Determining the Source of Excess Phosphatase in Hypothetical Patient
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Lauren Gray
Determining the source of excess phosphatase in hypothetical patient
Abstract
Enzymes are diverse, biological catalysts that induce and regulate chemical reactions. Assays can be performed to determine the specific biochemical properties of enzymes that regulate the same reactions but have slightly different amino acid sequences. In stressed biological systems, enzymatic activities fluctuate and the source of a medical condition can be linked to abnormal concentrations of the specific isozyme that functions in an area of the body. The purpose of this experiment was to determine the source of excess phosphatase in a patient suffering from one of six potential conditions. To distinguish between bacterial, fungal, prostate-specific, hepatic, renal and intestinal phosphatases three assays were performed. The unknown phosphatases optimal pH and response to potential inhibitors 10mM EDTA and 1mM Levamisol were determined and compared to given information on the expected response of each isozyme. The unknown phosphatase was found to be optimally basic and was partially inhibited by both EDTA and Levamisol determining it to be hepatic in excess due to early-stage cirrhosis of the liver. Enzymes assays are simple, efficient and yet accurate tools for the diagnosis and monitoring of medical conditions. Understanding the biochemical properties of isozymes, also presents new possibilities for treatment by induced regulation of enzyme activity.
Keywords: enzyme kinetics, alkaline phosphatase (ALP), hepatic isoenzymes (isozymes), EDTA, Levamisol
Introduction
The enzymatic active site is the protein region responsible for reaction catalyzation and its specific shape is determined by the chemical interactions of amino acid side chains (Johnson 2007). To control reaction rates, cells can manipulate active site conformation by regulating environmental pH and temperature, or substrate concentration with the addition of potential inhibitors. Phosphatases, which catalyze reactions that cleave inorganic phosphates from molecules, have specific functional characteristics related to the areas of the body in which they are active (Johnson 2007). Extensive research has been performed to determine the properties of isozymes because of their importance in determining medical diagnosis and prognosis. For example, Marikuma et al (1993) found ALP activity was positively correlated to the degree of malignancy in meningiomas and astrocytomas. Also, understanding isozyme properties is vital in treatment. Recent studies suggest hepatic metabolism regulation by the inhibition of NF-κB activity can improve glucose tolerance and may provide a new treatment option for type 2 Diabetes (Tamura et al 2007) and EDTA can reactivate aminopeptidase N activity suppressed by metal ion chelators (Vanderheyden et al 2006). In our experiment, the source of excess phosphatase was determined for a patient with unknown symptoms based on the biochemical properties of the isozyme using different assays based on pH and reaction to potential inhibitors. Enzymatic activity was determined after incubation with pNPP substrate by the addition NaOH, which caused the reaction product nitrophenol to turn yellow. The relative amount of nitrophenol present was determined by Spectrophotometer. Sample LPT-0222 was determined to be one of four possible alkaline phosphatases (renal, bacterial, intestinal, or hepatic) based on its optimal enzymatic activity in pH 10 buffer. It was demonstrated that the enzymatic activity of the unknown phosphatase was halved, but not fully inhibited by both 10mM EDTA and 1mM Levamisol. The results correlated to the expected biochemical properties of a hepatic isozyme suggesting the source of excess phosphatase in the unknown patient sample LPT-0222 to be early stage cirrhosis of the liver.
Materials and Methods
The optimal pH of the phosphatase in excess for the unknown serum sample was determined using two different pH buffers. The relative enzymatic activities of stock alkaline, stock acid, unknown serum, and normal serum phosphatases were tested in both pH 4 buffer and pH 10 buffer. Each assay was performed in triplicate with constant buffer volume (4.0mL), enzyme concentration (.5mL), substrate concentration (.5mL of 10mM pNPP), and incubated for 10 minutes until enzymatic activity was stopped using .5mL of 2M NaOH. Absorbance was measured by Spectrophotometer, which was set at 420nm and blanked with pH 4 buffer before assays at pH 4 were measured and blanked with pH 10 buffer before pH 10 assays were measured. The relative enzymatic activities of alkaline, acid, unknown, and normal patient phosphatases were determined in the presence of potential inhibitor EDTA. The control for the unknown serum contained 4.0mL of pH 10 buffer and .5mL of unknown serum enzyme and no inhibitor. Absorbencies were determined by Spectrophotometer, which was blanked using pH 10 buffer. The relative