HPLC Methods for Analysis of Porphyrins in Biological Media

James S. Woods1, P. Lynne Simmonds1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.9
DOI:  10.1002/0471140856.tx0809s07
Online Posting Date:  May, 2001
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Abstract

Changes in porphyrin concentrations in biological media may serve as biological indicators of exposure and toxicity from a wide variety of drugs and chemical agents. This unit describes procedures for quantitative extraction of porphyrins from urine, feces, blood, and biological tissues as well as their separation and analysis by HAPLY spectrofluorometrc techniques.

     
 
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Table of Contents

  • Basic Protocol 1: Separation and Quantitation of Porphyrins by HPLC
  • Sample Collection Procedures for Porphyrins
  • Support Protocol 1: Animal Urine
  • Support Protocol 2: Human Urine
  • Support Protocol 3: Feces
  • Support Protocol 4: Blood
  • Porphyrin Extraction Procedures
  • Support Protocol 5: Urine Porphyrin Extraction
  • Support Protocol 6: Tissue and Fecal Porphyrin Extraction
  • Support Protocol 7: Blood Porphyrin Extraction
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Separation and Quantitation of Porphyrins by HPLC

  Materials
  • Porphyrin extract (see Support Protocol protocol 65, protocol 76, or protocol 87)
  • Combined porphyrin standard: Porphyrin Acid Chromatographic Marker Kit (Porphyrin Products)
  • HPLC‐grade methanol
  • 0.05 M sodium phosphate buffer, pH 3.5 (see recipe)
  • Helium (for sparging or degassing mobile phase solutions)
  • HPLC system equipped with two programmable solvent‐metering pumps, a gradient programmer, an automatic variable volume sample injector, and a fluorescence detector
  • C‐18 250 × 4.6–mm 5‐µm analytical HPLC column (e.g., Alltech Econosphere) and SS prefilter element (frit)
  • C‐18 guard column
  • Column heater
  • Amber HPLC vials, caps and 300‐ to 500‐µl inserts (size appropriate for specific instrument employed by the investigator)
  • Filters for mobile phase solvents (Millipore, type HVLP, 0.45‐µm)

Support Protocol 1: Animal Urine

  Materials
  • Sodium bicarbonate
  • Disodium EDTA
  • Test animals
  • Distilled water
  • Metabolism cages (Hoeltge, Inc.) that permit separation of urine from feces and other solid materials
  • Graduated polypropylene flasks (50‐, 100‐ml, or larger depending of size of animal used)

Support Protocol 2: Human Urine

  Materials
  • Sodium bicarbonate
  • Disodium EDTA
  • Human subject(s)
  • 100‐ml clinical urine collection cups with orange screw‐on lids (one per subject per void; Starplex Scientific, Fisher)
  • 2.5‐liter dark brown polypropylene bottles with screw‐on lids (one per subject per 24‐hr period; Biomedical Polymers, VWR)
  • Female urinals (as required; Commode Specimen Collection System, Fisher)

Support Protocol 3: Feces

  Materials
  • Metabolism cages (Hoeltge, Inc.) that permit separation of urine from feces and other solid materials or Commode Collection System (for human feces; Fisher)

Support Protocol 4: Blood

  Materials
  • Sterile 10‐ml syringe and needle or 10‐ml Vacutainer tube (Becton Dickinson) with EDTA as anticoagulant

Support Protocol 5: Urine Porphyrin Extraction

  Materials
  • HPLC‐grade methanol
  • 10 mM sodium phosphate buffer, pH 3.5 (see recipe)
  • 1 N and 6 N HCl
  • Urine sample (see Support Protocol protocol 21 or protocol 32)
  • 35% (v/v) HPLC‐grade methanol in 10 mM sodium phosphate buffer, pH 3.5 (see recipe for buffer)
  • 10 mM sodium phosphate buffer, pH 7.5 (see recipe)
  • 80% (v/v) HPLC‐grade methanol/20% (v/v) 10 mM sodium phosphate buffer, pH 7.5 (see recipe for buffer)
  • Nitrogen source
  • C‐18 Bond Elute columns with 10‐ml reservoirs (size 10 ml/250 mg; Varian) or C‐18 Sep‐Pac columns (Waters) and 10‐ml plastic syringes to be used as column barrel
  • Vacuum manifold system: e.g., Baker SPE 10 vacuum extraction system or Millipore 1225 Sampling Manifold
  • 15‐ml conical polystyrene centrifuge tubes with screw‐on caps
  • Adjustable pipettor (Gilson 5000‐µl) and tips to fit or 10‐ml serological pipets and electric pipettor (Drummond)
  • Beckman J2‐27 or equivalent centrifuge
  • 20‐ml glass or plastic scintillation vials with Mylar/Teflon‐lined lid (Research Products International)
  • 60°C water bath or heating block
  • Glass 5¾‐in. Pasteur pipets
  • 4‐mm Millex‐HV 0.45‐µm membrane filters (Millipore)
  • 1‐ml plastic syringe
  • 10‐ml plastic syringe plunger
  • Amber HPLC vials to fit autosampler used (e.g., 1‐ml amber shell vials for 96‐position autosampler tray) and 300‐ to 500‐µl inserts with bottom springs (Alltech Associates or Waters)

Support Protocol 6: Tissue and Fecal Porphyrin Extraction

  Materials
  • HPLC‐grade acetonitrile
  • HPLC‐grade distilled H 2O
  • Fecal (see protocol 4) or tissue sample
  • 1 N HCl
  • 50:50 1 N HCl acetonitrile mixture
  • Nitrogen source
  • C‐18 Bond Elute columns with 10‐ml reservoirs (size 10 ml/250 mg; Varian) or C‐18 Sep‐Pac columns (Waters) and 10‐ml plastic syringes to be used as column barrel
  • Vacuum manifold system: e.g., Baker SPE 10 vacuum extraction system or Millipore 1225 Sampling Manifold
  • Brinkman Power‐Gen homogenizer (VWR) or equivalent
  • 15‐ml glass round‐bottom centrifuge tubes, for homogenization
  • Beckman J2‐27 or equivalent centrifuge
  • Adjustable pipettor (Gilson 5000‐µl) and tips to fit or 10‐ml serological pipets and electric pipettor (Drummond)
  • 50‐ml round bottom test tubes
  • 32°C water bath or heating block
  • Glass 5¾‐in. Pasteur pipets
  • 50‐ml glass round‐bottom centrifuge tubes, for drying
  • 4‐mm Millex‐HV 0.45‐µm membrane filters (Millipore)
  • 1‐ml plastic syringe
  • 10‐ml plastic syringe plunger
  • Amber HPLC vials to fit autosampler used (e.g., 1‐ml amber shell vials for 96‐position autosampler tray) and 300‐ to 500‐µl inserts with bottom springs (Alltech Associates or Waters)

Support Protocol 7: Blood Porphyrin Extraction

  Materials
  • Blood sample (see protocol 5)
  • 3:1 (v/v) ethyl acetate/glacial acetic acid containing 50 pmol mesoporphyrin IX (Porphyrin Products) as internal standard
  • Amber HPLC vials to fit autosampler used (e.g., 1‐ml amber shell vials for 96‐position autosampler tray) and 300‐ to 500‐µl inserts with bottom springs (Alltech Associates or Waters)
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Figures

Videos

Literature Cited

Literature Cited
   Bowers, M.A., Aicher, L.D., Davis, H.A., and Woods, J.S. 1992a. 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.
   Bowers, M.A., Luckhurst, C.L., Davis, H.A., and Woods, J.S. 1992b. Investigation of factors influencing urinary porphyrin excretion in rats: Strain, gender and age. Fundam. Appl. Toxicol. 19:538‐544.
   Carlson, R.E. and Dolphin, D. 1976. High pressure liquid chromatographic techniques for the separation of complex mixtures of occurring porphyrins naturally. In Porphyrins in Human Diseases (M. Doss, ed.) 462‐471. S. Karger, Basel.
   Ford, R.E., Ou, C.N., and Ellefson, R.D. 1981. Liquid‐chromatographic analysis for urinary porphyrins. Clin. Chem. 27:397‐401.
   Ho, J.W. 1990. Determination of porphyrins in human blood by high performance liquid chromatography . J. Liq. Chromatogr. 13:2179‐2192.
   Kennedy, S.W. and Maslen, A.L. 1989 Separation of porphyrin isomers by high‐performance liquid chromatography. J. Chromatogr. 493:53‐62.
   Li, F., Lim, C.K. and Peters, T.J. 1986. Analysis of urine and faecal porphyrins by HPLC coupled to an advanced automated sample processor. Biomed. Chromatogr. 1:94‐95.
   Lim, C.K. and Peters, T.J. 1984. Urine and faecal porphyrin profiles by reversed‐phase high‐performance liquid chromatography in the porphyrias. Clin. Chim. Acta 139:55‐63.
   Maines, M.D. 1984. New developments in the regulation of heme metabolism and their implications. CRC Crit. Rev. Toxicol. 12:241‐314.
   Marks, G.S. 1985. Exposure to toxic agents: The heme biosynthetic pathway and hemoproteins as indicator. CRC Crit. Rev. Toxicol. 15:151‐179.
   McColl, K.E.L. and Moore, R. 1981 The porphyrias. An example of pharmacogenetic disease. Scott. Med. J. 26:32‐40.
   Nacht, C., San Martin De Vaile, L.C., and Grinstein, M. 1970 Human porphyria cutanea tarda. Isolation and properties of the urinary porphyrins. Clin. Chim. Acta 27:445‐452.
   Schreiber, W.E., Raisys, V.A., and Labbe, R.F. 1983. Liquid‐chromatographic profiles of urinary porphyrins. Clin. Chem. 29:527‐530.
   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.
   Seubert, A. and Seubert, S. 1982. High‐performance liquid chromatographic analysis of porphyrins and their isomers with radial compression columns. Anal. Biochem. 124:303‐307.
   Woods, J.S. 1995. Porphyrin metabolism as indicator of metal exposure and toxicity. In Handbook of Experimental Pharmacology: Toxicology of Metals‐Biochemical Aspects, Vol. 115 (R.A. Goyer and M.G. Cherian, eds.) 119‐125. Springer‐Verlag, Berlin.
   Woods, J.S. and Miller, H.D. 1993. Quantitative measurement of porphyrins in biological tissues and evaluation of tissue porphyrins during toxicant exposures. Fundam. Appl. Toxicol. 21:291‐297.
   Woods, J.S., Eaton, D.L., and Lukins, C.B. 1984. Studies on porphyrin metabolism in the kidney. Effects of trace metals and glutathione on renal uroporphyrinogen decarboxlase. Mol. Pharmacol. 26:336‐341.
   Woods, J.S., Bowers, M.A., and Davis, H.A. 1991. Urinary porphyrin profiles as biomarkers of trace metal exposure and toxicity: studies on urinary porphyrin excretion patterns in rats during prolonged exposure to methyl mercury. Toxicol. Appl. Pharmacol. 110:464‐476.
Key References
   Bowers, M.A. 1992a See above
   Contains detailed information on HPLC analysis of human and rat urinary porphyrins and describes the effects of prolonged lead and mercury exposure on urinary porphyrin excretion.
   Bowers, M.A. 1992b See above.
  Describes the influence of age, sex, and strain on urinary porphyrin excretion in rats.
   Martin, M.D., McCann, T., Naleway, C., Woods, J.S., Leroux, B.G., and Bollen, A.M. 1996 The validity of spot urine samples for low‐level occupational mercury exposure assessment and relationship to porphyrin and creatinine excretion rates. J. Pharmacol. Exp. Ther. 277:239‐244.
  Describes diurnal variations in urinary excretion of porphyrins among men and women and establishes the validity of random (“spot”) sampling for assessment of 24‐hr porphyrin excretion rates.
   Woods and Miller, 1993. See above.
   Contains detailed information on HPLC analysis of tissue porphyrins and the effects of drug and metal treatments on liver and kidney porphyrin concentrations in rats.
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