New Strategy for in Vitro Determination of Carbonic Anhydrase Activity from Analysis of Oxygen-18 Isotopes of CO2

摘要

The oxygen-18 isotopic (O-18) composition in CO2 provides an important insight into the variation of rate in isotopic fractionation reaction regulated by carbonic anydrase (CA) metalloenzyme. This work aims to employ an O-18-isotope ratio-based analytical method for quantitative estimation of CA activity in erythrocytes for clinical testing purposes. Here, a new method has been developed that contains the measurements of O-18/O-16 isotope ratios during oxygen-18 isotopic exchange between (COO)-C-12-O-16-O-16 and (H2O)-O-18 of an in vitro biochemical reaction controlled by erythrocytes CA and estimation of enzymatic activity of CA from the isotopic composition of CO2. We studied the enrichments of O-18-isotope of CO2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, (H2O)-O-18, and cellular produced CO2 on this reaction, we performed the in vitro experiments in closed containers with variations of those parameters. Finally, we mimicked the exchange reaction at 5% [CO2], 5 parts per thousand [(H2O)-O-18], pH of 7.4, and temperature of 37 degrees C to create the physiological environment equivalent to that of the human body and monitored the exchange kinetics with variations of CA activities, and subsequently, we derived the quantitative relation between the O-18-isotope of CO2 and CA activity in erythrocytes. This assay may be applicable for rapid and simple quantification of carbonic anhydrase activity which is very important to prevent the carbonic-anhydrase-associated disorders in human.