Quickscan

Separation of serum LDH isozymes was performed by electrophoresis using Quickgel LD (Helena Laboratories Japan Co, Saitama, Japan) and an exclusive electrophoresis chamber (Helena Laboratories Japan Co) according to the manufacturer’s instructions. To obtain a sharply defined electrophoretic profile, samples with tLDH activity values higher than 1000 IU/L were diluted with saline to below 1000 IU/L. LDH zymograms were stained with TitanGel S-LD reagent (QG) (Helena Laboratories Japan Co) according to the manufacturer’s instructions. Stained zymograms were scanned, and the fraction rate was quantified using Jokoh densitron CR-20 (JOKOH Co., LTD. Kikukawa, Shizuoka, Japan) and QuickScan (Helena Laboratories Japan Co). The high correlativity of measurement values between CR-20 and QuickScan has been confirmed by the measurement of 67 samples (data not shown). Thus, we pooled the measurement values obtained from both densitometers for statistical analysis in this study. Each isozyme activity value was obtained by multiplying the fraction rate of each isozyme by tLDH activity value.

Plasma pyruvate concentrations were measured by a previously described method (Czok and Lamprecht 1974 ) and lactate concentrations were measured with a commercial kit (Lactate Assay Kit-WST, Dojindo, Tokyo, Japan). Plasma LDH (Kaloustian et al. 1969 (link)) and MDH (Bergmeyer and Bernet 1974 ) activity were measured by previously described methods. The plasma M/L ratio was calculated as MDH activity divided by LDH activity. Plasma LDH isozyme patterns were detected by the biphasic agarose gel electrophoresis utilizing commercial Quick LD gels (Helena Laboratories, Saitama, Japan) (Hirakawa et al. 2012 (link)). The LDH fraction was assessed and analyzed using Quick Scan (Helena Laboratories, Saitama, Japan).
These measurements were carried out by one person in the Laboratory of Veterinary Biochemistry, Nippon Veterinary and Life Science University.