생체 시료 중 아세틸콜린 및 콜린에 대한 효소-분광학적 정량분석

Enzymatic Spectrophotometric Determinations of Acetylcholine and Choline in the Biological Samples

In order to determine acetylcholine and choline in the biological samples, the specific enzymes of acetylcholinesterase (AChE) and choline oxidase (ChO), which utilize acetylcholine and choline as substrates, were employed to convert substrates to H2O2. The produced H2O2 was coupled to 4-aminoantipyrine/phenol with peroxidase (PO) yielding quinoneimine dye which was measured at 508 nm. In the present enzymatic spectrophotometric analysis the product at the equilibrium state was measured considering accuracy, precision, time and cost of the analysis. The developed analytical method yielded good linearity (calibration curve; A508=9534[acetylcholine]+0.009, correlation coefficient (R2); 0.999) with detection limit of 1.11×10-7 M, reasonable precision (relative standard deviation; 0.10~1.62% at 2.5×10-6 M~1.25×10-4 M) and accuracy (relative error; -0.24~0.97% at 4.13×10-6M~1.01×10-4 M) for acetylcholine chloride standard solution. The concentrations of acetylcholine and choline in human serum were found as 3.20×10-5 M and 1.14×10-4 M, respectively. The brain tissues of Sprague-Dawley strain rat contained 9.82 μg/g of acetylcholine and 6.53 μg/g of choline in the cerebrum, while 7.37 μg/g of acetylcholine and 5.34 μg/g of choline in the cerebellum.