Fluorometric micro determination of citrate in tissue extracts.

An enzymic procedure has been developed for the determination of micro quantities of tissue citrate. The reactions involve the cleavage of citrate by citrate lyase to oxalacetate and its subsequent oxidation of NADH by malic dehydrogenase. The NAD+ thus produced is cycled with lactate, α-ketoglutarate, and, ammonium ion, in the presence of the enzymes lactate dehydrogenase and glutamic dehydrogenase. Conversion of the cycling product (i.e., pyruvate) plus NADH to NAD+ and lactate leads to a strongly fluorescent substance when heated in strong alkali. The fluorescence thus produced from NAD+ can be equated by means of known standards, with the citrate originally present in tissue samples. Data obtained from several tissues are presented to demonstrate the potential applicability of the method to very small tissue samples and even to single cells.