224 P. SOUPART 
3) Dispersion of the cell sediment, breaking thoroughly the buffy coat, in an equal 
volume of a buffered solution at pH 7.3* containing 2% polyvinylpyrrolidone 
(Société parisienne d’expansion chimique, Paris, France). Spontaneous sedimentation 
of the erythrocytes for 60 min, the centrifuge tube being placed at 45° angle in a 
37° water bath. Collection of the supernatant, which contains leucocytes and platelets, 
and storage in a plastic centrifuge tube in the ice box at 4°. This spontaneous sedi- 
mentation is repeated twice, all supernatants being poured together. 
4) Two washings of the erythrocytes with saline and sampling of two aliquots of 
packed red cells, respectively of 4 and 8 g, which are then lysed by 6 and 12 ml 
of distilled water. Each of the lysates is deproteinized by 5 vol. of 1% picric acid 
solution by the method used for plasma. 
5) Separation of the leukocytes from the platelets in the polyvinylpyrrolidone 
supernatant by means of repeated centrifugations of short duration: 1.5 min at less 
than 500 x g in the refrigerated centrifuge at 4°. This procedure, which may be 
repeated three to four times, is controlled by checking the supernatant and sediment 
contents by examination in phase microscopy after each step. Only the first and 
second sediments, which contain mainly leucocytes but a very few platelets and red 
cells, are collected and quantitatively washed in another centrifuge tube, previously 
weighed, with pH 7.3 buffered isotonic solution without polyvinylpyrrolidone. The 
washing of the leucocytes sediment is repeated twice, centrifuging each time for 
2.5 min at less than 500 x g at 4°. The supernatant is then poured off, drained 
thoroughly with filter paper and the weight of the wet sediment is recorded. The 
cells are lysed in 5 ml of distilled water, the solution is quantitatively transferred 
in a medium-size tissue grinder and the final volume made up to ro ml with the 
washings. The solution is then ground to insure complete rupture of the cells, and 
deproteinized by 5 vol. of 1° picric acid solution. After centrifugation, the super- 
natant is deep-frozen pending desalting and analysis. 
6) The supernatant obtained in 5), which contains platelets is centrifuged at 
5000 < g for 30min at 4° in a plastic centrifuge tube previously weighed. The 
sediment is washed twice in the same way in pH 7.3 isotonic buffered solution 
without polyvinylpyrrolidone, and the last sediment is processed the same way 
as the leucocyte sediment. 
After desalting to remove picric acid’, all deproteinized extracts are concen- 
trated under reduced pressure in a Craig rotary evaporator!’ to a volume of approx. 
1 ml. The extracts of the 4 and 8 g of red cells are brought to pH 2.0 and quanti- 
tatively transfered to 150 and 50-cm ion-exchange columns, respectively. As glu- 
tathione is eluted in the range just in between those of threonine-glutamine and 
glutamic acid an alternative procedure is to transform it into the glutathione S- 
sulfonate, by means of treatment by 0.5 M sodium sulfite solution as advised by 
MoorE, SPACKMAN AND STEIN!®, This substance will be eluted with the front of 
the eluent and will appear at the very beginning of the elution curve, before glycero- 
phosphoethanolamine (GPE). Owing to the amount of glutathione usually present 
in 4 g of packed red cells, 1.5 ml of the 0.5 M solution of sodium sulfite is sufficient 
for complete transformation into glutathione S-sulfonate under conditions des- 
* Buffered isotonic solution at pH 7.3. NaCl, 7.65 g; KCl, 0.20g; sodium acetate, 1.50 g; 
NaH,PO,, 0.05 g; KH,PO,, 0.10 g; NaHCOg, 0.70 g; dextrose, 1.00 g; distilled water tooo ml. 
References p. 261/262 
