Plasma storage at −80 °C does not protect matrix metalloproteinase-9 from degradation

Recently, matrix metalloproteinase-9 (MMP-9) has been identified as a cardiovascular risk marker and is increasingly being determined in clinical studies. Among other matrix metalloproteinases, MMP-9 is known to be self-activable, as the cleavage of the propeptide leads to the formation of an active enzyme. In such a case the issue of storage of biological samples such as plasmas is of outstanding importance, as an enzymatic activity, although minimal, may remain at common storage temperature, i.e., −80 °C. Since 2000 our institute has created a plasma library from patients presenting with acute myocardial infarction. Recently, the evaluation of the MMP-9 led to the surprise of finding a dramatically low level of detectable enzyme in the oldest plasma samples. By using zymography, enzyme-linked immunosorbent assay and Western blots, we evaluated new and old samples and found that MMP-9 degrades over time. After 2 years, the detectable total MMP-9 dropped by 65%, and the asymptotic profile of the curve reached a residual 1% level after 43 months. These results were confirmed by zymography and western blotting. TIMP-1, the natural inhibitor of MMP-9 and MMP-2, remained rather stable over time. The results suggest that human plasma MMP-9 levels should be determined as soon as possible after sampling.