CLINICA CHIivlICA ACTA 81

CCA 5021

DIRECT COLORI~~ETRIC DET~R~~INATION OF PHOSPHORUS IN SERUM

AND URINE

PATRICIA A. DREWES
Research Department, Rio-Science Laboratories, 7600 T?iro?ze Avenue, vavan.
hTzcys, Calif. 9I405
(U.S.A.)

(Received December rg, 1971)

SUMMARY

A simple, sensitive and reliable method is presented for the direct photometric
measurement of inorganic phosphate in serum or urine. Reagents are stable, inex-
pensive and minimal in number. The phosphomolybdate complex formed in an acid
medium is reduced by ~-methylaminophenol sulfate; subsequent alkalinization with
monoethanolamine produces a clear blue color, which is read at 660 nm. Results
compare favorably with the Goldenberg-Fernandez manual method, which requires
prior serum deproteinization, and the Kessler automated method. Inter-run precision
is $: 3% (2 C.V.%). Bilirubin interference is slight and minimal hemolysis can be
tolerated. Elevated serum protein levels do not interfere with the method.

INTRODUCTION

The majority of methods for determination of inorganic phosphorus in serum

and other biological materials are based on the formation of phosphomolybdate com-
plexes, followed by reduction to molybdenum blue in a variety of ways. Until recently
it has been customary to perform the test after prior removal of protein, usually by
acid precipitation.
In 1969 the idea of a direct method was reintroduced by two groups. Goodwin1g2
formed the phosphomolybdate complex in the presence of borate, reduced with as-
corbic acid, then solubilized the protein with carbonate. Gindler and Ishizak? formed
the pllosphomolybdate complex in the presence of a non-ionic surfactant and reduced
with ~-methylanlinophenol sulfate (Elon) while solubilizing the protein with aqueous
monoethanolamine. Both methods resemble but extend the older method of Raabe4,
in which the phosphomolybdate was reduced by hydroquinone and sodium ascorbate,
then the color was read directly upon clearing of the solution with a sodium carbonate-
sulfite reagent.
The Gindler-Ishizaki procedure has greater simplicity in terms of the number
and composition of reagents. The advantages of Elon as reductant have already been
pointed out by Gomori5; namely, its excellent stability, low blanks and resistance to

Cl&. Chim. A&z, 39 (1972) 81-88

82 DREWES

interference from oxalate, citrate, fluoride, etc. Elon was also the reductant of choice
in the Delsal-Manhouri procedure, which does, however, involve prior preparation
of a protein-free filtrate.
The method of Gindler and Ishizaki has not yet been published in full. Our
initial attempts to set it up with the aid of one of the authors7 revealed several areas
for improvement. These were (I) large and variable blanks, (2) instability of color
during calorimeter readings, (3) necessity for frequent preparation of reagents, (4)
interference by elevated protein levels. Modifications to overcome these problems
and additional comparison studies with existing manual and automated methods are
presented here. This is a reliable, direct micromethod for either serum or urine
specimens, with few reagents, all of them stable, a linear standard curve, and the
capacity for handling elevated levels of protein in the sample.

EXPERIMENTAL

Phosphorus standard. Dissolve 0.2197 g KH,PO, (A.R., Mallinckrodt) and

2.5 ml cont. hydrochloric acid (B 8r A quality, Allied Chemical) in distilled water and
bring to a total volume of I 1. This contains the equivalent of 5 mg inorganic phos-
phorus per IOO ml. Stable indefinitely.
Reducing reagent. Dissolve 5 gp-methylaminophenol sulfate (“Elon” from J. T.
Baker) and 15 g sodium bisulfite powder (A.R., Mallinckrodt) in distilled water and
bring to a total volume of I 1. Stable for months. A slight increase in absorbance with
time will not affect results.
Acid-molybdate reagent. Dissolve 44 g (NH,),Mo,0,,.4H20 crystals (A.R., Mal-
linckrodt) and go ml cont. sulfuric acid (B & A quality, Allied Chemical) in distilled
water and bring to a total volume of I 1. Stable for several months in a polyethylene
container. Discard if it turns blue from phosphate contamination.
Monoethanolamine. (J. T. Baker). Slight discoloration with time does not affect
the test. Variations in sensitivity may occur between different lots but do not other-
wise affect results since samples and standard are similarly affected.

Procedwe
(I) Set up a series of test tubes, or more conveniently, screw-cap vials, with 3.0
ml of reducing reagent.
(z) Add 0.05 ml of phosphorus standard, water (blank) *, test serum or diluted
test urine** specimen and mix well. An M.L.A.*** automatic pipettor was used for
the sample and standard additions in our studies.
(3) Add 1.0 ml of acid-molybdate reagent to all tubes and mix well.
(4) Noting the starting time, add 0.5 ml of monoethanolamine to all tubes,
mixing well after addition.
(5) Allow tubes to stand undisturbed at room temperature for IO min for full
color development.

* Addition of such a small volume of water can be omitted without affecting the results.
** Twenty-four hour specimen is well mixed, but not centrifuged. If alkahne, it is adjusted to pH
1-2 with HCI. 4 I : IO or I : 20 dilution is made with water.
* ** Medical Laboratory Automation, Inc., 520 Nuber i\venue, Mount Vernon, New York rogjo.

Cli%. Chim. ACta, 39 (1972) 81-88

PHOSPHORUSIN SERUM 83

(6) Read the absorbance of the clear solutions at 660 nm (use an appropriate
filter if a filter photometer is used) *, against the blank, between IO and 25 min after
addition of the alkali.
(7) Unknowns are calculated by reference to the standard, with appropriate
corrections for dilution factors.

RESIJLTS

~~~od~~cat~o~and de~e~~~~en~ of fhe G~ndZey-Is~~~uk~method. In the original

method, the reducing reagent contained Bibn NE-g (9-ethyleneoxide adduct of p-
nonylphenol from Pierce Chemical), acting as a stabilizer of the final molybdenum
blue sol. However, after two different batches of Bion NE-9 resulted in large and
variable blanks (amounting to an absorbance equivalent to the standard itself), it
was considered better to omit it. Blanks were then minimal and no additional insta-
bility of the final color was noted in the absence of the nonionic surfactant than in its
presence.
The original acid-molybdate reagent was found to lack the stability for a
manual method which might be used only on a stat basis. Not only did a precipitate
form in the reagent, but the blanks showed increasing absorbance with time. Several
authors~~8~9 have reported excellent stability for a reagent similar in composition but
of greater concentration, particularly with respect to the acid. The proposed method
utilizes one of these more concentrated reagentsa, which we found to be stable for at
least 3 months (even at 5~~) while still yielding low blanks (< 1.5 absorbance x 100).
The use of stronger acid in the step in which proteins are precipitated along
with the formation of phosphomolybdate complexes, necessitated a corresponding
increase in the alkalinity of the monoethanolamine used to redissolve the protein.
A study of the effects of variable pH after addition of molybdate and after addition
of alkali is summarized in Table I. As acidity of the Gindler-Ishizaki reagent was in-
creased to approach the level of the Goldenberg-Fernandez reagent, the final pH
dropped from IO to 8.7 and further to 5.9 with increased molybdate. Concurrently,
the absorbance of the final color was decreased. Although there was some fading of
color while readings were being taken at pH higher than 8.7, these conditions yielding
the best sensitivity were selected for the test. A final pH approaclling IO (9.9) was
achieved by increasing the alkali to full strength, as seen in last line of the table.
Under these conditions, absorbance was proportional to concentration when
readings were taken immediately upon inserting the cuvet, but the downward drift of
the needle as one waited was objectionable. Substituting Baker’s Elon for Pierce’s
product somewhat improved matters, but stability was only attained for sure when
the reaction mixture was mixed, allowed to stand undistul-bed for color development,
and read without any further mixing or inverting. The phenomenon appears to be
related to oxidation of the chromophore by aeration during the transfer. The sug-
gested 5 min for complete color development was not sufficient for all samples; there-
fore a ro-min interval was chosen, The color is stable an additional 15 min.

* Jn this study a Diagnostest Photoelectric Calorimeter, Dow Diagnostics, P.O. Box 1656, In-
dianapolis, Indiana, was used. Screw-cap vials from this company can be used directly as cuvettes
in this instrument. Solutions must not be disturbed by transferring to a cuvet before reading ab-
sorbance.

Clin. Chim. Acta, 3~ (1972) 81-88

 84 DREWES

TABLE I

EFFECT cwpH ON COLOR REACTION

Reference Acid Zolybdate Teagent Monoethanolamine ,4 bsovbance Fading

Ammoniulrz pH after COWL. pH after x 100 Of
Hz=‘,
nzolybdate addition additiolz color
g/IO0 ml ml~roo 1121 9);

7 3 2 I.7 40 IO.0 29.0 Yes

3 I.4 4o 9.7 21.5 Yes
3 : I.1 4o 9.5 LO.2 Yes
3 9 I.0 4o 8.7 20.8 No
8 4.4 9 I.0 4o 5.9 IO.5 No
This method 4.4 9 1.1 100 9.9 29.7 Yes

3.0 ml reducing reagent, 50 ~1 of IO mg/roo ml Pi standard, 1.0 ml of acid-molybdate reagent of varying com-
position based on references as shown, 0.5 ml of monoethanolaminc of strength as shown.

Characteristics of proposed modified method. Linearity. Gindler and Ishizaki re-

ported linearity up to 8 mg/roo ml. In the modified method, employing a smaller
sample to reagent ratio, Beer’s Law is obeyed up to at least IO mg/roo ml inorganic
phosphorus. If 0.1 ml sample is used, then our data indicates agreement with the
8 mg/roo ml figure. Fig. I shows color-concentration relationship for phosphorus
standard, for serum and for urine, using 0.05 ml samples. Beyond the linear range,
it is necessary to repeat the test on a sample (further) diluted with water.
Comparison with reference methods. Twenty-four serum specimens were analyzed
by both the proposed method and Kessler’s automated modification10 of the Kuttner-
Cohen methodll. The data presented in Fig. z shows that there is no significant differ-
ence between the two methods (t = -1.903, critical t = 2.069, at the 95% confidence
limits). Using the same serum samples, a comparison was also made between the
proposed method and the Goldenberg and Fernandez method (Fig. 3). The methods

25-

mg Pi/ 100 ml

Fig. I. Linearity of proposed phosphorus method. l = phosphorus standards in 0.5% HCl.

x = Warner-Chilcott Calibrate Reference Standards: A set of 3 control sera (3.0, 5.0 and 8.0
mg/roo ml phosphorus). o = urine specimen at various dilutions. The three curves were run on
different days. DIAGNOSTXST vials and photoelectric calorimeter are designed for absorbance
numerically equivalent to absorbance in Beckman DU with I cm cells.

Clin. Chim. Acta, 39 (1972) 81-88

PHOSPHORUS IN SERUM 85

IOr Ior
G
2
f
E 8-
0

FoF 2 4 6 8 IO
mg Pc/100ml serum (Automated method) mg FjjOOml serum (Goldenberg-Fernandez)

Fig. 2. Comparison between proposed method and Kessler automated method. Serum sampIes and
two control sera (x) : Versatoi A and Hyland Normal.
Fig. 3. Comparison between proposed method and Goldenberg-Fernandez indirect method. Serum
samples and two control sera (x) : Versatol A and Hyland Normal.

are significantly different according to the t test (t = 3.274, critical t = z.og3, at the
95% confidence limits). The direct method is lower by no more than 4% (regression
constant Y = o.ggog7 for the line shown in the figure, where y = 0.10486+0.9492x).
Fig. 4 shows a scattergram of 22 urine values for phosphorus obtained by the
proposed method and by the automated method. There is no statistical difference
between the methods (t = 1.342, critical t = 2.080, at the 95% confidence limits).
The method was further evaluated by analysis of several commercial sera for
phosphorus content. For Hyland, Versatol, Metrix and Warner-Chilcott control sera

0 / t I L t t

20 40 60 80 100 120
mg PiflOOml urine (Automated method1

Fig. 4. Comparison between proposed method and Kessler automated method. Urine samples
diluted I : 10 or I : 20 with distilled water for assay.

Clin. Chim. Acta, 39 (1972) 81-88

86 DREWES

RECOVERY OF ADDED INORGANIC PHOSPHORUS

I I : 20 122,s ‘71.3 48.8 97.6

2 1:20 97.9 147.3 494 98.8
3 I : 20 72.2 123.3 51.1 102.2
4 x:10 37.3 86.7 494 98.8
5 1: IO 3I.S 78.2 46.7 93.4
Two aliquots of each urine specimen were diluted to the stated amount with water. Phosphorus
standard equivalent to 50 mg/Ioo ml was added to one of each pair of aliquots just before bringing
to final volume.

the results came in range of stated values. The latter company’s “calibrate” with
low, medium and high levels was also particularly useful in demonstrating linearity.
Precision. Twenty-four sera containing between 2.8 and 5.S mg inorganic phos-
phorus per IOO ml were analyzed in inter-run duplication by theproposedmethod.
Statistical analysis of the series of duplicates yielded a mean of 3.58, with a standard
deviation of & 0.054. The coefficient of variation was & I.~I%. Thus between-run
precision at the 95% confidence limits was :C 3.304.
Recovery. Recovery of the method was found to be about 9So/b (93-r02~4 on
the basis of five urine specimens to which a known amount of phosphorus standard
had been added (Table II).
Interfereme by bilimbin. -4 serum pool was loaded with increasing known quan-
tities of bilirubin (Pfanstiel, meeting specifications of joint committee on bilirubin
standardizationl~ and all levels were analyzed for phosphorus. The presence of bili-
rubin tends to decrease the phosphorus result but the error is negligible (< 5%) until
levels exceed IO mg bilirubin per IOO ml serum. At 20 mg bilirubin per IOO ml serum
the error is about -9%.
E$cct of CXC~SS jmtein. Since this is a direct method without prior removal of
protein, it was important to test the capacity of the method to handle sera of high
protein content. Five serum specimens selected for elevated total protein were
analyzed for gamma globulin and inorganic phosphorus (both automated method
and the proposed method). In allcases, the concentration of alkali was sufficient to
immediately redissolve the entire precipitate at Step 4 of the procedure. Table III
shows that the phosphorus results were not affected by the presence of such large
quantities of protein; phospl~~~rus by the direct method is in agreement with phos-
phorus by the automated procedure which includes prior dialysis of the serum.
interference by hemolysis. It is well established that serum must be separated
from red cells as far as possible and hemolysis avoided, because of the interference
from acid-hydrolyzable phosphates in red cells. The extent of this effect was tested
by lysing hemoglobin whole blood control (Hyland), adding it in known amounts of
llellloglobin to a serum pool and deternlining the phosphorus. Table IV demonstrates
the considerable degree to which hemolysis increases the apparent phosphorus level.
However, it may be observed that the amount of red cell leakage evidenced by mild
hemolysis to the extent of IOO mg hemoglobin/roe ml will not alter the phosphorus
determination by more than 5 ‘$&and therefore can be tolerated by the proposed method.
E#ect of detergents. Glassware must be scrupulously clean for phosphorus deter-

Cl?%&.
C/ii?iZ.
Act@“, 39 (1972) 81-88
PHOSPHORUS IN SERUM 87

TABLE III

EFFECT OF HIGH PROTEIN CONTENT ON DIRECT PHOSPHORUS DETERMINATION

Serum Total protein y-Globulin “tg inorg. P/IO0 mE

g/I00 mE g/loo mE Zutomated This rn~th~~ -

I 9.0 3.7 53 5.7

2 9.3 4.1 3.7 3.8
3 II.0 5.5 3.7 3.4
4 9.1 2.7 5.0 4.5
5 9.4 3.3 3.3 3.3

Total protein was determined by Biuret; gamma globulin by electrophoresis on cellulose acetate.

mination. This precaution applies also to reagent bottles, particularly for the acid-
molybdate solution. Phosphates from detergents cause large errors; however, an
occasional contaminated test tube is easily spotted when duplicates are routinely run.
It is best to use disposable glassware if possible.

TABLE IV
EFFECT OF HEMOLYSIS ON PROPOSED METHOD

Hemoglobin Inorganic P 0/OInterference

mg/.roo ml mg/Ioo ml
- 6.04 -
IO0 6.30 i-4
200 6.48 -t7
400 7.23 420
600 7.87 +30
a00 8.47 Jr 40

Hemoglobin whole blood control was lysed by 3 cycles of freezing (dry ice and alcohol) and thawing
then diluted to concentrations of 1.1, 2.2, 4.4, 6.6. and 8.8 g hemoglobin/Ioo ml. -4ddition of rooyl
of each dilution (or water for the control) to I ml of serum provided the samples for Pi analysis.

DISCUSSION

The described method for direct measurement of phosphorus utilizes only stable
reagents. In this respect, modification of the acid-molybdate solution and utilization
of undiluted mono~thanala~ne are an improvement over the original procedure3,7.
The method is less time-consuming than those previously available, and the
reagents are easily prepared.
The color formed in the reaction obeys Beer’s law up to about IO mg Pi/roe ml.
Consequently, there is no need for more than one standard. As further simplification,
the proposed method does not appear to have any requirement for correction such
as including albumin in the standard (CL Ref. z). The total elapsed time from starting
to reporting a single stat serum specimen would be about 15-20 min. Additional speci-
mens would drastically cut down the time per specimen.
The sensitivity of the method allows for the accurate determination of patholo-
gically low specimens. The major factor affecting sensitivity is the batch to batch
variation of the monoethanolamine. An occasional batch has been encountered which
contains su~cient impurities (ferrous ion) to almost double the absorbance of the
final color indicated in Fig. I. On the other hand, purification of the monoethanol-
amine by glass-distillation provides a reagent of lower but more reproducible sensitiv-
Clin. Chim. Ada, 39 (1972) aI-88
88 DREWES

itv. In this case, subnormal serum levels are better repeated with IOO ,LL~.If the IOO ,~l
specimen is used routinely, it must be remembered that the linear response would be
more limited in scope.
Comparisons show the new method to agree closely with the Kessler automated
procedure and reasonably well with the Goldenberg and Fernandez procedure. These
methods are known to agree with the more cumbersome reference methodss~13. The
automated method employs dialysis and hence does not introduce volume error into
the determination. Any precipitation method would be expected to diverge a minute
amount from the ideal situation of direct determination. The fact that such a small
discrepancy was uncovered by the data attests to the precision of both methods. The
classic methods have necessarily been based on precipitation of protein prior to esti-
mation of phosphorus; however, a direct method is theoretically more correct in that
no difference in manipulation of sample and standard exists.
A direct phosphorus method must be able to handle the elevated levels of pro-
tein found in specimens from multiple myeloma cases. The presence of such high
concentrations of protein, especially globulin, was not found to cause any turbidity
and had no deleterious effect on the estimated phosphorus content by the procedure
described.
The proposed modified form of the Gindler and Ishizaki method for phosphorus
in serum and urine is not only rapid and reliable, but utilizes reagents well suited to
long-term storage.

Sotc added in proof: Subsequent to submission of this manuscript, Dr. E. Melvin

Gindler informed me that high blanks are not due to the presence of phosphorus con-
tamination in the BION NE-g. He has been able to avoid high blanks by adjustment
of reagents, while still gaining the sensitivity inherent in smaller dilution of sample in
the presence of the surfactant.

ACKNOWLEDGEMENTS

I wish to thank Dr. E. Melvin Gindler for his interest and helpful communica-
tions during this work, Dr. William Stavropoulos for identifying and removing the
iron contamination in one of the reagents, and Mr. Wilbert Zell and Mr. Adalbert
Kowalski for their expert technical assistance.

REFERENCES

I J. F. GOODWIN, Cl&. Chew., (Abstr.), I5 (1969) 806.

2 J. F. GOODWIN, Clin. Chenz., 16 (1970) 776.
3 E. X GIXDLER AXD R. T. ISHIZAKI, Clin. Chew., (Abstr.), ~j (1969) 807.
4 S. RAABE, Rec. TVLZV.Chim., 74 (1955) 6j2.
5 G. GOMORI, J. Lab. Clin. Med., 27 (1942) 955.
6 DELSAL AND iLZASHOuR1, in I. D. P. WOOTOK (Ed.), Micro-Analysis in Medical Biochemistry,
4th Ed., Churchill, London, 1964, p. 77.
7 E. 112.GISDLER, personal communication.
8 H. GOLDEXBERG AXD A. FERXAP~DEZ, Clin. Chem., 12 (1966) 871.
9 C. H. FISKE .~KD Y. SUL~BAROW, J. Biol. Chem.. 66 (1925) 375.
IO G. KESSLER, Adu. Clin. Chew, IO (1967) 45.
II T. KUTTNER AND H. R. COHEN, J. BioZ. Chew, 7j (1927) 517.
12 Recommendation on a Uniform Bilirubin Standard, Clin. Chem., 8 (1962) 4oj.
13 R. L. DRYER, A. R. TAMMES AND J. I. ROUTH, J. Biol. Chew, 225 (1957) 177.

Clin. Chim. Acta, 39 (1972) 81-88