Effect of anticoagulants and cell separation media as preanalytical determinants on zymographic analysis of plasma matrix metalloproteinases.

To the Editor: Matrix metalloproteinases (MMPs) are calcium/zinc-dependent endoproteinases involved in physiologic and pathologic processes, modulating extracellular matrix degradation (1 ). MMP-2 (EC 3.4.24.24) and MMP-9 (EC 3.4.24.35) circulating in the peripheral blood (PB) of patients with neoplasia showed contrasting results, revealing the preanalytical issue of whether the method of PB sampling influences MMP concentrations (2 ) and their zymographic profiles (3 ). We therefore analyzed the effects of anticoagulants and cell separation media on PB gelatinolytic profiles. PB samples from 30 healthy volunteers were collected into Vacutainer Tubes with clot activator (SST), lithium heparin (LH), dipotassium EDTA (K2E), sodium fluoride/potassium oxalate (NaF/KOx), and buffered/acidic citrate [natrium citrate (9NC), acid-citrate-dextrose (ACD), and citrate-phosphate-dextrose-adenine (CPDA); Becton Dickinson]. After centrifugation at 500g for 15 min at 4 °C, the supernatants and buffy coats were collected and analyzed. Leukocyte subpopulations were obtained after Lympholyte gradient (5.64% Nycograde Polysucrose 400, 9.65% sodium diatrizoate; Cedarlane), and their subset recovery was tested through cytometric analysis (4 ). Gelatinases from leukocytes and plasma samples were analyzed by gelatin zymography, with 150 g of total protein loaded on the gel (3 ). Calibrators were prepared from capillary PB (5 ). MMP-2 and MMP-9 were measured by ELISA (5 ). Differences were compared using the Mann–Whitney Utest and the paired t-test; P values 0.05 were statistically significant. The present work was carried out in accordance with the ethics standards of the Helsinki Declaration of 1975, as revised in 1983. We found in plasma a 72-kDa constitutive gelatinase that was produced by nonproteolytic activation of MMP-2 with sodium dodecyl sulfate, and additional MMP-9 forms at 92, 130, and 225 kDa. Western blot analysis, Ca /Zn dependence, and p-aminophenyl-mercuric acetate activation (data not shown) identified the plasma gelatinases as fibroblast-derived proMMP-2 and neutrophil-derived proMMP-9, circulating as latent activatable proenzymes. MMP-9 was significantly higher in Fig. 1. Gelatin zymograms of plasma and serum MMPs from the same healthy individuals (median age, 38 years).