Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing.
Abstract: Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivocal evidence of an overall performance-enhancing effect of insulin, in human sports the misuse of insulin preparations is reported among elite athletes. The desired effects of insulin include the increase of muscular glycogen prior to sports event or during the recovery phase, in addition to a chalonic action, which increases the muscle size by inhibiting protein breakdown. In the present study urinary insulin was detected in equine samples and differences between equine insulin, human insulin, as well as rapidly acting recombinant insulin variants were examined. The method was based on sample purification by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC), and subsequent analysis by microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using top-down sequencing for the determination of various insulins. Product ion scan experiments of intact proteins and B-chains enabled the differentiation between endogenously produced equine insulin, its DesB30 metabolite, human insulin and recombinant insulin analogs, and the assay allowed the assignment of individual product ions, especially those originating from modified C-termini of B-chains.
Copyright (c) 2008 John Wiley & Sons, Ltd.
Publication Date: 2008-01-09 PubMed ID: 18181226DOI: 10.1002/rcm.3360Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research analyses the presence of different types of insulin in horse urine in relation to doping control in horse-racing. It utilises methods like solid-phase extraction, immunoaffinity chromatography, and tandem mass spectrometry to identify insulin variants in the urine samples.
Background
- Insulin, a peptide hormone with two peptide chains, is produced in different forms (long-, intermediate-, and rapid-acting) for improved pharmacokinetic action in diabetes treatment. The B-chain’s C-terminal end of insulin plays a significant role.
- The rarity of equine type I diabetes suggests that use of insulin therapeutically in racing horses is rare. However, the misuse of insulin preparations as performance-enhancing substances is reported among elite human athletes.
- Insulin can increase muscle glycogen, beneficial prior to sports events, and contribute to a chalonic action—increasing muscle size by inhibiting protein breakdown.
Purpose and Methods
- The study aims to identify urinary insulin in equine samples and distinguish between equine insulin, human insulin, and the fast-acting recombinant insulin variants.
- The researchers used solid-phase extraction (SPE) to purify the urine samples and immunoaffinity chromatography (IAC) to separate the different types of insulin. Following purification, they used microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) for analysis.
Findings
- The method allowed for the differentiation of endogenously produced equine insulin, its DesB30 metabolite, human insulin, and recombinant insulin analogs.
- The assay permitted the assignment of individual product ions, particularly those coming from the modified C-termini of B-chains. These findings suggest the presence of different types of insulin can be detected and differentiated in horse urine, which is significant for doping control in horse-racing.
Cite This Article
APA
Kuuranne T, Thomas A, Leinonen A, Delahaut P, Bosseloir A, Schänzer W, Thevis M.
(2008).
Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing.
Rapid Commun Mass Spectrom, 22(3), 355-362.
https://doi.org/10.1002/rcm.3360 Publication
Researcher Affiliations
- Institute of Biochemistry - Center for Preventive Doping Research, German Sports University Cologne, Cologne, Germany.
MeSH Terms
- Animals
- Chromatography, Affinity / methods
- Chromatography, High Pressure Liquid / methods
- Doping in Sports / prevention & control
- Horses
- Immunoassay / methods
- Insulin / urine
- Reproducibility of Results
- Sensitivity and Specificity
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods
- Substance Abuse Detection / methods
- Substance Abuse Detection / veterinary
- Urinalysis / methods
Citations
This article has been cited 3 times.- Go YY, Hazard NW, Balasuriya UBR, Chapman AM, Fitton NS, Kenéz Á, Andrews FM. Clinical evaluation of the Immulite® 1000 chemiluminescent immunoassay for measurement of equine serum insulin. Front Vet Sci 2023;10:1018230.
- Yuan M, Breitkopf SB, Asara JM. Serial-omics characterization of equine urine. PLoS One 2017;12(10):e0186258.
- Warnken T, Huber K, Feige K. Comparison of three different methods for the quantification of equine insulin. BMC Vet Res 2016 Sep 9;12(1):196.
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