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Clinical Chemistry and Laboratory Medicine (CCLM)

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

Editor-in-Chief: Plebani, Mario

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

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IMPACT FACTOR 2016: 3.432

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1437-4331
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Volume 43, Issue 2 (Apr 2005)

Issues

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

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.

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

References

  • 1

    Piomelli S. A micromethod for free erythrocyte porphyrins: the FEP test. J Clin Med 1973, 81: 932–40. Google Scholar

  • 2

    Chisholm JJ, Brown DH. Microscale photofluorimetric determination of “free erythrocyte porphyrin”. Clin Chem 1975; 12: 1669–81. Google Scholar

  • 3

    Gunter EW, Wayman E, Turner WE, Huff DL. Investigation of protoporphyrin IX standard materials used in acid extraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX. Clin Chem 1989; 35: 1601–8. Google Scholar

  • 4

    Doss MO. Porphyrins and porphyrin precursors. In: Curtius HC, Roth M, editors. Clinical biochemistry – principles and methods, vol. 2. New York: de Gruyter, 1974:1323–71. Google Scholar

  • 5

    Sassa S, Granick JL, Granick S, Kappas A, Levere RD. Studies in lead poisoning. I. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorimetry in the detection of chronic lead intoxication in the subclinical range. Biochem Med 1973; 8: 135–48. CrossrefGoogle Scholar

  • 6

    Wranne L. Free erythrocytes copro-and protoporphyrin. A methodological and clinical study. Acta Pediatr Scand Suppl 1960; 124: 1–78. Google Scholar

  • 7

    Grinstein M, Wintrobe MM. Free erythrocyte protoporphyrin. J Biol Chem 1948; 172: 460–7. Google Scholar

  • 8

    Allen WM. A simple method for analyzing complicated absorption curves, of use in the colorimetric determination of urinary steroids. J Clin Endocrinol 1950; 10: 71–83. CrossrefGoogle Scholar

  • 9

    Rimington C. Spectral absorption coefficients of some porphyrins in the Soret-band region. Biochem J 1960; 175: 620–3. Google Scholar

  • 10

    With TK. Porphyrin concentration with ultraviolet extinction. A note on the calculation. Scand J Clin Lab Invest 1955; 7: 193–4. CrossrefGoogle Scholar

  • 11

    Martin RF. General Deming regression for estimating systematic bias and its confidence interval in method-comparison studies. Clin Chem 2000; 46: 100–4. Google Scholar

  • 12

    WSLH. National EP Proficiency Testing Program. Madison, WI, USA: Environmental Health Division, Wisconsin State Laboratory of Hygiene. Project H46 MC00123. Personal communication. Google Scholar

  • 13

    Schlegel H, Kufner G. Long-term observation of biochemical effects of lead in human experiments. J Clin Chem Clin Biochem 1979; 17: 225–33. Google Scholar

  • 14

    Falk H. Chemistry of phenanthroperylenchinone. Angew Chem 1999; 111: 3306–26. Google Scholar

  • 15

    Gunter EW, Lewis BG, Koncikowski SM. Laboratory procedures used for the Third National Health and Nutrition Examination Survey (NHANES III) 1988–1994. Atlanta, GA, USA: Centers for Disease Control. Centers for Disease Control and Prevention, 1996. Google Scholar

  • 16

    Bailey GG, Needham LL. Simultaneous quantification of erythrocyte zinc protoporphyrin and protoporphyrin IX by liquid chromatography. Clin Chem 1986; 32: 2137–42. Google Scholar

  • 17

    Piomelli S, Lamola AA, Poh-Fitzpatrick MF, Seaman C, Harber LC. Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. II. Different binding of erythrocyte protoporphyrin to hemoglobin. J Clin Invest 1975; 56: 1528–35. Google Scholar

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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©2005 by Walter de Gruyter Berlin New York. Copyright Clearance Center

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