Quantitative measurement of porphyrins in biological tissues and evaluation of tissue porphyrins during toxicant exposures

Abstract

Porphyrins are formed in most eukaryotic tissues as intermediates in the biosynthesis of heme. Assessment of changes in tissue porphyrin levels occurring in response to the actions of various drugs or toxicants is potentially useful in the evaluation of chemical exposures and effects. The present paper describes a rapid and sensitive method for the extraction and quantitation of porphyrins in biological tissues which overcomes difficulties encountered in previously described methods, particularly the loss of porphyrins during extraction and interference of porphyrin quantitation by coeluting fluorescent tissue constituents. In this procedure 8- through 2-carboxyl porphyrins are quantitatively extracted from tissue homogenates using HCl and methanol and are subsequently separated from potentially interfering contaminants by sequential methanol/phosphate elution on a C-18 preparatory column. Porphyrins are then separated and measured by reversed-phase high-performance liquid chromatography and spectrofluorometric techniques. Recovery of tissue porphyrins using this method is close to 100% with an intraassay variability of less than 10%. We have employed this procedure to measure liver and kidney porphyrin concentrations in male Fischer rats and to define the distinctive changes in tissue porphyrin patterns associated with treatment with the hepatic and renal porphyrinogenic chemicals, allylisopropylacetamide, and methyl mercury hydroxide, respectively. This method is applicable to the measurement of tissue porphyrin changes resulting from drug or toxicant exposures in clinical, experimental or environmental assessments.