A F Hunter

F H T Lowry

J W Kip

JR. Irvine

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RADIOACTIVE TRACER STUDIES ON ARSENIC INJECTED AS
POTASSIUM ARSENITE
II. CHEMICAL DISTRIBUTION IN TISSUES
0. H. LOWRY, F. T. HUNTER, A. F. KIP, AND J. W. IRVINE, JR.
Received for publication July 18, 1942
The preceding paper has shown that small amounts of arsenite tagged with
As74, when injected into the body, become distributed in the various tissues at
concentrations more or less characteristic for each tissue. These arsenic con-
centrations, in general, are much greater in the tissues than in the serum. This
suggested that a non-diffusible combination of the injected arsenic is formed
within the cells. The present paper presents the results of our investigations of
the manner in which arsenic is held by the tissues after its administration.
Because of its extreme sensitivity the radioactive tracer method has proved of
great advantage in this work. For example, following the administration of a
non-toxic amount of arsenite containing As74, a sample of liver had so much
radioactivity that only 3 mgm. of tissue, or an equivalent fraction of a larger
tissue sample, were required for analysis. This amount of tissue contained only
106 mgm. of arsenic.
Voegtlin, Dyer and Leonard (6) suggested that the sulfhydryl groups in re-
duced glutathione and in proteins combine with trivalent arsenic, and Rosenthal
(7) observed the in vitro combination of arsenoxide (3-amino-4-hydroxyphenyl
arsenious oxide) with proteins in parallel with their content of free sulfhydryl
groups. However, it was not possible for them to demonstrate that arsenic
combined exclusively with sulfhydryl groups. The further possibility exists
that arsenic might replace phosphorus, since arsenate will apparently substitute
for phosphate in at least one step in the glycolytic cycle, and it has been reported
that liver nucleoproteins will take up arsenic (8). To investigate the relationship
of arsenic to phosphorus was one object of our work.
The first step in tracing the arsenic was to divide the tissues into acid-soluble,
lipid, and protein fractions. The acid-soluble fraction should contain (a)
inorganic arsenic, (b) arsenic which might have replaced phosphorus in the acid-
soluble organic phosphorus compounds, and (c) any arsenic in combination with
reduced glutathione. Following this general fractionation, the protein portion,
which was found to contain the bulk of the arsenic, was further fractionated,
chiefly with ammonium sulfate. Analyses for phosphorus as well as for arsenic
were made on the various fractions to see if any correlation could be observed
in the distribution of these two elements.
Although the arsenic did not prove to be confined exclusively to any group of
proteins, definite differences were observed in the amount of arsenic in different
protein fractions.
METHODS. The tissue acid-extracts were prepared with 0.75 M nitric acid. The lipids
were extracted from the acid-insoluble residue with alcohol and ether. The lipid-free
221
222 LOWRY, HUNTER, KIP AND IRVINE
residue was taken as the “protein” fraction although it obviously would contain some
glycogen. These fractions were then digested with sulfuric and fuming nitric acids, a
definite volume of 3% sodium bicarbonate was added to each sample, and aliquots were
taken for measuring the radioactivity and phosphate (9).
For the protein fractionation, the tissue was minced and then thoroughly broken up in
a Waring blender with ice cold 1% sodium chloride solution at pH 7.4. The portion of the
tissue in solution after this treatment was used for fractionation of the proteins with dif-
ferent concentrations of ammonium sulfate (6#{176}C.).
In order to precipitate each fraction a number of times with ammonium sulfate without
too much 1058, a scheme of precipitation was used which was analogous to the procedure
employed in making repeated fractional distillations. After making the initial successive
precipitations (la, ib, etc.) , the fraction la, insoluble in the lowest salt concentration
(0.6 M (NH4)SO4), was dissolved insofar as possible in water, centrifuged, and precipitate
a obtained by adding to the separated supernatant fluid a sufficient quantity of ammonium
sulfate to bring the salt concentration to 0.6 M once more. At this point instead of dis-
carding the resultant supernatant, it was used to redissolve precipitate ib, previously
obtained, with the next higher salt concentration (1.2 M). Any of this precipitate which
failed to redissolve in the 0.6 M ammoniu.m sulfate was combined with precipitate a for
succeeding reprecipitations. Meanwhile precipitate b was obtained by adding ammonium
sulfate to the supernatant liquid to obtain a concentration of 1 .2 M. This general proce-
dure was followed with each of the other fractions obtained with increasing am.monium
sulfate concentrations. By thus retaining all of the supernatant fluids, the only protein
lost was that which failed to dissolve in water at one end of the scale, and that which failed
to precipitate with the highest ammonium sulfate concentration (3.6 M) at the other.
Each fraction was precipitated four times, following which it was washed with 10% tri-
chloracetic acid, then with a 1 :3 alcohol-ether mixture, dried, weighed, wet-ashed, and
finally analyzed for phosphorus and arsenic.
In addition several samples of nucleoprotein were prepared . The method of Greenstein
(10) was followed initially, but since this involves bringing the preparation to pH 10.5, and
since at this pH arsenic was found to be partially split from the proteins, the procedure was
modified to avoid pH’s above 8.5. As a result, the yield was decreased, but the phosphorus
content was increased from about 1% to over 4%.
RESULTS. In Table 1 is recorded the distribution of arsenic and phosphorus
in the acid-soluble, lipid, and “protein” fractions obtained from five tissues and
the serum of a guinea pig, and from the liver and kidney of a chimpanzee and of
a baboon. The guinea pig had been injected 8 times with arsenite over a period
of 4 days, the chimpanzee had received a single injection 24 hours previously,
while the baboon had been injected one week before the samples were obtained.
With the exception of the serum, the protein fraction appeared to contain the
bulk of the arsenic, with a variable amount in the acid-soluble fraction and a
negligible amount in the lipid fraction.
Not all of the acid-soluble arsenic was dialyzable through a cellophane mem-
brane. It may be mentioned at this point that in contrast to the findings in
regard to the tissues, in urine, serum, and bile practically all of the arsenic was
found to dialyze readily through a cellophane membrane.
In the case of liver and kidney, the tissue proteins were further fractionated
to see if the arsenic was concentrated in any particular group. Some typical
data for the liver of the same chimpanzee mentioned above are shown in Table 2.
Per gram of protein there was three times as much arsenic in the less soluble pro-
DISTRIBUTION OF RADiOACTIVE ARSENIC 223
TABLE 1
Distribution of arsenic and phosphorus in fractions of various tis8ues (values reported per kg.
offresh tissue)
ACW-SOLUBLE LIPID PROTEIN
As 10 P 101 As/P
mol mol xlO’
As 10’ P 101 As/P
mol mol xlO’
As 107 P 101 As/P
mol mol xlO’
Guinea pig
Liver
Kidney
Spleen
Brain
Muscle
Serum
3.5 36.1 .097
2.7 26.2 .103
<.3  39.5 <.01
24.9 .016
2 59.4 .004
8 .70 1.1
.5
.7
<.3
.1
.2
.1
37.0 .01 32.6 31.4
26.6 .03 29.0 28.4
17.5 <.02 8.4 50.8
59.7 .002 11.7 15.5
10.9 .02 9.1 4.8
.32 .3 .06 .06
1.04
1.02
.14
.75
1.90
1.0
Chimpanzee 1 12
Liver
Kidney
4.0
3.7
32.6 .12
38.3 .10
.1
.1
24.8 .006 10.6 25.3 .42
23.0 .004 5.9 15.1 .39
Baboon
Liver
Kidney
.9
1.9
34.0 .03 .4 5.7 23.4 .24
8.1 26.8 .30
TABLE 2
Distribution of arsenic and phosphorus in protein fractions of the liver
PROTEIN FRACTION
PER CENT As 10 MOLS
OF TOTAL PER KG.
PROTEIN PROTEIN
P 10-3 MOLS
PER KG.
PROTEIN
As/P xIO’
Chimpanzee  112
Whole liver
Sol. in 1% NaCI
So!. 1% NaC1; ins. H,O
So!. 1120; ins. .6 M
So!. .6 M; ins. 1.2 M5
So!. 1.2 M; ins. 1.8 M*
So!. 1.8 M; ins. 2.7 1W
So!. 2.7 M; ins. 3.6 M5
So!. 3.6 M; ins. 10% CC13COOHt .
100
82
4
13
15
19
17
2
4
56
60
76
69
61
43
28
23
27
133
152
163
226
61
62
61
40
.42
.50
.42
.27
.70
.45
.38
.67
Baboon
Whole liver 100
Nucleoprotein .7
30
28
123 .24
1350 .02
* Molarity of ammonium sulfate.
t Precipitate with trichioracetic acid from the last supernatant removed from previous
fraction.
224 LOWRY, HUNTER, KIP AND IRVINE
tein fractions than in the more soluble fractions. Similar results were observed
for other liver samples and a kidney specimen. Although a relationship seemed
to exist between solubility and arsenic content, the proteins initially insoluble
in 1 % saline were not found to be any higher in arsenic than those fractions
initially soluble in the dilute sodium chloride solution. The tendency for the
values for arsenic and phosphorus to parallel each other in the liver (table 2)
was not found when kidney tissue was investigated, for in that tissue the highest
phosphorus values were obtained in the most soluble fractions.
In regard to nucleoprotein, the arsenic concentration did not appear to be any
. higher than the average obtained for other proteins (see Table 2).
TABLE 3
Removal of arsenic and phosphorus from liver proteins under various conditions
TREATMENT As PER CENT P PER CENT
INITIAL VALUE INITIAL VALUE
Chimpanzee  86
Rapid isolation with 10% CC1,COOH
10% CC1,COOH 18 hours 25#{176}C
N/10 HC1  hour 100#{176}C
N/10 HC1 1 hour 100#{176}C
4% Borax 1 hour 25#{176}C .
N/l0 NaOH 1 hour 25#{176}C
100
105
48
33
55
4
100
32
70
47
80
98
Baboon
Rapid isolation with 10% CC!,COOH
pH 7.4 1 hour 25#{176}C.. . . .
pH 8.0 1 hour 25#{176}C
pH 8.5 1 hour 25#{176}C
pH 9.0 1 hour 25#{176}C
pH 9.7 1 hour 25#{176}C
pH 12.5 1 hour 25#{176}C
N/10 HC1 0.16 hour 100#{176}C
N/10 HC1 0.5 hour 100#{176}C
N/l0 HC! 1.0 hour 100#{176}C
N/10 HC! 2.0 hours 100#{176}C.....
100
97
91
85
75
60
35
56
41
38
35
100
101
87
56
47
34
34
Evidence against the replacement of phosphorus in proteins by arsenic may be
found in the difference in the behavior of the two elements on hydrolysis (Table 3).
In contrast to phosphorus, arsenic appears to be very susceptible to the action
of cold alkali, and insensitive to cold acid, a behavior compatible with a combina-
tion with sulfhydryl groups. On the other hand, both arsenic and phosphorus
show a similar behavior toward hot acid, for after a decrease to about one-third
the original value a limit appears to be reached. This suggests the presence of
two types of bonds for both elements in their combination with protein.
SUMMARY
1. Following the administration of arsenite tagged with As74, the distribution
of arsenic in various fractions of mammalian tissues has been investigated.
DISTRIBUTION OF RADIOACTIVE ARSENIC 225
2. The bulk of the arsenic was found in the protein fraction, with a much
smaller amount in the acid-soluble portion and only an insignificant amount in
the lipid fraction.
3. The arsenic was not evenly distributed throughout the proteins, but in
general appeared to be more concentrated in those tissue proteins which are
precipitated by less than half-saturated ammonium sulfate.
4. The nucleoproteins did not take up more arsenic than proteins in general.
5. From the comparative distribution and behavior on hydrolysis of phos-
phorus and arsenic, there is little evidence for the replacement of phosphorus by
arsenic in the tissues.
Acknowledgment. To the many individuals who gave assistance or advice
we wish to extend a word of thanks. Particularly, we are indebted to Dr. John
D. Ferry for hemoglobin fractionations, to Dr. A. Baird Hastings for helpful
advice and suggestions leading to the second portion of this paper, and to Dr.
John F. Fulton and Dr. Margaret A. Kennard of New Haven, who gave us the
opportunity for studying higher apes, and generously donated their time. Dr.
Maurice Fremont-Smith and Mr. Edwin S. Webster were of great help in ob-
taming financial backing, and our competent technician Miss Patria M.
Rodriguez proved indispensable. Finally, to Professor Robley D. Evans and
Dr. James H. Means, for assistance in ways too numerous to mention, we owe
a deep debt of gratitude.
REFERENCES
1. FORKNER, C. E., AND Sco’rT, T. F. M.: J. A. M. A., 97: 3, 1931.
2. HUNTER, F. T., AND Kip, A. F.: J. App!. Phys., 12:324, 1941.
3. IRvINE, J. W., JR.: Ibid., 1.2: 347, 1941.
4. KOLTOFF, I. M., AND AMDUR, E.: md. Eng. Chem.-Anal. Ed., 12: 177, 1940.
5. Kip, A. F., AND EVANS, R. D.: Physical Review, 59: 920 A, 1941.
6. VOEGTLIN, C., DYER, H. A., AND LEONARD, C. S.: Pub. Health Rep., 38: 1882, 1923.
7. ROSENTHAL, E. M.: Ibid., 47: 241, 1932.
8. SLOWTZOFF, B.: Beitr. z. Chem. Physiol. u. Pathol., 2: 307, 1902.
9. FISKE, C. H., AND SUBBAROW, Y.: J. Biol. Chem., 66: 375, 1925.
10. GREENSTEIN, J. P.: J. Nat. Cancer Inst., 1: 91, 1940.


RADIOACTIVE TRACER STUDIES ON ARSENIC INJECTED AS POTASSIUM ARSENITE II. CHEMICAL DISTRIBUTION IN TISSUES

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RADIOACTIVE TRACER STUDIES ON ARSENIC INJECTED AS POTASSIUM ARSENITE II. CHEMICAL DISTRIBUTION IN TISSUES

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