Kai M, Ishida J, Ohkura Y
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
J Chromatogr. 1988 Sep 9;430(2):271-8. doi: 10.1016/s0378-4347(00)83162-x.
Fluorimetrically reactive leucine-enkephalin, one of the opioid peptides in rat brain tissues such as the striatum, cortex and hypothalamus, was assayed by reversed-phase high-performance liquid chromatography with fluorescence detection based on pre-column derivatization of the tyrosyl residue. The tyrosine-containing peptides extracted from the tissue were first formylated with chloroform in an alkaline medium, and the resulting aldehydes were then converted into highly fluorescent derivatives by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative of leucine-enkephalin-like peptide in tissue was separated from various other fluorescent compounds on a reversed-phase column (TSK gel ODS-120T) by isocratic elution and detected by fluorimetry. The concentrations of the leucine-enkephalin-like peptide in the tissues were 20-245 pmol/g. The method is sensitive enough to permit the quantitative determination of the endogenous peptide at concentrations as low as 5.6 pmol/g in brain tissues.
采用基于酪氨酸残基柱前衍生化的反相高效液相色谱荧光检测法，对大鼠脑组织如纹状体、皮质和下丘脑等中的阿片肽之一——荧光反应性亮氨酸脑啡肽进行了测定。从组织中提取的含酪氨酸肽首先在碱性介质中用氯仿进行甲酰化，然后通过与1,2 - 二氨基 - 4,5 - 二甲氧基苯反应将生成的醛转化为高荧光衍生物。组织中亮氨酸脑啡肽样肽的衍生物通过等度洗脱在反相柱（TSK凝胶ODS - 120T）上与各种其他荧光化合物分离，并通过荧光法进行检测。组织中亮氨酸脑啡肽样肽的浓度为20 - 245 pmol/g。该方法灵敏度足够高，能够定量测定脑组织中低至5.6 pmol/g浓度的内源性肽。
