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Effect of anticoagulants on the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 344 (2005) 147–149 www.elsevier.com/locate/yabio
Notes & Tips
EVect of anticoagulants on the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities Raquel F. Gerlach a, Juliana A. Uzuelli b, Caroline D. Souza-Tarla b, Jose E. Tanus-Santos b,¤ a
Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto and Paulo, 14049 Ribeirao Preto, SP, Brazil b Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, 14049 Ribeirao Preto, SP, Brazil Received 10 March 2005 Available online 17 May 2005
Matrix metalloproteinases (MMPs)1 are zinc-dependent enzymes involved in the degradation of components of the extracellular matrix during physiological and pathological processes. SpeciWcally, MMP-2 (gelatinase A, EC 3.4.24.24) and MMP-9 (gelatinase B, EC 3.4.24.35) are involved in many physiological processes, and increased expression and activity of these enzymes have been reported to play roles in a variety of pathological conditions including neoplastic [1], cardiovascular [2], and respiratory [3] diseases. Importantly, the circulating level of MMP-9 has been suggested to be a blood borne biochemical marker helpful in the diagnosis of neoplastic and cardiovascular diseases[1,2,4–6]. Moreover, MMP-9 plasma levels were shown to predict mortality in patients with coronary artery disease [2]. Taken together, these Wndings are consistent with the notion that plasma MMP-9 and MMP-2 may have diagnostic and prognostic value. While commercially available enzyme immunoassays (ELISA) have been used in many previous studies, quantitative gel zymography is thought to compare favorably with ELISA because it allows the detection of both the latent and the active forms of both MMP-2 and MMP-9 in concentrations as low as 1 nM [7]. However, there are controversial data on the eVects of diVerent anticoagulants as preanalytical determinants of plasma MMP activities by quantitative gel zymography [8–10]. For example, lower MMP-2 activity was *
Corresponding author. Fax: +55 16 633 2301. E-mail address: [email protected] (J.E. Tanus-Santos). 1 Abbreviations used: MMPs, matrix metalloproteinases; ELISA, enzyme-linked immunosorbent assay. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.04.038
found in EDTA plasma than in heparin plasma or serum [8]. Conversely, another study carried out by the same group showed increased plasma MMP-2 activity and decreased MMP-9 activity with increasing amounts of EDTA during blood collection [9]. While the explanation for such a discrepancy is not known, the use of citrate as anticoagulant has been shown to minimize MMP-9 release during blood collection [10]. In addition, signiWcantly lower MMP-9 activity was shown in citrate plasma compared with either heparin or EDTA plasma [10]. This is an important issue because it casts doubts upon the validity of a number of previous studies using anticoagulants other than citrate. In this study we examined whether diVerent anticoagulants aVect plasma MMP-2 and MMP-9 activities. In addition, we evaluated the eVects of increasing concentrations of anticoagulants and examined whether there is correlation between MMPs activities measured in serum and those measured in plasma samples separated from blood drawn into tubes with diVerent anticoagulants. To assess whether diVerent anticoagulants aVect plasma MMPs activities, venous blood samples were collected from healthy subjects (N D 8) into standard vacutainer tubes (Becton–Dickinson, Brazil) containing either sodium/potassium EDTA, sodium citrate, lithium heparin, or no anticoagulants. To assess the eVects of increasing concentrations of anticoagulants on plasma MMPs activities, we added either the recommended volume of blood (1X), one half of that volume (2X), or one third of that volume (3X) to tubes with EDTA, citrate, or heparin. Blood samples were centrifuged (1000g for 15 min) no later than 20 min after
148
Notes & Tips / Anal. Biochem. 344 (2005) 147–149
blood drawing, and serum/plasma aliquots were immediately assayed for gelatin zymography of MMP-2 and MMP-9. To assess MMP-2 and MMP-9 activities, 10 and 30 L of each plasma sample were diluted in 190 and in 170 L of sample buVer (2% SDS, 125 mM Tris– HCl, pH 6.8, 10% glycerol, and 0.001% bromophenol blue), respectively [11]. Thereafter, 10 L of each plasma solution was loaded in each gel lane and gelatin zymography of MMP-2 and MMP-9 was performed in duplicate as previously described [11,12]. BrieXy, plasma solutions were subjected to electrophoresis on 12% SDS–PAGE copolymerized with gelatin (1%) as the substrate. After electrophoresis was complete, the gel was incubated for 1 h at room temperature in a 2% Triton X-100 solution and incubated at 37 °C for 16 h in Tris–HCl buVer, pH 7.4, containing 10 mmol/L CaCl2. The gels were stained with 0.05% Coomassie brilliant blue G-250 and then destained with 30% methanol and 10% acetic acid [7]. Gelatinolytic activities were detected as unstained bands against the background of Coomassie-blue-stained gelatin. Enzyme activity was assayed by densitometry using a Kodak Electrophoresis Documentation and Analysis System 290 (Kodak, Rochester, NY). The pro and active forms of MMP-2 and MMP-9 were identiWed as bands at 72 and 67 kDa and at 92 and 87 kDa respectively, by the relation of log Mr to the relative mobility of Sigma SDS–PAGE LMW marker proteins. The results are expressed as means § SE. One-way analysis of variance for repeated measurements followed by the Bonferroni test (StatView for Windows, Cary, NC, USA) was used to analyze diVerences in MMPs’ activities. A probability value <0.05 was considered the minimum level of statistical signiWcance. Fig. 1 shows a representative zymogram of serum and plasma samples. Fig. 2 shows no signiWcant diVerences in Pro-MMP-9 (Fig. 2A) and in Pro-MMP-2 (Fig. 2C) activities when serum was compared with EDTA, citrate, and heparin plasma (both p > 0.05). However, signiWcantly higher MMP-9 activity was observed in serum compared with EDTA, citrate, or heparin plasma (Fig. 2B; all p < 0.05). While our results conWrm previous Wndings showing increased MMP-9 activity in serum compared with plasma [10], the diVer-
ences that we found here are much less important than those previously reported [10]. In addition, although citrate-treated plasma samples showed signiWcantly lower pro-MMP-9 activity than either EDTA- or heparin-treated samples, we found signiWcant correlations (all Pearson r values above 0.85) between MMP-9 activities in EDTA, citrate, and heparin plasma (all p < 0.05). These results suggest that the diVerent anticoagulants studied here may have no major consequences in the comparative evaluation of pathological and normal blood samples drawn during a single study, as long as only one anticoagulant is used in the study. Importantly, these Wndings suggest that the validity of previous studies using anticoagulants other than citrate may not be aVected by the diVerences in MMP-9 activity associated with use of diVerent anticoagulants. This is because the diVerent anticoagulants cause proportional changes in MMP-9 in all samples. The use of serum samples to assess MMP-9 activity, however, is probably not recommended because artiWcially higher levels would be measured. Further supporting this suggestion, we found no signiWcant correlation between MMPs activities in serum and in EDTA, citrate, or heparin plasmas (all p > 0.05). Finally, increasing the concentrations of the three anticoagulants produced no eVects on MMPs activities (Fig. 2, all p > 0.05), except for a signiWcant decrease in MMP-9 activity when increasing concentrations of citrate were studied (Fig. 2B; p < 0.05). We are not aware of any possible explanation for such a Wnding. These Wndings suggest that increasing the concentrations of the anticoagulant does not signiWcantly aVect the diVerences observed among anticoagulants. In conclusion, our results suggest that similar MMPs activities are found in plasma samples separated from blood collected into EDTA, citrate, or heparin tubes. While citrate plasma presents lower proMMP-9 levels compared with EDTA or heparin plasma, there is high correlation among MMPs activities measured in the three anticoagulants, thus suggesting that either citrate, EDTA, or heparin could be used to assess plasma MMPs activities. Serum samples, however, should not be used to assess MMPs activities.
Fig. 1. Representative zymogram of serum and plasma samples separated from blood drawn into tubes with increasing concentrations of anticoagulants (1X, 2X, or 3X). The bands corresponding to pro-MMP-9, MMP-9, and pro-MMP-2 are shown.
Notes & Tips / Anal. Biochem. 344 (2005) 147–149
149
Acknowledgments This study was supported by Fundação de Amparo à Pesquisa do Estado de Sao Paulo, Conselho Nacional de Desenvolvimento CientíWco e Tecnológico, and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior.
References
Fig. 2. (A, B, and C) Mean (§SE) values (N D 8) of pro-MMP-9, MMP-9, and pro-MMP-2 activities, respectively, in the serum and plasma samples separated from blood drawn into tubes with increasing concentrations of anticoagulants (1X, 2X, or 3X). *p < 0.05 for the indicated comparison.
[1] E. Farias, S. Ranuncolo, C. Cresta, S. Specterman, E. Armanasco, M. Varela, J. Lastiri, M.G. Pallotta, E. Bal de Kier JoVe, L. Puricelli, Plasma metalloproteinase activity is enhanced in the euglobulin fraction of breast and lung cancer patients, Int. J. Cancer 89 (2000) 389–394. [2] S. Blankenberg, H.J. Rupprecht, O. Poirier, C. Bickel, M. Smieja, G. Hafner, J. Meyer, F. Cambien, L. Tiret, Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease, Circulation 107 (2003) 1579–1585. [3] H. Ohbayashi, Matrix metalloproteinases in lung diseases, Curr. Protein Pept. Sci. 3 (2002) 409–421. [4] P. Altieri, C. Brunelli, S. Garibaldi, A. Nicolino, S. Ubaldi, P. Spallarossa, L. Olivotti, P. Rossettin, A. Barsotti, G. Ghigliotti, Metalloproteinases 2 and 9 are increased in plasma of patients with heart failure, Eur. J. Clin. Invest. 33 (2003) 648–656. [5] S.M. Ranuncolo, E. Armanasco, C. Cresta, E. Bal De Kier JoVe, L. Puricelli, Plasma MMP-9 (92 kDa-MMP) activity is useful in the follow-up and in the assessment of prognosis in breast cancer patients, Int. J. Cancer 106 (2003) 745–751. [6] J.R. Lynch, R. Blessing, W.D. White, H.P. Grocott, M.F. Newman, D.T. Laskowitz, Novel diagnostic test for acute stroke, Stroke 35 (2004) 57–63. [7] D.E. Kleiner, W.G. Stetler-Stevenson, Quantitative zymography: detection of picogram quantities of gelatinases, Anal. Biochem. 218 (1994) 325–329. [8] F. Mannello, EVects of blood collection methods on gelatin zymography of matrix metalloproteinases, Clin. Chem. 49 (2003) 339–340. [9] F. Mannello, F. Luchetti, B. Canonico, S. Papa, EVect of anticoagulants and cell separation media as preanalytical determinants on zymographic analysis of plasma matrix metalloproteinases, Clin. Chem. 49 (2003) 1956–1957. [10] G.S. Makowski, M.L. Ramsby, Use of citrate to minimize neutrophil matrix metalloproteinase-9 in human plasma, Anal. Biochem. 322 (2003) 283–286. [11] C.D. Souza-Tarla, J.A. Uzuelli, A.A. Machado, R.F. Gerlach, J.E. Tanus-Santos, Methodological issues aVecting the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities, Clin. Biochem. 38 (2005) 410–414. [12] A.C.T. Palei, R.A.G. Zaneti, G.M. Fortuna, R.F. Gerlach, J.E. Tanus-Santos, Hemodynamic beneWts of matrix metalloproteinase-9 inhibition by doxycycline during experimental acute pulmonary embolism, Angiology (2005) in press.
Notes & Tips
EVect of anticoagulants on the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities Raquel F. Gerlach a, Juliana A. Uzuelli b, Caroline D. Souza-Tarla b, Jose E. Tanus-Santos b,¤ a
Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto and Paulo, 14049 Ribeirao Preto, SP, Brazil b Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, 14049 Ribeirao Preto, SP, Brazil Received 10 March 2005 Available online 17 May 2005
Matrix metalloproteinases (MMPs)1 are zinc-dependent enzymes involved in the degradation of components of the extracellular matrix during physiological and pathological processes. SpeciWcally, MMP-2 (gelatinase A, EC 3.4.24.24) and MMP-9 (gelatinase B, EC 3.4.24.35) are involved in many physiological processes, and increased expression and activity of these enzymes have been reported to play roles in a variety of pathological conditions including neoplastic [1], cardiovascular [2], and respiratory [3] diseases. Importantly, the circulating level of MMP-9 has been suggested to be a blood borne biochemical marker helpful in the diagnosis of neoplastic and cardiovascular diseases[1,2,4–6]. Moreover, MMP-9 plasma levels were shown to predict mortality in patients with coronary artery disease [2]. Taken together, these Wndings are consistent with the notion that plasma MMP-9 and MMP-2 may have diagnostic and prognostic value. While commercially available enzyme immunoassays (ELISA) have been used in many previous studies, quantitative gel zymography is thought to compare favorably with ELISA because it allows the detection of both the latent and the active forms of both MMP-2 and MMP-9 in concentrations as low as 1 nM [7]. However, there are controversial data on the eVects of diVerent anticoagulants as preanalytical determinants of plasma MMP activities by quantitative gel zymography [8–10]. For example, lower MMP-2 activity was *
Corresponding author. Fax: +55 16 633 2301. E-mail address: [email protected] (J.E. Tanus-Santos). 1 Abbreviations used: MMPs, matrix metalloproteinases; ELISA, enzyme-linked immunosorbent assay. 0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.04.038
found in EDTA plasma than in heparin plasma or serum [8]. Conversely, another study carried out by the same group showed increased plasma MMP-2 activity and decreased MMP-9 activity with increasing amounts of EDTA during blood collection [9]. While the explanation for such a discrepancy is not known, the use of citrate as anticoagulant has been shown to minimize MMP-9 release during blood collection [10]. In addition, signiWcantly lower MMP-9 activity was shown in citrate plasma compared with either heparin or EDTA plasma [10]. This is an important issue because it casts doubts upon the validity of a number of previous studies using anticoagulants other than citrate. In this study we examined whether diVerent anticoagulants aVect plasma MMP-2 and MMP-9 activities. In addition, we evaluated the eVects of increasing concentrations of anticoagulants and examined whether there is correlation between MMPs activities measured in serum and those measured in plasma samples separated from blood drawn into tubes with diVerent anticoagulants. To assess whether diVerent anticoagulants aVect plasma MMPs activities, venous blood samples were collected from healthy subjects (N D 8) into standard vacutainer tubes (Becton–Dickinson, Brazil) containing either sodium/potassium EDTA, sodium citrate, lithium heparin, or no anticoagulants. To assess the eVects of increasing concentrations of anticoagulants on plasma MMPs activities, we added either the recommended volume of blood (1X), one half of that volume (2X), or one third of that volume (3X) to tubes with EDTA, citrate, or heparin. Blood samples were centrifuged (1000g for 15 min) no later than 20 min after
148
Notes & Tips / Anal. Biochem. 344 (2005) 147–149
blood drawing, and serum/plasma aliquots were immediately assayed for gelatin zymography of MMP-2 and MMP-9. To assess MMP-2 and MMP-9 activities, 10 and 30 L of each plasma sample were diluted in 190 and in 170 L of sample buVer (2% SDS, 125 mM Tris– HCl, pH 6.8, 10% glycerol, and 0.001% bromophenol blue), respectively [11]. Thereafter, 10 L of each plasma solution was loaded in each gel lane and gelatin zymography of MMP-2 and MMP-9 was performed in duplicate as previously described [11,12]. BrieXy, plasma solutions were subjected to electrophoresis on 12% SDS–PAGE copolymerized with gelatin (1%) as the substrate. After electrophoresis was complete, the gel was incubated for 1 h at room temperature in a 2% Triton X-100 solution and incubated at 37 °C for 16 h in Tris–HCl buVer, pH 7.4, containing 10 mmol/L CaCl2. The gels were stained with 0.05% Coomassie brilliant blue G-250 and then destained with 30% methanol and 10% acetic acid [7]. Gelatinolytic activities were detected as unstained bands against the background of Coomassie-blue-stained gelatin. Enzyme activity was assayed by densitometry using a Kodak Electrophoresis Documentation and Analysis System 290 (Kodak, Rochester, NY). The pro and active forms of MMP-2 and MMP-9 were identiWed as bands at 72 and 67 kDa and at 92 and 87 kDa respectively, by the relation of log Mr to the relative mobility of Sigma SDS–PAGE LMW marker proteins. The results are expressed as means § SE. One-way analysis of variance for repeated measurements followed by the Bonferroni test (StatView for Windows, Cary, NC, USA) was used to analyze diVerences in MMPs’ activities. A probability value <0.05 was considered the minimum level of statistical signiWcance. Fig. 1 shows a representative zymogram of serum and plasma samples. Fig. 2 shows no signiWcant diVerences in Pro-MMP-9 (Fig. 2A) and in Pro-MMP-2 (Fig. 2C) activities when serum was compared with EDTA, citrate, and heparin plasma (both p > 0.05). However, signiWcantly higher MMP-9 activity was observed in serum compared with EDTA, citrate, or heparin plasma (Fig. 2B; all p < 0.05). While our results conWrm previous Wndings showing increased MMP-9 activity in serum compared with plasma [10], the diVer-
ences that we found here are much less important than those previously reported [10]. In addition, although citrate-treated plasma samples showed signiWcantly lower pro-MMP-9 activity than either EDTA- or heparin-treated samples, we found signiWcant correlations (all Pearson r values above 0.85) between MMP-9 activities in EDTA, citrate, and heparin plasma (all p < 0.05). These results suggest that the diVerent anticoagulants studied here may have no major consequences in the comparative evaluation of pathological and normal blood samples drawn during a single study, as long as only one anticoagulant is used in the study. Importantly, these Wndings suggest that the validity of previous studies using anticoagulants other than citrate may not be aVected by the diVerences in MMP-9 activity associated with use of diVerent anticoagulants. This is because the diVerent anticoagulants cause proportional changes in MMP-9 in all samples. The use of serum samples to assess MMP-9 activity, however, is probably not recommended because artiWcially higher levels would be measured. Further supporting this suggestion, we found no signiWcant correlation between MMPs activities in serum and in EDTA, citrate, or heparin plasmas (all p > 0.05). Finally, increasing the concentrations of the three anticoagulants produced no eVects on MMPs activities (Fig. 2, all p > 0.05), except for a signiWcant decrease in MMP-9 activity when increasing concentrations of citrate were studied (Fig. 2B; p < 0.05). We are not aware of any possible explanation for such a Wnding. These Wndings suggest that increasing the concentrations of the anticoagulant does not signiWcantly aVect the diVerences observed among anticoagulants. In conclusion, our results suggest that similar MMPs activities are found in plasma samples separated from blood collected into EDTA, citrate, or heparin tubes. While citrate plasma presents lower proMMP-9 levels compared with EDTA or heparin plasma, there is high correlation among MMPs activities measured in the three anticoagulants, thus suggesting that either citrate, EDTA, or heparin could be used to assess plasma MMPs activities. Serum samples, however, should not be used to assess MMPs activities.
Fig. 1. Representative zymogram of serum and plasma samples separated from blood drawn into tubes with increasing concentrations of anticoagulants (1X, 2X, or 3X). The bands corresponding to pro-MMP-9, MMP-9, and pro-MMP-2 are shown.
Notes & Tips / Anal. Biochem. 344 (2005) 147–149
149
Acknowledgments This study was supported by Fundação de Amparo à Pesquisa do Estado de Sao Paulo, Conselho Nacional de Desenvolvimento CientíWco e Tecnológico, and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior.
References
Fig. 2. (A, B, and C) Mean (§SE) values (N D 8) of pro-MMP-9, MMP-9, and pro-MMP-2 activities, respectively, in the serum and plasma samples separated from blood drawn into tubes with increasing concentrations of anticoagulants (1X, 2X, or 3X). *p < 0.05 for the indicated comparison.
[1] E. Farias, S. Ranuncolo, C. Cresta, S. Specterman, E. Armanasco, M. Varela, J. Lastiri, M.G. Pallotta, E. Bal de Kier JoVe, L. Puricelli, Plasma metalloproteinase activity is enhanced in the euglobulin fraction of breast and lung cancer patients, Int. J. Cancer 89 (2000) 389–394. [2] S. Blankenberg, H.J. Rupprecht, O. Poirier, C. Bickel, M. Smieja, G. Hafner, J. Meyer, F. Cambien, L. Tiret, Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease, Circulation 107 (2003) 1579–1585. [3] H. Ohbayashi, Matrix metalloproteinases in lung diseases, Curr. Protein Pept. Sci. 3 (2002) 409–421. [4] P. Altieri, C. Brunelli, S. Garibaldi, A. Nicolino, S. Ubaldi, P. Spallarossa, L. Olivotti, P. Rossettin, A. Barsotti, G. Ghigliotti, Metalloproteinases 2 and 9 are increased in plasma of patients with heart failure, Eur. J. Clin. Invest. 33 (2003) 648–656. [5] S.M. Ranuncolo, E. Armanasco, C. Cresta, E. Bal De Kier JoVe, L. Puricelli, Plasma MMP-9 (92 kDa-MMP) activity is useful in the follow-up and in the assessment of prognosis in breast cancer patients, Int. J. Cancer 106 (2003) 745–751. [6] J.R. Lynch, R. Blessing, W.D. White, H.P. Grocott, M.F. Newman, D.T. Laskowitz, Novel diagnostic test for acute stroke, Stroke 35 (2004) 57–63. [7] D.E. Kleiner, W.G. Stetler-Stevenson, Quantitative zymography: detection of picogram quantities of gelatinases, Anal. Biochem. 218 (1994) 325–329. [8] F. Mannello, EVects of blood collection methods on gelatin zymography of matrix metalloproteinases, Clin. Chem. 49 (2003) 339–340. [9] F. Mannello, F. Luchetti, B. Canonico, S. Papa, EVect of anticoagulants and cell separation media as preanalytical determinants on zymographic analysis of plasma matrix metalloproteinases, Clin. Chem. 49 (2003) 1956–1957. [10] G.S. Makowski, M.L. Ramsby, Use of citrate to minimize neutrophil matrix metalloproteinase-9 in human plasma, Anal. Biochem. 322 (2003) 283–286. [11] C.D. Souza-Tarla, J.A. Uzuelli, A.A. Machado, R.F. Gerlach, J.E. Tanus-Santos, Methodological issues aVecting the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities, Clin. Biochem. 38 (2005) 410–414. [12] A.C.T. Palei, R.A.G. Zaneti, G.M. Fortuna, R.F. Gerlach, J.E. Tanus-Santos, Hemodynamic beneWts of matrix metalloproteinase-9 inhibition by doxycycline during experimental acute pulmonary embolism, Angiology (2005) in press.