Running title: Carnosine and β-alanine in exercise and training Word count: 6227 2 Acknowledgements:

Carnosine (beta-alanyl-L-histidine) is present in high concentrations in human skeletal muscles. The oral ingestion of beta-alanine, the rate-limiting precursor in carnosine synthesis, has been shown to elevate the muscle carnosine content both in trained and untrained humans. Little human data exist about the dynamics of the muscle carnosine content, its metabolic regulation, and its dependence on muscle fiber type. The present study aimed to investigate in three skeletal muscle types the supplementation-induced amplitude of carnosine synthesis and its subsequent elimination on cessation of supplementation (washout). Fifteen untrained males participated in a placebo-controlled double-blind study. They were supplemented for 5-6 wk with either 4.8 g/day beta-alanine or placebo. Muscle carnosine was quantified in soleus, tibialis anterior, and medial head of the gastrocnemius by proton magnetic resonance spectroscopy (MRS), before and after supplementation and 3 and 9 wk into washout. The beta-alanine supplementation significantly increased the carnosine content in soleus by 39%, in tibialis by 27%, and in gastrocnemius by 23% and declined post-supplementation at a rate of 2-4%/wk. Average muscle carnosine remained increased compared with baseline at 3 wk of washout (only one-third of the supplementation-induced increase had disappeared) and returned to baseline values within 9 wk at group level. Following subdivision into high responders (+55%) and low responders (+15%), washout period was 15 and 6 wk, respectively. In the placebo group, carnosine remained relatively constant with variation coefficients of 9-15% over a 3-mo period. It can be concluded that carnosine is a stable compound in human skeletal muscle, confirming the absence of carnosinase in myocytes. The present study shows that washout periods for crossover designs in supplementation studies for muscle metabolites may sometimes require months rather than weeks.

The role of the presence of carnosine (β-alanyl-L-histidine) in millimolar concentrations in human skeletal muscle is poorly understood. Chronic oral β-alanine supplementation is shown to elevate muscle carnosine content and improve anaerobic exercise performance during some laboratory tests, mainly in the untrained. It remains to be determined whether carnosine loading can improve single competition-like events in elite athletes. The aims of the present study were to investigate if performance is related to the muscle carnosine content and if β-alanine supplementation improves performance in highly trained rowers. Eighteen Belgian elite rowers were supplemented for 7 wk with either placebo or β-alanine (5 g/day). Before and following supplementation, muscle carnosine content in soleus and gastrocnemius medialis was measured by proton magnetic resonance spectroscopy ((1)H-MRS) and the performance was evaluated in a 2,000-m ergometer test. At baseline, there was a strong positive correlation between 100-, 500-, 2,000-, and 6,000-m speed and muscle carnosine content. After β-alanine supplementation, the carnosine content increased by 45.3% in soleus and 28.2% in gastrocnemius. Following supplementation, the β-alanine group was 4.3 s faster than the placebo group, whereas before supplementation they were 0.3 s slower (P = 0.07). Muscle carnosine elevation was positively correlated to 2,000-m performance enhancement (P = 0.042 and r = 0.498). It can be concluded that the positive correlation between baseline muscle carnosine levels and rowing performance and the positive correlation between changes in muscle carnosine and performance improvement suggest that muscle carnosine is a new determinant of rowing performance.

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects' age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.

The oral ingestion of beta-alanine, the rate-limiting precursor in carnosine synthesis, has been shown to elevate the muscle carnosine content. Carnosine is thought to act as a physiologically relevant pH buffer during exercise but direct evidence is lacking. Acidosis has been hypothesised to influence oxygen uptake kinetics during high-intensity exercise. The present study aimed to investigate whether oral beta-alanine supplementation could reduce acidosis during high-intensity cycling and thereby affect oxygen uptake kinetics. 14 male physical education students participated in this placebo-controlled, double-blind study. Subjects were supplemented orally for 4 weeks with 4.8 g/day placebo or beta-alanine. Before and after supplementation, subjects performed a 6-min cycling exercise bout at an intensity of 50% of the difference between ventilatory threshold (VT) and VO(2peak). Capillary blood samples were taken for determination of pH, lactate, bicarbonate and base excess, and pulmonary oxygen uptake kinetics were determined with a bi-exponential model fitted to the averaged breath-by-breath data of three repetitions. Exercise-induced acidosis was significantly reduced following beta-alanine supplementation compared to placebo, without affecting blood lactate and bicarbonate concentrations. The time delay of the fast component (Td(1)) of the oxygen uptake kinetics was significantly reduced following beta-alanine supplementation compared to placebo, although this did not reduce oxygen deficit. The parameters of the slow component did not differ between groups. These results indicate that chronic beta-alanine supplementation, which presumably increased muscle carnosine content, can attenuate the fall in blood pH during high-intensity exercise. This may contribute to the ergogenic effect of the supplement found in some exercise modes.