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`
`Synthesis of Cobalamin Coenzymes by
`Human Lymphocytes In Vitro and the Effect of
`Folates and Metabolic Inhibitors
`
`By E. V. Quadros, D. M. Matthews, A. V. Hoffbrand, and J. C. Linnell
`
`The uptake of 57Co-cyanocobalamin (CN(cid:173)
`Cbl) and its conversion to S-deoxyadeno(cid:173)
`sylcobalamin
`(Ado-Cbl), methylcobala(cid:173)
`min
`(Me-Cbl), and hydroxocobalamin
`(OH-Cbl) has been studied
`in phyto(cid:173)
`hemagglutinin (PHA)-transformed
`lym(cid:173)
`phocytes from normal subjects and pa(cid:173)
`tients with pernicious anemia. Uptake and
`conversion were much greater by PHA(cid:173)
`stimulated lymphocytes than by mature
`non-transformed lymphocytes. In normal
`cells, uptake of 57Co_CN_Cbl and synthesis
`of the cobalamin coenzymes were approxi(cid:173)
`mately linear between 3 and 48 hr incuba(cid:173)
`tion. Ado-Cbl was the major cobalamin
`
`formed, and after 72 hr the cells contained
`about twice as much Ado-Cbl as Me-Cbl.
`Uptake by
`lymphocytes from patients
`with untreated pernicious anemia (PA)
`was greater than that by normal lympho(cid:173)
`cytes, but the proportions of Ado-Cbl
`and Me-Cbl synthesized by each were
`similar. Folic acid and methyltetrahydro(cid:173)
`folate enhanced synthesis of Me-Cbl both
`in normal and in PA cells, while metho(cid:173)
`trexate and S-fluorouracil depressed it.
`This depression was overcome by 5-
`formyltetrahydrofolate, suggesting that an
`uninterrupted folate cycle may play an
`important role in Me-Cbl synthesis.
`
`M AMMALIAN CELLS already used in studies of vitamin B12 metabolism
`
`include HeLa cells, Ehrlich ascites carcinoma cells, kidney cells, skin
`fibroblasts, and various hemopoietic cells, and all have been shown to take up
`cobalamins in vitro. The immature dividing cells of bone marrow take up
`cyanocobalamin (CN-Cbl) by an active, calcium-dependent process, requiring
`cellular respiration, oxidative phosphorylation, and the presence of free sulf(cid:173)
`hydryl groups,' whereas in erythrocytes and reticulocytes uptake is mainly by
`energy-independent adsorption at the cell surface.2 In lymphocytes, uptake of
`CN-Cbl is greater in phytohemagglutinin (PHA)-transformed cells than in
`mature, nontransformed cells.3 Lymphocytes stimulated with PHA show in(cid:173)
`-7 which precede blastogenesis
`creases in RNA, DNA, and protein synthesis4
`and mitosis.8.9 These changes occur in lymphocytes from normal subjects and
`also in lymphocytes from patients with megaloblastic anemia due to B12 or
`folate deficiency, producing cells which are very similar to mega lob lasts. This
`finding suggests that PH A-transformed lymphocytes provide an excellent model
`for studies of cobalamin metabolism in proliferating human cells.
`Synthesis of cobalamin coenzymes has been studied in HeLa cells10 and in
`human fibroblasts,'I.28 but not, so far as we are aware, in any hemopoietic cell
`in vitro. We have estimated the uptake of 57Co-CN-Cbl and measured the
`
`From the Department of Experimental Chemical Pathology. Vincent Square Laboratories of
`Westminster Hospital. and the Department of Haematology. Royal Free Hospital. London. England.
`Submitted April 19.1976: accepted June 21. 1976.
`Supported by the WeI/come Trust and an award to E. V.Q. from the Ministry of Overseas Develop(cid:173)
`ment.
`Address for reprint requests: Dr. J. C. Linnell. Department of Experimental Chemical Pathology.
`Vincent Square Laboratories. 124 Vauxhall Bridge Road. London SWI V 2RH. England.
`© 1976 by Grune & Stratton. Inc.
`
`Blood, Vol. 48, No.4 (Odober), 1976
`
`609
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`
`610
`
`QUADROS ET AL.
`
`cellular synthesis of 51Co-Me-Cbl and 51Co-Ado-Cbl in PHA-transformed
`lymphocytes from healthy normal subjects and patients with pernicious anemia.
`Since cobalamins have been implicated in both folate metabolism and DNA
`synthesis, we have also studied the effects of various folates, antifolate com(cid:173)
`pounds, and other metabolic inhibitors on the synthesis of the cobalamin co(cid:173)
`enzymes. An abstract of part of these studies has been publishedY
`
`MATERIALS AND METHODS
`
`Heparinized venous blood (50 ·60 ml) was taken from healthy adult volunteers and from pa(cid:173)
`tients in whom a diagnosis of untreated pernicious anemia (PA) had been established on the
`basis of megaloblastic anemia, low serum vitamin B\2 levels, normal serum folate levels, and
`malabsorption of radioactive BI2 corrected by intrinsic factor. Lymphocytes were separated on a
`Triosil-Ficoll gradient13 and cultures set up as previously described!4 The lymphocytes were
`suspended in TC 199 medium (Wellcome) containing 200 III homologous plasma and II III phyto(cid:173)
`hemagglutinin (PHA-Wellcome) per I x 106 cells, in a total volume of I ml. Three milliliters
`of the cell suspension was dispensed into a sterile 5 ml Bijou bottle. 51Co_CN_Cbl (specific
`activity 100-150 IlCi//lg) was diluted to 20 nglml, and 100 III added per 3 x 106 cells. Cells were
`then incubated in darkness for periods between! and 72 hr at 37°C.
`51Co-CN-Cbl and folate analogues or metabolic inhibitors were added to further aliquots of
`cells and incubated in darkness for 72 hr at 37"C. Concentrations were as follows: pteroylglu(cid:173)
`tamic acid (Sigma, PteGlu); 10-5 M; DL-N 5methyltetrahydrofolic acid, barium salt (Sigma,
`methyl H 4PteGlu), 2 x 10- 5 M; calcium leucovorin (Lederle, formyl H 4 PteGlu), 10- 5 M;
`methotrexate (Lederle, MTX), 10-5 M; 5-fluorouracil (Roche, 5-FU), 10- 5 M; hydroxyurea
`(Squibb, HU), 10- 3 M. After incubation, the cells were centrifuged down, washed three times
`with approximately 6 ml normal saline, then resuspended in I ml glass-distilled water and stored
`at -20°C.
`Total cell uptake of 51 Co-CN-Cbl was estimated by measuring the activity of each sample
`(foil-wrapped to prevent photolytic loss of cobalamin coenzymes) in an aUlO-"),-spectrometer
`(Packard). Cobalamins were then extracted with hot ethanol and separated by two-dimensional
`chromatography and bioautography as previously described!5.16 The extracts applied to each
`thin-layer plate were overspotted with markers (50 pg each of Me-Cbl, CN-Cbl, Ado-Cbl, and
`OH-Cbl), since the Escherichia coli mutant used for the later bioautographic location of the
`separated zones was not sufficiently sensitive to respond to the very small quantities of each
`labeled cobalamin present in the cell extracts. Bioautogram zones were then excised, and the radio(cid:173)
`activity in each was measured in an auto-"),-spectrometer. The amounts of Ado-Cbl, Me-Cbl, and
`OH-Cbl formed by the lymphocytes were calculated from the total cell uptake, and the proportion
`of radioactivity recovered in each bioautogram zone. Initially, the whole bioautogram was divided
`into small areas, and the radioactivity in each area was counted; 87°;, 94°0 of the radioactivity ap(cid:173)
`plied to the chromatogram was located in zones corresponding to the four cobalamins. while
`radioactivity recovered at the origin ranged from 0.5"'0-1.9''0.
`
`RESULTS
`
`Effects of PH A Stimulation
`A comparison of the total uptake of 51Co-CN-Cbl and synthesis of the co(cid:173)
`enzymes by lymphocytes with and without PHA stimulation is shown in
`Table I. Total uptake of radioactivity after 72-hr incubation was much higher
`in cells stimulated with PHA, and synthesis of Ado-Cbl and Me-Cbl was ap(cid:173)
`proximately five times as great as that in untreated cells from the same sub(cid:173)
`ject. In unstimulated lymphocytes, there was very little conversion of CN-Cbl
`to other cobalamins. More than 80~~ of the radioactivity remained as CN-Cbl
`after 72-hr incubation, similar to the proportion recovered as CN-Cbl from
`chromatograms of aqueous 51Co-CN-Cbl (Table I). Stimulating "normal"
`
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`
`SYNTHESIS OF COBALAMIN COENZYMES
`
`611
`
`Table 1. Effect of PHA Stimulation on Uptake and Conversion of 57 Co-CN-Cbl
`After 72 hr by Lymphocytes From a Normal Subject
`
`Chromatogram
`Zone
`
`PHA·stimulated
`Cells
`
`Unstimulated
`Cells
`
`Aqueous
`S7 CO CN.Cbl-
`
`Total Radioactivity Recovered From Chromatogram (0/0)
`
`Me·Cbl
`CN-Cbl
`Ado-Cbl
`OH-Cbl
`Origin
`
`Total uptake
`(pg/l 0 6 cells)
`Range in 15 nor-
`mal subjects
`
`21.4
`37.1
`26.6
`13.7
`1.2
`
`3.1
`
`2.2-9.4
`
`* As added to lymphocyte cultures.
`
`3.9
`82.8
`5.8
`6.5
`1.0
`
`1.8
`
`0 .8
`87.1
`3.5
`3.7
`4.9
`
`lymphocytes with PHA for 18 or 69 hr before adding 57Co-CN-Cbl appeared
`to decrease rather than to increase CN-Cbl uptake and conversion. By com(cid:173)
`parison with cells to which PHA was added only half an hour before addition
`of 57Co-CN-Cbl, total uptake was halved, and the proportion converted to
`Ado-Cbl and Me-Cbl was slightly reduced (Table 2).
`Uptake of radioactivity by PHA-transformed cells from normal subjects was
`initially rapid, and approximately a quarter of the final uptake at 72 hr oc(cid:173)
`curred within the first 3 hr (Fig. 1). Between 3 and 48 hr the increase was
`approximately linear, but thereafter uptake virtually ceased. Chromatography
`revealed that during the first 3 hr unchanged CN-Cbl accounted for much of
`the uptake of radioactivity (Fig. 1). Between 3 and 72 hr the cellular concen(cid:173)
`tration of this cobalamin altered little, the increase in radioactivity corre(cid:173)
`sponding to increasing amounts of newly formed cobalamins_
`
`Synthesis of the Cobalamin Coenzymes
`The results of estimating 57Co-labeled Ado-Cbl, Me-Cbl, and OH-Cbl
`in lymphocytes following incubation with 57Co-CN-Cbl for 0.5- 72 hr are
`shown in Fig. 2. For much of this time, the rates of interconversion were ap(cid:173)
`proximately linear, the proportion of CN-Cbl falling as that of the other
`cobalamins increased. There was virtually no Me-Cbl, Ado-Cbl, or OH-Cbl
`formed during the first half hour, since the values obtained for each cobalamin
`
`Table 2. Effect of PHA Stimulation for!. 18. or 69 hr Prior to Uptake
`and Conversion of 57 Co-CN-Cbl by Lymphocytes From Normal Subjects
`
`PHA
`Stimulation (hr)*
`
`Tota~Uptake/
`10 Cellst
`
`1
`"2
`18
`69
`
`+77.8
`+35.7
`+35.9
`
`Total Radioactivity Recovered (%)
`
`Me-Cbl
`
`CN-Cbl
`
`Ado-Cbl
`
`1.9 ± 0.7t
`1.5
`1.0
`
`80 ± 4.9
`90
`84
`
`6.6 ± 1.7
`4.6
`3.7
`
`OH-Cbl
`
`11 ± 3.4
`3.7
`11
`
`* Delay between stimulation with PHA and addition of 57 Co CN-Cbl.
`tPer cent increase in total 57 Co CN-Cbl uptake between ~ and 3 hr.
`tMean ± SEM (n = 4) .
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`
`612
`
`QUADROS ET Al.
`
`.12
`
`·n
`
`I~ .
`t
`
`.0'
`
`22
`
`Hour. of incubation
`
`••
`
`n
`
`Fig. 1. Total uptake and convenion of 57Co-CN_Cbl between 0.5 and 72 hr by PHA-trans(cid:173)
`formed lymphocytes from normal subjects. The mean ± SEM is shown at each point. The number
`of experiments is indicated in parentheses. -e-, total uptake of radioactivity; - x -, radioactiv(cid:173)
`ity recovered as CN-Cbl; -0 - , 57 Co-CN-Cbl converted to other cobalamins.
`
`were almost identical to those from chromatograms of the aqueous 57Co-CN(cid:173)
`Cbl initially added to the cell cultures. During the early stages of incubation,
`the formation of OH-Cbl was rapid, and after 3 hr, its proportion was ap(cid:173)
`proximately five times that of Me-Cbl, and almost twice that of Ado-Cbl. After
`48 hr, Ado-Cbl was the major cobalamin synthesized (mean 31 ~o), though OH(cid:173)
`Cbl and CN-Cbl accounted for approximately a quarter of the total labeled
`cobalamin. Between 48 and 72 hr, the proportions of Ado-Cbl and CN-Cbl
`remained virtually unchanged, but Me-Cbl synthesis continued, apparently at
`the expense of OH-Cbl so that after 72 hr the cells contained almost as much
`Me-Cbl (mean 18%) as OH-Cbl.
`The total uptake of 57Co-CN-Cbl after 72 hr incubation was almost twice as
`high in cells from untreated PA patients (0.2 ± SEM 0.02% of the added radio(cid:173)
`activity) as that in cells from normal subjects (0.1 ± 0.01%) (Table 3). Though
`the proportion of each labeled cobalamin was very similar in PA cells to that
`in normal cells, the actual amounts of Me-Cbl, Ado-Cbl, and OH-Cbl formed
`were higher in PA cells, probably as a result of the greater total uptake of
`57Co-CN_Cbl.
`
`Effects of Folate Analogues and Antimetabolites
`MTX is known to inhibit dihydrofolate reductase and thereby depletes the
`supply of reduced folates. 5-FU inhibits thymidylate synthetase directly, which
`leads to a reduction in DNA synthesis. Indirectly, it will interrupt cycling of
`
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`
`SYNTHESIS OF COBALAMIN COENZYMES
`
`613
`
`100 6)
`
`(4)
`
`60 l~~
`
`0
`
`---c
`'1
`u
`#
`
`II
`.:It
`II
`Q.
`:0
`
`it .. o .. ... o
`o .. .. o
`o .. Co
`
`c
`
`Co
`
`20
`
`(9)
`T
`
`,.
`0------: ,..
`::r----~' 1
`T
`::
`;:
`i
`
`• .!.
`
`•
`
`~ 3
`
`22
`
`48
`
`72
`
`hours of incubation
`
`Fig. 2. Formation of 57Co-labeled Ado-Cbl (-o-), Me-Cbl (-o-), and OH-Cbl (-e-I from
`57Co-CN_Cbl (-... -1 in PHA-transformed lymphocytes from normal subjects (mean ± SEMI. The
`number of experiments is indicated in parentheses.
`
`the folate coenzymes. We attempted to assess the influence of these inhibitors
`and folates on cobalamin coenzyme synthesis.
`The effect of MTX on the uptake and conversion of 57Co-CN-Cbl in cells
`from normal subjects is shown in Table 4. Me-Cbl synthesis was significantly
`reduced by more than a third, but at the concentration used (10- 5 M), MTX
`had no significant effect either on total uptake or on formation of s7Co_OH_
`
`Table 3. Uptake and Conversion of 57 Co-CN-Cbl After 72 hr by PHA-transformed
`lymphocytes From Normal Subjects and Patients With Pernicious Anemia
`Labeled Cobalamin Formed/l 06 Cells
`Ado-Cbl
`
`Me-Cbl
`
`OH-Cbl
`
`Subjects
`
`Normal controls
`(n = 15)
`
`PA patients
`(n = 6)
`
`T olal'pplake/
`10 Cells
`(pg)
`
`5.3'
`±0.6
`
`p < 0.01
`
`9.0
`± 1.0
`
`(pg)
`
`1.2
`±0.3
`
`(%)
`
`18.1t
`±2_2
`
`pg
`
`1.6
`±0.3
`
`(%)
`
`27.5
`±2.7
`
`pg
`
`1.2
`±0.2
`
`(%)
`
`19.8
`±2.1
`
`2.0
`±0.4
`
`21.1
`±2.6
`
`2.5
`±0.4
`
`26.5
`±2.0
`
`2.0
`±0.3
`
`22.9
`±2.4
`
`'Mean ± SEM.
`tCalculated from total activity on chromatogram.
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`
`Table 4. Effect of Methotrexate on the Uptake and Conversion of 57 Co-CN-Cbl in
`PH A-transformed Lymphocytes From Nine Normal Subjects
`
`Labeled Cobalamin Formed/lOb Cells
`
`Total.}lptake/
`10 Cells
`(pg)
`
`5.0'
`±0.9
`
`Me·Cbl
`
`(%)
`
`18.9
`±2.8
`
`(pg)
`
`1.0
`±0.2
`
`p < 0.01
`
`p < 0.Q1
`
`Ado·Cbl
`(pg)
`
`(%)
`
`1.5
`±0.3
`
`27.0
`±3.3
`
`OH·Cbl
`
`(pg)
`
`1.1
`±0.3
`
`(%)
`
`18.8
`±2.6
`
`4.6
`±0.9
`
`0.6
`±0.1
`
`12.9
`±1.4
`
`1.5
`±0.4
`
`28.2
`±3.1
`
`1.1
`±0.3
`
`21.7
`±2.3
`
`Methotrexate
`Concentration
`
`0
`
`10- 5 M
`
`'Mean ± SEM.
`
`"
`
`l
`~
`",.
`'"
`~ I
`i
`
`>.
`
`~
`
`~
`
`!i!
`
`~~ l l !
`'"
`~l fi!i
`
`'"
`
`J,-'l
`
`i
`5l-'l
`
`fi!£
`->.
`,H
`
`"
`~
`Ii:
`
`0
`
`200
`
`180 .
`
`160
`
`140
`
`•
`
`00
`
`120
`
`0
`0
`
`0
`
`0
`
`•
`-
`•
`
`•
`• - -
`•
`•
`• 0
`
`• 0
`
`•
`•
`•
`-~.w - . ~ - _ .. - . - I-
`•
`•
`•
`8 • ·0
`•• 0
`•
`•
`
`k
`~
`C
`0
`U
`~ 100
`
`'0
`
`0 .,
`
`~
`.>:
`
`0-
`
`" ~
`"
`" ~
`0 ...
`
`0
`0
`
`.0
`
`80
`
`60
`
`0
`
`40
`
`20
`
`o
`
`Fig. 3. Total uptake of 57 Co-CN-Cbl by PH A-transformed lymphocytes in the presence of folate
`compounds and metabolic inhibitors. e, normal subjects; 0, PA patients.
`
`614
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`
`SYNTHESIS OF COBALAMIN COENZYMES
`
`615
`
`180
`
`160
`
`140
`
`120
`
`• .::
`!: . 0 u
`
`~ >
`"0
`
`~
`100 _Q..-
`
`~
`~ ..
`f
`
`0
`
`i
`
`• 0
`
`0
`
`• 0
`
`~
`
`~
`
`iii
`
`•
`•
`
`•
`•
`-0 - --- - -- - --
`•
`~
`c:e
`~
`
`0
`
`0
`
`•
`
`~
`0
`~
`:;;
`u
`I
`
`~ ..
`
`80
`
`60
`
`40
`
`0
`
`Fig. 4. Effect of folates and meta(cid:173)
`bolic
`inhibitors on
`the formation of
`57 Co-Me-Cbl by PH A-transformed lym(cid:173)
`phocytes. e, normal subjects; 0, PA pa(cid:173)
`tients.
`
`~
`
`Cbl or s7Co-Ado-Cbl. Addition of PteGlu, methyl H4 PteGlu, formyl H4 PteGlu,
`5-FU, HU, and MTX either singly or in combination did not appear to have
`any consistent effect on total cell uptake ofs7Co-CN-Cbl (Fig. 3).
`The effect of folate compounds or metabolic inhibitors on the synthesis of
`Me-Cbl is shown in Fig. 4. Addition of PteGlu or methyl H4 PteGIu increased
`both the proportion and amount of Me-Cbl in cells from normal subjects. In
`lymphocytes from patients with pernicious anemia, PteGlu increased the for(cid:173)
`mation of Me-Cbl in five of six subjects, but in the sixth, Me-Cbl synthesis was
`depressed to 68~%, of the control value. This subject had clinical signs of
`subacute combined degeneration and a lower serum BI2 (20 pg/ml) than any of
`the other patients, a serum folate of 15 ng/ml, and a red cell folate of 99 ng/ml.
`Methyl H4PteGIu apparently stimulated Me-Cbl synthesis in cells from one
`of the two PA patients studied.
`When MTX or 5-FU were included in the culture medium with cells from
`normal subjects, the proportion of s7Co-Me-Cbl fell to about 50o/~ of that
`produced in the absence of these inhibitors. A similar effect was observed in
`cells from patients with pernicious anemia. In contrast, HU did not decrease
`either the proportion or amount of Me-Cbl formed in the two experiments
`with cells from normal SUbjects. Since we found that addition of MTX and
`5-FU reduced Me-Cbl synthesis, we used two folates, methyl H4 PteGIu and
`
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`
`616
`
`QUADROS ET AL.
`
`+80
`
`+60
`
`+40
`
`+20
`
`0
`
`-20
`
`n
`
`L..
`
`-40 .
`
`I-
`
`III'X
`
`III'X
`
`ro
`
`ro
`
`...
`0 ... ...
`" 0 "
`"" " cd
`... .. ., ...
`" ., .,
`... .,
`D .,
`" " ., ... .,
`... ... ....
`..
`
`iO
`
`Q
`
`lID -
`
`Control
`
`fPGA~ Test
`
`(n)
`
`(3)
`
`(1)
`
`III'X
`IIeFGA
`
`(2)
`
`(2)
`
`(2)
`
`Influence of methyl H4 PteGIu (MePGA) and formyl H4 PteGIu (fPGA) on synthesis of
`Fig. 5.
`57 Co-Me-Cbl in the presence of metabolic inhibitors. Hatched bar, proportion of Me-Cbl; open
`bar, amount of Me-Cbl (pg/l 0 6 cells).
`
`formyl H4PteGlu, to see whether either compound could bypass the effect
`of MTX and 5-FU. Figure 5 shows that in cells cultured with MTX and 5-FU,
`methyl H4 PteGIu failed to stimulate Me-Cbl synthesis, but when formyl
`H4 PteGIu was added with either MTX or 5-FU, there was an increase both in
`the proportion and amount of 57Co-Me-Cbl. HU, which inhibits the enzyme
`ribonucleotide reductase and has no direct effect on folate metabolism, failed
`to reduce Me-Cbl synthesis (Fig. 4), and the addition of formyl H4PteGIu did
`not affect its action in either of the cases in which Me-Cbl synthesis was in(cid:173)
`creased (Fig. 5).
`
`DISCUSSION
`PH A-transformed lymphocytes have been shown to take up more CN-Cbl
`than mature nontransformed lymphocytes when incubated for short periods. 3
`The present results indicate that the same occurs when cells are incubated with
`CN-Cbl for much longer periods of up to 72 hr. In addition, we have found that
`mature lymphocytes have very little ability to convert CN-Cbl to OH-Cbl,
`Ado-Cbl or Me-Cbl, unlike PH A-stimulated cells which show active conversion
`of CN-Cbl to the cobalamin coenzymes.
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`SYNTHESIS OF COBALAMIN COENZYMt5
`
`617
`
`In an earlier study, it was observed that perniCIOUS anemia lymphocytes
`which had first been incubated with PHA for 72 hr took up less CN-Cbl in 1 hr
`than did similarly treated lymphocytes from normal subjects. 3 The present
`study showed that when PH A-transformed lymphocytes were incubated with
`CN-Cbl for longer periods of up to 72 hr, uptake by PA cells was not lower but
`significantly higher than by normal cells, as observed in human bone marrow. I
`This difference may be due to the lower cobalamin content of PA cells,15.16 or to
`the greater unsaturation with endogenous cobalamin of the B12-binding pro(cid:173)
`teins in PA plasma, which may therefore transfer more 57Co-CN-Cbl to the cells
`at higher specific activity than is transferred by transcobalamins in normal
`plasma. In all cases, an excess of labeled B12 was added to saturate the trans(cid:173)
`cobalamins in order to ensure maximal uptake of B12 by the lymphocytes in cul(cid:173)
`ture. Since the proportions of each cobalamin in PA lymphocytes were very
`similar to those in normal lymphocytes, it was unlikely that B 12-depletion pro(cid:173)
`duced any significant imbalance of the cobalamin-synthesizing enzyme systems.
`In Ehrlich ascites carcinoma cells 17 and in E. coli cells,18 cobalamin uptake
`in vitro involves at least two independent steps: (I) a primary rapid uptake due
`to adsorption at the cell surface which is complete within seconds or minutes,
`and (2) a slower secondary phase requiring several hours for completion and
`which depends on metabolic energy. Hence it is unlikely that increases in up(cid:173)
`take occurring beyond 30 min are due to adsorption at the cell surface. Our
`results show that between 30 min and 22 hr of incubation, both total cell radio(cid:173)
`activity and CN-Chl concentration increase, suggesting that CN-Cbl is trans(cid:173)
`ported into the cell intact. CN-Cbl has previously been shown to appear in
`blood following oral doses,15 and in breast milk after parenteral administra(cid:173)
`tion, 19 indicating transport of unchanged CN-Cbl. Preliminary results with high
`specific activity 57Co-Me-Cbl isolated from Streptomyces griseus 20 suggest that
`this cobalamin also is taken up intact by PHA-stimulated lymphocytes and is
`then converted to OH-Cbl and Ado-Cbl.
`Intracellular changes with increases in the amount of nuclear protein have
`been observed in lymphocytes within 20 min of incubation with PHA.21 Our
`finding that approximately 12% of the CN-Cbl is converted into OH-Cbl within
`3 hr may not, therefore, be surprising and suggests rapid intracellular decyana(cid:173)
`tion prior to synthesis of the two cobalamin coenzymes. The continuing con(cid:173)
`version of CN-Cbl to other cobalamins between 3 and 48 hr is unlikely to have
`been due to a time-dependent increase in cell metabolic activity caused by PHA
`activation, but rather to the length of time cells were incubated with 57Co-CN(cid:173)
`Cbl, since lymphocytes preincubated with PHA for 18 or 69 hr took up less
`CN-Cbl than did cells to which PHA and CN-Cbl were added almost simul(cid:173)
`taneously. The greater rate of Ado-Cbl synthesis is compatible with the ob(cid:173)
`servation that in almost all human tissues concentrations of Ado-Cbl are very
`much higher than those of Me_CbI. 16.23
`to
`The methylfolate transferase-dependent conversion of homocysteine
`methionine requires Me-Cbl as cofactor, but little is known about factors af(cid:173)
`fecting the synthesis of this coenzyme. The increase in Me-Cbl in certain
`neoplasms22 and the high proportion of Me-Cbl in fetal 0;gans23 parallel high
`methyl H 4 PteGIu homocysteine methyltransferase activity in rapidly dividing
`tissues in which general cellular activity and DNA metabolism are also in-
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`
`618
`
`QUADROS ET AL.
`
`creased. 24.25 Lindstrand 26 found that folate deficiency lowered the proportion
`of Me-Cbl in the livers of week-old chicks. Recent studies in patients with PA
`showed that shortly after feeding formyl H 4 PteGlu, plasma methyl H 4 PteGlu
`rose and levels of Me-Cbl were increased in bone marrow, red cells, and
`plasma. z7 The present studies with PH A-transformed lymphocytes showed
`that PteGlu and methyl H 4 PteGlu each increased Me-Cbl synthesis, while
`MTX and 5-FU depressed it. The failure of HU to decrease the rate of forma(cid:173)
`tion of this cobalamin coenzyme suggested that reduction of Me-Cbl synthesis
`by MTX and 5-FU was not simply due to inhibition of cell proliferation but
`was related to a disturbance in folate coenzyme recycling. This finding was
`supported by the correction by formyl H 4 PteGlu of the defect in Me-Cbl syn(cid:173)
`thesis produced by both MTX and 5-FU. The failure of methyl H 4 PteGlu to
`have a similar correcting effect is difficult to explain. It may be due to instability
`of methyl H 4 PteGlu in the 72 hr of culture. It may also be related to differences
`in the way methyl H 4 PteGlu enters the cell folate coenzyme pool.
`Our results suggest that in PA patients treated with high doses of folate com(cid:173)
`pounds, Me-Cbl synthesis may be stimulated at the expense of Ado-Cbl syn(cid:173)
`thesis. Ado-Cbl is required for the isomerisation of methylmalonate to suc(cid:173)
`cinate and, hence, could influence myelin synthesis. An imbalance in cobalamin
`coenzyme synthesis might therefore explain the precipitation of neurologic
`sequelae in PA patients when treated with folates. At all events, it does appear
`from these studies that an uninterrupted folate cycle is a prerequisite for nor(cid:173)
`mal Me-Cbl synthesis in man.
`
`REFERENCES
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`Matthews DM: Chromatographic and bio-
`
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`
`SYNTHESIS OF COBALAMIN COENZYMES
`
`619
`
`autographic estimation of plasma cobalamins
`in various disturbances of vitamin B 12 metab(cid:173)
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`HA-A, Wise IJ, Matthews DM: Tissue distribu(cid:173)
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`17. Parenchych W, Cooper BA: Factors in(cid:173)
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`(vitamin B12 ) by Ehrlich ascites carcinoma
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`J Clin PathoI24:449-455, 1971
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`DM, Linnell JC: Isolation of 51 Co-labelled
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`1975, pp 287-333
`24. Sauer H, Wilms K, Wilmanns W,
`Jaenicke L: Die activitlit der methioninsynthe(cid:173)
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`5.6,7.8-tetrahydrofolsliure:
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`the synthesis of
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`feeding DL-5-formyl-tetrahydrofolate in perni(cid:173)
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`28. Linnell JC, Matthews DM, Mudd SH,
`Uhlendorf BW, Wise IJ: Cobalamins in fibro(cid:173)
`blasts cultured from normal control subjects
`and patients with methylmalonicaciduria.
`Pediatr Res 10: 179-183, 1976
`
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`blood® 197648609-619
`
`Synthesis of cobalamin coenzymes by human lymphocytes in vitro and the
`effect of folates and metabolic inhibitors
`
`EV Quadros, DM Matthews, AV Hoffbrand and JC Linnell
`
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