Vitamin B12 and folate activity in normal human platelets.

Preparation of platelets. Venous blood from normal male and female subjects, ages 20-40, was mixed (9: 1) with 1 per cent EDTA in 0.7 per cent saline in siliconized tubes and centrifuged at 2500 rpm in a PR-2 centrifuge for 3 minutes to obtain platelet-rich plasma (PRP). Subsequent centrifugations were at 3000 rpm and 4 C. for 30 minutes. A platelet button was obtained by centrifuging the PRP, and the platelets were washed 7 times with one-half the plasma volume of cold .05M imidazole-buffered isotonic saline, pH 7.3, containing 0.1 per cent EDTA, in siliconized, graduated McNaught tubes. Where necessary, contaminating red cells were removed during the washings. Vitamin B12 studies. The washed, packed platelets from each of 16 normal subjects were suspended in isotonic saline in a final concentration of 1 per cent (V/V) and the platelets counted by phase microscopy. The suspension contained no leukocytes and 0-30 red cells per 10 platelets. In order to extract vitamin B12 in a soluble form suitable for microbiological assay, the platelets were disrupted by ultra-sonic vibration. This was achieved by sonicating 2 ml. of the platelet suspension, immersed in ice, for 5 minutes at maximal amplitude.0 After this procedure, no platelets or platelet fragments were seen by phase microscopy. The sonicate was then centrifuged at 24,000 g for 30 minutes at 4 C. and the supernatant removed and stored for 2-10 days at -20 C. Vitamin B12 activity in the supernatant was assayed microbiologically, in a 1: 10 flask dilution, using Euglena gracilis as the test organism1 and the B12 activity in platelets expressed as either pg per 108 cells or pg per ml. of packed platelets. Folate. Washed, packed platelets were suspended, in a 1 per cent V/V concentration, in 0.05 M phosphate buffer, pH 6.2, containing 0.5 per cent ascorbate. The suspensions were sonicated, centrifuged and the supernatants stored as described for B,2 assay. Folate