Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride.
Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride.
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DOI：
10.1006/abio.1995.1480
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发表时间：
1995-09
影响因子：
2.9
通讯作者：
Y. Yatomi;F. Ruan;J. Ohta;R. Welch;S. Hakomori;Y. Igarashi
中科院分区：
文献类型：
--
作者：
Y. Yatomi;F. Ruan;J. Ohta;R. Welch;S. Hakomori;Y. Igarashi
我们在这里详细描述的方法来定量测量1-磷酸鞘氨醇（Sph-I-P），一种生物活性鞘脂的发展。Sph-I-P首先在碱性条件下通过Folch相分离从细胞中萃取到上层水相中，然后在酸性条件下再萃取到下层氯仿相中。通过用[3 H]乙酸酐进行N-酰化，将提取的磷酸化鞘氨醇碱定量转化为N-[3 H]-乙酰化Sph-I-P，即[3 H]C2-神经酰胺1-磷酸（C2-Cer-1-P）。通过薄层色谱法分离酰化形成的[3 H]C2-Cer-1-P，用放射自显影法检测，并通过刮取相应条带并用闪烁计数器计数其放射性来定量。该测定允许在至少100 pmol（通常为30 pmol）至10 nmol（测试的最高水平）的范围内定量Sph-1-P。使用人血小板证明了我们的测定的实用性和有效性。血小板提取物中Sph-1-P的量与细胞数成比例，计算为141 +/- 4 pmol/10（8）个细胞（平均值+/- SD，n = 3），约为鞘氨醇的4倍。强效激动剂凝血酶不影响血小板悬液中总的Sph-1-P量，但诱导储存在细胞中的Sph-1-P释放到培养基中。
We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.