Angus Lindsay, Tejraj Janmale, Nick Draper and Steven P. Gieseg

Measurement of changes in urinary neopterin and total neopterin in body builders using SCX HPLC

Open Access
De Gruyter | Published online: July 23, 2014


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 [6] 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 [15]. 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 [15]. 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) [23] and coronary artery disease [24]. 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.

HPLC analysis

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 [36]. 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 [37] as it is fast, reliable and accurate [38]. 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 [38]. 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 [29]. It is common practice in medicine to use creatinine as a marker of urine volume correction [41] as it is released at a fairly constant rate [42]. 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.

Table 1

Subject characteristics.

Subject Age Height, m Weight, kg Phase Calories/day Experience
S1 25 1.68 70.0 Mass gain 2000 Novice
S2 46 1.70 92.0 Mass gain 3500 Novice
S3 25 1.77 77.1 Competition prep 1900 Multiple shows
S4 22 1.68 76.0 Mass gain 2000 1 show
S5 26 1.91 88.0 Competition prep 2500 Novice
S6 23 1.74 80.0 Mass gain 4000 Novice
S7 34 1.63 58.5 Competition prep 1300 Multiple shows
S8 19 1.78 79.0 Competition prep 2500 Novice
Mean 27.5 1.74 77.6 2462
SD 8.6 0.09 10.3 889

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.

Experimental design

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.

Sample preparation

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 [43].

(1) S G - c o r r e c t e d c o n c . = (1 .020 1)/(SG sa m ple 1) × [ n e o p t e r i n ]/nmol  (1)

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.

Statistical analysis

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.

Figure 1 SCX chromatography analysis of neopterin in a control subject’s urine. The neopterin was detected by it native fluorescence (Ex 353 nm Em 438 nm) at 7.4 min as indicated by the arrow.

Figure 1

SCX chromatography analysis of neopterin in a control subject’s urine. The neopterin was detected by it native fluorescence (Ex 353 nm Em 438 nm) at 7.4 min as indicated by the arrow.

Figure 2 Elution of creatinine from the same control subject’s urine shown in Figure 1. HPLC analysis was the same as in Figure 1 but this chromatograph is from the spectrophotometer positioned after the fluorescence detector. The chromatograph shows the absorbance of the eluent at 234 nm with the creatinine indicated by the arrow eluting at 20.4 min.

Figure 2

Elution of creatinine from the same control subject’s urine shown in Figure 1. HPLC analysis was the same as in Figure 1 but this chromatograph is from the spectrophotometer positioned after the fluorescence detector. The chromatograph shows the absorbance of the eluent at 234 nm with the creatinine indicated by the arrow eluting at 20.4 min.

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.

Table 2

Control subjects (C) and mean daily body building subjects’ neopterin and total neopterin concentrations corrected with creatinine and specific gravity.

Day NP/CR, μmol/mol TNP/CR, μmol/mol NP/SG, nmol/SG1.020 TNP/SG, nmol/SG1.020
C 93±39 175±64 1233±681 2234±1028
1 124±58 360±173a 1345±673 3840±1864a
2 140±56a 343±84a 1530±566 3793±1294a
3 131±75 318±139a 1263±467 3308±1630a
4 142±68 344±138a 1946±818 4699±1449a
5 145±62a 341±105a 1636±562 3794±808a
6 158±79a 385±168a 1803±958 4508±2563a
7 185±79a 485±231a 2729±2106 6944±5413a
8 156±69a 470±176a 2160±1607 6493±4878a

Values are mean±SD. adenotes statistically different from control levels, p<0.05.

Figure 3 Mean urinary neopterin and total neopterin concentrations for the group over 8 days of training with Saturday and Sunday left as rest days (no training). The data shows neopterin and total neopterin (neopterin+7,8-dihydroneopterin) corrected for urine volume using either creatinine (A) or specific gravity (B). There was no significant change in the percentage of neopterin to total neopterin (C) over the week of analysis for the group data whether it was corrected using creatinine or specific gravity.Error bars show standard deviation of the data.

Figure 3

Mean urinary neopterin and total neopterin concentrations for the group over 8 days of training with Saturday and Sunday left as rest days (no training). The data shows neopterin and total neopterin (neopterin+7,8-dihydroneopterin) corrected for urine volume using either creatinine (A) or specific gravity (B). There was no significant change in the percentage of neopterin to total neopterin (C) over the week of analysis for the group data whether it was corrected using creatinine or specific gravity.

Error bars show standard deviation of the data.

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 [29] 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.

Figure 4 Urinary neopterin concentration (A) and total neopterin (B) corrected with creatinine for individual body builders. The percent neopterin as a measure of oxidation of 7,8-dihydroneopterin to neopterin is displayed for the individuals over the course of training, using creatinine to correct for urine volume.

Figure 4

Urinary neopterin concentration (A) and total neopterin (B) corrected with creatinine for individual body builders. The percent neopterin as a measure of oxidation of 7,8-dihydroneopterin to neopterin is displayed for the individuals over the course of training, using creatinine to correct for urine volume.


The method commonly employed for urinary neopterin detection is based on the reverse phase method developed in 1979 [37]. 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 [29]. 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 [44], 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 [29], 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 [29]. Other research has identified “normal” NP/CR as 294.6±178.6 [47] 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 [48]. 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 [8]. 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 [51]. 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 [15] whilst several reviews have stated that TNF-α does not change in response to resistance training when CRP decreases [52]. 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 [53], the sampling time and number of training days remain different. Some studies have measured before and after a training session [54] whilst others have used a 7-week training period followed by an acute bout of resistance exercise to differentiate any changes in stress markers [55]. 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.


1. Gibala M, MacDougall J, Tarnopolsky M, Stauber W, Elorriaga A. Changes in human skeletal muscle ultrastructure and force production after acute resistance exercise. J Appl Physiol 1995;78:702–8. Search in Google Scholar

2. Newham D, Jones D, Clarkson P. Repeated high-force eccentric exercise: effects on muscle pain and damage. J Appl Physiol 1987;63:1381–6. Search in Google Scholar

3. Roth SM, Martel GF, Ivey FM, Lemmer JT, Metter EJ, Hurley BF, et al. High-volume, heavy-resistance strength training and muscle damage in young and older women. J Appl Physiol 2000;88:1112–8. Search in Google Scholar

4. Staron RS, Hikida RS, Murray TF, Nelson MM, Johnson P, Hagerman F. Assessment of skeletal muscle damage in successive biopsies from strength-trained and untrained men and women. Eur J Appl Physiol Occup Physiol 1992;65:258–64. Search in Google Scholar

5. Howatson G, Van Someren KA. The prevention and treatment of exercise-induced muscle damage. Sports Med 2008;38:483–503. Search in Google Scholar

6. Calle MC, Fernandez ML. Effects of resistance training on the inflammatory response. Nutr Res Pract 2010;4:259–69. Search in Google Scholar

7. Radom-Aizik S, Leu S-Y, Cooper DM, Zaldivar F Jr. Serum from exercising humans suppresses t-cell cytokine production. Cytokine 2007;40:75–81. Search in Google Scholar

8. Petersen AM, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol 2005;98:1154–62. Search in Google Scholar

9. Miles MP. How do we solve the puzzle of unintended consequences of inflammation? Systematically. J Appl Physiol 2008;105:1023–5. Search in Google Scholar

10. Honda H, Kimura H, Rostami A. Demonstration and phenotypic characterization of resident macrophages in rat skeletal muscle. Immunology 1990;70:272. Search in Google Scholar

11. Lowe DA, Warren GL, Ingalls CP, Boorstein DB, Armstrong R. Muscle function and protein metabolism after initiation of eccentric contraction-induced injury. J Appl Physiol 1995;79:1260–70. Search in Google Scholar

12. McLennan IS. Resident macrophages (ED2-and ED3-positive) do not phagocytose degenerating rat skeletal muscle fibres. Cell Tissue Res 1993;272:193–6. Search in Google Scholar

13. Nieman DC. Prolonged aerobic exercise, immune response, and risk of infection. In: Hoffman-Goetz, editor. Exercise and immune function. Boca Raton, FL: CRC Press, 1996:143–62. Search in Google Scholar

14. Nieman D, Johanssen L, Lee J, Arabatzis K. Infectious episodes in runners before and after the Los Angeles Marathon. J Sports Med Phys Fitness 1990;30:316. Search in Google Scholar

15. Olson T, Dengel D, Leon A, Schmitz K. Changes in inflammatory biomarkers following one-year of moderate resistance training in overweight women. Int J Obes 2007:31:996–1003. Search in Google Scholar

16. Tomaszewski M, Charchar FJ, Przybycin M, Crawford L, Wallace AM, Gosek K, et al. Strikingly low circulating CRP concentrations in ultramarathon runners independent of markers of adiposity. Arterioscler Thromb Vasc Biol 2003;23:1640–4. Search in Google Scholar

17. Damas P, Ledoux D, Nys M, Vrindts Y, De Groote D, Franchimont P, et al. Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann Surg 1992;215:356. Search in Google Scholar

18. Ronsen O, Lea T, Bahr R, Pedersen BK. Enhanced plasma IL-6 and IL-1ra responses to repeated vs. single bouts of prolonged cycling in elite athletes. J Appl Physiol 2002;92:2547–53. Search in Google Scholar

19. Breen E, Rezai A, Nakajima K, Beall G, Mitsuyasu R, Hirano T, et al. Infection with HIV is associated with elevated IL-6 levels and production. J Immunol 1990;144:480–4. Search in Google Scholar

20. Müller MM, Curtius HC, Herold M, Huber CH. Neopterin in clinical practice. Clin Chim Acta 1991;201:1. Search in Google Scholar

21. Wachter H. Neopterin: biochemistry, methods. Clinical Application: Berlin-New York: de Gruyter, 1992. Search in Google Scholar

22. Gieseg S, Crone E, Flavall E, Amit Z. Potential to inhibit growth of atherosclerotic plaque development through modulation of macrophage neopterin/7, 8-dihydroneopterin synthesis. Br J Pharmacol 2008;153:627–35. Search in Google Scholar

23. Fuchs D, Chiodi F, Albert J, Asjö B, Hagberg L, Hausen A, et al. Neopterin concentrations in cerebrospinal fluid and serum of individuals infected with HIV-1. Aids 1989;3:285–8. Search in Google Scholar

24. Garcia-Moll X, Cole D, Zouridakis E, Kaski J. Increased serum neopterin: a marker of coronary artery disease activity in women. Heart 2000;83:346–50. Search in Google Scholar

25. Widner B, Mayr C, Wirleitner B, Fuchs D. Oxidation of 7, 8-dihydroneopterin by hypochlorous acid yields neopterin. Biochem Biophys Res Commun 2000;275:307–11. Search in Google Scholar

26. Gieseg SP, Maghzal G, Glubb D. Protection of erythrocytes by the macrophage synthesized antioxidant 7, 8 dihydroneopterin. Free Radic Res 2001;34:123–36. Search in Google Scholar

27. Gieseg SP, Maghzal G, Glubb D. Inhibition of haemolysis by the macrophage synthesized antioxidant, 7, 8-dihydroneopterin. Redox Rep 2000;5:2–3. Search in Google Scholar

28. Gieseg SP, Whybrow J, Glubb D, Rait C. Protection of U937 cells from free radical damage by the macrophage synthesized antioxidant 7, 8-dihydroneopterin. Free Radic Res 2001;35:311–8. Search in Google Scholar

29. Wachter H, Fuchs D, Hausen A, Reibnegger G, Werner ER. Neopterin as marker for activation of cellular immunity: immunologic basis and clinical application. Adv Clin Chem 1989;27:1–141. Search in Google Scholar

30. Sprenger H, Jacobs C, Nain M, Gressner A, Prinz H, Wesemann W, et al. Enhanced release of cytokines, interleukin-2 receptors, and neopterin after long-distance running. Clin Immunol Immunopathol 1992;63:188–95. Search in Google Scholar

31. Schobersberger W, Sumann G, Mittermayr M, Griesmacher A, Falkensammer G, Greie S, et al. Muscle trauma and immune activation after a downhill marathon (Tyrolean Speed Marathon). Pteridines 2007;17:121–8. Search in Google Scholar

32. Gunga HC, Machotta A, Schobersberger W, Mittermayr M, Kirsch K, Koralewski E, et al. Neopterin, IgG, IgA, IgM, and plasma volume changes during long-distance running. Pteridines 2002; 13:13–20. Search in Google Scholar

33. Tilz GP, Domej W, Diez-Ruiz A, Weiss G, Brezinschek R, Brezinschek HP, et al. Increased immune activation during and after physical Eexercise. Immunobiology 1993;188:194–202. Search in Google Scholar

34. Flavall EA, Crone EM, Moore GA, Gieseg SP. Dissociation of neopterin and 7, 8-dihydroneopterin from plasma components before HPLC analysis. J Chromatogr B 2008;863:167–71. Search in Google Scholar

35. Fuchs D, Granditsch G, Hausen A, Reibnegger G, Wachter H. Urinary neopterin excretion in coeliac disease. Lancet 1983;322:463–4. Search in Google Scholar

36. Westermann J, Thiemann F, Gerstner L, Tatzber F, Kozák I, Bertsch T, et al. Evaluation of a new simple and rapid enzyme-linked immunosorbent assay kit for neopterin determination. Clin Chem Lab Med 2000;38:345–53. Search in Google Scholar

37. Wachter H, Hausen A, Grassmayr K. Increased urinary excretion of neopterin in patients with malignant tumors and with virus diseases. Hoppe Seyler’s Z Physiol Chem 1979;360:1957. Search in Google Scholar

38. Fuchs D, Hausen A, Reibnegger G, Wachter H. Automatized routine estimation of neopterin in human urine by HPLC on reversed phase. Biochem Clin Aspect Pteridines 1982;1:67–79. Search in Google Scholar

39. Schulze JJ, Lundmark J, Garle M, Skilving I, Ekström L, Rane A. Doping test results dependent on genotype of uridine diphospho-glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. J Clin Endocrinol Metabol 2008;93:2500–6. Search in Google Scholar

40. Fuith LC, Hetzel H, Fuchs D, Hausen A, Reibnegger G, Werner ER, et al. Urinary neopterin excretion in patients with uterine sarcomas. Cancer 1990;65:1228–31. Search in Google Scholar

41. Iizuka T, Minatogawa Y, Suzuki H, Itoh M, Nakamine S, Hatanaka Y, et al. Urinary neopterin as a predictive marker of coronary artery abnormalities in Kawasaki syndrome. Clin Chem 1993;39:600–4. Search in Google Scholar

42. Shaffer P. The excretion of kreatinin and kreatin in health and disease. Am J Physiol 1908;23:1–17. Search in Google Scholar

43. Goldberger B, Loewenthal B, Darwin WD, Cone EJ. Intrasubject variation of creatinine and specific gravity measurements in consecutive urine specimens of heroin users. Clin Chem 1995;41:116–7. Search in Google Scholar

44. Cone EJ, Caplan YH, Moser F, Robert T, Shelby MK, Black DL. Normalization of urinary drug concentrations with specific gravity and creatinine. J Anal Toxicol 2009;33:1–7. Search in Google Scholar

45. Decombaz J, Reinhardt P, Anantharaman K, Von Glutz G, Poortmans J. Biochemical changes in a 100 km run: free amino acids, urea, and creatinine. Eur J Appl Physiol Occup Physiol 1979;41:61–72. Search in Google Scholar

46. Anderson RA, Polansky MM, Bryden NA, Roginski EE, Patterson KY, Reamer DC. Effect of exercise (running) on serum glucose, insulin, glucagon, and chromium excretion. Diabetes 1982;31:212–6. Search in Google Scholar

47. de Castro MR, Di Marco GS, Arita DY, Teixeira LC, Pereira AB, Casarini DE. Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection. J Biochem Biophys Methods 2004;59:275–83. Search in Google Scholar

48. Fuchs D, Milstien S, Krämer A, Reibnegger G, Werner ER, Goedert JJ, et al. Urinary neopterin concentrations vs total neopterins for clinical utility. Clin Chem 1989;35:2305–7. Search in Google Scholar

49. Nieman DC, Davis J, Brown VA, Henson DA, Dumke CL, Utter AC, et al. Influence of carbohydrate ingestion on immune changes after 2 h of intensive resistance training. J Appl Physiol 2004;96:1292–8. Search in Google Scholar

50. Peake J, Nosaka KK, Muthalib M, Suzuki K. Systemic inflammatory responses to maximal versus submaximal lengthening contractions of the elbow flexors. Exerc Immunol Rev 2006;12:72–85. Search in Google Scholar

51. Beavers KM, Brinkley TE, Nicklas BJ. Effect of exercise training on chronic inflammation. Clin Chim Acta 2010;411: 785–93. Search in Google Scholar

52. Ploeger HE, Takken T, De Greef M, Timmons BW. The effects of acute and chronic exercise on inflammatory markers in children and adults with a chronic inflammatory disease: a systematic review. Exerc Immunol Rev 2009;15:6–41. Search in Google Scholar

53. Stewart LK, Flynn MG, Campbell WW, Craig BA, Robinson JP, Timmerman KL, et al. The influence of exercise training on inflammatory cytokines and C-reactive protein. Med Sci Sports Exerc 2007;39:1714. Search in Google Scholar

54. Peake JM, Suzuki K, Wilson G, Hordern M, Nosaka K, Mackinnon L, et al. Exercise-induced muscle damage, plasma cytokines, and markers of neutrophil activation. Med Sci Sports Exerc 2005;37:737. Search in Google Scholar

55. Izquierdo M, Ibañez J, Calbet JA, Navarro-Amezqueta I, González-Izal M, Idoate F, et al. Cytokine and hormone responses to resistance training. Eur J Appl Physiol 2009;107:397–409. Search in Google Scholar

56. Schraufstätter I, Browne K, Harris A, Hyslop PA, Jackson JH, Quehenberger O, et al. Mechanisms of hypochlorite injury of target cells. J Clin Invest 1990;85:554. Search in Google Scholar

Received: 2014-2-25
Accepted: 2014-4-8
Published Online: 2014-7-23
Published in Print: 2014-7-1

©2014 by Walter de Gruyter Berlin/Boston

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