Enzymatic detection and quantification assay of isatin, a putative stress biomarker in blood.

Abstract

Isatin is an endogenous inhibitor of monoamine oxidase B and is found in human blood and tissue. Increased levels of isatin have been linked to stress and anxiety in rodents and humans; however, the metabolism of isatin in humans is largely unknown. We have developed a fluorescence-based enzymatic assay that can quantify isatin in blood samples. A phase extraction of isatin followed by a second phase extraction combined with an enzymatic reaction performed by an isatin hydrolase is used to extract and quantify isatin in whole blood samples. This results in a purity of more than 95% estimated from RP-HPLC. The hydrophobic molecule isatin is in equilibrium between an organic and aqueous phase; however, conversion by isatin hydrolase to the hydrophilic product isatinate traps it in the aqueous phase, making this step highly specific for isatin. The described protocol also offers a novel method for fast and efficient removal of isatin from any type of sample. The isolated isatinate is converted chemically to anthranilate that allows fluorescent detection and quantification. Pig plasma isatin levels are quantified to a mean of 458 nM ± 91 nM. Biophysical characterization of the isatin hydrolase shows enzymatic functionality between pH 6 and 9 and at temperatures up to 50 °C. Isatin hydrolase is highly selective for manganese ions with a dissociation constant determined to be 9.5 μM. We deliver proof-of-concept for the enzymatic quantification of isatin in blood and provide a straightforward method for further investigation of isatin as a biomarker in human health.