Ohta H, Ruan F, Hakomori S, Igarashi Y
Biomembrane Institute, Seattle, Washington 98119.
Anal Biochem. 1994 Nov 1;222(2):489-94. doi: 10.1006/abio.1994.1522.
A simple and sensitive method for quantification of sphingosine in cellular lipid extracts was developed. The assay is based on quantitative conversion of sphingosine to N-[3H]acetylated sphingosine ([3H]C2-ceramide) by N-acylation with [3H]acetic anhydride under certain conditions. Sphingosine was extracted from cultured cells with chloroform and methanol and then treated with base to remove interfering glycerolipids having reactive amino groups (e.g., phosphatidylethanolamine or phosphatidylserine). Sphingosine was acylated with [3H]acetic anhydride in the presence of 0.004 N NaOH. Acylation was complete in 1 h at 37 degrees C when sphingosine was present in the picomole range. After the acylation, samples were treated with NaOH to reduce background radioactivity by removing the remaining [3H]acetic anhydride and hydrolyzing any ester linkages formed during the acylation and resolved by thin-layer chromatography. [3H]C2-ceramide converted from sphingosine with the acylation was detected with radioautography and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. [3H]C2-ceramide formed was quantitatively measured. This assay allows quantification of sphingosine over a range of 10 to 1500 pmol. The amount of sphingosine in lipid extracts from cultured cells was proportional to the number of cells. Sphingosine levels in human gastric cancer KATO III cells, human promyelocytic leukemic HL60 cells, and human monoblastic U937 cells, determined by this method, were 26.6 +/- 2.2, 6.3 +/- 0.4, and 6.8 +/- 0.6 pmol per 10(6) cells, respectively. Our new procedure allows quantification of sphingosine levels present in the low picomole range in lipid extracts from biological samples.
我们开发了一种简单且灵敏的方法用于定量细胞脂质提取物中的鞘氨醇。该测定法基于在特定条件下，通过用[³H]乙酸酐进行N-酰化反应，将鞘氨醇定量转化为N-[³H]乙酰化鞘氨醇（[³H]C2-神经酰胺）。用氯仿和甲醇从培养细胞中提取鞘氨醇，然后用碱处理以去除具有反应性氨基的干扰性甘油脂质（如磷脂酰乙醇胺或磷脂酰丝氨酸）。鞘氨醇在0.004N NaOH存在下用[³H]乙酸酐进行酰化。当鞘氨醇存在于皮摩尔范围内时，在37℃下1小时酰化反应完成。酰化反应后，用NaOH处理样品以通过去除剩余的[³H]乙酸酐并水解酰化过程中形成的任何酯键来降低背景放射性，并通过薄层色谱法进行分离。通过放射自显影检测经酰化反应从鞘氨醇转化而来的[³H]C2-神经酰胺，并通过刮下相应条带并用闪烁计数器计数其放射性进行定量。对形成的[³H]C2-神经酰胺进行定量测量。该测定法可对10至1500皮摩尔范围内的鞘氨醇进行定量。培养细胞脂质提取物中鞘氨醇的量与细胞数量成正比。用该方法测定的人胃癌KATO III细胞、人早幼粒细胞白血病HL60细胞和人单核细胞U937细胞中的鞘氨醇水平分别为每10⁶个细胞26.6±2.2、6.3±0.4和6.8±0.6皮摩尔。我们的新方法能够对生物样品脂质提取物中低皮摩尔范围内的鞘氨醇水平进行定量。
