Division of Animal and Nutritional Sciences, West Virginia University, Morgantown 26505.
Division of Animal and Nutritional Sciences, West Virginia University, Morgantown 26505.
J Dairy Sci. 2017 Oct;100(10):8602-8608. doi: 10.3168/jds.2016-12538. Epub 2017 Jul 26.
The hepatic synthesis and export of ceramide is enhanced in diabetic monogastrics. Moreover, ceramide in lipoproteins can mediate the development of insulin resistance. We have previously demonstrated that circulating ceramide increases during the progression of insulin resistance in postpartum dairy cows. Considering that the origins of circulating ceramide required investigation, our objective was to develop a method to characterize the ceramide profile of lipoprotein fractions collected from dairy cows. Serum was collected from 4 nonpregnant and nonlactating Holstein dairy cows. Serum lipoproteins were isolated using size exclusion chromatography by fast protein liquid chromatography (SEC-FPLC). Measurement of triacylglycerol (TAG), phospholipid, total cholesterol, and protein was performed using standard colorimetry practices. Following lipid extraction, fractions were analyzed using electrospray ionization tandem mass spectrometry. Data were analyzed as repeated measures using a mixed model. Lipoprotein isolation using SEC-FPLC and subsequent colorimetric analyses confirmed the presence of 4 distinct fractions: TAG-rich, low density (LDL), and large (buoyant) and small (dense) high density lipoprotein (HDL) subclasses. As expected, the fraction representing mixed very low density lipoproteins and chylomicrons primarily contained TAG. Low density lipoprotein fractions were equally enriched with cholesterol and phospholipid. Buoyant HDL contained elevated levels of cholesterol, phospholipid, and protein. In contrast, the fraction containing dense HDL primarily contained protein. Our method revealed that LDL are enriched with ceramides. Ceramides were also compartmentalized to a lesser extent within both HDL subclasses and TAG-rich lipoproteins. Comparable to whole serum, C16:0-ceramide was the predominant ceramide quantified in all lipoprotein subclasses. Interestingly, the proportion of C24:0-ceramide to total ceramide was elevated in TAG-rich lipoproteins, relative to all other lipoprotein subclasses. We conclude that SEC-FPLC coupled with mass spectrometry is a means to quantify ceramides in lipoprotein fractions. Moreover, ceramides are enriched within bovine LDL, and lipoprotein ceramide profiles reflect levels observed in whole serum. Future investigation will need to determine the biological importance of lipoprotein ceramides with distinct C-chains at amide residues.
在糖尿病单胃动物中，肝合成和输出神经酰胺增加。此外，脂蛋白中的神经酰胺可以介导胰岛素抵抗的发展。我们之前已经证明，在产后奶牛胰岛素抵抗进展过程中，循环神经酰胺增加。考虑到需要研究循环神经酰胺的来源，我们的目标是开发一种方法来描述从奶牛中分离的脂蛋白级分的神经酰胺谱。从 4 头非妊娠和非泌乳的荷斯坦奶牛中采集血清。使用快速蛋白质液相色谱法（SEC-FPLC）通过大小排阻色谱法分离血清脂蛋白。使用标准比色法测量三酰基甘油（TAG）、磷脂、总胆固醇和蛋白质。脂质提取后，使用电喷雾串联质谱法分析各馏分。使用混合模型对数据进行重复测量分析。使用 SEC-FPLC 分离脂蛋白并随后进行比色分析，证实存在 4 种不同的馏分：富含 TAG 的、低密度（LDL）、大（浮）和小（密）高密度脂蛋白（HDL）亚类。正如预期的那样，代表混合极低密度脂蛋白和乳糜微粒的馏分主要含有 TAG。LDL 部分富含胆固醇和磷脂。浮式 HDL 含有较高水平的胆固醇、磷脂和蛋白质。相比之下，含有密式 HDL 的馏分主要含有蛋白质。我们的方法表明，LDL 富含神经酰胺。神经酰胺也在 HDL 亚类和富含 TAG 的脂蛋白中较少地分隔。与全血清类似，C16:0-神经酰胺是所有脂蛋白亚类中定量的主要神经酰胺。有趣的是，与所有其他脂蛋白亚类相比，富含 TAG 的脂蛋白中 C24:0-神经酰胺与总神经酰胺的比例升高。我们得出结论，SEC-FPLC 与质谱联用是定量脂蛋白级分中神经酰胺的一种方法。此外，神经酰胺在牛 LDL 中富集，脂蛋白神经酰胺谱反映了全血清中观察到的水平。未来的研究需要确定酰胺残基中具有不同 C 链的脂蛋白神经酰胺的生物学重要性。
