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Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage
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Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage
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Angus Lindsay a, Sam Carr a, Nick Draper b, Steven P. Gieseg a, c, * a b c
Free Radical Biochemistry Laboratory, School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand College of Life and Natural Sciences, University of Derby, Derby DE22 1GB, UK Department of Radiology, University of Otago, Christchurch 8011, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2015 Received in revised form 1 September 2015 Accepted 1 September 2015 Available online xxx
This study investigated a means of quantifying urinary myoglobin using a novel reverse-phase highperformance liquid chromatography (RPeHPLC) method that is an alternative measure of exerciseinduced muscle damage. It also investigated the effect of storage and alkalization on urinary myoglobin stability issues. An RPeHPLC method was validated by precision and repeatability experiments. Myoglobin stability was determined through spiked urine samples stored at various temperatures over an 8-week period using alkalization and dilution in a pH 7.0 buffer. The method was validated with urine collected from mixed martial arts fighters during a competition and training session. The method produced linearity from 5 to 1000 mg/ml (R2 ¼ 0.997), intra- and inter-assay coefficients of variation from 0.32 to 2.94%, and a lower detection limit of 0.2 mg/ml in the final dilution and 2 mg/ml in the original urine sample. Recovery ranged from 96.4 to 102.5%, myoglobin remained stable at 4 C when diluted in a pH 7.0 buffer after 20 h, and a significant increase (P < 0.01) and an identifiable peak were observed following a mixed martial arts contest and training session. Storage length and conditions had significant effects (P < 0.05) on stability. The method's simplicity and noninvasive nature means it can be used as an alternate noninvasive muscle damage assay following exercise and trauma. © 2015 Elsevier Inc. All rights reserved.
Keywords: HPLC Myoglobin Mixed martial arts Stability Muscle damage Exercise
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Myoglobin, the hemoprotein that facilitates oxygen storage for sustained oxidative phosphorylation in muscle fibers, is a key marker of muscle damage that has notably been employed to assess the severity of injury and monitor the recovery of individuals suffering from rhabdomyolysis [1], myocardial infarction [2], accidental trauma [3], and exercise [4,5]. Muscle damage quantification is also pertinent to the diagnosis of exercise-induced physiological stress for the management of athlete recovery and performance. To this end, myoglobin has been routinely measured in several exercise studies ranging from rugby union [6,7] to ultra-endurance exercise [8]. The methodologies employed for myoglobin measurement include sensitive and expensive immunoassays, dipsticks,
Abbreviations: ELISA, enzyme-linked immunosorbent assay; RPeHPLC, reversephase high-performance liquid chromatography; UV, ultraviolet. * Corresponding author. Free Radical Biochemistry Laboratory, School of BiologQ1,2 ical Sciences, University of Canterbury, Christchurch 8140, New Zealand. E-mail address: [email protected] (S.P. Gieseg).
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histochemical staining, radioimmunoassays, and relatively inexpensive liquid chromatograph assays [6,9,10]. Enzyme-linked immunosorbent assays (ELISAs) are capable of quantifying myoglobin concentrations through their rapid and sensitive processing time. However, de Waard and van't Sant [11] highlighted ELISAs' instability, inaccuracy, and limited detection range (~1 mg/ ml), suggesting that all immunoassay-based myoglobin assays are unsuitable. Similarly, urinary dipsticks are unable to differentiate clearly between myoglobin and hemoglobin and do not offer a quantification method [12]. When concentrations range from 3.2 to 3000 mg/L following serious trauma, myocardial infarction, and professional rugby [2,4,13], repeated handling and the inability to specifically quantify muscle damage severity make immunoassay options rather impractical. Likewise, compounding effects such as pH, temperature, freezing, and unidentified urinary compounds smaller than 10 kDa routinely affect stability and the ability to accurately quantify myoglobin [14]. The need for a noninvasive, reliable, repeatable, simple, and cost-effective alternative assay for the quantification of muscle
http://dx.doi.org/10.1016/j.ab.2015.09.001 0003-2697/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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damage following physical exercise is essential. Creatine kinase and lactate dehydrogenase are routinely measured in exercise research through plasma analysis [15,16] but can be severely limited by its delayed response to the muscle-damaging exercise [17] and its invasive approach, providing robust reasoning for the requirement of an assay that can provide an immediate noninvasive quantification of muscle damage. Furthermore, several size exclusion, anion exchange, and reverse-phase high-performance liquid chromatography (RPeHPLC) techniques have been used for protein and myoglobin determination [2,18,19]; however, they are somewhat outdated, coelute proteins of similar size, are unvalidated in urine, or require more than one preparative process. RPeHPLC methods commonly use water and an organic solvent for separation and identification that have shown a recovery of approximately 100% following addition to an extract [20]. We have adopted this methodology to develop, validate, and report a similarly reliable RPeHPLC method that quantifies myoglobin in urine by simple ultraviolet (UV) detection that negates the use of invasive procedures. The aims of this research were to develop a simple and noninvasive RPeHPLC method that can reliably quantify urinary myoglobin following exercise-induced muscle damage as an alternative to commonly used invasive approaches and to investigate the effect of alkalization and sample dilution on urinary myoglobin stability for sample preparation optimization. Materials and methods All solutions and reagents were prepared with water purified using the NANOpure ultrapure water system from Barnstead/ Thermolyne (Dubuque, IA, USA). Chemicals and reagents were supplied from Sigma Chemical (St. Louis, MO, USA), Abcam (Melbourne, Australia), and BDH Chemicals New Zealand (Auckland, New Zealand). Precision studies Spectral analyses of commercially supplied pure human (Abcam, ab96036) and equine (SigmaeAldrich, M0630) skeletal myoglobin were conducted to identify the optimal absorbance level of myoglobin, whereas spectral analysis at 210, 280, and 400 nm were conducted by RPeHPLC on spiked myoglobin urine samples to identify any interference from other urinary compounds. Intraassay precision was evaluated using 20 replicates of a single urine sample in a single analytical run spiked with 10, 100, and 1000 mg/
ml myoglobin standard. Inter-assay precision was evaluated using 20 replicates of a single urine sample on 4 consecutive days for each of the three concentrations while a calibration curve was established using myoglobin standards ranging from 5 to 1000 mg/ml for both human and equine skeletal myoglobin. The lower detection limit for the assay was established using spiked urine samples where the peak was three times greater than the baseline noise, as described previously [19]. Recovery and stability The recovery and stability of the myoglobin was investigated by spiking a urine sample with known concentrations of human myoglobin standard (10e1000 mg/ml). Standards ranging from 10 to 1000 mg/ml were added to urine samples before being diluted 1:10 with 10 mM ammonium acetate (pH 7.0) and analyzed every 4 h for a 28-h period at 4 C. This was to replicate standard urine collection protocols. This time period was specifically chosen due to the length of each assay (32 min) while accounting for as many as 40 samples in any one analytical run. Because myoglobin is notoriously unstable in urine when measured by ELISA [14], the stability of myoglobin in a urine sample over an extended period of time was examined to determine percentage recovery. To a urine sample, 10, 100, and 1000 mg/ml human myoglobin standard was added and diluted 1:10 with 10 mM ammonium acetate (pH 7.0) before being either (i) frozen at 80 C, (ii) frozen at 20 C, or (iii) refrigerated at 4 C for 1, 2, 4, and 8 weeks before thawing and analysis to determine stability and degradation kinetics. All experimental research was conducted in triplicate. Clinical subjects and experimental design This analysis methodology was validated from urine collected from 14 mixed martial artists (178.4 ± 8.4 cm, 84.3 ± 12.9 kg, 26.6 ± 6.2 years), following a simulated contest (n ¼ 10) and contest preparation training session (n ¼ 13), who were informed of the risks involved in the study before their written consent was obtained. The simulated contest consisted of three rounds of 5 min with 60 s between rounds, whereas the training session consisted of individual fighting for three rounds of 5 min followed by sparring, wrestling, and fitness for a total session time of approximately 75e90 min. Samples were collected using a 70-ml collection pot midstream before, post, and 1 and 2 h post each event. At the time of collection, all samples were pH corrected and diluted with 10 mM ammonium acetate (pH 7.0) (1 ml urine:4 ml buffer) and
Fig. 1. Individual subjects' urinary myoglobin concentrationetime course changes for the MMA contest (A) and training session (B).
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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66 67 68 69 70 HPLC analysis 71 72 HPLC measurement of myoglobin was performed using a Shi73 madzu Sil-20A HPLC device with autosampler and an RF-20Axls 74 and SPD-20A photo diode array detector. A sample (10 ml) was 75 injected onto a Jupiter C5 300 Å column (5-mm, 150 4.6 mm). The 76 mobile phase was generated by mixing 0.1% (v/v) trifluoroacetic 77 acid (pH 2.5) in water (A) with 100% acetonitrile (B) by pre-pump 78 mixing to give 10% A/90% B at 1 ml/min. The myoglobin was 79 eluted with a linear gradient from 10 to 70% B over 15 min and was 80 followed by 70% B for a further 5 min. The column was returned to 81 the starting condition by 70 to 10% B gradient over 2 min and 82 holding at 10% B for 10 min, making a total run time of 32 min. 83 Myoglobin was detected by absorbance at 400 nm. All analytes 84 were quantified by peak area using Shimadzu Class VP software and 85 were conducted in duplicate. 86 87 Statistical analysis 88 89 Statistical analysis was conducted using the Statistical Package 90 for Social Sciences version 20. A two-way analysis of variance 91 (ANOVA) using Tukey's test for post hoc analysis was conducted on 92 all recovery and time course experiments (Fig. 1). Statistical sig- Q5 93 nificance was set at 0.05. 94 95 96 Results 97 98 Equine skeletal myoglobin provided near identical properties to 99 human skeletal myoglobin as determined by spectrophotometry 100 and HPLC analysis with spectrophotometric detection. Therefore, 101 we suggest the use of equine skeletal myoglobin as a standard for 102 clinical measurements by HPLC due to its relative availability and 103 lower purchase price in comparison with human myoglobin. 104 Urinary myoglobin was detected at 400 nm by RPeHPLC eluting 105 at approximately 15 min with no visible tailing of the peak or 106 interference from neighboring peaks. After spectrophotometer 107 analysis identified 210, 280, and 400 nm as the optimal absorbance 108 wavelengths (data not shown), spectral analysis of a urine sample 109 spiked with myoglobin by HPLC found that 400 nm provided the 110 highest absorbance units and lowest signal-to-noise ratio and did 111 not interfere with any other urinary compound at that wavelength, 112 although 280 nm also provided a clear and visible peak. 113 The method was validated in all 14 urine samples that were 114 collected following both events. Myoglobin was detectable in 5 of 10 subjects following the fight (0.95e13.24 mg/ml) (Fig. 2A and 11 of Q6 115 116 14 subjects following the training session (0.19e63.3 mg/ml) 117 (Fig. 2B). All subjects had undetectable concentrations (lower 118 detection limit of 0.2 mg/ml) before the competition or training 119 session. For one of the selected fighters, myoglobin was undetect120 able pre-fight (Fig. 3A), rose to 3.82 mg/ml immediately post-fight 121 with the identification of a single peak (Fig. 3B), peaked 122 (13.24 mg/ml) 1 h post-fight (Fig. 3C), and dropped to 1.24 mg/ml 2 h 123 post-fight (Fig. 3D). For a selected subject participating in the 124 contest preparation training session, myoglobin was undetectable 125 immediately pre (Fig. 3E), rose to 5.83 mg/ml immediately post 126 (Fig. 3F), peaked 1 h post at 63.3 mg/ml (Fig. 3G), and was still 127 detectable (7.91 mg/ml) 2 h post (Fig. 3H). 128 The intra- and inter-assay coefficients of variation for 10, 100, and 129 1000 mg/ml were 1.49 and 2.94%, 0.39 and 0.70%, and 0.32 and 0.79%, 130 respectively, whereas the assay presented linearity in both human placed on ice immediately at location. They were transported back to the laboratory for immediate analysis. This was approved by the Health and Disability Ethics Committee (Wellington, New Zealand).
Fig. 2. (A) Calibration curve for human and equine skeletal myoglobin at concentrations ranging from 5 to 1000 mg/ml detected at 400 nm. (B,C) Lower detection limit for the RPeHPLC method as indicated by the arrow. An unspiked urine sample (B) and a spiked urine sample (2 mg/ml) before the 1:10 dilution (C) at the same sensitivity are shown. Lower detection was calculated when the peak was three times the size of the baseline noise.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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Fig. 3. RPeHPLC chromatographs of two selected mixed martial arts fighters following a simulated contest (AeD) and a competition preparation training session (E, F). Pre (A, E), post (B, F), 1 h post (C, G), and 2 h post (D, H) contest/training session chromatographs indicating myoglobin (arrow) eluting at approximately 15 min at an absorbance wavelength of 400 nm are shown.
(R2 ¼ 0.998) and equine (R2 ¼ 0.997) over the range of 5e1000 mg/ml (Fig. 2A) with a lower detection limit of 0.2 mg/ml (Fig. 2B and C). Myoglobin was more stable following alkalization and dilution with 10 mM ammonium acetate (pH 7.0) over a 28-h analytical run in comparison with purified water (data not shown). Therefore, it was decided to pursue further stability experiments using this protocol. Myoglobin recovery from spiked urine samples ranged from 96.4 to 102.5%, whereas its stability over 28 h in a urine sample diluted with 10 mM ammonium acetate (pH 7.0) at 4 C decreased by 9.71, 7.12, and 2.12% for 10, 100, and 1000 mg/ml, respectively, at the 28-h time point (Fig. 4). Meanwhile, storage length and conditions had significant effects (P < 0.05) on myoglobin stability in urine (Table 1). The longer a sample was stored, the more degradation occurred. However, this loss was attenuated by the storage temperature (P < 0.01), whereas the greater the concentration, the slower the degradation process (P < 0.01).
suitable, reliable, and rapid (32 min) alternative RPeHPLC method for the quantification of urinary myoglobin following exerciseinduced muscle damage. The concentrations observed following each of the mixed martial arts events suggest that a training session is of greater intensity than a simulated contest, most likely a result of the difference in duration. Moreover, it is comparable to other
Discussion Myoglobin is typically quantified in urine through sensitive and rapid immunoassays [21] as well as several forms of HPLC [2]. With intra- and inter-assay variations in accordance with the accepted range for a reliable and repeatable assay, we have demonstrated a
Fig. 4. Myoglobin recovery and stabilization over 28 h from urine samples diluted 1:10 with 10 mM ammonium acetate (pH 7.0) and spiked with concentrations ranging from 10 to 1000 mg/ml at 4 C. All analyses were completed in triplicate, and data are presented as means ± standard deviations.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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A. Lindsay et al. / Analytical Biochemistry xxx (2015) 1e6 Table 1 Storage effect on myoglobin in a pH corrected and diluted urine sample spiked with known concentrations. [Myoglobin] (mg/ml)
Time point (weeks)
Conditions 4 C
20 C
80 C
10
1 2 4 8 1 2 4 8 1 2 4 8
5.3 ± 0.2 4.6 ± 0.3 3.1 ± 0.1 2.7 ± 0.1 81.0 ± 0.3 83.4 ± 0.1 77.3 ± 0.8 72.3 ± 0.1 975.3 ± 1.2 963.4 ± 20.6 947.9 ± 4.6 924.5 ± 18.0
6.3 ± 0.3 5.1 ± 0.2 4.2 ± 0.01 4.2 ± 0.02 81.2 ± 0.04 83.2 ± 0.7 71.6 ± 3.9 67.0 ± 1.4 998.6 ± 22.0 980.9 ± 7.6 952.7 ± 6.8 941.9 ± 7.5
7.3 ± 0.01 6.1 ± 0.5 6.0 ± 0.2 6.4 ± 0.01 86.0 ± 0.1 91.4 ± 2.0 82.3 ± 0.1 77.0 ± 0.5 987.5 ± 36.3 1020.4 ± 1.1 980.0 ± 2.2 977.0 ± 17.1
100
1000
Note. Three replicates of each concentration at each time point were analyzed. Data are means ± standard deviations (mg/ml).
high-impact sports that have shown similar changes in urinary myoglobin [4,5], which demonstrate the severity of the sport that has been widely neglected in exercise research. Detection of myoglobin at an absorbance of 400 nm produced a clear visible peak with no observed tailing or interference from neighboring urinary analytes allowing for clear interpretation, whereas the dilution in 10 mM ammonium acetate (pH 7.0) seemed to alleviate any stabilization concerns that had been previously noted over a 24-h period [14]. The mechanism of how this alkalization improves myoglobin stability has not yet been fully elucidated. However, it is believed to revolve around myoglobin's isoelectric point, where increasing the pH beyond 6.9 reduces its precipitation [22] and further prevents the dissociation of myoglobin into its smaller nephrotoxic components [1]. This issue becomes particularly imperative when assessing exercise-induced muscle damage and the potential for false-negative results arising from myoglobin degradation in the bladder [11]. In spite of relative stability over 28 h using this protocol, which alleviates any potential issue arising from the extended HPLC assay time, the time course experiments suggest that urinary myoglobin be analyzed immediately following collection and alkalization to avoid any potential degradation loss when concentrations are not known. These results expand on the previous findings of de Waard and van't Sant [11], who found that alkalized urine stored at 4 C decreases by up to 50% within a week. Whereas some compounds remain stable for months at 80 C [23], urine samples containing <1000 mg/ml myoglobin clearly do not remain stable for long periods of time and, therefore, should avoid freezeethawing for optimal analysis. Samples should be collected, alkalized, and analyzed or, if need be, diluted accordingly with a pH 7.0 buffer and stored promptly at 80 C on presentation. The dilution factor can, of course, be altered depending on myoglobin concentration or concern for column life expectancy. Myoglobin's detection and recovery from urine samples using this method is comparable to that using similar RPeHPLC methods [20] that provide a simple and cost-effective assay using UV detection for values in a clinical and exercise relevant range. Although it is relatively economical to run, setup costs for HPLC are quite expensive compared with traditional ELISA kits. Its detection limit is substantially lower (30 mg/ml) than that provided for myoglobin by size exclusion HPLC [19], whereas the ease of preparation for quantification provides a more simple option than previous methods [2,18]. Its use in a clinical and exercise context for diagnosis and observational purposes should, therefore, consider sample pH stabilization and dilution in 10 mM ammonium acetate (pH 7.0) followed by analysis within 20 h at 4 C to use this method
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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Conclusion 101 102 The quantification of urinary myoglobin using the described 103 RPeHPLC method provides a precise, simple, and cost-effective 104 method for muscle damage quantitation in a clinical and exercise 105 relevant range. The detection of myoglobin using this method in 106 mixed martial arts competitors identifies severe structural damage 107 following a contest, validating the usefulness of this method, 108 whereas immediate alkalization and dilution in a pH 7.0 buffer al109 leviates any stabilization concerns over 24 h. Through its simplicity, 110 noninvasiveness, and speed of analysis, this method can further be 111 used for evaluating the degree of muscle damage following 112 exercise-induced activities and sports. 113 114 Acknowledgments Q7 115 116 The authors thank St. George's Hospital, Heart Centre, and the 117 University of Canterbury for funding this project, Strikeforce Can118 terbury Gym, Karl Webber and each of the subjects for their plan119 ning and participation, and Matt Walters for his expertise in the 120 preparation of the figures. 121 122 References 123 124 [1] I. Alterman, A. Sidi, L. Azamfirei, S. Copotoiu, T. Ezri, Rhabdomyolysis: another 125 complication after prolonged surgery, J. Clin. Anesth. 19 (2007) 64e66. 126 [2] S. Powell, E. Friedlander, Z. Shihabi, Myoglobin determination by highperformance liquid chromatography, J. Chromatogr. A 317 (1984) 87e92. 127 [3] A. Genthon, S.R. Wilcox, Crush syndrome: a case report and review of the 128 literature, J. Emerg. Med. 46 (2014) 313e319. 129 [4] A. Lindsay, J. Lewis, C. Scarrott, N. Gill, S.P. Gieseg, N. Draper, Assessing the 130 effectiveness of selected biomarkers in the acute and cumulative physiological to its fullest capacity. This is highly feasible considering the noninvasive, nonburdening nature of this method with no need for venipuncture, the faster elimination kinetics of myoglobin compared with the sometimes delayed response of common muscle damage makers such as creatine kinase [24], and the ease of installation of the measurement apparatus within a laboratory or hospital setting. With commercial ELISAs having upper detection limits of approximately 1 mg/ml, the linearity of this method from 5 to 1000 mg/ml and the lower detection limit of 0.2 mg/ml provide a significant alternative for the quantification of a muscle damage marker. These properties are similar to those reported by anion exchange [2] and two-dimensional liquid chromatographyeelectrospray ionization mass spectrometry [25], which negates the dilution requirement for absolute concentration determination and which also avoids unnecessary repeated analysis. Although it does not have the sensitivity limit of 10 ng/ml that prior chemiluminescence detection has shown [26], the concentrations in the linear range are more clinically relevant and similar to those noted previously [2]. Feinfeld and coworkers [9] observed patients with acute rhabdomyolysis exceeding concentrations of 1 mg/ml, whereas previous studies have suggested that patients with values exceeding 20 mg/ml are at more of an increased risk of decreased renal function [21,27]. In addition, values as high as 32.9, 410, and 3000 mg/ml have been observed following crush trauma [3], strenuous military training [13], and myocardial infarction [2], respectively, whereas values reaching 52 and >100 mg/ml have been observed in competitors following a marathon [28] and professional rugby [5], respectively. Therefore, in an exercise and/or serious trauma scenario where muscle damage is expected to rise significantly, this assay may provide the most cost-effective and operator-friendly option for simple and rapid quantitation for both small and large cohorts suffering mild to severe skeletal muscle stress in a range that is widely accepted as “clinically relevant.”
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
stress response in professional rugby union through non-invasive assessment, Int. J. Sports Med. 36 (2015) 446e454. A. Lindsay, J. Healy, W. Mills, J. Lewis, N. Gill, N. Draper, S.P. Gieseg, Impactinduced muscle damage and urinary pterins in professional rugby: 7,8-dihydroneopterin oxidation by myoglobin, Scand. J. Med. Sci. Sports (2015), http:// dx.doi.org/10.1111/sms.12436. A. Lindsay, J. Lewis, C. Scarrott, N. Draper, S.P. Gieseg, Changes in acute biochemical markers of inflammatory and structural stress in rugby union, J. Sports Sci. 33 (2014) 882e891. A. Lindsay, J. Lewis, N. Gill, S.P. Gieseg, N. Draper, Effect of varied recovery interventions on markers of psychophysiological stress in professional rugby union, Eur. J. Sport Sci. 15 (2015) 543e549. L. Wallberg, C.M. Mattsson, J.K. Enqvist, B. Ekblom, Plasma IL-6 concentration during ultra-endurance exercise, Eur. J. Appl. Physiol. 111 (2011) 1081e1088. D. Feinfeld, J. Cheng, T. Beysolow, A.M. Briscoe, A prospective study of urine and serum myoglobin levels in patients with acute rhabdomyolysis, Clin. Nephrol. 38 (1992) 193e195. J. Royds, S. Variend, W. Timperley, C.B. Taylor, Comparison of beta enolase and myoglobin as histological markers of rhabdomyosarcoma, J. Clin. Pathol. 38 (1985) 1258e1260. H. de Waard, P. van't Sant, Use of serum myoglobin assays for urine myoglobin measurements can cause false-negative results, Clin. Chem. 55 (2009) 1240e1241. E.A. Anigilaje, O.T. Adedoyin, Correlation between dipstick urinalysis and urine sediment microscopy in detecting haematuria among children with sickle cell anaemia in steady state in Ilorin, Nigeria, Pan Afr. Med. J. 15 (2013) 135. R.F. Smith, Exertional rhabdomyolysis in naval officer candidates, Arch. Intern. Med. 121 (1968) 313e319. Z. Chen-Levy, M.H. Wener, B. Toivola, P. Daum, M. Reyes, J.S. Fine, Factors affecting urinary myoglobin stability in vitro, Am. J. Clin. Pathol. 123 (2005) 432e438. R.D. Johnston, T.J. Gabbett, A.J. Seibold, D.G. Jenkins, Influence of physical contact on neuromuscular fatigue and markers of muscle damage following small-sided games, J. Sci. Med. Sport 17 (2014) 535e540. T.K. Singh, K.J. Guelfi, G. Landers, B. Dawson, D. Bishop, A comparison of muscle damage, soreness, and performance following a simulated contact and non-contact team sport activity circuit, J. Sci. Med. Sport 14 (2011) 441e446.
[17] B. Cunniffe, A.J. Hore, D.M. Whitcombe, K.P. Jones, J.S. Baker, B. Davies, Time course of changes in immuneoendocrine markers following an international rugby game, Eur. J. Appl. Physiol. 108 (2010) 113e122. [18] D. Sviridov, B. Meilinger, S.K. Drake, G.T. Hoehn, G.L. Hortin, Coelution of other proteins with albumin during size-exclusion HPLC: implications for analysis of urinary albumin, Clin. Chem. 52 (2006) 389e397. [19] H. Liang, M.K. Scott, D.J. Murry, K.M. Sowinski, Determination of albumin and myoglobin in dialysate and ultrafiltrate samples by high-performance sizeexclusion chromatography, J. Chromatogr. B 754 (2001) 141e151. [20] I.M. Oellingrath, A. Iversen, G. Skrede, Quantitative determination of myoglobin and haemoglobin in beef by high-performance liquid chromatography, Meat Sci. 28 (1990) 313e320. [21] A. Wu, I. Laios, S. Green, T.G. Gornet, S.S. Wong, L. Parmley, A.S. Tonnesen, B. Plaisier, R. Orlando, Immunoassays for serum and urine myoglobin: myoglobin clearance assessed as a risk factor for acute renal failure, Clin. Chem. 40 (1994) 796e802. [22] T.W. King, O.Z. Lerman, J.J. Carter, S.M. Warren, Exertional compartment syndrome of the thigh: a rare diagnosis and literature review, J. Emerg. Med. 39 (2010) e93ee99. [23] A. Garde, Å. Hansen, Long-term stability of salivary cortisol, Scand. J. Clin. Lab. Invest. 65 (2005) 433e436. €ri, Elimination kinetics of [24] H. Lappalainen, E. Tiula, L. Uotila, M. M€ antta myoglobin and creatine kinase in rhabdomyolysis: implications for follow-up, Crit. Care Med. 30 (2002) 2212e2215. € pl, E. Lange, C. Klein, [25] B.M. Mayr, O. Kohlbacher, K. Reinert, M. Sturm, C. Gro C.G. Huber, Absolute myoglobin quantitation in serum by combining twodimensional liquid chromatographyeelectrospray ionization mass spectrometry and novel data analysis algorithms, J. Proteome Res. 5 (2006) 414e421. [26] V. Maltsev, T. Zimina, A. Khvatov, B.G. Belenkii, Determination of myoglobin in human serum by high-performance liquid chromatography with chemiluminescence detection, J. Chromatogr. B 416 (1987) 45e52. [27] B. Loun, R. Astles, K.R. Copeland, F.A. Sedor, Adaptation of a quantitative immunoassay for urine myoglobin: predictor in detecting renal dysfunction, Am. J. Clin. Pathol. 105 (1996) 479e486. [28] H.B. Schiff, E.T. MacSearraigh, J.C. Kallmeyer, Myoglobinuria, rhabdomyolysis, and marathon running, Q. J. Med. 47 (1978) 463e472.
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Q9
Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage
Q8
Angus Lindsay a, Sam Carr a, Nick Draper b, Steven P. Gieseg a, c, * a b c
Free Radical Biochemistry Laboratory, School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand College of Life and Natural Sciences, University of Derby, Derby DE22 1GB, UK Department of Radiology, University of Otago, Christchurch 8011, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2015 Received in revised form 1 September 2015 Accepted 1 September 2015 Available online xxx
This study investigated a means of quantifying urinary myoglobin using a novel reverse-phase highperformance liquid chromatography (RPeHPLC) method that is an alternative measure of exerciseinduced muscle damage. It also investigated the effect of storage and alkalization on urinary myoglobin stability issues. An RPeHPLC method was validated by precision and repeatability experiments. Myoglobin stability was determined through spiked urine samples stored at various temperatures over an 8-week period using alkalization and dilution in a pH 7.0 buffer. The method was validated with urine collected from mixed martial arts fighters during a competition and training session. The method produced linearity from 5 to 1000 mg/ml (R2 ¼ 0.997), intra- and inter-assay coefficients of variation from 0.32 to 2.94%, and a lower detection limit of 0.2 mg/ml in the final dilution and 2 mg/ml in the original urine sample. Recovery ranged from 96.4 to 102.5%, myoglobin remained stable at 4 C when diluted in a pH 7.0 buffer after 20 h, and a significant increase (P < 0.01) and an identifiable peak were observed following a mixed martial arts contest and training session. Storage length and conditions had significant effects (P < 0.05) on stability. The method's simplicity and noninvasive nature means it can be used as an alternate noninvasive muscle damage assay following exercise and trauma. © 2015 Elsevier Inc. All rights reserved.
Keywords: HPLC Myoglobin Mixed martial arts Stability Muscle damage Exercise
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Myoglobin, the hemoprotein that facilitates oxygen storage for sustained oxidative phosphorylation in muscle fibers, is a key marker of muscle damage that has notably been employed to assess the severity of injury and monitor the recovery of individuals suffering from rhabdomyolysis [1], myocardial infarction [2], accidental trauma [3], and exercise [4,5]. Muscle damage quantification is also pertinent to the diagnosis of exercise-induced physiological stress for the management of athlete recovery and performance. To this end, myoglobin has been routinely measured in several exercise studies ranging from rugby union [6,7] to ultra-endurance exercise [8]. The methodologies employed for myoglobin measurement include sensitive and expensive immunoassays, dipsticks,
Abbreviations: ELISA, enzyme-linked immunosorbent assay; RPeHPLC, reversephase high-performance liquid chromatography; UV, ultraviolet. * Corresponding author. Free Radical Biochemistry Laboratory, School of BiologQ1,2 ical Sciences, University of Canterbury, Christchurch 8140, New Zealand. E-mail address: [email protected] (S.P. Gieseg).
Q4
histochemical staining, radioimmunoassays, and relatively inexpensive liquid chromatograph assays [6,9,10]. Enzyme-linked immunosorbent assays (ELISAs) are capable of quantifying myoglobin concentrations through their rapid and sensitive processing time. However, de Waard and van't Sant [11] highlighted ELISAs' instability, inaccuracy, and limited detection range (~1 mg/ ml), suggesting that all immunoassay-based myoglobin assays are unsuitable. Similarly, urinary dipsticks are unable to differentiate clearly between myoglobin and hemoglobin and do not offer a quantification method [12]. When concentrations range from 3.2 to 3000 mg/L following serious trauma, myocardial infarction, and professional rugby [2,4,13], repeated handling and the inability to specifically quantify muscle damage severity make immunoassay options rather impractical. Likewise, compounding effects such as pH, temperature, freezing, and unidentified urinary compounds smaller than 10 kDa routinely affect stability and the ability to accurately quantify myoglobin [14]. The need for a noninvasive, reliable, repeatable, simple, and cost-effective alternative assay for the quantification of muscle
http://dx.doi.org/10.1016/j.ab.2015.09.001 0003-2697/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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damage following physical exercise is essential. Creatine kinase and lactate dehydrogenase are routinely measured in exercise research through plasma analysis [15,16] but can be severely limited by its delayed response to the muscle-damaging exercise [17] and its invasive approach, providing robust reasoning for the requirement of an assay that can provide an immediate noninvasive quantification of muscle damage. Furthermore, several size exclusion, anion exchange, and reverse-phase high-performance liquid chromatography (RPeHPLC) techniques have been used for protein and myoglobin determination [2,18,19]; however, they are somewhat outdated, coelute proteins of similar size, are unvalidated in urine, or require more than one preparative process. RPeHPLC methods commonly use water and an organic solvent for separation and identification that have shown a recovery of approximately 100% following addition to an extract [20]. We have adopted this methodology to develop, validate, and report a similarly reliable RPeHPLC method that quantifies myoglobin in urine by simple ultraviolet (UV) detection that negates the use of invasive procedures. The aims of this research were to develop a simple and noninvasive RPeHPLC method that can reliably quantify urinary myoglobin following exercise-induced muscle damage as an alternative to commonly used invasive approaches and to investigate the effect of alkalization and sample dilution on urinary myoglobin stability for sample preparation optimization. Materials and methods All solutions and reagents were prepared with water purified using the NANOpure ultrapure water system from Barnstead/ Thermolyne (Dubuque, IA, USA). Chemicals and reagents were supplied from Sigma Chemical (St. Louis, MO, USA), Abcam (Melbourne, Australia), and BDH Chemicals New Zealand (Auckland, New Zealand). Precision studies Spectral analyses of commercially supplied pure human (Abcam, ab96036) and equine (SigmaeAldrich, M0630) skeletal myoglobin were conducted to identify the optimal absorbance level of myoglobin, whereas spectral analysis at 210, 280, and 400 nm were conducted by RPeHPLC on spiked myoglobin urine samples to identify any interference from other urinary compounds. Intraassay precision was evaluated using 20 replicates of a single urine sample in a single analytical run spiked with 10, 100, and 1000 mg/
ml myoglobin standard. Inter-assay precision was evaluated using 20 replicates of a single urine sample on 4 consecutive days for each of the three concentrations while a calibration curve was established using myoglobin standards ranging from 5 to 1000 mg/ml for both human and equine skeletal myoglobin. The lower detection limit for the assay was established using spiked urine samples where the peak was three times greater than the baseline noise, as described previously [19]. Recovery and stability The recovery and stability of the myoglobin was investigated by spiking a urine sample with known concentrations of human myoglobin standard (10e1000 mg/ml). Standards ranging from 10 to 1000 mg/ml were added to urine samples before being diluted 1:10 with 10 mM ammonium acetate (pH 7.0) and analyzed every 4 h for a 28-h period at 4 C. This was to replicate standard urine collection protocols. This time period was specifically chosen due to the length of each assay (32 min) while accounting for as many as 40 samples in any one analytical run. Because myoglobin is notoriously unstable in urine when measured by ELISA [14], the stability of myoglobin in a urine sample over an extended period of time was examined to determine percentage recovery. To a urine sample, 10, 100, and 1000 mg/ml human myoglobin standard was added and diluted 1:10 with 10 mM ammonium acetate (pH 7.0) before being either (i) frozen at 80 C, (ii) frozen at 20 C, or (iii) refrigerated at 4 C for 1, 2, 4, and 8 weeks before thawing and analysis to determine stability and degradation kinetics. All experimental research was conducted in triplicate. Clinical subjects and experimental design This analysis methodology was validated from urine collected from 14 mixed martial artists (178.4 ± 8.4 cm, 84.3 ± 12.9 kg, 26.6 ± 6.2 years), following a simulated contest (n ¼ 10) and contest preparation training session (n ¼ 13), who were informed of the risks involved in the study before their written consent was obtained. The simulated contest consisted of three rounds of 5 min with 60 s between rounds, whereas the training session consisted of individual fighting for three rounds of 5 min followed by sparring, wrestling, and fitness for a total session time of approximately 75e90 min. Samples were collected using a 70-ml collection pot midstream before, post, and 1 and 2 h post each event. At the time of collection, all samples were pH corrected and diluted with 10 mM ammonium acetate (pH 7.0) (1 ml urine:4 ml buffer) and
Fig. 1. Individual subjects' urinary myoglobin concentrationetime course changes for the MMA contest (A) and training session (B).
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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66 67 68 69 70 HPLC analysis 71 72 HPLC measurement of myoglobin was performed using a Shi73 madzu Sil-20A HPLC device with autosampler and an RF-20Axls 74 and SPD-20A photo diode array detector. A sample (10 ml) was 75 injected onto a Jupiter C5 300 Å column (5-mm, 150 4.6 mm). The 76 mobile phase was generated by mixing 0.1% (v/v) trifluoroacetic 77 acid (pH 2.5) in water (A) with 100% acetonitrile (B) by pre-pump 78 mixing to give 10% A/90% B at 1 ml/min. The myoglobin was 79 eluted with a linear gradient from 10 to 70% B over 15 min and was 80 followed by 70% B for a further 5 min. The column was returned to 81 the starting condition by 70 to 10% B gradient over 2 min and 82 holding at 10% B for 10 min, making a total run time of 32 min. 83 Myoglobin was detected by absorbance at 400 nm. All analytes 84 were quantified by peak area using Shimadzu Class VP software and 85 were conducted in duplicate. 86 87 Statistical analysis 88 89 Statistical analysis was conducted using the Statistical Package 90 for Social Sciences version 20. A two-way analysis of variance 91 (ANOVA) using Tukey's test for post hoc analysis was conducted on 92 all recovery and time course experiments (Fig. 1). Statistical sig- Q5 93 nificance was set at 0.05. 94 95 96 Results 97 98 Equine skeletal myoglobin provided near identical properties to 99 human skeletal myoglobin as determined by spectrophotometry 100 and HPLC analysis with spectrophotometric detection. Therefore, 101 we suggest the use of equine skeletal myoglobin as a standard for 102 clinical measurements by HPLC due to its relative availability and 103 lower purchase price in comparison with human myoglobin. 104 Urinary myoglobin was detected at 400 nm by RPeHPLC eluting 105 at approximately 15 min with no visible tailing of the peak or 106 interference from neighboring peaks. After spectrophotometer 107 analysis identified 210, 280, and 400 nm as the optimal absorbance 108 wavelengths (data not shown), spectral analysis of a urine sample 109 spiked with myoglobin by HPLC found that 400 nm provided the 110 highest absorbance units and lowest signal-to-noise ratio and did 111 not interfere with any other urinary compound at that wavelength, 112 although 280 nm also provided a clear and visible peak. 113 The method was validated in all 14 urine samples that were 114 collected following both events. Myoglobin was detectable in 5 of 10 subjects following the fight (0.95e13.24 mg/ml) (Fig. 2A and 11 of Q6 115 116 14 subjects following the training session (0.19e63.3 mg/ml) 117 (Fig. 2B). All subjects had undetectable concentrations (lower 118 detection limit of 0.2 mg/ml) before the competition or training 119 session. For one of the selected fighters, myoglobin was undetect120 able pre-fight (Fig. 3A), rose to 3.82 mg/ml immediately post-fight 121 with the identification of a single peak (Fig. 3B), peaked 122 (13.24 mg/ml) 1 h post-fight (Fig. 3C), and dropped to 1.24 mg/ml 2 h 123 post-fight (Fig. 3D). For a selected subject participating in the 124 contest preparation training session, myoglobin was undetectable 125 immediately pre (Fig. 3E), rose to 5.83 mg/ml immediately post 126 (Fig. 3F), peaked 1 h post at 63.3 mg/ml (Fig. 3G), and was still 127 detectable (7.91 mg/ml) 2 h post (Fig. 3H). 128 The intra- and inter-assay coefficients of variation for 10, 100, and 129 1000 mg/ml were 1.49 and 2.94%, 0.39 and 0.70%, and 0.32 and 0.79%, 130 respectively, whereas the assay presented linearity in both human placed on ice immediately at location. They were transported back to the laboratory for immediate analysis. This was approved by the Health and Disability Ethics Committee (Wellington, New Zealand).
Fig. 2. (A) Calibration curve for human and equine skeletal myoglobin at concentrations ranging from 5 to 1000 mg/ml detected at 400 nm. (B,C) Lower detection limit for the RPeHPLC method as indicated by the arrow. An unspiked urine sample (B) and a spiked urine sample (2 mg/ml) before the 1:10 dilution (C) at the same sensitivity are shown. Lower detection was calculated when the peak was three times the size of the baseline noise.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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Fig. 3. RPeHPLC chromatographs of two selected mixed martial arts fighters following a simulated contest (AeD) and a competition preparation training session (E, F). Pre (A, E), post (B, F), 1 h post (C, G), and 2 h post (D, H) contest/training session chromatographs indicating myoglobin (arrow) eluting at approximately 15 min at an absorbance wavelength of 400 nm are shown.
(R2 ¼ 0.998) and equine (R2 ¼ 0.997) over the range of 5e1000 mg/ml (Fig. 2A) with a lower detection limit of 0.2 mg/ml (Fig. 2B and C). Myoglobin was more stable following alkalization and dilution with 10 mM ammonium acetate (pH 7.0) over a 28-h analytical run in comparison with purified water (data not shown). Therefore, it was decided to pursue further stability experiments using this protocol. Myoglobin recovery from spiked urine samples ranged from 96.4 to 102.5%, whereas its stability over 28 h in a urine sample diluted with 10 mM ammonium acetate (pH 7.0) at 4 C decreased by 9.71, 7.12, and 2.12% for 10, 100, and 1000 mg/ml, respectively, at the 28-h time point (Fig. 4). Meanwhile, storage length and conditions had significant effects (P < 0.05) on myoglobin stability in urine (Table 1). The longer a sample was stored, the more degradation occurred. However, this loss was attenuated by the storage temperature (P < 0.01), whereas the greater the concentration, the slower the degradation process (P < 0.01).
suitable, reliable, and rapid (32 min) alternative RPeHPLC method for the quantification of urinary myoglobin following exerciseinduced muscle damage. The concentrations observed following each of the mixed martial arts events suggest that a training session is of greater intensity than a simulated contest, most likely a result of the difference in duration. Moreover, it is comparable to other
Discussion Myoglobin is typically quantified in urine through sensitive and rapid immunoassays [21] as well as several forms of HPLC [2]. With intra- and inter-assay variations in accordance with the accepted range for a reliable and repeatable assay, we have demonstrated a
Fig. 4. Myoglobin recovery and stabilization over 28 h from urine samples diluted 1:10 with 10 mM ammonium acetate (pH 7.0) and spiked with concentrations ranging from 10 to 1000 mg/ml at 4 C. All analyses were completed in triplicate, and data are presented as means ± standard deviations.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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A. Lindsay et al. / Analytical Biochemistry xxx (2015) 1e6 Table 1 Storage effect on myoglobin in a pH corrected and diluted urine sample spiked with known concentrations. [Myoglobin] (mg/ml)
Time point (weeks)
Conditions 4 C
20 C
80 C
10
1 2 4 8 1 2 4 8 1 2 4 8
5.3 ± 0.2 4.6 ± 0.3 3.1 ± 0.1 2.7 ± 0.1 81.0 ± 0.3 83.4 ± 0.1 77.3 ± 0.8 72.3 ± 0.1 975.3 ± 1.2 963.4 ± 20.6 947.9 ± 4.6 924.5 ± 18.0
6.3 ± 0.3 5.1 ± 0.2 4.2 ± 0.01 4.2 ± 0.02 81.2 ± 0.04 83.2 ± 0.7 71.6 ± 3.9 67.0 ± 1.4 998.6 ± 22.0 980.9 ± 7.6 952.7 ± 6.8 941.9 ± 7.5
7.3 ± 0.01 6.1 ± 0.5 6.0 ± 0.2 6.4 ± 0.01 86.0 ± 0.1 91.4 ± 2.0 82.3 ± 0.1 77.0 ± 0.5 987.5 ± 36.3 1020.4 ± 1.1 980.0 ± 2.2 977.0 ± 17.1
100
1000
Note. Three replicates of each concentration at each time point were analyzed. Data are means ± standard deviations (mg/ml).
high-impact sports that have shown similar changes in urinary myoglobin [4,5], which demonstrate the severity of the sport that has been widely neglected in exercise research. Detection of myoglobin at an absorbance of 400 nm produced a clear visible peak with no observed tailing or interference from neighboring urinary analytes allowing for clear interpretation, whereas the dilution in 10 mM ammonium acetate (pH 7.0) seemed to alleviate any stabilization concerns that had been previously noted over a 24-h period [14]. The mechanism of how this alkalization improves myoglobin stability has not yet been fully elucidated. However, it is believed to revolve around myoglobin's isoelectric point, where increasing the pH beyond 6.9 reduces its precipitation [22] and further prevents the dissociation of myoglobin into its smaller nephrotoxic components [1]. This issue becomes particularly imperative when assessing exercise-induced muscle damage and the potential for false-negative results arising from myoglobin degradation in the bladder [11]. In spite of relative stability over 28 h using this protocol, which alleviates any potential issue arising from the extended HPLC assay time, the time course experiments suggest that urinary myoglobin be analyzed immediately following collection and alkalization to avoid any potential degradation loss when concentrations are not known. These results expand on the previous findings of de Waard and van't Sant [11], who found that alkalized urine stored at 4 C decreases by up to 50% within a week. Whereas some compounds remain stable for months at 80 C [23], urine samples containing <1000 mg/ml myoglobin clearly do not remain stable for long periods of time and, therefore, should avoid freezeethawing for optimal analysis. Samples should be collected, alkalized, and analyzed or, if need be, diluted accordingly with a pH 7.0 buffer and stored promptly at 80 C on presentation. The dilution factor can, of course, be altered depending on myoglobin concentration or concern for column life expectancy. Myoglobin's detection and recovery from urine samples using this method is comparable to that using similar RPeHPLC methods [20] that provide a simple and cost-effective assay using UV detection for values in a clinical and exercise relevant range. Although it is relatively economical to run, setup costs for HPLC are quite expensive compared with traditional ELISA kits. Its detection limit is substantially lower (30 mg/ml) than that provided for myoglobin by size exclusion HPLC [19], whereas the ease of preparation for quantification provides a more simple option than previous methods [2,18]. Its use in a clinical and exercise context for diagnosis and observational purposes should, therefore, consider sample pH stabilization and dilution in 10 mM ammonium acetate (pH 7.0) followed by analysis within 20 h at 4 C to use this method
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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Conclusion 101 102 The quantification of urinary myoglobin using the described 103 RPeHPLC method provides a precise, simple, and cost-effective 104 method for muscle damage quantitation in a clinical and exercise 105 relevant range. The detection of myoglobin using this method in 106 mixed martial arts competitors identifies severe structural damage 107 following a contest, validating the usefulness of this method, 108 whereas immediate alkalization and dilution in a pH 7.0 buffer al109 leviates any stabilization concerns over 24 h. Through its simplicity, 110 noninvasiveness, and speed of analysis, this method can further be 111 used for evaluating the degree of muscle damage following 112 exercise-induced activities and sports. 113 114 Acknowledgments Q7 115 116 The authors thank St. George's Hospital, Heart Centre, and the 117 University of Canterbury for funding this project, Strikeforce Can118 terbury Gym, Karl Webber and each of the subjects for their plan119 ning and participation, and Matt Walters for his expertise in the 120 preparation of the figures. 121 122 References 123 124 [1] I. Alterman, A. Sidi, L. Azamfirei, S. Copotoiu, T. Ezri, Rhabdomyolysis: another 125 complication after prolonged surgery, J. Clin. Anesth. 19 (2007) 64e66. 126 [2] S. Powell, E. Friedlander, Z. Shihabi, Myoglobin determination by highperformance liquid chromatography, J. Chromatogr. A 317 (1984) 87e92. 127 [3] A. Genthon, S.R. Wilcox, Crush syndrome: a case report and review of the 128 literature, J. Emerg. Med. 46 (2014) 313e319. 129 [4] A. Lindsay, J. Lewis, C. Scarrott, N. Gill, S.P. Gieseg, N. Draper, Assessing the 130 effectiveness of selected biomarkers in the acute and cumulative physiological to its fullest capacity. This is highly feasible considering the noninvasive, nonburdening nature of this method with no need for venipuncture, the faster elimination kinetics of myoglobin compared with the sometimes delayed response of common muscle damage makers such as creatine kinase [24], and the ease of installation of the measurement apparatus within a laboratory or hospital setting. With commercial ELISAs having upper detection limits of approximately 1 mg/ml, the linearity of this method from 5 to 1000 mg/ml and the lower detection limit of 0.2 mg/ml provide a significant alternative for the quantification of a muscle damage marker. These properties are similar to those reported by anion exchange [2] and two-dimensional liquid chromatographyeelectrospray ionization mass spectrometry [25], which negates the dilution requirement for absolute concentration determination and which also avoids unnecessary repeated analysis. Although it does not have the sensitivity limit of 10 ng/ml that prior chemiluminescence detection has shown [26], the concentrations in the linear range are more clinically relevant and similar to those noted previously [2]. Feinfeld and coworkers [9] observed patients with acute rhabdomyolysis exceeding concentrations of 1 mg/ml, whereas previous studies have suggested that patients with values exceeding 20 mg/ml are at more of an increased risk of decreased renal function [21,27]. In addition, values as high as 32.9, 410, and 3000 mg/ml have been observed following crush trauma [3], strenuous military training [13], and myocardial infarction [2], respectively, whereas values reaching 52 and >100 mg/ml have been observed in competitors following a marathon [28] and professional rugby [5], respectively. Therefore, in an exercise and/or serious trauma scenario where muscle damage is expected to rise significantly, this assay may provide the most cost-effective and operator-friendly option for simple and rapid quantitation for both small and large cohorts suffering mild to severe skeletal muscle stress in a range that is widely accepted as “clinically relevant.”
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[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
stress response in professional rugby union through non-invasive assessment, Int. J. Sports Med. 36 (2015) 446e454. A. Lindsay, J. Healy, W. Mills, J. Lewis, N. Gill, N. Draper, S.P. Gieseg, Impactinduced muscle damage and urinary pterins in professional rugby: 7,8-dihydroneopterin oxidation by myoglobin, Scand. J. Med. Sci. Sports (2015), http:// dx.doi.org/10.1111/sms.12436. A. Lindsay, J. Lewis, C. Scarrott, N. Draper, S.P. Gieseg, Changes in acute biochemical markers of inflammatory and structural stress in rugby union, J. Sports Sci. 33 (2014) 882e891. A. Lindsay, J. Lewis, N. Gill, S.P. Gieseg, N. Draper, Effect of varied recovery interventions on markers of psychophysiological stress in professional rugby union, Eur. J. Sport Sci. 15 (2015) 543e549. L. Wallberg, C.M. Mattsson, J.K. Enqvist, B. Ekblom, Plasma IL-6 concentration during ultra-endurance exercise, Eur. J. Appl. Physiol. 111 (2011) 1081e1088. D. Feinfeld, J. Cheng, T. Beysolow, A.M. Briscoe, A prospective study of urine and serum myoglobin levels in patients with acute rhabdomyolysis, Clin. Nephrol. 38 (1992) 193e195. J. Royds, S. Variend, W. Timperley, C.B. Taylor, Comparison of beta enolase and myoglobin as histological markers of rhabdomyosarcoma, J. Clin. Pathol. 38 (1985) 1258e1260. H. de Waard, P. van't Sant, Use of serum myoglobin assays for urine myoglobin measurements can cause false-negative results, Clin. Chem. 55 (2009) 1240e1241. E.A. Anigilaje, O.T. Adedoyin, Correlation between dipstick urinalysis and urine sediment microscopy in detecting haematuria among children with sickle cell anaemia in steady state in Ilorin, Nigeria, Pan Afr. Med. J. 15 (2013) 135. R.F. Smith, Exertional rhabdomyolysis in naval officer candidates, Arch. Intern. Med. 121 (1968) 313e319. Z. Chen-Levy, M.H. Wener, B. Toivola, P. Daum, M. Reyes, J.S. Fine, Factors affecting urinary myoglobin stability in vitro, Am. J. Clin. Pathol. 123 (2005) 432e438. R.D. Johnston, T.J. Gabbett, A.J. Seibold, D.G. Jenkins, Influence of physical contact on neuromuscular fatigue and markers of muscle damage following small-sided games, J. Sci. Med. Sport 17 (2014) 535e540. T.K. Singh, K.J. Guelfi, G. Landers, B. Dawson, D. Bishop, A comparison of muscle damage, soreness, and performance following a simulated contact and non-contact team sport activity circuit, J. Sci. Med. Sport 14 (2011) 441e446.
[17] B. Cunniffe, A.J. Hore, D.M. Whitcombe, K.P. Jones, J.S. Baker, B. Davies, Time course of changes in immuneoendocrine markers following an international rugby game, Eur. J. Appl. Physiol. 108 (2010) 113e122. [18] D. Sviridov, B. Meilinger, S.K. Drake, G.T. Hoehn, G.L. Hortin, Coelution of other proteins with albumin during size-exclusion HPLC: implications for analysis of urinary albumin, Clin. Chem. 52 (2006) 389e397. [19] H. Liang, M.K. Scott, D.J. Murry, K.M. Sowinski, Determination of albumin and myoglobin in dialysate and ultrafiltrate samples by high-performance sizeexclusion chromatography, J. Chromatogr. B 754 (2001) 141e151. [20] I.M. Oellingrath, A. Iversen, G. Skrede, Quantitative determination of myoglobin and haemoglobin in beef by high-performance liquid chromatography, Meat Sci. 28 (1990) 313e320. [21] A. Wu, I. Laios, S. Green, T.G. Gornet, S.S. Wong, L. Parmley, A.S. Tonnesen, B. Plaisier, R. Orlando, Immunoassays for serum and urine myoglobin: myoglobin clearance assessed as a risk factor for acute renal failure, Clin. Chem. 40 (1994) 796e802. [22] T.W. King, O.Z. Lerman, J.J. Carter, S.M. Warren, Exertional compartment syndrome of the thigh: a rare diagnosis and literature review, J. Emerg. Med. 39 (2010) e93ee99. [23] A. Garde, Å. Hansen, Long-term stability of salivary cortisol, Scand. J. Clin. Lab. Invest. 65 (2005) 433e436. €ri, Elimination kinetics of [24] H. Lappalainen, E. Tiula, L. Uotila, M. M€ antta myoglobin and creatine kinase in rhabdomyolysis: implications for follow-up, Crit. Care Med. 30 (2002) 2212e2215. € pl, E. Lange, C. Klein, [25] B.M. Mayr, O. Kohlbacher, K. Reinert, M. Sturm, C. Gro C.G. Huber, Absolute myoglobin quantitation in serum by combining twodimensional liquid chromatographyeelectrospray ionization mass spectrometry and novel data analysis algorithms, J. Proteome Res. 5 (2006) 414e421. [26] V. Maltsev, T. Zimina, A. Khvatov, B.G. Belenkii, Determination of myoglobin in human serum by high-performance liquid chromatography with chemiluminescence detection, J. Chromatogr. B 416 (1987) 45e52. [27] B. Loun, R. Astles, K.R. Copeland, F.A. Sedor, Adaptation of a quantitative immunoassay for urine myoglobin: predictor in detecting renal dysfunction, Am. J. Clin. Pathol. 105 (1996) 479e486. [28] H.B. Schiff, E.T. MacSearraigh, J.C. Kallmeyer, Myoglobinuria, rhabdomyolysis, and marathon running, Q. J. Med. 47 (1978) 463e472.
Please cite this article in press as: A. Lindsay, et al., Urinary myoglobin quantification by high-performance liquid chromatography: An alternative measurement for exercise-induced muscle damage, Analytical Biochemistry (2015), http://dx.doi.org/10.1016/j.ab.2015.09.001
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