An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials
An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials
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DOI：
10.1007/s002100000365
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发表时间：
2001-03-01
影响因子：
3.6
通讯作者：
Kleuser, B
中科院分区：
文献类型：
--
作者：
Ruwisch, L;Sch채fer-Korting, M;Kleuser, B
鞘氨醇-1-磷酸（SPP）被认为是细胞内的第二信使和通过特定的细胞表面受体作为细胞外介质。随着细胞功能的多样化，迫切需要定量内源性和外源性SPP的方法。在这里，我们报告了一种快速高效液相色谱方法，允许定量的SPP在皮摩尔范围内，甚至在复杂的生物系统。两步脂质提取用于从大多数干扰磷脂和鞘脂中分离SPP。重要的是，二氢鞘氨醇-1-磷酸（dihydrospp）在所有培养细胞和大量生物样品中都检测不到，具有相同的提取特性，因此是理想的内标。提取后，邻苯二醛（OPA）将SPP和二氢SPP转化为荧光异吲哚衍生物，并以甲醇和0.07 M K2HPO4为洗脱剂，采用梯度程序进行HPLC分离。使用该方法，我们能够在0.5 pM至0.2 nM的宽范围内获得可重复的SPP测量值。用离子对试剂硫酸四铵确定了SPP和二氢-SPP的同一性，引起了两个峰的移位，但没有改变峰的面积。此外，牛肠黏膜碱性磷酸酶（AP）对鞘氨醇和鞘氨氨酸的酶转化排除了重叠化合物的存在。SPP的水平被测定在各种生物样品，如血清、血小板、原代角质形成细胞和一些细胞系。此外，我们能够检测暴露于1 α，25-二羟基维生素D-3 (1,25-(OH)(2)D-3)后，人角化细胞细胞内SPP水平的增加，其中鞘氨酸激酶活性的刺激已被确认。
Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracellular second messenger and as an extracellular mediator via specific cell surface receptors. Based on the increasing diverse cellular roles methods to quantify endogenous and exogenous SPP are highly required.Here, we report a rapid HPLC method that allows quantification of SPP in the picomolar range even in complex biological systems. A two-step lipid extraction serves to separate SPP from most interfering phospholipids and sphingolipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not detectable in all cultured cells and biological samples in considerable amounts, possesses equal extraction properties and therefore is an ideal internal standard. Following extraction SPP and dihydro-SPP are converted to fluorescent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated by HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro-SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover, enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucose alkaline phosphatase (AP) excluded the existence of overlapping compounds.Levels of SPP were determined in a variety of biological samples like serum, thrombocytes, primary keratinocytes and several cell lines. Furthermore, we were able to detect increases of intracellular SPP levels in human keratinocytes after exposure to 1 alpha ,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) for which a stimulation of sphingosine kinase activity has been recognized.