Abstract

Background

Lipid hydroperoxides decompose to reactive aldehydes, such as acrolein. Measurement of oxidative stress markers in the clinic could improve risk stratification for patients.

Methods

To aid the development of diagnostic oxidative stress markers, we defined the acrolein modifications of haemoglobin using mass spectrometry.

Results

Acrolein modifications have little effect on the secondary structure of haemoglobin. They do not disrupt the quaternary structure, but instead promote crosslinked octamers. For acrolein modified haemoglobin the response to O₂ binding is altered such that cooperativity is lost. Mass spectrometry experiments at a 1:1 acrolein:haemoglobin molar ratio demonstrate that the α-chain quickly forms an aza-Michael adduct (+56 Da), which then forms a more stable adduct, Nε-(3-methylpyridinium)lysine (MP-lysine, +76 Da) over 7 days. The β-chain remains relatively unchanged over the duration of the 7 days and the aza-Michael adduct is dominant. At 2:1 and 5:1 molar ratios the α-chain was consistently modified at K7, H20, H50, and the β-chain at C93 and H97 with the aza-Michael adduct. Beyond 5 h, an MP-adduct (+76 Da) was located predominantly at K7 of the α-chain, while an FDP-adduct (+94 Da) was observed at K95 of the β-chain.

Conclusions

We have generated qualitative evidence identifying the acrolein target sites on haemoglobin, a potential oxidative stress marker that is easily measured in circulation.

General significance

We provide data for the community to develop targeted mass spectrometric or immunometric assays for acrolein modified haemoglobin to further validate the potential of haemoglobin as an oxidative stress marker in patients .

中文翻译：

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脂质过氧化副产物丙烯醛修饰血红蛋白的反应动力学和残留物鉴定

摘要

背景

脂质过氧化氢分解成反应性醛，例如丙烯醛。在临床上测量氧化应激标志物可以改善患者的风险分层。

方法

为了帮助开发诊断性氧化应激标志物，我们使用质谱法定义了血红蛋白的丙烯醛修饰。

结果

丙烯醛修饰对血红蛋白的二级结构影响不大。它们不会破坏四元结构，而是促进交联八聚体。对于丙烯醛修饰的血红蛋白，对 O _2的反应绑定被改变，从而失去了协同性。丙烯醛：血红蛋白摩尔比为 1:1 的质谱实验表明，α 链迅速形成氮杂迈克尔加合物 (+56 Da)，然后形成更稳定的加合物 Nε-(3-甲基吡啶鎓)赖氨酸 (MP -赖氨酸，+76 Da) 超过 7 天。β-链在 7 天期间保持相对不变，并且 aza-Michael 加合物占主导地位。在 2:1 和 5:1 的摩尔比下，α-链在 K7、H20、H50 和 C93 和 H97 的 β-链与 aza-Michael 加合物一致地被修饰。超过 5 小时，MP 加合物（+76 Da）主要位于 α 链的 K7，而 FDP 加合物（+94 Da）主要位于 β 链的 K95。

结论

我们已经产生了确定血红蛋白上丙烯醛靶位点的定性证据，血红蛋白是一种在循环中很容易测量的潜在氧化应激标志物。

一般意义

我们为社区提供数据，以开发针对丙烯醛修饰的血红蛋白的靶向质谱或免疫测定法，以进一步验证血红蛋白作为患者氧化应激标志物的潜力。