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DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.


INTRODUCTION
Skeletal muscle overproduces reactive oxygen species (ROS) during contractile activity, compared to the lower -but still significantlevels found at rest [1]. However, depending on the prevalent energy-demanding activity (aerobic/endurance or anaerobic/resistance), intensity, and duration, intracellular ROS sources other than mitochondria might be significantly involved in the process [2].
The enzyme xanthine oxidase, found in muscle and vascular endothelium, is activated in response to the intermittent (pseudo) ischaemia-reperfusion that occurs during anaerobic/resistance exercise coupled with severe energy depletion in exhausted fast-twitch type-II fibres (AMP accumulation) [3]. This enzyme has long been known to catalyze the conversion of hypoxanthine (from inosine) into uric acid (UA) by the purine catabolic pathway, with the concomitant generation of superoxide radicals (O 2 • -) and hydrogen peroxide (H 2 O 2 ) [4]. In turn, O 2 • and H 2 O 2 yield the highly reactive hydroxyl (HO • ) and lipid-alkoxyl (RO • ) radicals via several iron-catalyzed reactions. In the worst-case scenario, iron overload in plasma and muscles has been associated with strenuous/intense exercise [5].

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST
logical systems: prevention, interception/scavenging, and repair [9].
Among several antioxidants in plasma, UA has been suggested as a powerful biological iron-chelating agent to provide protection against oxidative injury [10]. There is significant current interest in redox biomarkers in plasma and their relationship to exercise and other physiological alterations and/or pathologies, such as atherosclerosis, obesity, and cardiopathies [11]. Moreover, fast, simple, and low-cost analytical tests have been prioritized in sports science for practical and logistic reasons. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress after an exhaustive anaerobic 30 s cycling sprint test (Wingate test).

Experimental protocol
The protocol for the Wingate test was that described by Dotan and Bar-Or [4], and was carried out with a Cybex cycle ergometer, Model Metabolic Systems (Division of Lumex, Ronkonkoma, NY, USA).
After each set of maximal effort, the workload was adjusted to accommodate the active recovery mode (no resistance, 80 rpm, for 3 min).

Sample collection and analyses
Blood samples (5 mL) were withdrawn from the forearm cubital vein of the volunteers immediately before, 5 min after, and 60 min after the Wingate test, using EDTA-containing Vacutainer ® kits. Heparinized tubes were avoided for blood sampling since physiological haem iron was also measured in plasma. Blood samples were immediately centrifuged for 5 min at 4 × g (RT), and plasma fractions were isolated and stored at -80°C until analysis.

Total iron determination
The total iron concentration in plasma was assayed using a specific

DISCUSSION
Iron is a crucial microelement for several physiological/biochemical processes in the human body and regarding exercise, the disruption of iron homeostasis can lead to reduction in oxygen transport capacity, diminished energy supply for muscle contractions, and oxidative stress [15]. As shown in Figure 1A Uric acid as an antioxidant after a wingate test from deleterious Fenton-type and other radical reactions [12].
As shown in Figure 5A Figure 6A).
Interestingly, the concentration of UA -one of the major antioxidants in human plasma -did not vary immediately after exercise, but showed a late onset accumulation (29% compared to baseline; p = 0.0135) during the following recovery period (Figure 2A-B).
Thus, we can assume that the antioxidant accumulation of UA in plasma was important to sustain FRAP activity during the recovery period (from 5 to 60 min after exercise). This hypothesis was reinforced by the relatively high correlation between UA TOTAL released in plasma and FRAP TOTAL (Adj. R-Square = 0.636; inset, Figure 6B).
The reasons for the delayed increase of UA concentration in plasma or (ii) MDA, immediately after moderate and intense exercises [18].
It is worth mentioning that aldehyde derivatives from lipid peroxidation -most of the TBARS compounds, including MDA -promptly react with proteins and nucleic acids in plasma to form adducts that, thus, interfere in the precise determination of free MDA concentration in plasma [19]. Finally, it is possible that the Wingate protocol applied was not intense enough to induce maximal (or detectable) redox imbalances in all subjects, especially considering simple, fast, and low-cost assays as proposed here. We aimed to monitor exerciseassociated oxidative stress by simple methods in order to discuss (or start discussing) sampling and redox analysis in situ, which would provide crucial information for adequate post-exercise supplementation, energy supply, etc. However, an evaluation regarding method sensibility, technique costs, and data accuracy undoubtedly deserves further investigation.

CONCLUSIONS
The late response of UA following the exhaustive Wingate test is here suggested as a secondary antioxidant frontline that limits iron availability, sustains higher antioxidant activity (FRAP), and limits lipid peroxidation in plasma during the recovery period.