TECHNIQUES FOR IN VIVO TRACER STUDIES WITH RADIOACTIVE CARBON”

BY HOWARD E. SKIPPER, CARL E. BRYAN, LOCKE WHITE, Ja., AND OLIVIA S. HUTCHISON

(From the Southern Research Institute, Birmingham, Alabama)

(Received for publication, November 7, 1947)

In view of the increasing importance of radioactive Cl4 in biological studies and the lack of extensive literature on methods for assaying this isotope in animal tissues and fluids, it is felt that such methods as have been found useful might be worthy of recording. Described herein are techniques for the collection of excretory products suspected of containing isotopic carbon from mice, quantitative oxidation of tissues and excretory products prior to activity assay, and an application of the method of Miller (1) with slight variations for assay of BaC03 resulting from the above oxidation.

EXPERIMENTAL

Metabolism Chamber for Collection of Excretory Products-In an attempt to carry out a balanced experiment in tracer biochemistry with carbon isotopes, it is of course necessary to include the respiratory system as a possibly important excretory route. This fact, along with such others as economy of carbon isotope and dilution factors related to animal weight, may influence one to choose the mouse as an experimental animal, at least in exploratory studies.

In order to measure the quantity and rate of excretion of active carbon by the respiratory route, it is necessary to utilize an absorption line in which the CO2 exhaled by the animal can be quantitatively absorbed for subsequent precipitation and weighing prior to radioactive assay. A sketch of the apparatus employed in our laboratory for this purpose is presented as Fig. 1. By means of the legend on the sketch, the operation of the chamber may be described as follows:

Room air is drawn through a wet test meter, A, and successively through absorption bottles containing 10 per cent sodium hydroxide, B, saturated barium hydroxide, C, and saturated sodium chloride, D. The carbon dioxide is removed from room air in B; the barium hydroxide absorption bottle, C, is used simply as an indicator of the adequacy of B. The satu- rated sodium chloride solution, D, is employed for equilibration of humidity

* This work was supported by grants from Mr. Ben May, Mobile, Alabama, and the American Cancer Society.

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372 TRACER STUDIES WITH RADIOACTIVE CARBON

in the mouse chamber, E, to approximately 75 per cent. Feces are collected on a wire screen; urine samples are collected in tube F. Water is supplied to the mouse from bottle G. The carbon dioxide exhaled by the test animal in the glass chamber E is absorbed in COs-free sodium hydroxide, 11. Absorption bottle I, containing saturated barium hydroxide, is used as an absorption efficiency indicator. Air flow is maintained at a constant rate of about 300 ml. per minute by means of a critical orifice, J, constructed of capillary tubing, one end of which has been constricted by fire polishing until the proper line air flow is obtained. A pressure drop across the orifice is maintained at greater than the critical pressure for air by means of a vacuum pump. This requires a pressure differential equal to or greater t,han 14.5 inches of mercury.

In view of the fact that the respiration of a 20 gm. mouse is of the order of 22 ml. per minute and the CO2 content of the expired air is approxi-

+VACUUY PUMP

FIG. 1. Apparatus for collecting respiratory carbon dioxide and other excretory products. A, wet test meter; B, C, and D, absorption bottles containing 10 per cent

sodium hydroxide, saturated barium hydroxide, and saturated sodium chloride; E, mouse chamber; F, urine collection tube; G, water bottle; H and I, absorption bottles containing COz-free sodium hydroxide and saturated barium hydroxide; J, orifice.

mately 4 per cent, it may be estimated that about 0.05 mole of carbon dioxide will be expired by a 20 gm. mouse in 24 hours.

Typical data, obtained with the absorption line, to collect COz expired by a 25 gm. mouse are given in Table I.

It is important to maint.ain an air flow in the mouse chamber great enough to prevent accumulation of COZ, which might affect respiratory rate. Excessively high flow rates, however, result in incomplete absorption. When short collection periods were used, we have increased our chamber air flow for a few seconds at the end of each period so that the time for reaching theoretical clearance of the chamber is very short as calculated by Silver’s equation t, = K (a/b) where 2 is the per cent of nominal con- centration attained in time t (minutes), K is a constant, depending on the desired degree of equilibration (4.6 for 99 per cent), a is the volume of the chamber, and b is the volume of air passing through the chamber each minute (2).

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SKIPPER, BRYAN, WHITE, AND HUTCHISON 373

Oxidation of Animal Tissues for Determination of Radioactive Carbon- Recause of the low energy radiation of Cl4 and absorption factors, it is impossible to carry out activity assays in tissues per se. This difficulty can be overcome by oxidizing the tissues to carbon dioxide and counting in this form or as barium carbonate. The procedure we have found satis- factory for the oxidation of tissues is an adaptation of the method of Van Slyke and Folch (3) for manometric carbon determination. The method depends on combustion in a mixture of chromic, iodic, sulfuric, and phos- phoric acids, and the preparation of the reagents has been described by these authors. In the present work, the carbon dioxide is not determined manometrically but is absorbed in sodium hydroxide solution and pre- cipitated as barium carbonate. The latter is weighed in order to calculate

TABLE I Carbon Dioxide Output of 86 Gm. Mouse

Absorption period

min.

O-10 10-20 20-30 30-60

hrs. l- 2 2- 3 3- 4 4- 5 5-6 6-24

Barium carbonate recovered

Per period

P. k-m.

0.1841 0.1841 0.1203 0.3044 0.1647 0.4691 0.4086 0.8777

0.6700 1.5477 0.6783 2.2260 0.6597 2.8857 0.6724 3.5581 0.6243 4.1824

10.0879 14.2703 -

Cumulative

the total carbon content of the tissues and is then reconverted to carbon dioxide for determination of the specific activity in the manner described in another section of this report. The reasons for this precipitation of COz and subsequent regeneration for activity assay are that (a) it is con- venient for different operators to carry out the oxidations and the activity assays, (b) it is desirable to save samples for future check, and (c) it is not feasible to allow the acid vapors from the tissue oxidation to enter the Geiger tube. The apparat.us for oxidation of tissues and absorption of COz is shown in Fig. 2. The procedure used is giyen as follows:

The sample and potassium iodat,e are placed in small flask E, carbon dioxide-free 10 per cent sodium hydroxide in absorption cell B, and barium chloride solution in tube D. The small tube A contains saOurated barium hydroxide solution as an indicator for completeness of carbon dioxide

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374 TRACER STUDIES WITH RADIOACTIVE CARBON

absorption. While a slow stream of nitrogen (or carbon dioxide-free air) is passed through the system, the combustion fluid is added slowly from funnel C. The flask is warmed gently and then gradually heated to boiling. The carbon dioxide formed by the oxidation passes into the absorption cell, B, through t,he sintered glass plate, P, which breaks it into fine bubbles. After the oxidation is complete, the apparatus is flushed with nitrogen and the sodium hydroxide is run through stop-cock d into tube D where the carbonate is precipitated as barium carbonate. The walls of B are carefully washed with water. Stop-cock a is opened to allow the washing of the inlet tube.

FIG. 2. Apparatus for tissue oxidation. A, t.ube containing saturated barium hy- droxide; B, absorption cell containing Cop-free 10 per cent sodium hydroxide; C, funnel; D, tube containing barium chloride solution; E, flask containing sample and potassium iodate; P, sintered glass plate; a, b, c, d, and e, stop-cocks.

The bottom of tube D is connected to the top of a small, tared, sintered glass funnel and the mixture in D filtered by suction through stop-cock e (which has a wide bore) with the careful exclusion of air. Particles of the precipitate are washed from D onto the funnel and the latter is washed until free of alkali. The funnel containing the barium carbonate is dried for a short time in an oven at 110’ and then in a vacuum desiccator before weighing.

The entire operation is carried out under a hood to avoid breathing any of the carbon dioxide which might possibly escape.

Results from the oxidation of samples of known composition are recorded in Table II, while those from the tissue combustions are given in Table III.

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SKIPPER, BRYAN, WHITE, AND HUTCHISON 375

Determination of Radioactive Carbon-The specific activity of barium carbonate samples is determined by a slight modification of the method of Miller (l), in which the carbonate is converted to carbon dioxide and the latter introduced into the counter tube.

The apparatus is diagrammed schematically in Fig. 3. Essentially, the procedure involves the liberation of carbon dioxide from the barium car- bonate by addition of perchloric acid, the removal of water by condensation at about -8O”, the collection of the carbon dioxide by condensation at. about - 195”, the volumetricmeasurement of the quantity of carbon dioxide, the addition of a predetermined quantity of carbon disulfide, the condensa-

TABLE II Analysis of Samples of Known Composition

Weight of B&O: sample ombustion

Tie?

Bensoic acid

Cystine

Fatty acidt

“~!xf EIPX C

Calculated Found -- -

0.:94 0 .:28 mtn. per cent min.

0.3320 0.24 10-12 0.0268 0.3033 0.2987 1.50 lo-12 0.0250 0.2830 0.2801 1.02 lo-12 0.0255 0.1257 0.1238 1.51 8 0.0271 0.1336 0.1361 1.87 8 0.0248 0.3088 0.2950 4.46 5 0.0309 0.3844 0.3639 5.33 6 0.0307 0.3828 0.3687 3.68 6 0.0240 0.2991 0.3055 2.14 7 0.0264 0.3329 0.3243 2.58 7 0.0330 0.4113 0.4068 1.10 8 0.0285 0.3552 0.3495 1.60 10

- - * Period of heating. t Neo-fat-l-65, Armour and Company; stearic acid 90 per cent, oleic acid 4 per

cent, palmitic acid 6 per cent. Microanalysis showed 75.8 per cent carbon.

tion of the resulting mixture in the Geiger-Miiller tube, the thorough mixing of the gases after evaporation, and the measurement of the counting rate.

Certain details of the apparatus warrant further mention. The water reservoir, Q, for the carbon dioxide generator, 0, is provided for ease in rinsing down the walls after each generation; if the water is drawn in while the generator is under reduced pressure, thorough rinsing occurs. The water trap, N, is of concentric tube design. Its outer jacket is removable at a ground glass joint for convenient drainage of collected water. The volume of the “dose? bulb, K, is such that, when it is filled with carbon disulfide vapor in equilibrium with the liquid at the ice point, it contains the proper amount to produce 2 cm. of carbon disulfide pressure in the counter

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TABLE III

Oxidation of Mouse Tissues Tissue A, normal mouse, 25 gm.; Tissues B and C, mice, 25 gm. each, injected

with radioactive urethan (35 mg., 2.49 microcuries).

Tissue.. . A B C T-

1. Blood ............ 2. Spleen. ........... 3. Adrenals. ......... 4. Kidneys ........... 5. Liver .............. 6. Testes ............ 7. Thymus ........... 8. Heart ............. 9. Lungs .............

10. Lymph nodes ...... 11. Brain. ............ 12. Muscle. ........... 13. Stomach and small

intestine. ....... 14. Large intestine .... 15. Skin and hair. ....

16. Carcass.

17. Bone. 18. Urine (bladder).

“ (collected). “ “

Weight C :ombustion time Carbon

m. sm.

0.790 0.935 0.230 0.130 0.010 0.010 0.385 0.310 1.365 1.470 0.150 0.160 0.040 0.020 0.100 0.100 0.135 0.140 0.040 0.020 0.390 0.430 0.070 0.240

1.855* 1.440” 2.99t 1.27 0.50 0.46 0.76 9.7211 1.31 8.41

1.900* 1.650* 3.141 1.72 1.42

8.2Oli 2.76 1.80 1.80 1.84

1 2

0.030 0.440

-

lw.

1.640 3.090 3.010 1.350 1.500 3.140 I.020 3.090 3.140 3.060 3.430 3.530

1.430* 2.750* 3.24s 3.45

9.200

1.400 0.670

- B

h.

25 10 5

10 20 10 8

10 10 10 11 10

rim.

30 10 5

10 30 10 7 7 7 7

15 6

25 30 20 30

30 28

16 18

18 60 45 45 45

3 10

c tin.

15 5 5

10 30 10 6 8

10 6

10 11

30 40

20

10

A

er cent 11.5 10.8 13.6 11.9 18.0 9.9

11.9 14.0 13.2 16.2 10.8 16.9

10.9 11.0

24.8 26.2

13.9

B C

er mm ‘er cent

11.6 10.1 13.3 13.9 18.1 33.3 9.6 18.4 6.1 14.5 8.7 10.4

23.2 30.6 14.8 18.3 13.5 13.7 23.6 16.7 8.4 13.0

12.3 12.0

4.3 7.4

22.0 30.4

11.2 10.2

29.2

10.8

10.0 7.7 3.0 2.6

3.4

* Covered with 85 per cent phosphoric acid before combustion fluid was added. t Divided into four portions for oxidation. The ebullition of carbon dioxide was

beyond control in the oxidation of the first two portions. The third and fourth parts were covered with 85 per cent phosphoric acid before combustion fluid was added; oxidation proceeded smoothly then.

$ Divided into two parts for oxidation; each was covered with 85 per cent phos- phoric acid before combustion mixture was added.

$ Only a small portion oxidized; covered with 85 per cent phosphoric acid before oxidation.

I/ Oxidized in two portions. Samples covered with 85 per cent phosphoric acid before combustion mixture was added. Reaction violent in the case of the larger sample because of its extreme size; absorption of carbon dioxide incomplete.

1 Oxidized and absorbed in four portions; combined again for precipitation of barium carbonate. Samples placed in 85 per cent phosphoric acid before oxidation.

376

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SKIPPER, BRYAX, WHITE, AND HUTCHISON 377

tube. The reservoirs, H and I, are of 500 and 1000 ml. capacity; one is usually used to store a blank sample for background measurements, and the other is used to store discarded samples, so that many such samples can be disposed of simultaneously. Although diagrammed otherwise, the leveling bulb, G, is permanently sealed just under the mercury reser- voir, and the level of the mercury is controlled byvacuum, asiscustomary with McLeod gages.

The counter, A, is of conventional design, having a copper cathode ap- proximately 2.0 cm. in diameter and 20 cm. long and a central mire of 0.004 inch tungsten. It is mounted vertically, with about 2 inches of lead shield- ing on top and about 1% inches on the sides. The shielding on the sides extends about, 5 inches below the cathode, but there.is no shielding directly

kUUM P Q R

.I$- N 0

FIG. 3. Apparatus used in the determination of radioactive carbon. A, Geiger- illtiller tube; B, trap; F, mixing bulb; G, leveling bulb; Hand I, carbon dioxide reser- voirs; J, manometer; K, “dose? bulb; L, carbon disulfide reservoir; M and N, traps; 0: generator; P, perchloric acid reservoir; Q, water reservoir.

below the tube. Under these conditions, the background is about 0.75 count per second.

Because the voltages required for counting with carbon dioxide are higher than are available in commercial Geiger-Miiller counter circuits, a 4000 volt supply has been prepared, according to the circuit, of Fig. 4. Exceptional stabilization was not attempted. Instead, a constant voltage transformer is used ahead of the supply; the stabilization is adequate to control the small fluctuations which pass through the tra.nsformer. In the circuit., transformer Trl and potentiometer RI are ganged. The maximum rotation possible with the potentiometer is such that only 115 volts can be applied to the primary of high voltage transformer Trz, when the minimum is about 10 volts. Under these conditions, the voltage applied to the regulator tube TP is about 600 volts when the output voltage is 3500, and

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378 TRACER STUDIES WITH RADIOACTIVE CARBON

is less at lower output voltages. With the voltage available from this supply, it is possible to count samples at pressures up to 35 cm. of carbon dioxide.

Since most of the samples were expected to have very low activities, of the order of background, classical resistor-condenser quenching was con- sidered satisfactory. The quenching resistor is 500 megohms, with a 5 .micro-microfarad condenser in parallel and a 20 megohm resistor in series to supply the signal to the preamplifier. A commercial scale of 32 circuit is employed.

FIG. 4. Schematic diagram of high volt.age supply. Tt, OCR/VRIOB; 7’2. 6SF5; To, 2X2/879; T,. 5Y3; Tr,, Variac. type 201)B connected for 0 to 135 volt secondary; Trs, high voltage transformer rated at 6000 volt,s, 2 milliamperes; ‘f’ra, Stancor P6289; Tr,, Stancor C1420; C, and Ct. 0.1 microfarad, 7000 volts; C’S and C., 8 microfarads, 450 volts; RI, 20,000 ohms, general radio type 301; R2, 120,000 ohms, 1 watt; Ra, 8 X 470,000 ohms. 1 watt; R,, 5 megohms, 1 watt; R6, 5000 ohms. 10 watts; M, 0 to l~milliampere. (In addition, the input to Cl is through a 203,000 ohms, 1 watt, resistor .)

As is to be expected with resistor-condenser quenching, the net counting rates on the plateaus of different samples do not increase linearly with the specific activities. A calibration curve is used to correct for these losses due to the dead time of the counter. The system is missing about 10 per cent of the counts when the measured counting rate is 20 counts per second, a loss rate of the correct order of magnitude for the time constant of the quenching circuit.

We have observed a second source of lost counts at counting rates above 10 to 15 counts per second. Apparently due to some sort of overdriving of the scaling and recording circuits, there results a distortion of the plateau, which, at lower rates, is usually several hundred volts in width. The

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SKIPPER, BRYAN, WHITE, AND HUTCHISON 379

counting rate rises rapidly to a peak at a voltage a little above threshold (about 60 volts above) and then drops off with increasing voltage. The greater the true counting rate, the more rapidly the rate falls off with in- creasing voltage above the peak. In a typical case, at about 26 counts per second the counting rate dlops over 10 per cent in the 120 volts just above the voltage corresponding to the peak.

Use of the low voltage peak counting rate obviates losses due to the over- driving. The negative slope of the plateau can be eliminated by decreasing the input resistance to the preamplifier as the overvoltage is increased, but this is not as convenient as to locate the peak. In our procedure, an operator counts at 120 volts above the threshold for 5 minutes. He then counts at 240 volts above the threshold for as much longer as is necessary to give the required precision, a time which he determines with the help of a family of curves giving the total number of counts required for several specified degrees of precision as functions of the ratio of total to background counts. If the counting rates at the two voltages agree within statistical expectations, he accepts the indicated rate. If they disagree, he counts further. From the additional counting, he may learn that the plateau is as flat as normal, in which case he accepts the corresponding counting rate, or that the plateau has a negative slope, in which case he locates the peak and uses its counting rate for calculations.

The detailed procedure for generation of CO2 (Fig. 3) is as follows: The sample of barium carbonate having been introduced into generator 0 and trap N having been cooled to -8O”, the generating system is evacuated roughly. Trap M is cooled to - 195” and the evacuation is continued until the water in the generator begins to boil. Perchloric acid, which is used because its barium salt is soluble, is then introduced slowly from reservoir P. When the liberation is complete, t,he system is again evacuated until the water boils. Three 1 ml. doses of air are admitted through R, and the evacuation is repeated, followed by a second dosing with air and another evacuation. The stop-cock between traps 251 and N is closed and the condensed carbon dioxide is exposed to the high vacuum for 3 minutes. The carbon dioxide is then allowed to evaporate into the system, measured volumetrically, and transferred to the counter for measurement of radio- activity.

Usually the dose of carbon disulfide is condensed into trap B during the generation of carbon dioxide. The quantity of carbon dioxide is measured in the manifold, which has a volume of about 80 ml. When both carbon dioxide and carbon disulfide have been allowed to evaporate from trap B into the counter tube, the mixture is not uniform, the more volatile oxide having evaporated faster than the sulfide. It has been found that five mixings by expansion into reservoir F produce a satisfactory mixture for counting.

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380 TRACER STUDIES WITH RADIOACTIVE CARBON

Because of the long time constant of the quenching circuit, it is neces- sary to dilute very strongly samples of higher activity, such as those from respiration and urine in the case of radioactive urethan. As much as 100-fold dilution is sometimes required. In such a case, the use of a conveniently weighable quantity of radioactive sample necessitates using perhaps 1 liter of diluent carbon dioxide at atmospheric pressure. In order to insure the homogeneity of such a sample, the gas is tediously twice evapo- rated int(o one of the reservoir flasks and recondensed before a portion is removed for counting.

The anticipated variation between experimental animals minimized t.he precision required of the apparatus, and exhaust,ive tests of the precision have not been made. The following indications have been obtained. (1) The volume of gas, presumably carbon dioxide, recovered from barium carbonate samples and from C.P. calcium carbonate is usually within 2 per cent of that calculated on the assumption of complete purity. It should be noted, however, that complete recovery is not particularly important in this procedure, since specific activity is the property determined. For diluted samples, it is necessary only that recovery be the same for both active sample and inactive diluent. It has been our practice to generate the carbon dioxide from sample and diluent simultaneously, and to cal- culate the dilution factor from their weights. (2) In a series of 50 measure- ments of specific activity by three operators, using different quantities of two samples of barium carbonate, the results were self-consistent within a probable error of 1.9 per cent. (3) In quadruplicate determinations on each of three samples involving extreme dilution, the probable error was 1.2 per cent.

In the determinations quoted above, the probable error associated with the number of counts involved was rarely much less than 1 per cent.

DISCUSSION

Although one must be prepared to expect vast differences in rates of excretion, routes of excretion, and accumulation of active molecules or ions in particular tissues, depending on the compound in question and its metabolic processes, we have found that starting with activities of 2.5micro- curies per mouse contained in active urethan we have been able to deter- mine with a fair degree of accuracy the amount of Cl4 in any 59 mg. of tissue 24 hours after injection. This has been possible with a compound which breaks down fairly rapidly (at least 85 to 95 per cent in 24 hours) and when the Cl4 is fairly evenly distributed throughout the normal animal.

Using the techniques described, we have been able to obtain reproducible tracer data on radioact,ive urethan with the active carbon in the carbony, group. Results of the urethan experiments are being published elsewhere

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SKIPPER, BRYAN, WHITE, AND HUTCHISON 381

NO attempts will be made at this time to compare the accuracy or rapidity of the assay for Cl4 in the gaseous state as opposed to measurement) in the form of BaC03, nor to discuss the relative advantages of the Geiger counter verstls the electroscope.

Our principal reasons for adoption of the present procedure were (1) a desire to keep animal radiation exposure t’o a minimum, in which case the added sensitivity of the gaseous measurement is an advantage, and (2) the fact that. without previous experience in the field and without pro- nounced preferer-ces as to method we mere influenced by immediate avail- ability of necessary equipment. During the past, 6 months of nearly continuous use of the described techniques, we have found them satis- factory. It has been possible to account for all of the radioactive car- bon injected with an over-all accuracy of about f10 per cent.

SUMMARY

1. An apparatus designed t.o absorb mouse respiratory carbon dioxide suspected of containing radioactive Cl4 is described.

2. A procedure is outlined for oxidizing tissues prior to radioactive carbon assay.

3. A method is given for determination of radioactive carbon in the gaseous state.

BIBLIOGRAPHY

1. Miller, W. W., Science, 106, 123 (1947). 2. Silver, S. D., J. Lab. and Clin. Med., 31. 1153 (1946). 3. Van Slyke, D. D., and Folch, J., J. Biol. Chem., 136, 509 (1940).

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White, Jr. and Olivia S. HutchisonHoward E. Skipper, Carl E. Bryan, Locke

CARBONSTUDIES WITH RADIOACTIVE

TECHNIQUES FOR IN VIVO TRACER

1948, 173:371-381.J. Biol. Chem. 

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