The oxygen-18 isotopic (O) composition in CO 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-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/O isotope ratios during oxygen-18 isotopic exchange between COO and HO of an in vitro biochemical reaction controlled by erythrocytes CA and estimation of enzymatic activity of CA from the isotopic composition of CO. We studied the enrichments of O-isotope of CO with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, HO, and cellular produced CO 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% [CO], 5‰ [HO], 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 the O-isotope of CO 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.
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