Participants and ethical approval

A total of 24 young men and women were included in the study (Table 1). Two participants withdrew from the study, due to busy time schedules. Results presented are from the remaining 22 participants (Fig. 1). All participants underwent a medical screening before entering the study. To take part participants had to be healthy and without any injuries to the musculoskeletal system that could interfere with the execution of training. Individuals with lactose intolerance, milk allergy or using any dietary supplements were excluded. Participants were strength trained (minimum one leg strength training session per week for the last six months) sport science students. The study was approved by the Regional Ethics Committee for Medical and Health Research of South-East Norway (2014/834/REK sør-øst C) and performed in accordance with the Declaration of Helsinki. All participants signed a written informed consent form before entering the study. The trial was registered at clinicaltrials.gov as NCT02968888.

Table 1 Participant characteristics Full size table

Study design

This study was a double blinded, partial crossover, randomized control trial (Fig. 2). Each participant was assigned to one of two groups. The randomization was stratified based on lean body mass. The milk group did the protocol once, whereas the whey group was exposed to the protocol two times, once consuming WPC-80 and once consuming native whey, in a randomized order, approximately two weeks apart. 3 h after a standardized breakfast participants performed an intense bout of high-load leg-resistance exercise. 20 g of protein from milk, WPC-80 or native whey, was ingested both immediately after, and again 2 h after exercise. Blood samples were collected from an antecubital vein to measure changes in blood concentrations of amino acids, glucose, insulin, urea and creatine kinase (CK). MPS and related intracellular signalling were measured during a 5-h recovery period combining biopsies and tracer infusion of [2H 5 ]phenylalanine. In addition, we measured recovery of muscle force-generating capacity by maximal isometric voluntary contractions (MVC) for 24 h after exercise.

Familiarization

During the two weeks prior to the study, participants met twice in the lab to establish their 8 repetition maximum (RM) in bilateral leg press and knee extension, and to perform familiarization to the standardized workout and the MVC-test. All participants were asked to refrain from physical exercise for 48 h prior to the experiments.

Diet

At each familiarization session participants completed a 24-h dietary recall interview. A trained dietician conducted the recall interviews and analysed dietary nutrient content using the software Mat på Data 5.1 (Mattilsynet, Oslo, Norway, 2009). To standardize the diet participants were provided with a diet plan and pre-packaged food for the day before the experiment, and for the rest of the experimental period (2.5 days in total). The diet plan was individualized relative to body mass and provided participants with 40 kcal/kg and 1.5 g protein/kg per day. The standardized breakfast contained 23 kJ, 0,11 g protein, 0,30 g fat and 0.58 g carbohydrates per kg body mass.

Infusion and exercise protocol

Participants arrived in the lab after an overnight fast. A cannula was inserted into a forearm vein in both arms. A baseline blood sample was drawn before participants ingested a standardized breakfast. The breakfast, containing 0.14 g of protein • kg body mass−1, was to be consumed within 20 min. Thirty minutes after the baseline blood sample a primed continuous infusion of [2H 5 ]phenylalanine (0.05 μmol·kg−1·min−1; 2 μmol·kg−1 prime; Cambridge Isotopes Laboratories, Andover, MA, USA) was started. In addition to the constant infusion of [2H 5 ]phenylalanine a bolus of [13C 6 ]phenylalanine and [15 N]phenylalanine, not related to the results of this study, were infused at 170 and 200 min, respectively. Biopsies and blood samples were collected according to Fig. 2. The exercise session consisted of 4 sets of 8 repetitions to failure (8 RM sets) of leg press and knee extension, with a new set starting every 3 min. Warm-up sets of 10 repetitions at 50% and 80% of the 8RM loads were carried out in leg press.

Supplements

Tine ASA (Oslo, Norway) produced the milk and whey supplements for this study. In order to match all drinks on macronutrients, cream (Tine, Norway), lactose (Arla food ingredients, Denmark), and water was added to WPC-80 and native whey (Table 2). Native whey and WPC-80 contained whey protein only, whereas milk contained 20% whey and 80% casein. Drinks were enriched with 6% [2H 5 ] phenylalanine in order to maintain the plasma enrichment after intake. All drinks were matched for appearance and flavour.

Table 2 Amino acid and macronutrient content in supplements Full size table

Dual-energy X-ray absorptiometry

Body composition was assessed by dual energy X-ray absorptiometry (Lunar iDXA GE Healtcare, Madison, Wisconsin, USA, using the enCORE Software Version 14.10.022). After an overnight fast, before one of the familiarization sessions, participants were scanned from head to toe in a supine position, providing values for lean tissue, fat mass and bone mineral content. The coefficient of variation (CV) for the assessment of lean tissue was <1.1%.

Maximal strength

Unilateral maximal knee extension strength was assessed in an isometric voluntary maximal contraction (MVC) in a custom-made knee-extension apparatus (Gym2000, Geithus, Norway). Participants were seated in a chair with a four-point belt fixing the chest and hips, with 90° in the hip and knee joints. Three attempts of 5 s with 1 min rest between were given to reach MVC force. Force was measured with a force transducer (HMB U2 AC2, Darmstadt, Germany). MVC was tested after a 5 min warm up on a cycle ergometer, except for 10 min after the workout. MVC was tested unilaterally and no effect of the biopsy procedure was observed.

Blood analyses

Blood serum was obtained by clotting for 30 min at room temperature before centrifugation. Plasma was obtained by immediate centrifugation in lithium heparin tubes, respectively. Both serum and plasma were centrifuged at 4 °C for 10 min at 1300 g, and stored at −80 °C until analysis. Serum samples were analyzed for creatine kinase (CV: 2.8%) and urea (2.2%) at Fürst Medical Laboratory (Oslo, Norway). Plasma samples were analyzed for concentrations of insulin (6.8%) using enzyme-linked immune sorbent assay (Alpco, Salem, NH, USA), and glucose (2.1%) using a Cobas clinical analyzer (Cobas 6000, Roche Diagnostics, Indianapolis, IN, USA). Amino acid concentrations were measured in plasma as described earlier [16] with a EZfaast amino acid analysis kit (Phenomenex®, Torrance, CA, USA) and gas chromatography/mass spectrometry (Shimadzu QP-2010 Ultra GCMS, Shimadzu Scientific Instruments, Columbia, MD).

Biopsy collection and pre-analytical processing

Muscle biopsies were collected from the mid portion of m. vastus lateralis with a modified Bergström technique with suction. Pre-analytical processing of muscle tissue was performed as per Paulsen and colleagues [26]. In short specimens were used to make a homogenate of soluble protein for Western blotting analyses and for analysis of MPS.

Western blot

Samples for Western blot were treated as previously described [26], quantified with ChemiDoc MP (BioRad Laboratories, CA, USA) and analyzed with Image Lab (v5.1, BioRad Laboratories, CA, USA). All samples were run in duplicate. Antibodies against p70S6K and phosphor-p70S6K Thr389, Eukaryotic elongation factor 2 (eEF-2), phospho-eEF-2Thr56, Eukaryotic initiation factor 4E–binding protein 1 (4EBP-1), phospho-4EBP-1Thr37/46, and secondary anti-rabbit were purchased from Cell Signalling Technology (Beverly, MA, USA).

Blood, muscle protein-bound and intracellular free phenylalanine enrichment

Plasma from blood samples for the measurement of phenylalanine enrichment was analysed as previously described [43]. Briefly, plasma was deproteinised with 500 μl 15% sulfosalicylic acid, and amino acids were purified using cation exchange chromatography (AG 50 W-8X, 100–200 mesh H+ form; Bio-Rad Laboratories, Richmond, CA, USA). Purified amino acids were dried under vacuum (Vacuum Dry Evaporator System, Labconco, Kansas City, MO, USA) and thereafter derivatised with 80 μl (1:1, v/v) acetonitrile: N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA, Sigma-Aldrich) for 45 min at 100 °C. Isotopic enrichments of the plasma samples were determined on the tert-butyl dimethylsilyl (TBDMS) derivatives using gas chromatography/mass spectrometry (Shimadzu QP-2010 Ultra GCMS, Shimadzu Scientific Instruments, Columbia, MD, USA) and selected ion monitoring [43]. Enrichments were expressed as tracer-to-tracee ratios. Appropriate corrections were made for overlapping spectra [43].

Plasma from blood samples for the measurement of phenylalanine enrichment was analyzed as previously described [7]. Briefly, plasma was deproteinized by adding 20% perchloric acid and centrifuged at 1000×g at 4C for 10 min. Supernatant was removed, and the mixed plasma protein pellet was washed three times with 2% PCA, two times with ethanol, and one time with diethyl ether, dried overnight at 50 °C and hydrolysed overnight in 6 N HCl at 110 °C. The hydrolysed mixed plasma protein samples were then processed using the same method as muscle protein bound samples.

Twenty-five to thirty mg of muscle was placed in 800 μl 10% perchloric acid (PCA), homogenized and centrifuged. The supernatant was collected for measurement of intracellular amino acid enrichment. The remaining pellet (bound protein) was washed three times with 2% PCA, two times with ethanol, and one time with diethyl ether, dried overnight at 50 °C and hydrolysed overnight in 6 N HCl at 110 °C. Amino acids from the bound and intracellular fractions were then purified by cation exchange chromatography and thereafter derivatised in the same way as for the blood samples. Isotopic enrichment of TBDMS-phenylalanine in the bound protein was determined by gas chromatography-combustion-isotope ratio/mass spectrometry (GC-C-IRMS) set to high temperature conversion (HTC) mode for analysis of deuterium (Delta V Advantage Isotope Ratio Mass Spectrometer with GC Isolink, Thermo Scientific, West Palm Beach, FL, USA). Enrichment is calculated from the relative ratios of hydrogen (mass 3/mass 2) corrected for natural abundance of deuterium, divided by fraction of atoms that could be labelled (5/39).

Calculations

Baseline muscle fractional synthesis rate (FSR) was calculated using the precursor product method [43]:

FSR (%h−1) = E p2 – E p1 / (E pre x t) × 100.

The product is the difference in enrichment of the bound protein pool (E p2 ) and the mixed plasma proteins (E p1 ). The precursor (E pre ) is the average plasma free or muscle free D 5 phenylalanine enrichments to estimate the upper (muscle free) and lower (plasma free) limits of the true muscle protein FSR. The tracer incorporation time is denoted by t.

Skeletal muscle fractional synthesis rate (FSR) was calculated (as a measure of MPS) according to the precursor product method where the precursor is the mean enrichment of the intracellular pool (E IC ) of biopsies being analysed [43]. The product is the difference in enrichment of the bound protein (E BP ) pools of the two muscle biopsies being analysed. Skeletal muscle FSR is expressed as per cent per hour: FSR (%/hour) = ((E BPt2 -E BPt1 )/(E IC ·(t 2 -t 1 )))·100.

The baseline MPS was only calculated during the first experiment for participants in the whey group, and this value was used as a baseline for both supplements in this group.

Statistics

Non-normally distributed data (D’Agostino and Pearson omnibus normality test) were log-transformed prior to statistical analysis. All data are illustrated in original form. As the main goal of the study was the comparison of WPC-80 with native whey, these data were analysed by one-way repeated measures ANOVA. Comparisons against milk were done with a two-way ANOVA with repeated measures (time x group), with group as non-repeated and time as repeated. A Tukey’s and Dunnett’s test was used as post hoc tests to specify the significant differences between trials and time points (within groups), respectively. Subject characteristics and AUC responses between groups were analysed with a one-way non-repeated measure ANOVA. A sample size calculation was conducted using a power of 80% based on mixed muscle FSR results from an earlier study comparing whey and casein in young men [36] StatMate, Graphpad Software, San Diego, CA, USA). Based on the power calculation the goal was to include 10 subjects in each group. Statistical analyses were made using Prism Software (Graphpad 6, San Diego, CA, USA), All results are expressed as means ± standard deviation (SD). Statistical significance level was set at p ≤ .05.