The Calculation of Hemoglobin Saturation in Single Erythrocytes

Abstract

Spectrophotometric measurement of pigments has been used to monitor biological systems since the 1930’s.¹ Recent methodology separates myoglobin (Mb) and hemoglobin (Hb) spectra spatially and calculates Mb saturation in subcellular volumes of frozen tissue. Using this methodology, results suggest a large RBC to tissue oxygen gradient at the capillary.² To measure this gradient directly the Hb saturation of single erythrocytes in capillaries and Mb saturation in the adjacent cells must be determined simultaneously. Hb saturation in unfrozen whole blood can be measured using commercial equipment. Determination of the Hb half-saturation (P₅₀) in unfrozen single erythrocytes has been attempted using a two wavelength method based on the Lambert-Beer law.³ Because of the potential difficulties related to light scattering in those experiments, we undertook to determine single-erythrocyte Hb saturation using the multicomponent wavelength analysis of Wodick and Lübbers⁴ as modified by Hoffmann and Lübbers (personal communication). The principal question we sought to answer was: Is there a variation amongst erythrocytes in the half-saturation of Hb (P₅₀)? Additionally, the sensitivity of this methodology to changes in measuring parameters was investigated.