REVERSE ISOTOPE-DILUTION ANALYSIS OF (CO2)-C-13 USING GAS-CHROMATOGRAPHY ISOTOPE RATIO MASS-SPECTROMETRY - APPLICATION TO THE QUANTITATIVE-DETERMINATION OF (CO2)-C-13, RELEASED BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES

We propose a new method for the quantitative determination of carbon dioxide released by a biological microgenerator: a suspension of human polymorphonuclear leukocytes (PMNL). This method is based on the reverse isotope dilution analysis by gas chromatography/isotope ratio mass spectrometry of (CO2)-C-13 released by PMNL in a controlled isotope abundance atmosphere containing 3% CO2. (CO2)-C-13 release is effective after PMNL stimulation in the presence of [C-13]glucose, labeled on positions 1, 2 or 6. The validation of this method is carried out by the measurement of the isotope ratio (CO2)-C-13/(CO2)-C-12 using known amounts of [C-13]sodium hydrogen carbonate and the comparison with theoretical isotope abundances derived from various CO2 equilibria. Complete release of CO2 is achieved by the acidification of the medium. This method requires only few cells, displays high sensitivity and specificity and can be applied to the analysis of large series of samples using an automatic sample injector. in addition, this method can also be applied to other types of biological microgenerators of carbon dioxide.