Measurement of Erythrocyte Protoporphyrin Concentration by Double Extraction and Spectrofluorometry

Patrick J. Parsons1

1 Wadsworth Center, New York State Department of Health, and The University at Albany, Albany, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.8
DOI:  10.1002/0471140856.tx0808s02
Online Posting Date:  May, 2001
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Abstract

Quantification of the level of erythrocyte protoporphyrin can be used as a screening assay for lead exposure. In this method, porphyrins and heme compounds are extracted from whole blood using ethyl acetate, and porphyrins are then separated from heme by back‚Äźextraction with HCl solution. The extracted porphyrins are then quantified using a spectrofluorometer calibrated with protoporphyrin IX standard solutions.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Erythrocyte Protoporphyrin Concentration
  • Identifying and Resolving Problems with Ethyl Acetate Quality
  • Support Protocol 1: Testing Ethyl Acetate Stock for Quenching Impurities
  • Support Protocol 2: Ethyl Acetate Cleanup by Glass Distillation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring Erythrocyte Protoporphyrin Concentration

  Materials
  • 5‐µg protoporphyrin IX standards (Porphyrin Products)
  • Protosolv (Porphyrin Products)
  • 1.5 M HCl (see recipe)
  • Blood specimens in 10‐ml evacuated collection tubes with EDTA anticoagulant (purple‐cap Vacutainers, Becton Dickinson or equivalent)
  • Quality control (QC) samples
  • 4:1 (v/v) ethyl acetate/acetic acid solution (see recipe)
  • 50‐, 100‐, and 200‐µl fixed‐volume micropipets
  • 10‐ml glass volumetric pipet (class A preferable)
  • Light‐tight cabinet or box suitable for storing a rack(s) of culture tubes protected from light
  • UV/visible absorption spectrophotometer equipped with standard 1‐cm2 quartz cells
  • 2‐ml glass volumetric flasks (class A preferable) wrapped in aluminum foil
  • Adjustable or fixed‐volume micropipets in the range 400 to 1600 µl
  • 10 × 75–mm borosilicate glass culture tubes (Kimble, Corning or Scientific Products)
  • Microplate with 0.5‐ml wells
  • 1‐liter actinic glass bottle‐style pipets (Repipet, Barnstead/Thermolyne or equivalent) with 1‐ml glass dispensers
  • Small benchtop centrifuge suitable for 10 × 75–mm glass culture tubes (Sero‐Fuge, Clay Adams or equivalent)
  • Photographic dark room fluorescent tube filters (e.g., Macolite Toob Gard or EncapSulite safelite filters or equivalent)
  • Spectrofluorometer equipped with a red‐sensitive photomultiplier tube (Hamamatsu R 928 or equivalent) and a cell holder modified to accommodate standard 10 × 75–mm glass culture tubes (Fig. )
NOTE: All plastic materials provided with the dispenser (i.e., uptake tubes and/or dispensing tips of the Repipet dispenser) must be replaced with glass devices to ensure the integrity of the ethyl acetate. This may require the services of a glassblower to modify commercially available devices.NOTE: Standards and samples must be protected from exposure to bright light as much as possible throughout the procedure. Use yellow photographic filters for fluorescent tube lights. These tube‐style filters are placed over standard fluorescent tube lights to reduce exposure from UV wavelengths.

Support Protocol 1: Testing Ethyl Acetate Stock for Quenching Impurities

  Materials
  • Potassium iodide
  • Ethyl acetate to be tested

Support Protocol 2: Ethyl Acetate Cleanup by Glass Distillation

  Materials
  • Ethyl acetate
  • 2‐liter round‐bottom glass boiling flask (standard taper 24/40)
  • Teflon boiling stones
  • Column and condenser apparatus, including:
  •  ∼15‐inch glass fractionation column with standard taper 24/40 ground‐glass joints, wrapped in insulation and aluminum foil
  •  ∼15‐inch glass condenser with elbow to connect with collection vessel (standard taper 24/40)
  •  2 ring stands and a lab jack
  •  500‐W heating mantle (Glas‐Col or equivalent)
  •  Rheostat (0‐120 V output, 1500 W; Glas‐Col or equivalent)
  •  2‐liter glass collection bottle with standard taper 24/40 ground glass joint opening
CAUTION: This is a hazardous procedure that must be done under a laboratory hood. Ethyl acetate and its vapors are highly flammable. When a bad batch of ethyl acetate has been identified, all pipet bottles should be washed with a clean batch to prevent cross‐contamination.
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Figures

Videos

Literature Cited

Literature Cited
   Blumberg, W.E., Eisinger, J., Lamola, A.A., and Zuckerman, D.M. 1977a. The hematofluorometer. Clin. Chem. 23:270‐274.
   Blumberg, W.E., Eisinger, J., Lamola, A.A., and Zuckerman, D.M. 1977b. Zinc protoporphyrin level in blood determined by a portable hematofluorometer: A screening device for lead poisoning. J. Lab.Clin. Med. 89:712‐723.
   Bowers, M.A., Aicher, L.D., Davis, H.A., and Woods, J.S. 1992. Quantitative determination of porphyrins in rat and human urine and evaluation of urinary porphyrin profiles during mercury and lead exposures. J. Lab. Clin. Med. 120:272‐281.
   CDC (Centers for Disease Control). 1975. Increased lead absorption and lead poisoning in young children [Report]. U.S. Department of Health, Education, and Welfare, Atlanta, Ga.
   CDC (Centers for Disease Control). 1978. Preventing lead poisoning in young children [Report]. U.S. Department of Health, Education, and Welfare, Atlanta, Ga.
   CDC (Centers for Disease Control). 1985. Preventing lead poisoning in young children [Report]. U.S. Department of Health and Human Services, Atlanta, Ga.
   CDC (Centers for Disease Control). 1991. Preventing lead poisoning in young children [Report]. U.S. Department of Health and Human Services, Atlanta, Ga.
   Chiba, M. and Sassa, S. 1982. Analysis of porphyrin carboxylic acids in biological fluids by high‐performance liquid chromatography. Anal. Biochem. 124:279‐285.
   Chisolm, J. Jr. and Brown, D.H. 1975. Micro‐scale photofluorometric determination of “free erythrocyte pophyrin” (protoporphyrin IX). Clin. Chem. 21:1669‐1682.
   Doran, D. and Mitchell, D.G. 1984. Problems in the determination of erythrocyte protoporphyrin by ethyl acetate‐acetic acid extraction. Ann. Clin. Biochem. 21:141‐145.
   Gunter, E.W., Turner, W.E., and Huff, D.L. 1989. Investigation of protoporphyrin IX standard materials used in acid‐extraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX. Clin. Chem. 35:1601‐1608.
   Ho, J., Guthrie, R., and Tieckelmann, H. 1987. Quantitative determination of porphyrins, their precursors and zinc protoporphyrin in whole blood and dried blood by high‐performance liquid chromatography with fluorimetric detection. J. Chromatogr. 417:269‐276.
   Kammholz, L.P., Thatcher, L.G., Blodgett, F.M., and Good, T.A. 1972. Rapid protoporphyrin quantitation for detection of lead poisoning. Pediatrics 50:625‐631.
   NCCLS (National Committee for Clinical Laboratory Standards). 1996. Erythrocyte protoporphyrin testing; Approved guideline. 1 pp. NCCLS Document C42‐A, Wayne, Pa.
   Parsons, P.J., Reilly, A.A., and Hussain, A. 1991. Observational study of erythrocyte protoporphyrin as a screening test for detecting lead exposure in children: Impact of lowering the blood lead action threshold. Clin. Chem. 37:216‐225.
   Piomelli, S. 1973. A micromethod for free erythrocyte porphyrins: The FEP test. J. Lab. Clin. Med. 81:932‐940.
   Sagen, E. and Romslo, I. 1985. Determination of porphyrins by high performance liquid chromatography: Fluorescence detection compared to absorbance detection. Scand. J Clin. Lab. Invest. 45:309‐314.
   Sassa, S., Granick, J.L., Granick, S., Kappas, A., and Levere, R.D. 1973. Studies in lead poisoning. I. Microanalysis of erythrocyte protoporphyrin levels by spectrophotometry in the detection of chronic lead intoxication in the subclinical range. Biochem. Med. 8:135‐148.
   Sato, H., Ido, K., and Kimura, K. 1994. Simultaneous separation and quantification of free and metal‐chelated protoporphyrins in blood by three‐dimensional HPLC. Clin. Chem. 40:1239‐1244.
   Schwartz, S., Berg, M.H., Bossenmaier, I., and Dinsmore, H. 1960. Determination of porphyrins in biological materials. In Methods of Biochemical Analysis, Vol. VIII (D. Glick, ed.) pp. 221‐294. Interscience Publishers, New York.
   Scoble, H.A., McKeag, M., Brown, P.R., and Kavarnos, G.J. 1981. The rapid determination of erythrocyte porphyrins using reversed‐phase high performance liquid chromatography. Clin. Chim. Acta 113:253‐265.
   Smith, R.M., Doran, D., Mazur, M., and Bush, B. 1980. High‐performance liquid chromatographic determination of protoporphyrin and zinc protoporphyrin in blood. J. Chromatogr. 181:319‐327.
   Yip, R., Johnson, C., and Dallman, P.R. 1984. Age‐related changes in laboratory values used in the diagnosis of anemia and iron deficiency. Am. J. Clin. Nutr. 39:427‐436.
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