2.5. Lactate Dehydrogenase (LDH) Activity Assay

The LDH activity of 14 plasma samples prepared after the indicated time delay (0, 60, 120, or 180 min after the blood collection) was addressed spectrophotometrically using a Lactate Dehydrogenase Activity Assay Kit (MAK066, Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, for each well, 25 µL of plasma was mixed with 25 µL of assay buffer and added to 50 µL of reaction mix (consisting of substrate mix diluted in the assay buffer). On each microtiter plate, duplicates of 3 µL of positive control were included. After the initial read, the plate was read continuously every five minutes for the total length of 20 min. Absorption values obtained after 10 min of incubation (per each time point) were all in the linear range and were selected for quantification.