Reactive aldehydes derived from reducing sugars and lipid peroxidation play a critical role in the formation of advanced glycation end (AGE) products and oxidative tissue damage. We have recently proposed another mechanism for aldehyde generation at sites of inflammation that involves myeloperoxidase, a heme enzyme secreted by activated phagocytes. We now demonstrate that human neutrophils employ the myeloperoxidase-H 2 O 2 -chloride system to produce ␣ -hydroxy and ␣ , ␤ -unsaturated aldehydes from hydroxy-amino acids in high yield. Identities of the aldehydes were established using mass spectrometry and high performance liquid chromatography. Activated neutrophils converted L -serine to glycolaldehyde, an ␣ -hydroxyaldehyde which mediates protein cross-linking and formation of N ⑀ -(carboxymethyl)lysine, an AGE product. L -Threonine was similarly oxidized to 2-hydroxypropanal and its dehydration product, acrolein, an extremely reactive ␣ , ␤ -unsaturated aldehyde which alkylates proteins and nucleic acids. Aldehyde generation required neutrophil activation and a free hydroxy-amino acid; it was inhibited by catalase and heme poisons, implicating H 2 O 2 and myeloperoxidase in the cellular reaction. Aldehyde production by purified myeloperoxidase required H 2 O 2 and chloride, and was mimicked by reagent hypochlorous acid (HOCl) in the absence of enzyme, suggesting that the reaction pathway involves a chlorinated intermediate. Collectively, these results indicate that the myeloperoxidase-H 2 O 2 -chloride system of phagocytes converts free hydroxy-amino acids into highly reactive ␣ -hydroxy and ␣ , ␤ -unsaturated aldehydes. The generation of glycolaldehyde, 2-hydroxypropanal, and acrolein by activated phagocytes may thus play a role in AGE product formation and tissue damage at sites of inflammation. ( J. Clin. Invest. 1997. 99:424-432.)

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We have recently demonstrated that neutrophils oxidize nearly all of the amino acids commonly found in plasma to a corresponding family of aldehydes in high yield. The reaction is mediated by hypochlorous acid (HOCl), the major oxidant generated by the myeloperoxidase-H 2 O 2 -Cl ؊ system of phagocytes. We now present evidence for the underlying mechanism of this reaction, including the structural requirements and reaction intermediates formed. Utilizing mass spectrometry and isotopically labeled amino acids, we rule out hydrogen atom abstraction from the ␣-carbon as the initial event in aldehyde formation during amino acid oxidation, a pathway known to occur with ionizing radiation. Aldehyde generation from amino acids required the presence of an ␣-amino moiety; ␤-and ⑀-amino acids did not form aldehydes upon oxidation by either the myeloperoxidase system or HOCl, generating stable monochloramines instead. UV difference spectroscopy, high pressure liquid chromatography, and multinuclear ( 1 H, 15 N) NMR spectroscopy established that the conversion of ␣-amino acids into aldehydes begins with generation of an unstable ␣-monochloramine, which subsequently decomposes to yield an aldehyde. Precursor product relationships between ␣-amino acid and ␣-monochloramine, and ␣-monochloramine and aldehyde were confirmed by high pressure liquid chromatography purification of the reaction intermediate and subsequent We have recently shown that the enzyme can use H 2 O 2 and chloride ions (Cl Ϫ ) to oxidize nearly all of the common amino acids into a family of aldehydes (3). Because these products react readily with biological constituents and may be generated in significant quantities at sites of inflammation, we set out to elucidate the chemical mechanism by which they are generated.Our study explored two potential mechanisms (Scheme I). Pathway A, known to be a route by which amino acids are oxidized by ionizing radiation, involves initial hydrogen atom abstraction to generate an ␣-carbon-centered radical (4 -9). This short-lived intermediate decomposes into carbon dioxide and an imine, which then may form an aldehyde in the presence of H 2 O 2 through liberation of ammonia (4 -9). Metalcatalyzed oxidation of amino acids may similarly convert amino acids into aldehydes (9, 10), as first suggested by Dakin (11-13) nearly a century ago. Pathway B involves initial chlorination of the ␣-amino moiety, generating an ␣-monochloramine. This reaction is plausible because myeloperoxidase generates hypochlorous acid (HOCl) from H 2 O 2 and Cl Ϫ (14), and monochloramines form readily when HOCl reacts with amines (15-17). Zgliczynski and colleagues (18) first suggested that monochloramines of common amino acids might serve as precursors in aldehyde formation. These conclusions, however, were drawn from indirect evidence and confirmed neither the structures of products formed nor the putative monochloramine intermediates. In fact, monochloramines are generally thought to be relatively stable compounds under physiological conditions (15-17...

13-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the 18-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Verylong-chain acyl-CoA dehydrogenase [VLCAD; very-long-chainacyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in 13-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1 --a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C'837 > T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid 13-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the mitochondrial (3-oxidation spiral that supplies the majority of energy in mature heart and is crucial to intermediary metabolism in the liver (1-3). This spiral requires four enzymatic activities, an initial fatty acyl-CoA dehydrogenase, a 2,3-enoyl-CoA hydratase reaction, a 3-hydroxy-acyl-CoA dehydrogenase step, and the final 3-ketoacylCoA thiolase cleavage step. Because fatty acids with different chain lengths are substrates in these reactions, several enzymes, encoded by distinct nuclear genes, are required for each step. For the initial fatty acyl-CoA dehydrogenase step, four enzymes with overlapping, but different, chain length specificities have been identified and designated as VLCAD, which acts on substrates of 14-20 carbons in length (1); long-chain acyl-CoA dehydrogenase (LCAD); medium-chain acyl-CoA dehydrogenase (MCAD); and short-chain acyl-CoA dehydrogenase (SCAD). Biochemical characterization of the last three soluble, matrix enzymes (4) revealed similar structures consisting of four identical 42-kDa subunits and FAD as a cofactor. The cloning of mRNAs encoding the subunits of MCAD, LCAD, and SCAD revealed similarity of their primary sequences (5, 6). The discovery, characterization, and cloning of rat VLCAD (1) demonstrated significant differences from the other three members of this CAD family. VLCAD is a homodimer of 70-kDa subunits and is associated with the inner mitochondrial membrane.The importance of the f-oxidation pathway for energy production is emphasized by inherited deficiencies of these enzyme activities (2, 3). Individuals with these disorders frequently become critically ill in infa...