Factors influencing analysis of prolyl endopeptidase in human blood and cerebrospinal fluid: increase in assay sensitivity.

Prolyl endopeptidase (EC 3.4.21.26) (PEP) is present in nearly all investigated mammalian cells and biological fluids and might be involved in the degradation of physiologically important neuropeptides. To be able to investigate the variation of PEP in blood and cerebrospinal fluid (CSF) in human disease, the factors influencing analysis of PEP in these body fluids must be determined. The purpose of the present work was to study the influence of storage conditions, anticoagulation additives, freezing and thawing and substrate solvent on determination of PEP in blood plasma/serum and CSF. It was found that the PEP activity was about 10% higher in plasma (with EDTA and heparinate for anticoagulation) than in serum. Storage at room temperature (20 degrees C) caused a rapid decline in enzyme activity, which was smaller but still considerable at 4 degrees C. Storage at -20 degrees C and -70 degrees C did not decrease the PEP activity. Freezing and thawing of plasma/serum samples showed that the first freeze-thawing cycle produced a 20% reduction in enzyme activity but little further decrease was observed during subsequent cycles of freeze-thawing. In conclusion, PEP activity should preferably be measured within one hour after sampling using EDTA- or heparinate plasma. For long-term storage, samples should be immediately frozen and stored at -20 degrees C or colder. The selection and amount of the organic solvent used to dissolve the fluorogenic substrate strongly influenced the sensitivity of the assay. By developing an optimal solvent system an increase in assay sensitivity of about 400% could be obtained, which for the first time allowed measurement of the PEP activity in CSF.