36 year old, 162 cm tall, 60.9 Kg body mass weight, 24 h race champion runner signed a written consent term after being informed of all the procedures involved in the study. The Federal University of São Paulo Ethics Committee on Human Research approved all experimental procedures (Protocol number: 2061/07).

The athlete was clinically evaluated by a medical doctor and, considered fit to participate in the study, was submitted to a muscular biopsy taken from the distal vastus lateralis muscle region. The methodology employed was that proposed by Dubowitz using the Schmidt “dança dos farabeufs” technique [1-3]. A small fragment of approximately three square millimeters was taken and the PAS muscular glycogen identification histochemical technique applied. The muscle fragment extracted was immediately fixed and frozen in n-hexane, refrigerated in liquid nitrogen and properly stored at -80ºC for future analysis (Figure 1a).

Figure 1a: Pre exercise biopsy of vastusl ateralis colored histochemically with PAS. x125. Normal PAS.

A thirty-day recovery period was proposed, after which time the athlete could perform the six hour running session. The running procedure was performed on a RT 250 pro São Paulo Brazil Movement® treadmill at a 1% gradient. The suggested running speed was ten kilometers per hour (10 km/h), the same speed comfortably maintained by the athlete in competitions. An S120 Polar Finland heart rate monitor was used to monitor effort intensity. The athlete was advised to eat properly as well as to be adequately hydrated during the days leading up to the exercise session.

On the test day the athlete attended the exercise session venue 3 h after a standard breakfast similar in content and composition to that which he would normally ingest before competition. Our athlete’s heart rate at rest was recorded along with weight on 2096 PP Toledo® São Paulo-Brazil scales which weighed up to 200 kg in 50 g graduations. The athlete was wearing only running clothes, which included a pair of running shoes (Table 1).

Table 1: Time (h): Time in hours elapsed throughout of the test, HR (bpm): heart rate in beats per minute, and Vel (km/h): velocity in kilometers per hour.

Following a proper warm up the running session was begun. Throughout the exercise period only water was offered to the athlete, such that no energetic supplement was used. Every full hour the runner came down from the treadmill to urinate. Subsequently body mass and heart rate were recorded to monitor for dehydration.

After six hours the athlete was immediately submitted to a vastus lateralis muscle biopsy on the opposite limb had been used in the first biopsy. The fragment was immediately frozen and later prepared and histochemically stained to detect the presence of glycogen (Figure 1b).

Figure 1b: Post exercise biopsy of vastusl ateralis colored histochemically with PAS. There was a difference of color intensity in post exercise. However, large amounts of glycogen still remain in depleted cells at the present slice. x125.

The analysis of the biopsies pre and post ultra-endurance exercise, shown in Figures 1a and 1b, respectively, reveal a discreet reduction of intramuscular glycogen in Figure 1b, evidenced by slight color reduction in some muscle cells.

Although there are studies in the scientific literature that have observed muscle metabolism behavior in ultra-prolonged exercise, ultra exercise effects with no caloric supplementation to intramuscular glycogen reserves have not yet been reported [4,5]. In our research we decided to evaluate muscular metabolism performance under conditions never previously investigated. The athlete underwent a sixhour running session on a treadmill in a post absorptive state while receiving no caloric supplementation. Despite these conditions large amounts of muscular glycogen were observed in muscle cells (Figure 1b). The assumptions that may explain this intramuscular glycogen presence are as follows:

Firstly, after the beginning of the exercise and upon gradual decrease in intramuscular glycogen, a heterotrimeric serine/treonine protein kinase, recently described as 5’AMP-activated protein kinase (AMPK), acts as an important intracellular AMP/ATP and creatine/ phosphocreatine rate sensor [6]. There is evidence that it is regulates according to the variation in muscular glycogen reserves [7]. AMPK is found in its active form during exercise and increases with effort intensity [8-15]. Its function is probably to translocate the GLUT4 carrier protein to the cell membrane, where GLUT4 is the most important glucose carrier isoform during exercise [16]. This capture is particularly important since blood flow to active muscles can increase up to twenty fold during intense exercise [17]. Initially, this process probably prevents a critical reduction of intramuscular glycogen reserves, since it increases in the same way as glyconeogenic activity in the liver [5]. However, during ultra-prolonged exercise glycemic levels would be significantly reduced if it were not for the glucose/fatty-acids cycle modulation known as the “Randle Cycle”, which increases fattyacid oxidation due to the increase in fatty-acids supply at the cellular level [18]. The blood glucose supply constitutes a 2nd hypothesis. Defined as blood flow product divided by glucemic concentration, this is related to the amount of glucose carried by the blood which can reach the muscle arteries in the active muscles while facilitating its capture [17].

The 3rd assumption is that some intramuscular glycogen “granules”, also called glycosomes, perform a different role. These granules play a structural protein role participating in myofilament sliding and persist even after their death [7,19]. This fact alone would prevent PAS from showing a negative result for the intramuscular glycogen presence.

We conclude that six-hour ultra-prolonged exercise does not exhaust intramuscular glycogen on PAS even in the absence of caloric replacement during exercise. The most likely reasons for this persistence are: AMPK influence on GLUT4; initial plasmatic glucose levels; modulation of acetyl-CoA Rundle Cycle entry and glycosomes presence which plays a structural role in myofilament adherence.

Biochemical analyses through glycogen digestion using KOH and Antrona seems to be the best mean of identifying glycogen.