TY - JOUR
T1 - New Strategy for in Vitro Determination of Carbonic Anhydrase Activity from Analysis of Oxygen-18 Isotopes of CO2
AU - Ghosh, Chiranjit
AU - Mandal, Santanu
AU - Pal, Mithun
AU - Pradhan, Manik
N1 - Publisher Copyright:
© 2017 American Chemical Society.
PY - 2018/1/16
Y1 - 2018/1/16
N2 - The oxygen-18 isotopic (18O) 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 18O-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 18O/16O isotope ratios during oxygen-18 isotopic exchange between 12C16O16O and H218O 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 18O-isotope of CO2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, H218O, 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 18O], 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 18O-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.
AB - The oxygen-18 isotopic (18O) 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 18O-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 18O/16O isotope ratios during oxygen-18 isotopic exchange between 12C16O16O and H218O 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 18O-isotope of CO2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, H218O, 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 18O], 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 18O-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.
UR - https://www.scopus.com/pages/publications/85040730970
UR - https://www.scopus.com/inward/citedby.url?scp=85040730970&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.7b04572
DO - 10.1021/acs.analchem.7b04572
M3 - Article
C2 - 29244947
AN - SCOPUS:85040730970
SN - 0003-2700
VL - 90
SP - 1384
EP - 1387
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 2
ER -