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THE TAGGINGOF RED CELLS ANDPLASMAPROTEINS WITHRADIOACTIVE CHROMIUM1

By SEYMOURJ. GRAYAND KENNETHSTERLING 2

(From the Biophysical Laboratory and the Department of Medicine, Harvard Medical School,and the Medical Clinic, Peter Bent Brigham Hospital, Boston)

(Submitted for publication July 31, 1950; accepted, September 21, 1950)

Relatively few reports have appeared on thechromium content of biological material. Pre-vious studies were devoted primarily to the analy-sis of plants and soils ( 1 ).

In preparation for the present study, arc spec-trography 3 was used to demonstrate the presenceof trace amounts of chromium in human tissues,including the blood. Tissue analyses were per-formed on samples of normal human blood bymeans of a colorimetric method (2) with the find-ing of mean values of 20 y % for packed red cellsand 14 y % for plasma.

The present report is concerned with a newbiological tracer, radioactive chromium (Cr51),which is bound by the red cells and plasma pro-teins. Both anionic hexavalent (Na2Cr51O4) andcationic trivalent (Cr51Cl3) states of the elementhave been studied.

Cr51 is a soft X-ray emitter with a half-life of26.5 days. It disintegrates by K capture withtransmutation to vanadium and emission of X-rays of 4.92 Kev plus a few per cent of 0.237Mev gammarays (3).

To prepare Cr51 from Cr50 in the pile with suf-ficiently high specific activity,'4 it was necessary toenrich ordinary chromic oxide (Cr2,O) electro-magnetically. Naturally occurring chromium con-tains 4.49 %o Cr50; the enriched chromium, 41.2%o.This enriched mixture was then irradiated in theOak Ridge pile for two months, at the end of which

' This work was supported in part by the United StatesAtomic Energy Commission, the Office of Naval Re-search, and the National Cancer Institute, U. S. PublicHealth Service.

2This work was done in part during the tenure of aU. S. Public Health Service Postdoctorate ResearchFellowship.

3 Courtesy of Harold C. Harrison.4The chromium 50 and 51 used in this investigation was

supplied by the Isotopes Division, U. S. Atomic EnergyCommission.

time 100 y of chromium contained 13 to 23 micro-curies.

METHODS

Preparation of Na2Cr5104 (anionic hexavalentCr51)

The chromium obtained from Oak Ridge was thedark green oxide (Cr2O3). The oxide was trituratedin a platinum crucible with a 1: 5 mixture of NaNO,and Na2CQ3 and fused several times by heating to red-ness with two Bunsen burners. After approximatelytwo hours of fusion, the resulting yellow melt was dis-solved in distilled water and filtered. The yellow alkalineNa2Cr10O solution was adjusted to pH 7.4 by the drop-wise addition of concentrated HCI.

Preparation of Cr51Cl3 (cationic trivalent Cr511)

Na2Cr'0, solution was acidified with excess concen-trated HCI and reduced with formaldehyde by boiling tofumes in a beaker on a hot plate. Distilled water wasadded to the dry salt and the resulting green solution ofCr'Cl, was filtered and then adjusted to pH 4 by theaddition of NaOH.

Measurement of radioactivityAn X-ray end window Geiger-Muller counter 5 was

used for counting. It was tested with a standard Fe'source having 26,180 disintegrations per minute of whichit recorded 368, or a counting efficiency of 1.4%. Usingthe same geometry and assuming the same counting ef-ficiency for the soft X-rays of Fe' and Cr', 1 microcurieof chromium was computed to approximate 31,000 countsper minute.

The concentrated radioactive chromium solutions werediluted 1: 500, and 1 cc. aliquots were pipetted into plan-chets and dried overnight. These planchets were retainedas standards and were counted daily. The half-life ob-tained was in good agreement with the published valueof 26.5 days (3). All samples were corrected for self-absorption and radioactive decay.

On receipt from Oak Ridge, 100 - of chromium emitted403,000 to 713,000 counts per minute (13 to 23 micro-curies). This amount of chromium was present in 0.18cc. to 0.46 cc. of the original concentrated chromiumsolution.

5 North American Phillips, tube type 62017-Amperex.1604

RADIOCHROMIUMTAGGING OF RED CELLS AND PLASMA

UPTAKE OF ANIONIC Cr51 (Na2Cr5104)BY HUMAN RED BLOOD CELLS

iU 100-jwQ 90a

cw 80

X 70IL

z 60wv* 50

I'- 40z00 30-i4O 200

a.-o 0

15 15. 30 4.5 Ihr.mtn.ml n. mm. m in.menI ',hrs.

TIMEFIG. 1

Procedure for counting radioactivity of tagged redcells and plasma

Red cells: Red blood cells were dried and ground to afine powder prior to counting. Whole blood or suspen-sions of red cells in saline were placed in 4 cc. hematocrittubes and centrifuged at approximately 2,700 r.p.m. (741g) for 60 minutes at 40 C. The plasma or saline waswithdrawn and the packed red blood cells were pouredinto unweighed aluminum planchets and dried overnight

in an oven at 600 C. This temperature was not exceeded,since chromium may sublime at 830 C. Constant weightwas achieved by this procedure. The dried caked redcell mass was ground to a fine powder with a pestle in afolded filter paper and counted in a weighed planchet.

One cc. of packed red blood cells, centrifuged to con-stant volume and measured in a calibrated pipette wasfound to weigh 0.35 gm. when dried to constant weightin an oven at 600 C. The counts per cc. of packed redcells was computed from the counts contained in 0.35gm. of dried ground red cells.

Plasma: One cc. of plasma was pipetted into a weighedplanchet, dried overnight at 600 C, and counted.

I. UPTAKE OF RADIOACTIVE CHROMIUMBY THE

RED BLOODCELL

A. Anionic hexavalent chromium (Na2Cr5104)The marked affinity of erythrocytes for chromate

anion was demonstrated by the rapid uptake ofradioactive chromium following the addition ofNa2Cr51O4 to saline suspensions of washed redcells (Figure 1). Eighty-six y of chromium, asthe chromate, was added to a series of 5 cc.aliquots of a saline suspension of washed erythro-cytes with a hematocrit adjusted to 40. Aftermixing in a mechanical shaker for periods rangingfrom one minute to two hours at room tempera-ture, the suspensions were rapidly centrifuged;the supernatant saline and the red cells, washedthree times, were prepared and counted. In aseries of such experiments, 80-90%o of the isotopewas bound by the red cells within two hours (Fig-ure 1).

A similar experiment, carried on for 43 hours,demonstrated the prolonged retention of radio-

UPTAKE OF ANIONIC Cr5' (No2?Cr51O4) BY HUMANRED BLOOD CELLSDo~~~~~~~~)0 r

RED BLOOD CELLS

30

60 '

50

30 \

10 '-__, SUPERNATANT SALINEC _ _A-*-.A-.

5m. 30min. I hr. 2hrs. 3hrs. 17hrs. 23hrs. 43hrs.

FIG. la

/RD BLOODQ /{ ~~~~CELLS

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'0%%%" SUPERNATANT

%% SALINE

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1605

SEYMOURJ. GRAY AND KENNETHSTERLING

UPTAKE OF ANIONIC Cr5' (Na2Cr5'O4) BY HUMANRED BLOOD CELLS

10 SUSPENDED IN SALINE

90

80

*70

2.65 r 26.5r

O.IXO6

132.5rw Cr ADDED AS Na2CrO4

COUNTSADDEDFIG. 2

265r

1.1 XIO'

activity by the red cells without significathe saline medium (Figure la). Slighthad begun to occur at the end of this pe

Varying amounts of radioactive chroxadded to red cell suspensions and the r

UPTAKE OF ANIONIC Cr5' (Na2BY HUMAN RED BLOOD CEL

I00

o 90

> 8070

z 604a- 50OI-z 40

30I-It0 20I -

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26.5rY 132.5 YY Cr added as Na2CrO4

I . 48,490 42,450

Counts added

FIG. 2a

ant loss to uptake determined at the end of two hours. Therehemolysis was an inverse ratio between the amount of chro-riod. mium added and the percentage uptake by the redmate were blood cells (Figure 2). The addition of 2.65 y)ercentage of chromium to 5 cc. of red cell suspension re-

sulted in 97.4%o uptake of the total counts added.,Cr5' 04) Successively larger amounts of chromium resultedLS in proportionately smaller percentage of red cell

uptake, although the absolute amount of chromiumbound was larger. Addition of 265 y of chromiumresulted in the uptake of 66.4%o in the erythro-cytes. These erythrocytes contained approxi-mately 400 times the quantity of chromium foundby chemical analysis in untreated red cells (2).One cc. of these packed red cells emitted 735,000counts per minute or 23.7 microcuries.

Simultaneous uptake curves were determinedwith red cells in saline and whole blood from thesame individual. The uptake by red cells in salinewas appreciably greater than in whole blood(Figure 2a). The difference between the curveswas attributed to binding of some of the chromiumby the plasma proteins.

265 y A similar affinity of the red cell for anionichexavalent chromium was demonstrated in vivo

84,900 by the intravenous injection of Na2Cr51O4 in thedog.

Figure 3 illustrates graphically the distribution

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RED BLOOD CELLS~- JN SALINE

1606

RADIOCHROMIUMTAGGING OF REDCELLS AND PLASMA

of Cr5' in the red cells and plasma of a normalmongrel dog weighing 15.5 kilograms after the in-travenous injection of 603 'y of chromium asNa2Cr5104 emitting 4,788,800 counts per minute(155 microcuries). The findings are typical offour such experiments. The radioactivity of theblood was predominantly within the red cells. Theplasma activity declined rapidly, and after twodays the plasma contained only a small fraction ofthe radioactivity of the erythrocytes. The redcells, however, retained their radioactivity for aprolonged period, declining gradually but notshowing a single exponential decay. There wasa fall of 76% of the erythrocyte activity from thesecond to the 52nd day, which is approximatelytwice the rate of decline that could be attributedto the death of the red cell assuming a life span of120 days (4) and the tagging of a mixed popula-tion of cells.

In view of the marked retention of radioactivityby the red cells, erythrocytes were tagged in vitroand re-injected into the same dog to determinewhether the radioactivity would remain in the redcells in the animal's circulation.

Tagged red cells were prepared by the additionof 7-26 microcuries of Na2Cr51O4 to red cellswith mixing for one hour at room temperature.The cells were then centrifuged and washed threetimes with cold physiological saline solution andfinally resuspended in plasma from the same dog.The plasma radioactivity was found to be less than0.5%o of that of the red blood cells.

After the intravenous injection of this recon-stituted whole blood containing the tagged red

DISTRIBUTON OFIN THE RED BLOODCEL

700006 ~~~~~~~~REDBLOOD CELLS

400z 0

cells, consecutive blood samples were taken fromthe dog to determine the counts in the red cellsand plasma. Figure 4 illustrates graphically thered cell radioactivity in one of ten such experi-ments. In all cases, the plasma contained no sig-nificant counts above background.

The activity of the red cells during the courseof the day of injection agreed within the limits ofthe method. In ten dogs studied, the consecutivered cell activities at various intervals during thefirst day were in satisfactory agreement (5). Agradual decline of red cell activity was observedafter the first day. The fall in 20 days was 53.6%o.This decline was again greater than that calculatedfrom the life span of the red cell and apparentlyexcludes the adaptation of the method to the directmeasurement of red cell survival time.

Since the tagged red cells retained their radio-activity without significant loss to the plasma, amethod for measuring the circulating red cell vol-ume with Cr5' was devised, utilizing the quotient:

total counts injected in tagged red cellscounts per cc. packed red cells in sample'

The stability of the red cell tagging was sufficientfor such experiments to be continued for a periodof 24 hours (5, 6).

B. Cationic trivalent chromium (Cr5"C13)The distribution of Cr5lCl8 in blood was found

to differ considerably from that of Na2Cr5tO4.When varying amounts of CrP5Cl3 from 8-y to

250 y were added to 5 cc. aliquots of whole bloodin uitro, almost all of the radioactivity (94-99%o)

AMONIC Cri CNo3Cr" O.)LLS AND PLASMAOF THE DOG

FIG. 3Counts per cc. in the ordinate signifies counts per minute.

1607


RADIOACTIVITY OF INJECTED RED BLOOD CELLS TAGGEDWITH ANIONIC Cr5' (Na2Cr5lO4)

300

200\

I 3 5 7 f11 13 15 1f7 1f9DAYS


remained in the plasma and only insignificantcounts were retained in the erythrocytes aftersaline washing. Similar results were obtainedin vivo. After intravenous injection of Cr51C1lthe radioactivity remaining in the animal's circula-tion was found exclusively in the plasma; the redcells manifested no significant uptake.

Figure 5 illustrates the distribution of radio-

600C

500C

40

19

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03100

400(

300(

200C

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activity between the red cells and plasma of a

normal mongrel dog weighing 15.0 kilograms afterthe intravenous injection of 906 y of chromium as

Cr51CI8, emitting 6,324,150 counts per minute(204 microcuries). The initial plasma activitywithin the first four hours after injection was

more than 25 times greater than that of the washedred cells; the latter fell to negligible levels within

1608

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DISTRIBUTION OF CATIONIC Crs, (Crl"CI3)IN THE RED BLOODCELLS AND PLASMAOF THE DOG~~~PLASMA

RED BLOOD CELLS jS u_6 8 10 12 14 16 la 22 52

HOURS DAYS



four hours. From the second day on the plasmaactivity approximated an exponential decay witha three day half-time.

The inability of the erythrocyte to take upCr51C13 could be explained by the relative im-permeability of the red cell membrane to cationicchromium or the competitive binding by plasmaproteins of this form of the isotope. To excludethe latter possibility, Cr5lCl8 was added to redcell suspensions in an isotonic acetate buffer atpH 5.5.6

After two hours of mixing at room temperature,the washed erythrocytes contained only 1.6-2.7%oof the counts added; approximately 98%o of theradioactivity was recovered in the supernatantbuffer.

II. TAGGING OF PLASMAPROTEINS WITHRADIOACTIVE CHROMIUM

A. Cationic trivalent chromium (Cr5lC13)

In vivo experiments in dogs indicated that whenCr1C13 was injected intravenously, essentially allof the counts in the blood were retained in theplasma with insignificant activity within theerythrocytes. The fact that the plasma remainedradioactive for periods exceeding ten days sug-gested the investigation of the plasma proteinbinding of Cr50C13.

A satisfactory protein for study was crystallinebovine albumin (Armour) since at its isoelectricpoint of 4.9 the chromic cation was in the solubleform. Radioactive GrHCQ was added to a seriesof albumin solutions, each containing 5 micromoles(3.45 gm. %). The molecular ratios of chro-mium to albumin in the series were 0.6: 1, 1: 1,5:1, 10:1, 100:1. After four hours of mixingin the cold (40 C), the albumin solutions weredialyzed against 2 liters of isotonic saline solutionwhich was changed daily for three days.7 The

6 The acid acetate buffer was used instead of saline toprevent the precipitation of Cr(OH)3 which occurs at ahigher pH, resulting from the buffering action of theerythrocytes. The pH was maintained at 5.5 for theduration of the experiment and the precipitation ofCr(OH). was avoided. This acid pH was shown notto prevent the usual erythrocyte uptake of anionic hexa-valent chromium.

7A control dialysis of comparably dilute Cr'Cl, solu-tions revealed that the dialysis was not complete, evenafter three days with mixing and daily change of dialy-

TABLE I

Binding of chromium (Cr'1 Cl3) by crystaUine bovine albumin

Moles of Cr Countspercc. Counts percc. p of Moles of Cradded per of albumin of albumn Pcent bound per

mole of before after couned mole ofalbumin dialysis dialysis rined albumin

0.6 14,640 9,970 68.1 0.41 21,400 14,950 69.9 0.75 94,800 57,300 60.4 3.0

10 48,200 26,800 55.6 5.6100 31,900 12,500 39.2 39.2

radioactivity per cc. of albumin solution was de-termined before and after dialysis (Table I). Asincreasing amounts of chromium were added, theper cent of radioactivity retained after dialysis de-creased, although the absolute amount of chro-mium bound increased. No stoichiometric rela-tionship was observed; the number of moles ofchromium bound per mole of albumin dependedupon the amount of chromium added. When 1mole of chromium was added to 1 mole of albumin,approximately 70%o of the chromium was bound.

The ability of the albumin to retain its radio-activity over a period of time was determined byprolonged dialysis over periods up to 17 days.There was little or no further decline in radio-activity following additional dialysis.

The addition of excess non-radioactive chro-mium to the fluid outside the dialyzing bag pro-duced only a very small loss of radioactivity. Ap-proximately 2%o of the counts were recovered inthe outside fluid. This further illustrates the firm-ness of the chromium-protein bond, since thebound radioactive chromium did not exchangeappreciably with the newly added non-radioactivechromium.

Visible changes indicating denaturation, such asdiscoloration and solidification, occurred afterdialysis of 100 moles of chromium and 10 moles ofchromium mixed with 1 mole of albumin. Solu-tions containing 5 moles or less of chromium with1 mole of albumin revealed no change in appear-ance after several months' storage at 40 C.

Physicochemical analyses of chromium-taggedalbumin did not disclose significant alterationsfrom the native state of the protein. A sample ofcrystalline bovine albumin which had bound 0.5

sate. The errors to be anticipated because of this wereof the order of 10%o or less. In contrast, Na2Cr'O, wasdialyzed completely by this technique.

1609


mole of chromium per mole of albumin was ana-lyzed in the ultracentrifuge and by electrophoresis.8In the ultracentrifuge, the tagged albumin washomogenous and showed no change from un-treated albumin. The electrophoretic mobility was- 7.6 x 10r5 cm2/volt/sec. Samples taken fromportions of the electrophoretic cell from whichthe protein had migrated revealed no radioactivity,indicating that no chromium had been detachedfrom the protein during electrophoresis. Theultracentrifugal and electrophoretic data for thetagged albumin did not differ from the nativebovine albumin. One gm. of this Cr5l-taggedalbumin emitted 925,000 counts per minute (29.9microcuries).

In addition to albumin, other protein fractionsincluding gamma globulin, beta metal combiningprotein, and beta lipoprotein were found to bindtrivalent chromium. Chromium-tagged crystallinebovine albumin was mixed with an equal amountof untagged bovine gammaglobulin, and the mix-ture was allowed to stand 22 days in the cold.When the proteins were separated by electro-phoresis, almost all of the radioactivity remainedin the albumin with only 2.7%o in the gammaglobulin fraction.

Binding of chromic ion to protein may involveinitially a polar attraction by carboxyl groups.The firm attachment may be explained by coor-dinate covalent bonds of the type of Cr(NH.)6+++between the chromium and terminal amino groupsof lysine or other basic groups of the protein.This would be analogous to hypotheses of thelinkage of chromium to collagen in leather tan-ning (7).

B. Anionic hexavalent chromium (Na2Cr5104)The addition of the anionic hexavalent form

of chromium (Na2Cr51O4) to proteins also re-sulted in tagging, but to a considerably less extentthan was achieved with the cationic trivalent form(Cr51Cl3). When Na2Cr51O4 was added to crys-talline bovine albumin and dialyzed, the bindingwas only 14%o of that attained by the addition ofan equal amount of Cr5lCl3.

The diminished uptake of Na2Cr51O4 by theerythrocytes in whole blood as compared to redcell suspensions in saline (Figure 2a) further sug-

sCourtesy of Charles Gordon, M. J. E. Budka andDr. J. L. Oncley.

gested plasma protein binding. The possibilitythat hexavalent chromium may be reduced to thetrivalent state in the process of protein bindingwill be discussed below.

III. MECHANISMOF REDCELL UPTAKEOF

CHROMIUM

A. Effect of non-radioactive chromate upon redcell tagging

The firm binding of Na2Cr5104 by red bloodcells was investigated further by determining theeffect of non-radioactive chromate upon the radio-activity of the tagged cells.

Radioactive Na2Cr51O4 (106 y of chromium)was mixed with a red cell suspension for twohours at room temperature and the counts per cc.of packed red blood cells were determined. Anexcess of non-radioactive chromate (200 y of chro-mium) was then added to a duplicate suspension,and the red cell activity was determined one hourlater. There was no loss of radioactivity after theaddition of non-radioactive chromate to the redcell suspension, indicating that anionic chromium,once bound by the erythrocyte, did not leave thecells and exchange with the outside medium forthe duration of the experiment. The marked con-trast between the firm erythrocyte binding of Cr51and the rapidly reversible exchange observed withp82 (8, 9) suggests a different mechanism oftagging.

B. Kinetics of uptake of Na2Cr51O4 by red cells

The kinetics of the uptake of Na2Cr51O4 by redcells in saline (Figure 1) were analyzed and sug-gested that sodium chromate diffused through thecell membrane and was then bound firmly. Whenthe dotted line curve of Figure 1 representingcounts remaining in the saline solution was plottedon semi-logarithmic paper, the points approximateda straight line (Figure 6). The relation may beexpressed by:

C-= CTe\t,where Cs = counts remaining in saline phase at

time "t"CT = total counts in the systemA = rate of binding of chromium by the

red cellst = time in minutes.

1610


Adherence to this standard equation for firstorder reactions indicated that under the condi-tions of the experiment, i.e., excess of red cellscompared to chromium concentration, the rate ofdisappearance of chromium from the saline wasproportional to the concentration of chromium re-maining in the saline. The slope of the line wasan index of the rate of binding of chromium bythe red cells. The fall of activity of the saline to10%o of the initial level in two hours suggestedfirm binding of chromium within the red cells.Two %o of the chromium remaining in the salinewas bound per minute, as computed from Figure 6.The half-time of the chromium binding was 38minutes. These findings were typical of five suchexperiments performed under the same condi-tions.

C. Fractionation of red blood cells presiouslytagged with Na2Cr51O4

To ascertain what component of the erythrocytecontained the radioactivity, red blood cells pre-viously tagged with Na2Cr5104 were fractionatedinto stroma-free hemoglobin, globin HCl, hemin,and washed stroma 9 (10-12) and the radioactivity

9 Courtesy of Dr. R. B. Pennell.

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TABLE II

Fractionation of red blood cells previouslytagged with Na2Cr"I04

Counts calculatedon the basis of Per-cent

Blood fraction 1 cc. packed red cells of counts

per centRed blood cells 19,700Hemoglobin (stroma-free) 19,150 97

Globin HCI 15,400 78Hemin 0 0

Washed stroma 398 2

of the intact red cells and the red cell fractionswas compared. The counts were calculated onthe basis of 1 cc. of packed red cells 10 (Table II).

The activity of the hemoglobin (stroma-free)was 97%o of that of the red cells, indicating thathemoglobin was the important factor in red cellbinding (Table II). The recovery of 78%o of theactivity in the globin fraction and only a trace inhemin suggests binding of the chromium by theglobin portion of hemoglobin. (Approximately19%o of the activity was lost in fractionating thehemoglobin to globin and hemin.) The presenceof only 2%o of the radioactivity in the washedstroma confirms the importance of hemoglobin inred cell uptake.

When stroma-free hemoglobin was preparedfrom red cells previously tagged with Na2Cr5104and then dialyzed against saline for three days,68.5%5b of the radioactivity remained with thehemoglobin within the bag. The tagged hemo-globin, moreover, retained its activity after 24hours of mixing with a combination of anionic andcationic exchange resins capable of removing allof the chromium present.'1

D. Uptake of radioactive chromium by red cellfractions

The mechanism of erythrocyte uptake was in-vestigated further by the addition of radioactivechromium to the stroma and hemoglobin fractionsof red cells (Table III). The quantitative rela-tionships of radioactive chromium to intact red

10 The weight of hemoglobin was taken as 98%7o of theweight of dried red cells, and the stroma as 2%o. Sincehemin with a molecular weight of 615 constitutes 9.05 X10' X weight of hemoglobin (molecular weight 68,000),the weight of globin was considered essentially the same

as hemoglobin.11 Ionac C240, A-300 (Cyanamid Company), Dowex

and Amberlite.

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T I M E IN MINUTES


1611

SEYMOUTRJ. GRAY AND KENNETHSTERLING

TABLE III

Uptake of radioactive chromium by red cellfractions

Counts added to the Counts bound by theRed cell fraction Form of isotope added pH after dialysis fraction present in 1 cc. of fraction present in 1 cc. of Per cent uptake

packed red cells packed red cells

Stroma Na2Cr5lO4 5,399 158.5 2.9Hemoglobin Na2Cr51O4 7.2 6,136 1,388 22.6Hemoglobin Na2Cr5104 4.5 6,360 2,088 32.8Hemoglobin Cr61Cl3 4.5 7,840 4,528 57.8

cells described previously (Figure 1) were es-sentially maintained in all the experiments witherythrocyte fractions.

The stroma, washed free of hemoglobin, did notbind significant amounts of chromium (Table III).The addition of 53 y of chromium as Na2Cr51O4 tothe stroma content of 1 cc. of packed red cells(7 mg. of stroma) resulted in the uptake of only2.9%o of the counts.

The binding capacity of the hemoglobin for theanionic and cationic forms of chromium was stud-ied by determining the radioactivity of taggedhemoglobin solutions after dialysis.

Radioactive chromium (Na2Cr510O and Cr5lCl)was added to 7%o hemoglobin solutions in concen-trations of 150y of chromium per gram of hemo-globin. The solutions were mixed at room tem-perature for two hours and then dialyzed in thecold for three days with daily changes of dialysate.The solution tagged with Cr51C13 was dialyzedagainst acetate buffer at pH 4.5 to prevent pre-cipitation of Cr(OH)8, and the hemoglobin solu-tions containing Na2Cr5104 were dialyzed againstthe same buffer at pH 4.5 and against saline at pH7.2.

Hemoglobin, in contrast to stroma, revealed asignificant affinity for radioactive chromium (TableIII). It is of considerable interest that the uptakewas greater with Cr5lCl than with Na2Cr51O4,emphasizing again that the inability of Cr51C15 totag the intact red cell is a reflection of the relativeimpermeability of the red blood cell to cations.

COMMENT

A marked affinity of the red cell for anionichexavalent chromium (Na2Cr5104) has been dem-onstrated both in vitro and in uivo. The site oftagging appeared to be within the hemoglobin ofthe erythrocyte, in particular the globin fraction;only 2%o of the counts were recovered in the

washed stroma. Although the intact red cell didnot take up a significant amount of cationic triva-lent chromium (Cr5lCl) in vivo or in vitro, thehemoglobin solution was capable of binding 57.8%oof the Cr51C18 added to it. The failure of Cr51C15to tag the intact erythrocyte is attributed to therelative impermeability of the red cell membraneto cations.

Comparison of the binding capacity of hemo-globin for cationic trivalent and anionic hexavalentchromium, moreover, revealed a significantlygreater uptake of cationic Crl5CI5. In view of therapid and firm binding of anionic Na2Cr51O4 bythe erythrocyte, it is suggested that anionic hexa-valent chromium diffuses readily through the cellmembrane and is bound by the hemoglobin, prob-ably after reduction to the cationic trivalent statewithin the red cell.

In the foregoing experiments with red cellfractions, the hemoglobin was not necessarily freeof other protein components of the red cell, includ-ing the enzymes. It was, however, considered un-likely that enzymes played a major role in the ac-tual binding by the red cell. In the usual red celluptake studies, 120-170y of chromium were addedper gram of hemoglobin, resulting in an uptake of0.13-0.23 mole of chromium per mole of hemo-globin. It was not believed likely that minutequantities of enzymes could bind this much chro-mium, but a possible enzymatic reduction of hexa-valent to trivalent chromium within the erythro-cyte was not excluded.

Further studies of the metabolism of chromiumand its distribution in the body are in progress.

SUMMARY

1. A new biological tracer, radioactive chro-mium (Cr51) with a half-life of 26.5 days, wasfound to tag both red blood cells and plasmaproteins.

1612


2. The anionic hexavalent form of the isotope(Na2Cr5104) labelled red blood cells while thecationic trivalent form (Cr5"Cl3) was firmly boundby plasma proteins.

3. The addition, in vitro, of 86 -y of chromium asthe chromate to a 5 cc. suspension of red cells insaline resulted in an uptake of 80-90%o of theradioactivity within two hours.

4. The intravenous injection of Na2Cr5O4 indogs resulted in a rapid tagging of the red cellswhich retained their radioactivity for a prolongedperiod, while the plasma activity fell rapidly.

5. Washed red cells, tagged with Na2Cr5104 invitro, were injected intravenously into dogs andretained their activity without significant loss tothe plasma for approximately 24 hours.

6. The site of tagging of the erythrocyte withNa2Cr5104 appeared to be the globin portion ofhemoglobin.

7. Hemoglobin demonstrated a significantlygreater binding capacity for Cr51C13 than forNa2Cr5104.

8. Cationic trivalent chromium, in contrast toanionic hexavalent chromium, was not taken upby the red blood cells, presumably because of therelative impermeability of the cell membrane tothe cation.

9. It is suggested that anionic hexavalent chro-mium diffuses through the red cell membrane andis bound by hemoglobin within the cell probablyafter reduction to the cationic trivalent state, re-sulting in the firm tagging of the erythrocyte.

10. An albumin solution labelled with Cr5Cl8contained 29.9 microcuries per gram after dialysisand did not differ from native albumin by ultra-centrifugal and electrophoretic analyses.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the helpful sug-gestions of Doctors Arthur K. Solomon, Charles V.Robinson, Isadore S. Edelman, Walter L. Hughes, Jr., andJohn L. Oncley.

Harold A. Papazian and Madison B. Whittier renderedvaluable technical assistance.

BIBLIOGRAPHY

1. Monier-Williams, G. W., Trace Elements in Food.John Wiley & Sons, New York, 1949, pp. 439-443.

2. Gray, S. J., Mitchell, A. M., and Sterling, K., Thecolorimetric determination of trace amounts ofchromium in tissues. To be published.

3. Bradt, H., Gugelot, P. C., Huber, O., Medicus, H.,Preiswerk, P., and Scherrer, P., Die radioaktiveUmwandlung des Cr'. Helvet. physiol. acta, 1945,18, 252.

4. Bale, W. F., Yuile, C. L., De La Vergne, L., Miller,L. L., and Whipple, G. H., Hemoglobin labeled byradioactive lysine; erythrocyte life cycle. J. Ex-per. Med., 1949, 90, 315.

5. Gray, S. J., and Sterling, K., Determination of cir-culating red cell volume by radioactive chromium.Science, 1950, 112, 179.

6. Sterling, K., and Gray, S. J., Determination of thecirculating red cell volume in man by radioactivechromium. J. Clin. Invest., 1950, 29, 1614.

7. Gustavson, K. H., Some protein-chemical aspects oftanning processes, in Advances in Protein Chem-istry, edited by Anson, M. L., and Edsall, J. T.Academic Press, New York, 1949, Vol. 5, p. 354.

8. Hevesy, G., Koster, K. H., Sorensen, G., Warburg,E., and Zerahn, K., The red corpuscle content ofthe circulating blood determined by labeling theerythrocytes with radio-phosphorus. Acta med.Scandinav., 1944, 116, 561.

9. Reeve, E. B., and Veall, N., A simplified method forthe determination of circulating red-cell volumewith radioactive phosphorus. J. Physiol., 1949, 108,12.

10. Anson, M. L., and Mirsky, A. E., Protein coagula-tion and its reversal; preparation of insoluble globin,soluble globin and heme. J. Gen. Physiol., 1930,13, 469.

11. Bernstein, S. S., Jones, R. L., Erickson, B. N., Wil-liams, H. H., Avrin, I., and Macy, I. G., A methodfor the preparation of posthemolytic residue orstroma of erythrocytes. J. Biol. Chem., 1937-38,122, 507.

12. Pennell, R. B., Smith, W. E., and Werkheiser, W. C.,Preparation of hemoglobin solutions containinghemoglobin reducing enzymes. Proc. Soc. Exper.Biol. & Med., 1947, 65, 295.

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