Published online before print November 29, 2007, doi: 10.1161/ATVBAHA.107.158790
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© 2008 American Heart Association, Inc.
Letters to the Editor |
Matrix Metalloproteinase-8 and Tissue Inhibitor of Metalloproteinase-1 in Serum Do Not Reflect the Analytes Circulating in Blood
Department of Urology, Research Division, University Hospital Charité, Berlin, Germany
Key Words: matrix metallproteinases • preanalytical impact • sample processing • serum • plasma
To the Editor:
It was with great interest that I read the article by Tuomainen et al1 recently published in this journal on the association of matrix metalloproteinase (MMP)-8 and tissue inhibitor of MMP-1 (TIMP-1) with cardiovascular diseases. The authors measured both analytes in serum and concluded from their results that serum MMP-8 and the ratio of MMP-8 to TIMP-1 were useful biomarkers with prognostic and diagnostic significances in men with cardiovascular disease, especially in those with prevalent or subclinical arteriosclerosis.1 Although I am not experienced in this clinical field, I believe it might be meaningful to make the readership of this journal aware of an important issue that was obviously not considered by Tuomainen et al in their study design. It refers to the influence of blood sample processing as essential precondition for the correct determination of circulating MMPs and TIMPs in peripheral blood. The effect of the type of blood sample, either collected as serum with or without clot activator or as plasma with the anticoagulants heparin, citrate, or EDTA, on the measurement of these analytes was discussed in detail both in analytical and clinical journals.2–9 For example, serum samples are less suitable to measure circulating MMP-9 or TIMP-1 in blood because these components are released from platelets and leukocytes during coagulation or blood collection.2,8,9 Tuomainen et al used serum samples for their measurements. But no informative details regarding the blood sample collection and handling, either with or without clot activator, were given in the description of the methods in that article. On the other hand, there is as yet no information available from literature on the effect of blood sampling on the circulating MMP-8. To clarify this aspect of a rather insufficient consideration of preanalytical interfering factors which may lead to misinterpretation of data, I should like to demonstrate that problem by summarizing some of our own experiments.
Serum and plasma samples from 10 healthy adults for MMP-8 and from 8 adults for TIMP-1 were prepared in plastic tubes (Monovette Systems, Sarstedt AG) by centrifugation of the collected blood samples at 1600g for 15 minutes within 30 minutes after venipuncture. Tubes without additives or with kaolin-coated plastic granulate as coagulation accelerator were used to prepare native serum (serum(–)) and serum after enhanced coagulation (serum(+)), respectively. Tubes coated with lithium heparin, sodium citrate, or dipotassium EDTA were used to prepare corresponding plasma samples. The supernatants were carefully removed and stored at –80°C until analysis. MMP-8 measurements were performed with the Fluorokine MultiAnalyte Profiling assay (R&D Systems) on a Luminex 100 Bioanalyzer (Luminex Corp). The assay detects pro-, mature, and TIMP-1 complexed MMPs with less than 0.5% cross-reactivity between the different MMPs. TIMP-1 was determined using the TIMP-1 ELISA from Amersham. GraphPad Prism 5.01 for Windows (GraphPad Software) was used for statistical analyses using ANOVA and Student paired t test. Statistical significances were considered with P<0.05.
Data are summarized in the Figure with the following conclusions: (1) MMP-8 and TIMP-1 concentrations are several times higher in serum than in plasma samples; (2) MMP-8 showed distinctly higher values in serum(+) samples collected with clot activator as the conventional way to prepare serum for routine use compared with those in serum(–) samples collected without clot activator.
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Our results show that the measurement of MMP-8 and TIMP-1 in serum does not only detect these components as they are primarily circulating in blood but to an extremely high degree as released from platelets and leukocytes depending on the different blood collection procedures. MMP-8 and TIMP-1 are abundantly expressed in platelets and leukocytes.10,11 The data confirm the importance of measuring true concentrations of circulating MMPs and TIMPs in peripheral blood. The problem is not only restricted to MMP-9, which was recently studied in detail.9 Concerns have been raised that this high unspecific background of MMPs and TIMPs released from blood cells in serum could hamper the diagnostic trueness or might be the reason for a misinterpretation of data.12
In conclusion, clinicians should be aware of these preanalytical pitfalls of data if MMPs and TIMPs are used as diagnostic or prognostic biomarkers for clinical purposes. Therefore, it might be reasonable to reconsider the conclusions of Tuomainen et al also under this aspect.
Acknowledgments
I thank Silke Klotzek for excellent technical assistance.
Disclosures
None.
References
1. Tuomainen AM, Nyyssonen K, Laukkanen JA, Tervahartiala T, Tuomainen TP, Salonen JT, Sorsa T, Pussinen PJ. Serum matrix metalloproteinase-8 concentrations are associated with cardiovascular outcome in men. Arterioscler Thromb Vasc Biol. 2007; 27: 2722–2728.
2. Jung K, Nowak L, Lein M, Henke W, Schnorr D, Loening SA. What kind of specimen should be selected for determining tissue inhibitor of metalloproteinase-1 (TIMP-1) in blood? Clin Chim Acta. 1996; 254: 97–100.[CrossRef][Medline] [Order article via Infotrieve]
3. Jung K, Laube C, Lein M, Lichtinghagen R, Tschesche H, Schnorr D, Loening S. Kind of sample as preanalytical determinant of matrix metalloproteinases 2 and 9 (MMP2; MMP9) and tissue inhibitor of metalloproteinase 2 (TIMP2) in blood. Clin Chem. 1998; 44: 1060–1062.
4. Makowski GS, Ramsby ML. Use of citrate to minimize neutrophil matrix metalloproteinase-9 in human plasma. Anal Biochem. 2003; 322: 283–286.[CrossRef][Medline] [Order article via Infotrieve]
5. Mannello F, Luchetti F, Canonico B, Papa S. Effect of anticoagulants and cell separation media as preanalytical determinants on zymographic analysis of plasma matrix metalloproteinases. Clin Chem. 2003; 49: 1956–1957.
6. Meisser A, Cohen M, Bischof P. Concentrations of circulating gelatinases (matrix metalloproteinase-2 and -9) are dependent on the conditions of blood collection. Clin Chem. 2005; 51: 274–276.
7. Souza-Tarla CD, Uzuelli JA, Machado AA, Gerlach RF, Tanus-Santos JE. Methodological issues affecting the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities. Clin Biochem. 2005; 38: 410–414.[CrossRef][Medline] [Order article via Infotrieve]
8. Mannello F, Tonti GA. Gelatinase concentrations and zymographic profiles in human breast cancer: matrix metalloproteinases circulating in plasma are better markers for the subclassification and early prediction of cancer: the coagulation/fibrinolysis pathways alter the release, activation and recovery of different gelatinases in serum. Int J Cancer. 2007; 121: 216–218.[CrossRef][Medline] [Order article via Infotrieve]
9. Mannello F, Tonti GA, Tanus-Santos JE, Gerlach RF. Silicate increases the release of MMP-9 forms in peripheral blood: why gelatin zymography differs significantly in citrate plasma and serum obtained with or without clot activators. Clin Chem. 2007; 53: 1981–1982.
10. Santos-Martinez MJ, Medina C, Jurasz P, Radomski MW. Role of metalloproteinases in platelet function. Thromb Res. In press.
11. Owen CA, Hu Z, Lopez-Otin C, Shapiro SD. Membrane-bound matrix metalloproteinase-8 on activated polymorphonuclear cells is a potent, tissue inhibitor of metalloproteinase-resistant collagenase and serpinase. J Immunol. 2004; 172: 7791–7803.
12. Wu CY, Wu MS, Chiang EP, Chen YJ, Chen CJ, Chi NH, Shih YT, Chen GH, Lin JT. Plasma matrix metalloproteinase-9 level is better than serum matrix metalloproteinase-9 level to predict gastric cancer evolution. Clin Cancer Res. 2007; 13: 2054–2060.
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Arterioscler Thromb Vasc Biol 2008 28: 611-614.
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