Blood 1958 Van Putten 789 94

8/13/2019 Blood 1958 Van Putten 789 94

1/7

1958 13: 789-794

L. M. VAN PUTTEN and Fineke Croon

in Vivo32Labeling with DFPThe Life Span of Red Cells in the Rat and the Mouse as Determined by

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DCBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

For personal use only.by guest on April 8, 2013.bloodjournal.hematologylibrary.orgFrom
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlhttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests

8/13/2019 Blood 1958 Van Putten 789 94

2/7

T he L ife Span of R ed C ells in the R a t and th eM ou se as D eterm in ed by L ab e lin g w ith

D FP 32 in V ivo y L M . V A N P U T rE N

W ith the tech n ica l assL ita nce o f F ineke C roo n

S E V E R A L M E T H O D S have b een u sed to de te rm in e the life span of ra te ry th ro cy te s . P on tico rvo de te rm ined the d etite r ium con ten t o f hem in a fter

feed in g th is n uc ide to ra ts and es tim a ted the su rv iv al at 10 0 d ay s. B e rlin ,H uff and H ennessy 2 a rr ived at tile s am e e stim ation by de te rm in ing tile tu rn -O ve r o f F e59 in h em og lob in . In the sam e labo ra to ry , s tud ies w ith g lyc in e-C 4 -tagged h em oglo b in sho w ed a m ean su rv iva l o f 6 8 d ay s.3 F inch and co-w o rk ersdetern i ined th e red ce ll life span afte r transfusion s o f F e5 9-tag ged ce lls andfo tind it to be from 45 to 50 days.4 W ith the aid o f C r5 th ey es tim a ted theave rage su rv iva l tim e at days.5 T he ir w o rk is based o n the assu m ptio ntha t red cell de struc tion in the rat is a ran lofll phe IlO ille nO ll, a n assum ptiona t va riance w ith the o bse rv atio ns o f B erlin .1

F ew recen t d ata o n the life span of f l lOHSC red ce lls w ere fo und in thelitera ttire . D od , B ie rm an alld S ili lllk illa determ ine d th e life span fro m thed isapp ea ran ce o f su lfhem og lob in from the red ce lls an d fou nd a m ax im a lsu rv iv al o f ab ou t 19 d ay s. B u rn ell, B rick ley and F inch4 in jec ted F e5 9-tag gedred cells an d fo llow ed th e d isapp earance o f rad ioac tiv ity from the b lood . T h eyca lcu la ted an ave rage su rv iva l tim e of 2 0 to 30 d ay s.

T he use o f d iisop ro py lp hosp horo fluo rida te tag ged w ith rad ioactiv e p hos-ho rt is D F P 3 2 as a labe ling agen t fo r hum an b lood cells w as firs t in troducedb y C ohen an d W arringa7 in 1 954 , an d its su ccessfu l use has been rep orted bysevera l inv es tiga to rs.8 B ecause o f the s im p lic ity o f th is m eth od of labe lingcel ls 11 1 v iv o , its app licab ility fo r sm all laborato ry an im als w as invest igated .

MATERIALS AND M ETHOD S

Th e exp erim en ta l an in la ls w er e m a l e \\.A.C. ra ts an in i)red \V istar A lb in o s t r a in , oh -t a m e d ill 1953 fron l th e G lax o labo rato rie s ) w eig h in g 235 -25 0 Cm . an d olale F l m ice C B A x C 57B L ) w eigh ing 2 2-2 7 G ins . D F P 2 w as o bta ine d in i tia l ly fro m the M ed i ca lB iolog ical L ab ora to ry o f the N ationa l D efe nse R esearch C o unc il w here it wa s syn thesizedb y I)r . R . O os terhaa n . L ate r it w a s syn thes ized by D r. H . M . K lo uw en in ou r Inst itu te .D FP w ith a spe cif ic ac tiv i ty be tw e en 2 0 and 2 00 tic . /im ig . D F P w a s d isso lv ed in pea nu to il a nd ad m in is tere d by in tra m u scu lar in ject ion . T he re su ltan t ra d io act iv ity in th e red cel lsw as d ete rm ine d b y the m eth od o f C o hen e t a l.7 R ed cel ls from h ep arin ied b loo d w e rew ash ed 3 tin leS in at leas t 5 vo lum es of sa line an d h em oly zed in d istil led w ater . T hev o l u m e of th e hem olysa te w as m ad e up to I 0 n il. a nd the rad ioa ctiv ity com pa red in aliqu id G .M . co un ter w ith that of a stand ard , ob tain ed by h ydro ly zin g a k no w n am ou ntof D F P 32. A t le ast 400 0 c ounts from e ach sam ple w ere registered. T o t a l ni t roge n w as d ete r-m ine d in a po rtio n of e ac h h em oly sa te.

F ro m th e R ad iob io lo g ic al Inst itu te o f the N a tion al H ea lth R ese arc h C ou ncil , T N O ,R ijsw ijk , T he N eth erla nd s.

S ubm it ted N ov . 20 , 195 7; ac cep ted fo r publ ic a t ion F eb . 15 , 1 95 8 .78 9

http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtlhttp://bloodjournal.hematologylibrary.org/

8/13/2019 Blood 1958 Van Putten 789 94

3/7

79 0 L. M . V A N PU T T E N

The activ ity of the blo od samples w as ex pre sse (l in mic rog ram s l)FP3 ? per gram e rythro-cyte-nitrogen. ( One grain of nitroge n is c ontained in about 4 x1 0 rat erythroc ytes o r 5x 10m ous e e ry tllro cy te s. ) In a pre lim inary e xpe rim ent it w as found that the e xte nt o f labelingof re d ce lls in the rat w as inde pe nde nt of the do sag e betw ee n 30 and 1 00 g . D FP perrat.The Life Span of Rat Erythrocytes

Fo rty ,.tg . D FP32 w as adm inistered to e acil o f 40 rats . A t vario us tim es afterthe injec tion, groups of f ive rats w ere killed and the radioac tiv ity in the redce lls w as determ ined. The results are presented in table 1 and fig ure 1 . Inthe first w eek, the am ount o f in the red ce lls sho w ed a sharp decrease .A fter the e ighth day , how ev er, ti le los s of radioactiv ity w as macil slow er.The early steep fall cannot be due to red cell destructio n , < 1 day; thered ce ll co tints ill the peripheral blood of the rats did Ilo t v ary betw eel l c lay 1and day ) . It ca n only reflect lo ss o f part o f the ac tiv ity from the red ce lls .The slo w dec line after tile e ighth clay is linear w ith tim e . A s it is highlyim probable that this co uld represent e lutio n of tile labe l from the cells , it isasstim ed that this part o f the curve reflects the disappearance of labe led redce lls fro m the circulation. The regression formula fo r the data after c lay 5 ,as calculated by the m ethod o f least squares , is Y = I . 09 5 - 0 .01 82 5 X , andthe life span of the red cells is 60 3. 2 S . D . ) days.The Life Span of M ouse Erythrocytes

Fifteen i.tg . D FP32 w as adm inistered to each of 30 m ice . A t various timesafter injec tion, gro ups o f 5 m ice w ere killed and the radioac tiv ity in the redcells w as de term ined. The results are g iven in table 2 and fig ure 2 . The dis-appearance curve is linear w ith tim e and there is nO ev idence o f e lution orrando m destruc tion of ery thro cy tes . The regression fo rm ula calculated by them ethod o f least sqtiares is Y = 3 .706 - 0 .0911 X , alld tile red ce ll life spanis 40 .7 1. 9 S . D . ) day s. In a second experim ent w itil m ice, a me tho d ofdeterlninillg the red ce ll life 5P 1 1 il l il ldiV idual al linlals w as investigated. Fif-teen i.tg . o f DFP:t2 w as adm iniste red to each of 10 m ice . A t 5 aild at 18 daysafter this injec tion the anim als w ere bled to determ ine the radi oac ti vi ty inthe red cells . The am ount o f blood w ithdraw n at 5 days did not exceed 0 .05m l., w hich is e stim ated to correspo nd to 3 per cent o f the to tal bloo d vo lum e .From the dec line in radioac tiv ity the life 5P 11 o f tile red ce lls w as calculated

T A B L E 1. Phosphorus F ound in Erythrocrjtes of Rats After Injection of 40 g. D F PD ays af t er in j ect ion - g. D FP/G m. er yt hr ocy te-n i t rogen

1 2 .1 8 0 .104 1 .3 0 0 .038 0 .9 4 0 .03

1 5 0 .80 0 .0322 0 .7 0 0.0 329 0 .5 5 0 .0336 0 .48 0 .0 143 0 .31 0 .01

Each figure ex pre sse s the ineaii value ( S .E.) for 5 rats.


8/13/2019 Blood 1958 Van Putten 789 94

4/7

d R a tsY 1 0 9 5 0 0 0 1 8 2 5 XL = 60 d a y s

U0.ca

I

L I F E SPA N OF RED CEL L S I N R A T A ND M OUSE 79 1

1 0 15 20 2 5 30 35 h O 4 5 5 0 5 5 60D a y s a fte r in je c tio n o f 4 0 m ic ro g ra m s 0 F 3 2 P

Fic. 1.-A m ou nt of P32 f ou nd in rat ery thro cy tes af ter in jec tio n of D FP3 ex pressed asg. D F P /G m . ery th roc y te -nitrog en) . T he v ertical bars giv e the s tand ard e rro rs of them ean f o r eac h gro up o f 5 rats. T he straigh t line has bee n calculated acc ord ing to them etho d of least squares f rom the data af ter th e f if th day .

f o r each anim al. T he resu ltin g v alues w ere: 28 , 41 , 41 , 4 2 , 42 , 44 , 4 5 , 50 , 54an d 59 day s; m ean and S .D . w ere 44 .6 8 .1 .

D I S C U S S I O NT he f in d ing of an elation o f tile labe l f ro n l rat ery th rocy tes deserv es corn-

m ent becatise th is phen om eno n has no t b een rep orted b e fore w ith D FP as alab elin g agent. A pre lim in ary ex p erim ent in the rat, using a dose o f 1 0 0 jz g .D FP, resu lted in a sim ilar tag ging of the red ce lls as repo rted abo v e: the to talam ou nt fo und in the red cells af te r 1 day w as 2 .07 ,sg ./G m . n itro gen , and theam ou nt o f labe l w hich w as no t sub jec t to elation w as ex trapolated at 1 .1 1i tg IGm nitrogen . In the ex perim en t w ith 40 ,sg . D FP per rat th ese v aluesw ere 2 .18 and 1 .09 5 g ./G m . n itrog en , respec tiv e ly .

T A B L E 2 Phosphorus F ound in Er ythr ocytes of M ice After inject ion of 15 j ig D FPD ays af t er in j ect ion d ig. DFP/Gm . er y t h r ocyt e-n i t r ogen

1 3.72 0 .0 65 3.23 0.068 2 .9 5 0 .0 9

11 2.590.0621 1 .85 0 .032 8 1 .1 6 0 .0 5

E a ch f ig ure e x presses the m ean v alu e S E .) f o r 5 m ice .


8/13/2019 Blood 1958 Van Putten 789 94

5/7

79 2 L . M . V A N PU T FEN

ci M i c eC V.3 .706-0 ,0911 X

\\\\\f\ L= h 0 .7 d a ys

r { 1 7 6 }

1 NN \\

1. 0 -

1 5 O 5Day s a f t e r i n je c t i o n o f 5 m i c r o g r a m F32P

Fic. 2.-Anlount of P found in m imo use erythroc yte s m fte r i nje cti on of l )FP ( expres sed asp.g { 1 49} FP/G nl. ery thro cy te-nitro ge n ) . T ime ve rtic al i)ars g ive the s tandard e rro rs of thenean fo r e ach g ro up of 5 m ice .

The c lose sim ilarity ill tile e xte nt o f labelimlg o f rat erv throcv tes in time tw oexperim ents covering rather dif ferent dosages suggests tilat a saturation hasoccurred fo r bo th rnechanisllls by w hich D FP is I)aulldl I)\ the ce ll, a stableco m binatio n and a labile one \vhic il is rev ersible ill v iv o . It has been calculate dthat each rat ery throcyte bimlds irreversibly 900 0 m o lecule s o f D FP, anum ber w hich is low er than that fo und in tile m ouse at le ast 23 ,0 00 ) andin m an at least 12 ,500 ) . In m an the dosag e used for labe ling red ce llsand thro m bo cy tes has been m uch low er than 0 .1 m g /Kg ., the clo se at w hichsaturatio n o f rat ery throcyte s is just approached. It is thus po ssible that humanred cells are able to bind an apprec iably hig her am o unt o f D FP. ) A bout theac tual site s o f binding of D FP in rat ery throcyte s, flO definite data are available .

The linearity o f the second part o f tile disappearance curve for the ery thro-cy te s o f the rat conform s to the ex istence of a de finite life span and bearsev idence against random destruc tion o f ce lls . The calculated life span is inreasonable agreem ent w ith the m o re recent values in the literature .35

In the case of the m ouse the ex perim ental results are strong ly sug gestive o fa true life span w itho ut random destructio n.


8/13/2019 Blood 1958 Van Putten 789 94

6/7

L IFE SPAN O F RED CELLS IN RA T AND MOUSE 793

T he linearity of the normal disappearance curve may be of great advantagefor determinations of the life span of red cells in pathologic conditions in thisspecies. T he finding of random destruction by earlier workers is probablydue to the use of less suitable tagging methods. I t seems unlikely that it wouldbe due to the use of different mouse strains.

S U M M A R Y AN D CosciusloNsT he red cells of rats and mice were tagged in vivo by injection of diiso-

propyiphosphorofluoridate D FP , labeled with p: and the disappearanceof radioactivity from the circulating red cells was determined. From the dataobtained, it is concluded that the disappearance of the red cells is linear w ithtime and that the red cells in both rats and mice have a true life span withoutmeasurable random destruction.

T he life span of the erythrocytes was foulld to 1w 60.0 : 3.2 S.D. daysin the rat and 40.7 1.9 S.D. days in the mouse.

Especially in the mouse tile determination of the red cell life span withD FP32 is a simple procedure.

SUMM AR IO IN INTERL INGUA

L e erythrocytos de rattos e muses esseva marcate in V IV Per Ic injectiontie di-isopropyiphosphorofluoridato a p32, I c disparition del radioactivitateab le erythrocytos circulante esseva determinate. Super le base del resultatosobtenite, le conclusion es formulate que le disparition del erythrocytos eslinear con le tempore e que le erythrocytos tanto in rattos como etiam inmuses ha un ver duration vital sin mesurabile destruction al hasardo. L e dora-tion vital del erythrocytos esseva 60,0 dies in Ic ratto e 40,7 dies in I c rntis condeviationes standard de 3,2 e 1,9 d ie s, resp ect iv emen te .

L e determination del duration vital de erythrocytos per medio de istemethodo-specialmente in muses-es tin simple manovra.

REFERENCES1. Pomiticorvo, L ., R ittenherg, D . and Blocim, K . : Th e utihzatioml of acetate for the syn-

thesis of fatty acids, cholesterol and protoporphyrin. J. Biol. Chemn. 1 79:839, 1949.2. Berlin, N . I ., H uff, R . L . and H ennessy, T . G . : I ron metabolism in the cobalt-polycy-

themic rat. j Biol. C hem. 188:445, 1951.3. --. M eyer, L . M . and L azarus, M . : L ife-span of rat red cells as determined by glycine-

2-C4. A m. J. Physiol. 165:565 1951.4 B urnell, E. L ., Brickley, B. A . and Finch, C . A . : Erythrocyte l ife span in small animals;

comparison of two methods employing radioiron. A m. j. Physiol. 172:718, 1953.5. G iblett, E. R., M otulsky, A . C. , Casserd, F., H oughton, B . and Finch, C. A . : Studies

on the pathogenesis of splenic anemia. Blood 1 1 :1 1 1 8 , 19 56 .6. D od, K . S., Bierman, H . R. and Shinlkin, M . : T he disappearance of sulfhemoglohin in

the blood as a measure of the life span of erytllrocytes in normal rabbits and 1111CCand in tumor-bearing mice. J. N at. C ancer Inst. 11:1093, 1951.7. Cohen, j. A . and W arringa, M ia G .P.J.: T he fate of P labelled diisopropylfluoro-phosphonate in the human body and its use as a labelling agent in the study ofthe turnover of blood plasma and red cells. J. Clin. I nvest. 33:459, 1954.

8. L eeksma, C. H . W . and Cohen, J. A .: D etermination of the l ifespan of human bloodplatelets using labelled diisopropylfiuorophosphonate. N ature 175 :5 5 2 1 9 5 5 .


8/13/2019 Blood 1958 Van Putten 789 94

7/7

79 4 L . M . V AN PUT T EN

9. and : Determination of the life span o f human blo od platele ts using labelle ddiisopropylfluorophosphonate. J. C lin. Invest. 3 5 :964 , 195 6 .

1 0 . M ay er, K. and Le y, A . B . : Tile ev aluatio n and interpre tation of iso tope-labe lled e rythro -cy te surv iv al s tudies. Internat. J. A ppl. R adiatio n and Iso topes 1:124 , 195 6 .

1 1 . Pollyc ove , M ., dal S anto , C . and Law renc e, H . H.: S imultaneo us m easurem ent of leuko-cy te , plate let and ery throcy te surv ival w ith diisopropylf iuo ropho sphate 2 (D FP) .Clin. R esearc h Proc . 5 :15 5 , 1 957 .

12 . B ove , J. R. and Ebaug h, F. G .: The use of diiso pro pylfluoro pho sphate 3 (D FP2) fo rthe in v ivo determ inatio n o f re d cel l life span and plasm a pro tein turno ve r in nonnalman. C lin. R esearch Proc . 5 :1 43 , 1957 .

13 . A thens, J. W ., M aim er, A . M ., A shenhruc ker, H. and Cartw right, G . E.: Labe lling ofle uko cy tes in v iv o w ith I)FP. J. C li n. Inve st. 6 :8 70 , 19 57 .


Documents

Blood 1958 Van Putten 789 94