**5. Detection and quantification of lactate dehydrogenase isoenzymes**

As many proteins were separated by electrophoresis, lactate dehydrogenase isoenzymes were detected (stained) specifically using a colorimetric method with an assay system in 0.1 M Gly-NaCl-NaOH, pH 8.3: 1.0 mol/l sodium lactate (1.0 ml), 10 mg/ml NAD<sup>+</sup> (1.5 ml), 1.0 mg/ml nitro blue tetrazolium (NBT) (6.0 ml), and 2.0 mg/ml phenasine methosulfate (PSM) (0.6 ml) up to 30 ml with 0.1 M Gly-NaCl-NaOH, pH 8.3. In this reaction mixture, the gels were incubated at 37°C for 20–30 min. Then, they were immersed into 7.5% (v/v) acetic acid to stop the reaction. To detect lactate dehydrogenase isoenzyme zones on the electrophoreogram, we avoid using Tris-HCl buffer as it produced quite an intensive background on the gel. PhastImage system (Pharmacia LKB, Sweden) served for densitometric scanning (613 nm) of the pattern, and for the determination of relative distribution (%) of the isoenzyme fractions, GEL LOGIC 100 IMAGING SYSTEM with Kodak 1D Image Analysis Software (Japan) was used as well. To identify individual bird LDH isoenzymes on the electrophoreogram, a principle commonly used for the identification of mammalian LDH isoenzymes was applied: the fraction nearest to the anode was designated LDH1 and that nearest to the cathode LDH5 .
