CF and exercise-induced oxidative stress and inflammation
A total of 6 studies looked into the effects of CF supplementation and exercise on parameters of oxidative stress (Wiswedel et al. 2004; Fraga et al. 2005; Singh et al. 2006; Allgrove et al. 2011; Davison et al. 2012; Taub et al. 2016). Two studies analysed the effects of acute intake of CF, 2 h prior to an exercise bout. Davison et al. (2012) found that a single supplementation of 248 mg CF (39.1 mg epicatechin) resulted in increased total antioxidant status (TAS) pre-exercise compared to baseline or to PL in trained men. Moreover, the increase in free F2-isoprostanes after prolonged steady state (SS) exercise (2.5 h cycling at 60% VO2max) tended to be lower due to CF intake. The exercise-induced increase in vitamin C was not different between the two trials. Wiswedel et al. (2004) found that the acute intake of 186 mg CF (no details on epicatechin) tempered the increase in F2-isoprostane and relative MDA after 29 min cycling in healthy untrained men, while they did not observe any differences in TAC. Allgrove et al. (2011) studied the effects of a 2-week intake of 40 g of dark chocolate (38.7 mg epicatechin) or PL in 20 male participants. On the testing day, participants consumed a ‘double dose’ 2 h before exercise. Exercise consisted of a SS cycling exercise at 60% VO2max for 1.5 hr, in which every 10 min the exercise intensity increased until 90% VO2max for 3s, followed by a time-to-exhaustion trial (90% VO2max). CF intake caused a significant smaller increase in (free) F2-isoprostane, oxidized low-density lipoproteins (LDLs) and F2-isoprostane after the time-to-exhaustion trial and 1 h post-recovery compared to PL. Singh et al. (2006) examined the effects of 7-day CF (240 mg CF) or PL supplementation in 8 healthy trained and 8 healthy untrained participants. They did not find any differences between CF and PL in TAS, before 1 h of cycling exercise at 70% VO2max. Fraga et al. (2005) examined the combined effects of 2-week CF (or PL) intake (186 mg CF or <5 mg CF) and exercise training (3x football/week) in 28 healthy male football players on oxidative stress. This study demonstrated a 12% decrease in MDA after 14 days of CF intake compared to baseline, in contrast with a 10% increase in MDA in the PL trial. Moreover, after 2 week CF intake, urate was decreased and vitamin E/cholesterol and β-carotene were increased, while these parameters did not change after PL intake. However, no difference between PL and CF was found for plasma TAC, vitamin E, lycopene and co-enzyme Q10. In the study of Taub et al. (2016), 3 months of CF intake (175 mg CF, 26 mg epicatechin) resulted in an increased ratio of reduced vs. oxidized gluthathione (GSH:GSSG) and decreased protein carbonylation in the M. vastus lateralis compared to PL in untrained men at rest. Only the results of the study of Taub et al. showed a large effect size, while all other reported improvements had small effects. Only 2 studies examined the effect of CF intake on exercise-induced inflammation. Neither the study of Allgrove et al. (2011) nor the study of Davison et al. (2012), found effects of (acute nor sub-chronic) CF supplementation on exercise-induced increases in leukocyte count, neutrophil count, plasma IL-6 and IL-10 concentrations.
CF, exercise and vascular function
Six studies examined the effects of CF and exercise on vascular function using different techniques in obese and healthy participants: (i) BP (Fraga et al. 2005; Davison et al. 2008; Berry et al. 2010; Patel et al. 2015), (ii) Flow mediated dilation (FMD) (Davison et al. 2008; Berry et al. 2010) and (iii) platelet count and volume (Singh et al. 2006; Soleimani et al. 2013). The acute intake of CF (701 mg, 139 mg epicatechin) increased FMD and tempered the exercise-induced increase in mean BP (by 14%) and diastolic BP (by 68%) compared to PL in 21 overweight or obese men and women (Berry et al. 2010). Davison et al. (2008) looked at the concomitant effects of 12-week exercise training and CF supplementation on FMD and BP in overweight or obese participants. The four interventional groups included in this study were (i) CF (=902 mg), (ii) CF + exercise (12 weeks, 3 x 45 min at 75% of the predicted HRmax), (iii) PL (=36 mg); and (iv) PL + exercise. Exercise alone neither improved vascular function nor decreased BP. In contrast, 12-week CF intake resulted in a decreased diastolic BP and mean BP and enhanced vascular function (increase in FMD (1.6%)), while exercise training did not augment this effect.
The acute intake of 5 mg/kg CF (~360 mg) 2 h prior to an exhaustive ‘Bruce aerobic exercise test’ decreased blood platelets, mean platelet volume (MPV) and platelet distribution width (PDW) in healthy, young, male soccer players compared to PL (Soleimani et al. 2013). On the other hand, no beneficial effects of 7-day CF intake (240 mg) on acute exercise-induced changes in total platelet count and MPV were found in 16 healthy men (8 trained; 8 untrained) (Singh et al. 2006). The effect sizes of the beneficial influence of CF intake on BP, FMD, cerebral blood flow and platelet function reported in the studies of Berry, Davison, Fraga and Soleimani were large.
CF, exercise and carbohydrate and lipoprotein metabolism
Five studies examined lipid and/or carbohydrate metabolism in response to exercise and CF intake. Two studies examined the acute and sub-chronic effects of CF on carbohydrate and fat metabolism during exercise (Allgrove et al. 2011; Stellingwerff et al. 2014). Two studies examined the effects of a combined intervention of CF intake and exercise training on body composition and carbohydrate metabolism at rest in well-trained and untrained participants (Fraga et al. 2005; Davison et al. 2008). The study of Taub et al. (2012) examined the effect of 3-month CF intake on skeletal muscle metabolic changes at rest (prior to a cycling test). Stellingwerff et al. (2014) found significant changes in carbohydrate and fat metabolism during an acute exercise, consisting of a cycling SS (2.5 h) and 15’ TT, after acute CF intake (262 mg CF, 89 mg epicatechin) compared to low CF intake. These authors demonstrated an increase in glucose concentration and decrease in glucagon during exercise, with a concomitant increase in insulin concentration during recovery. However, the effect sizes were small. Allgrove et al. (2011) found a 21% increase in free fatty acids (FFA) during exercise after a 2-week CF intake (98.7 mg CF, 77.4 mg epicatechin) compared to PL in healthy participants. Levels of glucose, insulin, glucagon or cortisol were not affected. Two studies examined the effects of sub-chronic CF intake in combination with exercise training. Fraga et al. (2005) found that 2-week CF intake (186 mg) in combination with football training decreased cholesterol and LDL cholesterol, while this effect was not evident after PL intake. However, the effect size was small. In a population of overweight participants, Davison et al. (2012) found that 2-week CF supplementation in combination with or without exercise training (3 x 45 min per week) improved insulin resistance. Exercise alone led to increased fat oxidation and decreased abdominal body fat, while CF intake did not augment these effects and did not affect these parameters in the absence of exercise. In contrast, insulin resistance was lowered by CF intake, but this improvement was not altered by concomitant exercise training.
Taub et al. (2016) aimed to investigate the effects of 3-month CF intake, without exercise training, on cardio metabolic changes, skeletal muscle metabolic changes and exercise performance in sedentary untrained participants. Three-month CF intake (175 mg CF, 26 mg epicatechin) resulted in increased high density lipoprotein (HDL) cholesterol and decreased triglycerides (TG), while glucose, cholesterol, LDL, CRP and Hemoglobin-A1c (HBA1c) were not altered (Taub et al. 2016). Moreover, the authors found that the expression and activation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and its upstream regulators AMP-activated protein kinase α (AMPK) and Liver kinase B1 (LKB-1) in the muscle were upregulated by CF intake. CF intake did not affect mitochondrial volume, but increased citrate synthase, a marker of mitochondrial function. These results had a large effect size.
CF and exercise performance and recovery
Seven studies examined the effect of acute (2 h pre-exercise), sub-chronic (2 weeks) or chronic (3 months) CF supplementation on exercise performance in healthy participants (Fraga et al. 2005; Allgrove et al. 2011; Davison et al. 2012; Stellingwerff et al. 2014; Peschek et al. 2014; Patel et al. 2015; Taub et al. 2016). The acute intake of CF (ranging from 246.8-262 mg CF and 89-97 mg epicatechin) 2h pre-exercise did not result in any significant changes in TT performance (15’ TT after 2.5 h SS cycling), HR, RPE, VO2max and RER during SS exercise in trained athletes (Davison et al. 2012; Stellingwerff et al. 2014). Only one randomized, single blinded cross-over study examined the effects of acute CF supplementation (350 mg CF/serving; no details on epicatechin) on exercise recovery by examining a 5-km running TT, 48 h after a downhill running protocol which induced muscle soreness, in a rather small population of 8 healthy endurance trained athletes (Peschek et al. 2014). However, these authors could not find any significant improvements in running performance, whole blood CK-concentration, muscle function and self-perceived muscle soreness. Two-week intake of CF (256 mg CF, 46 mg epicatechin) resulted in an 11% increase of gas exchange threshold compared to the low CF placebo (PL) during a VO2max test in 9 moderate trained healthy men (Patel et al. 2015). Moreover, a 6% increase in 2’ cycling TT performance compared to baseline and PL was shown. However, the authors could not find further significant changes in O2 cost, VO2max, RER and lactate concentrations during the 20’ cycling SS preceding the 2’ TT (Patel et al. 2015). Allgrove et al. (2011) did not detect any changes in HR, RPE, VO2max during a 1.5 h SS and performance on the subsequent time-to-exhaustion test, following 2-week CF intake (98.7 mg CF, 77.2 mg epicatechin). Fraga et al. (2005) did not find any beneficial effects on the VO2max during a shuttle run test in trained football players after 2 weeks of CF intake (186 mg CF, 39 mg epicachin + catechin) in trained athletes. In contrast, chronic intake (3 months) of CF (26 mg epicatechin) increased VO2max and power output during an incremental cycle test in untrained participants (Taub et al. 2016).
Functional NIRS, a non-invasive optical imaging technique, was used to assess acute changes in local cerebral blood volume (reflecting CBF) and oxygenation (Oxymon continuous-wave NIRS (CW-NIRS) system (ArtinisMedical Systems B.V.). Following introduction of near-infrared light through the skull, HbO2 and HHb absorb light at slightly different wavelengths (800-940 nm and 600-750 nm, respectively) allowing the measurement of their relative concentrations in the cerebral blood (Perrey 2008). These concentration changes are the result of the interplay between regional cerebral blood flow, blood volume and metabolic rate of oxygen. Whereas HbO2 and HHb reflect the balance of O2 delivery and extraction, the sum of both, total haemoglobin (Hbtot) is an index of changes in regional blood volume (Oussaidene et al. 2015).
Intrinsically adjusted for the baseline measurement (at the start of application), this system measures concentration changes across a single recording session. Two nominal wavelengths of light (~765 and 855 nm) were emitted with 4 cm distance between an emitter and receptor and the differential path-length factor was adjusted according to the subjects’ age. The modified Beer-Lambert law was used by the software to calculate relative changes in the concentrations of HbO2, HHb and Hbtot (Oussaidene et al. 2015). Data were collected with a sampling frequency of 5 Hz and were down sampled with factor 5 for analysis.
The emitter/receptor optode pair was positioned over the left prefrontal cortical area between Fp1 and F3, according to the modified international EEG 10-20 system (Rupp and Perrey 2008). The NIRS emitter/receptor optodes pair was set up on the prefrontal cortex at baseline and the position was marked. Ninety min after the CF intake, the optode pair was repositioned and was kept in position until the end of the post-exercise CT. A black cloth was placed over the optode pair to prevent interference of external light and a dark elastic band was wrapped around the head to keep the NIRS-optode pair in place. When the NIRS measurement started, subjects sat still without speaking or moving for 2 min (reference measurement). All NIRS measurements until the end of the time trial, were normalized to reflect changes from this 2-min reference measurement to express the magnitudes of changes. Immediately after exercise, subjects sat still without speaking or moving for 2 min once again before the post-exercise CT started (post-exercise reference measurement). Post-exercise NIRS values were normalized to reflect changes from this post-exercise reference measurement. Mean concentration changes (ΔHbO2, ΔHHb and ΔHbtot) were calculated for the two parts of each Stroop task (baseline, pre-exercise and post-exercise CT). ΔHbO2, ΔHHb and ΔHbtot were calculated during the first (“start) and last (“end”) 30 seconds of the TT. The use and limitations of NIRS for monitoring cerebral regional hemodynamics and oxygenation have been extensively reviewed (Rooks et al. 2010).
Blood sampling and determination of serum BDNF concentration
A catheter was placed in the forearm upon arrival at the lab. Venous blood samples were collected at baseline, 90 min after consuming the chocolate drink (pre-exercise) and immediately after the time trial. Blood was collected in 8-ml anticoagulant-free tubes and centrifuged (10 min at 3000 RPM, 4 °C) after 30 min at room temperature to allow clotting to obtain serum. Serum was aliquoted and stored at -80 °C until analysis. Serum BDNF was analysed using a commercially available ELISA kit (ChemiKine® BDNF kit, Millipore®, Temecula,CA, USA). The kit has a detection range from 7.8 pg/mL to 500 pg/mL. Intra-assay and inter-assay variations are ± 3.7% (125 pg/ mL) and ± 8.5% (125 pg/mL) respectively. Data were corrected for changes in plasma volume using the determination of haematocrit and the concentration of haemoglobin according to Dill and Costill (Dill and Costill 1974). Pre- and post-exercise values were normalized to the baseline values and expressed as percentage change to baseline
Table of contents :
Table of contents
Chapter 1. General introduction
1.1 Food supplements
1.2 Nutritional supplements and the brain in sports performance (partly based on: Nutritional supplements and the brain. Meeusen and Decroix 2018)
1.4 Cocoa flavanols
1.4.1 Origin and components
1.4.3 General health benefits
184.108.40.206 Promoting cardiovascular health
220.127.116.11 Neurological effects
18.104.22.168 Other functions
1.4.4 Hypothetical benefits for sports performance
1.5 Purpose of the PhD
1.6 General methods
1.6.1 Cocoa flavanol intervention
1.6.2 Environmental stress: hypoxia
1.6.3 Methods to measure exercise performance
1.6.4 Methods to assess cognitive performance
1.6.5 Methods to study the brain
1.6.6 Methods to measure oxidative stress and NO metabolism
Chapter 2. Cocoa flavanol supplementation and exercise: a systematic review
2.3 Materials & Methods
2.4.1 Study selection and characteristics
2.4.2 CF and exercise-induced oxidative stress and inflammation
2.4.3 CF, exercise and vascular function
2.4.4 CF, exercise and carbohydrate and lipoprotein metabolism
2.4.5 CF and exercise performance and recovery
Chapter 3. Acute cocoa flavanol improves cerebral oxygenation without enhancing executive function at rest or after exercise
3.3 Materials & Methods
3.3.2 Study design
3.3.4 Statistical analyses
3.4.1 Effect of CF and exercise on cognitive function
3.4.2 Effect of CF and exercise on CBF and oxygenation (NIRS)
3.4.3 Effect of CF and exercise on serum BDNF
Chapter 4. Acute cocoa flavanol intake improves cerebral hemodynamics while maintaining brain activity and cognitive performance in moderate hypoxia
4.3 Materials & Methods
4.3.3 Cognitive test
4.3.4 fNIRS measurements
4.3.5 EEG and Sloreta
4.4.1 Physiological measures
4.4.2 Cognitive performance
Chapter 5. The effect of acute cocoa flavanol intake on the bold response and cognitive function in type 1 diabetes: a randomized, placebo-controlled, double-blind cross-over study.
5.3 Materials & Methods
5.3.2 Study design
5.3.3 Nutrition and CF supplementation
5.3.4 Procedure during the hospital visit
5.3.5 Outcome measurements
5.4.1 Cognitive function
Chapter 6. Acute cocoa flavanols intake has minimal effects on exercise-induced oxidative stress and nitric oxide production in healthy cyclists: a randomized controlled trial.
6.3 Materials & Methods
6.3.2 Study design
6.3.4 Sample size calculation and statistical analysis
6.4.1 Epicatechin/catechin serum concentrations
6.4.2 Plasma concentrations of mediators of the NO-pathway
6.4.3 Markers of oxidative stress
6.4.4 Plasma markers of inflammation
6.4.5 Impact of CF intake on exercise performance: TT1 performance and pacing strategy
6.4.6 Impact of CF intake on exercise recovery: performance on TT2
Chapter 7. One week CF intake increases prefrontal cortex oxygenation at rest and during moderate-intensity exercise in normoxia and hypoxia
7.3.2 Study design
7.3.4 The four interventional trials
7.3.6 Statistical analyses
7.4.1 Subject characteristics
7.4.2 Effects of CF intake on (−)-epicatechin and (+)-catechin
7.4.3 Effect of CF intake on NO availability during exercise in H
7.4.5 Exercise tolerance and performance
Chapter 8. General discussion
8.1 Cocoa flavanols, the brain and cognitive performance
8.2 Cocoa flavanols, oxidative stress, NO availability and exercise performance .
8.3 Strengths and limitations of this dissertation
8.4 Guidelines for further research