Gieseg S P, Maghzal G, Glubb D
Free Radical Biochemistry Laboratory, Department of Zoology, University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
Free Radic Res. 2001 Feb;34(2):123-36. doi: 10.1080/10715760100300121.
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.
新蝶呤及其还原形式7,8 - 二氢新蝶呤（78NP）是体内巨噬细胞受到γ - 干扰素刺激后释放的蝶啶。尽管由于新蝶呤具有很强的荧光特性，已在临床上用作免疫细胞活化的标志物，但78NP在炎症反应中的作用尚不清楚。我们以红细胞作为细胞模型，证明微摩尔浓度的78NP可以抑制或减少由2,2'-偶氮二异丁脒盐酸盐（AAPH）、过氧化氢或次氯酸盐诱导的红细胞溶血。100微摩尔的78NP在次氯酸浓度高达5微摩尔次氯酸/10⁷个红细胞的情况下可防止次氯酸溶血。50微摩尔的78NP可使2毫摩尔过氧化氢引起的溶血减少39%，而相同浓度的78NP能完全抑制2.5毫摩尔AAPH诱导的溶血。以高效液相色谱 - 硫代巴比妥酸反应物（HPLC - TBARS）测量的脂质过氧化水平不受78NP添加的影响。脂质氧化与细胞溶血之间没有相关性，这表明脂质过氧化对于溶血并非必不可少。在接触过氧化氢6小时和12小时后进行的共轭二烯测量支持了TBARS数据。细胞膜蛋白的凝胶电泳表明78NP可能抑制蛋白质损伤。以二酪氨酸作为蛋白质损伤的指标，我们证明200微摩尔的78NP可使过氧化氢/亚铁处理的红细胞膜中双酪氨酸的形成减少30%。高效液相色谱分析表明78NP与所有三种氧化剂之间存在直接反应。2毫摩尔过氧化氢每分钟氧化119纳摩尔的78NP，而1毫摩尔AAPH每分钟仅氧化50纳摩尔78NP，这表明78NP对溶血的抑制作用并非由于78NP清除主要起始反应物。相反，次氯酸与78NP之间的反应几乎是瞬间的。AAPH和过氧化氢将78NP氧化为7,8 - 二羟基蝶呤，而次氯酸盐氧化产生新蝶呤。78NP的细胞抗氧化特性表明它可能在炎症过程中保护免疫细胞免受自由基损伤方面发挥作用。
