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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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