Protection of erythrocytes by the macrophage synthesized antioxidant 7,8 dihydroneopterin.
Neopterin and the reduced form, 7,8-dihydroneopterin (78NP) are pteridines released from macrophages when stimulated with gamma-interferon in vivo. The role of 78NP in inflammatory response is unknown though neopterin has been used clinically as a marker of immune cell activation, due to its very fluorescent nature. Using red blood cells as a cellular model, we demonstrated that micromolar concentrations can inhibit or reduce red blood cell haemolysis induced by 2,2'-azobis(amidinopropane)dihydrochloride (AAPH), hydrogen peroxide, or hypochlorite. One hundred microM 78NP prevented HOCl haemolysis using a high HOCl concentration of 5 micromole HOCl/10(7) RBC. Fifty microM 78NP reduced the haemolysis caused by 2 mM hydrogen peroxide by 39% while the same 78NP concentration completely inhibited haemolysis induced by 2.5 mM AAPH. Lipid peroxidation levels measured as HPLC-TBARS were not affected by addition of 78NP. There was no correlation between lipid oxidation and cell haemolysis suggesting that lipid peroxidation is not essential for haemolysis. Conjugated diene measurements taken after 6 and 12 hour exposure to hydrogen peroxide support the TBARS data. Gel electrophoresis of cell membrane proteins indicated 78NP might inhibit protein damage. Using dityrosine as an indicator of protein damage, we demonstrated 200 microM 78NP reduced dityrosine formation in H(2) O(2) /Fe(++) treated red blood cell ghosts by 30%. HPLC analysis demonstrated a direct reaction between 78NP and all three oxidants. Two mM hydrogen peroxide oxidised 119 nM of 78NP per min while 1 mM AAPH only oxidised 50 nM 78NP/min suggesting that 78NP inhibition of haemolysis is not due to 78NP scavenging the primary initiating reactants. In contrast, the reaction between HOCl and 78NP was near instant. AAPH and hydrogen peroxide oxidised 78NP to 7,8-dihydroxanthopterin while hypochlorite oxidation produced neopterin. The cellular antioxidant properties of 78NP suggest it may have a role in protecting immune cells from free radical damage during inflammation.
Free Radical Biochemistry Laboratory, Department of Zoology, University of Canterbury, Private Bag 4800, Christchurch, New Zealand. s.gieseg@zool.canterbury.ac.nz
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D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects*; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D019798:Neopterin / Q000031:analogs & derivatives; D010084:Oxidation-Reduction; D011621:Pteridines / Q000494:pharmacology*; D013552:Swine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000097:blood
D000578:Amidines; D001798:Blood Proteins; D011621:Pteridines; C000976:7,8-dihydroneopterin; D014443:Tyrosine; D019798:Neopterin; D006997:Hypochlorous Acid; C046728:2,2'-azobis(2-amidinopropane); D007371:Interferon-gamma; D006861:Hydrogen Peroxide; C007543:dityrosine