Body building is a sport where ultrastructual damage to muscle fibres aids the development of dense muscle layers. Using a new strong cation exchange (SCX) based chromatography technique to measure neopterin and 7,8-dihydroneopterin, we investigated whether this muscle damage caused increased levels of inflammation. Urine samples were collected over eight consecutive mornings from 10 natural competitive body builders. Samples were analysed using SCX high performance liquid chromatography (HPLC) with urine volume corrected for creatinine and specific gravity (SG). The majority of subjects showed large changes in both neopterin and total neopterin (7,8-dihydroneopterin+neopterin) levels, though the mean data for the group showed no significant change over the week. There was no evidence of the high intensity resistance training causing an accumulation of inflammation as the values for all the body builders returned to close to the starting values after 2 days rest. The SCX analysis had an intra-specific viability of 3.04% and the inter-specific viability was 5.42%. Urine volume correction with SG was found to give the same values as using creatinine. Creatinine and specific gravity are both reliable methods for correcting for urine volume while SCX HPLC provides a new means of measuring urinary neopterin and total neopterin.
Body-building is a sport where competitors are judged based upon their muscular physique. Multiple years of preparation and dedication are required for the retention of strict training and nutrition regimes that are crucial for success within such a sport. Training typically consists of year round, high intensity and high frequency hypertrophy and strength training which cause ultrastructural muscle damage through eccentric muscle actions [1–5]. Frequent damage to the muscle results in the invasion of inflammatory cell populations that may last days or sometimes weeks while repair, regeneration and growth occur. A host of cytokines are released into the surrounding area which facilitate the arrival of lymphocytes, macrophages and neutrophils  and begin the process of inflammation. Other physiological factors such as exercise intensity, oxidative stress, acidosis, heat, intensity, duration, recovery between sets and training status can influence cytokine secretion rate and concentration [7–9].
Neutrophils initially invade the damaged muscle which may be phagocytic in nature and help with cellular debris degradation, while macrophages provide several functions including debris removal from an injured muscle which can effect muscle cell differentiation and proliferation [10–12]. Managing the damage, repair and adaption process is critical for continual year-round training and progression. It is a significant concern for body builders who rely on recovery to ultimately build larger, denser muscle and repeat this process on a weekly basis. The accumulation of repetitive high intensity exercise can potentially lead to an increased susceptibility to infection [13, 14], and chronic inflammation . With common training regimes of 5 days training and 2 days off, the persistent eccentric loading could develop the onset of inflammation and immune system activation. Biomarkers such as C-reactive protein (CRP) and interleukin-6 (IL-6) are common markers often used for inflammation detection [16, 17] and have been demonstrated to possess elevated levels following resistance training . Identifying the level of potential inflammation accumulation over a week of high intensity resistance training can lead to increasingly effective training protocols to maximize muscle growth and recovery.
Neopterin and 7,8-dihydroneopterin
Significant attention has been given to CRP, IL-6, IL-1 β, IL-8, IL-1 ra, IL-10, IL-15, tumor necrosis factor alpha (TNF-α) and its soluble receptor (sTNF-αR1) in response to diseases and exercise that elicits an inflammatory response, all of which have been shown to increase in response to resistance training [18, 19]. Neopterin and 7,8-dihydroneopterin are pteridine compounds synthesized and released from activated macrophages upon stimulation with gamma interferon [20–22]. They have been found to be elevated in patients with human immunodeficiency virus (HIV)  and coronary artery disease . GTP-cyclohydrolase, an enzyme upregulated by γ-interferon, catalyses the breakdown of GTP to 7,8-dihydroneopterin triphosphate. In primate macrophages, the accumulation results in the diffusion of 7,8-dihydroneopterin out of the activated macrophage and into the intracellular spaces and finally the plasma.
Some of the 7,8-dihydroneopterin is oxidized into the highly fluorescent neopterin by hypohalous acids such as HOCl [25–28]. The communication of T cells and macrophages and the subsequent release of neopterin makes it an effective marker of immune system activation and inflammation [21, 29]. Several studies have identified the rise of neopterin in plasma and urine following exercise [30–33]. Our thought is that the damage to muscle as a result of eccentric loading and hypertrophy training will increase the amount of macrophages present at these sites, and subsequently produce higher concentrations of 7,8-dihydroneopterin which can be oxidized to neopterin. Therefore, the high frequency training philosophy of many body builders should elicit an increase in measureable 7,8-dihydroneopterin and neopterin.
Due to its high fluorescence, neopterin is relatively easy to detect in plasma and urine using reverse phase HPLC [22, 34, 35], though many clinical laboratories use enzyme linked immunosorbent assays as well . Although immunoassays are ideal due to ease of use and rapid processing time, the immunoassays developed for neopterin are expensive. Reverse phase separation of urine components for neopterin analysis is the most commonly reported method used  as it is fast, reliable and accurate . We have found the reverse phase method gives poor separation and resolution of neopterin from other urinary components. Following advice from Schirks Laboratory in Switzerland, we have developed and report on here an ion exchange based separation method using SCX HPLC chromatography with ultraviolet excitation at 353 nm and emission at 438 nm. Currently, a combination of neopterin and 7,8-dihydroneopterin have only been measured in plasma in relation to atherosclerotic plaque formation [26, 27] not exercise. The majority of reports on exercise have focused specifically on urine and plasma neopterin levels . While this provides information about an acute inflammatory response and the level of oxidative stress, it does not provide information about the total inflammatory response. Oxidation of neopterin to 7,8-dihydroneopterin in vitro allows the quantification of total inflammation and immune system activation inclusive of the level of oxidation present.
Urine volume normalization
Urine volume normalization is critical to determining an analyte concentration. Creatinine and specific gravity are the two methods currently employed [39, 40]. The upper limits of normal μmol neopterin/mol creatinine for women range as follows: 208 (18–25 years), 209 (26–35 years), 239 (36–45 years), 229 (46–55 years), 249 (56–65 years), 251 (older than 65 years). In men, upper limits for normalcy are slightly lower: 195 (18–25 years), 182 (26–35 years), 176 (36–45 years), 197 (46–55 years), 218 (56–65 years), 229 (older than 65 years). These upper limits include 97.5% of healthy controls . It is common practice in medicine to use creatinine as a marker of urine volume correction  as it is released at a fairly constant rate . For doping tests, specific gravity is the method of choice for the World Anti-Doping Agency (WADA) but it is not widely used due to a lack of data comparing creatinine to specific gravity.
This research aims to provide information regarding the potential elevated concentrations of neopterin and 7,8-dihydroneopterin over the course of a week of high intensity resistance training in competitive, natural (no use of banned substances) body building and to provide an alternative HPLC technique using SCX based chromatography with urine volume correction and utilizing both creatinine and SG.
Materials and methods
All solutions and reagents were prepared with water purified using a NANOpure ultrapure water system from Barnstead/Thermolyne (Dubuque, IA, USA). Chemicals and reagents were supplied from Sigma Chemical Company (Auckland, New Zealand)or BDH Chemicals limited (Auckland, New Zealand) unless otherwise stated and 7,8-dihydroneopterin was supplied by Schircks Laboratories (Jona, Switzerland).
Ten healthy controls with an average age of 32±8 years, height of 181±8.1 cm and weight of 80.6±6.7 kg, and eight competitive natural body builders that train and compete in Christchurch and New Zealand volunteered for this study. The characteristics of the subjects enrolled in the study, including their experience in the sport and training phase are in Table 1. The experimental protocol was approved by the University of Canterbury Human Ethics Committee, Christchurch, New Zealand and all subjects were informed of the risks involved in the study before their written consent was obtained.
|Subject||Age||Height, m||Weight, kg||Phase||Calories/day||Experience|
|S3||25||1.77||77.1||Competition prep||1900||Multiple shows|
|S4||22||1.68||76.0||Mass gain||2000||1 show|
|S7||34||1.63||58.5||Competition prep||1300||Multiple shows|
Each subject would train one to two body parts per day, three-six exercises per body part, three-six sets per exercise and eight-12 reps per set. All subjects took a range of nutritional supplements that were not monitored due to the large variation and quantity of products taken and were of satisfactory health during and after the study based on a questionnaire.
The inclusion criteria of subjects for this study was for the subject to be a natural competitive body builder with a training schedule of 5 days training and 2 days off. Control subjects provided a single urine sample for comparison. Each body-building subject was provided with 8 urine canisters and asked to provide a sample mid-stream upon the second time they needed to pass urine. The sample was either frozen immediately at the subject’s home or stored in a cooler box provided filled with ice. The first sample was collected on the morning after their scheduled 2 days of rest, and each morning for the following seven mornings. Samples were collected at the cessation of the 8 days, transported to the laboratory, aliquoted into four 1.8 mL centrifuge tubes per sample, and frozen at –80 °C until analysis.
Samples were prepared in darkness where possible to prevent oxidative loss of 7,8-dihydroneopterin from UV light. Samples were thawed and diluted to 1 in 40 with phosphate buffer (20 mM (NH4)3 PO4 pH 2.5). For neopterin and creatinine determination, 100 μL was transferred to an autosampler vial for HPLC analysis. For total neopterin analysis, an oxidation step was included to convert 7,8-dihydroneopterin to the fluorescent neopterin. 20 μL of acidic iodide solution (5.4% I2/10.8% KI in 1 M HCl) was added to 100 μL of the 1 in 40 diluted urine sample and incubated for 15 min at room temperature in the dark. 10 μL of 0.6 M ascorbate was then added to reduce the tri-iodine before HPLC analysis.
HPLC analysis and SG
HPLC measurement of neopterin and creatinine was performed using a Shimadzu Sil-20A HPLC with autosampler, RF-20Axls fluorescence detector and a SPD-20A photo diode array detector. A total of 10 μL of sample was injected onto a Luna 5 μm SCX 100Å, 250×4.6 mm column (supplied by Phenomenex NZ Ltd) with a mobile phase of 20 mM ammonium phosphate pH 2.5 pumped at 1 mL/min. Neopterin was detected by its native fluorescence at 353 nm for excitation and 438 nm for emission and creatinine at its natural absorbance of 234 nm. The concentration and identity of the eluted neopterin and creatinine was confirmed by comparison to a standard. These were made up daily using 1.5–2 mg of neopterin or creatinine and dissolved and diluted down to 50 nM and 100 μM respectively using 20 mM ammonium phosphate pH 6 and quantified by peak area using the software Shimadzu Class VP. All analysis was conducted in duplicate and data is displayed as the mean±the standard deviation. SG was calculated using a hand-held refractometer (Atago). 50 μL of each sample was added to the refractometer and calculated using distilled water as a zero standard. SG was calculated using the following formula based on the normal population SG1.020 .
Precision studies and recovery
Intra-assay precision was evaluated using 20 replicates of a single urine sample in a single analytical run. Inter-assay precision was evaluated using 20 replicates of a single urine sample on 4 consecutive days.
A calibration curve was established using neopterin standards of 25, 50, 100, 500 and 1000 nmol/L.
Data was analysed using repeated measures ANOVA for neopterin and total neopterin corrected for creatinine and SG.
Urinary neopterin and creatinine were detected in the same HPLC run and elute at 7.4 and 20.5 min, respectively (Figures 1 and 2). Each peak stands alone, is clearly visible and is sharp with no visible tailing. The intra-assay coefficient of variation for neopterin was 3.04% while the inter-assay coefficient of variation was 5.42%. Over the range of standards (25–1000 nmol/L), the assay presented a linear response.
Control subject values for neopterin per mole of creatinine (NP/CR), total neopterin per mole of creatinine (TNP/CR), neopterin corrected by specific gravity (NP/SG) and total neopterin corrected by specific gravity (TNP/SG) are presented in Table 2. There was no statistically significant difference in the level of neopterin or total neopterin on days during the week of training for the body builders when corrected for creatinine (Figure 3A) or SG (Figure 3B). There is a trend towards an increasing inflammatory response near the end of the training week which is observed for both creatinine and SG correction.
|Day||NP/CR, μmol/mol||TNP/CR, μmol/mol||NP/SG, nmol/SG1.020||TNP/SG, nmol/SG1.020|
Values are mean±SD. adenotes statistically different from control levels, p<0.05.
There are significant differences (p>0.05) in the body building group compared to controls on certain days for NP/CR and every day for TNP/CR and TNP/SG as highlighted in Table 2. All NP/CR values remained within the upper limits of normal  for the healthy population, however some subjects had values as high as 360 μmol/mol creatinine.
The amount of oxidation during the training week did not significantly change for creatinine or SG (Figure 3C). Each urine volume correction method demonstrated very similar values while the level of oxidation between subjects was significantly different with values ranging from 20.9% to 92.1%. There is a large individual variation between subjects (Figure 4). The general trend is toward an increasing concentration of neopterin and total neopterin when urine volume is corrected with both creatinine and SG. Although some subjects demonstrate greater increases compared to other, some show little day to day variation.
The method commonly employed for urinary neopterin detection is based on the reverse phase method developed in 1979 . de Castro et al. (2004) developed a similar reverse phase method with an intra assay CV of 12.9% and inter assay CV of 12.5%. The intra-assay CV of the SCX method described in this work was 3.04% and the inter-assay CV 5.42%. Our SCX ion exchange method which is described here provides a method similar in reliability, repeatability and reproducibility to the C18 method that also allows for the simultaneous detection of both urinary neopterin and creatinine. Results from the control subjects are similar to the published normal range of urinary neopterin in healthy individuals . This demonstrates the reliability of this method for providing an alternative to reverse phase. Additionally, the current method is able to identify the neopterin and creatinine peaks with no interference from neighbouring peaks (Figures 1 and 2) which makes the detection and quantification extremely easy.
This study is the first to report urinary neopterin concentrations whilst correcting for SG in comparison to creatinine. While other drugs have been corrected for using SG , the nearly identical patterns and trends observed for neopterin (Figures 3A, B and 4C) provide evidence of a reliable alternative method for urine volume normalization. The removal of the need to measure creatinine drastically reduces the required run times for the urine neopterin analysis and decreases the chance of false-negative results following exercise which is known to increase creatinine concentrations by as much as 50%–100% [45, 46].
Due to subjects having NP/CR values within previously stated normal values and with a sample size of eight , conclusions cannot be drawn in terms of inflammation and immune system activation during a week of competitive natural body building training. This study does report TNP/CR, NP/SG and TNP/SG values which are significantly higher than in control subjects (Table 2) suggesting the training regime of a competitive natural body builder is sufficient to cause the individual to be in a continual state of immune system activation. This is the first study to analyse such training in order to provide insight into the intensity of high frequency and highly concentrated resistance training and the effects it has on the immune system.
Although there is a statistical difference between the control group and body builder group on specific days indicating immune system activation (Table 2), levels are still all within previously stated values of normality . Other research has identified “normal” NP/CR as 294.6±178.6  which indicates more research may be required using different methods, to better understand the “normal” level. Additionally the level of neopterin produced by a macrophage is solely dependent on the level of oxidation within the body. The TNP/CR and TNP/SG may provide a more effective representation of the total inflammatory response that is not altered by the level of oxidative stress present. This is highlighted by the subject variability (20.9%–92.1%). Previous research has suggested a 3:1 ratio of total neopterin to neopterin . While some individuals show this ratio, the majority do not. This may be due to the type of subjects involved in this study. By measuring both 7,8-dihydroneopterin and neopterin by oxidising the 7,8-dihydroneopterin to neopterin with acidic tri-iodide in vitro, the level of total macrophage activation in states of immune system activation and inflammation can be assessed. Moreover, there are more significant difference observed in the concentrations of the body builders group to that of controls when total neopterin is analysed throughout the week in comparison to neopterin alone. This data provides two potential conclusions. The level of oxidation does not change when performing resistance training of a body-building nature, regardless of the total amount 7,8-dihydroneopterin being produced, i.e., the amount oxidizing to neopterin does not change. Or alternatively, body building training elicits a significant inflammatory event as a consequence of the ultrastructural damage to muscles.
Time of collection is an important aspect for determining the acute inflammatory response. Resistance training has been shown to elicit muscle damage and provoke an inflammatory reaction as measured by several other cytokines . The current study identified a lack of statistical change throughout the week of training, suggesting that no one day provided more inflammation than another, even though they were statistically elevated above control values on specific days. Previous studies have identified immediate increases in plasma neopterin and other inflammatory cytokines post-exercise [31, 49, 50] suggesting the delay in sample collection in the current study may have masked the full extent of total immune system activation. Since muscle damage and subsequently inflammation occur following resistance exercise, the delay in sample collection still allows communication between T-cells and macrophages and the production of neopterin and 7,8-dihydroneopterin at sites of muscle damage. The significant change in comparison to the control group might suggest the intensity was high enough to cause structural integrity loss to the muscle, but the exposure of competitive body builders to such training has shown that the human body is able to adapt to the rigors of such exercise and why values are still within the previously stated “normal” range due to the rapid repair process. Similar responses have been postulated in chronic inflammatory diseases where the use of resistance training decreases the level of circulating inflammatory biomarkers . One study that investigated resistance training over a one year period identified no change in IL-6 basal levels. The study also found a reduction in plasma CRP  whilst several reviews have stated that TNF-α does not change in response to resistance training when CRP decreases . Furthermore, the 24 h between training sessions may be sufficient to allow the repair process to complete and thusly, for inflammation to become undetectable.
Two trends are evident as a result of this research. There is an increasing concentration of neopterin and total neopterin toward the end of the training week, and an observable difference between day one and days seven and eight. This suggests that there may be an accumulation of inflammation as a result of 5 days of intense resistance training (Figure 3A and B) and that some subjects might not have been fully recovered before the commencement of a new training week. This information might be crucial for some individuals to tailor their training specifically around their needs rather than conforming to what is considered the “best” strategy or “norm” for developing muscle.
The research to date remains equivocal with regard to long term resistance training and its effects on physiological and biochemical markers of stress. Whilst some use traditional resistance training of 2–3 days a week at 80% 1RM to monitor inflammation , the sampling time and number of training days remain different. Some studies have measured before and after a training session  whilst others have used a 7-week training period followed by an acute bout of resistance exercise to differentiate any changes in stress markers . These approaches are not in accordance with common body building or weight lifting protocols and thus cannot be used to definitively define the stress accompanying commonly used training programs. Studies must differentiate the foci of the research between an acute or chronic exercise focus in order to produce a clear and relevant approach to data collection and analysis. This study determined the chronic exposure to high intensity resistance training and subsequently allowed the observation of delayed onset muscle soreness and chronic inflammation. With the highest values observed in the days of rest, the data indicate increased levels of inflammation as a result of five days of resistance training. Peaks may have been observed immediately post exercise, and the fact that these high levels were seen following days of rest suggests that there are still sites of muscle damage present that are invaded by activated macrophages producing 7,8-dihydroneopterin and neopterin, resulting in values significantly higher than in the controls.
Of most significance to a sport like body building and similarly weight lifting is the overall stress due to continued resistance training and whether or not an individual’s body is recovering on a weekly basis. Studies have highlighted the decrease in circulating levels of CRP following a year of training. When a competitive athlete trains, adapts and subsequently increases the intensity of their workouts as a result of larger muscle fibres, they are continuously pushing their body to a new limit through the course of training. Sample size and several other markers of inflammation should consequently be considered in evaluating chronic inflammation in body building training. Individual differences observed in this research (Figure 4) highlight the importance of separating research based on group or population data with variable individual response. Although there were no significant differences between any days of training or rest when the mean values for the group are compared, some specific patterns are observed for certain individuals (Figure 4). Based upon questionnaires gathered from each subject, none exhibited any cold or flu symptoms. Neither did they exhibit any symptoms during the week following training. This suggests the values reported are indeed a true representation of the training week and imply that such training (5 days training, 2 days rest) results in increased levels of inflammation. Other individuals are able to cope with this intensity and can subsequently continue with their current training regime without a hindrance to recovery or an over-active immune system.
The level of oxidation throughout the week (Figures 3C and 4C) suggests that the level of oxidation remained constant regardless of the day of the week, and that resistance training of this nature does not induce an increased flux of free radicals. Individually, there were significant differences between subjects which further highlights the need to observe the individual and their unique response to stress rather than a group. It is difficult to ascertain whether the total amount of 7,8-dihydroneopterin being produced or the amount of oxidative stress present are the limiting factor of 7,8-dihydroneopterin oxidation to neopterin, based on the lack of change in neopterin or total neopterin within the group. On an individual basis, the level of oxidation changes dramatically with each person which could potentially be attributed to either 7,8-dihydroneopterin production or oxidative stress accumulation (Figure 4C). The source of plasma and urinary neopterin is poorly understood. Hydrogen peroxide and other reactive oxygen species generate dihydroxanthopterin, not neopterin, from 7,8-dihydroneopterin. Hypochlorite, which is generated by activated macrophages and neutrophils during inflammation, does oxidise 7,8-dihydroneopterin to neopterin, but whether this occurs in vivo has not been determined [25, 27, 28, 56]. Resultantly, though it is likely that there are other mechanisms for neopterin generation during inflammation in addition to hypochlorite, they have yet to be described. Further debate also continues about the rate order kinetics of 7,8-dihydroneopterin to neopterin conversion and whether the concentration of oxidants is the limiting step in 7,8-dihydroneopterin oxidation or whether the total amount of 7,8-dihydroneopterin present limits this conversion. Further research is required to identify the actual pathways that generate neopterin.
This study indicates that resistance training of this nature attributed to a group does not cause an increase in oxidative stress, but the level of oxidative stress is observably different for each individual as every individual physically responds and manages stress uniquely.
A new, reliable, and repeatable method for simultaneous detection of urinary neopterin and creatinine has been demonstrated using ion exchange SCX analytical chromatography. Neopterin and 7,8-dihydroneopterin are sensitive markers of immune system activation and inflammation in body building – a sport governed by high intensity resistance training. The training itself shows a trend toward increasing levels of inflammation even though values are within the upper limits of normality for signifiers of healthiness and the individual response of each subject should be meticulously considered. Creatinine and SG were found to be both reliable and effective means of correcting for urine volume.
This work was supported by the Free Radical Biochemistry Laboratory in the School of Biological Sciences, University of Canterbury.
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