Chromatographic determination of Evans blue in plasma and serum.
The methods at present available for the determination of the dye, Evans blue, in plasma have certain disadvantages. Direct colorimetry of dyed plasma is inapplicable where the plasma is haemolyzed, turbid or lipaemic. The butanol extraction procedure of Harington, Pochin & Squire (1940) is troublesome and time-consuming where many analyses must be carried out; its accuracy is lower than those of other methods (Bowler, Crooke & Morris, 1944); it is liable to interference by haemolysis; and the modification suggested for haemolyzed plasma has not proved satisfactory in this laboratory. The method of Crooke & Morris (1942), which involves precipitation of plasma proteins and pigments with a hydrochloric acid-ethanolic phosphotungstic acid mixture, also has its disadvantages. It is likewise liable to interference-by haemolysis, although this can be obviated by an optical correction method (Morris, 1944); and in certain specimens of phosphotungstic acid there is a substance which brings about the photochemical oxidation of Evans blue to a colourless compound. This difficulty can be overcome by working in artificial light. Certain plasma samples do not give an absolutely clear supernatant fluid on precipitation, and the occurrence of this effect, although uncommon, cannotbe predicted. In Phillips's (1943) method the effect of interfering substances is allowed for by measuring residual light absorption after the Evans blue has been reduced to a colourless compound with sodium hydrosulphide. The method gives satisfactory results in the presence of a considerable degree of haemolysis, but the reduction requires 12 hr. for completion, and a supply of pure carbon monoxide is necessary. In the course of an investigation of the homogeneity of commercial samples of .Evans blue, it was observed that the dye was adsorbed from aqueous solution by aluminium hydroxide. This suggested that it might be possible to effect a quantitative separation of the dye from. plasma or serum by chromatography. The adsorption behaviour of Evans blue in solution in plasma is, however, quite different from that of the dye in aqueous solution. Evans blue is only adsorbed from plasma at approximately pH 10. At this pH it is not adsorbed from aqueous solution. Ogston (1943) has shown by means of the ultracentrifuge Biochem. 1944, 38 that Evans blue combines with the piasma proteins, and it is therefore probable that the difference in adsorption behaviour, and also certain differences in light absorption (Phillips, 1943), are due to the existence of an Evans blue protein complex. With suitable conditions, however, it is possible to separate the dye from plasma by chromatographic means, the plasma proteins and pigments passing directly through the adsorption column. If the sample is haemolyzed, a portion of the alkaline haematin formed on adjusting to pH 10 is adsorbed on the column, but this can be removed by elution with acetic acid-ethanol. The dye is subsequently eluted from the adsorbent with hydrochloric acidethanol and the concentration in the eluate determined colorimetrically. This method permits a complete separation of Evans blue from all other light-absorbing substances present in, plasma. If it is necessary to measure very low dye concentrations, it is possible to achieve a high degree of accuracy by concentrating the dye from larger plasma samples than those usually employed.