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The myeloperoxidase system of human phagocytes generates Nε-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation
Melissa M. Anderson, … , Suzanne R. Thorpe, Jay W. Heinecke
Melissa M. Anderson, … , Suzanne R. Thorpe, Jay W. Heinecke
Published July 1, 1999
Citation Information: J Clin Invest. 1999;104(1):103-113. https://doi.org/10.1172/JCI3042.
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The myeloperoxidase system of human phagocytes generates Nε-(carboxymethyl)lysine on proteins: a mechanism for producing advanced glycation end products at sites of inflammation

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Abstract

Reactive aldehydes derived from reducing sugars and peroxidation of lipids covalently modify proteins and may contribute to oxidative tissue damage. We recently described another mechanism for generating reactive aldehydes from free α-amino acids. The pathway begins with myeloperoxidase, a heme enzyme secreted by activated neutrophils. Conversion of α-amino acids to aldehydes requires hypochlorous acid (HOCl), formed from H2O2 and chloride by myeloperoxidase. When L-serine is the substrate, HOCl generates high yields of glycolaldehyde. We now demonstrate that a model protein, ribonuclease A (RNase A), exposed to free L-serine and HOCl exhibits the biochemical hallmarks of advanced glycation end (AGE) products — browning, increased fluorescence, and cross-linking. Furthermore, Nε-(carboxymethyl)lysine (CML), a chemically well-characterized AGE product, was generated on RNase A when it was exposed to reagent HOCl-serine, the myeloperoxidase-H2O2-chloride system plus L-serine, or activated human neutrophils plus L-serine. CML production by neutrophils was inhibited by the H2O2 scavenger catalase and the heme poison azide, implicating myeloperoxidase in the cell-mediated reaction. CML was also generated on RNase A by a myeloperoxidase-dependent pathway when neutrophils were activated in a mixture of amino acids. Under these conditions, we observed both L-serine–dependent and L-serine–independent pathways of CML formation. The in vivo production of glycolaldehyde and other reactive aldehydes by myeloperoxidase may thus play an important pathogenic role by generating AGE products and damaging tissues at sites of inflammation.

Authors

Melissa M. Anderson, Jesús R. Requena, Jan R. Crowley, Suzanne R. Thorpe, Jay W. Heinecke

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Figure 9

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Reaction requirements for generation of CML on RNase A by activated huma...
Reaction requirements for generation of CML on RNase A by activated human neutrophils in medium supplemented with plasma concentrations of free amino acids. Isolated neutrophils (106/mL) were activated with 200 nM PMA in medium B supplemented with 1 mg/mL RNase A, 260 μM L-glutamate, 210 μM L-alanine, 200μm L-serine, 175 μM glycine, 165 μM L-valine, 100 μM L-proline, and 100 μM L-lysine. When indicated, L-serine was omitted (–Ser) from the medium, and sodium azide (Azide; 5 mM) or 3-aminotriazole (ATA; 10 mM) was included. After a 1-hour incubation at 37°C, cells were removed by centrifugation, and the medium was incubated for another 16 hours at 37°C. Reaction mixtures were then subjected to analysis for CML by negative-ion electron capture isotope dilution GC/MS with selected ion monitoring. Values are corrected for the endogenous CML content (21.0 μmol/mol lysine) of RNase A and represent the mean ± SEM of 6 determinations.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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Referenced in 8 patents
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