j. biol. chem. 1946 simonsen 747 55

8/13/2019 J. Biol. Chem. 1946 Simonsen 747 55

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M. Westover and John W. MehlDaisy G. Simonsen, Maxine Wertman, LeolaMOLYBDIVANADATE METHOD

PHOSPHATE BY THE

THE DETERMINATION OF SERUM

ARTICLE:

1946, 166:747-755.J. Biol. Chem.

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THE DETERMINATION OF SERUM PHOSPHATE BY THEMOLYBDIVANADATE METHODBY DAISY G. SIMONSEN, MAXINE WERTMAN, LEOLA M. WESTOVER, AND

JOHN W. MEHL(From the Departments of Medicine and Biochemistry, School of Medicine, University ofSouthern California, and the Los Angeles County General Hospital, Los Angeles)

(Received for publication, August 22, 1946)One of the commonly used methods for the calorimetric determination ofphosphorus is based upon the reduction of molybdic acid to molybdenumblue and subsequent estimation of the intensity of the blue color formed..An excellent discussion of the molybdenum blue reaction and its limitationsis given by Woods and Mellon (1). In using this reaction over a period ofyears the authors have been increasingly aware of its shortcomings.Acid stannous chloride was used as the reducing agent and the pro-cedures of Kuttner and Cohen (2), Woods and Mellon (l), and Fontaine (3)

were carefully followed. In addition, modifications in concentration andtype of both acid and molybdate have been studied. The results obtainedwere in accord with those of Woods and Mellon (1) who found that therelative amounts of acid and molybdate were important since, if the ratioof acid to molybdate was too low, some of the molybdate was reduced alongwith the heteropoly acid, and if too high, there was a marked decrease inthe intensity of the color. In general, the difficulties encountered in ourlaboratory with the use of chlorostannous acid as the reducing agent were(1) control of concentration of acid and molybdate, (2) instability of thecolor after a short time interval, (3) non-conformity with Beers law whenthe concentration of phosphorus was above 4 parts per million, (4) rapiddeterioration of the stannous chloride solution, particularly of the dilutereagent, and (5) inability to obtain duplicate results from day to day whenstandard solutions of phosphate were used.Three recent methods for the determination of phosphate have made useof ammonium vanadate with subsequent calorimetric estimation of theyellow molybdivanadophosphoric acid formed. Willard and Center (4)described a method for the determination of phosphorus in iron ore, Koenigand Johnson (5) one for the determination of phosphorus in foods and bio-logical material, and Kitson and Mellon (6) a method used for the deter-mination of phosphorus in carbon and low alloy steels. Since the pro-cedure seemed to offer distinct advantages from the standpoint of stabilityof the final color and simplicity, the application of the method of Kitsonand Mellon to the determination of serum phosphate has been investigated.

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748 DETERM INATION OF SERUM PHOSPHATE

Reagents-Procedure

1. Redistilled water (from an all-glass still) or distilled water known to bephosphate-free. This water was used for the preparation of all reagents.

2. Trichloroacetic acid, 7.5 and 10 per cent solutions.3. Ammonium molybdate, 5 per cent aqueous solution.4. Ammonium vanadate,l 0.25 per cent solution. Dissolve 2.5 gm. of

ammonium vanadate in approximately 500 ml. of boiling water, coolslightly, and then add 20 ml. of concentrated nitric acid. Allow the mix-ture to cool to room temperature and make up to a volume of 1 liter.

5. Nitric acid (1: 2) ;2 1 volume of concentrated nitric acid plus 2 volumesof water.

6. Phosphate standard. Dissolve 0.4389 gm. of reagent grade potassiumdihydrogen phosphate (previously dried to constant weight) in 7.5 per centtrichloroacetic acid and make to a volume of 1 liter (with the same strengthacid). 1 ml. of this solution contains 100 y of phosphorus. Suitabledilute standards (in 7.5 per cent trichloroacetic acid) are prepared from thestock solution to give a range of 5 to 80 y of phosphorus in the final 5 ml.,which was adopted as the standard volume for this procedure.

Procedure for Klett-Summerson Photoelectric Calorimeter-Measure into acalibrated Klett tube (preferably graduated at 5 ml.) 3 ml. of a suitable dilutestandard or sample. Add 0.5 ml. of 1: 2 nitric acid and mix by thoroughshaking. Then add 0.5 ml. of 0.25 per cent ammonium vanadate solutionand mix by shaking. Finally add 0.5 ml. of 5 per cent ammonium molyb-date, make to a volume of 5 ml. with water, and mix by inversion. Afterthe mixture has stood 5 minutes, read in the calorimeter with the No. 42(blue) filter. A blank is prepared with 3 ml. of 7.5 per cent trichloroaceticacid and the reagents added in the same amounts and order as for the stand-ard. In every case the calculations are based upon the reading after sub-traction of the blank reading.

Procedure for Serum-To 6 ml. of 10 per cent trichloroacetic acid in a 15ml. centrifuge tube add 2 ml. of serum. Mix by inversion, allow to stand15 minutes, and then centrifuge for 7 to 10 minutes at 2500 to 3500 R.P.M.or until the supernatant liquid is clear. Measure 3 ml. of the supernatant

1 May be obtained from Eimer and Amend, New York.* After the larger part o f the experiment.s had been completed, it was found that thevanadate and nitric acid could be combined by dissolving the ammonium vanadate asdirected, but adding 350 ml. of concentrated nitric acid instead of 20 ml., and dilutingto 1 liter. 0.5 ml. oi this combined reagent is used in place of 0.5 ml. each of the orig-inal ammonium vanadate and 1:2 nitric acid. The strongly acid vanadate solutionhas given the same results as those obtained with the original reagents, and has beenstable for the 2 months that it has been in use.

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SIMONSEN, WERTM AN, WESTOVER, AND MEHL 749fluid (equivalent to 0.75 ml. of serum) into a calibrated Klett tube, andproceed in the same manner described for the standard solutions. Thenumber of micrograms of phosphorus, taken from the calorimeter readingof the standard curve, multiplied by 0.133 equals the number of mg. ofphosphorus per 100 ml. of serum.

Inasmuch as the nitric acid and vanadate could be combined as one rea-gent, the procedure for the determination of serum phosphorus was alteredas follows: To 4 ml. of the trichloroacetic acid filtrate add 0.5 ml. of thecombined vanadate-nitric acid reagent, then 0.5 ml. of ammonium molyb-date, and mix by inversion. Read in the calorimeter after a 5 minute inter-val.

It is advisable to run one or two standards with each set of unknowns inorder to check for contamination and stability of reagents and any varia-bili ty of the calorimeter. A blank determination is also made each time.

Spectrophotometric Determinations-Spectrophotometric measurementswere also made in certain instances. The determinations were made in theBeckman quartz spectrophotometer, with 1.00 cm. Corex cells. The resultsare reported as the observed optical densities of the solutions.

ResultsPhosphate Standards-The phosphate standards were routinely made up

in trichloroacetic acid of the same concentration as that in the serum fi l-trates, since some effect of trichloroacetic acid was noted in the higher eon-centrations of phosphate. The calibration curves are given in Fig. 1. Thecurve of corrected readings against concentration is not strictly linear whenthe amount of phosphate corresponds to 5 to 80 y of P in the f inal 5 ml.

This deviation from a straight line is doubtless attributable to the spectralcharacteristics of the filter and the response of the photocell. The opticaldensities of these same solutions, measured at 440, 420, 400, and 380 rnp,are given in Fig. 2. In each case the absorption of the blank at the cor-responding wave-length has been subtracted. The solutions will be seen tofollow Beers law.

It is also evident that the absorption increases with decreasing wave-length. Since it was not clear from previous reports whether there mightbe an absorption peak which could be used for spectrophotometric studies,the values of the optical densities of the blank and of solutions containing0.1 and 0.2 mg. of P per 100 ml. were determined at several wave-lengthsand are given in Fig. 3. It was not possible to determine the position ofthe maximum because of the very great absorption of the blank at theshorter wave-lengths, but it is evident that there is little to be gained byusing wave-lengths below about 380 rnp, since the blank is increasing muchmore rapidly than the value of E:?,..

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750 DETERMINATION OF SERUM PHOS PHAT EI I I I I I I

POqP, Y 20 40 60 80FIG. 1. Curves of Klett-Summerson calorimeter readings (corrected for the blank)

for various amounts of phosphate P in the final 5 ml. of solution. 0, phosphate solu-tions in water; 0, phosphate solutions in 7.5 per cent trichloroacetic acid.

POqP, Y 20 40 60 80FIG. 1. Curves of Klett-Summerson calorimeter readings (corrected for the blank)

for various amounts of phosphate P in the final 5 ml. of solution. 0, phosphate solu-tions in water; 0, phosphate solutions in 7.5 per cent trichloroacetic acid.

PO4-P, Y 20 40 60 80FIG. 2. Optical densities of solutions (corrected for the blank) for variousamounts of phosphate in the final 5 ml. o f solution, at 440,420,400, and 330 ~UL. Allphosphate solutions were made in 7.5 per cent trichloroacetic acid.

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SIMONSEN, WERTMAN, WESTOVER, AND MEHL 751Some attention was given to the possibility of reducing the blank by de-creasing the concentration of the reagents employed. This attempt was notsuccessful, since the high blank is largely due to the molybdate, and colordevelopment does not take place in the absence of the large excess of molyb-date specified in the procedure.Although greater sensitivity can be obtained with the spectrophotometer,the results to be presented were obtained with the calorimeter, since thisinstrument is more likely to be used in routine procedures.

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340 mu 380 420 460FIG, 3. Optical densities of the blank (Curve A), a solution containing 0.1 mg. of

Pod-P per 100 ml. (Curve B), and of a solution containing 0.2 mg. of Pod-P per 100ml. (Curve C), as a function df the wave-length.The color of the blanks and the test solutions is entirely reproducible and

is stable for periods of at least 24 hours. Duplicate determinations didnot vary more than 3 divisions on the calorimeter scale, and were usually inperfect agreement. The blank gives a reading of 10 to 11 divisions, andthe increase over the blank is 26 to 30 divisions per microgram of P per ml.,depending upon the concentration.After the color has been developed, it is not possible to dilute the sampleswith water or 7.5 per cent trichloroacetic acid in order to obtain a lowercalorimeter reading. Readings of the diluted samples give a positive de-viation, of as much as 12 per cent in the higher concentrations, when thedilution factor is taken into account. Thus, if the unknown solution con-tains too high a concentration of phosphorus to be calculated from the cali-bration curve, one must repeat the procedure with a smaller sample.

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752 DETERM INATION OF SERUM PHOSPHATE

or2.55.07.510.0

12.515.017.522.525.0

TABLE IRecovery of Phosphate Added to Serum

741.343.546.548.951.254.356.953.464.066.4

_ .Y per cent

43.8 99.346.3 100.448.8 100.251.3 99.853.8 100.956.3 101 .o58.8 99.363.8 100.366.3 100.1

AveraReerror.................................................... ho.5

TABLE IISerum Phosphate Values of Normal Adults

Patient No.

1234567891011

12131415161718192021

Average. . . . . . . . . . . . . . . .

IFemales M S

w.4.13.64.04.93.94.63.94.33.94.44.03.54.03.94.13.94.53.73.6

m .3.23.13.64.33.53.93.02.84.33.63.34.33.64.54.84.53.54.34.03.33.93.8.0

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SIMONSEN, WERTMAN, WESTOVER, AND MEIIL 753Recovery of Phosphate Added to Serum-Various phosphate standards

were added to 3 ml. samples of pooled serum so that the added phosphorusranged from 10 to 100 y per 3 ml. of serum. Sufficient water and 20 per centtrichloroacetic acid were then added so that the final volume and aciditywere identical with those used in the determination of serum phosphate.TABLE III

Serum Phosphate Values of Children P

1234567891011

121314151617181920212223

sex

F.M.IIF.M.LI

F.IIIII

P per looml. seNmyrr. w.11 5.53 5.613 5.913 7.213 5.99 5.112 5.110 5.1

13 7.19 7.29 6.011 5.112 6.48 5.311 5.211 5.110 4.69 5.911 4.97 5.69 6.112 6.712 5.7

Average................... 5.7--

Diagnosis

Controlled diabetesProbable infectious mononucleosisAcute rheumatic fever I

I I Streptococcus throatSickle cell anemiaProbable subacute glomerulonephritisI Acute glomerulonephritisUndiagnosed but possible anemiaCellulitisPossible rheumatic fever1 Mumps or recurrent parotitiaUpper respiratory infectionTonsillitisLobar pneumonia Acute glomerulonephritisRheumatic fever

The phosphorus was then determined in 3 ml. of the acid filtrate accordingto the standard procedure. The recoveries are given in Table I.The results show an excellent recovery of added phosphorus. The errorsare no greater than those that would be obtained by setting up and readinga series of different sera or a set of standards, and give an average deviationof ~0.5 per cent from the calculated value.Serum Phosphate of Normal Adults-Blood samples were obtained frommembers of the laboratory staff which included chemists, physicians, tech-nicians, medical students, and general laboratory assistants. There were

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754 DETERMINATION OF SERUM PHOSPHATEforty adults in the group, ranging in age from 20 to 60 years. Sex distribu-tion showed nineteen females and twenty-one males. The values obtainedranged from 2.8 to 4.9 mg. of phosphorus per 100 ml. of serum. The resultsare given in Table II.

Serum Phosphate of Children-A group of twenty-three hospitalizedchildren was used for the study of phosphorus values of children. Patientswith bone disease were excluded; otherwise the cases were selected at ran-dom. Since the majority were near the end of convalescence and ready tobe dismissed from the hospital, the values approximate those of normalchildren. The values obtained range from 4.6 to 7.2 mg. of phosphorus per100 ml. of serum. The difference between the lowest values of children andadults was 1.8 mg. and that of the highest values was 2.3 mg. of P per 100ml. of serum. Based on the average values of the two groups, the childrenhad a serum phosphate higher by 1.8 mg. of P per 100 ml. of serum than theadults. The complete results are given in Table III.

DISCUSSIONThe method is based upon the yellow color formed when an excess of

molybdate is added to an acidif ied solution of an orthophosphate and avanadate. The exact nature of the reaction is uncertain, but presumablydepends upon the formation of a heteropoly complex, (NH&*POa.NH,VOs- 16Mo03, such as that formulated by Mission (7). This methodis less sensitive than the molybdenum blue methods, but is adequate formost biological applications, and has the great advantage of providing astable solution for calorimetry. We feel that this advantage, together witha lower sensitivity to changes in final acid concentration, outweighs theadvantages of the molybdenum blue methods for orthophosphate deter-minations.

SUMMARY1. The molybdivanadate method for orthophosphate, as described by

Kitson and Mellon, is suitable for the determination of serum phosphate.2. The solutions obey Beers law, and the method may be used spectro-photometrically for concentrations of PO,-P between 0.5 and 16 y per ml.

3. The increase in calorimeter readings was not found to be strictly pro-portional to the concentration of phosphate, but this appeared to be due tothe characteristics of the instrument. The method is applicable to thephotoelectric calorimeter for the range of concentrations from 2 to 16 yof P per ml.

4. The serum phosphate in twenty-one normal, adult males was found toaverage 3.8 mg. of P per 100 ml., with an extreme range of 2.8 to 4.8 mg.per 109 ml. The values in ninetcen females averaged 4.0 mg. per 100 ml.,with a range from 3.5 to 4.9 mg. per 100 ml.

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SIMONSEN, WERTM AN, WESTOVER, AND MEHL 7555. The serum phosphate in twenty-three children had an average value of5.7 mg. of P per 100 ml., and varied from 4.6 to 7.2 mg. per 100 ml.

BIBLIOGRAPHY1. Woods, J. T., and Mellon, M. G., Ind. and Eng. Chem., Anal. Ed., 13,760 (1941).2. Kuttner, T., and Cohen, H. R., J. Biol. Chem., 76,517 (1927).3. Fontaine, T. D., Innd. and Eng. Chem., Anal Ed., 14,77 (1942).4. Willard, H. H., and Center, E. J., Ind. and Eng. Chem., Anal. Ed., 13,81 (1941).5. Koenig, R. A., and Johnson, C. R., Ind. and Eng. Chem., Anal. Ed., 14,155 (1942).6. Kitson, R. E., and Mellon, M. G., Ind. and Eng. Chem., Anal. Ed., 16,379 (1944).7. Mission, G., Chem. Ztg., 32,633 (1908).

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