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JOURNAL OF NUCLEAR MEDIC INE 7 :58 3-5 88, 1 966

Cobalt-57 Labeled Vitam in B-12 Plasma Levels for the

D ifferential D iagnosis of M acrocytic A nem ias15

J os ep h B . W o rk m an M .D .1 â €¢ 4n d E dw ar d R usc he M .D .:1

Baltimore Maryland

The value of isotopically labeled Vitam in B-12 as a diagnostic agent in the

study of Pernicious Anem ia and related disorders, is now well established. The

work done during the early part of the past decade by Heinle 1 ) and Schilling

2) in this country, and Mollin 3) and Booth 4) in England, has advanced

the basic understanding of the role of Vitam in B-12 in the pathogenesis of Per

nicious A nem ia as w ell as in other disorders of intestinal absorption.

Several m ethods have been developed for the determ ination of intestinal

absorption of orally adm inistered radiocyanocobalam in. Successful m ethods in

use are: the fecal recovery procedure 1); measurem ent of hepatic uptake 5);

urinary excretion technic 2); and the measurement of blood levels of labeled

V itam in B-12. The test m ost popular in the United States had been the urinary ex

cretion procedure as described by Schilling 2). The necessity for patient coop

eration and the difficulties inherant to any com plete 24 hour urine collection es

pecially in the elderly, have lead to a search for additional m ethods of evaluating

V itam in B -1 2 i nte stin al a bs orp tio n.

1F rom the D epartm ent of M edicine, R adioisotope Laboratory, U niversity of M aryland

S ch oo l o f Me di ci ne , B alt im or e, Ma ry la nd .

?AsspcÃŽÃ¢{Ãª ftoÃ¯ÃªssÃ´rÃ•Med icineand Director,Radioisotope aboratory,

Instruetorn Med ieine,

4Beprintequ estsshou ldbe direÂ§tede Bf:W orkman;

â€¢Â»Presented,n part ,at the 7th annualmeet ingel the leeiety ef NuelearMedieinei tes

Park,Colorado,uneÂ§3,960,

m

by Indonesia: J of Nuclear Medicine Sponsored on June 6, 2016. For personal use only. jnm.snmjournals.orgDownloaded from

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5 8 W O R K M AN A ND R U SC H E

The measurement of blood levels of orally administered cobalt labeled

V itamin B-12 w ould appear to be an obvious answ er to the problem of urinary

collection. U nfortunately , the problems created by the large tracer amounts of

cobalt-58 or cobalt-6 0 labeled V itamin B -12 necessary to produce significant

blood radioactivity have not allow ed this procedure to be used routinely. The

recent av ailability o f ano ther co balt iso to pe, cy clo tro n pro duced, 2 70 day half-

life cobalt-57, has made larger tracer doses possible. The fact that cobalt-57

emits no particulate energy and but one gamma-ray (0.123 MeV) makes it

a sui table mate rial f or plasma level de te rminations .

M E TH O DS AN D M AT ER IAL S

This report presents our experiences w ith cobalt-57 labeled V itamin B -12

for the determination of plasma levels and urinary excretion in 98 hemato-

logically normal individuals and in 38 patients w ith Pernicious A nemia. The

diag no sis o f Pernicio us Anemia was established by clinical histo ry and phy sical

ex aminatio n, peripheral blo od and bone marrow studies, g astric analy sis and, in

a few patients, by measurement of serum V itamin B -1 2. The urinary excretio n

procedure w as performed on 150 hematolo gically normal individuals and in 3 8

patients w ith Pernicious A nemia. A ll of the latter patients w ere tested initially

w ithout and again w ith the addition o f purifie d Intrins ic Fac to r c onc entrate . The

original lot of cobalt-57 labeled Vitamin B-121 had a specific activity of 600

m ic ro curie s pe r m illig ram . H ighe r spe cific ac ti vity mate rial (one m ic ro curie pe r

microgram) became available later in the study and it w as this material w hich

was used for the plasma level de te rminations .

UR I N RY EX RET I ON PRO EDURE

The method is essentially that of Schilling (2) modified to our own labor

atory equipment. Cobalt-57 labeled V itamin B -12 , (0.56 /Â ¿gB -1 2 and 0.5 6 ^C

cobalt-57 was given to the fasting patient in a capsule. A 24 hour urine col

lection w as begun simultaneously w ith administration of the tracer dose. Tw o

hours late r, one m ill ig ram o f nonradioac tiv e V itam in B -12 was g iv en parente rally

to saturate the B -12 binding protein of the blood and allow the labeled material

to be ex crete d in the urine. A t the end o f the co llectio n perio d, to tal urine v olume

was recorded and a 100 ml aliquot placed in a volumetric flask and counted on

top of a 2 x 2 inch crystal w ell-type scintillation detector. The corrected count

w as then applied to the total urine volume and divided by the counts obtained

from a 1 00 do se standard co unted under similar co nditio ns. Counting erro r was

less than five per cent using this method. Counting efficiencies for three cobalt

iso to pes co unted in this manner w ere calculated; co balt-6 0 = 1 .2 ?; co balt-5 8 = 4

and cobalt-5 7 = 7 per cent.

The per cent o f administered dose appearing in the urine in a 24 hour period

in 1 50 hemato lo gically no rmal indiv iduals rang ed from 7 - to -3 7 (Me an = 3 0 ).

Obtained from Mr. Geoffrey Gleason, Abbott Laboratories, Oak Ridge, Tennessee.

(Oc tobe r, 1 959)




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DIF F E R E N T IAL DIA G NO SIS O F M AC R O C YT IC A NE M IA S 585

N one of the patients w ith Pernicious A nemia excreted more than one per cent

o f the administere d do se prio r to the adm inistratio n o f Intrinsic Facto r co nc en

trate. When the test w as repeated w ith the addition of 60-to -100 mg purified In

trinsic Factor concentrate to the test dose, the per cent of administered dose ap

pearing in the urine in 24 hours in the Pernicio us A nemia patients rose to w ithin

the normal range .

P L ASM A R AD IO AC T IV IT Y P R O CE DU RE

Nine ty -e ight hemato lo gic ally no rmal indiv iduals re ce iv ed o ral trac er do se s

of 1 .0 fiC of cobalt-57 labeled V itamin B -12 in capsule form. The test material

had a spe cif ic ac tiv ity o f 1 .0 /Â¿Ce r ju.Cand was g iv en w ith the patie nt in a fas ting

state. N o no nradioactive V itamin B -12 w as administered to these patients. Ini

tially, heparinized blo od samples w ere obtained at 2, 4, 6, 8, 10, 12, and 24 hours

after the tracer do se, and 4 ml aliquots of plasma counted for a significant num

ber of counts in a standard w ell-type scintillation detector w ith a medical spec

trometer1 set to discriminate against gamma energies below 50 keV and abo ve

150 keV . Later in the study, only the 8, 10 and 12 hour samples w ere found to be

nece ssary . An aliquo t o f the 1 00 3sdo se s tandard was co unted in a similar manner

and results expressed as per cent of administered dose per liter of plasma. The

counting efficiencies for cobalt-57 = 90 and for cobalt-58 = 45 using this

procedure . Cobalt-60 was not tested.

Eig hty o f the 9 8 no rmal indiv iduals had max imum concentratio n o f plasma

activity in the eight-hour sample w hile the remaining 18 reached peak activity

at 1 0 o r 1 2 hours. The curv e o f plasma radio ac tiv ity is no t s ig nificantly different

from that reported for cobalt-58 Vitamin B-12 (8). The results obtained in

normals are summariz ed in Table I.

TABLEI

PLASMALEVELSOF CoBALT-57 VITAMINB-12 IN 98 NORMALINDIVIDUALS

(PER CENTOF DOSEPER LITER PLASMA)

Tim e inoursMean

( )Range

( )8

Hours0.760.55

0.9510

Hours1.050.88

1.2712

Hours0.950.78

1.15

The results obtained w hen the 38 patients w ith Pernicious A nemia w ere

tested, are show n in Table II. The values obtained for the eight-hour samples

from the group of normal individuals and from those w ith Pernicious A nemia

w ere s ubjected to statistical analy sis. The analy sis o f v ariance and reg ressio n o f

values betw een the tw o groups w as found to be significant ( p = 0.0001 ). In ad-

1Nuc le ar-Ch ic ago Corporati on Mode l 1 32B Analyz er-Compute r.




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586 W O R K M AN AND R U SC H E

dition, there w as no evidence of crosso ver betw een the normal and Pernicious

A nemia groups using the eight-hour plasma sample as a point of reference.

TABLEII

PLASMALEVELSOF CoBALT-57 VITAMINB-12 IN 38 PATIENTSWITH

PERNICIOUSANEMIA

(PERTime

inoursMean

( )Range

( )CENT

OFOSE8

Hours0.160.00

0.19PER

LITERLASMA )10

Hours0.210.00

0.2312

Hours0.220.00

0.25

Each of the patients w ith Pernicious A nemia w as given a second 1.0 micro-

curie tracer dose of cobalt- (57) V itamin B -12 along w ith a capsule of 6 0-to-100

mg o f purified Intrinsic Facto r Concentrate1 and heparinized blo od samples o b

tained at 8, 10 and 12 hours. Four milliliter aliquots of plasma, as w ell as a 100

dose standard w ere co unted in a manner similar to the initial test and the results

expressed as per cent of dose per liter of plasma. A s noted in Table III, all pa

tients tested in this manner demonstrated an increase in plasma conc entratio n

o f labe led V itamin B -1 2 o ve r the initial lev els, altho ugh seldom equal to the lev el

attained by the normal s.

TABLE III

PLASMALEVELSOF CoBALT-57 VITAMINB-12 IN 38 PATIENTSWITHPERNICIOUS

ANEMIAFOLLOWINGADDITIONOF 60-To- lOO MG INTRINSICFACTORCONCENTRATE

(PERTime

inoursMean

( )Range

( )CENT

OFOSE8

Hours0.570.48

0.71PER

LITERLASMA)10

Hours0.840.62

1.2712

Hours0.810.69

1.03

1Obtained from E. R. Squibb & Sons, N ew B runsw ick, N ew Jersey.




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D IF FE RE NT IAL DI AG NO SI S O F M AC R OC YT IC AN EM IA S

58 7

DISCUSSION

The superiority of cobalt-57 labeled Vitamin B-12 over the other com

monly emplo yed co balt iso to pes, is no t limited to de terminatio n o f plasma lev els.

Prior to 1956, the biologic half-life of Vitamin B-12 in man was thought to be

8-to-1 0 days. A dditional basic information concerning bo dy distribution and

bio lo gic half-life o f V itamin B -1 2 hav e been supplied by the work o f Reizenstein

(8) and Schloesser (9). The more generally accepted figure for biologic half-

life of V itamin B-12 is now 200-to-400 days averaging about one year. In addi

tio n, it has been w ell established that abo ut 40 o f an abso rbed dose of labeled

V itamin B-12 concentrates in the liver w here it remains throughout decay. If

this knowledge is applied to cobalt-60 labeled Vitamin B-12 with its five

year physical half-life, a value for effective half-life for this material becomes

328 days. Cons idering that w ith each di sintegration, cobal t- 60 emi ts be ta parti cl es

with an average energy of 0.306 MeV and two gamma rays of 1.172 and 1.332

MeV energies, the value of cobalt-60 V itamin B -12 as a routine testing agent in

man, appears to be l im ited.

TABLEIV

RAD IATIONDOSEDELIVEREDTOTHELIVER BY ONE MICROCURIEOF

COBALTLABELEDVITAMINB-12

IsotopeCobalt 57Cobalt 58Cobalt 60Half Life

hysical270721900in

Days

Effective15560329Energies

eta0.480.310.48in

M eV

Gamma0.1230.510.811.171.33Liver

Dose

in M r ad s4 85 12 6 46

SUMMARY

The re sults o f s tudie s o f pl asma le ve ls and urinary e xc re tion value s fo llowing

o ral adm inis tration o f trac er quantitie s o f ano the r c obalt is otope , the 270 day half-

life cobalt-57, are presented. Tests w ere performed in 98 normal indiv iduals

and in 38 patients w ith Pernicious A nemia, w ithout and w ith the addition o f In

trinsic Facto r Concentrate the y remained lower than the no rmal v alues. S ig nifi

cant statistical s eparatio n betw een no rmals and Pernic io us Anem ia g ro ups was

demonstrated using the eight-hour plasma sample as a reference. Of the co balt

isotopes in general use, cobalt-57 delivers by far the smallest radiation dose to

the liv er and has the bes t co unting e fficiencies when standard ho spital radio iso -

tope laboratory equipment i s used.




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588 W O R K M AN AND R USC H E

A C K N O W L E D G E M E N T S

We would like to ex pres s o ur appreciatio n to the Univ ersity Hospital Radio -

isotope Laboratory Technicians and especially to M iss Emilie Larsen and Mr.

Abraham Samuels who perfo rme d the majo rity o f the pro cedures repo rted in this

manuscript. W e are also indebted to D r. Merrill J. Snyder fo r the statistical an

alyses and to Commander Thomas Mitchell, U SN MSC for the liver dosimetry

calculations.

REFERENCES

1. HEIN LE, R. W., WELCH, A . D ., S CHA RF, V., MECHAM, G . C. A ND PHU SOFF, W. H.:

Studies of Excretion (and Absorption) of Cobalt60 Labeled Vitamin B-12 in Pernicious

A nemia. Tr. Am. Phy sicians 6 5:2 14 , 1 95 2.

2. SCHILLING,R. F.: Intrinsic Factor Studies; II: The Effect of Gastric Juice on the

U rinary Ex cre tio n o f Radio ac tiv ity after the Oral A dministration o f Radio activ e V itamin B -1 2.

J r. L ab. an d C linic al M ed icin e 42 :8 60 1 95 3.

3. MOLLIN , D . L., A ND B AKER, S.J.: The A bsorption and Excretion of V itamin B-12 in

M an. Proceedings of the B iochemical S ociety, London, 337th M eeting, page 28, February 19,

1955.

4. B OOTH, C . C., WA NPOEL, A . A ND MOLLIN , D . L.: Serum Radioactivity after Single

Oral D ose of Cobalt Labeled V itamin B-12 of High S pecific A ctivity. The Proceedings of the

International Conference on Peaceful U ses of A tomic Energy. U nited N ations pp. 475-481,

N ew Y ork, 1 956 .

5 . GLASS , G . B . J., BOYD, L. J., AND STAPHANSON ,L.: Inte stinal A bs orptio n o f V itamin

B -12 in Humans Studied by Isotope Techniques. Proc. Soc. Exp. B iol. and Med. 86:522, 1954.

6. ELLEN BOGEN ,L., WILLIAM, W . L., RA BIN ER, S. F. A ND LICHTMAN, H . C. : A n Im

proved Urinary Excretion Test as an Assay for Intrinsic Factor. Proc. Soc. Exp. Biol. and

M e d. 8 9: 35 7 1 95 5.

7. GOLDBERG,S . R., B HRAT, T. A ND OLIN EH, L.: Radioactive B -12 S tudies. Jr. Lab and

C li ni ca l M e di ci ne 4 9: 58 3 1 95 7.

8. REIZENSTEIN, P.: Body Distribution of Vitamin B-12 Preparations In Man. Acta

M e di ca S ca nd in av ia 1 65 :4 67 1 95 9.

9 . S cHLOESSEH ,L. L ., ANDSCHILLING,R . F.: B io lo gic Hal f- Li fe o f H epati c Radi oac ti vity

from Cobalt80 V itamin B -12. S ixth Congress, International S ociety of Hematology B oston,

A bstract 2 35 , page 24 5, 1 95 6.

1 0. WORKMAN ,J. B . ANDRUSCHE, E.: Cobalt Labeled V itam in B -1 2 Plasma Le vels fo r

the D if fe re nti ati on D iag no sis o f Mac ro cy tic Anemi as . Jr. Nuc le ar Medi ci ne 1 :1 31 , (Abs trac te d) .

1960.




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1966;7:583-588.J Nucl Med.

Joseph B. Workman and Edward Rusche Macrocytic AnemiasCobalt-57 Labeled Vitamin B-12 Plasma Levels for the Differential Diagnosis of

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