Introduction: Determination of methemoglobin (Met-Hb) in blood is important for the diagnosis of poisoning by oxidants, such as nitrite, nitrate, chlorate, chlorite, alkyl nitrites, nitroglycerin, aniline and other compounds. In 1938, Evelyn and Malloy [1] had devised a photoelectric method for determination of Met-Hb in blood. Minor modifications of this method were made by several researchers to increase sensitivity [2–4]. These methods are based on a phenomenon that the absorbance maximum of weakly acidic Met-Hb at 630 nm disappears by addition of cyanide. Met-Hb concentration can be easily determined as the ratios of the absorbance changes induced by cyanide before. | Methemoglobin by Keizo Sato Introduction Determination of methemoglobin Met-Hb in blood is important for the diagnosis of poisoning by oxidants such as nitrite nitrate chlorate chlorite alkyl nitrites nitroglycerin aniline and other compounds. In 1938 Evelyn and Malloy 1 had devised a photoelectric method for determination of Met-Hb in blood. Minor modifications of this method were made by several researchers to increase sensitivity 2-4 . These methods are based on a phenomenon that the absorbance maximum of weakly acidic Met-Hb at 630 nm disappears by addition of cyanide. Met-Hb concentration can be easily determined as the ratios of the absorbance changes induced by cyanide before and after addition of potassium ferricyanide. In forensic science practice there are many cases in which Met-Hb has to be measured for blood specimens containing high concentrations of carboxyhemoglobin CO-Hb . especially in the cases of fire and exhaust gas poisoning 5 . However the above methods 1-4 are not suitable for specimens containing high levels of CO-Hb. In 1981 the author et al. 6 developed a modification of the methods which enabled accurate determination of blood Met-Hb even in the presence of high concentrations of CO-Hb by using about a 100-fold excess of potassium ferricynade to be added to the Hb iron. In this chapter a simple procedure of the modified spectrophotometric method for Met-Hb is briefly described. Reagents and their preparation Potassium ferricyanide 4 w v in distilled water. It should be stored in a dark bottle and be prepared monthly. Phosphate buffer solution M pH . Phosphate buffer solution 40 mM pH . It can be prepared by dilution of two parts of the above reagent with three parts of distilled water. Potassium cyanide 5 w v dissolved in the above 40 mM phosphate buffer solution. It should be prepared just before use. Analytical instrument A Hitachi 557 dual-wavelength spectrophotometer Hitachi Ltd. Tokyo Japan . Procedure i. A .
