THE FLUOROPHOTOMETRIC DETERMINATION OF URANIUM IN BIOLOGICAL MATERIAL*

BY W. F. NEUMAN, R. W. FLEMING, A. B. CARLSON, AND N. GLOVER

(From the Division of Pharmacology and Toxicology, Department of Radio&y, School of Medicine and Dentistry, The University of Rochester,

Rochester, New York)

(Received for publication, December 5, 1947)

For a comprehensive study of the excretion and distribution of uranium in animals, a sensitive and accurate analytical procedure for the determina- tion of uranium in biological material was necessary. Among the existing methods were a permanganate titration (l), a magneto-optic procedure (2), a radioactivity method (3), and the fluorescent technique of Hoffmann (4).

The fluorescent method was chosen for further study because it offered three advantages: specificity, sensitivity, and economy of equipment. In addition, Bloor’sl investigations had indicated that the accuracy of the fluorescent techniques of Hoffmann (4) could be improved.

Briefly, Bloor’s procedure was as follows: The uranium sample was fused with sodium fluoride in a platinum dish, the fusion illuminated with ultra- violet light, and the intensity of fluorescence determined by measuring the depth of a methylene blue solution required (Duboscq calorimeter) t,o ab- sorb the emitted light. Since the sample was compared with a blank in total darkness, it was necessary that the investigator’s eyes be dark- adapted.

To facilitate the measurement of fluorescent intensities and to increase the accuracy of the measurement, a fluorophotometer was constructed. This instrument permitted the accurate analysis of quantities of uranium as low as 0.0005 y. However, the presence of even small amounts of extraneous inorganic material was found to give erroneous results, usually by quenching the fluorescence (5). Therefore, it became necessary to dc- vise a procedure for the isolation of uranium to make the method applic- able to biological specimens.

The affinity of protein for uranium, even at its isoelectric point, served as the basis of the isolation procedure developed. In conjunction with electrolysis for the removal of heavy elements other than uranium, the isolation procedure permitted the accurate analysis of uranium in concen- trations less than 1 part per million of fresh tissue by fluorophotometry.

* This paper is based on work performed under contract No. W-7401-Eng-49 for the Manhattan Project at the University of Rochester.

1 Bloor, W. R., unpublished results. 41

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42 DETERMINATION OB URANIUM

EXPERIMENTAL

Fluorometrg

Fluorophotometer-Recently, while this report was in preparation, a re- port appeared (6) describing an apparatus similar in many respects to that presented here. The instrument and procedure (6), however, lack the sensitivity and precision required for biological study.

Sample Holder-Bloor’s technique’ of fusing the uranium-sodium fluoride flux in platinum foils was adopted. The sample cup was made of 0.13 mm. (0.005 inch) platinum foil, 20 by 24 mm., having a circular indenta-

la TO? SIDE

VIEW VIEW

PLATlNUM CUP

KNOB

((? SPR;NG STOP

NOTCHES

SAMPLE HOLDER

FIG. 1. Perspective drawings of the platinum fusion cup and sliding brass sample holder.

tion 11 mm. in diameter and approximately 1.5 mm. deep at the center, as indicated in Fig. 1. In the fluorophotometer, the foils were brought int.0 reading position by means of a sliding brass bar (Fig. 1) which accommo- dated five samples and was equipped with a spring stop mechanism to facilitate centering of the specimens.

IZZumination-Several types of ultraviolet sources were tested: General Electric bulb BH4, Mineralight model V41, a mercury arc, and others. All of these activated the uranium-sodium fluoride flux to a bright, yellow-green fluorescence, indicating that the wave-length of the ultraviolet radiation was not critical. For the fluorophotometer, the Conti-Glo lamp (Conti-

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NEUMAN, FLEMING, CARLSON, AND GLOVER 43

nental Lithograph Company, Cleveland) equipped with a clear glass bulb (General Electric, AH4) was the most satisfactory. To minimize varia- tions in radiation output, the lamp was supplied with a constant voltage transformer (Sola, model 30808). Corning Filter 5874 (heat-resistant) and several thicknesses of Filter 9863 were interposed between the lamp and sample holder to remove visible radiation. A cooling system, a fan or water jacket, removed the heat generated by this lamp.

Photometer-The wave-length of the emitted fluorescent light has been reported (5) to be principally 555 rng. To isolate this wave-length, Corn- ing Filters3484 and 9780were placed above the illuminated sample. Above these filters was fixed a light-tight box housing the phototube, as indicated in Fig. 2. The current of the phototube, after amplification, operated a

U.V. LIGHT

/

/LIGHT - TIGHT

HOUSING

-PHOTOTUSE

>CORNING FILTERS

U.v: FILTERS

FIG. 2. Section drawing of the fluorescence chamber. For details, refer to the text.

galvanometer. The wiring diagram of the amplifier is given in Fig. 3. It is a slight modification of a standard D.C. amplifier circuit (7). By the use of three separate grid resistors of different resistances and an optional galvanometer shunt, the analytical range could be varied 1000-fold. Since the phototube currents were very small, the stability was improved by keeping the connections to the control grid of the amplifier tube as short as possible. Accordingly, the amplifying tube and grid resistors mere mounted in the phototube housing. It was necessary to keep a fresh sup- ply of silica gel in the chamber to avoid instability caused by humidity. In operation, the circuit was adjusted to give a 0.6 volt drop across the filament and a plate current of 100 microamperes.

Procedure-Because of the small amounts of material taken for analysis,

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44 DETERMINATION OF URANIUM

all samples were prepared in solution form, adjusted to a range in con- centration of uranium from 0.01 to 20 y per ml. To prevent the adsorp- tion of the small quantities of uranium on the walls of vessels, etc., the solutions were made at least 0.1 N with respect to free nitric acid.

A 0.1 ml. aliquot of the solution to be analyzed was pipetted into a platinum cup. The cup was placed on an asbestos board and the contents evaporated to dryness on an electric hot-plate. If organic material was present, the temperature was raised until charring took place. The as- bestos board and cup were then removed and allowed to cool. Approxi-

P2 R4 R8 Re

R8

FIG. 3. Wiring diagram of amplifier circuit. !I’,, No. lLN5; TZ, No. 929, base re- moved; BI, 24 volt storage battery; Bz, 22.5 volt B battery; St, single pole, single throw toggle switch; Sz, special switch; Ml, 200 microampere meter; ilIt, galvanom- eter, 6 microamperes full scale; PI. 60 ohms; Pz, 200 ohms; Pa, 10,000 ohms; Pa, 1000 ohms (wire-wound); RI, 100,000 megaohms; RP, 10,000 megaohms; R8, 1000 mega- ohms; Rd, 200 ohms; Rs, 82 ohms; Re, 5.6 ohms; R7,lOOO ohms; Rs, 220 ohms.

mately 80 mg. of sodium fluoride (Baker and Adamson, reagent grade) were added to the cup and the sample twice fused and read in the fluoro- photometer.

Fusion-The reading given by a known quantity of uranium was rela- tively unaffected by some variation in the amount of sodium fluoride em- ployed which made it convenient to measure the fluoride by means of a small scoop which delivered 80 f 20 mg. This quantity gave best results with the size of the platinum cup previously described. Two samples were fused simultaneously by placing them side by side on a nichrome wire rack above a Meker burner.

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NEUMAN, FLEMING, CARLSON, AND GLOVER 45

The use of a large diameter pipe (1.5 inch) connected directly to the gas main, or to a constant pressure head, obviated changes in the gas pressure, which insured an even fusion temperature.

Reading-All samples were analyzed in quadruplicate. A convenient set would, for example, consist of

1 blank x4= 4 3 standards X 4 = 12

20 samples X4=80 - 96 fusion cups

Such a test took one operator 6 to 7 hours working time. Before a series of readings, the fluorophotometer was given about $ hour

to reach thermal equilibrium. By adjustment of the zero controls, the galvanometer was set to give a reading of 10 for the first blank. This blank was kept in the sample holder and used as a reference point for the reading of all subsequent samples.

Contamination Control-To analyze extremely small quantities of ura- nium, it was necessary to exercise a rigorous control over operating con- ditions. No dry uranium compounds were permitted in the analytical room. All glassware for quantitative use was left overnight in cleaning solution and, immediately before use, rinsed thoroughly in 1 per cent nitric acid followed with distilled water. The platinum cups were boiled in 1 per cent nitric acid and rinsed several times with distilled water, reshaped, and kept under distilled water until ready for use. Immediately before the samples were to be analyzed, the cups were again boiled with dilute nitric acid and rinsed several times with distilled water. Extreme care was exercised during the period in which samples were pipetted, dried, and fused.

Results-A linear relationship between galvanometer deflection and the quantity of uranium present was observed throughout the analytical range (0.001 to 3.0 7).

Analysis of Pure Solutions of Uranyl Nitrate-To obtain a statistical analysis of the accuracy of the method, a series of unknowns was prepared by a disinterested person and analyzed in the routine manner. The results of this study ase presented in Table I.

It was shown that the absolute error of the method increased with in- creasing quantities of uranium, .giving a fairly consistent per cent error. Because of this fact, and because of convenience of the term “per cent error,” the standard deviation was calculated as a percentage. The results of the statistical analysis of the data in Table I are presented in Table II.

It can be seen from Table II that the standard error of the fluorophoto-

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46 DETERMINz4TION OF URANIUM

metric method, with pure solutions of uranium, is less than 3 per cent. Of twenty-seven samples analyzed, the largest error observed was 10 per cent.

Analysis of Tissue Specimens-Poor recoveries were observed when known quantities of uranium were placed in porcelain crucibles. Accord- ingly, platinum ware was employed. All tissue specimens were dry-ashed in a muffle furnace at temperatures not exceeding 750”. At lower tempera- tures, ashing was incomplete; at higher temperatures, the ash of some tissues (urine especially) tended to crawl out of the crucibles.

The ash of all tissues reduced the intensity of the fluorescence of uranium. Typical of the results obtained are the data presented in Table III in which

TABLE I

Results of Fluorophotometric Analysis of Uranyl Nitrate Solutions

Contained Found Contained Found

Y Y Y Y

0.228 0.238 0.040 0.040 0.228 0.229 0.040 0.042 0.228 0.229 0.040 0.042 0.228 0.225 0.0228 0.021 0.170 0.170 0.0228 0.023 0.170 0.174 0.0228 0.023 0.170 0.175 0.0228 0.023 0.170 0.170 0.020 0.020 0.160 0.159 0.020 0.020 0.160 0.152 0.020 0.020 0.160 0.163 0.010 0.011 0.080 0.083 0.010 0.011 0.080 0.078 0.000 0.000 0.080 0.077 0.000 0.002 0.080 0.079 0.000 0.000

fluorescence was reduced approximately 20 per cent by microgram quanti- ties of either bone ash or ferric iron.

When applied to the analysis of biological samples, a 17 per cent in- hibition was observed when the concentration of uranium in bone ash was 2 parts per thousand. Obviously, to determine uranium in lower concen- trations (1 p.p.m.), some method for the isolation of uranium was neces- sary.

Isolation of Uranium from Biological Material

The observations of Dounce2 that uranium forms a. stable complex with proteins served as the basis for further experimentation, which led to the development of the isolation procedure described below.

2 Dounce, A. L., unpublished results.

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NEUMAN, FLEMING, CARLSON, AND CLOVER 47

Procedure-The tissue ash was dissolved in a minimum of 2 N hydro- chloric acid and an aliquot containing 50 to 100 mg. of ash transferred to a centrifuge tube graduated at 10 and 30 ml. 2 drops of brom-cresol green indicator were added and the solution neutralized to pH 4.5 (green color) with 1 N sodium hydroxide. 3 ml. of 1 N sodium acetate-acetic acid buffer (pH 4.5) and then a solution containing 100 mg. of one of the protein preparations described below were added. The resultant solution was diluted to 30 ml. and, after thorough mixing, the tube was immersed in a hot water bath at 80” for 45 minutes. The coagulum which formed was separated by centrifugation, washed once with fresh 0.1 N acetate buffer

TABLE II

Statistical Analysis of Accuracy of Fluorophotometric Method

No. of samples.. . . . . . . . . . . . . . . . . _. Concentration range, p.p.na......................................................... Coefficient of correlation.. . . . . . . . . . . . .

Rangeofrecovery............................................. Meanrecovery................................................. Standarddeviation............................................

“ error................................................

27 0.1-2.9 0.999

)Lw cent

92-110 101

3.9 2.6

TABLE III Inhibition of Uranium Fluorescence

Bone ash, 0.2 y U per sample Ferric chloride, 0.1 y U per sample

Added Inhibition

Y per cent

0.0 0 0.1 0 1.0 5

10.0 11 100.0 17

Y 0.0 0.4 0.8 4.0

-

-

Inhibition

)l?Y ctmt

0 5

12 25

(pH 4.5), and again centrifuged. The washed precipitate was dissolved in concentrated nitric acid and made up to a volume of 10 ml. 0.1 ml. ali- quots were then taken for fluorophotometric analysis. “Blank” deter- minations were carried through the procedure with each set of samples as a check against contamination of reagents.

E$ect of Protein Concentration-In the preliminary studies, crystalline egg albumin was employed. Because of the expense and trouble involved in its preparation, the crystalline protein was discarded in favor of com- mercial egg albumin, precipitated twice with ammonium sulfate, and di- alyzed free of salt. Later, it was found that a preparation of bovine al-

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48 DETERMINATION OF URANIUM

bumin (Fraction V from bovine plasma, Armour Laboratories, Chicago) could be used directly without preliminary purification or manipulation.

In Table IV are presented the results of two experiments designed to determine the effect of varying quantities of protein on the recovery of

TABLE IV

Effect of Varying Protein Concentration on Recovery of 10 7 of Uranium

Concentration of protein

I Test 1

n&g. per mt. 0.63 0.85 1.25 1.58 1.88 2.00 2.50 3.00

per cent

62 72 86 88 91 89 94

102

Test 2

per cent

64 78 83 93 92

104 100

IL 0

. 0 l 00

so- 0 t

E

6 0

& 0

85-

0 ii 0 0

:

l?J 0

i-5 80

a. 4.0 4.5 5.0 5.5

PH OF ACETATE BUFFER

FIG. 4. Effect of pH on the pre&itation of the uranium-protein complex

uranium. It is evident that, with the volumes employed, the protein concentration should be in excess of 2.5 mg. per ml.

Eflect of pH--Below pH 4.0 and above pH 5.0 it is difficult to obtain the complete coagulation of the protein which is essential for good recoveries of uranium. In Fig. 4 are presented the results of studies designed to determine the optimal pH for recovery of uranium. These data indicate

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NEUMAN, FLEMING, CARLSON, AND GLOVER 49

that pH 4.5 is optimal, but a change of f 0.2 pH unit does not seriously reduce the recovery.

Solubility of Ash-Bone ash was assumed to be the least soluble ash encountered in biological material. Accordingly, “solubility” experiments were conducted to determine the maximum quantity of bone ash soluble in acetate buffer at pH 4.5. This was found to be approximately 4.0 mg. per ml. However, as is indicated in Table V, heating, which was necessary to coagulate the protein, reduced the “solubility” to 3.3 mg. per ml. Since a volume of 30 ml. was customarily used, the maximum quantity of ash taken for analysis was 100 mg.

Eficiency of Isolation-An experiment was conducted to determine how completely uranium was separated from tissue ash and to determine how much tissue ash carried through the isolation. The standard procedure

TABLE V Effect of Temperature on Solubility of Bone Salt

- denotes complete “solubility,” + denotes precipitation.

Tiie

min. “C.

0 55 18 60 22 65 28 70 36 76 43 SO 73 90

Temperature i

3.3 mg. per ml.

Concentration of bone salt

3.5 mg. per ml. 3.8 mg. per ml. 4.0 mg. per ml.

- -

- -

- -

- - -

- +

+ + +

+ ++ +++

was applied to two solutions of pure uranyl nitrate (10 7) and to six solu- tions to which 100 mg. of bone ash had been added. The ash content of the resulting washed protein precipitates was determined. In the ex- perimental samples, containing 100 mg. of bone ash, 4.4, 4.1, 4.4, 3.9, 4.0, and 3.7 mg. (average 4.1 mg.) of ash were found, and in the control samples, containing no bone ash, 5.4 and 5.6 mg. (average 5.5). These results indicated that the ash content of the uranium-protein complex is less in the presence of bone salt than in its absence. This seemingly para- doxical result is best explained by the fact that, in the presence of bone ash, the protein coagulum is more dense and occupies a smaller volume than when salt is absent (familiar phenomenon of t,he salting-out of pro- teins). Consequently, a smaller volume of wash solution containing 8 mg. per ml. of sodium acetate was retained by the protein coagulum when

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50 DETERMINATION OF URANIUM

bone salt was present. Under the circumstances, it seems likely that very little, if any, bone ash carries through the isolation procedure.

TABLE VI

Results Obtained with Protein Isolation

Bone samples ____-- ‘-

No. of samples.. . . _. _. _. 16 Concentration range, p.*.nz.. . l-1000

-- -

per cent

Meanrecovery............................. 96 Standard deviation.. . . . / 9.1 Range of recovery.. . .I 70-104

TABLE VII

/ I

Pure solutions

16 0.01-10

per cent

92 7.0

77-102

Soft tissues ___-

28 0.5-50

per cent

90 5.8

83-105 -

Recoveries of Uranium from Liver, Spleen, and Blood Both by Electrolysis and by Protein Isolation

No. of samples.. . . . . . . . . . . . Concentration range, p.p.m.. . . .

17 2-25

per cent

Mean recovery................................................ 92.5 Standard deviation......... ..___....................... 7.7 Range of recovery. . 75103

TABLE VIII

Over-All Summary of Analytical Results I

Pure solutions

No. of samples. 16 Concentration range, f5.p.m.. 0.01-10

-___

per cent

Mean recovery....... .._................ 92.0 Standard deviation......................... 7.0 I Range of recovery.......................... 70-102

_____

Tissues

61 0.5-1000

per cent

92.3 7.0

70-105

Results--The results obtained from the analysis of pure solutions of uranium and of samples of bone and soft tissue3 to which known quantities of uranium were added are presented in Table VI.

Not included in Table VI are the results obtained with liver, blood, and

3 The following tissues were taken: kidney, bladder, genitals, heart, lung, stolnach and contents. intestines, leg muscle, and skin and hair.

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NEUMAN, FLEM.INQ, CARLSON, AND BLOVER 51

spleen, from which recoveries as low as 38 per cent were observed. This difficulty was overcome by electrolyzing such samples in a small mercury cathode cell prior to isolation of the uranium by the protein met,hod.

To the HCl-ash solution, perchloric acid was added to give a final con- centration of 3 per cent and current (10 volts, D.c.) passed through for 1 hour. The electrolyzed solutions were then subjected to the isolation procedure described above.

With the electrolysis, good recoveries were obtained from liver, spleen, and blood, as indicated in Table VII.

The over-all average recoveries are presented in Table VIII. It is clearly evident from these data that uranium can be recovered from tissue equally well as from distilled water. Although the recoveries were not strictly quantitative (92 per cent), they were consistent (standard devia- tion, 7 per cent) and within the accepted limits of biological variation.

DISCUSSION

The fluorophotometric method for the determination of uranium offers extraordinary specificity, sensitivity, and, for the quantities analyzed, unusual accuracy. The ultimate limit of sensitivity has not been deter- mined. If higher value grid resistors are employed, the fluorophotometer can be adjusted so that uranium can be detected in even smaller quantities than reported above. In our experience, slight impurities in the best grade of reagent sodium fluoride set a lower detection limit of 0.0005 y of uranium per fusion cup. Translated in terms of tissue, this is equivalent to 0.005 y per gm. of fresh, soft tissue.

The use of organic reagents in microanalytical procedures is gaining in popularity. Proteins, however, represent a group of organic reagents rarely employed. The point of interest in this particular application is the fact that the combination with uranium is practically quantitative at the isoelectric point. Good evidence2 has been obtained that it is a direct combination with protein and not the usually observed coprecipitation phenomenon. Since, at the isoelectric point, electrovalent combinations with both cations and anions are minimal, it is probable that the uranium- protein complex which forms is a covalent type of combination. It is possible that the technique employed in the development of the isolation procedure may be applied to (CL) the isolation of other metals and (b) the evaluation of the type of linkage between protein and various ions.

SUMMARY

With a protein isolation, and with electrolysis when necessary, uranium can be quantitatively determined fluorophotometrically in tissue samples in concentrations as low as 0.005 y per gm. of soft tissue.

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52 DETERMINATION OF URANIUM

BIBLIOGRAPHY

1. Gustafson, G., and Koletsky, S., AA. Int. Med., 74, 416 (1944). 2. Jones, H., and Goslin, R., Am. J. Ph@oZ., 105, 693 (1933). 3. Holman, R., and Douglas, W., Proc. Sot. Exp. Biol. and Med., 57, 72 (1944). 4. Hoffmann, J., Biochem. Z., 315, 26 (1943). 5. Nichols, E. I,., and Slattery, M. K., J. Optical Sot. America, 12, 449 (1926). 6. Gibb, T. R. P., Jr., and Evans, H. T., Jr., Science, 105, 72 (1947). 7. DuBridge, I,. A., Phys. Rev., 37, 392 (1931).

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and N. GloverW. F. Neuman, R. W. Fleming, A. B. Carlson

BIOLOGICAL MATERIALDETERMINATION OF URANIUM IN

THE FLUOROPHOTOMETRIC

1948, 173:41-52.J. Biol. Chem. 

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