Methodological issues affecting the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities

Objectives: To examine whether a linear relationship exists between the volume of plasma loaded in each gel lane and the gelatinolytic activities of plasma matrix metalloproteinase (pMMP)-2 and pMMP-9, and the influences of plasma storage temperature, fasting, and a variable number of repeated freeze–thaw cycles. Design and methods: Plasma from venous blood collected into EDTA tubes was stored for 1 month at −20°C or at −70°C until assayed for gelatin zymography of pMMP-2 and pMMP-9. The effects of food intake, repeated freeze–thaw cycles, and the gelatinolytic activity produced by serial dilutions of plasma were examined. Results: Storage temperature, fasting, and up to five repeated freeze–thaw cycles had no effect on the activities of pMMPs (all P< 0.05). A linear relationship was found with plasma volumes below 0.75 μL/lane for MMP-2, and from 0.75 μL/lane to 2 μL/lane for MMP-9. Conclusions: Different volumes of plasma should be studied when pMMP-2 and pMMP-9 activities are studied. Storage temperature, repeated freeze–thaw cycles, and food intake do not affect the activities of pMMPs