1. The uptake of 14C from [methyl-14C]methyltetrahydrofolate was significantly reduced in the phytohaemagglutinin (PHA)-stimulated lymphocytes from nine patients with untreated pernicious anaemia compared with the uptake in seven normal subjects.2. The uptake of I4C from [14C]methyltetrahydrofolate by the lymphocytes from seven of the patients with pernicious anaemia was consistently increased by addition of vitamin B,, in vitro.3. The proportion of 14C taken up from ['4C]methyltetrahydrofolate transferred to non-folate compounds was found to be significantly reduced in the PHA-stimulated lymphocytes from nine patients with untreated pernicious anaemia compared with the proportion transferred in the PHA-stimulated lymphocytes from seven normal subjects. Addition of vitamin BIZ in vitro consistently increased the transfer in vitamin B,,-deficient cells but had no consistent effect in normal cells. Normal and vitamin B,,-deficient PHA-stimulated lymphocytes took up[3H]folic acid and after 72 h incubation converted this largely into pteroylpolyglutamate forms.5. The proportion of labelled lymphocyte folate as pteroylpolyglutamate after incubation with [3H]folic acid was the same in vitamin B,,-deficient as in normal lymphocytes and the proportion of pteroylpolyglutamates formed in vitamin BIZdeficient lymphocytes was unaffected by addition of vitamin B,, in uitro. 6. No radioactivity could be decteted in pteroylpolyglutamates after incubating normal PHA-stimulated lymphocytes with [14C]methyltetrahydrofolate for 72 h, suggesting that pteroylpolyglutamate forms of folate cannot be made directly from methyltetrahydrofolate. 7. These results are consistent with the 'methyltetrahydrofolate trap' hypothesis Correspondence: Professor A. V. Hoffbrand, Department of Haematology, The Royal Free Hospital, Pond Street, Hampstead, London NW3 2QG. 617

No abstract

It has been established that three binders of vitamin B,, can be separated by DEAEcellulose column chromatography following addition of labelled vitamin to normal serum (1-4). All 3 binders are present in trace amounts. They are known as transcobalamin I (TC I ) , transcobalamin I1 (TC 11), and transcobalamin I11 (TC 111) or "main protein peak binder" (MPPB) . TC I1 and TC I11 have an electrophoretic mobility of a-globulin, while TC I has the mobility of ,&globulin (1, 2).Labelled vitamin B,, is cleared rapidly from intravenously administered TC I1 5 7 C~ B,, but much more slowly from TC I 5 7 C~ B,, (2,5,6). The differences in clearance are less marked, however, when the transcobalamins are labelled with orally administered labelled vitamin BI2. The rapid clearance of 57C0 B12 from intravenously administered B,,--TC I1 has been attributed to partial denaturation of the protein during its purification in vitro (7). Finkler and Hall (8) reported that HeLa cells take up TO B,, from TC I1 in vitro but not from TC I while Retief et al. (9) observed that TC I1 delivers vitamin B,z to reticulocytes at a faster rate than TC I. Uptake by perfused rat liver of labelled vitamin B,, from 5 7 C~ B,, labelled TC I1 and TC I11 from serum of normal donors and pernicious anemia patients was substantially greater than from 5 7 C~ B,, labelled TC I (10). The exact role of TC 111 vita-1In receipt of a grant from the Leukemia Research Fund.747 min B,, in plasma is still uncertain. Chanarin and co-workers have recently shown that following oral administration of 5 7 C~ BIZ, the labelled vitamin is taken up simultaneously by all 3 serum binders (7, 11). These authors also showed immunological identity of TC I and TC 111. However, the ability of TC I11 to deliver vitamin B,, to cells is still unknown.H o a r a n d et al. (12) used phytohemagglutinin (PHA) -transformed lymphocytes as a model cell system to investigate uptake of serum bound radioactive vitamin B,, by normal proliferating human hemopoietic cells. In the present study we have used this system to compare the ability of human TC I11 with that of TC I and TC I1 to deliver labelled vitamin B,, to human cells. Materials and Methods. Preparation of vitamin B,, binders.This has been reported in detail elsewhere (13). Thirty ml of normal serum were used in each column chromatographic separation, Serum used in each experiment was drawn from an individual donor considered to be hematologically normal. Five hundred pg of 5 7 C~ B,, (specific activity 120 mCi/mg; purchased from the Radiochemical Centre, Amersham, Amersham, England) were added per ml to 30 ml of serum. The solution was allowed to stand at 37" for 20 min, then dialysed at 4" against 4 litres of 0.0175 M sodium phosphate buffer, pH 6.3, for 48 hr. The buffer was changed twice during this period.DEAE-cellulose ( Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 and 0.95 mEq/g) was packed after

Summary Rice et al. (1986) have described a flow cytometric method where the non-fluorescent probe monochlorobimane (mBCl) forms a fluorescent adduct with cellular glutathione (GSH) under the action of glutathione-S-transferase. We show here that for EMT6 carcinosarcoma cells there is a close correlation between mean cell fluorescence, expressed as a ratio to that of fluorescence calibration beads, and biochemically determined GSH content over the range 0.2 -2.0 fmol cell' . Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCI and examined by flow cytometry. There was a wide range in individual cell fluorescence, which in contrast to EMT6 cells was not strongly correlated with (Ozols et al., 1987;Lee et al., 1987;Skapek et al., 1988 Clinical samples A total of 14 clinical samples were examined. Seven of these were obtained by 23 gauge needle aspiration from patients who gave verbal informed consent for the procedure, one consisted of peripheral blood lymphoblasts separated by density-gradient centrifugation from a patient with end-stage T-cell acute lymphoblastic leukaemia, and the remainder were fresh surgical specimens which were mechanically disaggregated using crossed scalpel blades. Cell suspensions were filtered through gauze, washed and counted. If necessary, clumps were dispersed by syringing through 25 gauge needles. After adjusting to a final concentration of I x 106ml-', cells were filtered through 70 tm plankton netting and stained using 40 JiM mBCI for exactly 5 min at 2O°C. Flow cytometry was done exactly as for cultured cells, except that the machine was triggered on Coulter volume, set at a threshold level of 300 jtm3 to exclude red blood cells and cellular debris. In addition to fluorescence, the ratio of cell fluorescence to Coulter volume was obtained using the electronics of the FACS Analyser. Results Monochlorobimane staining of EMT6 CellsThe effects of mBCI concentration and time on the staining intensity of EMT6 cells are shown in Figure 1

No abstract