Concentration and composition of free ceramides in human plasma
Concentration and composition of free ceramides in human plasma
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DOI：
10.1055/s-2002-33597
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发表时间：
2002-08-01
影响因子：
2.2
通讯作者：
Namiot, Z
中科院分区：
文献类型：
--
作者：
Górska, M;Dobrzyn, A;Namiot, Z
方法本研究方案经Biaesthystok医学科学院人类和动物研究伦理委员会批准。从三组受试者中采集血样：第1组，年龄为22-26岁的献血者;第2组，来自Biaystok的Sniadecki地区医院内科的年龄为60-64岁的患者;第3组，来自相同科室的年龄为78-82岁的患者。每组有10名受试者。第2组和第3组的受试者诊断如下：结肠息肉（2，3）、功能性消化不良（2，1）、二尖瓣脱垂（2，2）、食管裂孔疝（3，2）和单纯性甲状腺肿（1，2）。括号中的数字分别表示第2组和第3组中的受试者数量。患者一般状况良好，无血脂异常。他们没有接受任何会干扰血浆脂质代谢的药物。禁食一夜后，出院时抽血。将血样采集至肝素化试管中。分离血浆并储存在-80 ℃.用氯仿-甲醇提取血浆脂质[1]。甲醇含有丁基化羟基甲苯（Sigma）作为抗氧化剂。如前所述[6]，使用薄层色谱法在硅胶板（硅胶60，0.25 mm，Merck）上分离神经酰胺。首先，在由氯仿/甲醇/25% NH3（20/5/0.2 v/v/v）组成的溶剂中，将平板展开至平板总高度的三分之一。接下来，将板干燥并在由庚烷/异丙醚/乙酸（60/40/3 v/v/v）组成的溶剂中展开。然后将板在室温下干燥。然后，用0.5%的2′ 7′-二氯荧光素甲醇溶液喷雾，暴露于氨蒸气，在紫外光下观察条带。根据神经酰胺标准品（非羟基脂肪酸神经酰胺，Sigma）鉴定含神经酰胺的条带，所述标准品与样品一起沿着在每个板上运行。图[1]显示了含有个体脂质标准品和血浆脂质的平板图片。将含有神经酰胺的条带从板上刮下并转移到含有甲基十五烷酸（C：15; Sigma）作为内标的管中。按照莫里森和史密斯的方法，使用14%的三氟化硼甲醇溶液[4]，在100 ℃沿着凝胶将神经酰胺脂肪酸甲基化90分钟。使用气液色谱法（Hewlett-Packard）鉴定和定量所得脂肪酸甲酯。使用50 μ m毛细管柱（HP-INNOWax）。用FID检测脂肪酸。该系统允许分离和鉴定C14-C24碳数范围内所有可能的脂肪酸。由神经酰胺标准品制备脂肪酸甲酯的效率为81- 85%。使用未配对数据的学生t检验对结果进行统计学比较。
MethodsThe protocol was approved by the Ethical Committee for the Human and Animal Studies in the Medical Academy of Białystok. Blood samples were taken from three groups of subjects: group 1, blood donors aged 22-26 yrs; group 2, patients aged 60-64 yrs from the Departments of Internal Diseases of the SŽniadecki Regional Hospital in Białystok; and group 3, patients aged 78-82 yrs from the same departments. There were ten subjects in each group. The subjects in groups 2 and 3 were diagnosed as follows: colonic polyp (2, 3), functional dyspepsia (2, 1), mitral valve prolapse (2, 2), hiatal hernia (3, 2), and simple goitre (1, 2). The numbers in brackets denote a number of subjects in group 2 and 3, respectively. The patients were in very good general condition and did not have plasma lipid disorders. They were not receiving any drugs that would interfere with the plasma lipid metabolism. Blood was taken at discharge after an overnight fast. Blood was sampled into heparinised tubes. The plasma was separated and stored at-80 C. Plasma lipids were extracted with chloroform-methanol [1]. Methanol contained butylated hydroxytoluene (Sigma) as an antioxidant. Ceramides were isolated using thin-layer chromatography on silica plates (Silica gel 60, 0.25 mm, Merck) as previously described [6]. First, the plates were developed to one-third of the total height of the plate in a solvent composed of chloroform/methanol/25% NH 3 (20/5/0.2 v/v/v). Next, the plates were dried and developed in a solvent composed of heptan/isopropyl ether/acetic acid (60/40/3 v/v/v). The plates were then dried at room temperature. Next, they were sprayed with a 0.5% solution of 2′ 7′-dichlorofluoresceine in methanol, exposed to ammonia vapours, and the bands were visualised under UV light. Ceramide-containing bands were identified according to the standard of ceramide (Non-Hydroxy Fatty Acid Ceramide, Sigma), which was run on each plate along with the samples. Fig.[1] shows a picture of a plate with individual lipid standards and plasma lipids. Bands containing ceramides were scraped off the plates and transferred into tubes containing methylpentadecanoic acid (C: 15; Sigma) as an internal standard. Ceramide fatty acids were methylated for 90 min at a temperature of 100 C along with gel according to Morrison and Smith, using 14% solution of boron trifluoride in methanol [4]. The resultant fatty acid-methyl esters were identified and quantified using gas-liquid chromatography (Hewlett-Packard). A 50 m capillary column (HP-INNOWax) was used. Fatty acids were detected with FID. This system allows separation and identification of all the possible fatty acids in the C14-C24 carbon number range. The efficiency for the preparation of fatty acid methyl esters from ceramide standard was 81-85%. Student's t-test for unpaired data was used for statistical comparison of the results.