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The Effect of Sports Injury on Insulin-Like Growth Factor-I and Type 3 Procollagen: Implications for Detection of Growth Hormone Abuse in Athletes

Endocrinology and Metabolism Unit, Developmental Origins of Health and Disease Division, School of Medicine (I.E.-M., C.M., R.S., B.C., V.W., K.H., P.H.S., R.I.G.H.), University of Southampton, Southampton SO16 6YD, United Kingdom; Institute of Mathematics, Statistics, and Actuarial Science (E.E.B.), University of Kent, Canterbury, Kent CT2 7NZ, United Kingdom; and Department of Forensic Science (C.B., D.C.), Drug Monitoring, Drug Control Centre, King’s College London, London SE5 9RS, United Kingdom

Address all correspondence and requests for reprints to: Dr. Richard I. G. Holt, The Institute of Developmental Sciences (IDS Building), MP887, University of Southampton, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom. E-mail: r.i.g.holt{at}soton.ac.uk.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: A method to detect exogenously administered growth hormone (GH) based on the measurement of two GH-dependent markers, IGF-I and type 3 procollagen (P-III-P) has been proposed. Skeletal or soft tissue injury may alter these markers. Elevations in either of these proteins after injury might lead to a false accusation of doping with GH.

Objective: The objective of the study was to assess the effect of musculoskeletal or soft tissue injury on IGF-I and P-III-P concentrations in amateur and elite athletes and assess the effect of injury on the proposed GH detection method.

Design: This was a longitudinal observational study after sporting injury.

Setting: The study was conducted at Southampton General Hospital and British Olympic Medical Centre.

Subjects: Subjects included elite and amateur athletes after an injury.

Intervention: Interventions included measurement of IGF-I and P-III-P and application of the GH-2000 discriminant function score up to 84 d after an injury as well as classification of injury by type and severity.

Outcome Measures: IGF-I and P-III-P concentration and ability to detect GH abuse in athletes without the risk of false accusation because of an injury were measured.

Results: There was no change in IGF-I concentration after an injury. By contrast, P-III-P concentrations rose by 41.1 ± 16.6%, reaching a peak around 14 d after an injury. The rise in P-III-P varied according to injury type and severity. This rise had a trivial effect on the GH-2000 discriminant function score, and no subject reached the threshold needed for a doping offense.

Conclusions: Although there was a rise in P-III-P after injury, this was insufficient to invalidate the GH-2000 detection method based on IGF-I and P-III-P concentrations.

	Introduction

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The detection of exogenously administered GH in professional athletes poses a formidable challenge. A multinational research group, named the GH-2000 team proposed a method based on the measurement of IGF-I and type 3 procollagen (P-III-P) (1) in conjunction with gender-specific discriminant functions. There are concerns that skeletal injury may affect the test because P-III-P increases after bony fracture (2, 3, 4).

We therefore assessed the effect of a musculoskeletal injury on serum IGF-I and P-III-P and the GH-2000 detection method in amateur and elite sportsmen and women.

	Subjects and Methods

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Subjects

One hundred twenty-seven male (aged 29.6 ± 0.9 yr, range 17–68 yr) and 30 female (aged 32.0 ± 2.1 yr, range 19–63) amateur athletes were recruited from the Accident and Emergency outpatient clinic or orthopedic fracture clinic at the Southampton University Hospitals Trust (United Kingdom). Sixteen male (aged 23.8 ± 1.4 yr) and 10 female (aged 24.7 ± 1.6 yr) professional athletes were recruited from the British Olympic Medical Centre. Subjects were recruited within 10 d of a bony or soft tissue musculoskeletal injury of sufficient severity to prevent training for at least 3 d. Exclusion criteria included neoplastic disease, diabetes, pregnancy or lactation, and any condition likely to affect the GH-IGF axis.

The protocol was approved by the Southampton and South West Hampshire Local Research Ethics Committee. All subjects gave written informed consent, and the study was conducted in accordance with the Declaration of Helsinki and good clinical practice guidelines.

Methods

A brief medical and sporting history was obtained including gender, race, age, and sporting category. The nature of the injury was classified as either soft tissue (joint dislocations, ligament or tendon injury, sprain injury, or bruises) or bony (fractures, cracks, or chips). Injury severity was classified by a blinded physician as severe (fracture of a major long bone or disruption of a major joint or multiple injuries), moderate (fracture of a cancellous bone, wrist or forefoot fracture, ligament damage), or mild (digit fracture or minor soft tissue injury).

A 10-ml venous blood sample was obtained. Follow-up blood samples were scheduled on 7 ± 3, 14 ± 3, 21 ± 3, 28 ± 3, 42 ± 7, and 84 ± 14 d after the injury (target day ± acceptable range).

Blood samples were collected and were centrifuged within 12 h of collection. The serum was stored at –80 C until analysis at the World Anti-Doping Agency (WADA) accredited laboratory at the Drug Control Centre, King’s College (London, UK).

Assays

All samples were analyzed in duplicate.

Serum IGF-I was measured by the DSL-5600 Active IGF-I immunoradiometric assay (IRMA; Diagnostic Systems Laboratories, Inc., Webster, TX). The manufacturer’s reported intra-assay coefficient of variation (CV) at concentrations of 61 ng/mL, 292.5 ng/mL, and 547.9 ng/mL was 4.6%, 3.3% and 4.1% respectively with an inter assay CV of 15.8%, 10.3%, and 9.3% at concentrations of 60.1 ng/mL, 312.1 ng/mL, and 594.3 ng/mL respectively.

Serum P-III-P was measured by a two-stage sandwich RIA (CIS Biointernational, Oris Industries, Gif-Sur-Yvette Cedex, France). The lower and upper limits of detection are 0.1 and 14 U of P-III-P per milliliter, respectively. The reported intraassay variability at 0.8, 1.5, and 4.0 U/ml is 2.9, 2.9, and 4.0%, respectively. The interassay variability at 0.25, 1.5, and 5.6 U/ml is 11.3, 7.8, and 9.3%, respectively.

Data analysis

Statistical analysis was performed using the SAS software (SAS Institute, Inc., Cary, NC). IGF-I and P-III-P were log transformed. The changes in the biomarkers and GH-2000 score, compared with baseline values, was assessed by ANOVA by injury type and severity, after adjustment for age and gender. Data are mean ± SEM.

Adjusting for IGF-I assay differences

In the GH-2000 study, serum IGF-I was determined by a hydrochloric acid-ethanol extraction RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA) (5). Initially it was planned that identical assays would be used for the GH-2004 Injury study, but in 2005, Nichols Institute Diagnostics ceased trading, and we needed to use an alternative IGF-I assay. To apply the GH-2000 detection method, we had to ensure that values of serum IGF-I measured in this study were on the same scales as those used by the GH-2000 group.

To adjust for assay differences between the GH-2000 and GH-2004 studies, 73 GH-2000 samples previously analyzed using the Nichols RIA were reanalyzed using the DSL-5600 IRMA. A conversion factor was estimated to convert measured concentrations from the DSL-5600 scales to the measurement scales of the Nichols RIA assay: GH-2000 RIA = 0.660 x DSL-5600 IRMA.

No adjustment for P-III-P was needed because no significant adjustments had been made to the assay structure and methodology.

Effect of injury on the GH-2000 detection method

The previously published GH-2000 discriminant function formulae are (5): Male score = –6.586 + 2.905 x log(P-III-P) + 2.100 x log(IGF-I) – 101.737/age; and Female score = –8.459 + 2.454 x log(P-III-P) + 2.195 x log(IGF-I) – 73.666/age.

To assess whether injury affected the GH-2000 score significantly, the score for each participant was calculated at every time point using the measured P-III-P and assay adjusted IGF-I and subject age.

A cutoff point for these GH-detection formulae has not been agreed by the World Anti-Doping Agency. It has been suggested that a possible cutoff point should be at the value of 3.7; equivalent to a false-positive rate of approximately 1 in 10,000 tests.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Baseline characteristics

Most volunteers were Caucasian (n = 174). There were five Afro-Caribbean individuals, one Indo-Asian volunteer, one Oriental subject, and two subjects of mixed ethnic origin. Seventy-five subjects had sustained soft tissue injuries and 108 bony injuries. Some volunteers had both soft tissue and bony injuries and were classified under the bony injury group. The injury severity was classified as mild (n = 92), moderate (n = 48), or severe (n = 43). A total of 482 blood samples were taken, representing an average of 2.63 ± 0.14 samples per subject.

Effect on IGF-I

Baseline IGF-I was 522.6 ± 21.8 ng/ml. There was no difference between injury types or severity. There was no significant change in IGF-I during the study either in the whole population or when analyzed separately according to severity or injury type.

The intraindividual variability of IGF-I was estimated as 24.3 ± 8.5%.

Effect on P-III-P

The baseline P-III-P concentration was 0.42 ± 0.02 U/ml. There was no significant difference in P-III-P at baseline between injury types or severity.

In contrast to IGF-I, there was a significant rise in P-III-P after both soft tissue and bony injuries. The pattern of response differed between soft tissue and bony injury, and therefore, the two types of injury were analyzed separately. After a soft tissue injury, there was a rise in P-III-P that peaked 14 d after the injury, the magnitude of which varied between 41.1 ± 16.6% and 44.3 ± 19.2%, according to injury severity (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Box plot showing change in P-III-P concentration over 84 d after injury according to injury type and severity. The median and upper and lower 25% centiles are represented by the box. The whisker represents the range, except where there are outliers. Mild outliers are represented by circles and extreme outliers are represented by stars.

The intraindividual variability of the P-III-P concentrations for this study period was estimated as 33.1 ± 11.1%.

Although we do not have the statistical power to assess differences between elite and amateur athletes, the patterns of change of P-III-P and IGF-I in the nine elite athletes with multiple samples appeared no different from amateur athletes.

Effects on discriminant formulae

The changes in male GH-2000 and female score with time after injury are shown in Fig. 2. There was no significant change in either male or female score with time after an injury (P = 0.78).

View larger version (10K): [in this window] [in a new window]   FIG. 2. A, Box plot showing change in GH-2000 score in men over 84 d after injury. The median and upper and lower 25% centiles are represented by the box. The whisker represents the upper and lower 95% confidence intervals. Mild outliers are represented by circles and extreme outliers are represented by stars. B, Box plot showing change in GH-2000 score in women over 84 d after injury. The median and upper and lower 25% centiles are represented by the box. The whisker represents the range, except where there are outliers. Mild outliers are represented by circles and extreme outliers are represented by stars.

All samples from both men and women were below the 3.7 cutoff limit. Thus, no individual would have been falsely accused of GH doping.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Our study assessed the effect of injury on the serum IGF-I and P-III-P and the performance of the proposed GH-2000 test. Because injuries are common in sports and may cause an elevation in P-III-P (4, 7) and IGF-I (3), it is vital to ensure that no athlete will be falsely accused of GH doping under the proposed methodology because of an injury. Equally important is the need to eliminate a defense argument that an accused athlete had been falsely accused of doping because of an injury.

Although fluctuations were found in IGF-I concentrations during the study period, these were small and no larger than the intraindividual variation among healthy elite and amateur athletes. The finding that IGF-I did not change after injury was unexpected because it is recognized that IGF-I falls with acute illness (8). The reason for this may relate to the lesser severity of the injury, compared with the illness needed to result in a fall in IGF-I on the intensive care unit.

P-III-P rose significantly after injury, the magnitude and duration of which varied according to the injury type and severity. Previous studies have showed similar increases in P-III-P after fracture. In one study, P-III-P reached a peak 2 wk after a malleolar fracture, whereas P-III-P peaked after 12 wk after a tibial fracture (9). Two further studies of subjects with tibial fractures showed maximum P-III-P after 2 and 8 wk (10). Poorly healing fractures has been associated with elevated P-III-P for up to 10 wk (11). There are no previous reports of the change in P-III-P after soft tissue injury.

Fracture healing is divided into three distinct phases: inflammation, regeneration, and remodeling. During the initial inflammatory phase, type III collagen production increases in the initial hematoma, which is subsequently replaced by fibrous tissue containing predominantly type III collagen (9). Time for fracture healing and extent of changes in markers of bone metabolism are mainly dependent on fracture size.

Although P-III-P rose significantly, the magnitude (41–75%) was considerably lower than the rise in P-III-P after recombinant human GH administration (299% over a 14 d period) (12). Consequently, no athlete exceeded the provisional cutoff point for a positive test and reinforces the conclusion that injury would not adversely affect the performance of this detection method.

The study has a number of limitations. There were few female and elite subjects in this study, and a bigger sample size may have improved the power to detect differences. A second criticism was the need to make an adjustment for the change in the IGF-I assay. By using a direct comparison between assays, we were, however, able to make appropriate assay adjustments.

In conclusion, although IGF-I does not change significantly, P-III-P rose after injury. This rise, however, is insufficient to invalidate the performance of the proposed GH-2000 test.

	Acknowledgments

 
We thank Rod Park, the GH-2004 project manager, for his administrative support for the study. We also thank the nurses of the Wellcome Trust Clinical Research Facility at the University of Southampton for their assistance in recruitment and follow-up of the subjects. We acknowledge the support of Dr. Richard Budgett, Director of Medical Services for the British Olympic Association. We also acknowledge the legacy of the GH-2000 Project (a European Union Biomed 2 Project BMH4 CT950678).

	Footnotes

 
The GH-2004 study was supported by the World Anti-Doping Agency and U.S. Anti-Doping Agency, without which this study would not have been possible.

Disclosure Statement: All authors have nothing to declare.

First Published Online April 15, 2008

Abbreviation: P-III-P, Type 3 procollagen.

Received December 20, 2007.

Accepted April 9, 2008.

	References

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