August 1983
`
`,n health
`
`tOWDLE 105-110
`
`usion in
`
`.VOORT-
`OUDIER 111-116
`
`: partial
`
`IAMOTO 1 I 7-] 20
`
`?OLMAN i 21-126,
`
`of brain
`
`9STNOV 127-!35
`
`RRARA,
`IANCINI 137-141
`
`~on the
`; ill man
`lODGER 143-147
`
`,’sio n of
`IS
`DAVIES 149-153
`
`h~ the
`
`IALLEY 155-157
`
`iodel of
`
`R, LEE 159-164
`
`man
`tNSTON 165-t75
`
`177-184
`
`t-IFIELD 185-191
`
`muscle
`
`WARDS 193-201
`
`its with
`
`;SAY RE 203-205
`
`ion
`JONES,
`LXlGHT 207-208
`
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`
`

`
`ethod for studying splenic
`uetion in sickle cel! disease
`application: a brief report.
`
`~s, E,A., Schwartz. A.D,,
`.L. & Spencer, R.P, (1970)
`7ction.’d asplenia in young
`lctnia. A"ew England Jot~r~at
`
`¯ P. & Comdius, E.A. ~1969)
`le~e~l anemia. NewEngtand
`923-926,
`R & We~thcrall, D.J. t1972)
`n of small amounts of
`~aturation, ,lountM of Clinb
`3.
`& Lewis. S.M. (1976) Rate
`n of heat-damaged erythro-
`’athology, 29, 852-854.
`t1975) Practh’al Haemato.
`471. Omrchill Livingstone,
`
`& Szur. L. t1966) The use
`~aged red cells to study
`tion of the met]~od. British
`2, 161-166.
`atlstical Methods, 5th cdn.
`
`>osptenism. dowwat of ~he
`ion~ 157, 613.
`r, F.H, (1970) Acquisition
`by truman erythrocytes,
`~n. Blood. 36,566-575.
`1979t RBC surLace pits in
`s. American Jou~nal of
`;26-527.
`SP. & Baum. S. (1973)
`ism in sickle celbC disease.
`~ne, 55, 720-722.
`
`Clinical Science t1983165, 303-305
`
`303
`
`Increased folate catabolism in mice with ascitic tumours
`
`D, A. KELLY, J. M. SCOTT AND D, G. WEIR
`
`Departmcnt,v of Ct[~lic’af Med£’iue and Bt’ocItemL~ty3", Tritfftv College. Dublin. k’eland
`
`(Received l l M~vember 1982/15 FebruaO, 1983; accepted 14 March 1983)
`
`Sttmmary
`
`1. Folate deficiency is reported in association
`with certain malignant turnouts, and it has been
`su~ested that this arises from increased folale
`turnover and catabolism in such circumstances.
`2, Using an experimental anima[ model to
`delermine Ihe rate of calabolism
`glutamate ( folio acid ) by the quantitative estimation
`of the two urinary cata~olitesp-[Sttlaminobenzoyl-
`glutamate and [~H]acetamJdobenzoylglutanaate, we
`have measured the rate of lblate catabolism in
`mice wRh ascitic turnouts,
`3. There was a significanl increase in the rate
`of catabolism in the mice with turnouts compared
`with controls over a 10 day period. Tlnis was
`associated with lhe accumulation of ascitic fluid
`and an increase in the number of tumour calls in
`the treated animals.
`4. The increase in catabolism appeared to be
`due to increased cell lurnover ~" the turnout rather
`than an increase in cell I]]as5, as the hlcrease ill
`mass of the IUr~IOLIr WaS negligible.
`
`Key words: ascitic turnout, folate, pteroylgluta-
`mate. turnouts.
`
`Introduction
`
`Folale deficiency is known to develop in palicnts
`willn certain tumours [t, 2] and is presumed 1o be
`due to the increased demand for relate coenzynles
`in rapt(cid:128)fly dividing cells. Further support for tiffs
`hypothesis was obtained when an abnormal pteri.
`dine. 6-hydroxymethylpterin. which is a product
`of folale catabolism, was demonstrated botlt in
`tumour cell cuhures and in the urine or’cancer
`palients I31.
`Correspondence: Professor B. G. Weir. Depart-
`merit of Gastroenterology, Sir Palrick DLnl’s
`Hospital, Dublin 2, Ireland.
`
`The aim of this study was to investigate the
`possibility that [l~e tblate deficiency associated
`wilh malignant tumor.Its is due to increased folate
`catabolism. The rate of catabolism of {aH] pteroy[.
`glutamate ( [aH]PteGh0 in mice with ascitic turnout
`ceils was measured by estimating quantitatively the
`two main urinary catabolites, p-aminobenzoyiglu’ca.
`mate (.PABGlu} and acetamidobenzoylgk~tamate
`(.APABGIu} [4, 5],
`
`Meth od s
`
`Radiochemicals
`
`The tL~llowing radiochemicals (with their cor-
`responding specific radioactivitiesl were supplied
`by Amersham International. Amersham. Bucks.,
`U K.: [aH } pteroylg~ ttt~ mate ( [- , 5.7.9 ( n baH ~ Ptc-
`Glu: 93 m(’i,,"mg) >05% pure: [3H]hexadecane
`{2,17 mCi/mg): ~Cr,labelled EDTA (700~Ci/mg}.
`
`Animals and pn>ce~ho’e
`
`BDF ~C67BL/6 x DBA,,"21 mice of the mate sex
`were rand~m~ly assigued ~o groaps of five per meta-
`bolic cage. These mice were specially inbred and
`arc particular]y suitable for evaluation of the P388
`lymphatic leukaemia ~6, 71. All mice were fed on a
`standard diet containing 0.5 mg of folio acid/kg
`body wt, [81. and their daily weight and dietary
`intake and fluid balauce were measured. All mice
`received a single inttaperitoneal injection of 4
`of [3H]PtcGR~ and 5 nCi of ~Ct-labelled EDTA,
`and the pooled urine from each cage was collected
`daily for 10 days.
`~tmoto" mice. Four groups of five mice each
`received a single intraperitoneal iujection of 100~1
`of Locke-Ringer soiulion conlainhag 106 P388
`lymphoid teukaemia cells. This lumour l~ne was
`induced in 1955 in a DBA/2 mouse [b]. I! is
`maintained m DBA/2 mice 171. At 10 days the
`
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`
`304
`
`D. A. Kelly et al.
`
`(4.9 +- 0.5 } × 104c.p.in. (conlrols) and (11.4+ 2.8)x
`104 c,p.m, ltreated) (Y< 0.00J.). Mean (-+ SEM)
`cumulative excretion from day 4 ~o day 10 was
`(3.6+0.5) x 10So.p.m, (controls) and (5,5 -+0,5) x
`I 0s c.p,m. {treated} (P < 0,00i ),
`
`Estimation o.F[ aH]PAB carabolites
`Estimatim~ of [aH]PAB calabolites was highest
`in the first 3 days, slabilizing after this time (Fig.
`1). Ttiere was a definite increase in these catabolites
`from day 4 onward in the treated group. Mean
`(.+- sE.’,.O daily excretion in the treated group was
`(2.6 a 0.25) x 10~ c.p.m, aud in lhe control group
`(1.7 -+ 0.21} x 104c.p.m. (PK0.01L Mean (-+ SEMI
`
`7
`
`6
`
`5
`
`105
`
`9
`8
`
`5
`
`4
`
`mice were killed by cervical dislocation; the
`ammmt of ascitic fluid was measured and the
`tu]m)ur cells were separaLed, weighed and counted
`with a haemocytome~er [6].
`ConFol mice. Two groups of five mice each
`received a single intraperitoneal injeclk)n of 100~1
`of Locke-Ringer solution alone.
`
`Estimation of [ aH]PABGIu and [ aH]APABG&
`
`Esthnation of tt~ese cataboliles was performed
`by alkaline hydrolysis of all p-aminobenzoic acid
`([aH]PAB)-eomaining caabolites to [aH]PAB
`which was extracted by a previously described
`procedure and the radioactivity estimated [5]. The
`completeness of the injection and collection tech-
`niques was monitored by use of S~Cr-labelled
`EDTA [5].
`
`The difference between the mean daffy excre-
`tion and cumulative excretion of [aH]PAB cata-
`boli*es in the control and the Iumonr groups was
`determined [91.
`
`Results
`
`Estimation of tumottr growth
`
`In the firsl 3 days each grottp off mice lost
`weight in the metabolic cages: mean weight loss
`per cage of five mice was 3 g in the controls and
`9.8 g in the lreated mice. Subsequently over
`next 7 days all mice gained weight: mean weight
`gain per cage of five mice was 1,2 g per cage
`the controls and 17,2 g per cage in the treated
`mice. The increase in weight in the lreated mice
`was partly due to an increase in the weight of the
`turnout cells (5,0 g per cage (~t five miceL in turn
`maii~]y dtm to lhe accumLitation of ascitic fluid
`{ 11.6 g per cage of five mice). The lumour cells
`accotmted for a YX increase in body weight,
`The number of turnout ceils also increased from
`the inocu]um of 10~ Pag8 lymphoid leukaemia
`cells per mouse to 330 x 10a per mouse or l.b x]O
`per cage of five mice. There was no accumulation
`of ascitic fluid in the control group.
`
`[’2~Hmat ion of to tai radioactiviO,
`
`Tola[ excreted radioactivily was highest in the
`first 3 days in all groups, stabilizing aher this time
`to a slow decline [~i- There was a significant in-
`crease in total excretion of radioadivi~y in the
`trealed group compared with controls fiom day
`4 onwards, Mean (~S[,:M) daily excretion was
`
`"[04 l t~ t ~t 1 i t i i~
`4
`6
`8
`2
`10
`
`I-line
`lq(;. 1, Rate of folatc catabolism in mice with
`ascitic turnouts compared with controls. ~, Mean ~
`SEN of l~ur groups of five mice with ascitic
`turnouts; .., meank SEM Of ~wo groups of five
`control mice,
`
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`
`

`
`Folate catabolism b~ mice with ascitic turnouts
`
`305
`
`cumulative excretion from day 4 to day 10 in the
`treated group was (1.80- 02)x !0s c~p.m÷ and in
`the control group was (1.20 + 0.2) x lOs c.p.m.
`(P < 0.001 ).
`
`however, that rapid cell division, dependent as it
`is on folate participation, mighl lead to irtcreased
`turnover of the vitamins.
`
`Discussion
`
`This study demonstrates that there is a 5074 in.
`crease in the rate o~" folate catabolism in mice with
`ascific tnmours. The increased rate of catabolism
`coincided wilh the accumulation of ascitic fluid
`in the treated animats and a substantial increase
`in both the number and the weight of tile malig-
`nant cells, implying an active turnout. Thlssuggests
`that the increased rate of catabolism was due
`either to an increased demand for rotate coenzymes
`in tire malignant ceils or m increased fi~[ate turn-
`over in the rapidly dividing cells. As there was a
`5% increase in the weight of the turnout and a 50%
`increase in catabolism, it is likely that the increased
`catabolism was associated with increased cell
`turnover rather than an increase in weight of the
`
`tuIrl o n[,
`These results conflict with a recent study which
`reported ~hat t’olate metabolism is decreased in
`tumour-bearing rats [10]. However. in the latter
`study the minary metabolites were measured at
`24 and 48 h after administration of radioactive
`PteGlu. It has been consistently shown that there
`is a complex pattern of excreted intact folates in
`the first 2 days after a radioactive dose and that
`the radioactive catabolic products are not fnund in
`the urine until after day 3 [4]. Furthermore, as in
`tidal study [!0] the two main catabolites, PABGlu
`and APABGlu, were not estimated after day 3
`it is unlikety that rotate catabolism was being
`measnred, Measurement of tmal excretion during
`this iMtial equilibration period cornpletely masks
`any difference that exists in the excretion of folatc
`catabolites [4], In addition, the study made no
`attempt to distinguish between excretion of intact
`folate and nf catabolites.
`Why the presence of tumot,r ceils, either in
`man [3] or in mice, should lead to increased folate
`breakdown remains unclear. It seems reasonable,
`
`Ackaaowledglnents
`
`We thank Dr M. L. Conalty for supplying the
`P388 lymphoid leukaemia cells and the BDF mice,
`Mrs E. Wilson for her technical assistance and Miss
`Mary Kavanagh for her expert typing.
`
`References
`
`!. Rose. D.P. 09661 Foli¢ acid deficiency in leukemia
`and lymphoma. Your~ta[ o.t Ch)~feal Patholo.,<r, 19,
`29-36.
`2. Hol~Srand, A.V., Hobbs, J.R., Fremench~.ky, S. &
`MollJn, I).L, ~1967) Incidence and pathogones~s o~
`megatoblastic erythropoiesis in multiple myeloma.
`Journal of Ch’n&d PaHto[ogg’, 20, 699-705,
`3. Sled. B., Blacklund, P.S., Berkey, P.B., Cho, A.K.,
`Halpern, B,C.. tlalpern, R.M. & Smifl~. R-A. {1978)
`f.’olate and pterin metabolism in cancer cells in cub
`lure. Calico" Research. 3& 2378-2384~
`4, Murphy, M.. Keati~g, M., Boyle, P., Weir, D.G.
`Scott. J.M. 11976) The elucidation of the mechanism
`of (cid:128)olate catabolism in the rat. Biochemical and
`physical Research Comm,mk.afions, 71, 1017-1024.
`5, Kelly. D.A., Reed, B.. Weir. D,G. & Scott. J.M. t1979~
`EfDct of anticonvulsant drags on the rate of folate
`catabo~sm in mice. Journal oJ Clinical
`64, 1089-109@
`6. Geran, Rd.. Greenbe~g. N.tl., MacDonald. N.N..
`Schum~er, A.M.& Abbot, B.V. {1972~ Lymptmcyfic
`leukemia P.388. Protocol 1.200. {}mcer Chemotherap.v
`RtT~orrs, 3, 9-10.
`7. Belton. J.G, Conalty, M.L. & O’Sullivan, J.F. I!9761
`Anticancer agents. XI. Antitumour activity of 4-
`amino-7-nitrobenzofuroxans a~d related ~olIlpOOl?dS.
`~oceedb~,¢s of thv Rqval tris’h Academy. 76, 133-
`149.
`8. Coates. M.E.. O’Donoghue, P.N.. Payne. P.R. &
`Ward, RA. ~1969) Dietary Stamlards ,¢br Laboratory
`Ro~s aml Mice, p. 17, Laboratory Animals Ltd,
`kondon.
`Snedccor, G~W. & (’ochran, W.G. {l 967) StaHstical
`Methods. 6fl~ edm pp. 285-288. Iowa Stale University
`Press, Ames, Iowa.
`Saleh. A.M., Pheasant, A.I.{_ & Blair, J.A. 11981)
`r:olatc catabo~sm in tumour-bearhlg rats and rals
`~reated with methotrexate. British goto’t*o[
`~, 700-768.
`
`~trols) and (11.4_+ 2.8)x
`0.001L Mean {_+ SEM}
`day 4 to day 10 was
`~trols.) and 15.5 -+ 0.5.) x
`.0011.
`
`abolires
`
`catabolites was highest
`ing ariel’ this time (Fig.
`cease in these cataboIites
`m treated group. Mean
`the treated group was
`ld in the contro| group
`< 0.01). Mean (_+
`
`I I I I, 9,
`8 10
`
`tdaysl
`
`labo[ism in mice with
`¯ ith controls. ~, Mean -+
`ive mice with ascilic
`two groups of five
`
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