Determination of the carboxyhaemoglobin saturation of blood by spectrophotometric analysis.

The spectrophotometric method of Commins and Lawther (I965) for the determination of carboxy-haemoglobin saturation in blood was found to lead to systematic undervaluation, because of dissociation of the carboxyhaemoglobin, and physical dissolution of the carbon monoxide in the solvent used for the analysis. The theoretical relationship between the true and apparent carboxyhaemoglobin saturations was calculated and was found to be in satisfactory agreement with the experimental findings, using samples of blood saturated with carbon monoxide, and aerated solvent. The error is much smaller when using oxygen-free solvent, but it is nevertheless appreciable at high carboxyhaemoglobin saturations. An elegant method for the determination of carboxyhaemoglobin saturation, using a very small sample of blood, was recently described in this journal by Commins and Lawther (i965). The method requires OOI ml. of blood, which is dissolved in IO ml. of 0-04% ammonia solution. Measurements of the optical density differences at 4I4 mL,, 4202 m,u, and 426 m,u between this solution and a portion thereof from which all carboxy-haemoglobin has been converted to oxyhaemoglobin by bubbling oxygen give an estimate of the carboxy-haemoglobin present in the original sample. The method is calibrated by using a part of the same solution which has been saturated with carbon monoxide. The purpose of this note is to point out that a systematic error occurs, because of dissociation of the carboxyhaemoglobin caused by physical dissolution of the carbon monoxide in the solution. This error can be serious, particularly at high carboxy-haemoglobin concentration. Commins and Lawther stated that preliminary experiments showed that the analysis gave the same result whether or not the solution was oxygen-free. This has not been the experience of the writer, who found an error which was in good agreement with prediction for air-saturated solutions. Consider a blood sample of volume Vb, with oxygen or carbon monoxide binding capacity of r ml. S.T.P.D. per ml. of blood. Let the carboxy-haemoglobin saturation be S0. Then the total quantity of carbon monoxide in this sample is: (I) This blood sample is now dissolved in ammonia solution to a volume V,. Let the new carboxy-haemoglobin saturation be S, and let 'a' be the Henry's law constant for carbon monoxide in the solution. Thus 'a' equals the volume, in ml., of carbon monoxide, at S.T.P.D., dissolved per ml. of solution, at i atmosphere pressure.