Cclii. Some Observations on the Estimation of Muscle

N-UMEROUS observations in the past decade, culminating in the isolation of the crystalline material and the study of its properties by Theorell [1932; 1934, 1, 2, 3, 4] have established the identity of muscle haemoglobin. Attempts to estimate it, however, have for the most part been concerned with it less as a chemical identity than as that haemoglobin which remains behind after perfusing the tissue apparently free of blood. The most notable of these is that of Whipple [1926]. Though his estimations show consistency, it is not certain that they represent the actual concentrations of muscle haemoglobin. There is the possibility that some haemoglobin has been lost in the perfusion of the tissue and uncertainty both as to whether there is an equal distribution of muscle haemoglobin between the tissue and the extracting fluid and as to the actual volume of the tissue. Further there is the possibility of other acid haematin compounds being extracted from the cell and introducing error into the subsequent acid haematin determination. Finally the conclusions drawn from the colorimetric comparison of the carboxy-compounds may be incorrect, for Theorell [1934, 2] has since shown that the light absorptions by the carboxyhaemoglobins of muscle and blood are different. In this paper is reported an attempt to develop a method for a more exact estimation by extracting the whole of the haemoglobin from the unperfused tissue and by determining the relative concentrations of muscle and blood haemoglobin in the extract from the mean position of the oc-band of the oxyhaemoglobin compounds as observed with the Hartridge reversion spectroscope and the determination of the total concentration of haemoglobin spectrophotometrically. Some observations on Whipple's [1926] method of extraction, the outcome of a comparison of it with the method developed, are also reported. Shenk et al. [1934] describe a method somewhat similar in principle. These authors are to be commended for their heroic effort in perfusing an ox. There exist, however, discrepancies between the spectrophotometric constants obtained with their extract of the perfused tissue and those obtained by Theorell [1934, 2] with the crystalline horse muscle haemoglobin. Though these may be due to the difference in the source of the haemoglobin, it has been the experience of the author that it is extremely difficult to perfuse a tissue entirely free of blood haemoglobin. Further, the same uncertainty as to the distribution of the haemoglobin between the tissue and the extracting fluid etc., exists as in Whipple's [1926] method. 6