Measurement of δ‐Aminolevulinate Dehydratase Activity

Hiroyoshi Fujita1

1 Hokkaido University School of Medicine, Sapporo
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.6
DOI:  10.1002/0471140856.tx0806s01
Online Posting Date:  May, 2001
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Abstract

Vertebrate ALAD is a cytosolic enzyme that catalyzes the second step of porphyrin formation, and it is expressed abundantly in liver and erythroid tissues as well as in other tissues. This unit describes a colorimetric assay for measuring ALAD activity in mammalian blood or tissues and the extent of reactivation of the enzyme in lead‐inhibited samples.

     
 
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Table of Contents

  • Basic Protocol 1: Colorimetric Measurement of ALAD Activity
  • Alternate Protocol 1: ALAD Assay for Detecting Lead Poisoning
  • Alternate Protocol 2: Measurement of ALAD Activity by HPLC
  • Alternate Protocol 3: Fluorimetric Determination of Erythrocyte ALAD Activity by HPLC
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Colorimetric Measurement of ALAD Activity

  Materials
  • Blood or tissue sample
  • 100 mM Tris‐acetate buffer, pH 7.2
  • Protein assay dye reagent (e.g., Bio‐Rad; see appendix 3A)
  • 100 mM δ‐aminolevulinic acid hydrochloride (ALA⋅HCl) in H 2O (Sigma; store up to 1 month at –20°C)
  • 10% (w/v) trichloroacetic acid (TCA)/0.1 M HgCl 2, ice‐cold (store at room temperature)
  • Modified Ehrlich's reagent (see recipe)
  • Heparinized Vacutainer
  • Heparinized precalibrated capillary tubes (Lancer)
  • Critoseal (Lancer)
  • Potter homogenizer
  • Microhematocrit centrifuge
  • 1‐ml semimicro cuvettes, 4‐mm wide with 10‐mm path length

Alternate Protocol 1: ALAD Assay for Detecting Lead Poisoning

  • 1 M DTT ( appendix 2A; store up to 1 month at −20°C)
  • 10 mM zinc acetate (store at room temperature)

Alternate Protocol 2: Measurement of ALAD Activity by HPLC

  Materials
  • 5 to 50 µM PBG standards
  • Internal standard: 2‐methyl‐3‐carbmethoxy‐4‐(3‐propionic acid)‐pyrrole (see recipe)
  • Mobile phase (see recipe)
  • Whole blood collected in heparinized Vacutainer
  • 0.1 M sodium phosphate buffer, pH 6.8 ( appendix 2A) containing 10 mM δ‐aminolevulinic acid hydrochloride (ALA⋅HCl; prepare just before use)
  • 10% (w/v) trichloroacetic acid (TCA)/0.1 M HgCl 2, ice‐cold (store at room temperature)
  • HPLC system with 5‐µm particle Hypersil‐SAS column (5 × 100–mm) and UV detector set at 240 nm and 0.02 AU (full scale; Shandon Southern)

Alternate Protocol 3: Fluorimetric Determination of Erythrocyte ALAD Activity by HPLC

  Materials
  • Heparinized whole blood
  • 0.2 M sodium phosphate buffer, pH 6.8 ( appendix 2A)
  • 20 mM δ‐aminolevulinic acid hydrochloride (ALA⋅HCl) in water
  • Authentic porphyrins: 500 µg/liter coproporphyrin I and appropriate concentration uroporphyrin I, dissolved in glacial acetic acid
  • 10% (w/v) trichloracetic acid
  • Mobile phase: 500:500:10 (v/v/v) acetonitrile/aqueous 10 mM KH 2PO 4/glacial acetic acid
  • HPLC system with:
  • 5‐µm particle Shim‐pack CLC‐ODS reversed‐phase column (6 × 150–mm; Shimadzu Ltd.)
  •  Fluorescence detector with excitation wavelength of 400 nm (bandwidth 18 nm) and emission wavelength of 620 nm (bandwidth 22 nm)
  • 100°C water bath
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Figures

Videos

Literature Cited

Literature Cited
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   Fujita, H., Sassa, S., Lundgren, J., Holmberg, L., Thunell, S., and Kappas, A. 1987b. Enzymatic defect in a child with hereditary hepatic porphyria due to homozygous δ‐aminolevulinic acid dehydratase deficiency: Immunochemical studies. Pediatrics 80:880‐885.
   Fujita, H., Bishop, T.R., and Ishida, N. 1994. Toxicology and molecular biology of δ‐aminolevulinate dehydratase. In Regulation of Heme Protein Synthesis (H. Fujita, ed.) pp. 27‐39. AlphaMed, Dayton, Ohio.
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   Mitchell, R.A., Drake, J.E., Wittlin, L.A., and Petering, H.G. 1978. Erythrocyte porphobilinogen synthase (delta aminolevulinic acid dehydratase) activity: A reliable and quantitative indicate of lead exposure in humans. Clin. Chem. 23:105‐111.
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   Sassa, S., Granick, S., Bickers, D.R., Levere, R.D., and Kappas, A. 1973. Studies on the inheritance of human erythrocyte δ‐aminolevulinate dehydratase and uroporphryrinogen synthetase. Enzyme 16:326‐333.
   Sassa, S., Fujita, H., and Kappas, A. 1990. Succinylacetone and δ‐aminolevulinic acid dehydratase in hereditary tyrosinemia: Immunochemical study of the enzyme. Pediatrics 86:84‐86.
   Sato, K., Sano, S., and Fujita, H. 1982. Increase in the amount of erythrocyte δ‐aminolevulinic acid dehydratase in workers with moderate lead exposure. Int. Arch. Occup. Enviorn. Health 50:287‐297.
   Shemin, D., Abramsky, T., and Rassel, C.S. 1954. The synthesis of protoporphyrin from δ‐aminolevulinic acid in a cell‐free extract. J. Am. Chem. Soc. 76:1204‐1205.
   Thunell, S., Holmberg, L., and Lundgren, J. 1987. Aminolevulinate dehydratase porphyria in infancy: A clinical and biochemical study. J. Clin. Chem. Clin. Biochem. 25:5‐14.
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   Wetmur, J.G., Bishop, D.F., Cantelmo, C., and Desnick, R.J. 1986. Human δ‐aminolevulinate dehydratase: Nucleotide sequence of a full‐length DNA clone. Proc. Natl. Acad. Sci. U.S.A. 83:7703‐7707.
   Wetmur, J.G., Kaya, A.H., Plewinska, M., and Desnick, R.J. 1991. Molecular characterization of δ‐aminolevulinate dehydratase 2 (ALAD2) allele: Implications for molecular screening of individuals for genetic susceptibility to lead poisoning. Am. J. Hum. Genet. 49:757‐763.
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Key Reference
   Fujita et al., 1994. See above.
   This article reviews not only toxicological but also molecular biological aspects of δ‐aminolevulinate dehydratase.
   Sassa, S. 1982. δ‐aminolevulinic acid dehydratase assay. Enzyme 28:133‐145.
   Good description of basic assay conditions for crude enzyme preparation.
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