Suarna C, Dean R T, Southwell-Keeley P T, Moore D E, Stocker R
Biochemistry Unit, Heart Research Institute, NSW, Australia.
Free Radic Res. 1997 Oct;27(4):397-408. doi: 10.3109/10715769709065779.
In previous work we demonstrated that up to 30% of cholesteryl linoleate in homogenates of advanced human plaque samples is present in oxidized forms. Here we show that the material from plaque hexane extracts which co-elutes with cholesteryl hydroxy-linoleate on reversed phase HPLC (Anal Biochem 1993;213:79), is composed of several isomers of cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. Enzymatic hydrolysis and measurement of liberated cholesterol and disappearance of the esters revealed that almost all of the material consisted of unoxidized cholesterol esterified to oxidized derivatives of octadecadienoate. Semi-preparative reversed-phase HPLC was used to obtain sufficient quantities of this co-eluting material to undertake normal phase HPLC separation of these components. The nature of such separated and isolated compounds was identified, by co-chromatography with authentic standards, UV spectroscopy and chemical ionization and electron impact mass spectrometry, as cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. These oxidized fatty acids have been observed previously in plaque, in agreement with our new unambiguous demonstration of their presence as cholesteryl esters. The application of the methods described for the separation of the various forms of oxidized cholesteryl octadecadienoate may aid mechanistic studies of in vitro and in vivo lipoprotein lipid oxidation.
在之前的研究中，我们证明了在晚期人类斑块样本匀浆中，高达30%的亚油酸胆固醇酯以氧化形式存在。在此我们表明，在反相高效液相色谱（《分析生物化学》1993年；213:79）上与羟基亚油酸胆固醇酯共洗脱的斑块己烷提取物中的物质，由羟基胆固醇酯和氧代十八碳二烯酸胆固醇酯的几种异构体组成。酶促水解以及对释放的胆固醇的测量和酯的消失情况表明，几乎所有该物质都由酯化到十八碳二烯酸氧化衍生物上的未氧化胆固醇组成。使用半制备反相高效液相色谱获得了足够量的这种共洗脱物质，以便对这些成分进行正相高效液相色谱分离。通过与标准品共色谱、紫外光谱以及化学电离和电子轰击质谱，将这种分离和分离出的化合物的性质鉴定为羟基十八碳二烯酸胆固醇酯和氧代十八碳二烯酸胆固醇酯。这些氧化脂肪酸此前在斑块中已被观察到，这与我们对它们作为胆固醇酯存在的新的明确证明一致。所描述的用于分离各种形式的氧化十八碳二烯酸胆固醇酯的方法的应用，可能有助于体外和体内脂蛋白脂质氧化的机制研究。
