New Strategy for in Vitro Determination of Carbonic Anhydrase Activity from Analysis of Oxygen-18 Isotopes of CO 2 .

The oxygen-18 isotopic (18 O) 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 an18 O-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 of18 O/16 O isotope ratios during oxygen-18 isotopic exchange between12 C16 O16 O and H2 18 O 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 of18 O-isotope of CO2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, H2 18 O, 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‰ [H2 18 O], pH of 7.4, and temperature of 37 °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 the18 O-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.