Comparison of Heat Fractionation and Gel Electrophoresis Methods for the Quantitative Determination of Alkaline Phosphatase Isoenzymes

Abstract Introduction Alkaline phosphatase (ALP) is important in the diagnostic work-up for hepatobiliary and bone diseases. ALP isoenzymes are expressed in the bone, liver, kidney, placenta, and intestine, and vary in heat stability and electrophoretic mobility. Distinguishing the different ALP isoenzymes is clinically important for the diagnosis of pathologies associated with elevated ALP activity. Current modalities available to measure ALP isoenzymes utilize the heat stability, electrophoretic mobility, and immunochemical properties of the isoenzymes. The differences inherent in these methods allow for unique benefits of each method in identifying ALP isoenzymes. The objective of this study was to compare bone, liver, and placental ALP isoenzyme results determined by heat fractionation and gel electrophoresis and to characterize the heat-stable non-liver fraction (t1/2 >11 min), reported by heat fractionation, using gel electrophoresis. Methods A total of 72 de-identified serum samples that span a wide range of known ALP isoenzyme concentrations and disease states were used to measure ALP using gel electrophoresis and heat fractionation. Heat fractionation was achieved by selective inactivation of the isoenzymes at 56 °C in 10, 15, and 20-minute intervals. Log-percent activity of the total and heat-inactivated fractions at each time point was plotted against time in minutes. The linear activity decay between 10 and 20 minutes determined the relative amount of liver isoenzyme activity and the slope of the line determined the half-lives of ALP isoenzymes. Electrophoresis was performed according to the manufacturer’s protocol using the Hydragel ISO-PAL gel to resolve ALP isoenzymes based on their electrophoretic mobility and interaction with lectin. ALP isoenzymes were quantified by densitometry. Results Our results show a significant correlation coefficient (r) of 0.98, Deming regression slope of 1.1, and bias of -1.2% for the liver isoenzyme (n=43). However, liver fractions are not distinguishable by heat fractionation when heat-stable isoforms are present. The bone fraction (n=43) showed a coefficient of correlation of 0.86, slope of 0.55, and bias of -31%. Although, with a small sample size (n=6), the placental isoenzyme showed a significant agreement between the two methods: r = 0.999, slope = 0.98, and a -3.5% bias. Of the non-liver fractions reported by heat fractionation (n=13, ALP >100 U/L) eleven (85%) showed distinct qualitative bands in the in