Spectrophotometry Studies on Blood Serum and Plasma: The Physical Determination of Hemoglobin and Bilirubin

Abstract

The absorption spectrum of normal blood serum shows a "crook" at 575 mu. Heilmeyer and Toop suspected this of being due to hemoglobin. The present investigation confirms this observation by additive and reduction experiments with pure hemoglobin; establishes a quantitative relationship between the hemoglobin concentration and the difference of the optical densities at 575 mu and at 560 mu; and demonstrates the effects of bilirubin, lipemia and other factors on this relationship. As a result of these experiments, plasma bilirubin concentration can be determined, without extraction or reagents, by optical density measurements at 2 wavelengths; plasma hemoglobin concentration, at 1 additional wavelength. The hemoglobin and bilirubin results obtained by the physical procedure on specimens with known concentrations demonstrated excellent recovery and compared favorably with those obtained by the benzidine and van den Bergh techniques. It was found that normal plasma contains less than 0.5 mg hemoglobin and 2.7 mg total hematin/100 ml. Plasma from bank whole blood between 5-21 days old contained 21-165 mg hemoglobin and 24-190 mg total hematin/100 ml. In such specimens, there was also evfdence of increase in hemoglobin breakdown products particularly after 1 week of storage. After a l l whole blood transfusion the hemoglobin and total hematin concentrations were 55 and 70 mg, respectively,/100 ml plasma. Details for the preparation of stable aqueous bilirubin solutions and for the application of the new recording Rapid Scanning Spectrophotometer (American Optical Company) are also presented.