Study population
A total of 52 subjects, 35 MHD patients from the Vanderbilt University Outpatient Dialysis Unit and 17 volunteers without kidney disease were recruited between November 2011 and December 2014. Primary inclusion criteria for MHD group was being on MHD therapy at least 6 months with well-functioning hemodialysis vascular access and, on a thrice-weekly MHD program receiving an adequate dose of dialysis (single pool Kt/V > 1.2). Exclusion criteria included patients with active infectious or inflammatory disease (i.e., vascular access infections, active connective tissue disorder, active cancer, HIV, and liver disease) and patients hospitalized within the last month prior to the study. Patients receiving steroids (>5 mg/day) and/or immunosuppressive agents and patients with Type 1 DM and type 2 DM who were using insulin or insulin-sensitizing medications were also excluded. The Institutional Review Board of Vanderbilt University approved the study protocol and written informed consent was obtained from all study patients.
Study procedures
Hyperinsulinemic-euglycemic (HEGC) clamp protocol
All studies were performed after an overnight 8-h fasting period at the General Clinical Research Center (GCRC) at Vanderbilt University Medical Center (VUMC). Clamp studies were performed on a non-dialysis day in order to eliminate the potential effects of the hemodialysis therapy.
On the morning of the clamp study, for the MHD patients, the dialysis shunt was accessed using 15-gauge fistula needles placed in opposite directions at least 4 fingerbreadths apart. The venous needle was used for the infusion of insulin and dextrose. Arterial blood samples were taken through the dialysis needle placed at the arterial side of the dialysis access. An IV was placed into the contralateral forearm vein (preferably in a retrograde fashion) to obtain samples of venous blood. For the control subjects, an IV was inserted into an antecubital vein for the infusion of all test substances. Second and third IVs were placed antegradely and retrogradely in the opposite arm for blood sampling. The antegrade IV was placed distal to the retrograde IV and the hand was kept in a heated box to achieve arterialization of the venous blood [11]. The remaining IV was used for venous sampling.
A schematic diagram of the metabolic study protocol is depicted in Fig. 1. After obtain blood and breath samples at time point 0, bolus injection of NaH13CO3 (0.12 mg/kg), L-(1-13C) Leucine (7.2 μmol/kg), L-(ring-2H5) Phenylalanine (3.6 μmol/kg), and 6,6-2H2 Glucose (3.6 mg/kg) to prime the CO2, Leucine, Phenylalanine, and Glucose pools and a continuous infusion of labeled Leucine (0.12 μmol/kg/min), Phenylalanine (0.06 μmol/kg/min) and Glucose (0.06 mg/kg/min) started and continued throughout the remainder of the study. The initial 2 h were for tracer equilibration. During the equilibration period, forearm blood flow was estimated by using capacitance plethysmography (model 2560 with URI/CP software version 3.0; Moro Bay, CA, USA). Metabolic cart (Med Graphics CPX/D metabolic cart; software version 6.1. St. Paul, MN) was used to measure simultaneous energy expenditure and respiratory quotient.
After equilibration period, baseline blood and breath sampling obtained between T120 min and T150 min for every 15 min. A primed infusion of crystalline insulin at the concentration of 2.0 mu/kg/min was then started at time point 150 min and maintained throughout the study procedure to obtain constant hyperinsulinemia. The goal plasma insulin concentration was 100μU/mL. The insulin dose used was determined by the required dose during HEGC study in patients with ESRD to suppress hepatic gluconeogenesis and allow the measurement of peripheral insulin resistance [12]. Following initiation of insulin, target plasma glucose levels were 90 ± 5 mg/dL, achieved by adjustment of 20 % dextrose infusion. Constant monitoring of plasma glucose concentration was performed every 5 min and of leucine levels every 10 min using rapid bedside high-performance liquid chromatography (HPLC) methodology. Once steady state was reached and confirmed for 90 min, GDR or the metabolizable glucose “M” value (mg/kg/min insulin mediated glucose disposal rate) was calculated from samples taken between time points 240 to 270 min. This served as an index of in vivo insulin sensitivity. For reasons of comparison, the M-value was normalized to body weight.
Hyperinsulinemic–euglycemic-euaminoacidemic (HEAC) clamp protocol
Once the HEGC was achieved, a continuous infusion of a balanced amino acid solution (Freeamine III 10 %, B. Braun Medical, Inc. Irvine, CA) (amino acid concentrations in Freeamine III: leucine, 910 mg; isoleucine, 690 mg; lysine, 730 mg; valine, 660 mg; phenylalanine, 560 mg; threonine, 400 mg; methionine, 530 mg; tryptophan 150 mg; alanine, 710 mg; arginine, 950 mg; glycine, 1400 mg; proline, 1120 mg; serine, 590 mg; histidine, 280 mg; cysteine, <16 mg; phosphoric acid NF, 120 mg; sodium bisulfite, <100 mg) was administered at variable dose to achieve and maintain the leucine concentration similar to baseline state, which was calculated as the average level of time points 120 min, 135 min, and 150 min.
The formula for computing the periodically adjusted leucine infusion rate for every 10 min is
$$ \mathrm{R}\ \left(\mathrm{ml}/\mathrm{h}\right) = \left({\mathrm{C}}_{\mathrm{d}}\hbox{-} {\mathrm{C}}_{\mathrm{c}}\right) \times k \times 6 $$
The individual components of this formula are as follows: (Cd-Cc) equals the leucine deficit or excess in μM/L where Cd is the desired plasma leucine concentration (μM/L). Multiplication by 6 converts the transfusion time from 10 min to one hour. Cc, is the actual plasma leucine concentration (μM/L) at any time point. Constant factor k is derived from the formula of
$$ \left|\frac{B\times BW}{100}\times \left(\frac{1-HCT}{100}\right)\right|\times \left|\frac{9.1}{131.17}\times 1000\right| $$
Total body blood volume is 8 % of body weight. Multiplication by 1 minus hematocrit equals to the total plasma volume of total body. 9.1 mg is the total leucine amount in 1 mL of Freeamine III solution. Division by 131.17 (molecular weight of leucine) and further multiplication by 1000 converts the leucine amount from milligram per liter to micromoles per liter.
When the steady state condition for leucine was achieved, the leucine infusion rate was assumed to be equal to the leucine incorporation rate into protein since endogenous leucine release from protein is assumed zero under steady state condition. Leucine disposal rate (LDR) was obtained by the leucine infusion rate (as in mg/min) during the final 30 min of the 390-min of study (steady state period for amino acids) and normalized by body weight (kg).
Body composition
Within one week of each study, participants underwent dual-energy x-ray absorptiometry (DEXA) offering a rapid, non-invasive three-compartment evaluation that quantifies fat mass, LBM, and bone mineral content with minimal radiation exposure. All DEXA measurements were completed on a non-dialysis day using a Lunar Prodigy iDEXA machine v.11.40.004 (software versions 2003 to 2011, General Electric, Madison, WI).
Laboratory analysis
All blood sampling was performed at the GCRC and analyzed at VUMC central laboratories. After blood draw was performed, samples were transported on ice and centrifuged at 3000 rpm for 15 min before being kept frozen at −80° Celsius. Plasma fasting glucose concentrations were analyzed using the glucose oxidase method (Glucose analyzer 2; Beckman Coulter, Brea, CA). Biochemistry measurements were analyzed at the VUMC Pathology Laboratory. High sensitivity C-reactive protein (hs-CRP) concentrations were measured by high-sensitivity particle-enhanced turbidimetric UniCel DxI Immunoassay system (Beckman Coulter).
Plasma amino acid measurements
Plasma arterial amino acid concentrations were determined by reverse phase HPLC after derivatization with phenyliosthiocynate. 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 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) [13].
Whole body protein turnover
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 [14]. Steady state conditions for KIC and CO2 enrichments were achieved as evidenced by slopes within each phase not significantly different than zero. Breath 13CO2 was determined by multiplying the total CO2 production rate by the breath 13CO2 enrichment. 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) [15] and by the plasma KIC enrichment. The leucine Rd, an estimate of whole-body protein synthesis, was determined indirectly by subtracting leucine oxidation from 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 non-oxidative leucine Rd, respectively, assuming that 7.8 % of whole-body protein is composed of leucine [16].
Statistical analysis
Descriptive statistics were expressed as mean ± SD or median (IQR) for continuous variables and as frequencies and percentage for categorical variables. Univariate analysis was performed to compare differences between cases and controls using Pearson’s χ
2 test for categorical variables and Wilcoxon rank-sum test for continuous variables. Spearman rank correlations were used to estimate the correlation coefficient between LDR and GDR. Linear mixed-effects models with random intercepts were fitted separately to estimate the differences in leucine, phenylalanine, BCAA, EAA, NEAA, and TAA levels at baseline, HEGC, and HEAC between groups. Analyses were performed using R, version 3.2.3 (http://www.r-project.org/). The 5 % significance level (2-sided) was used.