A Method for the Quantitative Determination of Hemoglobin and Related Heme Pigments in Feces, Urine, and Blood Plasma*

The purpose of the present study was to make available a simple quantitative procedure for total heme pigments in feces, urine, and blood plasma or serum. It was planned to convert these substances to pyridine ferrohemochromogen, which is known to have stronger absorption than other easily prepared heme compounds, and to measure this absorption under standard conditions with the Evelyn photoelectric calorimeter. Stokes (1) discovered the hemochromogen spectrum and Hoppe-Seyler (2) gave the substance its name. Anson and Mirsky (3) defined a hemochromogen as a subst,ance containing reduced heme and a nitrogen compound. Pyridine ferrohemochromogen has absorption bands at 5575 d. (maximum) and 5270 8. The first band is stronger than that of any other heme compound, having an extinction coefficient about one-third greater than that of oxyhemoglobin (4). An equilibrium is reached; so that it is necessary to have an excess of pyridine in order to complete the reaction (3). Hill (5) used a micro spectroscope mounted on a calorimeter to determine pyridine hemochromogen concentrations. He compared unknown solutions with a standard in order to get information about the equilibrium reactions of hemochromogen. Anson and Mirsky (6) also used the micro spectroscope and calorimeter for determining the amount of cytochrome in yeast. Lemberg and coworkers (7) used the same apparatus to follow t,he course of the coupled oxidation-reduction reaction of ascorbic acid and hemochromogens. Roetts (8) described a method of determining the content of hemoglobin and related substances in feces, urine, serum, and milk by forming pyridine hemochromogen and comparing the absorption with that of a standard solution.