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8/13/2019 Método Colorimétrico Para Determinación De Pi Usando Reactivo Taussky - Shorr
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A MICROCOLORIMETRIC METHOD FOR THEDETERMINATION OF INORGANIC
PHOSPHORUS*
BY HERTHA H. TAU SSKY AND EPHRAIM SHORR
WITH THE TECHNICAL ASSISTA NCE OF GLORIA KURZMANN
(From the Rus sell Sage Institute of Pathology, Department of Medicine, Cornell
University Med ical Colleg e, and The New York Ho spital, New York,
New York)
(Received for pub lication , November 28, 1952)
In 1944, Sumner (1) suggested reduction of the phosphomolybdic acid
formed during the first step in the analysis of inorganic phosphorus by
ferrous sulfate instead of by the various reducing agents, aminonaphthol-
sulfonic acid (2), stannous chloride (3, 4), or 2,4-diaminophenol hydro-
chloride (5) which had been conventionally employed for this purpose.
He also pointed out that when ferrous sulfate is used as a reducing sub-
stance the reaction can be carried out in a weakly acid solution, thereby
providing greater specificity with mixtures of inorganic phosphorus and
labile phosphate esters. Another distinct advantage is that the final color
produced with ferrous sulfate is developed with great rapidity and remains
stable for at least 2 hours. Rockstein and Herron (6) confirmed Sumner’s
observations.
We took advantage of the simplicity introduced by this reducing agent
to develop a method for the semiquantitative determination of phosphorus
in urine for clinical use in the management of renal phosphatic calculi byaluminum gels (7). This procedure has now been adapted to the quantita-
tive determination of inorganic phosphorus in serum, urine, spinal fluid,
and stool ash. Specific conditions were established which are optimal for
the analysis of 2 to 40 y of inorganic phosphorus, a convenient range for
biological material of this character. The necessary acidity for the pre-
cipitation of proteins, the concentration of molybdate, and the effect of
acidity on rapid color development were investigated. A Klett-Summer-
son calorimeter with a No. 66 filter was used for the color comparisons.
* Presented before the 122nd meeting of the American Chem ical Society, Atlantic
City, September, 1952. Th is research was supported in part by research grants from
the National Institute of Arthritis and Metabolic Disea ses of the National Institutes
of Health, United States Pub lic Health Service.
675
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676 MICRODETER MINATION OF INORGANIC P
EXPERIMENTAL
Reagents-
1. Potassium acid phosphate stock solution (should be kept in the re-
frigerator). 0.5853 gm. of KHzPOd are dissolved and diluted to 1 liter.
This solution contains 133.3 y of phosphorus per cc.
2. Trichloroacetic acid, 11.5 per cent (for use with the standard solu-
tions). 115 gm. of trichloroacetic acid are dissolved and diluted to 1 liter.
3. Trichloroacetic acid, 12 per cent (for use with serum). 120 gm. of
trichloroacetic acid per liter.
4. Trichloroacetic acid, 34 per cent (for use with urine and stool ash).
170 gm. of trichloroacetic acid per 500 cc.
5. Sulfuric acid, 10 N. 278 cc. of concentrated sulfuric acid are slowly
added to about 700 cc. of distilled water; after cooling, the solution is
further diluted to 1 liter.
6. Ammonium molybdate stock solution, 10 per cent. 50 gm. of (NH&-
Mo?Oz4.4Hz0 are weighed into a liter beaker and about 400 cc. of 10 N
sulfuric acid are added under constant stirring to prevent caking. When
completely dissolved, the solution is transferred to a 500 cc. volumetric
flask and washed in quantitatively with 10 N sulfuric acid to the 500 cc.
mark.
7. Ferrous sulfate-ammonium molybdate reagent (made up freshly be-
fore use). 10 cc. of ammonium molybdate stock solution are transferred
to a 100 cc. amber volumetric flask and diluted to about 70 cc. 5 gm. of
FeS04.7HzO are added, and the solution is made up to volume and shaken
until the crystals are dissolved.
Method
The steps in the analytical procedure are identical for serum, urine,
spinal fluid, and stool ash solutions except for the use of different dilution
factors. The sensitivity of the method is from 2 to 40 y. The diluted
samples are pipetted directly into calorimeter tubes and followed by the
addition of the ferrous sulfate-molybdate reagent. A blue color develops
maximally within 1 minute and is stable for at least 2 hours. The intensity
of the color is determined in a Klett-Summerson photoelectric calorimeterwith a No. 66 filter. There is a straight line relationship between the
calorimetric reading and the concentration of phosphorus.
Procedure for Determination of Phosphorus in Serum
0.2 cc. of serum is added to 3.5 cc. of 12 per cent trichloroacetic acid in a
15 cc. centrifuge tube. The mixture is well agitated, allowed to stand at
room temperature for about 10 minutes, and then centrifuged for the same
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H. H. TAUS SKY AND E. SHORR 677
period of time at about 1500 r.p.m. The protein precipitate packs well in
the tip of the centrifuge tube and 3 cc. of the supernatant fluid are readily
pipetted off and transferred to a calorimeter tube. 2 cc. of ferrous sulfate-
molybdate reagent are added and the intensity of the color is read in the
calorimeter with a No. 66 filter after 1 minute or within 2 hours.
Analysis of Standard Solutions-3 cc. aliquots of standard solutions con-
taining 4 y and 8 y of phosphorus are analyzed by the same procedure that
is used for serum filtrates. These solutions are prepared by appropriate
dilution and acidif ication of the aqueous stock solution in the following
way. 1 and 2 cc. of the stock solution are pipetted into 100 cc. volumetricflasks and diluted to volume with 11.5 per cent trichloroacetic acid. These
dilutions are stable for at least 3 weeks if the solutions are kept in the
refrigerator.
Calculation of Results for Serum-The need for the direct determination
of the relatively small reagent blank was avoided by analyzing two stand-
ards of different concentration for each series of determinations.
(1)
Reading of 8 y less reading of 4 y
4= reading with 1 y
117 - 63E.g. = 4 = 13.5 = 1 y’
(2) Read ing of 4 y les s (reading of 8 y les s reading of 4 y) = blank
E.g. 63 - (117 - 63) = 9 = blank
(3)Reading of unknown less blank
Reading with 1 yX 0.617 = mg. ‘% P in serum
Determination of Phosphorus in Spinal Fluid
The procedure is the same as for serum except that 0.4 cc. of spinal fluid
is taken for analysis. This changes the factor in Equation 3 from 0.617
to 0.326.
Procedure for Determination of Phosphorus in Urine
The usual range of urinary phosphorus values is dealt with by dilut ing
1 cc. of the acidified 24 hour specimen to 100 cc. If this dilution factoryields a final value above or below the range of accuracy of this method
(2 to 40 r), an appropriate dilution is selected. 2 cc. of diluted urine are
pipetted into a calorimeter tube. 1 cc. of 34 per cent trichloroacetic acid is
added and the solution is well mixed. 2 cc. of ferrous sulfate-molybdate
solution are added and the intensity of the blue color determined. Urine
containing proteins will show a distinct turbidity after the addition of
1 Thes e readings will vary with different instruments.
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678 MICRODETERM INATION OF INORGANIC P
trichloroacetic acid. In that case, the aliquots in the calorimeter tubes are
discarded and the procedure is changed in the following way: 4 cc. of
diluted urine are pipetted into a 15 cc. centrifuge tube, 2 cc. of 34 per
cent trichloroacetic acid are added, and the mixture is allowed to stand at
room temperature for about 10 minutes. After centrifuging for a few
minutes, 3 cc. of the supernatant fluid are pipetted into a calorimeter tube,
2 cc. of reagent are added, and the analysis is continued as above.
Calculation of Results for Urine-Equations 1 and 2 are calculated as for
serum. The final calculation is made as follows:
Reading of unknown less blankX
volume per 24 hrs.
Reading with 1 y 20 (for 1:lOO)= mg. P per 24 hrs.
Procedure for Determination of Phosphorus n Stool Ash Solutions
We are indebted to our associate Vincent Toscani for providing us with
stool ash solutions prepared as follows: 2 gm. of dried stool were first
ignited over a free flame and then ashed in a furnace at about 500-600”
until all carbon had disappeared. The white ash was dissolved by heating
with 10 cc. of water and 2 cc. of concentrated hydrochloric acid; this con-
centrated solution was diluted to 100 cc. in a volumetric flask. We further
diluted these solutions in most of the specimens 1 to 100 cc. As pointed
out above for urine, other dilutions may be necessary in stools of very
high or very low phosphorus content, with appropriate changes in the
equation given below. 2 cc. of the diluted solution are then pipetted into
a calorimeter tube. 1 cc. of 34 per cent trichloroacetic acid is added,
followed by 2 cc. of ferrous sulfate-molybdate reagent, as in the analysis of
serum or urine.Calculation of Results or Stool Ash SolutionsThe blank and the reading
with 1.0 y are calculated as for serum. The final step in the calculation is
as follows :
Reading of unknown less blank
Reading with 1 yX 2.5 (for 1:lOO) = mg. P per gm. stool
Application of Method to Determination of Alkaline and Acid Phosphatases
in Serum
The preparation of the substrates and the incubation periods were car-
ried out according to the procedures given by Hawk, Oser, and Summerson
S), which are modifications of the original methods of Bodansky 9-11)
and Shinowara, Jones, and Reinhart 12).
Procedure for Incubated Sample-Into a 15 cc. centrifuge tube are pi-
petted 2 cc. of substrate alkaline or acid), followed by 0.2 cc. of serum.
After incubation for 1 hour at 37”, the solution is cooled in ice water and
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H. H. TAUS SKY AND E. SHORR 679
1.5 cc. of 28 per cent trichloroacetic acid are added. After 10 minutes
standing at room temperature, the solution is centrifuged for about 10
minutes at about 1500 r.p.m. and the procedure is continued as described
under serum.
Procedure for Control Sample-This is similar to the procedure for the
incubated sample, except for the omission of the incubation period and the
addition of the 1.5 cc. of 28 per cent trichloroacetic acid preceding the
addition of the 0.2 cc. of serum.
Procedure for Standard Solutions-In order to maintain the same final
concentration of trichloroacetic acid and substrate in standard and un-known, the following dilutions are prepared: 3 and 6 cc. of the aqueous
stock phosphate solution are pipetted into 100 cc. volumetric flasks and
diluted to volume with 34 per cent trichloroacetic acid. 1 cc. of these
dilutions represents 4 and 8 y respectively. The substrate (alkaline or
acid) is further diluted: 8 cc. plus 2 cc. of water. 1 cc. of the standard
solution is pipetted into a calorimeter tube, followed by 2 cc. of the diluted
substrate, and the procedure continued as above. The calculation of the
mg. per cent of phosphorus before and after incubation is identical to thatfor serum inorganic phosphorus. Comparisons of alkaline and acid phos-
phatase in serum determined as described by Hawk, Oser, and Summerson
(8) and by our procedure were in good agreement.
DISCUSSION
Stability of Phosphorus in Stock Solution, Serum, Urine, and Stool Ash
Solutions-The aqueous stock solution is stable for at least 6 months.
Phosphorus values in serum remained constant for at least a week if the
samples were kept in the refrigerator. The analytical values for phosphorus
in urine were found to be reproducible for a period of more than 6 months
when 24 hour specimens were preserved with 2 cc. of concentrated hydro-
chloric acid per 100 cc. and kept under refrigeration. The same holds for
the phosphorus content in stool ash solutions.
Influence of Acidity. Sulfuric Acid-Sumner pointed out that the reduc-
tion with ferrous sulfate can be carried out in weakly acid solution and
that the lower the acidity, the less is the chance of labile esters splitting
under these experimental conditions. With this in mind we investigated
the effect of different concentrations of sulfuric acid in the ferrous sulfate-
molybdate reagent. In Fig. 1 are given the calorimeter readings for 6 y
of phosphorus at different normalities of sulfuric acid. No values could be
obtained below 1 N, since there was a spontaneous development of a blue
color in the reagent itself. The readings were unchanged from 1 to 2 N
sulfuric acid, whereas at 3 N the speed of color development was consider-
ably slower. We therefore selected 1 N sulfuric acid for the ferrous sulfate-
molybdate reagent.
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680 MICRODETERMINATION OF INORQANIC P
Trichloroucetic Acid-Regardless of the small amounts of serum taken
for analysis, it was found essential to use a concentration of at least 10
per cent trichloroacetic acid to obtain complete precipitation of the pro-
teins. With lower concentrations a slight turbidity appeared on addition
of the reagent. We investigated the use of higher concentrations of tri-
chloroacetic acid, 15 and 20 per cent, and obtained the same phosphorus
values in serum, but with a slightly higher blank. On the basis of these
findings, we selected a concentration of 12 per cent trichloroacetic acid for
the precipitation of the serum proteins.
Hydrochloric Ac&--Hydrochloric acid was used to dissolve the stoolashes and to preserve urine specimens. After appropriate dilution of these
solutions, the amount in the final aliquot taken for analysis was not greater
I,, ,
0.2 0.5 1.0 1.5 2.0 3.0
SULFURIC ACID NORMA LITY
FIG. 1. Color developed with 6 y of phosphorus as a function of the normality of
sulfuric acid used for the ferrous sulfate-molybdate reagent.
than 1 cc. of 0.005 N HCl; this concentration had no effect either on the
speed of the color development or on the actual final color. We further
investigated the addition of a larger amount of HCl and found that as
much as 1 cc. of 1 N HCl could be present without any interference. How-
ever, higher concentrations depress and delay the formation of the blue
color complex.
Efects of Molybdate Concentration-In Fig. 2 are given the calorimeterreadings for 10 to 40 y at different concentrations of ammonium molybdate.
It was noted that reduction of the concentration of ammonium molybdate
to 0.5 per cent decreased he speed of color development. A concentration
of ammonium molybdate above 1.5 per cent led to the spontaneous devel-
opment of a blue color in the reagent itself. The concentration of 1 per
cent was chosen as the lowest to secure rapid and maximal color develop-
ment for this range of phosphorus concentrations.
In Fig. 3 are presented the results obtained with 1 to 40 y of phosphorus.
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H. H. TAUS SKY AND E. SHORR 681
550
500
450
z 400
06
g 350
a?
3oc
W
4
$ 25C
0”
* 2oc
15c
1oc
40%O-0 0
/0
/
o-o26’6 o
0
o/o-o
13x o
10Xo-0-0 0
‘0.5 0.8 1.0 1.5
% AMMONIUM MOLYBDATE
FIG. 2. Color developed with 10 to 40 -y of phosphorus as a function of the concen-
tration of molybdate use d for the ferrous sulfate-mo lybdate reagent.
500
4oc
2(1
5 10 20 30 40
MICROGRAMS PHOSPHORUS
FIG. 3. Color developed as a function of the phosphorus concen trations over a
range of 1 to 40 y.
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682 MICRODETERM INATION OF INORGANIC P
Each point represents an arithmetic mean of four determinations at each
concentration, after the blank value for the reagents had been deducted.
The color produced obeys Beer’s law and the results are easily reproducible.
The blue color, due to inorganic phosphorus, develops within 1 minute and
is stable for at least 2 hours. These circumstances should be particularly
advantageous in the presence of those acid-labile phosphate esters whose
reaction velocity is slower. Their presence would be suspect if the color
intensity increased after 1 minute, the time at which the ful l intensity of the
TABLE I
Com parison of Phosphoru s by FeSO, and Fiske-Subbarow Methods
1
2
3
4
5
6
7
89
10
11
12
J,lny
4.8
3.6
3.2
5.3
6.0
4.4
1.5
3.84.3
3.6
2.7
5.3
, ntcn4”
4.8
3.6
3.1
5.4
6.1
4.3
1.6
3.84.4
3.7
3.0
5.7
er cm1
0
0
f3
-2
-2
+2-6
0-2
-3
-10
-7
Urine
ng. per24 hrs.
1530
1620
815
750
528
920
870
570312
760
n:;‘ff
1540
1615
830
750
518
872
864
590304
740
-
88ii
‘tr
R
mIX?81
-1
0
-2
0
+2
f5
fl
-3+3
+3
Stool ash
ng. 9er ng. )Cf
km. mm.
50.0 52.8
50.4 51.7
51.0 52.4
51.4 52.1
51.0 52.6
51.4 53.4
41.5 44.6
43.5 45.242.5 44.7
50.0 50.0
8Ba;2
w ncent
-5
-3
-3
-1
-3
-4
-7
-4-5
0
Spinal fluid
v;/ef ‘f;$
2.0 2.1
1.6 1.4
0.8 0.8
1.1 1.1
1.2 1.2
0.9 0.9
1.6 1.6
3.0 2.81.5 1.5
2.2 2.3
8B5
8
Be?cent
-5
1-14
0
0
0
0
0
t70
-4
color develops in the presence of inorganic phosphates alone. The problem
of the possible interference of acid-labile phosphate esters is dealt with
more specifically in the section on interfering substances.
Comparison of Fiske-Xubbarow and FeS04 Methods-Table I provides a
comparison of values obtained by the FeS04 method with a 0.2 cc. aliquot
of serum and by the Fiske-Subbarow method with a 1.0 cc. aliquot, of
urine and stool ash solutions with aliquots of 0.02 cc. by the FeS04 method
and 1.0 cc. by the Fiske-Subbarow method, and of phosphorus in spinal
fluid on 0.4 cc. aliquots for the FeS04 method and 3.0 cc. aliquots for the
Fiske-Subbarow method. Recovery experiments were carried out with
serum, urine, and stool ash solutions. With serum, the desired amount of
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H. H. TAUS SKY AND E. SHORR 683
phosphorus was incorporated in the trichloroacetic acid used for the pre-
cipitation of the proteins, and with urine and stool ash solutions, the
desired amount of phosphorus was added in making up the final dilutions.
These results are contained in Table II.
Consideration of Other Possible Reducing Substances-We have investi-
gated the possibility of substituting Fe(NH4)z(S04)2 or ascorbic acid for
FeS04 in the same concentrations as ferrous sulfate. Fe(NHd)z(S04)2 is
known to be a much more stable compound than FeSO+ both in the solid
state and in solution. However, solutions of Fe(NH&(SO&, after re-
TABLE II
Recovery of Added Phosphorus
Serum
%2?
1
1
2
2
3
3
4
4
5
56
6
Y 70 5.9
1.62 7.5
0 5.1
1.62 6.7
0 9.8
3.24 13.2
0 7.1
3.24 10.5
0 4.6
6.48 11.40 2.5
6.48 9.4
I
-I-
Phos-,horus
re-overed
wr cL?nt
99
99
104
104
105
106
ktlpkNO.
1
1
2
2
3
3
3
3
4
44
4
Phos- Phos->horus ,horus‘added
Ifound
Y Y
0 5.8
2.0 7.8
0 5.0
2.0 7.1
0 11.3
2.0 13.4
4.0 15.6
10.0 21.5
0 14.6
2.0 16.64.0 18.5
10.0 24.8
Phos-,horus
lx?-wered
er cent
100
105
105
107
102
10098
102
c
_-
Stool ash
kunpleNO.
1
1
2
2
3
3
3
3
4
44
4
x
-
Phos-,horusrtdded
7 Y0 10.2
2.0 12.2
0 10.3
8.0 18.6
0 8.4
2.0 10.5
4.0 12.4
10.0 19.0
0 9.1
2.0 11.14.0 13.3
10.0 19.6
Phos-,horus
re-wered
100
103
105
100
106
100105
105
maining at room temperature for a day or two, form the blue molybdate
complex of maximal intensity much more slowly, thereby introducing the
hazard of irregular results; hence, like the FeS04, it must be freshly pre-
pared. When freshly prepared, it was as effective as FeS04 with regard
to the speed of color development. Comparisons of phosphorus determina-tions in serum, urine, and stool ash solutions carried out with both these
reducing agents were in agreement. We have retained the FeS04 in this
procedure, since most of the analyses, comparisons, and investigations of
possible interfering substances were carried out with this reagent.
Ascorbic acid has been recommended by several investigators (13-15) as
a reducing agent in phosphorus determinations. We have studied its char-
acteristics under our experimental conditions with the following results.
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684 MICRODETERM INATION OF INORGANIC P
The maximal color intensity was about 4 times as great as with FeSOr.
The color developed slowly to reach a maximum at about 70 minutes and
remained stable thereafter for several days. When measurements were
made after 70 minutes, the results were in agreement with those obtained
with FeSO., and Fe(NH&(SO& for serum, urine, and stool ash solutions.
The slow color development would be a great disadvantage in solutions
which contain not only inorganic phosphorus, but labile phosphate esters
as well. In solutions containing inorganic phosphorus only, ascorbic acid
can replace ferrous sulfate, and is particularly useful because of the in-
tensity of the color produced, which greatly increases the sensitivity.Interfering Substances--A number of substances have been investigated
for their possible interference with the determination of inorganic phos-
phorus in amounts of 1 mg. added to 10 y of phosphorus. The following
did not interfere: creatine, glycocyamine, creatinine, calcium glycerophos-
phate, urea, uric acid, p-aminohippuric acid, inulin, glycogen, lith ium lac-
tate, thymol, toluene, acetone, dextrose, cysteine, cystine, aluminum chlo-
ride, sodium fluoride, and the following acids: acetylsalicylic, adenylic,
citric, fumaric, glutaric, cY-ketoglutaric, hippuric, malic, malonic, oxalic,pyruvic, and succinic. Lead acetate up to 5 mg. did not interfere with the
final calorimeter reading; a heavy precipitate was formed on addition of the
reagent, which settled nicely after centrifuging for a few minutes. The
following substances did not interfere when added in amounts of 100 y
to 4 to 10 y of phosphorus: sodium silicate, lead acetate, dipotassium glu-
cose-l-phosphate, barium fructose-l ,6-diphosphate, barium glucose-6-phos-
phate, and barium fructose-6-phosphate. Adenosinetriphosphate and ad-
enosinediphosphate in the above ratios to phosphorus gave slightly highercalorimeter readings; however, these readings did not increase on standing.
This suggested contamination with minute amounts of free inorganic phos-
phorus rather than splitting of the ester. On the other hand, acetyl phos-
phate and creatine phosphate, even in minimal concentrations, are rapidly
split under the conditions of our procedure and contribute to the reading
after 1 minute. Hence the analytical results with this procedure, as with
the Fiske-Subbarow method, will include whatever acetyl phosphate and
creatine phosphate may be present. Ascorbic acid in amounts of 25 y
added to 5 y of phosphorus did not interfere. This ratio of ascorbic acid
to phosphorus is at least 10 times that which would be expected in either
serum or urine.
SUMMARY
1. A micromethod has been described for the determination of inorganic
phosphorus in small samples of serum, urine, spinal fluid, and stool ash,
and for the analysis of alkaline and acid phosphatases.
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8/13/2019 Método Colorimétrico Para Determinación De Pi Usando Reactivo Taussky - Shorr
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H. H. TAUS SKY AND E. SHORR 685
2. This procedure is based on Sumner’s suggestion that the reduction of
phosphomolybdic acid be carried out by ferrous sulfate in weakly acidsolution.
3. The range of sensitivity of the method is from 2 to 40 y.
The results are in good agreement with those obtained with the method
of Fiske and Subbarow.
BIBLIOGRAPHY
1. Sumner, J. B., Science, 196, 413 (1944).
2. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925).3. Kuttner, T., and Cohen, H. R., J. Biol. Chem., 76, 517 (1927).
4. Kuttner, T., and Lichtenstein, L., J. Biol. Chem., 86, 671 (1930).
5. Allen, R. J. L., Biochem . J., 34, 858 (1940).
6. Rockstein, M., and Herron, P. W., Anal. Chem., 23, 1500 (1951).
7. Taussky, H. H., and Shorr, E., J. Ural., 69, 454 (1953).
8. Hawk, P. B., Oser, B. L., and Summerson, W. II. , Practical physiological chem-
istry, Philadelphia, 12th edition, 584 (1947).
9. Bodansky, A., J. BioZ. Chem., 99,197 (1932-33).
10. Bodansky, A., J. BioZ. Chem., 101, 93 (1933).
11. Bodansky, A., Am. J. CZin. Path., 7, Tech . S uppl., 1, 51 (1937).12. Shinowara, G. Y. , Jones, L. M., and Reinhart, H. L., J. BioZ. Chem., 142, 921
(1942).
13. Lowry, 0. H., and Lopez, J. A., J. BioZ. Chem., 162, 421 (1946).
14. Waygood, E. R., Canad. J. Res., Sect. C, 26,461 (1948).
15. Castella Bert&n, E., An. fucu ltad vet. univ. Madrid, 2, 50 (1950).
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