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.

To the Editor: Whether serum or plasma is the best specimen for determination of matrix metalloproteinases (MMPs) is a matter of debate, as are the influences of sample collection and processing on MMP concentrations (1–3). MMP-2 concentrations do not differ significantly in plasma and serum, whereas MMP-9 concentrations are significantly higher in serum than in plasma, and in plasma differently affected by anticoagulants (2, 3). Furthermore , both unexpected protein peaks and increased MMP-9 concentrations occur in serum samples collected in tubes coated with clot accelerators such as kaolin or silica gel (2, 4). To find an explanation of the differences in MMP-9 concentrations among citrate plasma, serum, and serum with clot accelerators (Sca), we analyzed the effects of silicate on MMP in whole blood (WB), plasma, serum, and buffy coat (BC), as well as in culture media of U-937 my-elomonocytic leukemia cells (SF U937). We obtained peripheral blood (PB) and BC samples from 30 healthy volunteers age 23–55 years (median 36 years). Samples were collected into plastic tubes (Vacutainer ® from Bec-ton Dickinson). After centrifugation at 500g for 15 min at 4 °C, the super-natants of cell culture, BCs, plasma, and serum with and without silicate were analyzed by use of Western blot (75-7F7 and GE-213 monoclonal anti-bodies against MMP-2 and-9, respectively ; Calbiochem) and gelatin zymography (7.5% polyacrylamide gels containing 2 g/L gelatin 90 Bloom type A from porcine skin; Sigma) (2). U-937 cells were cultured in serum-free conditions (to avoid endogenous bovine serum gelati-nases) and treated for 3 h with sili-cate (54 mg/L). Calibrators were prepared from capillary WB (2). The accuracy/precision of gelatinolytic activities were evaluated by zymo-gram densitometry with Image Pro-Plus software (Cybernetics) (4). Differences were compared using the Mann–Whitney U-test; P values Ͻ0.05 were considered statistically significant. All study participants gave informed consent, and the work was carried out in accordance with the ethics standards of the Helsinki Declaration of 1975, as revised in 1983. Western blots identified pro-MMP-2 (Gelatinase A, EC 3.4.24.24, 72 kDa) and pro-and complexed forms of MMP-9 (Gelatinases B, EC 3.4.24.35, of 92, 130, and 225 kDa) in whole PB (Fig. 1, lanes 2 and 3). Citrate plasma results showed that pro-MMP-2 expression did not differ significantly between serum and plasma, nor did it change with silicate treatment in any of the paired sample types. MMP-9 forms were found in significantly higher amounts in serum (mean 5-fold higher; P Ͻ0.001) …