Clin Chem Lab Med 2009;47(7):888–891  2009 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2009.203 2007/71 Article in press - uncorrected proof Letter to the Editor Positive correlations between serum and plasma matrix metalloproteinase (MMP)-2 or MMP-9 levels in disease conditions Raquel F. Gerlach 1 , Cesar A. Meschiari 1 , Andrea M. Marcaccini 1 , Ana C.T. Palei 2 , Valeria C. Sandrim 3 , Ricardo C. Cavalli 4 and Jose E. Tanus-Santos 3, * 1 Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil 2 Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil 3 Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil 4 Department of Gynecology and Obstetrics, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil Keywords: metalloproteinases; plasma; serum. Matrix metalloproteinases (MMPs) are zinc-depend- ent enzymes capable of degrading components of the extracellular matrix during physiological and patho- logical processes. Of particular importance, altered concentrations of MMP-2 (gelatinase A, EC 3.4.24.24) and MMP-9 (gelatinase B, EC 3.4.24.35) have been reported to reflect important pathophysiological alter- ations in many disease conditions and in response to drug therapy (1–6). However, while circulating levels of MMPs, especially MMP-9, have been suggested to have diagnostic and prognostic value in many disease conditions, many authors have neglected preanaly- tical factors that can significantly affect measured concentrations of circulating MMPs and their endo- genous inhibitors; the tissue inhibitors of metallo- proteinases (TIMPs) (7). This is an important issue and many publications cast doubt on the validity of many previous studies that used serum instead of plasma to assess MMPs and TIMPs concentrations (7). We along with others have shown artificially higher MMP- 9 concentrations in serum compared with plasma (7–9). Artificially higher MMP concentrations meas- ured in clinical studies could hamper their diagnostic *Corresponding author: Jose E. Tanus-Santos, MD, PhD, Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirao Preto, SP, Brazil Phone: q55 16 3602 3163, Fax: q55 16 3633 2301, E-mail: tanus@fmrp.usp.br; tanussantos@yahoo.com validity (7) and thus have detrimental impact on clinical decision-making. However, no previous study has examined whether serum MMP concentrations correlate with respective plasma MMP concentrations in patients. We examined whether serum concentrations of MMP-9, MMP-2, TIMP-1, and TIMP-2 correlate with respective plasma concentrations in two disease con- ditions associated with increased circulating MMP-9 concentrations. If significant correlation exists between MMP concentrations in serum and plasma samples, then assessment of MMP concentrations in serum samples would not preclude clinicians from making appropriate decisions, as long as serum is consistently used during follow-up of patients. The present work was carried out in accordance with the ethics standards of the Helsinki Declaration of 1975, as revised in 1983. Approval for use of human subjects was obtained from the Institutional Review Board of the University of Sao Paulo, Brazil. We studied 28 women with gestational hypertension (2). After completing studies of MMPs and TIMPs in this group of patients, we confirmed our findings by studying another small group of 13 patients with periodontal disease. Venous blood samples were collected into standard Vacutainer tubes (Becton- Dickinson, Sao Paulo, Brazil) containing either sodi- um/potassium EDTA (plasma) or no anticoagulants (serum). Samples were centrifuged (1000=g for 15 min) at 248C within 10 min following blood collec- tion, serum/plasma aliquots were separated, and then immediately frozen and stored at –708C until meas- urement of MMP-9, MMP-2, TIMP-1, and TIMP-2 concentrations. Serum and plasma concentrations of MMP-9, TIMP- 1, and TIMP-2 were measured using commercially available enzyme-linked immunosorbent assay kits (R&D Systems Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. MMP-2 concentrations in plasma and serum were determined using gelatin zymography as described previously (8). Briefly, plasma samples were subject- ed to electrophoresis on 7% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) co-polymerized with gelatin (1%) as the substrate. Fol- lowing electrophoresis, the gel was incubated for 1 h at room temperature in a 2% Triton X-100 solution, and then incubated at 378C for 16 h in Tris-HCl buffer, pH 7.4, containing 10 mmol/L CaCl 2 . The gels were stained with 0.05% Coomassie Brilliant Blue G-250, and then destained with 30% methanol and 10%