This study aimed at investigating serum AA changes related to BH-diving in elite BH-divers after an open sea training session under several aspects: energy need, fatigue tolerance, NO and antioxidant production, hypoxia tolerance.
The diving protocol was designed to expose all BH-divers to their maximum personal effort level during a “usual” free-diving training session, for which we did not impose any number of warm-up dives or maximum depth.
BH-diving-related physical effort can request an increase in catabolic metabolism to produce adequate amount of ATP. We observed a statistically significant decrease in several AA that can be used as substrate for energy need including ALA, LEU, ILE, VAL, HIS, THR, LYS, MET. ALA reduction can be the consequence of pyruvate production that is converted in acetyl-CoA to produce ATP (Krebs Cycle). ALA release should also lead to the muscle protein synthesis but, in this case, the Cahill cycle did not occur for the inhibition of proteosynthetic cascade .
During physical activity, BCAA (LEU, ILE,VAL) release, splanchnic bed rises and is accompanied by an elevated BCAA uptake by contracting muscles and by an enhancement of BCAA oxidation therein . In skeletal muscle, BCAA oxidation is catalysed by branched-chain α-keto acid dehydrogenase (BCKDH)  to use them as energetic substrate  providing about 3–6% of the total energy demand , according to some authors that observed a decrease in BCAA after prolonged effort such as a tennis tournament , a marathon  and a cyclist race , and LEU decrease in sprinters and jumpers when the muscles work in anaerobic conditions .
HIS decrease can be explained because it is converted into GLU, then in α-ketoglutarate to go into the Krebs cycle. THR is converted to pyruvate via threonine dehydrogenase. An intermediate in the THR catabolism can undergo thiolysis with coenzyme A (CoA) to produce acetil-CoA.
LYS is the precursor for carnitine  which transports fatty acids to the mitochondria, where they can be oxidised to produce acetil-CoA, involved in tricarboxylic acid (TCA) cycle . Finally, for this group, according to data obtained by other authors , MET decreased after prolonged physical activity. This reduction may reflect increased transmethylation in which DNA, histones and other macromolecules are methylated in response to exercise . PRO may also be related to the free fatty acids (FFA) release because some authors found a correlation between the decrease in PRO and the increase in FFA  as energy source during prolonged physical activity.
Physical activity led to release of antifatigue molecule precursors to improve the tolerance to physical effort . TYR, obtained from PHE, is decomposed to give acetoacetate and fumarate that go into the TCA cycle. Its slow recovery is due to the PHE reduction: PHE is used to produce catecholamines, as observed by Sponsiello et al.: urine catecholamine levels increased immediately after the dives, while we would have expected despite the participants were very expert BH-divers .
TYR decrease after the BH-diving session: this may be related to the stimulation of catecholamines (dopamine, norepinephrine, epinephrine) synthesis . Prolonged repetitive physical exercises may activate signalling testosterone and brain-derived neurotrophic factor (BDNF)-dependent pathways, leading to a raise of tyrosine hydroxylase activity and increasing catecholamine levels .
ORN seems to have an antifatigue effect increasing the efficiency of energy consumption and promoting the excretion of ammonia [29, 64]. Some authors found that ORN promotes fatty acid and protein catabolism improving physical performance and fatigue tolerance, especially in female athletes .
Also, we observed a reduction in CIT, used with aspartic acid to synthesize arginine-succinate that is a precursor for arginine, the primary substrate for NO biosynthesis. These data could be explained by the elevate increase in NO production in BH-divers . Indeed, NO plays a key role in the adaptation of subjects exposed to high hydrostatic pressure  and recent measurements taken in SCUBA and BH-divers at − 40 m depth showed remarkable increases in the plasma concentrations of NO derivatives [37, 44]. Particularly, NO is the principal molecule involved in the regulation of vasoconstriction/vasodilatation mechanism, necessary to adapt the endothelium to the increased ambient pressure and the related regional modifications .
On the other hand, the increase in pO2 triggers the formation of ROS and RNS leading to oxidative stress, . SCUBA and BH-divers can activate the endogenous antioxidant system to control vascular oxidative stress [43, 65]. This can explain the raise of serum CYST concentration: CYST is an important Cysteine source that is, with GLU and GLY, necessary for glutathione biosynthesis. GLY reduction may be due to the synthesis of glutathione: this is also confirmed by the increase in the glutathione peroxidase whose main biological role is to protect the organism from oxidative damage [43, 66, 67].
Finally, hypoxia occurs during the final part of BH-diving (ascent phase) . SER is involved in the protection from hypoxia: mitochondrial serine catabolism protects from hypoxia maintaining mitochondrial redox balance and cell survival . PRO decrease may be also related to the production of GLY, involved in the synthesis of antioxidants. The production of TAU precursors and TAU would underlie the tolerance to hypoxia: some authors registered a dose-dependent protective effect of TAU on the synaptic function of rat hippocampal slices exposed to a hypoxic insult [68, 69]. A similar protection mechanism may occur also in BH-diving despite the intermittent hypoxia. TAU decrease seems to be related to its antioxidant properties protecting tissues from highly toxic hypochlorite produced by inflammatory cells in the course of free radical processes  and other oxidative stress markers .
Our data seem to indicate a clear picture of the body adaption to hyperbaric exposure in BH-divers. From the interpretation of these results, it is clear that energetic metabolic request (for physical effort and for body adaptation to the extreme environmental) is in large part supported by AA used as substrate for fuel metabolism. The reduction in several AA involved in energy support at T1 seems to influenced by the characteristic of BH-diving, probably related to the “relax and comfort” training, diving experience and diving techniques adopted by expert BH-divers. Despite the absence of data related to serum AA changes in BH-diving, the major part of BH-Divers seems to perform their repetitive dives without an intensive muscle effort due to the correctness of the athletic gesture, the use of appropriate equipment and the adequate mental technique. Furthermore, it could be interesting to extend this test in other BH-diving specialty (static and dynamic apnoea) in which the use of muscle is absent (static apnoea) to use this model to understand better AA changes in BH-divers.
The short-term effect of serum AA profile changes found represents the most important data in our results and may indicate a muscle activity more intense than that usually BH-divers perceive/referred.
Data related to the NO production and antioxidant synthesis could explain the well-known BH-diving-related vascular adaptation and the response to oxidative stress during diving deep phase, as observed by SCUBA and BH-diving underwater blood draw studies [37, 44]. Finally, there are interesting data related to the hypoxia [72, 73] stimulus that indirectly may confirm that the muscle apparatus works under strong exposure conditions notwithstanding the very short/low intensity of exercise, due to the intermittent hypoxia caused by repetitive diving.
Obviously, the AA catabolism may be also explained in part by the increases in circulating AA besides cardiac and skeletal muscle work in the particular muscle activity conditions (increase in pressure, hypoxia in ascent phase, diving response) requiring several adaptation mechanisms including smooth muscle-mediated massive vascular response.
Even if it is well known that water immersion affects fluid balance, causing a redistribution of blood volume and an increase in urine production which results in fluid loss (dehydration). Our BH-divers did not show differences in blood volume, calculated by the Dill and Costill formula, between pre- and post-diving. This might be explained by the fact that all the BH-divers were expert instructors and/or high-level athletes that common drink adequate amount of water during the BH-diving training session.
Our results about changes in serum amino acid profile after repetitive breath-hold dives are an absolute novelty in this specific field, as far as we know, and could represent an interesting new approach in the study of BH-diving physiological adaptation and become very interesting to structure BH-diving much more specific protocols, than it was done in the first preliminary study, allowing to better select the numerous stimuli that a diver undergoes.
The two main limitations of this study are the reduced sample size, and the absence of data related to NO production and oxidative stress changes which would have allowed a more in-depth analysis of the results. Furthermore, it could be interesting in the future to compare our data to those obtained by volunteers performing static and dynamic apnoea to analyse the different risk factor separately.