A Method for the Determination of Ergothioneine in Blood* by Donald

One of the difficulties associated with research on ergothioneine has been the lack of completely reliable methods for the quantitative determination of the compound in blood and other tissues. Ergothioneine was originally discovered to be present in the red blood cell by Benedict et al. (1, 2) and by Hunter and Eagles (3, 4), some 25 years ago, because of its reducing action on the arsenophosphotungstate reagent devised by Benedict for t,he determination of uric acid, and the earlier quantitative methods (5, 6) for ergothioneine were based on the use of this or similar reagents. The nonspecificity of the uric acid reagents left much to be desired. In 1928 Hunter (7) reported that the coupled product of ergothioneine with diazotized sulfanilic acid yields a characteristic magenta color in the presence of strong alkali. This reaction, which is highly sensitive for ergothioneine, shows excellent specificity (8, 9) and appears to be the most suitable basis for a quantitative method. The method proposed by Touster (lo), based on the oxidation of the sulfur of ergothioneine to sulfate by means of bromine, suffers from lack of specificit,y and non-quantit,ative recovery of ergothioneine added to blood. The difficulties involved in applying t.he diazo reaction to the analysis of ergothioneine in natural materials are due to the fact that many substances (e.g., tyrosine) yield yellow to orange colors with the diazo reagent and thereby mask the magent,a color given by ergothioneine, while other substances (e.g., glutathione) inhibit the color formation from ergot.hioneine itself. Compounds of both classes occur in blood filtrates. The usefulness of the diazo reaction is therefore dependent on the availability of an effective procedure for the removal of the interfering substances before the diazo reaction is applied. The difficulties in developing such a procedure are complicated by the variations in the amounts of interfering substances present, not only in blood from different species, but even in blood from different individuals within the same species. Hunter’s original diazo method is deficient in this respect. Modifications of the method, recently published by Hunter (11) and by Lawson, Morley, and Woolf (12), are attempts to overcome this difficulty. Hunter uses basic lead acetate to re-