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1958 13: 789-794

The Life Span of Red Cells in the Rat and the Mouse as Determined by Labeling with DFP 32 in Vivo
L. M. VAN PUTTEN and Fineke Croon

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The

Life Span Mouse as

of Red Determined DFP32

By

Cells in by Vivo

in

the Rat and Labeling with

the

With

the

technical

L. M. VAN assLitance

PUTrEN

of Fineke

Croon

S
Huff Over

EVERAL erythrocytes. feeding this and of Fe59

METHODS have been used to determine Ponticorvo determined the detiterium nucide to rats and estimated the survival arrived at In
tile same estimation

the life span of rat content of hemin after at 100 days. Berlin, determining with Finch and Fe59-tagged they on the
an

Hennessy2

by

tile

turn-

in hemoglobin. red from time destruction with the

the

same

laboratory,

studies

glycine-C4co-workers cells and the assumption

assumption

tagged
deterniined

hemoglobin the it to be survival

showed a mean cell life span 45 to 50 at 50 in the observations the life

alld

survival of 68 days.3 after transfusions of With Their of Berlin.1 the work aid is based

fotind average that

days.4 days.5 rat

span
Sililllkilla

of Cr5
pheIlOillenOll,

estimated

red

cell

is a ran(lofll
of
fllOHSC

at variance Few literattire.

recent data on Dod, Bierman

red

determined

cells the

were found in life span from

the the

disappearance of sulfhemoglobin from the red cells and found survival of about 19 days. Burnell, Brickley and Finch4 injected red cells and followed the disappearance of radioactivity from the calculated The hortis by cells Cohen
111 vivo,

a maximal Fe59-tagged blood. They phosintroduced

an use of ( DFP32 and

average

survival

time
agent

of 20 to 30 days. tagged
for human blood

diisopropylphosphorofluoridate

with
cells was

radioactive
first

as a labeling

Warringa7
its applicability

in 1954,
Because for
MATERIALS

and
of small the

its successful
simplicity laboratory
AND METHODS

use
of animals this

has
was

been
method

reported
of labeling

by

several

investigators.8

investigated.

The tamed

experimental ill

aninlals

were

male laboratories Gins. was

\\.A.C.
) DFP2

rats weighing was Research by 200 The al.7 I0

( an

ini)red 235-250

\Vistar
Cm.

Albino and from it

was

strain,

ohmice

1953

fronl

the of

Glaxo 22-27 the Later

olale the our the

Fl synthesized

( CBA
Biological by I)r. and

x C57BL) Laboratory R. Oosterhaan.

weighing

obtained Council Dr. H. M. DFP from the by

registered.

initially where Klouwen was

Medical
Institute. in red peanut cells were The in amount was detera

National it between of 5 volumes made of

from

Defense synthesized 20 Cohen of up a standard, each sample to and et injection. saline

in dissolved in distilled a
nitrogen

DFP
oil was washed

with
determined 3 G.M. At in

a specific
by tinleS counter least 4000 a portion in the at

activity by method least with

counts

tic. /imig. resultant Red and cells and

administered

intramuscular

radioactivity heparinied in radioactivity hydrolyzing

blood water. compared known

hemolyzed

volume
liquid

of the

hemolysate

was
that

nil. obtained were

of

DFP32.

Total

mined

of each

hemolysate.

From Rijswijk, Submitted

the The

Radiobiological Netherlands. Nov. 20, 1957;

Institute accepted

of for

the
publication

National Feb.

Health 15,

Research 1958.

Council,

TNO,

789

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790
The
mouse of red activity erytllrocytes. cells in the of the blood grain of samples
nitrogen

L.

M.

VAN

PUTTEN

was
is

expresse(l contained experiment of the in dosage

in

micrograms about between 4x10 found 30

l)FP3? rat that and the

per extent 100

gram of sg.

erythroor DFP 5x10 per labeling

cyte-nitrogen.

( One

erythrocytes

)
rat

In was

a preliminary independent

it was

rat. The Life Forty the cells the was first Span of Rat was Erythrocytes administered of five The amount rats results of due loss eighth represent curve
of least

,.tg. DFP32 groups determined. week, eighth steep cotints

4

to were are in
tile

eacil

of

40 rats. the in table showed

At various radioactivity 1 and a sharp

was macil
,

times in the figure

after red 1. In
slower.

injection,

killed presented the

loss

and cells

the

red
of

decrease.
1

After the The early red and The asstimed cells

as

day, however, fall cannot be

ill

radioactivity

to red of part clay reflects

squares,

cell

destruction rats did with label for

=

(
Ilot vary

< betweell

day;
clay

the 1 cells.

cell day slow

the after this part

the

peripheral reflect
tile

blood

of the is

).

It can that

only could

of the linear of tile the

formula is Y

activity

from time. from the data

-

the As the
of

red is cells,

decline that this

by

it

highly it is
red

improbable from
calculated

elution

of the The cells

method

disappearance I .095

labeled

the

circulation. of the of red Mouse was

regression 3.2

after 0.01825

clay X, and

5,

the The

life Life Fifteen

span Span

is 60

( S.D. )

days.

Erythrocytes administered were are time 3.706 to killed given each

of 30 mice. At

i.tg. DFP32

various in the 2. The

of elution by

times red

after cells

injection, groups was determined.

of 5 mice The results

and the radioactivity in table 2 and figure is

X,
nO

disor

appearance random method

is

curve is linear with destruction of erythrocytes. of least sqtiares is Y =

and there The regression

-

evidence
tile

formula
alld witil

0.0911

calculated red cell

mice, a investigated.

life
method

the span
of

40.7

1.9 the
of

( S.D. )
red
was

deterlninillg teen i.tg.

days. In a second experiment cell life 5Pt11 ill illdiVidual allinlals

administered to

was
At the

Fifat 18 days in

after the ml.,

From

this

DFP:t2 injection

the

animals

were

each of 10 mice. bled to determine at 5 days cent of the red

5 aild

radioactivity

red cells. The amount of blood which is estimated to correspond the decline
1.-Phosphorus
Days after injection

withdrawn to 3 per the life

did not exceed 0.05 total blood volume. cells was

of 40

in radioactivity
Found in

5Pt11 of tile
of
g.

calculated
g. DFP

TABLE

Erythrocrjtes

Rats
DFP/Gm.

After

Injection
erythrocyte-nitrogen

1 4 8

2.180.10 1.300.03 0.94

15

0.80

0.03 0.03
0.03

22
29 36 43 Each
figure expresses the ineaii value (S.E.) for

0.70
0.55 0.48 0.31
5 rats.

0.03 0.01

0.01

-_______

_______

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LIFE

SPAN

OF

RED

CELLS

IN

RAT

AND

MOUSE

791

Rats Y= 1.095-0.001825
L=

60 days

.c

U 0

I- ----

--

10

15

20

25

30

35

hO

45

50

55

60

Days

after

injection

of 40 micrograms

0F32P

g.

Fic. 1.-Amount DFP/Gm.

for each

of group

P32 found of 5 rats.

in

rat The

erythrocytes The straight vertical line

after bars has

injection give been the

of calculated

DFP3

( expressed
errors of to

as
the the

erythrocyte-nitrogen).

standard

mean

according

method

of least

squares

from

the data

after

the fifth day. 41, 41, 42, 42, 44, 45, 50, 54

for each animal. The resulting and 59 days; mean and S.D.

values were: 28, were 44.6 8.1.

DISCUSSION

The ment labeling DFP, amount amount

finding becatise agent. resulted

of an this

elation

of tile has tagging

label not of the

fronl been in red

rat the cells

erythrocytes before using rat,

deserves with a dose above: DFP of

100

cornas
jzg.

phenomenon

reported

A preliminary in a similar

experiment

as reported

the and at

total the 1.11

found in the red cells after 1 day was 2.07 of label which was not subject to elation with 40 ,sg. respectively.
of Mice

,sg./Gm. nitrogen, was extrapolated DFP per rat these

i.tg.IGm. nitrogen. In the experiment were 2.18 and 1.095 tg./Gm. nitrogen,
TABLE

values

2.-Phosphorus
Days after injection

Found

in

Erythrocytes

After

injection
erythrocyte-nitrogen

of

15

jig.

DFP

dig. DFP/Gm.

3.72 3.23

0.06
0.06

5
8 11

2.95

0.09

2.590.06

21
28 Each
figure expresses the mean value

1.85
( SE.) for 5 mice.

0.03

1.160.05

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792 ci Mice
V.3.706-0,0911
X

L.

M.

VAN

PUTFEN

\\\\\f\

L=h0.7

days

r#{176}

NN\\

1.0

Days
Fic.

after

injection

of 15 microgram
mfter i)ars injection give
of

0F32P
l)FP

2.-Anlount

of group

found 5 mice.

in

mimouse )
.

erythrocytes vertical

( expressed
errors of

as the

p.g#{149} DFP/Gnl. nean for each

erythrocyte-nitrogen of

Time

the

standard

The
experiments

close for each rat which

similarity
covering

ill

tile

extent

of

labelimlg

of

rat

ervthrocvtes
tilat

in

time

two

rather

different

dosages

suggests

occurred
combination

both
and

rnechanisllls
a labile one
bimlds

by
\vhicil

which irreversibly found

DFP

is I)aulldl
ill

a saturation has I)\ the cell, a stable

been calculated

is reversible

that number in and red actual The cytes

erythrocyte is lower
least 12,500

vivo. 9000
used

It has molecules

of
23,000

DFP,

a and cells

than

that

in than

tile 0.1

mouse mg/Kg., It is thus amount

flO

( at
for

least

)
red

man

( at

)
been

( In
much

man

the lower

dosage

labeling the possible of DFP. data close that

thrombocytes of rat are linearity of the rat able of binding sites cells

has

at which
human

saturation

erythrocytes to bind of the an of DFP second to

is just

approached. higher

appreciably part the of tile existence

)
are

About available. erythroand

the

in rat erythrocytes, of

definite

disappearance a definite

curve life

for span

the

conforms

bears is in
of

evidence reasonable
In
a true

against random destruction agreement with the more

case of the mouse the

of cells. The recent values

results

calculated life span in the literature.35

are strongly suggestive

the

experimental

life

span

without

random

destruction.

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LIFE

SPAN

OF

RED

CELLS

IN

RAT

AND

MOUSE

793

The for due be species. due

linearity The use to the

of the finding of less use

normal life random of

disappearance span tagging mouse

AND

curve cells by

may earlier

be

of great conditions is that

advantage in this probably it would

determinations to the

of the

of red destruction

in pathologic It seems

workers unlikely

suitable

methods. strains. CosciusloNs tagged with cells and

of different
SUMMARY

The

red

cells

of rats from the

and

mice

were

in was

vivo
p:12

by and red a true

::

injection the cells life 3.2

life

of the

diisodata with

propyiphosphorofluoridate of radioactivity obtained, time and measurable The life in the DFP32 rat Especially that the it is concluded red cells that

( DFP
circulating the in both

),

labeled red rats was

disappearance From span is linear

determined. of the have

disappearance mice

without
days

random destruction. span of the erythrocytes and 40.7 in the

foulld

to

1w 60.0
the red

( S.D. )
span

1.9 mouse

( S.D. )
tile

days

in the

mouse.
of cell with

determination

is a simple

procedure.
SUMMARIO IN INTERLINGUA

Le tie

erythrocytos

de

rattos

e muses

esseva a p32,

marcate Ic disparition Super disparition tanto in destruction in Ic ratto

in

VIV()

Per

Ic injection

di-isopropyiphosphorofluoridato

del

radioactivitate es in
con

ab le erythrocytos circulante esseva determinate. obtenite, le conclusion es formulate que le linear con le tempore e que le erythrocytos muses tion Le deviationes
methodo-specialmente

le base del resultatos del erythrocytos rattos como etiam al hasardo. Le doradies per in Ic rntis medio de

ha vital

un ver del standard

duration de del

vital esseva

sin
e

mesurabile 60,0 dies dies, de

simple 1,9

erythrocytos

e 40,7

3,2

respectivemente. erythrocytos
manovra. iste

determination

duration in muses-es

vital
tin

REFERENCES
1. Pomiticorvo, thesis of L., fatty Rittenherg,
acids,

D. cholesterol

and and

Blocim,

K. : The G. : Iron of rat

utihzatioml

of Chemn.

acetate 1 79:839, the in

for

the 1949.

syn-

protoporphyrin.

J. Biol.
metabolism red cells

2. Berlin, N. I., Huff, R. L. and Hennessy, T. themic rat. j. Biol. Chem. 188:445, 1951. 3. Meyer, L. M. and Lazarus, M. : Life-span 2-C4. Am. J. Physiol. 165:565, 1951. 4. Burnell, E. L., Brickley, B. A. and Finch, C.
--.

cobalt-polycyby glycineanimals; 1953. A. : Studies in and

1111CC

as life

determined span Finch, of in 172:718, C. small

A. : Erythrocyte Am. 1 1 :1 1 18, : The erytllrocytes j. B.

1956.

comparison

of pathogenesis as A.

two Motulsky,

methods A. of splenic of

employing

radioiron.

Physiol. and

5. Giblett,
on the

E. R.,

C., Casserd,
anemia.

F.,
Blood

Houghton,

6. Dod,
the
7. Cohen,

K. S., Bierman,
blood
j.

H. R. and
the Mia

Shinlkin,
life span

M.
of

disappearance in normal

sulfhemoglohin rabbits

a measure and of Warringa,

and in tumor-bearing
phosphonate the turnover in the
blood

mice. human
plasma

J. Nat.
body
and

Cancer and
red

Inst.
The

G.P.J.:

11:1093, fate of P

1951. labelled

diisopropylfluoroin 1954. of human

1955.

its use as a labelling agent cells. J. Clin. Invest. 33:459,

Determination of the
Nature lifespan 175:552,

the

study

of

8. Leeksma,
platelets

C. H. W.
using

and

Cohen,

J. A.:

blood

labelled

diisopropylfiuorophosphonate.

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794

L.

M.

VAN

PUTTEN

9. 10. 11.

--

and

--

: Determination Ley, studies. dal Santo, and A. B. : Tile Internat. C. erythrocyte

of

the

life

span

of

human

blood

platelets of isotope-labelled 1:124,

using

labelled erythroof leuko-

diisopropylfluorophosphonate. Mayer, cyte Pollycove, cyte, K. and survival M., platelet

J. Clin.
evaluation

Invest.
and Radiation H. with of span H. Invest.

35:964,
interpretation and H.:

1956.
Isotopes 1956. (DFP). (DFP2) turnover G. E.: in Labelling nonnal of

J.
and

Appl. survival

Lawrence,

Simultaneous

measurement

diisopropylfiuorophosphate2 diisopropylfluorophosphate3 and and 6:870, plasma protein

12.

Clin. Research Proc. 5:155, 1957. Bove, J. R. and Ebaugh, F. G.: The use the in vivo determination of red cell life man. Clin. Research Proc. 5:143, 1957.
Athens, leukocytes J. W., Maimer, in vivo with A. M., I)FP. Ashenhrucker,

for

13.

Cartwright, 1957.

J. Clin.