A MICROCOLORIMETRIC METHOD FOR THE DETERMINATION OF INORGANIC

PHOSPHORUS*

BY HERTHA H. TAUSSKY AND EPHRAIM SHORR

WITH THE TECHNICAL ASSISTANCE OF GLORIA KURZMANN

(From the Russell Sage Institute of Pathology, Department of Medicine, Cornell University Medical College, and The New York Hospital, New York,

New York)

(Received for publication, 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 by aluminum 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 Chemical Society, Atlantic City, September, 1952. This research was supported in part by research grants from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service.

675

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

676 MICRODETERMINATION 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 calorimeter with 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

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

H. H. TAUSSKY 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 acidification of the aqueous stock solution in the following way. 1 and 2 cc. of the stock solution are pipetted into 100 cc. volumetric flasks 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 - 63 E.g. = 4 = 13.5 = 1 y’

(2) Reading of 4 y less (reading of 8 y less reading of 4 y) = blank E.g. 63 - (117 - 63) = 9 = blank

(3) Reading of unknown less blank

Reading with 1 y X 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 diluting 1 cc. of the acidified 24 hour specimen to 100 cc. If this dilution factor yields 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 These readings will vary with different instruments.

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

678 MICRODETERMINATION 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 blank X

volume per 24 hrs. Reading with 1 y 20 (for 1:lOO)

= mg. P per 24 hrs.

Procedure for Determination of Phosphorus in 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 for 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 y

X 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

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

H. H. TAUSSKY 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 that for 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.

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

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 stool ashes 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 NORMALITY

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 calorimeter readings 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 the 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.

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

H. H. TAUSSKY AND E. SHORR 681

550

500

450

!z 400 0 6 g 350

a

? 3oc W 4

$ 25C

0” * 2oc

15c

1oc

40% O-0 0

/ 0

/

o-o 26’6 o

0

o/o-o 13x o

10X o-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 used for the ferrous sulfate-molybdate reagent.

500

4oc

2( 1

5 10 20 30 40

MICROGRAMS PHOSPHORUS

FIG. 3. Color developed as a function of the phosphorus concentrations over a range of 1 to 40 y.

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

682 MICRODETERMINATION 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 full intensity of the

TABLE I

Comparison of Phosphorus by FeSO, and Fiske-Subbarow Methods

1 2 3 4 5 6 7 8 9

10 11 12

J,lny

4.8 3.6 3.2 5.3 6.0 4.4 1.5 3.8 4.3 3.6 2.7 5.3

, ntcn4”

4.8 3.6 3.1 5.4 6.1 4.3 1.6 3.8 4.4 3.7 3.0 5.7

er cm1

0 0

f3 -2 -2

+2 -6

0 -2 -3

-10 -7

Urine

ng. per 24 hrs.

1530 1620

815 750 528 920 870 570 312 760

n :;‘ff

1540 1615

830 750 518 872 864 590 304 740

-

8 8 !ii

‘tr R

m IX?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 45.2 42.5 44.7 50.0 50.0

8 B a; 2

w n cent -5 -3 -3 -1 -3 -4 -7 -4 -5

0

Spinal fluid

v;/lef ‘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 2.8 1.5 1.5 2.2 2.3

8 B 5 8

Be? cent

-5 1-14

0 0 0 0 0

t7 0

-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

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

H. H. TAUSSKY 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 5 6 6

Y 7

0 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 11.4 0 2.5 6.48 9.4

I .! -

- I-

Phos- ,horus $

re- overed

wr cL?nt

99

99

104

104

105

106

ktlpk NO.

1 1 2 2 3 3 3 3 4 4 4 4

Phos- Phos- >horus ,horus ‘added

I found

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 16.6 4.0 18.5

10.0 24.8

Phos- ,horus

lx?- wered

er cent

100

105

105 107 102

100 98

102

c

_-

Stool ash

kunple NO.

1 1 2 2 3 3 3 3 4 4 4 4

x

-

Phos- ,horus rtdded

7 Y

0 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 11.1 4.0 13.3

10.0 19.6

Phos- ,horus

re- wered

100

103

105 100 106

100 105 105

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.

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

684 MICRODETERMINATION 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, lithium 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 higher calorimeter 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.

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

H. H. TAUSSKY 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 acid solution.

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. Suppl., 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. fucultad vet. univ. Madrid, 2, 50 (1950). by guest on M

ay 18, 2018http://w

ww

.jbc.org/D

ownloaded from

the technical assistance of Gloria KurzmannHertha H. Taussky, Ephraim Shorr and With

INORGANIC PHOSPHORUSFOR THE DETERMINATION OF

A MICROCOLORIMETRIC METHOD

1953, 202:675-685.J. Biol. Chem. 

  http://www.jbc.org/content/202/2/675.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/202/2/675.citation.full.haccessed free atThis article cites 0 references, 0 of which can be by guest on M

ay 18, 2018http://w

ww

.jbc.org/D

ownloaded from