Eight healthy young men (mean age 27.3 years, range 21 to 33 years) with body mass index 23.9 kg/m2 (range 20.7 to 28.4 kg/m2), an unremarkable medical history and no regular medication use were included in the study after they had given informed oral and written consent. All participants underwent a thorough clinical examination, including blood analyses (haemoglobin, WBC and differential counts, C-reactive protein, blood glucose, electrolytes, and liver, kidney and thyroid function parameters) and electrocardiogram recording. All tests were normal. The Ethical Committee of the Capital Region, Denmark, approved the study ([H-KF] 01-144/98).
Glutamine was given as a dipeptide, consisting of alanine-glutamine (Dipeptiven; Fresenius Kabi, Uppsala, Sweden) in a stock concentration of 200 mg/ml of the dipeptide suspended in saline. The solution used for infusion was a 20% (volume/volume) solution of Dipeptiven and saline. Isotonic saline was used as a placebo. The dipeptide solution could not visually be distinguished from placebo, and the containers were covered in a way that prevented both the volunteers and the investigators from distinguishing between them. The glutamine solution and saline were prepared under sterile conditions on the day before each trial.
The study was a randomized, double-blinded, placebo-controlled, crossover trial. All volunteers participated on 2 trial days, separated by 30 days. The volunteers were randomly assigned to either infusion with alanine-glutamine on the first trial day and placebo on the second (n = 5) or vice versa (n = 3). The difference in the numbers of volunteers was due to one being placed in the wrong group (human error).
The volunteers reported to the research unit at 06:30 a.m. after an overnight fast. They were immediately placed in bed. Catheters were placed bilaterally into an antecubital vein; one catheter was used to draw blood samples and the other to infuse glutamine or placebo. The volunteers were monitored by continuous electrocardiography, noninvasive blood pressure monitoring (every 15 minutes) and continuous measurement of rectal temperature and venous oxygen saturation. At 07:00 a.m. (t = 0), infusion with glutamine or placebo was initiated and continued for 10 hours. The glutamine infusion rate was set at 0.025 g/(kg body weight × hour). This rate has been tested for safety [24
] and is comparable to the doses used in patient studies [5
]. Placebo was infused at the same rate. At 09:00 a.m., after 2 hours of alanine-glutamine or placebo infusion, steady state was assumed to have been achieved [24
] and volunteers were given an intravenous bolus injection with a standard reference E. coli
endotoxin (Lot EC-6; US Pharmacopeia Convention, Rockville, MD, USA) at a dose of 0.3 ng/kg body weight. The vounteers were then monitored for another 8 hours.
Blood sample and analysis of blood samples
Blood samples were drawn hourly from 0 hours and onward, with additional samples at time 2.5 and 3.5 hrs (corresponding to 0.5 and 1.5 hours, respectively, after endotoxin injection). Whole blood was analyzed for WBC and differential counts using standard biochemical procedures.
Samples for measuring plasma concentrations of glutamine, cytokines and cortisol were drawn into tubes containing EDTA and centrifuged; plasma was separated and immediately stored at -80°C. Plasma glutamine concentration was determined enzymatically using an automated analyzer (Cobas Fara; F. Hoffmann-La Roche, Basel, Switzerland). Plasma TNF-α, IL-6 and cortisol levels were determined using a commercially available enzyme-linked immunosorbent assay (Quantikine and Parameter; R&D systems, Minneapolis, MN, USA). All measurements were taken in duplicate and means were calculated for the subsequent statistical analyses. Blood for isolation of blood mononuclear cells (BMNC) was drawn into tubes containing heparin; BMNCs were isolated by density gradient centrifugation (Lymphoprep Nycomed Pharma AS, Oslo, Norway) on LeucoSep tubes (Greiner, Frickenhausen, Germany). After separation, the BMNCs were immediately suspended in a modified RIPA cell lysis buffer (50 mmol/l Tris-HCl [pH 7.4], 150 mmol/l NaCl, 1 mmol/l EGTA, 1 mmol/l EDTA, 0.25% NaDeoxycholate, 1% Triton X-100) containing complete protease inhibitor cocktail (Roche, Basel, Switzerland) and frozen at -80°C until measurement of HSP70 protein content using Western blotting, as described below.
HSP70 Western blotting
BMNCs resuspended in cell lysis buffer were thawed on ice, pulled through a small-gauge syringe four to five times, and centrifuged in a microcentrifuge at maximum speed at 4°C for 15 minutes. The supernatant was then transferred to a new Eppendorf tube, and protein concentrations in this BMNC lysate were determined using the BioRad DC kit (BioRad, Hercules, CA, USA) with bovine serum albumin as standard. All measurements were conducted in triplicate.
Ten micrograms of BMNC protein lysate per lane were boiled in Laemmli buffer and separated on 4% to 12% Bis-Tris gels (Invitrogen, Taastrup, Denmark) and transferred to polyvinylidene fluoride membranes (Hybond-P; GE Healthcare, Little Chalfont, UK). Membranes were blocked for 1 hour at room temperature in a blocking buffer (Tris-buffered saline [pH 7.6], 0.1% Tween-20, 5% skimmed milk). The membranes were then incubated overnight at 4°C in a blocking buffer containing a primary antibody against HSP70 (SPA-810; Stressgen, Victoria, BC, Canada) at a 1:1,000 dilution. Subsequently, they were washed three times for 5 minutes in a washing buffer (Tris-buffered saline with 0.1% Tween-20) and incubated for 1 hour at room temperature with a secondary antibody (Rabbit anti-mouse HRP, P0260; Dako, Glostrup, Denmark) at a 1:10,000 dilution in a blocking buffer, followed by three washes of 5 minutes each in washing buffer. The protein bands were detected using ECL (GE Healthcare) and quantified using CCD image sensor (ChemiDoc XRS; BioRad) and software (Quantity One; BioRad).
All statistical analyses were done using SAS 9.1 (SAS Institute Inc., Cary, NC, USA). Log values were used when considered appropriate to approximate the normal distribution. P < 0.05 was considered statistically significant. A two-way analysis of variance for longitudinal measures was performed on each investigated variable using a means model (SAS PROC MIXED) with the model TIME TREATMENT TIME*TREATMENT, and with SUBJECT as a random factor. An autoregressive covariate structure was assumed. Goodness-of-fit of the mixed model was assessed by investigating the distribution of the residuals. Significant changes in inflammatory markers from the time of endotoxin administration to the following time points were analyzed by paired t-tests using the Tukey-Kramer method to adjust for multiple comparisons. Fractional changes in glutamine levels from baseline to endotoxin injection and from endotoxin injection to 4 hours after endotoxin injection were calculated and evaluated by t-tests.