QUANTITATIVE STUDIES ON URINARY THIOSULFATE

EXCRETION BY HEALTHY HUMAN SUBJECTS*

BY JOSEPH H. CAST, KAZKO ARAI, AND FRANK L. ALDRICH

(Strom the Department of Biochemistry, Baylor University College of Medicine,
Houston, Texas)

(Received for publication, December 28, 1951)

Since Schmiedeberg (1) first reported the finding of thiosulfate in cat

and dog urine, its presence in normal human urine has been the subject of
considerable controversy (2, 3). Many of the earlier reports (4, 5) leave
the impression that its occurrence was irregular or that it was present in
amounts below the sensitivity of the methods in use at that time. More-

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

over, it has been known since 1861 that thiosulfate was partly oxidized to
sulfate when fed to normal men and that the degree of oxidation varied
inversely with the amount administered (6). This was confirmed by Lasch
(5) and Nyiri (7), but Fromageot recently reported that, in a limited
number of subjects, small amounts of thiosulfate are normally present in
human urine (8).
All of the quantitative procedures that have been suggested for thiosul-
fate determination involve complex reactions carried out directly in the
urine (3, 9-11). None of these workers succeeded in separating thiosul-
fate from the other reducing substances present. In 1932, Spacu and Spacu
(12) demonstrated that triethylenediamine nickel(I1) was a specific pre-
cipitant for thiosulfate, and Chamot and Brickenkamp (13) used it in their
analytical scheme for sulfur anions.
This reagent was adapted to the quantitative determination of thio-
sulfate in rabbit urine (14) and applied by Vassel, Partridge, and Crossley
to dog urine (15). Preliminary reports on the application of this proce-
dure to the quantitative determination of thiosulfate in human urine (16)
and the results in a limited number of subjects (17) have been made. This
report is an extension of this series in normal, healthy adults in order to
provide a better background for the evaluation of the metabolic origin of
thiosulfate.

Methods
The 24 hour specimens were preserved with chloroform and after col-
lection were refrigerated until the analyses were completed. No attempt
* This paper is based on work performed under contract No. AT-(40-l)-285 of
Baylor University College of Medicine with the United States Atomic Energy Com-
mission.
875
876 URINARY THIOSULFATE E:XCRlCTION

was made to record or control the dietary intake of these subjects, but, in
order to evaluate the variations in their diet and metabolism, determina-
tions were made of total urinary nitrogen by the Koch-McMeekin micro-
Kjeldahl procedure (18) and of total urinary sulfur by adaptation of Denis
and Reed’s method (19) to the photoelectric calorimeter.

Thiosulfate Determination
Reagents-
Triethylenediamine nickel(H) nitrate. The crystalline compound was
prepared according to the method of Werner (20) by the addition of ethyl-
enediamine (Eastman Kodak 95 to 100 per cent) to nickel nitrate hexahy-
drate (Raker’s c.p. grade).’ The complex salt was recrystallized four

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

times from aqueous solutions, air-dried, pulverized, and stored in a desic-
cator over solid sodium hydroxide.
Wash solution. A 1: 1 mixture of absolute ethanol and water2 contain-
ing 50 mg. per ml. of triethylenediamine nickel nitrate was prepared and
kept refrigerated.
Dilute ammonium hydroxide. 5 ml. of concentrated ammonium hy-
droxide (c.p.) were diluted to 100 ml. with water.
Approximately 9 N sulfuric acid. A 1: 3 dilution of concentrated sul-
furic acid (c.p.) is used.
Dilute sulfuric acid. 6.13 gm. of sulfuric acid (c.p.) per liter.
Potassium iodide solution. 5 gm. of potassium iodide (Baker’s c.p.)
diluted to 100 ml. with water and prepared fresh whenever required.
Stock potassium iodate solution. 0.446 gm. of potassium iodate (Baker’s
c.p.; oven-dried and stored in a desiccator) is dissolved in water and made
to 100 ml. in a volumetric flask.
Dilute potassium iodate (working standard). Stock potassium iodate
is diluted lo-fold with water.
Standard iodine solution. 5 ml. of the dilute potassium iodate are added
directly to IO ml. of 5 per cent potassium iodide and 1.0 ml. of dilute sul-
furic acid, the flask is swirled, and the volume made up to 1 liter. At
this concentration, complete iodine liberation is not attained if the iodate
is added after partial dilution. This solution deteriorates slowly when
stored in a dark bottle, but its optical density is routinely measured each
time before use and the new factor determined from the curve for concen-
tration against optical density prepared from the same batch of stock
potassium iodate or by serial dilution with blank solution.
Blank solution. This is a reagent blank containing the same concentra-
tions of potassium iodide and sulfuric acid as the standard iodine solution
1 Freshly distilled ethylenediamine is not required, as the principal impurity
appears to he cobalt from the nickel nitrate.
2 Wherever water is indicated, fresh, double distilled water was used.
 J. H. GAST, K. ARAI, AND F. L. ALDRICH 877

but with the iodate omitted. This solution is stable for months if kept
in a stoppered container in a dark cabinet when not in use and not exposed
to sunlight.
Double strength blank solution. 20 ml. of 5 per cent potassium iodide
and 2 ml. of dilute sulfuric acid are diluted to 1 liter. This must be
prepared fresh when needed.
Absolute ethanol. United States Industrial Alcohol (U. S. P.) as pur-
chased in 1 gallon cans or bottles. Aldehyde-free ethanol prepared ac-
cording to the method of Palmer (21) may be used.
Chloroform, c.p.
Indicator papers. pH papers covering the range from 1.0 to 3.0 and
5.0 to 8.0 are used.

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

Procedure
Refrigerated specimens are allowed to come to room temperature and,
when sufficient sample is available, a portion is removed for adjustment
of pH with ammonium hydroxide to 7.0 to 7.5 by use of indicator papers.
Alkaline urines are used without further treatment.
5 ml. aliquots are pipetted into conical Pyrex centrifuge tubes (pI$ can
be adjusted here if insufficient material is available for other analyses).
5 ml. of absolute ethanol are added, the solutions are thoroughly mixed,
and the tubes are stoppered and refrigerated between 5-10” for a minimum
of 1 hour. After centrifugation at 2500 to 3000 r.p.m. (5.5 inch radius)
for 5 minutes, the supernatant solutions are decanted into other conical
centrifuge tubes correspondingly marked and 100 mg. portions of powdered
triethylenediamine nickel(I1) nitrate are added to each tube. Complete
solution and mixing are effected with glass rods kept separate for subse-
quent use in washing and titration. A mauve-colored precipitate usually
begins to form upon addition of the precipitating reagent to the urine-
alcohol mixture. Complete precipitation is obtained by placing the corked
centrifuge tubes in the refrigerator overnight.
After refrigeration the tubes are centrifuged at high speed for 5 minutes,
or until the precipitate is firmly packed, and the supernatant fluid is de-
canted and discarded.3 The tubes are inverted over a piece of absorbent
paper and allowed to drain for 10 minutes. The precipitates are each
now washed once with 1 ml. of cold wash solution, with stirring rods to
effect thorough washing, and the tubes refrigerated for 0.5 hour before
3 Where low concentrations of sulfate and thiosulfate are encountered, more
reproducible results are obtained by rubbing the crystals off the wall of the tube
with a rubber policeman before centrifuging and pouring the supernatant and wash
solution through a sintered glass funnel which is subsequently washed with the
solution used to dissolve the precipitate in the tubes. The reason for this precaution
is the tendency of the small crystals to float or remain suspended.
878 URINARY THIOSULFATE EXCRETION

recentrifugation. The wash solution is discarded and the tubes inverted

to drain free of excess wash solution.
The complex salt is dissolved in a small measured volume of blank4
solution which can be varied in accordance with the amount of thiosulfate
expected or found on a trial run, and a drop or two of 9 N sulfuric acid5
is added from a small diameter dropper to a pH of 1.5 to 2.0 (with indicator
paper). The mauve and blue colors of the different complexes are dis-
sipated and the pale green color of the nickelous ion appears. The amount
of acid required will depend upon the amount of precipitate, but seldom is
it more than several small drops (0.04 to 0.06 ml.).
Estimation of the thiosulfate content of the dissolved precipitate is
carried out immediately by the procedure of Sendroy and Alving (22),

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

except that we have used the Beckman model DU spectrophotometer with
1 cm. silica cuvettes at 355 mp. The steps usually followed are as follows:
5 ml. of standard iodine are diluted with 1 ml. of blank and the optical
density is measured as Rs.6 Next, two 1 ml. aliquots of the dissolved pre-
cipitate are pipetted into separate tubes. To one are added 5 ml. of blank
solution, the contents are mixed, and the optical density noted; this is
Rb.’ To the remaining tube are added 5 ml. of standard, and after mixing,
its optical density, R,, is immediately read.
4 The precipitate may be dissolved in either blank solution or water. If the
latter is used, a volume of double blank equal to that of the water must be added.
This is necessary to maintain the same iodide concentration as is present in the
standard iodine solution; otherwise the “effective” color varies, as shown by Sendroy
and Alving (22). We have found greater solution stability at lower iodide concen-
trations than they recommend, and since we did not require the color augmentation
they obtained, we sacrificed increased optical density for solution stability.
5 Due to coprecipitation of triethylenediamine nickel(I1) sulfate by ethanol, the
recovery of thiosulfate is improved but the bulk of the precipitateis larger. Addi-
tional acid is therefore required to dissolve it and to obtain the pH necessary for
stoichiomctric reaction of iodine and thiosulfate at the concentrations used. When
phosphoric acid was used as recommended in the original procedure (22), the excess
acid required caused nickel phosphate precipitation; hence sulfuric acid was substi-
tuted throughout the procedure.
6 Serious errors may be introduced in measurements of the optical density of
iodine solutions in the ultraviolet region unless readings are made almost instan-
taneously. This is true both for the standard iodine solution and the residual iodine
unchanged by thiosulfate. Decreases in optical density at 355 m,u average about
2 per cent after 20 seconds and 6 per cent after 80 seconds in the light path. Such
slight errors can be an appreciable proportion of the final result which is obtained by
difference between the reading of the standard and the unknown.
7 Optical density readings made on acidified solutions of the precipitate disclosed
a significant absorption of light at the wave-length used due either to thiosulfate
itself or to substances adsorbed on the complex. Therefore all values reported in
this paper have been corrected by our Rb reading and no attempt has been made to
determine the cause.
 J. H. GAST, K. ARAI, AND F. L. ALDRICH 879

Calculations are the same as for any calorimetric determination with

suitable corrections for the volume aliquots used. A concentration-optical
density curve was run for each batch of standard iodine prepared and
found to be identical within the limits of error of measurement.
Testing Method-Since triethylenediamine nickel(I1) thiosulfate has a
solubility of 2.5 mg. per cent of thiosulfate sulfur in ethanol-water (1: 1)
at 20” (23), it was necessary to show that thiosulfate is completely pre-
cipitated under the conditions used in the determination; i.e., at lower
temperature, in the presence of excess precipitating agent and with the
coprecipitation of triethylenediamine nickel(I1) sulfate.
The data shown in Table I are typical of the results obtained from four

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

TABLE I
Recovery of Thiosulfate from Aqueous Solutions Containing Sulfate
Standard solutions of sodium thiosulfate containing 22.5 mg. per cent of sulfate
sulfur to insure complete thiosulfate precipitation are used. The theoretical con-
centration of thiosulfate sulfur present was determined by direct iodometric titra-
tion for comparison with the amount found by the method described herein. Con-
trol titrations with the sulfate solutions alone gave no iodine-titratable material.

Thiosulfate sulfur, mg. per cent

Per cent recovery
Present Found
I

0.44 0.40 91
0.22 0.20 90
0.10 0.11 110

series of recovery experiments carried out in duplicate at eleven different

concentrations over a range from 1.0 to 0.05 mg. per cent of thiosulfate
sulfur. The average of all the recoveries was 95 per cent and is indicative
of complete precipitation from water solutions in the presence of sulfate.
Similar experiments, without added sulfate, indicate complete recovery
only at concentrations of 0.5 mg. per cent of thiosulfate sulfur or more.
This was to be expected from the tendency of the complex thiosulfate salt
to become supersaturated (23) ; this appears to be prevented by the simul-
taneous precipitation of the complex sulfate. Coprecipitation of triethyl-
enediamine nickel(I1) sulfate is therefore essential to make the procedure,
as described, quantitative down to concentrations of 0.1 to 0.05 mg. per
cent of thiosulfate sulfur. The exact amount of sulfate necessary for this
purpose has not been determined, though it has been established that any
concentration from 5 to 50 mg. per cent of sulfate sulfur suffices for com-
plete precipitation of thiosulfate and, since the concentrations found in
880 URINARY THIOSULFATE EXCRETION

normal human urines are within this range, the problem was not pursued
further.
The limiting factor in the procedure as described is the calorimetric
iodine determination which can be extended by increasing the chromogenic
equivalence of the iodine, as described by Sendroy and Alving (22), or by
increasing the volume of urine from which the thiosulfate is precipitated.
The completeness of recovery of thiosulfate from human urine was proved
by the addition of radioactive thiosulfate as the triethylenediamine nickel
salt to urine and a determination of the radioactive thiosulfate made on an

TABLE II
Recovery of Urinary Thiosulfate Measured with Added Radioactive Thiosuljate

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

0.2 ml. of triethylenediamine nickel(I1) thiosulfate, labeled with Ss and con-
taining 4.56 X 10-r mg. per ml., was added to a 20 ml. aliquot of each specimen after
adjustment of the pH. 50 ~1. of this material gave 860 c.p.m. after background cor-
rection. Each recovery shown is an average of triplicate determinations on 5 ml.
aliquots of the specimens carried through the usual procedure. Self-absorption
corrections were made by comparison counts on similar aliquots of the dissolved
precipitate from another portiod of each specimen to which an equivalent amount
of radioactive material was added after the triethylenediamine nickel(I1) thiosulfate
was dissolved. All the counts were made with a thin, mica, end window Geiger-
Mtiller tube, on the same shelf, spread over the same area, and dried.

Specimen No. Observed Corrected Per cent recovery

c.g.m. c.p.m.

5 647 825 96
6 563 809 94
7 517 890 104
8 517 745 93
9 527 825 97

aliquot of the precipitate. Table II shows that precipitation of thiosul-

fate is quantitative under the conditions of our procedure.
Additional tests of the specificity of the procedure were attempted by
precipitation of the triethylenediamine nickel(I1) sulfate complex from
water solutions containing thirty different compounds (commercially avail-
able) of biological interest or containing chemical groups that might react
similarly. Thiosulfate was not added in these tests so that the values found
by iodimetric titrations would represent apparent thiosulfate or adsorbed
iodone-titratable material. Both singly and in groups the following com-
pounds gave negative results in concentrations shown in parentheses (as
mg. per cent) after each substance: acetoacetic acid (40), nn-alanine (15),
alloxan (20)) arginine hydrochloride (lo), ascorbic acid (40)) L-asparagine
(12), creatinine (40), cysteine hydrochloride (20), L-cystine (16), dithiodigly-
 J. H. GAST, K. ARAI, AND F. L. ALDRICH 881

co1 (saturated solution), fumaric acid (20), glutathione (lo), glycine (lo),
hippuric acid (25)) L-histidine hydrochloride (lo), m-homocystine (12)) in-
dole (lo), n-lysine hydrochloride (lo), methyldisulfide (saturated solu-
tion), methylmercaptan (saturated solution), niacin (38), sodium P-glycero-

TABLE III
24 Hour Urinary Thiosulfate, Total Sulfur, and Nitrogen Excretion by Healthy Adults

Total nitrogen
No. of sample!

I-
s203 Total

Average Range Average Range AXWage Range

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

mg. +w. %. %. m. gm.
8 11.5 14.9-9.6 1085 12oc-1000 14.35 17.2-10.8
8 9.8 16.6-6.2 937 987- 900 13.3 16.7- 5.7
11 10.3 14.8-5.1 858 89& 805 13.5 16.0- 9.6
9 10.2 13.3-5.7 727 760- 700 12.3 14.6- 9.3
12 7.8 12.3-4.7 641 680- 601 10.9 13.G 9.2
8 6.5 10.8-2.7 582 599- 560 10.2 14.8- 6.7
7 5.6 7.1-3.2 446 490- 404 9.6 12.G 7.7
7 5.3 9.Ck2.1 302 393- 210 8.8 12.9- 6.4
-
The correlation of thiosulfate sulfur with total nitrogen and total sulfur, assum-
ing multiple linear regression, was evaluated by the method of analysis of variance
(24) and the F value obtained was highly significant.

Analysis of Variance

SOUPX
! /
I-
D.P.

I-
Sum of
Sq”Fl*eS
M~kUl
square

Regression, R?W ..................................... 3894.86 1947.43

Error of estimate, (1 - R~)z& ...................... 2024.38 30.21

/ 69 / 5919.24

P = 1947.43/30.21 = 64.46; P < 0.01.

phosphate (34), sodium oxalate (20), sodium thiocyanate (300), taurine

(25)) thiamine hydrochloride (25)) L-tryptophan (12), L-tyrosine (lo), urea
(120), and uric acid (33).

Results
Table III shows the average values for 70 samples from twenty-eight
subjects grouped in descending order of total sulfur excreted. Above a
total sulfur excretion of 700 mg. a significantly higher excretion of thio-
882 URINARY THIOSULFATE EXCRETION

sulfate is usually observed. Below this value, with four exceptions out
of thirty-four samples, all the results lie well below the means in the first
four groups.
TABLE IV
Typical Effect of Varied Protein Intake on Thiosulfate Excretion
24 hour urine collections were made before the start of the diet period and 3 days
after returning to the usual diet. Diet period collections were started simultane-
ously with increased or decreased protein intake.

Subject Diet Total nitrogen

s203 Total

m. m.

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

w.
K. D. Normal 5.2 625 13.6
Decrease 2.7 560 11.3
“ 2.1 270 8.0
“ 2.9 580 7.3
Normal 5.7 720 13.1
J. H. “ 7.4 987 11.6
Increase 9.6 1010 13.6
“ 1200 14.1
12.3
“ 14.8 898 16.0
Normal 8.0 588 12.2

TABLE V
Comparison of Thiosulfate Concentration in Preserved and in Untreated Aliquots of
Same Specimen
Freshly excreted, random urine specimens from six individuals were pooled and
divided into two portions. Chloroform was added to one, which was immediately
refrigerated. The other stood at room temperature, which varied from 25-35”
during the period of observation.

Thiosulfate sulfur, mg. per cent

Time
Refrigerated Room temperature

dCZYS
0 0.328 0.325
1 0.325 0.308
2 0.291
4 0.319 0.262
5 0.251
6 0.328 0.236
21 0.275

Seven of these subjects were then asked to vary their protein intake.
Table IV shows typical results obtained in two instances during 3 day
periods of decreasing and increasing protein intake respectively. Again
 J. H. GAST, K. ARAI, AND F. L. ALDRICH 883

it is noted that the thiosulfate falls with lowered urinary sulfur and nitro-
gen and vice versa. The typical lag in nitrogen excretion is noted. A
sample on the 4th day following return to their customary diet clearly
indicates the dependence of the thiosulfate excretion on the dietary protein.
Typical results on the effect of chloroform and refrigeration for preser-
vation of the specimens are shown in Table V. Additional observations
have indicated that the preserved specimens retain their original thiosul-
fate concentration for 7 to 10 days. Thereafter the loss is slow and vari-
able with some specimens showing one-fourth of their original content as
long as 31 weeks later. Moreover, pooled, fresh specimens with chloro-
form added immediately show no difference in 24 to 48 hours from refrig-
erated portions. This parallels the conditions used herein. The decreased

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

content of unpreserved specimens with time and the lack of any increase
in 24 hours are interpreted as an indication that the thiosulfate found is
not the result of bacterial action in urine after excretion.

DISCUSSION

These data show that thiosulfate is uniformly present in the urine of

healthy adults but that there is a wide range of variability in the amount
excreted. These variations generally correlate with the protein metabo-
lism as reflected in the urinary excretion of nitrogen and sulfur. However,
no evidence has been obtained to explain why some individuals show lower
or higher thiosulfate values while excreting comparable levels of total
sulfur. This may be due to metabolic variations, varied activity of the
intestinal flora, or varied intake of non-thiosulfate-forming sulfur com-
pounds. Preliminary studies8 on increased thiosulfate excretion following
cystine and methionine ingestion support the thesis that the source of the
urinary thiosulfate is the sulfur amino acid content of the dietary protein.
Comparison of the range of values found in this series with those in the
literature shows all the previous results to be in the lower range of our
data. Three possibilities suggest themselves as explanations: (1) lower
protein sulfur intake; (2) bacterial oxidation in the samples during or after
collection; (3) differences in methods. Since no information is given on
the dietary protein or urinary nitrogen and sulfur by the earlier workers,
nor on the preservation of the urine, it is not possible to reconcile these
differences at this time.
While the number of female subjects is small compared to the males, no
consistent differences have been observed that cannot be explained by
the urinary total sulfur and nitrogen variations. Likewise in the age range
(20 to 43 years) of the subjects, no correlation with age was observed.
8 Gast, J. H., and Arai, K., unpublished observations.
884 URINARY THIOSULFATE EXCRETION

SUMMARY

Quantitative data have been presented which demonstrate the consistent

presence of 2 to 17 mg. of thiosulfate sulfur in 24 hour urine specimens of
healthy young adults. Concomitant studies on urinary total nitrogen and
sulfur indicate that the urinary thiosulfate sulfur probably has its origin
in the sulfur of the dietary protein and varies with the latter. A method
for the determination of thiosulfate in urine has been described.

BIBLIOGRAPHY

1. Schmiedeberg, O., Arch. Heilk., 8, 422 (1867).

2. HetIter, A., Arch. ges. Physiol., 38, 476 (1886).
3. Salkowski, E., Arch. ges. Physiol., 39, 209 (1886).

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

4. Presch, W., Virchows Arch. path. And., 119, 148 (1890).
5. Lasch, W., Biochem. Z., 97, 1 (1919).
6. Trachtenberg, W., Zur Frage iiber die Neutralisation tiberschiissiger Alkalien
im Blute, Dissertation, Dorpat (1861).
7. Nyiri, W., Die Thiosulfateprobe, Wien (1923).
8. Fromageot, C., and Royer, A., Enq/moZogia, 11, 361 (1945).
9. Dezani, S., Arch. jisiol., 16, 39 (1917).
10. Zorkendorfer, W., Biochem. Z., 278, 191 (1935).
11. Fromageot, C., and Royer, A., Enz?Jmologia, 11, 253 (1944).
12. Spacu, G., and Spacu, P., Z. anal. Chem., 89, 192 (1932).
13. Chamot, E. M., and Brickenkamp, It. W., Mikrochemie, 16, 121 (1934).
14. Cast, J. H., Publ. No. 214, University Microfilms (1940).
15. Vassel, B., Partridge, R., and Crossley, M. L., Arch. Biochem., 4, 59 (1944).
16. Arai, K., Aldrich, F. L., and Cast, J. H., Federation Proc., 9, 146 (1950).
17. Cast, J. H., Aldrich, F. L., and Arai, K., Federation Proc., 9, 175 (1950).
18. Koch, F. C., and McMeekin, T. L., J. Am. Chem. Sot., 46, 2066 (1924).
19. Denis, W., and Reed, L., J. Biol. Chem., 71, 205 (1926-27).
20. Werner, A., 2. anorg. Chem., 21, 201 (1899).
21. Palmer, L. S., Carotinoids and related pigments, American Chemical Society
monograph series, New York, 205 (1922).
22. Sendroy, J., Jr., and Alving, A. S., J. Biol. Chem., 142, 159 (1942).
23. Cast, J. H., and Aldrich, F. L., J. Am. Chem. Sot., 73, 3037 (1951).
24. Snedecor, G. W., Statistical methods, Ames, 4th edition, 350 (1950).
 QUANTITATIVE STUDIES ON
URINARY THIOSULFATE EXCRETION
BY HEALTHY HUMAN SUBJECTS
Joseph H. Gast, Kazko Arai and Frank L.
Aldrich
J. Biol. Chem. 1952, 196:875-884.

Access the most updated version of this article at

Downloaded from http://www.jbc.org/ by guest on April 3, 2019

http://www.jbc.org/content/196/2/875.citation

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail

alerts

This article cites 0 references, 0 of which can be

accessed free at
http://www.jbc.org/content/196/2/875.citation.full.h
tml#ref-list-1