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

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 intestinal lane on gel electrophoresis; however, quantitative values did not correlate between the two methods. Conclusion Our data support an agreement between the heat fractionation and gel electrophoresis methods for the quantitative determination of liver and placental alkaline phosphatase isoenzymes. Although there is an association between the two methods, the activity of the bone isoenzyme was underestimated by the gel electrophoresis method, likely due to saturation of the gel and densitometry scan because of elevated protein concentrations. The non-liver fractions were qualitatively identified as intestinal isoenzyme.

Serum Profile of Lactate Dehydrogenase (LDH) and Alkaline Phosphatase (ALP) in Alcoholic Liver Diseases

Background: Alcohol‐related liver diseases affect the capability of the liver for the synthesis of proteins including enzymes. Among enzymes two expose the liver specific isoenzymes. There are lactate dehydrogenase and alkaline phosphatase. The purpose of this study was to explore the profile of lactate dehydrogenase and alkaline phosphatase isoenzymes in alcoholic liver diseases.Methods: The serum profile of lactate dehydrogenase and alkaline phosphatase isoenzymes was studied in alcoholic cirrhosis and alcoholic hepatitis by electrophoresis method on agarose gel.Results: The relative concentration of LDH 1 isoenzyme is decreased in alcoholic hepatitis, but LDH 4 and LDH 5 isoenzymes are increased. In addition, the relative level of LDH 5 isoenzyme is increased in alcoholic cirrhosis. The concentrations of intermediate isoenzymes (LDH 2 and LDH 3) are reduced in alcoholic cirrhosis. There were also an increase of liver ALP isoenzyme and parallel decrease in its bone isoenzyme in alcoholic hepatitis and cirrhosis. The severity of liver cirrhosis, expressed as a Child-Pugh score, didn’t affect total activity of both enzymes but effects the relative concentration of LDH 2, LDH 3 and LDH 4 isoenzymes.Conclusions: The serum profile of lactate dehydrogenase and alkaline phosphatase isoenzymes alters in alcoholic liver diseases and differs between them. The differences can be explained by the tissue prevalence and metabolic preferences of isoenzymes.