Ohyama T, Matsubara C, Takamura K
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan.
Analyst. 1996 Dec;121(12):1943-7. doi: 10.1039/an9962101943.
A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform-methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane-diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform-methanol-water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue-green spots of molybdophosphate-Malachite Green aggregate on a yellow-brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2-100 pmol per spot, the RSD being 2% (n = 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.
本文报道了一种灵敏的薄层层析（TLC）方法，用于测定人泪液中的血小板活化因子（PAF）和其他磷脂。将吸附在滤纸上的泪液样品用氯仿 - 甲醇进行硅藻土柱萃取。将含有磷脂的洗脱液点样于硅胶板上。先用正己烷 - 乙醚（4 + 1 v/v）预展开除去非磷脂类脂质后，再用氯仿 - 甲醇 - 水（65 + 35 + 7 v/v）展开进行各磷脂的分离。在45℃下将磷脂酶C溶液和碱性磷酸酶溶液喷于TLC板上，使各磷脂释放出磷酸根。通过喷洒钼酸铵和孔雀石绿的混合溶液，释放出的磷酸根在黄棕色背景上呈现为磷钼酸 - 孔雀石绿聚集体的蓝绿色斑点。用密度计在620 nm处测量板上各斑点的吸光度。发现斑点的峰面积与每个斑点中PAF含量在2 - 100 pmol范围内呈线性关系，相对标准偏差（RSD）为2%（n = 7）。添加到泪液中的标准PAF回收率约为86%。用该方法还可高灵敏度地检测出人泪液中的溶血磷脂酰胆碱（lysoPC）、磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）。健康人泪液中各磷脂含量几乎恒定；PAF、lysoPC、PC和PE分别占26.2%、42.3%、10.0%和19.7%。
