Üllen Andreas, Nusshold Christoph, Glasnov Toma, Saf Robert, Cantillo David, Eibinger Gerald, Reicher Helga, Fauler Günter, Bernhart Eva, Hallstrom Seth, Kogelnik Nora, Zangger Klaus, Oliver Kappe C, Malle Ernst, Sattler Wolfgang
Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.
Christian Doppler Laboratory for Flow Chemistry, Institute of Chemistry, University of Graz, Graz, Austria.
Biochem Pharmacol. 2015 Feb 15;93(4):470-81. doi: 10.1016/j.bcp.2014.12.017. Epub 2015 Jan 6.
Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl(-) system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood-brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120min, decaying at a rate of 5.9×10(-3)min(-1). NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC-MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo.
作为试剂添加的次氯酸或由髓过氧化物酶(MPO)-H2O2-Cl(-)系统产生的次氯酸会氧化修饰脑醚磷脂(缩醛磷脂)。该反应生成sn2-酰基溶血磷脂和氯化脂肪醛。2-氯十六醛(2-ClHDA)是氯化长链脂肪醛的典型成员,具有通过造成血脑屏障(BBB)损伤而产生的强大神经毒性潜力。在早期研究中我们可以表明,二氢查耳酮型多酚根皮素可减轻2-ClHDA诱导的BBB功能障碍。为了阐明潜在机制,我们现在研究了2-ClHDA与根皮素之间形成共价加合物的可能性。在磷脂酰胆碱脂质体中共同孵育2-ClHDA和根皮素显示2-ClHDA的半衰期约为120分钟,以5.9×10(-3)分钟(-1)的速率衰减。核磁共振研究和焓计算表明,2-ClHDA-根皮素加合物的形成是通过亲电芳香取代,然后在根皮素的A环上形成半缩醛而发生的。通过高分辨率质谱对加合物进行表征证实了这些结果。与2-ClHDA不同,共价的2-ClHDA-根皮素加合物对MTT还原(代谢活性指标)、细胞腺嘌呤核苷酸含量和脑微血管内皮细胞(BMVEC)的屏障功能没有不良影响。值得注意的是,在BMVEC培养物中也观察到了2-ClHDA-根皮素加合物的形成。腹腔注射并随后对脑脂质提取物进行气相色谱-质谱分析表明,根皮素能够穿透C57BL/6J小鼠的血脑屏障。本研究数据表明,根皮素清除2-ClHDA,从而减轻2-ClHDA介导的脑内皮细胞功能障碍。我们在此确定了一种原型氯化脂肪醛(通过MPO轴产生)的解毒途径,该途径在体外和体内损害血脑屏障功能。