Quantificationof SerumInorganicPhosphorus,Phosphatase,and

UrinaryPhosphatewithoutPreliminaryTreatment
Jesse F. Goodwin

A direct method has been devised for determining serum and urinary inorganic phosphorus and serum phosphatase activity. Inorganic phosphorus forms a phosphomolybdate complex in the presence of borate. The
complex

is reduced

with ascorbic

acid. The resulting

suspension

is solu-

bilized with carbonate and the absorbance measured at 720 nm. Samples
containing low to moderate concentrations of bilirubin may be assayed by
the method, and it compares favorably with a p-semidine
reduction
procedure with use of a TCA filtrate. Alkaline phosphatase values obtained bythe
method in which a fl-glycerophosphate substrate is used compare favorably with a reference method in which p-nitrophenolphosphate
is used as a
substrate. Citrates, fluorides, nitrites, oxalates, and mannitol do not appreciably affect results obtained by the method.
Additional Keyphrases

p-nitrophenol

phosphate

method

micromet hod #{149}Ringbom plot #{149}bilirubin interference

phatase #{149} urinary P value for an infant

M serum

TECHNIQUES
for estimating
inorganic
phosphorus
(Ps) are based
on the formation
of a molybdophosphate
complex
by the reaction
of phosphate
with molybdate
in
an acid medium
and subsequent
reduction
to a
heteropoly
blue complex under controlled
conditions (1, 2). The phosphomolybdate
reaction
is
more commonly
performed
on trichloroacetic
acid
filtrates of serum (3-6).
P may also be estimated
by the formation
of a
molydovanadophosphoric
acid complex
(7-9). In
this ease, the endpoint
is a stable yellow color
formed by the reaction
between
orthophosphate
and vanadate
in an acid medium.
However,
this
technique
for the quantification
of phosphorus
has
not been widely used in routine clinical laboratory
procedures.
Two direct methods
for serum phosphorus
are
currently
commercially
available.
One (Hycel,
Inc., Houston,
Tex.) is based on the formation
of
the molybdophosphate
complex in the presence of
sulfuric acid and molybdate,
and its subsequent
reduction
with
ferrous
sulfate.
The resulting
greenish-blue
complex is suspended
in a medium
containing
a surface-active
detergent
and measured by its absorption
at 650 nm. The second
OST

CLINICAL

From the General

Clinical
Research
Center for Children
of the
Childrens
Hospital
of Michigan,
and Wayne
State
University
School of Medicine,
Detroit,
Mich. 48202.
Received
April 20, 1970; accepted
June 9, 1970.

776 CLINICAL CHEMISTRY, Vol. 16, No. 9, 1970

compared
#{149} possible
acid and alkaline phos-

commercial
procedure
(Monitor
Corp.,
Indianapolis, md.) is based on the formation
of a phosphomolybdate
complex and subsequent
reduction
with hydroxylamine
in the presence of an alkaline
buffer. Polyvinylpyrrolidone
is incorporated
with
the reducing
reagent
in this procedure,
and allegedly catalyzes the formation of a phosphomolybdate polymer.
This polymer is then reduced to a
molybdenum
blue complex for measurement.
However, polyvinylpyrrolidone
possibly serves primarily as a suspending
agent for the phosphomolybdate complex and protein.
My interest in a direct procedure
for serum P
estimation
stems from experience
with the procedure of Raabe (10), who used a reducing agent
consisting of Metol (p-methylaminophenol
sulfate)
and bisulfite
for molybdophosphate
reduction,
and suspended
the reduced complex in a basic medium before measurement.
Samples could he measured directly without
deproteinization
when the
Raabe method was used. The basic medium used
in this method appeared
to solubilize and suspend
the protein and reduced molybdophosphate
complex. Consequently,
I tried to devise an accurate,
simple, sensitive,
and direct procedure
for serum
and urine P and serum phosphatase
estimation.
Measurement
of serum alkaline and acid phosphatase
is a valuable
clinical diagnostic
aid, a
number of methods being available. The technique
of Bodansky
(11), in which fl-glycerophosphate
is
the substrate,
is considered
to be more specific for

phosphatase
activity
than less tedious and timeconsuming
procedures
(12-14).
Phosphate
is estimated
in phosphatase
procedures
in which glycerophosphate
is the substrate,
and so this
substrate
is ideally suited for the direct method.

Materials and Methods

Reagents
Unless otherwise
specified all chemicals
are reagent grade and all solutions aqueous.
Borate solution. Na2B4O7 10 H20, 38.14 g/liter.
Stock ammonium
molybdate.
(NH4)Mo7024
H20,
50 mmol/liter.
Stock sulfuric
acid, 5 mol/liter.
Prepare
from
concentrated
H2SO4 and standardize.
Acid molybdate solution. Prepare
by mixing 30
parts of stock molybdate,
and 30 parts of stock
sulfuric acid with 40 parts of water. Cool before
using. Stable indefinitely.
Acid solution.
Prepare
by adding 30 parts of
stock sulfuric acid to 70 parts of distilled water.
Ascorbic acid reagent, 1 g/100 ml. Prepare
as
needed. Stable for three days refrigerated.
Carbonate solution, Na2CO3. 2 mol/liter.
Phosphorus
stock standard,
1 mg of P1 per ml. Dissolve 4.394 g of potassium
phosphate,
monobasic
(KH2PO4) per liter of distilled water.
.

Phosphorus
dilute stock standard,
20 mg of P1 per
100 ml. Dilute the phosphate
stock standard
five-

fold. Store refrigerated.

Albumin,
human.
Prepare
a phosphate-free
portion of albumin by mixing 1 g of Dowex 1 resin
(chloride form, 50-100 mesh) with 100 ml of saltpoor human albumin, 10 g/100 ml. Shake overnight
at 4#{176}C
to remove phosphates,
and filter.
Phosphorus
working standards for serum P1 and
phosphatase.
Add 2.0, 4.0, and 8.0 ml, respectively,
of the phosphorus
dilute stock standard
to three
tubes, each containing
12 ml of albumin,
and mix.
Refrigerate
when not in use. These standards
represent a concentration
of 2.0, 4.0, and 8.0 mg of
P1 per 100 ml of serum.
Phosphorus
working standards, urinary P1. Prepare in the same manner
as indicated
for phosphorus working standards
for serum P1 and phosphatase,
except
substitute
distilled
water
for
albumin solution.
Buffered
substrate
for alkaline
phosphalase
stock
solution.
Place 5.0 g of sodium $-glycerophosphate

and 8.50 g of sodium diethylbarbiturate

in a 500ml volumetric
flask, dissolve the mixture in about
300 ml of water, and dilute to volume. Add 5 ml of
petroleum
ether to the flask, as preservative,
and
mix. Refrigerate.
Discard after 12 weeks.
Phosphatase
buffer, alkaline.
Prepare
as for the
working
alkaline
phosphatase
substrate
except
omit the fl-glycerophosphate.
Working alkaline phosphatase substrate, pH 10.8.
Dilute
100 ml of buffered
substrate
for alkaline

phosphatase
stock solution and 5.8 ml of NaOH,
100 mmol/liter,
to exactly 200 ml. If the pH is more
than 0.1 unit from 10.8, adjust with NaOH or lid,
0.1 mol/liter.
Add 5 ml of petroleum
ether to the
flask and mix. Refrigerate.
1)iscard
after two
weeks.
Buffered
substrate for acid phosphatase,
stock
solution. Dissolve 5.0 g of sodium $-glycerophosphate and 4.26 g of sodium diethylbarbiturate
and
dilute to 500 ml. Add 5 ml of petroleum
ether to
the flask and mix. Refrigerate.
Discard
after 12
weeks.
Phosphatase
buffer, acid. Prepare
as for the
stock acid phosphatase
substrate
except omit the
fl-glycerophosphate.
Working

acid

phosphatase

substrate,

pH

5.0.

Dilute 100 ml of buffered substrate

for acid phosphatase,
stock solution, and 10 ml of 1 mol/liter
acetic acid to 200 ml. Adjust the pH to 5.0 0.1
with NaOH,
100 mmol/liter,
or acetic acid, 1
mol/liter.
Add 5 ml of petroleum
ether to the
flask, and mix. Refrigerate.
Discard after 10 days.

Procedures
Serum inorganic phosphorus.
(a) Pipet 0.1 ml of
serum into each of two test tubes labeled test
and blank.
Prepare
standards
by treating
the
phosphorus
working
standards
for serum P1 and
phosphatase
in the same manner.
(b)Add
1.5 ml of borate solution to the sample,
standards,
and their respective blanks, and mix.
(c) Add 0.5 ml of acid molybdate
solution to the
test and standard
tubes and 0.5 ml of acid solution
to their respective
blanks, and mix.
(d) Add 1.0 ml of ascorbic acid reagent to the
standards
and sample tubes and 1.0 ml of distilled
water to their respective
l)lanks, mix, and allow
the tubes to stand for 15 mm.
(e) Add 3.0 ml of carbonate
solution to all tubes
and mix. Allow to stand 5 mm and read the absorhances
of the sample
and standard
against
their respective blanks at 720 nm.
(f) From a graph of absorhance
vs. concentration
obtain the concentration
of the samples.
Urinary
in organic
phosphorus.
Mix urine well
and make a 10-fold or 20-fold
dilution.
Treat
the diluted sample as for serum, beginning
with
step a under the procedure for serum P1. Substitute
phosphorus
working
standards
(urinary
P1) for
the serum P1 standards.
Read
the phosphorus
concentration
of the
sample
from a graph
of the concentration
vs. absorbance.
Multiply
the concentration
obtained
from the graph by the dilution
factor used, to
obtain
the
urinary
phosphorus
concentration
in mg/100

ml of urine.

Acid and alkaline phosphatase.

of working phosphatase
substrate

(a) Place 0.9 ml

(alkaline or acid)

CLINICAL CHEMISTRY, Vol. 16, No. 9, 1970 777

into a tube labeled enzyme

sample and 0.9 ml
of phosphatase
buffer (acid or alkaline)
into each
of two other tubes labeled enzyme
blank
and
sample blank.
(b) Incubate all tubes at 37#{176}C
for 60 mm.
(c) Remove the tubes from the water bath and
treat the enzyme sample and enzyme blank as
outlined
for test tube, beginning
with step b of
the procedure
for serum P1. Treat the sample blank
the same as the blank
tube, step b in the procedure for serum P1. Both enzyme sample and
enzyme
blank are read at 720 nm vs. the sample blank.
(d)
Obtain the concentration
of phosphorus
in
the enzyme
sample and enzyme
blank from a
concentration
vs. absorbance
graph as outlined
under the method for serum P1, but include 0.9 ml
of phosphatase
buffer (alkaline
or acid) in the
standards
to make up for volume differences.
The
phosphatase
concentration
(acid or alkaline)
in
Bodansky
units is the enzyme sample minus the
enzyme blank.
The concentration
of P, in the enzyme blank is
the serum P, concentration.

0.8
0.7
06
p-Se,,idine Method
0,.ct Method

05
C

0
04

03
02
01
320 360 400 440

480

520 560 600 640 680 720 760 800 840

nm

Fig. 1. Absorbance
spectra of the phosphomolybdate
complex, as measured
on a Beckman
DB spectrophotometer

(1-cm

light path)

C
0

Results and Discussion

Absorption
spectra of the color produced by the
direct method for serum P1 and an indirect
procedure with p-semidine
(15) as a reductant
are
shown in Figure 1. In both cases a broad maximum
is seen in the 600- to 700-nm portion of the spectrum, a higher maximum
in the near-ultraviolet
portion.
The near-ultraviolet
maximum
for the
direct method is located around 320 nm, somewhat
higher than that for the indirect
or filtrate procedure
using
p-semidine
reduction
(15). The
ultraviolet
maximum
for the indirect
method
is
located at 340 nm. I have chosen to use the higher
wavelength
(720 nm) for colorimetric
quantification in the direct method,
but the lower wavelength may be useful for a micromethod.
A reagent
containing
ammonium
molybdate
and sulfuric acid is used to form the phosphomolybdate complex.
The concentration
of both components is critical for maximum
stability,
sensitivity, and adherence
to the Beer-Lambert
law in
the concentration
ranges at which P1 is present in
serum. Although
maximum
sensitivity
is obtained
with a sulfuric acid concentration
of 0.35 mol/liter,
the color is not linear with concentration.
An acid
concentration
range of 1-2 mol/liter
produces
a
stable color that obeys the Beer-Lambert
law.
An acid concentration
of 1.5 mol/liter
was chosen
for the molybdate
reagent; higher acid concentrations tend to hydrolyze
some of the more stable
serum organic
phosphates.
The molybdate
concentration
for optimum measurements
ranged from
100-200
mmol/liter.
At higher molybdate
concentrations,
blanks readings were high.

778 CLINICAL CHEMISTRY, Vol. 16, No. 9, 1970

.125

.150

.175

.200

Bofots Conc.ntrotlon,Normolity

Figure

2. Effect

of borate

concentration

on the

direct

method for serum P

Absorbance measured in a coleman
105-mm cuvets)

ii spectrophotometer

(19 X

Borate was used in the Raabe method (10) for

serum P1 estimation.
The exact role of this ion
is not understood,
but in its absence sensitivity
and reproducibility
decrease. Sensitivity
increases
with borate concentration
(Figure 2). A reagent
containing
100 mmol/liter
was chosen
because
borate is poorly soluble.
Carbonate
solubilizes
the protein
and reduced
phosphomolybdate
complex,
the best carbonate
concentration
being 1-2.5 mol/liter.
However, the
best linearity
was obtained
with a 2 mol/liter
carbonate
concentration.
With the direct procedure,
the color is maximum
in 5 mm and remains stable for 2 h, then graduall
decreases. Minimum color (80%) is reached in 4 h.
A Ringbom
plot (16) of the color produced
by
the direct method at 720 nm is shown in Figure 3.
The physiological
range of P, encountered
in most
serum samples falls within the linear portion
of
this plot; this makes for a minimized spectrophotometric error.
Certain
compounds
interfere
with the more
classical
acid molybdate
deproteinization
proce-

dures f or serum P1 estimation:

citrates,
fluorides,
oxalates,
nitrites,
and mannitol.
To examine the
effect of these compounds
on the proposed
direct
method, I dissolved known amounts of these substances in albumin
solutions
containing
8 mg of
P1 per 100 ml. The solutions were then analyzed by
the proposed procedure.
Very little interference
is
produced
by any of these compounds
under the
conditions
of the reaction
(Table 1). Citrate
and
nitrite enhanced
color formation
slightly
at higher
concentrations.
Interference
by bilirubin
was examined
(Table
2). A known concentration
of purified bilirubin,
dissolved in a small concentration
of 0.5M Na2CO3,
was added to albumin containing
4.5 mg of P1 per
100 ml. The sample was then assayed for P1. As

Table 1. Effect of Certain Compounds on Direct

Estimation of Serum Inorganic Phosphorus
Concn,
ml

Sodium citrate

fluoride

8.00

100

8.17
8.65
8.00

108
100

8.17

102

200
50

8.50
8.00
8.00
8.00
8.00

106
100
100
100
100

100
200

8.25
8.60

103
107

50

8.00

100
200

8.00
8.25

100
100

50
100
200

Potassium

50

oxalate

100
Sodium

nitrite

10

ml

50
200

Potassium

mg/100

100

Man nitol
11111?

P1 found

mg/100
Compound

102

103

20-

30-

40-

50-

60-

P,

mg/100

80-

90-

20
Inorganic Phosphorus, mg/lOO
3456810

4.46
4.50

12.0

4.25

4.46

9.6

4.40
4.45
4.50
4.50
4.50

4.50
4.52
4.45

7.2
4.8
2.4
1.2
0

3.17
3.55

ml

Fig. 3. Ringbom plot of P1 values by the direct method.

Ordinate: transmittance,
%
Values for the above plot were measured at 720nm in a Beckman
DB spectrophotometer (1-cm light path)

Bilirubin,
mg/100 ml

3.80

2.21
2.53

liii
4060

method

43.2
38.4
33.6
28.8
24.0
19.2
14.4

3.00
I

ml, direct

1.58#{176}

1.90

70-

1001

Table 2. Effect of Bilirubin Added to Albumin

Solution, on the Direct Estimation of Inorganic
Phosphorus

480b

4.72
4.70
4.62
4.54
4.50

4.50

4.50*

Readings made as indicated in Procedure section.

6

//
/

,,

/,

Readings made with an albumin blank, free of bilirubin (*).

the P1 was estimated at 4.50

By an indirect method (1.5)

mg/lao ml in every sample.

,#{149}/

N24
Y-O.177+L076X

o385O.42

3.97!

3
4
p-S.eldln. M.thod,Inoegaric
umq/IOOmI (X)

5
P

0Ir.ctFence, Sulfat.

Fig. 4. Comparison of three methods

phorus determination

0.49

4
5
Msthod,Ir.aeqanic P

ag/lOOm1

(Xl

for serum

phos-

The direct method for serum P1 estimation is compared with a

method

usinga protein-free filtrate and p-semidine reduction

(left), and a direct method using ferrous sulfate to reduce the

phosphomolybdate
complex (right)

shown, as much as 9 mg of hilirubin per 100 ml of

serum may be tolerated
without
serious interference. When an albumin blank sample was used instead, as much as 20 mg of bilirubin per 100 ml was
tolerable.
The bilirubin-phosphomolybdate
complex formed in the reaction
absorbs maximally
at
about 464 nm when measured
against an albumin
blank containing
no bilirubin.
However,
the color
produced
in a sample blank containing
bilirubin,
when ascorbate
and molybdate
are absent, is not
additive.
Apparently,
the bilirubin-protein
mixture suppresses
the absorbance
of the proteinbilirubin-phosphomolybdate
complex.
Therefore,
a sample containing
much bilirubin,
read against
its own blank, yields a lower reading than the same
sample read against
a blank of similar protein
composition
without
bilirubin.
Also, in icteric

CLINICAL CHEMISTRY, Vol. 16, No. 9, 1970 779

100

80

0S

-C

a
0

60
0
0

50
40
30

1 2

3 4

5 6 7

8 9 10 11 1213 14 15 161/
Dy

Fig. 5. Urinary inorganic

phosphorus
direct and p-semidine methods
Urinary P1 values on a 6-month-old
days

Number

infant,

values
obtained

by the
during 20

Table 3. Alkaline Phosphatase Activity Found in

24 Serum Samples by the Present Method and by
a p-Nitrophenol Phosphate Method with
9-Glycerophosphate as a Substrate
p-Nitrophenol

method,

Direct method,
Bodansky
units

Range

2.95
0.8-11.4

SD

2.26

Mean

BLB units

2.55

1.3-7.1
1 38
0.981
1.60

Bessy-Lowry-Brock.

samples one obtains a more correct P1 value if an

albumin blank is used.
As much as 600 mg of added hemoglobin
per
100 ml does not seriously interfere with the method.
Serum P1 results by the direct method and an
indirect
procedure
with a TCA filtrate
(16) are
shown on the scattergram
on the left in Figure 4.
The two methods correlate well (r = 0.973). The
method
was also compared
with a commercial
direct-reduction
procedure
in which ferrous sulfate
is used (Hycel).
Results with the direct method
were slightly higher than those obtained
by the
commercial
procedure,
but results by the two methods correlate well.
The direct method may also be used to measure
P1 in urine. Results of a comparative
study on a
20-day urine collection from a 5-year-old child are
shown in Figure
5. Results
by the p-semidine
method for urinary phosphorus
(6) are compared.
Values by the two methods agree closely. Apparently, the procedure
is well suited to measurement
of urinary P1.
Serum alkaline phosphatase
values obtained
by
the direct method
and by a procedure
using
pnitrophenyl
phosphate
as a substrate
(17)
correlate well (Table 3). However, abnormal
values by
the direct method are generally higher than those

180 CLINICAL CHEMISTRY, Vol. 16, No. 9, 1970

measured by the p-nitrophenol

method. The range
for the $-glycerophosphate
method was also wider.
Acid phosphatase
values were also estimated
by
both methods.
The two methods correlated
poorly
(r = 0.515). The range and average for the Bodansky method (X = 0.98; range, 0.6 to 2.2 units)
was higher and included
more abnormal
values
than samples assayed by the p-nitrophenol
method
(X = 0.32;range,0.120
to 0.870 unit).
One advantage
of the direct method for serum
phosphatase
is that a value for P1 is also obtained
(enzyme
blank).
This method
is shorter
than
the classic Bodansky
method
(11) or its more
popular
modification
by Shinowara
et al. (12)
since deproteinization
centrifugation
steps
are
omitted.
This
NIH.

work

was

supported

by USPHS

Grant

FR-74

from

the

References
1. Woods, J. T., and Mellon,
M. U., Molybdetium
md. Eng. Chem., Anal. Ed. 13, 760 (1941).
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1). F., and

mination
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3. Fiske,

of phosphorus
as molybdiphosphoric
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acid). Anal. Cheni. 20, 749 (1947).
C. H., and Subbaliow,
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Ghem.

Mellon,

M. U.,

blue reaction.

Spectrophotometric

deter(phosphoJ.

Biol.

81, 629 (1929).

4. Gomori,
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U.,

A modification

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6. 1)ryer, u. L., Tammes,

A. H., and Itouth,
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estimation

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iii steel.

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J. Biol. Chern. 169, 39 (1947).
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M. A., Spectrophotometric
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Anal. Chem. 27, 1626 (1955).

deteracid.

10. Raabe, S., The importance

of phosphata.se
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Bee. Tray. Chirn. 74, 652 (1955).
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A., Phosphata.se
studies:
1)eterminatioii
of serum
phosphata.se
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influencing
the accuracy
of the determination.
J. Biol. Gheni. 101, 93 (1933).
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U. Y., Jones, L. M., and Reinhart,
H. L., Estimation of serum
inorganic
phosphate
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J. Biol. Chem. 142, 921 (1942).
13. Henry,
11. J., and Chiamori,
N., Variation
clinical
samples
as a source of error in enzyme
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B. S., Lemon,
M. K., Acid phosphata.ses.

H. I\1., 1)avison,
Amer. J. Clin.

of the pH
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M. M., and Schwartz,

PaIhol. 24, 807 (1954).

15. Goodwin,
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with
Ada 11, 153 (1954).

of

in

E., Theory
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Chim.

17. Andersch,
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as the substrate
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