Comparison of rat and human alkaline phosphatase isoenzymes and isoforms using HPLC and electrophoresis

Total activity of alkaline phosphatase (ALP) in serum represents the sum of activities of some isoenzymes and their numerous isoforms derived from various tissues of the organism. The aim of this work was to separate ALP isoenzymes and their isoforms in rat and human serum, compare the properties of serum ALP isoforms in rats and humans, and determine the usefulness of some analytical methods for specific measurements of ALP isoenzyme and isoform activities. Two methods of separation, i.e. high-performance liquid chromatography (HPLC) and agarose gel electrophoresis were chosen. The combination of HPLC with electrophoresis of the eluted fractions and with ALP inhibition methods (urea, l-phenylalanine), inactivation (heat) and precipitation (wheat-germ lectins) enabled the identification of isoenzymes and isoforms of ALP in serum. Using chromatography and a post-column reactor, three isoforms of the intestinal isoenzyme and one bone isoform of a tissue non-specific isoenzyme were detected. Rat serum differs significantly from human as regards activities of intestinal and hepatic isoforms, whereas the properties of the bone isoform are similar in both species. Our HPLC method offers a higher resolution than agarose gel electrophoresis with respect to ALP subfractions in rat serum.