INFLAMMATION

The inflammatory response is a non-specific integral part of the innate immune system responsible for clearing the inflamed area of infectious and toxic agents and tissue debris by phagocytic and non-phagocytic means (Sherwood 2008). During exercise and particularly impact related sport, tissue damage is a common theme that results in the activation of the inflammatory system.

1.4.1 Exercise-Induced Inflammatory Effect

Exercise of various intensities can cause a transient increase in markers of inflammation. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and CRP are exceptionally common markers that become elevated following exercise. Interleukin-6 has been shown to increase from 0.76 pg/mL to 10.89 pg/mL following a 24 hour simulated laboratory marathon as well as a six day adventure race (Wallberg et al., 2011). This is comparatively mild to the 8000-fold increase in IL-6 following a 246 km ultra-endurance marathon (Margeli et al., 2005). Additionally, IL-6 mRNA expression is up-regulated during exercise (Nieman et al., 2003b) which is intensity and duration dependent (Pedersen et al., 2003). However its specificity to exercise induced tissue damage is questionable as a result of its rise irrespective of muscle damage (Febbraio and Pedersen 2002) and its newly defined function as a “myokine” (Febbraio and Pedersen 2005). Similarly, IL-1β and TNF-α become elevated post-marathon (Moldoveanu et al., 2000; Ostrowski et al., 1999) and after short intense exercise (Espersen et al., 1990). C-reactive protein, the first acute phase protein to be described that is a sensitive marker of inflammation (Pepys and Baltz 1983), has been significantly increased following ultra-endurance exercise (Scherr et al., 2011; Tauler et al., 2013) rugby union (Cunniffe et al., 2010) and soccer (Ispirlidis et al., 2008).

1.4.2 Neopterin and 7,8-dihydroneopterin

Interferon-γ (IFN-γ), produced from NK and NK T cells and CD4+ and CD8+ cells once antigen-specific immunity develops (Schoenborn and Wilson 2007), stimulates macrophage production of 7,8-dihydroneopterin from guanosine tri-phosphate (GTP) via up-regulation of GTP-cyclohydrolase 1 (GTPCH-1) (Schoedon et al., 1986). 7,8-dihydroneopterin triphosphate; the enzymatic breakdown product of GTP (Schoedon et al., 1986) is an

intermediate in the production of tetrahydrobiopterin (BH4) that is cleaved by intracellular

phosphatases (Schoedon et al., 2005) in macrophages specifically (Wirleitner et al., 2002). 7,8-dihydroneopterin is secreted from the activated macrophage into the intracellular spaces and finally the plasma (Gieseg et al., 2008a), where proton abstraction from carbon-7 and nitrogen-8 signifies oxidation to the highly fluorescent neopterin (NP) by hypohalous acids such as hypochlorous acid (HOCL) (Fig. 1.3) (Gieseg et al., 2000; Schraufstätter et al., 1990; Widner et al., 2000).

Figure 1.3. Neopterin and 7,8-dihydroneopterin production.

Hypochlorite is generated by neutrophils and to a lesser extent macrophages during inflammation (Halliwell and Gutteridge 1999; Pullar et al., 2000) suggesting much of the NP

measured in plasma has come from these sites. To date it is the only known compound capable of oxidizing 7,8-dihydroneopterin to NP in vivo, although it is widely accepted other potential mechanisms exist that are yet to be described. One such mechanism that may be related to impact-induced muscle damage is the oxidizing potential of the heme containing protein myoglobin. With significant increases observed following rugby union (Takarada 2003) and its proven ability to cause lipid peroxidation (Giulivi and Cadenas 1998), myoglobin oxidation may be an alternative mechanism responsible for a hypothesized increase in NP following rugby union.

Regardless, the central role of IFN-γ communication between T-cells and macrophages with the subsequent release of 7,8-dihydroneopteirn and its oxidized form NP, make them ideal measurements for gauging immune activation (Wachter et al., 1989). An experiment found that direct injection of IFN-γ results in a significant and sustained rise in plasma NP (Müller et al., 1991) which provides further conclusive evidence of the mechanism.

Neopterin has been implicated in several pathological diseases and conditions including HIV, cancer, arthritis, infections, septicaemia, multiple sclerosis, atherosclerosis, allograft recipients and tuberculosis (Aulitzky et al., 1988; Baydar et al., 2009; Beckham et al., 1992; Eisenhut et al., 2011; Gieseg et al., 2008a; Hoffmann et al., 2003). To a lesser extent, it has also been reported to possess pro-oxidant properties (Hoffmann et al., 2003), where its balance with 7,8-dihydroneopterin in atherosclerotic plaques can influence its progression (Gieseg et al., 2008a; Gieseg et al., 2008b), while also being a significant tool for blood and organ donor screening (Fuchs et al., 1983). Its serum concentration meanwhile, is dependent on age and gender while being modified by race, body mass index (BMI) and body fat percentage (Spencer et al., 2010).

7,8-dihydroneopterin’s properties depend on the concentration, chemical environment and oxidants it can react with. It has been reported to have anti-oxidant and pro-oxidant properties (Gieseg et al., 1995). 7,8-dihydroneopterin inhibits oxidized low-density lipoprotein (oxLDL) induced cell death in U937, THP-1 and human macrophages (Baird et al., 2005; Gieseg and Cato 2003). It has been demonstrated to be a potent scavenger of superoxide, peroxyl and nitrogen centred radicals (Baird et al., 2005; Gieseg and Cato 2003; Oettl et al., 2004; Weiss et al., 1993) and shown to induce apoptosis (at high mM

concentrations only) in several cell lines due to increased oxidative stress (Wirleitner et al., 2003).

1.4.2.1 Detection Methodology

The quantification of NP and 7,8-dihydroneopterin can provide a total measurement of macrophage activation and the level of associated inflammation. As a diagnostic tool in several diseases and markers of exercise-induced immune activation, the measurement requires a rapid and cost-effective methodology. The detection of urinary NP was first described in the late 1970’s (Wachter et al., 1979) which was closely followed by Fukushima and Nixon (1980) who both used a C18 reverse phase method employing a phosphate buffer

and methanol/H2O mobile phase, respectively. They also measured the dihydro- forms

following acidic and basic iodine oxidation because of their lack of fluorescence.

Whilst several methods are available for NP and 7,8-dihydroneopteirn quantification including enzyme linked immunosorbent assay (ELISA), dipstick and radioimmunoassay (RIA) (Bührer-Sekula et al., 2000; Werner et al., 1987), their sensitivity and processing speed does not account for their relatively expensive nature. High performance liquid chromatography (HPLC) is the preferred method of detection due to its inexpensive sampling costs after initial set-up and auto-sampling ability. Urinary NP and 7,8-dihydroneopteirn detection are most commonly assayed using the reverse phase method developed by Hausen et al. (1982) which was later updated in 1992 (Fuchs et al., ). It uses a phosphate buffer between pH 6 to 7, where 100 µL of urine is diluted with 1000 mL of a 0.015 mol/L phosphate buffer (pH 6.4) containing 2 g/L ethylenediaminetetraacetic acid (EDTA). Chromatography is isocratically performed using the dilution buffer without the EDTA with monitoring of NP done by its native fluorescence at 353 nm excitation and 438 nm emission wavelengths with a flow rate of 1 mL/min. Pre-column sample preparation is kept to a minimum because of a lack of protein present. In comparison, plasma, serum and cerebrospinal fluid requires careful precipitation of proteins by ethanol (Krcmova et al., 2011) or acetonitrile (ACN) (Flavall et al., 2008). de Castro et al. (2004) proposed an alternative method that used two coupled reverse phase columns, 150 mM sodium phosphate buffer pH 4 at a flow rate of 0.8 mL/min using various UV wavelengths from 353 – 390 nm. While they claimed it was a reliable and efficient alternative, Schroecksnadel et al. (2006)

appealed the results stating the large variation in comparison to previously published work was due to the inability to distinguish NP from 7,8-dihydroneopterin.

1.4.2.2 Exercise Effect

Neopterin has been routinely measured in plasma and urine as a marker of immune activation following various forms of exercise including running (Rokos et al., 1987), rowing (Jakeman et al., 1995), cycling (Deetjen et al., 1997) and triathlon (Margaritis et al., 1997). Deetjan et al. (1997) monitored subjects in an eight hour alpine cycling race that resulted in significant and sustained (48 hours) increases in NP immediately post-race. Similar kinetics were observed following a two and a half hour run (Dufaux and Order 1989), a 20 kilometre run completed in under two hours (Sprenger et al., 1992), five hours intense running (Tilz et al., 1993) and 67 km ultra-marathon (Schobersberger et al., 2000). Schobersberger (2006) also observed a rise in serum NP, CK and CRP following a downhill marathon run, whilst NP also has the potential to identify over-training or reaching. Jakeman et al. (1995) observed one third of an increase in the NP/creatinine ratio compared to pre-training levels in 27 elite rowers whose training had increased in the four weeks leading up to the Olympics.

The measurement of NP in exercise has declined over the past decade. In an investigation into NP excretion in individuals who participated in the Race across America in 2007, NP was shown to increase steadily, peaking at day four. It then began to drop toward the end of the race where at day seven there were no differences between the competitor and support person. There was also large inter-individual differences and a correlation with power output indicating NP excretion is intensity dependent (Moser et al., 2008). Furthermore, NP in conjunction with muscle damage and oxidative stress markers were observed to significantly increase following a 90 kilometre marathon race in Brazil (Dantas de Lucas et al., 2014). In contrast, an investigation of 15 trained runners following a marathon observed no significant increase up to 34 hours afterwards (Gunga et al., 2002). Whilst this may be related to the fitness level of the individuals involved, no difference in the NP change was observed between trained and un-trained subjects following one hour of cycling at 60 % O2max

(Smith et al., 1992). This may of course be slightly altered because of the moderate intensity associated with that level of maximal O2 uptake.

Furthermore, there are several issues that can be drawn from the current literature regarding NP quantification measurement as an indicator of inflammation from exercise. Traditionally papers have investigated NP alone and neglected to measure 7,8-dihydroneopterin (Dantas de Lucas et al., 2014; Dufaux and Order 1989; Sprenger et al., 1992). To gain a true understanding of macrophage activation, total NP (NP + 7,8-dihydroneopterin) has to be measured. Therefore, the papers who have recognized a NP increase may in fact only be measuring a change in oxidative stress. With NP concentrations approximately a third of total NP (Fuchs et al., 1989b), it is highly possible the increased NP observed following exercise is a result of an increased oxidation of 7,8-dihydroneopterin and not a result of increased macrophage activation. The mentioned studies did however identify a simultaneous rise in TNF-α which subsequently suggests an inflammatory reaction has ensued as a result of the exercise. Meanwhile, NP and 7,8-dihydroneopterin have not been measured in a sport that is focused around high force impacts and collisions that are known to cause muscle damage and induce changes in inflammatory and endocrine markers (McLellan et al., 2010; Smart et al., 2008).