Using Zymography to Assess Circulating Matrix Metalloproteinase (MMP)-2 and MMP-9 in Clinical Samples

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that cleave a variety of different substrates and play key roles in both physiological and pathological processes. They are involved in the pathophysiology of many disease conditions, and for this reason, many clinical studies have been carried out to examine whether circulating MMP concentrations reflect the severity of disease conditions. This article presents detailed information that is critical to assess circulating MMP-2 and MMP-9 activity in clinical samples by using zymography, which is a powerful technique that has been used for more than three decades to detect picogram quantities of MMPs. Important methodological issues have been addressed in the past regarding this technique, and some analytical and preanalytical details may severely affect the results and thus hamper the diagnostic and prognostic value of such measurements. While fasting or storing plasma samples for 1 month at -20 °C or at -70 °C does not significantly affect plasma MMP-2 or MMP-9 activity, repeating freeze-thaw cycles of plasma samples decrease MMP-9 activity after seven cycles. Importantly, the appropriate volume to assess MMP-2 is significantly lower than the appropriate volume to assess MMP-9 activity. Finally, special concerns exist with respect to circulating MMP-9 (but not MMP-2) measurements, which are found at artificially higher concentrations in serum than in plasma (EDTA, citrate or heparin) samples, even though significant correlations exist between MMP-9 levels assessed in plasma and in serum samples.