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Lead Poisoning / Trace Elements Laboratory

Lead Poisoning / Trace Elements

Erythrocyte Protoporphyrin

Background

EP2 The erythrocyte protoporphyrin (EP) test was previously the recommended screening test for lead poisoning but it is not very sensitive for detecting the low level lead exposures now believed to be harmful to children (1). Thus, the EP test was superceeded by a direct blood lead test. However, EP is still used to assess the long-term effects of high level lead exposure, and is also used for monitoring patients receiving chelation therapy. It is also useful in diagnosing erythropoetic protoporphyria, a rare inherited disorder unrelated to lead poisoning.

Analytical Methodology

EP1In our laboratory, erythrocyte protoporphyrin is measured in whole blood using a reference method based on ethyl acetate-acetic acid extraction. The complete details of our method can be found in Current Protocols in Toxicology (1). Briefly, porphyrins and heme components are extracted from whole blood into an ethyl acetate-acetic acid mixture. Porphyrins are then separated from heme by back-extraction into a hydrochloric acid solution, and quantitatively determined by molecular fluorometry using a spectrofluorometer calibrated with protoporphyrin IX (PPIX) standard solutions; however, the exact concentration of the standards must first be established using molecular absorbance, Beer's Law and the millimolar absorptivity of PPIX. This method is based upon published methods (2-4), and methods developed by other public health laboratories in the U.S., including the Centers for Disease Control and Prevention (CDC) (5), and the Wisconsin State Laboratory of Hygiene (WSLH) (6). The method is very similar to that recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (7). The method is recommended for laboratory participants in the EP Proficiency Testing Program operated by the NYS DOH, and is also suitable for use in programs operated by the WSLH and the Pennsylvania Department of Health.

References

  1. Parsons, P.J. Measurement of Erythrocyte Protoporphyrin Concentration by Double Extraction and Spectrofluorometry, 8.8.1-8.8.12, In:Current Protocols in Toxicology edited by M. Maines, L.G. Costa, C. Klaassen, D.J. Reed and S. Sassa, J. Wiley & Sons, Inc., New York, 1999.
  2. Piomelli, S. A micro method for free erythrocyte protoporphyrin. J. Lab. Clin. Med. (1973) 81:932.
  3. Chisolm, J.J. and Brown, D.H. Micro-scale photofluorometric determination of "free erythrocyte protoporphyrin" (protoporphyrin IX). Clinical Chemistry (1975) 21:1669.
  4. Sassa S. Granick S, Kappas A, Levere RD. Microanalysis of erythrocyte protoporphyrin by spectrophotometry in the detection of chronic lead intoxication in the subclinical range. Biochem Med (1973) 8:135-48.
  5. Gunter, E.W., Tuner, W.E., Neese, J.W., Bayse, D.D. "Laboratory Procedures used by the Clinical Chemistry Division Centers for Disease Control for the Second Health and Nutrition Examination Survey, HANES II (1985) page 10 (C.D.C. Lab. manual).
  6. Santroch, J., Field, P., Stanton, N. Erythrocyte protoporphyrin extraction method. State Laboratory of Hygiene (1985) (Madison, Wisconsin).
  7. Erythrocyte Protoporphyrin Testing; Approved Guideline. NCCLS document C42-A (1996) (ISBN 1-56238-306-X), NCCLS Wayne, (1996) 39 pgs.

Proficiency Testing

For information on Proficiency Testing for EP, please see Proficiency Testing.

Reprints

To obtain reprints of articles authored by the NYS Lead Poisoning and Trace Elements Laboratory please go to the Reprint Request Form page.