Problems in the determination of erythrocyte protoporphyrin by ethyl acetate/acetic acid extraction. Ann Clin Biochem

Abstract

SUMMARY. Three problems with the ethyl acetate-acetic acid method of protopor-phyrin analysis were identified: (1) protoporphyrin extraction from whole blood may yield low recoveries in comparison with extraction from prediluted blood; (2) recovery of protoporphyrin is low with some batches of ethyl acetate, but such unsatisfactory reagent can be identified by an iodide oxidation test; (3) protopor-phyrin standards prepared with deionised water may be unstable. However, glass-distilled water is satisfactory. The determination of erythrocyte protopor-phyrin (EP) concentrations in whole blood is widely used to screen for and diagnose lead poisoning. The ingestion of lead may disturb intercellular iron transport or inhibit the enzyme ferrochelatase, thereby interfering with haem synthesis. Suppression of haem synthesis results in elevated EP levels as do certain genetic disorders. EP is generally determined either by ethyl acetate-acetic acid extraction 1-3 or with a haematofluorometer. 4. 5 In the former proce-dure porphyrins and haem are extracted into ethyl acetate-acetic acid; the porphyrins are then back-extracted into HCI solution to sepa-rate them from the strongly absorbing haem. Protoporphyrin is determined fluorometrically, with an excitation wavelength of 405 nm and an emission wavelength of 608 or 663 nm. For 7 years we have been routinely determin-ing EP and have been involved in testing the proficiency of other laboratories that determine EP. We have noted several ongoing problems with the extraction procedure which can, and often do, result in poor quality of analysis. In this report we specifically describe the effects of: (1) sample predilution prior to ethyl acetate-acetic acid extraction, (2) contamin-ated ethyl acetate, and (3) contaminated water. Materials and methods REAGENTS AND STANDARDS Glass-distilled water was used to prepare stan-dards. Ethyl acetate-acetic acid reagent (4:1) was prepared by mixing gas chromatographic (GC) spectrophotometric-grade ethyl acetate (Omni

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