Thin Layer Chromatography - Determination of the Platelet-Activating Factor and other Phopholipids in Human Tears 3


The volume of each enzyme solution used was equivalent to 2 ml/100 cm2 of plate.


Figure 25. A Typical Chromatogram of the Phospholipids in Human Tears


The two enzymatic reactions were allowed to take place for 10 and 15 min respectively at 45˚C. The plates were then sprayed with the Mo-MG reagent (200 ml of 38 mmol/l ammonium molybdate, 100 ml of 8.7 mol/l sulfuric acid and 200 ml of 3.2 mmol/l of malachite green and 500 ml of water) at a coverage of 2 ml/100 cm2 of plate. The phosphates from the phospholipids reacted with the reagent to produce the intense blue-green spot of molybdophosphate malachite green. After 5 min. the plates were scanned at 620 nm with a CS-9000 two-dimensional densitometer (Schimadzu, Kyoto, Japan). A trace of the absorbance profiles (the chromatogram) is shown in figure 25. The quantitative assessment was carried out using peak area measurements in conjunction with calibration curves from standard phospholipid solutions. The peak area was found to be linearly related to concentrations over the range of 2-100 pmol per spot (RSD was 2%, n=7) About 86% of the PAF was recovered. The proportion of the individual phospholipids appeared to be sensibly constant for healthy humans and PAF, LysoPC, PC and PE were present at 26.2, 42.3, 10 and 19.7% respectively.