Identification of cobalt protoporphyrin IX formation in vivo following cobalt administration to rats

An electron paramagnetic resonance method for the identification and quantitation of cobalt protoporphyrin IX has been developed to provide definitive evidence for the formation of the cobalt chelate of protoporphyrin IX in vivo. Both authentic and enzymatically generated cobalt protoporphyrin exhibited a characteristic primary resonance at g = 2.32 at 100° Kelvin (K) in the reduced state. At low concentrations, signal averaging was found to improve considerably the signal to noise ratio and thereby allow for the measurement of low concentrations of cobalt protoporphyrin. It was possible to determine concentrations of cobalt protoporphyrin IX as low as 0.5 μM in biologic preparations, and the signal height of the e.p.r. resonance was linear with cobalt protoporphyrin concentrations up to at least 8.0 μM. Following the administration of cobaltous chloride to rats, cobalt protoporphyrin IX was demonstrated in livers at times when alterations in heme biosynthesis are known to occur. Cobalt protoporphyrin IX at the level of 4.8 nmoles/g liver was detected 120 min after the administration of cobaltous chloride at a dose of 60 mg/kg of body weight. This finding supports the concept that cobalt protoporphyrin IX is rapidly produced in vivo after the administration of cobaltous chloride and may be responsible for the observed inhibition of hepatic heme biosynthesis.