Study Design. Twenty healthy, recreationally-trained men voluntarily participated in this double-blind, placebo-controlled, crossover, nutritional intervention study. The participants attended the Aspire Academy Sport Science Laboratory (Doha, Qatar) on five separate occasions. During the first session a maximal incremental oxygen uptake test (VO2max) was performed on a cycling ergometer. The second and third visits were reserved for familiarization of the cycling time trial (TT) protocol, consisting of 10 min warm up and 8 min TT. Supplementation conditions (CB or placebo soup) were performed once each, in a random order, on visit 4 and 5 with one week time in between. Allocation to the CB and placebo condition was done by an independent researchers, thereby guaranteeing that both subjects and investigators were blinded for the condition. Figure 1 illustrates the time schedule of visits four and five.
Subjects. Participants underwent a routine medical assessment by a medical doctor and an evaluation of their body mass index (BMI), age and lifestyle was done in the laboratory. A questionnaire was asked for the training rate, food habits, sleeping hours and lifestyle habits. Subject were used to train ~10 h/week on average (mixed: swimming-cycling-running-strength). Before the start of the study, a training log was taken and we performed a direct assessment of training status via VO2max test. The definition “recreationally-trained” was given after evaluation of the VO2max test results (< 55 mL/kg/min, except for 2 participants who were above that value). A food log was required to any participant for the whole duration of the study. Subjects were instructed to maintain their normal dietary habits except for the last meal before the test days where a vegetarian meal was proposed as the only alternative, not to bias the upcoming results on carnosine, anserine on the following day.
Inclusion criteria were males, non-vegetarian, not taking any drugs and/or food supplements 3 month prior and during the study period, medium-high weekly training habits. During the course of the study, 6 subjects dropped out (2 got sick, one relocated to another Country, one for an ankle injury, 2 for work reasons). Of the fourteen subjects that completed the study, subjects’ age, weight and relative maximal oxygen consumption were 37.2 ± 6.5 years, 76.2 ± 10.0 kg and 50.0 ± 5.5 mL/min/kg, respectively. They gave their written informed consent and the Local Ethical Committee (Anti-Doping Laboratory Qatar, ADLQ) approved the study.
VO2max test. The maximal incremental oxygen uptake test (VO2max) was performed on a cycling ergometer (SRM bike; Schoberer Rad Messtechnik, Germany) using the Oxycon Pro ergospirometry testing device (Cardinal Health Germany 234 GmbH Leibnizstrasse 7, Hoechberg, Germany). Throughout this cycling test, starting at 100 W for 2 min and with an incremental rate of 50 W/2 min, the subjects maintained a constant pedal cadence of 80 rpm until volitional exhaustion. Maximal oxygen uptake (VO2max) was identified through breath-by-breath gas exchange analysis.
Supplementation. Subjects were instructed to follow a vegetarian meal (thus free of HCDs) on the evening before the test day and to observe 12 h fasting before their arrival. Once in the laboratory, participants were questioned about the compliance of the dinner meal instruction and once assessed it, they received a HCD-free pre-trial meal, designed with standardized nutritional recommendations to help optimize performance during short-term, high intensity exercise (1.5 g/kg BW carbohydrate intake: white bread, jam, Nutella, butter and Gatorade). They ate their breakfast in a separate room. Two hours after their breakfast and 25 min prior to warm-up, subjects consumed 8 mL/kg BW of CB, thereby administering 46.4 mg/kg BW of HCD, or placebo soup similar in taste but without HCD (vegan soup, flavoured with chicken flavour stock powder from InaPaarman’s). CB intake was scheduled 25 min before the start of the exercise sequence (warm up + time trial) based on Everaert et al.  who found peak plasma anserine concentrations between 24 and 31 min following intake of nutritionally relevant doses of synthesized anserine. Subjects were allowed to drink water ad libitum throughout the experiment. One person accidentally received CB on both occasions, as clearly appeared from the plasma HCD analysis. This subject was therefore excluded from all analysis, resulting in a trial group of 13 subjects.
Time trial. The test was performed using the same SRM cycling ergometer as in the maximal incremental oxygen uptake test. The 10 min warm-up for the 8 min TT consisted of the following sequence: 3 min at 100 W, 3 min at 150 W, 3 min at 200 W, 15 s at 60 W, 15 s at 350 W sprint, 15 s at 60 W, 15 s at 350 W sprint. Following 5 min of rest, the 8 min TT was started, during which subjects were free to choose cadence and speed. Subjects received feedback only on the time using a stopwatch, whilst power and cadence were blind during the TT. Bike was set in a linear mode and the mean power during 8 min (expressed as W/kg) was the primary performance outcome measure. Percentage change between the two conditions was calculated. The performance outcome was checked for order effects. Overall, 7 subjects performed best on the first testday, 5 subjects performed best on the second testday, and 1 subject performed equally well on both testdays, assuring that no order effects were present.
Preparation of CB and placebo soup. The experimental chicken broth was prepared according to Harris et al.  with minor modifications. Fresh chicken breast meat was retrieved from a local international food store (Doha, Qatar), with the meat originating from The Netherlands (Europe). Chicken breast (skinned and boned) was finely chopped and boiled for 25 min with water (1 litre for every 1.5 kg of chicken). Residual chicken meat was removed by course filtration. The filtrate was flavoured by the addition of carrot, onion, celery, salt, pepper, basil, parsley and tomato puree, and re-boiled for a further 20 min and then cooled before final filtration through fine muslin at 4°C. The yield from 1.5 kg chicken in 1litre of water was 870 mL of stock. Placebo soup was prepared using the same flavouring agents and vegetables in boiling water, without adding any meat. Single portions of both CB and placebo soup were prepared according to any participant’s body weight (8 mL mL/kg BW) and frozen in plastic bottles at -80°C until consumption. Each bottle was placed in a closed freezing bag for hygienic conditions. Only the freezing bags containing the bottles were labelled in order to guarantee double-blinding during the test days.
HCD in CB and placebo soup. Carnosine and anserine levels were quantified in both CB and placebo soup preparations through HPLC-fluorescence on two representative samples of each soup, following the method described by Everaert et al. . The carnosine and anserine concentrations of the CB were 8.61 and 16.05 mmol/L respectively and found below the limit of detection in the placebo soup. The subjects thus consumed 1181 ± 156 mg carnosine (15.6 mg/kg BW), 2340 ± 308 mg anserine (30.8 mg/kg BW), totalling 3522 ± 464 mg of HCD (46.4 mg/kg BW) in a portion of 607 ± 80 mL chicken broth (8 mL/kg BW).
Blood gas parameters. Capillary blood samples (70 µL) were taken by finger pricking and analysed with a blood gas analyzer (ABL90 Flex; Radiometer, Brønshøj, Denmark) at five different time points: fasted (at arrival), before warm-up, following 8 min TT, after 5 min and 10 min recovery. The samples were analysed for glucose, pH, lactate, bicarbonate and electrolytes (K+, Ca2+, Cl- and Na+).
Plasma HCD levels. Venous blood samples (EDTA) were collected at three time points: fasted, pre-exercise and 5 min post-exercise recovery. Precooled (4°C) EDTA tubes were centrifuged immediately after blood collection at 4°C to separate plasma. Plasma samples were deproteinized with 35% sulfosalicylic acid (SSA) and stored immediately at -20°C until analysis. These samples were analysed for plasma carnosine and anserine by an in-house developed and validated UPLC-MS/MS method. The analytical standards of L-carnosine and L-anserine were chemically synthesized by Flamma S.p.a. (Chignolo d’Isola, Bergamo, Italy). The internal standard (IS), carnosine-D4, was purchased from Sanbio B.V. (Uden, The Netherlands). Acetonitrile (ACN), methanol and formic acid (FA) were ULC-MS grade and obtained from Biosolve (Valkenswaard, The Netherlands). A Milli-Q® water system (Merck Millipore, Darmstadt, Germany) was used to obtain ultrapure water. Deproteinized plasma was vortexed and 150 µL was added to 240 µL of methanol containing 1% formic acid and 10 µL IS (2.5 µM in water). Samples were vortexed (30 sec) before centrifugation (15 min, 4°C, 15000 g) and 350 µL of supernatant was evaporated (35°C, vacuum, Gyrovap). The remaining droplet of about 20 µL was redissolved in 90 µL ultrapure water and vortexed for 30 sec before being transferred to an autosampler vial (Filterservice, Eupen, Belgium). A 5 µL aliquot was injected onto the UPLC-MS/MS system.
Analyses were performed on an UPLC-MS/MS platform consisting of an Acquity H-Class Quaternary Solvent Manager and Flow-Through-Needle Sample Manager with temperature controlled tray (8 °C) and column oven (45 °C), all from Waters (Milford, MA, USA). Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (100 x 2.1 mm, dp: 1.8 µm, Waters) in combination with an Acquity HSS T3 1.8 µm Vanguard pre-column, both from Waters. Gradient elution was established with a mobile phase consisting of 0.1% (v/v) FA in water (solvent A) and 0.1% (v/v) FA in ACN (solvent B) at a flow rate of 0.4 mL/min. The following gradient was used: 0.0 – 1.5 min (99% A, 1% B), 1.5 – 2.0 min (linear gradient to 5% A, 95 % B), 2.0 – 3.5 min (5% A, 95% B), 3.5 – 4.0 min (linear gradient to 99% A, 1% B), 4.0 – 7.0 min (99% A, 1% B). The UPLC column effluent was interfaced to a Xevo TQ-XSÒ MS/MS system, equipped with an electrospray ionization (ESI) probe operating in the positive mode (all from Waters). A divert valve was used and the UPLC effluent was directed to the mass spectrometer from 0.2 to 3.0 min. Instrument parameters were optimised by direct infusion of working solutions of 100 ng/mL of carnosine, anserine and the IS, respectively, at a flow-rate of 10 µL/min and in combination with the mobile phase (50 % A, 50 % B, flow-rate: 200 µL/min). The settings on the Xevo TQ-XS® were as follows: desolvation gas flow rate: 800 L/h; desolvation temperature: 500°C; cone gas flow rate: 150 L/h; source temperature: 150°C. The capillary voltage was optimized at 3.00 kV for ESI in positive ionization mode. The detector was operating in multiple reacting monitoring mode, scanning the two most intense transitions of carnosine: m/z 227.2 > 110.1 (quantification ion, cone = 35 V; collision energy (CE) = 20 eV ) and m/z 227.2 > 156.1 (confirmation ion, cone = 35 V; CE = 13 eV), anserine: m/z 241.2 > 109.1 (quantification ion, cone = 30 V; CE = 23 eV ) and m/z 241.2 > 170.1 (confirmation ion, cone = 30 V; CE = 15 eV) and carnosine-D4 (IS): m/z 231.0 > 110.1 (quantification ion, cone = 30 V; CE = 22 eV ) and m/z 231.0 > 156.0 (confirmation ion, cone = 30 V; CE = 15 eV). Masslynx software 4.2 (Waters) was used for instrument control and data extraction.
Pooled EDTA plasma used to prepare the calibration curve and quality control samples was obtained from 2 healthy volunteers who followed a lacto-ovo-vegetarian diet free of HCD for 2 days prior to blood withdrawal. This plasma pool contained basal carnosine and anserine concentrations of 23.34 ± 2.78 nmol/L and 7.93 ± 4.40 nmol/L, respectively. For quantification, a 10-point calibration curve was prepared by spiking aliquots of the pooled deproteinized EDTA plasma with known concentrations of carnosine and anserine ranging between 5 and 15000 nmol/L. Calibration curves showed good linearity (r > 0.99 and goodness-of-fit coefficient < 20 %, Table 1). Quality control (QC) was performed by spiking pooled plasma with 50, 500 and 5000 nmol/L of the dipeptides. The results of these QC samples could also be used to evaluate the within-run and between-run precision of the LC-MS/MS method, which fell within the acceptance ranges (Table 2). Calculated limit of detection (LOD) values were 5.72 nmol/L and 10.61 nmol/L for carnosine and anserine, respectively (Table 1). No carry-over of the analytes of interest was observed on the UPLC-MS/MS instrument. Results are expressed as absolute concentrations and the change in carnosine and anserine concentrations in blood due to supplement intake was calculated by subtracting baseline values from the 5 min recovery values.
Glutathione levels. Heparin venous blood samples to determine glutathione levels were collected at arrival (fasted), before warm-up and following exercise. Heparinized blood was treated with BPDS (bathophenanthrolinedisulfonic acid disodium salt hydrate solution) 1 mM and after centrifugation, 300 µL red blood cells (RBC) were stored at -20°C until further analysis. Quantification of reduced and oxidized glutathione forms (GSH and GSSG, respectively) was based on methods previously described by Reed et al.  and Yoshida . In short, the derivatization procedure includes the reaction of iodoacetic acid with thiols to form S-carboxymethyl derivatives followed by chromophore derivatization of primary amines with Sanger’s reagent, 2,4-dinitrofluorobenzene at pH 8-9 overnight at 4°C. Samples were centrifuged, filtered (cellulose syringe filter of 0.20 µm) and transferred to a HPLC vial. Derivatives were separated on a 3-aminopropyl column (EC250/4.6 Nucleosil 120-7 NH2; C18 4.6 x 150 mm, 5µm) by reversed-phase ion-exchange HPLC (Agilent 1200 series). The separation was carried out at 1.50 mL/min and 40°C. The eluted derivatives were measured by detection at 365 nm (DAD detector). GSH and GSSG were identified by retention times of authentic internal and external standards.
Plasma CN1 activity. CN1 activity was determined on heparinized plasma from the fasted state of the placebo supplementation test day, according to the method described by Teufel et al. . Briefly, the reaction was initiated by addition of substrate (L-carnosine) to a heparinized plasma sample and stopped after 10 min of incubation at 37°C by adding 1% sulfosalicylic acid (SSA). Liberated histidine was derivatized with o-phthaldialdehyde, and the maximum increase was used for determining the maximum activity. Fluorescence was measured by excitation at 360 nm and emission at 460 nm.
Statistics. A paired T-test was used to evaluate the effect of CB or placebo ingestion on mean power output of the 8 min TT. A 2 x 3 repeated measures analysis of variance ANOVA was used to evaluate plasma carnosine and anserine levels and glutathione levels in RBC with condition (CB or placebo) and time (fasted, pre-exercise and 5 min recovery) as within factors. A 2 x 5 repeated-measures ANOVA was used to evaluate glucose, pH, lactate, bicarbonate and electrolytes with condition (CB or placebo) and time (fasted, pre-exercise, post-exercise, 5 min recovery, 10 min recovery) as within factors. In case of a significant interaction, a Tukey post hoc analysis was performed. In case of missing values, a mixed-effects analysis was used. Correlations between CN1 activity and performance output were obtained by means of Pearson correlations. All statistical analyses were performed with Graphpad Prism version 8.0. Values are presented as mean ± SD and statistical significance threshold was set at p ≤ 0.05.