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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Volume 43, Issue 2 (Apr 2005)


A spectrophotometric micromethod for determining erythrocyte protoporphyrin-IX in whole blood or erythrocytes

Guenther Kufner / Helmut Schlegel
  • Former Director of the Department of Occupational Health, Daimler-Benz AG, Sindelfingen, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Reinhard Jäger
Published Online: 2011-09-21 | DOI: https://doi.org/10.1515/CCLM.2005.031


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.

Keywords: absorption; dechelated zinc-protoporphyrin-IX; hemin; hydroquinone/formic acid; second derivative; unchelated protoporphyrin-IX (base)


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About the article

Corresponding author: Guenther Kufner, Occupational Health Service AMD-Linz, Kaplanhofstraße 1, Linz, Austria

Received: 2004-08-03

Accepted: 2004-11-10

Published Online: 2011-09-21

Published in Print: 2005-04-01

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2005.031.

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