In studies of biological systems involving citrate there often arises the need to estimate small quantities of this metabolite, and the emphasis is on methods of greater sensitivity. Apart from enzymic procedures, the principal method for estimating small quantities of citrate in deproteinized biological fluids has been to oxidize and then brominate this compound to pentabromoacetone (PBA) , which is extracted from an aqueous phase by means of a hydrocarbon solvent. The PBA forms a yellow complex with sodium sulfide (1) and thiourea (2), or a red color in the pyridine phase when heated with pyridine and an aqueous alkali (3-5). A third reagent has been sodium iodide (6) from which iodine is liberated equivalent to the PBA, but although the method is sensitive high blank values render the estimation difficult for small quantities of citrate.
Jacobs and Lee (7) used radioactive bromine in the bromination step, and measured the PBA produced by liquid scintillation counting, but the principal limitation to this method is the short half-life of Bra'. Choy, Quattrone, and Elefant ( 8) measured with moderate sensitivity the ultraviolet absorption of the product of reaction between pyridine and citric acid in a nonaqueous system without converting it into PBA.
The methods measuring the yellow product (X = 400-450 mp) of the reaction of PBA with sodium sulfide and thiourea, although giving stable colors, arc less sensitive (E = 190%2300).
Greater sensitivity was achieved by Ettinger et al. ( 5) using the Fujiwara (3) reaction between a solution of PBA in heptane, pyridine, and strong aqueous potassium hydroxide (X = 530 mp, c = 15,900). The production of a red color as a result of a reaction between polyhalogenated hydrocarbons and alkaline pyridine was noted by Fujiwara (3) and independently by Ross (4). By prolonged heating of the product and measuring the optical density at 366 mp., Friedman and Cooper (9), and also Mantel, Molto, and Stiller (lo), increased the sensitivity further (c = 28,000 for citric acid) ; and more