Shinozaki K, Kawasaki T, Kambayashi J, Sakon M, Shiba E, Uemura Y, Ou M, Iwamoto N, Mori T
Department of Surgery II, Osaka University Medical School, Japan.
Life Sci. 1994;54(6):429-37. doi: 10.1016/0024-3205(94)00701-2.
There is no satisfactory assay procedure of PAF in human whole blood in terms of sensitivity, reproducibility and simplicity. This is due to coexisting lipids from plasma and cellular membranes which inhibit measurement of PAF in various assay procedures, including bioassay. In the present study, an attempt was made to eliminate these interfering lipid inhibitors from blood samples. Lipids in human whole blood were extracted according to the method of Bligh & Dyer and the organic layer was dried under a stream of nitrogen. Then, the dried organic layer was dissolved in diethyl-ether and the solution was kept at -20 degrees C which was then centrifuged. The resulting supernatant was then applied to an anion-exchange column and the PAF fraction was obtained by step-wise gradient elution. The fraction was further purified by normal phase HPLC. Then PAF in the final sample was determined by sensitive bioassay using rabbit platelets containing fibrinogen and epinephrine. The recovery rate of PAF throughout this procedure was constant and satisfactory (37.4 +/- 9.7%), which was confirmed using [3H]-PAF. The lower limit of the present assay was estimated to be 5pg in 1 ml of blood and it was sensitive enough to detect PAF in blood samples from healthy volunteers and patients with sepsis or liver cirrhosis. Furthermore, attempts were made to compare the sensitivity and the recovery of our method with these of a commercially available radioimmunoassay (RIA) kit for PAF. However, it was not possible to detect any amount of authentic PAF added to whole blood.
就敏感性、可重复性和简便性而言，目前尚无令人满意的人全血中血小板活化因子（PAF）的检测方法。这是由于血浆和细胞膜中存在的共存脂质会在包括生物测定在内的各种检测方法中抑制PAF的测量。在本研究中，试图从血液样本中消除这些干扰性脂质抑制剂。按照布莱和戴尔的方法提取人全血中的脂质，有机层在氮气流下干燥。然后，将干燥的有机层溶解在乙醚中，溶液保存在-20℃，随后进行离心。所得上清液应用于阴离子交换柱，通过逐步梯度洗脱获得PAF馏分。该馏分通过正相高效液相色谱进一步纯化。然后，使用含有纤维蛋白原和肾上腺素的兔血小板通过灵敏的生物测定法测定最终样品中的PAF。使用[3H]-PAF证实，整个过程中PAF的回收率恒定且令人满意（37.4±9.7%）。本检测方法的下限估计为每1毫升血液中5皮克，灵敏度足以检测健康志愿者以及脓毒症或肝硬化患者血液样本中的PAF。此外，还尝试将我们方法的灵敏度和回收率与市售的PAF放射免疫分析（RIA）试剂盒的灵敏度和回收率进行比较。然而，无法检测到添加到全血中的任何量的纯PAF。
