Partly Substituting Whey for Collagen Peptide Supplementation Improves Neither Indices of Muscle Damage Nor Recovery of Functional Capacity During Eccentric Exercise Training in Fit Males.

Previous studies showed that collagen peptide supplementation along with resistance exercise enhance muscular recovery and function. Yet, the efficacy of collagen peptide supplementation in addition to standard nutritional practices in athletes remains unclear. Therefore, the objective of the study was to compare the effects of combined collagen peptide (20 g) and whey protein (25 g) supplementation with a similar daily protein dose (45 g) of whey protein alone on indices of muscle damage and recovery of muscular performance during eccentric exercise training. Young fit males participated in a 3-week training period involving unilateral eccentric exercises for the knee extensors. According to a double-blind, randomized, parallel-group design, before and after training, they received either whey protein (n = 11) or whey protein + collagen peptides (n = 11). Forty-eight hours after the first training session, maximal voluntary isometric and dynamic contraction of the knee extensors were transiently impaired by ∼10% (Ptime < .001) in whey protein and whey protein + collagen peptides, while creatine kinase levels were doubled in both groups (Ptime < .01). Furthermore, the training intervention improved countermovement jump performance and maximal voluntary dynamic contraction by respectively 8% and 10% (Ptime < .01) and increased serum procollagen type 1N-terminal peptide concentration by 10% (Ptime < .01). However, no differences were found for any of the outcomes between whey and whey protein + collagen peptides. In conclusion, substituting a portion of whey protein for collagen peptide, within a similar total protein dose, improved neither indices of eccentric muscle damage nor functional outcomes during eccentric training.

Exogenous ketosis elevates circulating erythropoietin and stimulates muscular angiogenesis during endurance training overload.

De novo capillarization is a primary muscular adaptation to endurance exercise training and is crucial to improving performance. Excess training load, however, impedes such beneficial adaptations, yet we recently demonstrated that such downregulation may be counteracted by ketone ester ingestion (KE) post-exercise. Therefore, we investigated whether KE could increase pro-angiogenic factors and thereby stimulate muscular angiogenesis during a 3-week endurance training-overload period involving 10 training sessions/week in healthy, male volunteers. Subjects received either 25 g of a ketone ester (KE, n = 9) or a control drink (CON, n = 9) immediately after each training session and before sleep. In KE, but not in CON, the training intervention increased the number of capillary contacts and the capillary-to-fibre perimeter exchange index by 44% and 42%, respectively. Furthermore, KE also substantially increased vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression both at the protein and at the mRNA level. Serum erythropoietin concentration was concomitantly increased by 26%. Conversely, in CON the training intervention increased only the protein content of eNOS. These data indicate that intermittent exogenous ketosis during endurance overload training stimulates muscular angiogenesis. This likely resulted from a direct stimulation of muscle angiogenesis, which may be at least partly due to stimulation of erythropoietin secretion and elevated VEGF activity, and/or an inhibition of the suppressive effect of overload training on the normal angiogenic response to training. This study provides novel evidence to support the potential of exogenous ketosis to benefit endurance training-induced muscular adaptation. KEY POINTS: Increased capillarization is a primary muscular adaptation to endurance exercise training. However, excess training load may impede such response. We previously observed that intermittent exogenous ketosis by post-exercise and pre-sleep ketone ester ingestion (KE) counteracted physiological dysregulations induced by endurance overload training. Therefore, we investigated whether KE could increase pro-angiogenic factors thereby stimulating muscular angiogenesis during a 3-week endurance training overload period. We show that the overload training period in the presence, but not in the absence, of KE markedly increased muscle capillarization (+40%). This increase was accompanied by higher circulating erythropoietin concentration and stimulation of the pro-angiogenic factors vascular endothelial growth factor and endothelial nitric oxide synthase in skeletal muscle. Collectively, our data indicate that intermittent exogenous ketosis may evolve as a potent nutritional strategy to facilitate recovery from strenuous endurance exercise, thereby stimulating beneficial muscular adaptations.

Exogenous ketosis increases blood and muscle oxygenation but not performance during exercise in hypoxia.

Available evidence indicates that elevated blood ketones are associated with improved hypoxic tolerance in rodents. From this perspective, we hypothesized that exogenous ketosis by oral intake of the ketone ester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE) may induce beneficial physiological effects during prolonged exercise in acute hypoxia. As we recently demonstrated KE to deplete blood bicarbonate, which per se may alter the physiological response to hypoxia, we evaluated the effect of KE both in the presence and absence of bicarbonate intake (BIC). Fourteen highly trained male cyclists performed a simulated cycling race (RACE) consisting of 3-h intermittent cycling (IMT180') followed by a 15-min time-trial (TT15') and an all-out sprint at 175% of lactate threshold (SPRINT). During RACE, fraction of inspired oxygen ([Formula: see text]) was gradually decreased from 18.6% to 14.5%. Before and during RACE, participants received either 1) 75 g of ketone ester (KE), 2) 300 mg/kg body mass bicarbonate (BIC), 3) KE + BIC, or 4) a control drink in addition to 60 g of carbohydrates/h in a randomized, crossover design. KE counteracted the hypoxia-induced drop in blood ([Formula: see text]) and muscle oxygenation by ∼3%. In contrast, BIC decreased [Formula: see text] by ∼2% without impacting muscle oxygenation. Performance during TT15' and SPRINT were similar between all conditions. In conclusion, KE slightly elevated the degree of blood and muscle oxygenation during prolonged exercise in moderate hypoxia without impacting exercise performance. Our data warrant to further investigate the potential of exogenous ketosis to improve muscular and cerebral oxygenation status, and exercise tolerance in extreme hypoxia.

Voluntary exercise does not improve muscular properties or functional capacity during C26-induced cancer cachexia in mice.

Exercise training is considered as a potential intervention to counteract muscle degeneration in cancer cachexia. However, evidence to support such intervention is equivocal. Therefore, we investigated the effect of exercise training, i.e. voluntary wheel running, on muscle wasting, functional capacity, fiber type composition and vascularization during experimental cancer cachexia in mice. Balb/c mice were injected with PBS (CON) or C26 colon carcinoma cells to induce cancer cachexia (C26). Mice had free access to a running wheel in their home cage (CONEX and C26EX, n = 8-9) or were sedentary (CONS and C26S, n = 8-9). Mice were sacrificed 18 days upon tumor cell injection. Immunohistochemical analyes were performed on m. gastrocnemius and quadriceps, and ex vivo contractile properties were assessed in m. soleus and extensor digitorum longus (EDL). Compared with CON, C26 mice exhibited body weight loss (~ 20 %), muscle atrophy (~ 25 %), reduced grip strength (~ 25 %), and lower twitch and tetanic force (~ 20 %) production in EDL but not in m. soleus. Furthermore, muscle of C26 mice were characterizd by a slow-to-fast fiber type shift (type IIx fibers: +57 %) and increased capillary density (~ 30 %). In C26 mice, wheel running affect neither body weight loss, nor muscle atrophy or functional capacity, nor inhibited tumor growth. However, wheel running induced a type IIb to type IIa fiber shift in m. quadriceps from both CON and C26, but not in m. gastrocnemius. Wheel running does not exacerbate muscular degeneration in cachexic mice, but, when voluntary, is insufficient to improve the muscle phenotype.

Ketone ester supplementation blunts overreaching symptoms during endurance training overload.

KEY POINTS: Overload training is required for sustained performance gain in athletes (functional overreaching). However, excess overload may result in a catabolic state which causes performance decrements for weeks (non-functional overreaching) up to months (overtraining). Blood ketone bodies can attenuate training- or fasting-induced catabolic events. Therefore, we investigated whether increasing blood ketone levels by oral ketone ester (KE) intake can protect against endurance training-induced overreaching. We show for the first time that KE intake following exercise markedly blunts the development of physiological symptoms indicating overreaching, and at the same time significantly enhances endurance exercise performance. We provide preliminary data to indicate that growth differentiation factor 15 (GDF15) may be a relevant hormonal marker to diagnose the development of overtraining. Collectively, our data indicate that ketone ester intake is a potent nutritional strategy to prevent the development of non-functional overreaching and to stimulate endurance exercise performance. ABSTRACT: It is well known that elevated blood ketones attenuate net muscle protein breakdown, as well as negate catabolic events, during energy deficit. Therefore, we hypothesized that oral ketones can blunt endurance training-induced overreaching. Fit male subjects participated in two daily training sessions (3 weeks, 6 days/week) while receiving either a ketone ester (KE, n = 9) or a control drink (CON, n = 9) following each session. Sustainable training load in week 3 as well as power output in the final 30 min of a 2-h standardized endurance session were 15% higher in KE than in CON (both P < 0.05). KE inhibited the training-induced increase in nocturnal adrenaline (P < 0.01) and noradrenaline (P < 0.01) excretion, as well as blunted the decrease in resting (CON: -6 ± 2 bpm; KE: +2 ± 3 bpm, P < 0.05), submaximal (CON: -15 ± 3 bpm; KE: -7 ± 2 bpm, P < 0.05) and maximal (CON: -17 ± 2 bpm; KE: -10 ± 2 bpm, P < 0.01) heart rate. Energy balance during the training period spontaneously turned negative in CON (-2135 kJ/day), but not in KE (+198 kJ/day). The training consistently increased growth differentiation factor 15 (GDF15), but ∼2-fold more in CON than in KE (P < 0.05). In addition, delta GDF15 correlated with the training-induced drop in maximal heart rate (r = 0.60, P < 0.001) and decrease in osteocalcin (r = 0.61, P < 0.01). Other measurements such as blood ACTH, cortisol, IL-6, leptin, ghrelin and lymphocyte count, and muscle glycogen content did not differentiate KE from CON. In conclusion, KE during strenuous endurance training attenuates the development of overreaching. We also identify GDF15 as a possible marker of overtraining.

Effect of Stacked Sodium Bicarbonate Loading on Repeated All-out Exercise.

The purpose of this study was to evaluate whether NaHCO3, administered via a 9-h stacked loading protocol (i.e. repeated supplementation with small doses in order to obtain a gradual increase in blood [HCO3 -]), has an ergogenic effect on repeated all-out exercise. Twelve physically active males were randomly assigned to receive either NaHCO3 (BIC) or placebo (PL) in a double-blind cross-over design. NaHCO3 supplementation was divided in three identical 3-h cycles: a 6.3 g bolus at the start, followed by 2.1 g doses at +1-h and +2-h, yielding a total NaHCO3 intake of 0.4 g·kg-1 BM over 9-h. At the end of each cycle, participants performed 2-min all-out cycling. Capillary blood samples were analyzed for [HCO3 -], pH and [La-]. Pre-exercise blood [HCO3 -] in PL decreased from 25.6±0.2 mmol·L-1 in bout 1 to 23.6±0.2 mmol·L-1 in bout 4, while increasing from 25.5±0.2 to 31.2±0.4 mmol·L-1 in BIC (P<0.05). Concomitantly, pre-exercise pH values gradually decreased in PL (from 7.41±0.00 to 7.39±0.01) and increased in BIC (from 7.41±0.01 to 7.47±0.01; P<0.05). Mean power output of the four bouts was higher in BIC (428±20 W) than in PL (420±20 W; P<0.05). The ergogenic effect on repeated all-out exercise occurred in the absence of gastrointestinal distress.

High-intensity interval training in hypoxia does not affect muscle HIF responses to acute hypoxia in humans.

PURPOSE: The myocellular response to hypoxia is primarily regulated by hypoxia-inducible factors (HIFs). HIFs thus conceivably are implicated in muscular adaptation to altitude training. Therefore, we investigated the effect of hypoxic versus normoxic training during a period of prolonged hypoxia ('living high') on muscle HIF activation during acute ischaemia. METHODS: Ten young male volunteers lived in normobaric hypoxia for 5 weeks (5 days per week, ~ 15.5 h per day, FiO2: 16.4-14.0%). One leg was trained in hypoxia (TRHYP, 12.3% FiO2) whilst the other leg was trained in normoxia (TRNOR, 20.9% FiO2). Training sessions (3 per week) consisted of intermittent unilateral knee extensions at 20-25% of the 1-repetition maximum. Before and after the intervention, a 10-min arterial occlusion and reperfusion of the leg was performed. Muscle oxygenation status was continuously measured by near-infrared spectroscopy. Biopsies were taken from m. vastus lateralis before and at the end of the occlusion. RESULTS: Irrespective of training, occlusion elevated the fraction of HIF-1α expressing myonuclei from ~ 54 to ~ 64% (P < 0.05). However, neither muscle HIF-1α or HIF-2α protein abundance, nor the expression of HIF-1α or downstream targets selected increased in any experimental condition. Training in both TRNOR and TRHYP raised muscular oxygen extraction rate upon occlusion by ~ 30%, whilst muscle hyperperfusion immediately following the occlusion increased by ~ 25% in either group (P < 0.05). CONCLUSION: Ten minutes of arterial occlusion increased HIF-1α-expressing myonuclei. However, neither normoxic nor hypoxic training during 'living high' altered muscle HIF translocation, stabilisation, or transcription in response to acute hypoxia induced by arterial occlusion.

Acute environmental hypoxia induces LC3 lipidation in a genotype-dependent manner.

Hypoxia-induced muscle wasting is a phenomenon often described with prolonged stays at high altitude, which has been attributed to altered protein metabolism. We hypothesized that acute normobaric hypoxia would induce a negative net protein balance by repressing anabolic and activating proteolytic signaling pathways at rest and postexercise and that those changes could be partially genetically determined. Eleven monozygotic twins participated in an experimental trial in normoxia and hypoxia (10.7% O2). Muscle biopsy samples were obtained before and after a 20-min moderate cycling exercise. In hypoxia at rest, autophagic flux was increased, as indicated by an increased microtubule-associated protein 1 light chain 3 type II/I (LC3-II/I) ratio (+25%) and LC3-II expression (+60%) and decreased p62/SQSTM1 expression (-25%; P<0.05), whereas exercise reversed those changes to a level similar to that with normoxia except for p62/SQSTM1, which was further decreased (P<0.05). Hypoxia also increased Bnip3 (+34%) and MAFbx (+18%) mRNA levels as well as REDD1 expression (+439%) and AMP-activated protein kinase phosphorylation (+22%; P<0.05). Among the molecular responses to hypoxia and/or exercise, high monozygotic similarity was found for REDD1, LC3-II, and LC3-II/I (P<0.05). Our results indicate that environmental hypoxia modulates protein metabolism at rest and after moderate exercise by primarily increasing markers of protein breakdown and, more specifically, markers of the autophagy-lysosomal system, with a modest genetic contribution.

A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

Androgens have well-established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell-specific AR-knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total-limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ∼10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (-52%). Thus, muscle AR is involved in fiber-type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen-responsive genes (6-fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn-knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.-Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan-Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

Acute systemic insulin intolerance does not alter the response of the Akt/GSK-3 pathway to environmental hypoxia in human skeletal muscle.

PURPOSE: To investigate how acute environmental hypoxia regulates blood glucose and downstream intramuscular insulin signaling after a meal in healthy humans. METHODS: Fifteen subjects were exposed for 4 h to normoxia (NOR) or to normobaric hypoxia (HYP, FiO2 = 0.11) in a randomized order 40 min after consumption of a high glycemic meal. A muscle biopsy from m. vastus lateralis and a blood sample were taken before (T0), after 1 h (T60) and 4 h (T240) in NOR or HYP and blood glucose levels were measured before exposure and every 30 min. RESULTS: In HYP, blood glucose was reduced 100 min (110.1 ± 5.4 in NOR vs 89.5 ± 4.7 mg dl(-1) in HYP) and 130 min (98.7 ± 3.8 in NOR vs 85.6 ± 4.9 mg dl(-1) in HYP) after completion of a meal, which resulted in an 83 % lower AUC in HYP compared to NOR (p = 0.006). This coincided with 40 % lower GLUT4 protein in the cytosolic fraction (p = 0.013) and a tendency to increase in the crude membrane fraction (p = 0.070) in HYP compared to NOR. At T240, blood glucose concentration was similar between HYP and NOR, whereas plasma insulin as well as phosphorylation of muscle Akt and GSK-3 was ~2-fold higher in HYP compared to NOR (p < 0.05). In contrast, Rac1 protein was less abundant in the membrane fraction in HYP compared to NOR (p = 0.003), reflecting lower activation. CONCLUSION: Acute environmental hypoxia initially reduced blood glucose response to a meal, possibly via an increase in GLUT4 abundance at the sarcolemmal membrane. Later on, whole body insulin intolerance developed independently of defects in conventional insulin signaling in skeletal muscle.

Cyclist drag in team pursuit: influence of cyclist sequence, stature, and arm spacing.

In team pursuit, the drag of a group of cyclists riding in a pace line is dependent on several factors, such as anthropometric characteristics (stature) and position of each cyclist as well as the sequence in which they ride. To increase insight in drag reduction mechanisms, the aerodynamic drag of four cyclists riding in a pace line was investigated, using four different cyclists, and for four different sequences. In addition, each sequence was evaluated for two arm spacings. Instead of conventional field or wind tunnel experiments, a validated numerical approach (computational fluid dynamics) was used to evaluate cyclist drag, where the bicycles were not included in the model. The cyclist drag was clearly dependent on his position in the pace line, where second and subsequent positions experienced a drag reduction up to 40%, compared to an individual cyclist. Individual differences in stature and position on the bicycle led to an intercyclist variation of this drag reduction at a specific position in the sequence, but also to a variation of the total drag of the group for different sequences. A larger drag area for the group was found when riding with wider arm spacing. Such numerical studies on cyclists in a pace line are useful for determining the optimal cyclist sequence for team pursuit.

Disruption of skeletal muscle mitochondrial network genes and miRNAs in amyotrophic lateral sclerosis.

Skeletal muscle mitochondrial dysfunction is believed to play a role in the progression and severity of amyotrophic lateral sclerosis (ALS). The regulation of transcriptional co-activators involved in mitochondrial biogenesis and function in ALS is not well known. When compared with healthy control subjects, patients with ALS, but not neurogenic disease (ND), had lower levels of skeletal muscle peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA and protein and estrogen-related receptor-α (ERRα) and mitofusin-2 (Mfn2) mRNA. PGC-1ß, nuclear respiratory factor-1 (NRF-1) and Mfn1 mRNA as well as cytochrome C oxidase subunit IV (COXIV) mRNA and protein were lower in patients with ALS and ND. Both patient groups had reductions in citrate synthase and cytochrome c oxidase activity. Similar observations were made in skeletal muscle from transgenic ALS G93A transgenic mice. In vitro, PGC-1α and PGC-1ß regulated Mfn1 and Mfn2 in an ERRα-dependent manner. Compared to healthy controls, miRNA 23a, 29b, 206 and 455 were increased in skeletal muscle of ALS patients. miR-23a repressed PGC-1α translation in a 3' UTR dependent manner. Transgenic mice over expressing miR-23a had a reduction in PGC-1α, cytochome-b and COXIV protein levels. These results show that skeletal muscle mitochondrial dysfunction in ALS patients is associated with a reduction in PGC-1α signalling networks involved in mitochondrial biogenesis and function, as well as increases in several miRNAs potentially implicated in skeletal muscle and neuromuscular junction regeneration. As miR-23a negatively regulates PGC-1α signalling, therapeutic inhibition of miR-23a may be a strategy to rescue PGC-1α activity and ameliorate skeletal muscle mitochondrial function in ALS.

Effects of high altitude and cold air exposure on airway inflammation in patients with asthma.

AIMS: Eighteen patients with asthma were evaluated during preparation to climb to extreme altitude in order to study the effects of low fractional inspired oxygen (FiO(2)), prolonged exposure to cold air and high altitude on lung function, asthma control and airway inflammation. METHODS: Spirometry and airway inflammation (fractional exhaled nitric oxide (FeNO) and induced sputum) were studied under different test conditions: hypoxic (FiO(2)=11%) exercise test, 24-hour cold exposure (-5°C) and before, during and after an expedition that involved climbing the Aconcagua mountain (6965 m). RESULTS: Forced expiratory volume in 1 s (FEV(1)) and FeNO values were slightly lower (p<0.04) after 1 h of normobaric hypoxia. FEV(1) decreased (p=0.009) after 24-hour cold exposure, accompanied by increased sputum neutrophilia (p<0.01). During the expedition FEV(1) and forced vital capacity decreased (maximum FEV(1) decrease of 12.3% at 4300 m) and asthma symptoms slightly increased. After the expedition the Asthma Control Test score and prebronchodilator FEV(1) were reduced (p<0.02), sputum neutrophil count was increased (p=0.04) and sputum myeloperoxidase levels, sputum interleukin 17 mRNA, serum and sputum vascular endothelial growth factor A levels were significantly higher compared with baseline. Patients with asthma with the lowest oxygen saturation during the hypoxic exercise test were more prone to develop acute mountain sickness. CONCLUSIONS: Exposure to environmental conditions at high altitude (hypoxia, exercise, cold) was associated with a moderate loss of asthma control, increased airway obstruction and neutrophilic airway inflammation. The cold temperature is probably the most important contributing factor as 24-hour cold exposure by itself induced similar effects.

Exogenous Ketosis Improves Sleep Efficiency and Counteracts the Decline in REM Sleep after Strenuous Exercise.

INTRODUCTION: Available evidence indicates that ketone bodies may improve sleep quality. Therefore, we determined whether ketone ester (KE) intake could counteract sleep disruptions induced by strenuous exercise. METHODS: Ten well-trained cyclists with good sleep quality participated in a randomized crossover design consisting of two experimental sessions each involving a morning endurance training and an evening high-intensity interval training ending 1 h before sleep, after which polysomnography was performed overnight. Postexercise and 30 min before sleeping time, subjects received either 25 g of KE (EX KE ) or a placebo drink (EX CON ). A third session without exercise but with placebo supplements (R CON ) was added to evaluate the effect of exercise per se on sleep. RESULTS: Blood d -ß-hydroxybutyrate concentrations transiently increased to ~3 mM postexercise and during the first part of the night in EX KE but not in EX CON or R CON . Exercise significantly reduced rapid eye movement sleep by 26% ( P = 0.001 vs R CON ) and increased wakefulness after sleep onset by 95% ( P = 0.004 vs R CON ). Interestingly, KE improved sleep efficiency by 3% ( P = 0.040 vs EX CON ) and counteracted the exercise-induced decrease in rapid eye movement sleep ( P = 0.011 vs EX CON ) and the increase in wakefulness after sleep onset ( P = 0.009 vs EX CON ). This was accompanied by a KE-induced increase in dopamine excretion ( P = 0.033 vs EX CON ), which plays a pivotal role in sleep regulation. In addition, exercise increased sleep spindle density by 36% ( P = 0.005 vs R CON ), suggesting an effect on neural plasticity processes during sleep. CONCLUSIONS: These data indicate that KE ingestion improves sleep efficiency and quality after high-intensity exercise. We provide preliminary evidence that this might result from KE-induced increases in dopamine signaling.

Exogenous ketosis increases circulating dopamine concentration and maintains mental alertness in ultra-endurance exercise.

Exogenous ketosis can improve psychocognitive functioning during exercise as well as stimulate postexercise muscular recovery. Therefore, we hypothesized that ketone ester (KE) supplementation can counteract the decline in psychocognitive functioning during ultra-endurance exercise and stimulate muscular recovery. Eighteen recreational runners participated in a full 100 km trail run (RUN, n = 8), or ran to premature exhaustion (80 km: n = 6; 60 km: n = 4). Before (25 g), during (25 g·h-1), and after (5 × 25 g in 24 h) RUN they received ketone ester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE, n = 9) supplements or a noncaloric placebo (CON, n = 9). Blood samples and muscle biopsies were taken, and mental alertness was assessed by a psychocognitive test battery at different times before, during, and up to 36 h after RUN. Compared with CON (<0.3 mM), in KE blood d-ß-hydroxybutyrate concentration was consistently elevated to ∼2-3 mM during RUN. In CON, RUN increased visual reaction times from 353 ± 53 to 419 ± 54 ms, and movement execution times from 174 ± 47 to 245 ± 64 ms. But this effect was fully negated by KE (P < 0.05). Plasma dopamine concentrations doubled in KE during RUN but remained stable in CON, resulting in higher concentrations after RUN in KE (4.1 ± 1.7 nM) than in CON (2.4 ± 0.8 nM, P = 0.048). KE also inhibited muscular infiltration of macrophages and suppressed AMPK phosphorylation status until 36-h postexercise (P < 0.05 KE vs. CON). In conclusion, KE increases circulating dopamine concentration and improves mental alertness, as well as improves postexercise muscular inflammation in ultra-endurance exercise.NEW & NOTEWORTHY Oral ketone ester ingestion elevates circulating dopamine concentration during ultra-endurance exercise. This is associated with improved mental alertness. Furthermore, ketone ester intake inhibits postexercise skeletal muscle macrophage infiltration, and counteracts the increase in AMPK phosphorylation after exercise, which indicates improved muscular energy status.

Simultaneous determination of nitrite and nitrate in human plasma by on-capillary preconcentration with field-amplified sample stacking.

A simple method for the determination of nitrite and nitrate in human plasma has been developed using CZE with minimal sample preparation. Field-amplified sample stacking (FASS) was used to achieve submicromolar detection by dilution of the plasma sample with deionized water. In CZE, the separation of nitrite and nitrate was achieved within 10 min without adding EOF modifier. The optimal condition was achieved with 50 mM phosphate buffer at pH 9.3. The ninefold diluted plasma samples were injected hydrodynamically for 40 s into a 60 cm×75 µm id uncoated fused-silica capillary. The separation voltage was 20 kV (negative potential) and UV detection was performed at 214 nm. The linearity curves for nitrite and nitrate were obtained by the standard addition method. The estimated LODs for nitrite and nitrate in ninefold diluted plasma sample were 0.05 and 0.07 µM, respectively. The LODs for nitrite and nitrate in original plasma samples were 0.45 and 0.63 µM. The intra- and inter-day precisions for both analytes were <2.6% and the recovery ranged between 92.3 and 113.3%. It was found that nitrite was more stable than nitrate in the plasma after the sample preparation. This proposed method was applied to a number of human plasma samples and the measured nitrite and nitrate concentrations in human plasma were consistent with the literature ranges.