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

ABSTRACT

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 degrees C or at -70 degrees 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 microL/lane for MMP-2, and from 0.75 microL/lane to 2 microL/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.