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 d ioxide released by a biological microgenerator: a suspension of human polymorphonuclear leukocytes (PMNL). This method is based on the rever se isotope dilution analysis by gas chromatography/isotope ratio mass spectrometry of (CO2)-C-13 released by PMNL in a controlled isotope ab undance atmosphere containing 3% CO2. (CO2)-C-13 release is effective after PMNL stimulation in the presence of [C-13]glucose, labeled on po sitions 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 am ounts of [C-13]sodium hydrogen carbonate and the comparison with theor etical isotope abundances derived from various CO2 equilibria. Complet e release of CO2 is achieved by the acidification of the medium. This method requires only few cells, displays high sensitivity and specific ity and can be applied to the analysis of large series of samples usin g an automatic sample injector. in addition, this method can also be a pplied to other types of biological microgenerators of carbon dioxide.