Thin Layer Chromatography - Determination of the Platelet-Activating Factor and other Phopholipids in Human Tears 1

 

Before the analytical development was commenced, a further clean up was carried out on the plate by developing the plate in 20 ml of diethyl ether for 17 min in a continuous horizontal tank (the eluent reached the top of the plate in about 3 min). Development was continued for about 14 min., and, as a consequence, there was no solvent front. The plate was dried in a darkened chamber for 3 min in a stream of nitrogen. The interfering substances were contained at the plate edge and were removed by scraping 2 cm of silica from the edge. The plate was then rotated and developed in a normal tank and developed for 20 min with 20 ml of chloroform-xylene-acetone mixture (6:3:1). The plate was again dried for 5 min and the bottom portion (1 cm) of the silica was removed to decrease the development time and increase the resolution. The plate development was then continued with the same solvent mixture for 16 min. The plate was finally dried in a fan-assisted oven at 100˚C and scanned for fluorescence by the reflectance mode using the TLC II scanner (Camag 76610) and the SP 4270 integrator (Camag 76650).

 

The procedure appears complicated and is certainly lengthy for this type of analysis, but recoveries in excess of 90% were readily obtained and detection limits for the aflatoxins AFB1, AFB2, AFG1 and AFG2 were found to be 3.7, 2.5, 3.0, and 1.3 mg/kg respectively. Compared with the British Standard Method, the procedure was found to be both more efficient and more precise.

 

Determination of the Platelet-Activating Factor and other Phopholipids in Human Tears

 

Phospholipids are essential components of biological systems and the platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is particularly important as it is an immunization agent for a range of different diseases. PAF is difficult to assay in serum and other biological fluids as, following its secretion, it is immediately hydrolyzed by acetylhydrolase to form 1-hexade-canoyl-sn-glycero-3-phosphocholine (LysoPAF). In tears, however, acetylhydrolase is possibly absent or at least present at very low levels as in saliva, nevertheless there are also very small amounts of phospholipids present that offer the possibility of determining PAF in such samples. However, due to the very low concentrations present, a concentrating technique is necessary to provide sufficient material for analysis. Ohyama et al. (14) developed a satisfactory analytical method employing a simple extraction procedure followed by a TLC separation and a plate scanning 'finish'. The inorganic phosphate was liberated from the phospholipid molecules by an enzyme reaction that was followed by a high sensitivity color reaction that produced an ammonium molybdate-malachite green aggregate.