A spectrophotometric micromethod for determining erythrocyte protoporphyrin-IX in whole blood or erythrocytes
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Guenther Kufner
Abstract
An increased concentration of erythrocyte protoporphyrin-IX in whole blood or erythrocytes is a valuable diagnostic indicator for acquired porphyrias, e.g., iron deficiency anemia and lead poisoning, and for inherited porphyrias. We developed a spectrophotometric micromethod for determining erythrocyte protoporphyrin-IX. In this method, exhaustive release of erythrocyte porphyrins is achieved using hydroquinone and formic acid. The clean-up procedure for 50μL of whole blood or erythrocytes covers three steps of liquid/liquid solvent partition: two partitions using diethyl and diisopropyl ether and HCl 2.5mol/L, and one buffered step using ammonium formate. Determinations of erythrocyte protoporphyrin-IX are possible by: (a) absorption using three wavelengths, Rimington's constant and a millimolar absorptivity coefficient mɛ 408.8=294.3L·mmol -1·cm -1 according to With; and (b) 2nd derivative, which is linked to mɛ 408.8. Determination of erythrocyte protoporphyrin-IX using a 2nd derivative algorithm showed better spectral resolution and higher sensitivity at a five-fold lower detection limit compared to absorption. Within-run precision of medium and high levels was found for absorption and for 2nd derivative with a coefficient of variation (CV) of 1.4–1.9% (n=10). Total precision evaluated was CV=2.5–8.3% (n=20). Levels of reference intervals could only be measured using the 2nd derivative (CV 2.9%). Linearity was proved to E=1.0. Recoveries of protoporphyrin-IX ranged from 95.3% to 103.0%. Method comparison was carried out using a fluorimetric reference method (Piomelli). Reference intervals for gender groups are discussed.
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