The Determination of Blood Glutathione

In discussions of the factors necessary for accuracy in blood glutathione determinations, far more consideration has been given to the method of estimation than to the type of blood filtrate used. It has been shown that either iodometric titration, under the proper conditions of pH, temperature, and potassium iodide concentration, or ferricyanide calorimetric estimation accurately determines glutathione in pure solution. Mason (1) has further shown that both methods agree fairly well when applied to the same blood filtrate. However, the use of different filtrates combined with various methods of estimation has led to varying results and, therefore, much controversy over the actual amount of glutathione present in blood. Two essential requirements for the blood filtrate are (1) that no reduced glutathione be lost by autoxidation, and (2) that the glutathione be completely recovered from the blood. In the calorimetric ferricyanide method developed by Mason (1) a tungstic acid blood filtrate is used. This method has recently been applied to human blood by two investigators, Downes, reported by Benedict (2), and Schelling (3). The results of both investigators would indicate that the blood glutathione is considerably less than generally supposed. According to the original intention of Folin and Wu (4) for the tungstic acid filtrate, 1:lO dilution of the blood, it is only slightly acid. The pH is about 5.5, varying slightly with each individual blood. With Schelling’s modification, 1: 5 dilution, the pH is about 4.9. It is a well known fact that glutathione undergoes autoxidation at a neutral or alkaline pH and even at slight acidity. Mason realized the possibility of autoxidation in such filtrates and in a later paper (5) sug-