simplified method fortheestimation ofinorganic phosphorus ......ports that phosphorus excretion...

Simplified Method for the Estimation of InorganicPhosphorus in Body Fluids

Harry Goldenberg and Alberto Fernandez

A simplified procedure is described for the determination of inorganic phosphate in

body fluids. The method employs two stable reagents and requires a minimum num-

ber of steps. Serum is deproteinized with trichioroacetic acid containing ferrous ion

and thiourea. The supernatant is decanted and mixed with a small volume of

molybdic acid. The phosphomolybdate formed is immediately reduced in situ by the

ferrous ion to produce a blue color that is stable for several hours. The intensity of

color is insensitive to changes in concentration of acid, molybdate, ferrous ion, and

thiourea, and to losses in decanting the serum supernatant. Excellent conformity to

Beer’s law is demonstrated over a wide range of phosphorus concentrations. Recoveries

of phosphorus added to serum and urine are shown to be quantitative. A comparison

is presented between this method and that of Fiske and SubbaRow (1).

PHOSPHORUS is distributed throughout every cell of tile body and plays

all important role in intermediary metabolism, skeletal formation,

dentition, and acid-base balance. It is present in blood in the form of

both inorganic and organic piloSphate. The inorganic phosphate occurs

almost exclusively in the plasma or serum, while the erythrocytes con-

tain most but not all of the organic phosphate. Clinical studies indi-

cate that the phosphate coiitent of body fluids varies in a characteristic

manner in health and disease. Serum and urinary phosphorus are de-

pressed in rickets, osteonlalacia, and idiopathic steatorrhea. Both in-

dexes are similarly depressed following the administration of insulin,

glucose, and adrenalin, and following anesthesia with ether or chloro-

form. Hypophosphatemia is also encountered in ilyperparatllyroidism,

but tile urinary phosphate excretion tends to increase. There are re-

ports that phosphorus excretion is increased in mental disease. Ele-

vated serum phosphorus levels are most generally encountered in

renal failure, less of ten ill healing fractures.

Numerous colorimetric nIetilods have been proposed for tile esti-

Froln the Bio-Science Laboratories, 7600 Tyrolle Ave., \all Nuys, Calif. 91405.

JTeeeived for publication May 23, 1966; accepted for publication Julie 28, 1966.

871

872 GOLDENBERG & FERNAND� Clinical Chemistry

mation of inorgallic phosphate. In these methods the phosphorus is

usually treated with molybdate and the phosphomolybdate, formed is

subsequently reduced to molybdeiium blue. The reducing agents em-

ployed by previous authors include aminonaphtholsulfonic acid (1),

stannous chloride (2), p-methylaminophenol (3), ascorbic acid (4),

ferrous sulfate (5, 6) and p-semidine (7). Experience has shown that

most reductants in use are not completely acceptable for the analysis

of phosphorus. Some reducing agents are unstable and have a short

shelf life. Others yield molybdenum blue colors that are unstable,

deviate from Beer’s law, or are sensitive to small changes in the acidity

of the medium. lii our study of various reducing agents, ferrous ion

was found to offer the greatest advantages for routine use. When em-

ployed in the form of Mohr’s salt the compound appears to suffer from

few or none of the objections to other reductants.

The present method is based on the use of Mohr’s salt and was de-

veloped to provide the clinical laboratory with a simplified procedure

for the determination of phosphorus in body fluids. Toward this end,

the analytical approach adopted by previous workers has been re-

appraised. It is customary to add the reductant after the molybdate.

Taussky and Shorr (6) combined their reductant and molybdate solu-

tions to give a mixed reagent that would react directly with phosphate.

This mixed reagent is not stable for more than several hours. in the meth-

od to be described here the Mohr’s salt is combined with the protein

precipitant (trichioroacetic acid, TCA) and stabilized by the addition

of thiourea. Only two reagents are required for the analysis. Serum is

deproteinized with the iron-TCA and the supernatant solution decanted

into a cuvet for color development with inolybdate. Because of the

small volume of molybdate reagent used, the sample loss sustained in

decantation is shown to have no effect on the final absorbance measure-

ment.

Material and MethodReagents

Iron-TCA, stabilized Transfer 50 gm. of trichloroacetic acid to

a 500-ml. volumetric flask with distilled water. Add 5 gm. of thiourea

(Eastman Kodak Co.)* and 15 gm. of Mohr’s salt (ferrous ammonium

sulfate hexahydrate), dissolve and dilute to mark with water. Store in

an amber bottle. A deposit of sulfur begins to form after a week but

�Some commercial preparations of thiourea may contain small anfounts of phosphorus.

Thiourea is purified by recrystallizing from water. Dissolve 60 gm. of the compound in 100 ml.

of hot distilled water, filter if necessary, and place overnight in the refrigerator. Tile Crys-

talline deposit is filtered off on a Buchner funnel, washed with a little cold distilled water,

and dried in a desiccator. Yield, 45 gm.

Vol. 12, No. 12, 1966 INORGANIC PHOSPHORUS 873

does not interfere with the analysis. The shelf life of this reagent is

6-12 months.Molybdate Add 45 ml. of concentrated 112S04 with cooling to a

500-ml. volumetric flask containing 200 ml. of cold distilled water. Add

22 gm. of ammonium molybdate, previously dissolved in 200 ml. of

water, dilute to mark, and mix. This reagent is stable for several years.

Phosphorus standard, 5 mg./100 ml. Dissolve 0.2197 gm. of pure,

anhydrous KH2PO4 (dried for an hour at 1100) in distilled water and

dilute to a liter. A small amount of chloroform is added as a preserva�

tive. Discard if there are signs of mold growth.

Procedure for Serum (or Plasma)

1. Place 0.2 ml. of serum in a test tube and add 5 ml. of iron-TCA

with shaking. Let stand for 10 mm. and centrifuge.

2. Decant the supernatant into a clean tube.

3. Prepare a blank containing 0.2 ml. of water + 5 ml. of iron-TCA,

and a standard containing 0.2 ml. of 5 mg./100 ml. P + 5 ml. of iron-

TCA.

4. Add 0.5 ml. of molybdate reagent to each tube and mix by in-

version. The color develops rapidly and is measured after 20 mm. or

within 2 hr.

5. Read the absorbance of the serum unknown (An) and standard

(A8) versus the blank at 660 m� (Klett filter No. 66) or at any desired

wavelength between 640 and 750 mjj.

Calculation

The amount of phosphorus in the serum or plasma is calculated by

the formula

A.

mg. phosphorus/100 ml. serum = -i-- X 5 (1)

Comments

In decanting the centrifugate, it is unnecessary to transfer the last

few drops of solution, since a loss of 0.5 ml. (10 drops) produces an

analytical error of only 1%.

The procedure as described above requires 0.2 ml. of serum but can

be readily adapted to other volumes. For 0.1 ml. of serum: Use 3 ml. of

iron-TCA, centrifuge, decant, and add 0.3 ml. of molybdate. The blank

contains 0.1 ml. Qf water + 3 ml. of iron-TCA + 0.3 ml. of molybdate;

the standard, 0.1 ml. of 5 mg./100 ml. of P + 3 ml. of iron-TCA + 0.3

ml. of molybdate. Read in a photometer and use equation 1. For 0.5 ml.

of serum: Use 10 ml. of iron-TCA, centrifuge, decant, and add 1 ml.

874 GOLDENBERG & FERNANDEZ Clinical Chemistry

of molybdate. The blank contains 0.5 ml. of water + 10 ml. of iron-

TCA + 1 ml. of molybdate; the standard, 0.5 ml. of 5 mg./100 ml. P +

10 ml. of iron-TCA + 1 ml. of molybdate. Read and substitute in Equa-

tion 1. It may be noted that a loss of 1 ml. iii decanting the supernatant

(from 0.5 ml. of serum) decreases tile photometer reading by only 0.9%.

The decantation procedure is recommended for accuracy as well as

speed of analysis. As an alternative, the analyst may withdraw an

aliquot of the supernatant and mix it with one-tenth its volume of

molybdate reagent for color development. If a centrifuge is not avail-

able, the deproteinized solutions are filtered and an aliquot mixed with

a one-tenth volume of molybdate.

Procedure for Urine

1. To 0.2 ml. of urine, diluted 1 to 20 with water, add 5 ml. of iron-

TCA and 0.5 ml. of molybdate reagent. Mix by inversion.

2. A blank and standard are prepared in like manner, using 0.2 ml.

of water and 0.2 ml. of phosphorus standard respectively in place of

the diluted urine.

3. Zero the photometer with the blank at 660 m� (or other selected

setting) and measure the absorbance of the urine unknown (An) and

standard (A8).

Calculation

The amount of phosphorus in the urine is calculated by the formula

111g. phosphorus/100 ml. urine = -�- X 100 (2)

Comments

The urine dilutions most commonly used are 1 to 20 or 1 to 10, but

this may be varied at the discretion of the analyst.

Urine specimens containing protein will develop a turbidity upon

addition of iron-TCA. If this happens, allow the protein to settle for

15 mill. before centrifuging. Decant the supernatant and add molybdate

(0.5 ml.) in tile usual manner.

Experiment Results

A detailed study of the parameters involved in the phosphorus analy-

sis was made in order to establish the optimum conditions of assay.

The reagent concentrations and procedure used in these experiments

were similar to those described above under “Material and Method,”

except for the parameter(s) under evaluation.

Fig. 1. Color intensity produced by

phosphorus (16 �tg.) as function of

amillolliuni molybdate reagent con-

centration. Readings were taken at

700 m�i in a Beckman DU spectro-

photometer.

0.4

0.3Lu0z

� 0.2lfl

0.1

% AMMONIUM MOLYBDATE


Molybdate Reagent

The influence of variations iii concentration of amnionium molybdate

on the intensity of the color is shown in Fig. 1. The absorbance reaches

a maximum at a reagent concentration of about 2.3% and remains on

a plateau through the highest value tested, viz., 10%. Molybdate is

employed in the stock reagent at a concentration of 4.4%, which can

be decreased by almost one-half or doubled with no effect on the color

intensity. This insensitivity to changes in concentration of the color

reagent is mandatory for a valid application of the decantation prin-

ciple. Unlike the substances present in the TCA supernatant (phos-

phorus, Mohr’s salt, and thiourea), tile molvbdate undergoes a large

dilution when mixed with sample, yielding a fluial concentration that

is dependent on the decantation loss. A ma,�or decantation loss, such

as 50%, would approximately double the fiuial niolybdate concentration.

As seen in Fig. 1, the system call tolerate this increase with no effect

on the intensity of color.

The ability of the system to tolerate increased amounts of molybdate

is intimately related to a factor not previously considered, namely

the concentration of acid iii the mixture. If insufficient acid is present,

the molybdate may undergo reduction to form molybdenuni blue in the

absence of phosphorus. The amount of acid required to give a colorless

blank increases with tile amount of molybdate employed for analysis.

The iron-TCA solution contains 10% (0.GN) TCA; this is sufficient to

prevent spontaneous development of color even when the one-tenth

volume of molybdate used contains distilled water, rather than sulfuric

acid, as the solvent. However, increasing tile ratio of unacidified

molybdate to iroii-TCA to 1 :5 results in a colored blank whose intensity


deepens on standing. The possible development of colored blanks is

eliminated by adding sulfuric acid (approximately 3N) to the

molybdate reagent. When prepared in this manner, the molybdate

reagent has a shelf life of several years.

The effect of sulfuric acid on the color reaction was studied by

varying the acidity of the molybdate reagent from 0 to 6N, which

corresponds to 0 to 0.55N in the final mixture, exclusive of the TCA

contribution. No significant differences in color intensity were noted

in any of the tubes over a 2-hr. period. If the acidity due to TCA

(0.53N) is included with the sulfuric acid, the permissible range of

acid used in the system varies from 0.53 to 1.06N or higher.

lron-TCA Reagent

Sumner (5) noted that the color obtained when ferrous ion is added

to phosphomolybdate is instantaneous and stable, increasing no more

than 2% in 2 hr. He reported the shelf life of his ferrous sulfate reagent

to be 2 hr. The ferrous molybdate reagent of Taussky and Shorr (6)

was also found to be unstable after several hours. In view of the

rapidity and stability of color development obtained using ferrous ion,

an effort was made to establish conditions for its stabilization.

Mohr’s salt is comparatively stable toward oxidation by air, and is

employed as a primary standard in quantitative analysis. Combining

Mohr’s salt with TCA provided a reagent with a shelf life of several

days. This was not satisfactory. Miscellaneous reducing agents and

antioxidants were therefore tested as possible stabilizing agents for

the ferrous ion. Hydroquinone and diphenylamine were not satisfac-

tory since on aging they imparted yellow or pink colors to the reagent.

Other agents were also tested, including hydroxylamine, hydrazine,

and sodium sulfite, but in each case the compound underwent oxidation

to produce a marked yellow or green color. Thiourea was found to give

the most satisfactory results. Its principal oxidation product, sulfur,

precipitates from solution, hence the reagent remains colorless. The

stabilized iron-TCA reagent assumes a yellow appearance on aging

but this is largely an illusion due to the sulfur precipitate. The reagent

was found to be usable for periods up to 1 year.

The optimum concentrations of thiourea and ferrous ion were de-

termined by studies that are summarized in Tables 1 and 2. For the

first study the Mohr’s salt was fixed at 3% and the thiourea varied

from 0 to 5% in the iron-TCA reagent (Table 1). Stable colors were

obtained in 10-40 mm. with no apparent effect by thiourea at concen-

trations up to 1%. At the higher concentrations of thiourea, small but

definite increases in readings were noted, approximating 2-4% over


the indicated 30-mm. interval. On the basis of these data, 1% thiourea

was selected as optimal. Table 2 illustrates the effect of increasing

ferrous ion in the presence of a 1% concentration of thiourea. It is

clear from the first line in the table that thiourea reduces phospho-

molybdate in the absence of iron, but the color formed is not stable.

Other concentrations of thiourea were also tested, with the same gen-

eral results. The effect of adding Mohr’s salt is to stabilize the color;

this is apparent at concentrations of 3, 5, and 10%. Five per cent

Mohr’s salt probably represents the optimum concentration. How-

ever, a 3% solution has been chosen to maximize the shelf life of the

reagent. The change (increase) in color intensity using 3% Mohr’s

salt is about 4% in 2 hr., as compared with a 2% increase for 5%

Mohr ‘s salt.

The question arises whether the Mohr’s salt or thiourea is to be

regarded as the reductant. The stability and magnitude of the color

intensities (Table 1) suggest that ferrous ion is the effective reducing

agent, with the thiourea serving as an antioxidant and supplementary

reductant.

Absorption Spectrum

The molybdenum blue chromophore has a broad absorption maxi-

mum between 700 and 800 m� with a peak at 725-750 m� (Beckman DU

spectrophotometer). The absorbance decreases to 90% of its peak

Table 1. EFFECT o� THIOUREA ON COLOR INPENSIPr (PHOSPHORUS = 20 Mo.)

Thiourea (%) #{149}

Ktett reading (rain.)

10 20 40

0

0.5

1.0

3.0

5.0

252

250

252

260

263

253

251

250

262

266

252

252

251

266

273

Table 2. EFFEcT 05’ FERROUS ION ON CoLoR INTENSITY (PHOSPHORUS = 20 Mo.)

Mohr’s salt (%)

Klett readi ng (mm.)

10 20 30 40

0 268 276 280 284

0.5 237 247 253 256

1.0 244 249 253 255

3.0 251 251 252 253

5.0 263 263 262 264

10.0 269 269 271 269

0 8 16 24 32 40

MICROGRAMS PHOSPHORUS


value at 660 m� and to 84% at 640 m� before it reaches a minimum at

450 m�. Wavelength settings above 700 m�t provide maximum sensi-

tivity for phosphorus analysis. However, settings at 660 or 640 m� are

also acceptable because they do not involve much loss of sensitivity

and offer the advantage of being compatible with the upper wave-

length limits of most clinical photometers.

Conformance to Beer’s Law

Phosphorus standards ranging in concentration up to 20 mg./100 ml.

were tested under the routine conditions of assay, and read after

20-30 � in 3 different photometers. The results are given in Fig. 2.

Each instrument provided a linear response with n� indications of

Lu0zH

H

Fig. 2. Evaluation of phosphorus

method for conformity to Beer’s law.

deviation from Beer’s law at the highest phosphorus concentration.

Conformity to Beer’s law was also obtained using an iron-TCA reagent

containing 5% Mohr’s salt.

Recovery Studies

Phosphorus was added in 10-pg. amounts (0.2 ml.) to 0.2 ml. of serum

or urine (diluted 1 :20) and analyzed in the routine manner. The re-

coveries obtained from 6 serums averaged 97.0%, falling in a range of

94.3 to 100%. The phosphorus recovered from 6 urine specimens varied


from 95.0 to 101%, with an average of 98.0%. Quantitative recoveries

from serum and urine were also obtained when the pilosphorus was

added in amounts of 4, 8, 12, 16, and 20 �g.

Comparison to Reference Methods

A series of 12 serums was analyzed in duplicate by the present pro-

cedure and by the method of Fiske and SubbaRow (1). As indicated in

Table 3, the 2 methods yielded comparable results, with a mean dif-

ference of 0.03 mg./100 ml. This difference is equivalent to 1% of the

average phosphorus value for serum. When the comparison of methods

was extended to urine analyses, the present method gave results 5%

higher than the Fiske-SubbaRow values (Table 4) and the difference

Table 3. COMPARATIVE RESULTS OP SERUM PHOSPHORUS ANALYSES

Sevom sample

Fiske.S,LbbaRo,v Present method De�’iat ion

(mg./I0O ml.) (mg/100 nil.) (mg/ICC ml.)

1 3.61 3.53 -0.08

2 3.36 3.27 -0.09

3 3.94 3.91 -0.03

4 3.78 3.75 -0.03

5 4.64 4.51 -0.13

6 3.80 3.79 -0.01

7 4.03 4.12 +0.09

8 3.23 3.26 +0.03

9 3.70 3.64 -0.06

10 3.92 3.99 +0.07

11 3.28 3.22 -0.06

12 3.75 3.71 -0.04

MEAN 3.75 3.72 -0.03

Table 4. COMPARATIVE RESULTS op URINE PHOSPHORUS ANALYSES

Urine sample

Deviation

Fiske-SubbaRon’ Present method

(mg/ICC ml.) (mg/ICC ml.) (mg/ICC ml.) (%)

1 59.4 62.7 +3.3 + 5.6

2 48.4 50.0 +1.6 + 3.�

3 50.3 53.2 +2.9 + 5.8

4 69.7 72.6 +2.9 + 4.2

5 34.2 35.5 +1.3 + 3.8

6 60.1 63.8 +3.7 + 6.2

7 48.5 50.0 +1.5 + 3.1

8 93.6 98.8 +5.2 + 5.6

9 49.2 50.9 +1.7 + 3.5

10 78.2 80.6 +2.4 + 3.1

11 8.9 10.1 +1.2 +13.5

12 47.1 48.6 +1.5 + 3.2

% MEAN DEVIATION + 5.1


was statistically significant by the t test (p < 0.001). Repetition of this

study at a later date led to similar findings.

The small but significant differences noted in the urine phosphorus

analyses prompted the use of a second reference method. The pro-

cedure of Taussky and Shorr (6) was adopted and found to give the

same results as the present method (p > 0.5). This reference method

is unlike the present one in a number of respects, but resembles it in

the use of ferrous ion as the reductant. The reductant employed in the

method of Fiske and SubbaRow is aminonaphtholsulfonic acid. It is

suggested that the 5% discrepancy in urine phosphorus values noted

above is attributable to the difference in reductants used in the methods.

The origin of this discrepancy has not been determined.

Effects of Anticoagulants

The effect of anticoagulants on the phosphorus determination was

tested using phosphorus standards and serum before proceeding with

the collection of plasma samples. Sodium heparin, potassium oxalate,

sodium citrate, and disodium ethylenediamine tetraacetate (EDTA)

were added in 1-5-mg. quantities (0.1 ml.) to 0.2 ml. of phosphorus

standard or serum. There was no evidence of interference by any of

these agents in the amounts used. Their influence on plasma phosphorus

values was evaluated by comparison with serum analyses on the same

blood specimens, which were distributed among 5 tubes. The anti-

coagulants were added in the following amounts: heparin, 0.2 mg./ml.

of blood; oxalate, 2 mg./ml.; citrate, 5 mg./ml.; and EDTA, 1 mg./ml.

The results obtained with heparinized plasma and serum were identical.

Plasma prepared with oxalate and citrate tended to yield somewhat

lower phosphorus values than did serum (average difference, 5%).

This effect by citrate and oxalate is a fairly common one for otherplasma components and may be attributed to a dilution caused by a

shift in water from erythrocytes to plasma. Using EDTA as the anti-

coagulant, erratic differences were noted between plasma and serum

values in a small series of tests (6 blood specimens).

Discussion

The simplicity of the phosphorus method has been achieved by com-

bining the reducing and deproteinizing agents, by employing the de-

cantation principle (8), by eliminating unnecessary dilutions, and by

eliminating volumetric flasks for final adjustment to mark. Quality

control studies indicate that these changes in the classical approach

to the analysis of phosphorus involve no sacrifice of accuracy or

reliability. These observations have been confirmed by 4 major lab-

Fig. 3. Analytical errors resulting

from application of decantation prin-

ciple to determination of serum phos-

phorus. Serum = 0.2 ml., iron-TCA =

S ml., molybdate = 0.5 ml.

z

5.

z5.

rI,

0

20 30 40

% LOSS OF SAMPLE DURING DECANTATION

Vol. 12, No. l�, 1966 INORGANIC PHOSPHORUS 881

oratories in New York that are now using the method. According to

private communicatioiis from Dr. Julius Carr* and Albert Haiiokt, tile

procedure can be readily adapted to tile determination of acid and

alkaline phosphatase in serum.

It is recognized that the analyst who is unfamiliar with the de-

cantation principle may have reservations about its validity. The

chemist, by training and experience, is inclined toward the use of

pipets for transfer of samples. Pipets or related devices are necessary

for measuring out serum and precipitant, but upon fixing the con-

centration of phosphate in the mixture the pipet becomes superfluous

for further sampling. Decantation offers greater speed than pipetting,

less chance of random contamination, and accuracy surpassing the

sensitivity of the clinical photometer.

The acceptability of sampling by decantation is contingent upon a

number of requirements (8). Foremost among these is that the super-

natant must not be diluted appreciably before the absorbance measure-

ment. If the color reagents are added in infinitesimally small volume,

theoretically 99% of the supernatant could be lost during decantation

with no effect on the analysis, provided enough sample remained for

the photometric reading. Under the conditions of the phosphorus

analysis the volume of color reagent (molybdic acid) used is finite, viz.,

one-tenth the volume of supernatant. As shown in Fig. 3, the analytical

error resulting from a 10% decantation loss is 1%; a 20% loss yields

about a 2% error. If the molybdate were double-strength and used in

one-half volume-i.e., one-twentieth the volume of supernatant-a 20%

loss of sample would result in an analytical error of only 1%.

Figure 3 was plotted from Equation 3, given below, and confirmed

*Methodist Hospital, Brooklyn, N. Y.

tBronx Municipal Hospital Center, Bronx, N. Y.


by experiment. In this equation V� represents the volume of color

reagent (molybdate) alid V�1 is the volume of the superiiatant.

Analytical error (%) = X decantation error (%) (3)

The validity of this expression is easily checked by an example. For

ease of calculation it is assumed tile supernatant is exactly 10 ml. and

that the procedure calls for 1 ml. (one-tenth volume) of color reagent.

Suppose that 20% of the supernatant is lost during transfer. An ana-

lytical error of 20% would result if the sample (8 ml.) and color reagent

(1 ml.) were diluted to a prescribed volume before reading in a pho-

tometer. On the other hand, the error would be zero if the 8-mi. sample

were mixed with 0.8 ml. of color reagent to give a final volume of 8.8 ml.

It may be seen from purely volumetric considerations that the analyti-

cal error made in mixing 8 ml. of sample with 1 ml. of color reagent is

not equal to the 20% sample loss, but instead corresponds to diluting

the 8.8 ml. of color mixture to 9.0 ml. with excess color reagent. The

error is clearly (0.2/9.0) X 100 = 2.2%. A similar result is obtained

from Equation 3 by substituting 1 ml. for V�, 8 ml. for V,1, and 20%

for the decantation error as follows: (1/9) X 20 = 2.2%.

The decantation formula can be derived intuitively by following the

reasoning used in the example just given, or it may be derived by ele-

mentary geometry (8). The ratio, Ve/(V(. + Vd), is referred to as the

telescoping factor. For example, when the volume of color reagent is

one-twentieth the supernatant, a decantation error of 40% is telescoped

into an analytical error of about 2%.

References

I. Fiske, C. 11., and SubbaRow, Y., The colorlinetric determination of phosphorus. J. Biol.

Chests. 66, 375 (1925).

2. Kuttner, T., and Cohen, H. R., Micro colorimetric studies. I. A molybdic acid, stannous

chloride reagent. The micro estimation of phosphate and calcium in pus, plasma, and

spinal fluid. J. Biol. Chem. 75, 517 (1927).

3. Gomori, G., A modification of the colorimetric phosphorus determination for use with the

photoelectric colorimeter. J. Lab. GUn. Med. 27, 955 (1942).

4. Lowry, 0. H., and Lopez, J. A., The determination of inorganic phosphate in the presence

of labile phosphate esters. J. Biol. Chen,. 162, 421 (1946).

5. Sumner, J. B., Method for the colorimetric determination of phosphorus. Science 100, 413

(1944).6. Taussky, H. H., and Shorr, E., A microcolorimetric method for the determination of in-

organic phosphorus. J. Bud. Chem. 202, 675 (1953).

7. Dryer, R. L., Tammes, A. B., and Routh, J. I., The determination of phosphorus andphosphates with N-phenyl-p-phenylenediamine. J. Biol. Chem. 225, 177 (1957).

8. Goldenberg, H., Decantation as a precision step in colorimetrie analysis. Anal. Chesn. 28,1003 (1956).

simplified method fortheestimation ofinorganic phosphorus ......ports that phosphorus excretion...

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