Department of Nutrition, Université de Montréal, Montreal, QC H3C 3J7, Canada.
Free Radic Biol Med. 2009 Nov 15;47(10):1375-85. doi: 10.1016/j.freeradbiomed.2009.08.002. Epub 2009 Aug 12.
Quantification of 4-hydroxy-2-nonenal (HNE) bound to circulating proteins may prove to be useful in evaluating the role of this bioactive lipoperoxidation by-product in the pathogenesis of various diseases. Recently, we developed a quantitative gas chromatography-mass spectrometry (GCMS) assay of total protein-bound HNE (HNE-P) in blood after reduction with NaB(2)H(4) and cleavage with Raney nickel. Whereas it has been assumed that Raney nickel cleaves only Michael adducts of HNE to cysteine via a thioether bond (HNE-SP), results from this study demonstrate that our GCMS method also detects with precision picomoles of HNE adducts via nitrogen residues (HNE-NP). Specifically, evidence was obtained using various study models, including polyamino acids consisting of cysteine, lysine, and histidine and a biologically relevant molecule, albumin. Furthermore, we show that dinitrophenylhydrazine treatment before Raney nickel treatment can be used to discriminate and quantify the various HNE-P molecular species in plasma and blood samples from normal rats, which range between 0.15 and 3 pmol/mg protein or 10 to 600 nM. However, whereas HNE-SP predominated in whole blood, we detected HNE-NP only in plasma. We also identified another significant MS signal, which we attribute to protein-bound 1,4-dihydroxynonane (DHN-P) presumably formed from the enzymatic reduction of HNE-P. The distribution profile of all these species in plasma differed from that observed when physiologically relevant concentrations of albumin and HNE were incubated in vitro. Furthermore, interestingly, hypercholesterolemic rabbits showed higher plasma levels of HNE-NP, but not of DHN-P. Beyond documenting the presence of various types of HNE-P in circulating proteins, our results emphasize the importance of enzymatic mechanisms in situ as a factor determining their distribution in the various blood compartments under various conditions.
定量检测血液中与蛋白质结合的 4-羟基-2-壬烯醛（HNE）可能有助于评估这种生物活性脂质过氧化产物在各种疾病发病机制中的作用。最近，我们开发了一种定量气相色谱-质谱（GCMS）测定方法，用于还原后用 NaB(2)H(4)和雷尼镍裂解的血液总蛋白结合 HNE（HNE-P）。虽然人们一直认为雷尼镍仅通过硫醚键（HNE-SP）裂解 HNE 与半胱氨酸的迈克尔加成物，但本研究结果表明，我们的 GCMS 方法还可以通过氮残基（HNE-NP）精确检测 picomoles 的 HNE 加合物。具体来说，使用包括半胱氨酸、赖氨酸和组氨酸组成的多氨基酸和一种生物相关分子白蛋白的各种研究模型获得了证据。此外，我们表明，在雷尼镍处理之前用二硝基苯肼处理可以用于区分和定量正常大鼠血浆和血液样本中的各种 HNE-P 分子种类，其范围在 0.15 和 3 pmol/mg 蛋白或 10 到 600 nM 之间。然而，尽管 HNE-SP 在全血中占主导地位，但我们仅在血浆中检测到 HNE-NP。我们还鉴定了另一个重要的 MS 信号，我们将其归因于 HNE-P 的酶还原可能形成的蛋白结合 1,4-二羟基壬烷（DHN-P）。所有这些物质在血浆中的分布谱与在体外孵育生理相关浓度的白蛋白和 HNE 时观察到的分布谱不同。此外，有趣的是，高胆固醇血症的兔子显示出更高的血浆 HNE-NP 水平，但没有 DHN-P。除了证明循环蛋白中存在各种类型的 HNE-P 外，我们的结果还强调了原位酶促机制作为决定它们在各种条件下在各种血液隔室中分布的重要性。
