Archives of Biochemistry and Biophysics 437 (2005) 128–137 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.03.007 Supraphysiological concentrations of 5-aminolevulinic acid dimerize in solution to produce superoxide radical anions via a protonated dihydropyrazine intermediate Gregory A. Hunter a,¤ , Edwin Rivera b , Gloria C. Ferreira a,c a Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA b Department of Chemistry, University of South Florida, Tampa, FL 33612, USA c H. Lee MoYtt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, USA Received 19 November 2004, and in revised form 9 March 2005 Available online 22 March 2005 Abstract 5-Aminolevulinic acid (ALA) is the committed biological precursor to porphyrins. At supraphysiological concentrations ALA can dimerize to form 3,6-dihydropyrazine-2,5-dipropanoic acid (DHPY), which transfers electrons to XTT in a reaction that does not require metal ions and is speciWcally inhibited by superoxide dismutase. The formation of DHPY from ALA follows dimeriza- tion kinetics with a pK of 7.8 § 0.1. At pH 11.2, DHPY is relatively stable, but when the pH is dropped to 6.0 rapid conversion to 2,5-(-carboxyethyl)pyrazine occurs via an intermediate with an absorption maximum of 370 nm. Formation of this intermediate is pH-dependent with a pK of 6.0 § 0.1. These data indicate that ALA dimerizes to produce superoxide from a protonated form of DHPY. The signiWcance of these results with respect to the concentrations of ALA used in photodynamic therapy, and the increased incidence of liver cancer in acute intermittent porphyria, is discussed.  2005 Elsevier Inc. All rights reserved. Keywords: Photodynamic; Superoxide; Dihydropyrazine; Porphyria; Aminolevulinate; Aminolevulinic acid; Cancer; Heart disease; Autoxidation 5-Aminolevulinic acid 1 (ALA) is the committed pre- cursor for biosynthesis of all porphyrins, including hemes and chlorophylls [1]. Upon exposure to exogenous ALA, transient stimulation of the porphyrin biosynthetic path- way occurs in both plants and animals [2,3]. This has given rise to a variety of promising biomedical and agri- cultural applications for ALA, including photodynamic therapy of cancers and cardiovascular disorders, as well as use as a plant crop fertilizer [3–5]. ALA is an aminoketone and as such is highly reactive and unstable in solution. In the presence of porphobilin- ogen synthase, two molecules of ALA are condensed to form porphobilinogen, but in the absence of this enzyme ALA can undergo spontaneous dimerization. At neutral to mildly basic pH spontaneous ALA dimerization yields DHPY, which under aerobic conditions irreversibly decomposes to 2,5-(-carboxyethyl)pyrazine (Scheme 1) [6–11]. The structures of the dihydropyrazine intermedi- ate and the pyrazine product have been assigned using 1 H and 13 C NMR [7,10], and a requirement for dioxygen to convert DHPY to the Wnal pyrazine product is also ¤ Corresponding author. Fax: +1 813 974 5798. E-mail address: ghunter@hsc.usf.edu (G.A. Hunter). 1 Abbreviations used: ALA, 5-aminolevulinic acid; AMPSO, 3-[(1,1- dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid; Caps, 3-[cyclohexylamino]-1-propanesulfonic acid; DFO, desferroxamine; DHPY, 3,6-dihydropyrazine-2,5-dipropanoic acid; EDTA, ethylenedi- aminetetraacetic acid; Hepes, N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid; Mes, 2-[N-morpholino]ethanesulfonic acid; Mops, 3-[N-morpholino]propanesulfonic acid; TAPS, N-tris[hydroxymeth- yl]methyl-3-aminopropanesulfonic acid: XTT, 2,3-bis (2-methoxy-4-ni- tro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide.