Spectrophotometric micromethod for the quantitative determination of the free erythrocyte protoporphyrin.

Interesthas developed recently (l-3) in the significance of the free protoporphyrin which van den Bergh and Hyman (4) reported in 1928 as being present in human erythrocytes. This “erythrocyte protoporphyrin” (EP) is identical with the protoporphyrin which, in combination with iron and globin, forms hemoglobin (1, 5). An increase in the quantity of free EP has been observed in immature erythrocytes (reticulocytes) (1, 3) in iron deficiency and in lead and gold poisoning (1) as well as in cases of anemia associated with infection (2). It has been postulated that the most common cause of increased EP is uncompleted hemoglobin synthesis, whether due to the immaturity of the red blood corpuscle or the consequence of failure to synthesize hemoglobin through lack of iron or other building stones, a fault in iron metabolism or interference with hemoglobin synthesis in some other way (3). Van den Bergh and Hyman (4) devised a method for the quantitative determination of this porphyrin which depended on its extraction from the red blood corpuscles with a mixture of 3 volumes of ethyl acetate and 1 volume of glacial acetic acid. The concentration of the porphyrin was determined by measuring in a stufenphotometer the intensity of fluorescence exhibited under ultraviolet light by a final 5 per cent HCl solution. Modifications of this method by van den Bergh and Grotepass (6), Lageder (7), Vigliani and Angeleri (8), Schumm (9), and Seggel (1.0) have been concerned mainly with the purification of the protoporphyrin extracted by van den Bergh’s procedure and the measurement of the concentration of the protoporphyrin in the final HCl solution. This concentration has been determined by measuring the intensity of fluorescence or the absorption within a given spectral range. All of these methods have one or more of the following disadvantages: too large a volume of blood required, difficulty in measuring the intensity of fluorescence by color matching in the stufenphotometer, or technical difficulties in determining the absorption in the ultraviolet, as is the case