinogen increased significantly during HD, but the enrichment of the plasma remained stable. FSR-A increased during HD and were higher than that of controls (P < 0.01; Table 5). FSR-F was higher during HD compared with pre-HD (P < 0.01) but not different from controls. The increment Ep of the enrichment of the muscle protein-bound phenylalanine increased during HD, and the intracellular phenylalanine enrichment decreased. FSR-M increased during HD compared with pre-HD and controls (P < 0.001). FSR-M in controls and ESRD patients pre-HD were comparable. There was no significant difference in albumin, fibrinogen, or muscle protein synthesis between diabetic and nondiabetic subjects.
Skeletal muscle protein synthesis was correlated with albumin (r2 = 0.23, P = 0.05) and fibrinogen fractional synthetic rates (r2 = 0.27, P < 0.02). There was a modest but significant correlation between albumin and fibrinogen synthesis (r2 = 0.28, P = 0.05). Plasma IL-6 levels correlated positively with albumin (r2 = 0.36, P < 0.01), fibrinogen (r2 = 0.26, P < 0.05), and muscle protein (r2 = 0.32, P < 0.01) synthesis rates.
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DISCUSSION |
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Albumin and fibrinogen are both synthesized in the liver. Although albumin is catabolized in a mass-dependent manner, the catabolic rate of fibrinogen remains constant, despite considerable fluctuation in circulating mass of fibrinogen. Experimental models of renal failure in rats, as well as studies in dialysis patients, have shown that renal failure per se does not suppress albumin synthesis (25). However, Kaysen and Schoenfeld (24) observed that albumin synthesis is lower in hypoalbuminemic ESRD patients compared with those with normal plasma albumin levels. Giordano et al. (17) reported that FSR-A and FSR-F are increased in ESRD patients with normal nutritional status. Although plasma albumin was significantly lower in ESRD patients than in controls, FSR-A pre-HD was comparable to that of controls in our study, indicating that decreased fractional synthetic rates alone cannot explain hypoalbuminemia in these patients. Caglar et al. (8) observed an increase in albumin and fibrinogen synthesis during dialysis. Although the intradialytic increase in albumin synthesis rate (64%) was higher than that of fibrinogen (34%) in their study, we found that the increase in FSR-F (53.5%) tended to be larger than that of FSR-A (38.6%). Similar to our study, Prinsen et al. (32) recently reported that the absolute synthesis rates of albumin and fibrinogen in ESRD patients on peritoneal dialysis are higher than those of controls. They also observed a correlation between albumin and fibrinogen synthesis rates. The specific enrichment of tracer in serum may not be reflective of intrahepatic tracer level. Intradialytic loss of tracer may lead to underestimation of intrahepatic enrichment. However, it is reasonable to assume that both tracer and tracee are lost in the dialysate at the same rate; hence, the error in estimation of intrahepatic tracer level, if it exists, is minimal.
Animal studies have indicated that muscle protein synthesis is decreased and catabolism is increased in uremia (1). On the other hand, despite clinical evidence of malnutrition, protein turnover studies in stable nonacidotic CRF patients have failed to demonstrate any consistent abnormality (12, 29). Garibotto et al. (16) observed that in CRF there is a parallel increase in protein synthesis and catabolism. In our study, the FSR-M pre-HD value was not different from that of controls. This may be due to the fact that the patients did not have metabolic acidosis and that they were consuming an adequate protein diet. The effect of hemodialysis on protein turnover was studied by Lim et al. (27). They reported normal basal leucine flux, a transient decrease in protein synthesis, and a negative protein balance during HD. Raj et al. (34) estimated protein turnover and amino acid transport kinetics in ESRD patients before and during HD by use of stable isotopes of phenylalanine, leucine, lysine, alanine, and glutamine (34). Both protein synthesis and catabolism increased during HD. However, the increase in protein catabolism was higher than in synthesis, resulting in net protein loss. The present study confirms the previous observation from our laboratory that HD promotes skeletal muscle proteolysis. Despite an increase in muscle breakdown, whole body proteolysis was not increased during HD. Dissociation between muscle protein turnover and whole body protein kinetics has been observed by other investigators also (21, 27). More studies specifically designed to address this disparity are warranted.
Hepatic and muscle protein synthesis rates seem to be interrelated. In healthy humans, albumin accounts for 50% and fibrinogen for 10% of total liver protein synthesis (30). However, in disease states, fibrinogen synthesis may briefly increase 10- to 20-fold and dominate liver synthesis. In vitro studies have shown that IL-6 increases fibrinogen synthesis but decreases albumin synthesis (11). However, exposure to endotoxin induces release of IL-6 and increases synthesis of total liver proteins, including albumin, suggesting that there may be a nonspecific activation of all mRNAs involved in hepatic protein synthesis (3). Jahoor et al. (22) demonstrated that inflammatory stress decreases FSR-M but increases FSR-A and FSR-F. Similarly, in head injury patients, muscle protein synthesis is lower, but rates of albumin and fibrinogen synthesis were higher than in controls (28). Despite the intradialytic loss of amino acids, there was a coordinated increase in albumin, fibrinogen, and muscle protein synthesis. We found a significant positive correlation between IL-6 and FSR-A, FSR-F, and FSR-M. We previously reported a correlation between genes promoting protein catabolism and IL-6 (33). These results are consistent with the hypothesis that an intradialytic increase in IL-6 induces hepatic and muscle protein turnover. A positive correlation between protein synthesis and catabolism has been reported from our laboratory (6, 34). Availability of precursor amino acids is a potent modulator of protein synthesis (26). It is possible that the intracellular increase in amino acids derived from muscle catabolism stimulates muscle protein synthesis (6). The utilization of amino acids, however, is less efficient during HD, resulting in an increase in net outward transport of amino acids into the vein (34). This is supported by the observation that amino acid delivery to the leg (Fin) decreased but efflux (Fout) increased during HD. Previous studies have demonstrated the flow of amino acids from the muscle to visceral organs (31, 35). The amino acids released from the muscle are taken up by the splanchnic bed to be utilized for acute-phase protein synthesis, including albumin and fibrinogen.
To summarize, this study demonstrates that FSR-A, FSR-F, and FSR-M in ESRD patients without metabolic acidosis and consuming an adequate protein diet are similar to those of controls. Hemodialysis induces a coordinated increase in synthesis rates of albumin, fibrinogen, and muscle protein synthesis. Preliminary evidence indicates that IL-6 induces protein turnover during HD, resulting in a net release of amino acids from the muscle. Intradialytic increases in FSR-A and FSR-F are thus facilitated by IL-6 and a constant supply of amino acids derived from skeletal muscle catabolism.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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