Interpretation of cardiac troponin T behaviour in size-exclusion chromatography
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Etienne C.H.J. Michielsen
Abstract
Background: Knowledge about the presence of intact cardiac troponin T (cTnT) and/or its immunoreactive fragments is of great value for the interpretation of cTnT clearance from the circulation. Until now there has been a lot of controversy about cTnT fragmentation. To provide an answer to this controversy, we investigated fragmentation of cTnT with size-exclusion chromatography (SEC), and confirmed our data using mass spectrometry.
Methods: A highly purified human cTnT standard, characterised using mass spectrometry as a single peak of 34,377 Da and using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as a single immunoreactive band (37 kDa), was incubated in serum for 0, 24 and 48h at 37°C and analysed using SEC. A troponin TIC complex standard, used in an earlier study, was also investigated.
Results: We demonstrated that, because of its rod-like shape, the molecular weight of cTnT cannot be estimated from SEC using the molecular weight of globular proteins as a reference. The Stokes radius of intact cTnT was calculated to be 33.7Å. Incubation of both cardiac troponin standards in troponin-free serum resulted in a time-dependent decrease in intact cTnT and a simultaneous increase in smaller immunoreactive fragments (13.4 and 22.4Å).
Conclusions: cTnT has a Stokes radius of 33.7Å. Compared with globular calibrator proteins, intact cTnT elutes earlier than expected based solely on its molecular weight. For non-globular or uncharacterised proteins, Stokes radii should be used for correct interpretation of SEC data. By doing so, we were able to clearly demonstrate cTnT fragments.
Clin Chem Lab Med 2006;44:1422–7.
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©2006 by Walter de Gruyter Berlin New York
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Articles in the same Issue
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- Blood transfusions in athletes. Old dogmas, new tricks
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- Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR
- Lipoprotein lipase gene polymorphism at the PvuII locus and serum lipid levels in Guangxi Hei Yi Zhuang and Han populations
- Interpretation of cardiac troponin T behaviour in size-exclusion chromatography
- Point-of-care C-reactive protein testing in febrile children in general practice
- Improvement in HPLC separation of porphyrin isomers and application to biochemical diagnosis of porphyrias
- Measurement of late-night salivary cortisol with an automated immunoassay system
- Combining markers of nephrotoxicity and hepatotoxicity for improved monitoring and detection of chronic alcohol abuse
- Stone or stricture as a cause of extrahepatic cholestasis – do liver function tests predict the diagnosis?
- Insulin resistance and enhanced protein glycation in men with prehypertension
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- Analytical performance and clinical utility of the INNOTEST® PHOSPHO-TAU(181P) assay for discrimination between Alzheimer's disease and dementia with Lewy bodies
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- National survey on the pre-analytical variability in a representative cohort of Italian laboratories
- 10% CV concentration for the fourth generation Roche cardiac troponin T assay derived from Internal Quality Control data
- Biological variation of non-SI traceable biological quantities: example of proteins
- Effect of tibolone therapy on lipids and coagulation indices
- Acknowledgement
- Contents Volume 44, 2006
- Author Index
- Subject Index
