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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 7

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Detection of the trifluoroacetyl methyl ester derivative of CML generate...
Detection of the trifluoroacetyl methyl ester derivative of CML generated by activation human neutrophils by electron ionization GC/MS with selected ion monitoring. Freshly harvested human neutrophils (106/mL) were incubated at 37°C in medium B supplemented with 1 mM L-serine. Cells were stimulated with 250 nM PMA and maintained in suspension by intermittent inversion. After a 45-minute incubation, cells were removed by centrifugation, and RNase A (1 mg/mL final concentration) was added. After a 96-hour incubation at 37°C, protein-bound CML in the incubation medium was detected by electron ionization GC/MS with selected ion monitoring. The ions at m/z 392 (M•+ – CH3OH) and m/z 305 (M•+ – 2 COOCH3 –H) represent the most abundant ions in the full-scan mass spectrum of CML. Note that the ions derived from d4-labeled CML at m/z 309 and m/z 396 elute slightly earlier than the corresponding ions of CML, as has been shown for many other deuterated compounds (45).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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Referenced in 8 patents
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