Quantitation of total carbon dioxide in nanoliter samples by flow-through fluorometry

A new ultramicrofluorometric assay for the measurement of picomole amounts of total CO2 is described. The assay is based on a sequence of two linked enzymatic reactions: HCO3 reacts with phosphoenolpyruvate forming oxaloacetate, and oxaloacetate is then converted to malate with associated oxidation of NADH to NAD+. The fluorescence of NADH is measured in a modified ultramicrofluorometer. The HCO3 content of the sample is proportional to the decrease in NADH fluorescence. The method can resolve differences as small as 5.5 pmol total CO2 in 3-nl samples containing 0-160 pmol. The assay is 5- to 10-fold more sensitive than the widely used microcalorimetric assay. The assay can also measure total CO2 in biological samples. The assay will be useful in measuring transepithelial total CO2 transport in short distal nephron segments with low rates of total CO2 transport without resorting to extremely long collection times.