Improved resolution of liver and bone alkaline phosphatase isoenzymes using wheat germ lectin or neuraminidase pretreatment with the introduction of some technical modifications

ABSTRACT

This study aims to establish a routine reliable electrophoresis method with improved separation of liver and bone alkaline phosphatase isoenzymes, which allows for their quantitation. Modifications of the already available techniques will also be studied. Samples were collected from patients with liver diseases [n = 26], with bone diseases and from children [n = 24] and from pregnant females in the third trimester [n = 10]. Control sera containing liver and intestinal isoenzymes were also used. Samples were subjected to liver function tests, calcium and phosphorus determination, cellulose acetate electrophoresis ordinary, with germ wheat lectin and with neuraminidase pretreatment. Agarose gel electrophoresis was done with and without lectin. Samples were also subjected to sequential heat inactivation. Results showed that cellulose acetate electro-phoresis gave better separation of liver and bone fractions when done with germ wheat lectin or when samples were pretreated with neuraminidase. Several modifications were suggested to improve the technique. Agarose gel affinity electrophoresis [i.e., with lectin] gave the best separation of liver and bone isoenzymes into sharply defined bands. Sequential heat inactivation was tedious and needed scrupulous control of time and temperature. It overestimated the liver isoenzyme due to inclusion of biliary and intestinal fractions in its estimation. Excellent correlation was found between the different methods used for both bone and liver isoenzymes, Biliary isoenzyme was best separated by ordinary electrophoresis whether on cellulose acetate or agarose gel. Placental isoenzyme separation required preheating the sample at 65°C for 10 minutes to destroy the bone fraction which had the same migration mobility as placental isoenzyme. It was concluded that agarose affinity gel electrophoresis gave the sharpest and clearest separation of liver and bone fractions. On the other hand, cellulose acetate electrophoresis was less expensive, more sensitive and precise. Both methods were more suitable than the heat separation analysis method: