CLIN.
CHEM.
20/3,
332-336
(1974)
Enzymatic Method for Determination of Inorganic Phosphate in
Serum and Urine with a CentrifugalAnalyzer
Michael A. Pesce, Selma H. Bodourian, and John F. Nicholson
We describe an enzymatic method for determining
inorganic phosphate in serum and urine, with use of
the “CentrifiChem.”
The sample is mixed with a
combined enzyme-substrate
system consisting of
the enzymes phosphorylase a, phosphoglucomutase,
and glucose-6-phosphate
dehydrogenase, the coenzyme NADP, the substrate glycogen, and adenosine
5’-monophosphate
as the activator for phosphorylase. The increase in absorbance at 340 nm as
NADPH is formed is linearly proportional to the concentration of inorganic phosphate to 15 mg P/dl.
This method circumvents
deproteinization
and requires only 10 l of sample. Results obtained with
the automated enzymatic method show good correlation with manual and automated molybdenum blue
methods.
Additional Keyphrases: CentrifiChem
methods
#{149}
direct
#{149}
comparison
of
spectrophotometry
In most clinical
laboratories,
inorganic
phosphate
is determined
by formation
of a phosphomolybdate
complex
and reduction
of this complex
to molybdenum blue, which is measured
colorimetrically.
Various agents are used to effect the reduction,
among
them
aminonaphthalene
sulfonic
acid (1), p-semidine hydrochloride
(2), ascorbic
acid (3), stannous
chloride-hydrazine
(4), ferrous
ammonium
sulfatethiourea
(5), p-phenylenediamine
(6), and p-methylaminophenol
sulfate (7).
Recently,
a method
(8) was proposed
for determining inorganic
phosphate
in serum with a centrifugal
analyzer
(“CentrifiChem”),
by measuring
the unreduced phosphomolybdate
complex
at 340 nm. Pesce
and Bodourian
(9) found that samples
from hyperbilirubinemic
infants
(bilirubin
>3 mg/dl),
analyzed
by this method,
gave falsely elevated
values for inorganic phosphate.
Division
of Clinical
Chemistry,
Babies Hospital,
The Childrens
Medical
and Surgical
Center of New York at Columbia-Presbyterian Medical
Center;
and the Department
of Pediatrics,
College
of Physicians
and Surgeons
of Columbia
University,
3975 Broadway, New York, N. Y. 10032.
Received Oct. 1, 1973; accepted
Dec. 6, 1973.
332
CLINICAL
CHEMISTRY,
Vol.
20, No. 3,
1974
An enzymatic
method
for determination
of inorganic phosphate
in serum was introduced
in 1966 by
Fawaz et al. (10), but they presented
little experimental data. This approach
was also used by Schulz
et al. (11) to determine
inorganic
phosphate
in
tissues. The method is based on the conversion
of the
substrate
glycogen in the presence
of inorganic
phosphate to glucose-i-phosphate
by phosphorylase
(a1,4-glucan:orthophosphate
glucosyltransferase,
EC
2.4.1.1),
conversion
of
glucose-i-phosphate
to
glucose-6-phosphate
by phosphoglucomutase
(aD-glucose-1,6-diphosphate
: cr-n-glucose-i-phosphate
phosphotransferase,
EC 2.7.5.1), and reaction
of glucose-6-phosphate
with NADP+ in the presence of glucose-6-phosphate
dehydrogenase
(D-glucose-6-phosphate:NADP
oxidoreductase,
EC 1.1.1.49)
to give
NADPH
and 6-phosphogluconate.
The amount
of
NADPH
formed is measured
at 340 nm, and is proportional
to the concentration
of inorganic
phosphate. The reaction scheme is:
glycogen
+ P
glucose-i-phosphate
phophorylae
‘
glucose-i-phosphate
pbo.phog1ucomutae
glucose-6-phosphate
glucose-6-phosphate
+
NADP
glucose-6-phoiphate
debydrogensee
6-phosphogluconate
+ NADPH
With this enzymatic
system,
deproteinization
is
not required.
Because
only a small sample volume is
required,
we believed
that this method
might
be
suitable
for use with a centrifugal
analyzer.
Therefore, we have determined
the optimum
conditions
for
the enzymatic
estimation
of inorganic
phosphate
in
serum and urine with the CentrifiChem.
Materials and Methods
Instrumentation
A centrifugal
Carbide Corp.,
analyzer,
Tarrytown,
the CentrifiChem
(Union
N. Y. 10591), was used.
0.7
Reagents
Triethanolamine
(0.1 mol/liter,
pH 7.2)
and ethylenediaminetetraacetic
acid (0.01 mol/liter).
Dissolve
14.9 g of triethanolamine
(Fisher Scientific
Co., Pittsburgh,
Pa. 15219) and 3.4 g of disodium
ethylenediaminetetraacetate
(Sigma
Chemical
Co.,,
St. Louis, Mo. 63178) in 800 ml of water. Adjust the
pH to 7.2 with concentrated
hydrochloric
acid and
dilute to 1 liter with water.
2. Magnesium
chloride,
0.3 mol/liter.
Dissolve
61.0 g of magnesium
chloride
(J. T. Baker Chemical
Co., Phillipsburg,
N. J. 08865) in 1 liter of water.
3. Adenosine
5’-monophosphate
disodium
salt, 0.1
mmol/liter.
Dissolve
50 mg of adenosine
5’-monophosphate
disodium
salt
(Boehringer
Mannheim
Corp., New York, N. Y. 10017) in 1 liter of water.
4. Nicotinamide
adenine
dinucleotide
phosphate,
disodium
salt, 24 mmol/liter.
Dissolve
18.9 mg of
this salt (Boehringer
Mannheim
Corp.) in 1 ml of
distilled water.
5. Glycogen,
100 g/liter.
Dissolve 0.5 g of glycogen
monosodium
salt (grade
1; Boehringer
Mannheim
Corp.) in 5 ml of distilled water.
6. Phosphorylase
a,
100
U/ml
(Boehringer
Mannheim
Corp.).
7. Phosphoglucomutase,
1000 U/ml
(Boehringer
Mannheim
Corp.).
8. Glucose-6-phosphate
dehydrogenase,
1750 U/ml
(grade 1; Boehringer
Mannheim
Corp.).
Working reagent.
Mix together
7.5 ml of reagent 1
300 1 of reagent
2, 150 l of reagent
3, 375 tl of reagent 4, 375 l of reagent
5, 150 cl of reagent 6, 8 cl
of reagent 7, and 15 zl of reagent 8. The final volume
of the working
reagent
(8.87 ml) is sufficient
for one
run of inorganic
phosphate
(29 positions)
on the
CentrifiChem.
The working reagent
is stable for one
day.
Stock phosphate
standard,
100 mg P/dl. Dissolve
458 mg of disodium
hydrogen
phosphate
(National
Bureau
of Standards,
Standard
Reference
Material
18611c) in 100 ml of distilled
water.
Working
(phosphate)
standards.
Dilute the stock
standard
to 10 and 5 mg P/dl with distilled
water.
0.6-
1. Buffer:
Procedure
A 10-al volume of serum, urine, and working standards with 50 l of diluent
(water)
is pipetted
into
the sample
cavities
and 250 tl of working reagent
is
pipetted
into the reagent cavities of the transfer
disc.
The reference
position
contains
250 zl of reagent
and
50 zl of water. A temperature
of 37 #{176}C
and a wavelength of 340 nm is used. An initial reading
is taken
3.0 s after starting
the rotor, a final reading
16 mm
later. The change of absorbance
between
3.0 s and 16
mm is used to determine
the phosphate
concentration, using simultaneously
determined
standards.
Results and Discussion
Figure
1 shows that
in all cases absorbance
is
maximum
by 16 mm and remains
constant
for 28
mm thereafter.
-
LA
::z
I,
Smg/di standard
42._6..8__A
TTI.
MINUTES
Fig. 1. Change in absorbance as a function of time, for
an inorganic
serum,
phosphate
standard
(5 mg phosphorus/dI),
and urine
1.0
l6mln.
0.8
07
0.6
0.4
0.3
0.2
0,I
4
8
2
6
PHOSPHORUS
STAN DARDS
(mq/dI)
Fig. 2. Standard curve for inorganic phosphate determination in concentration range 0.5 to 15 mg phosphorus/
dl
Temperature.
The reaction
rate depends
on the
temperature.
The final absorbances
are the same
whether
a temperature
of 25, 30, or 37 #{176}C
is used,
but the time required
to reach maximum
absorbance
is longer than 16 mm at a temperature
of 25 or 30
#{176}C.
Therefore,
the temperature
of 37 #{176}C
was chosen
for the test.
Linearity.
The standard
curve (Figure 2) is linear
from 0.5 to 15 mg P/dl.
Buffer
and pH value. The reaction
rate is not influenced
by the type of buffer
used, similar
rates
being obtained
in triethanolamine
and tris(hydroxymethyl)aminomethane
buffers.
When triethanolamme buffer is used, the pH-absorbance
curve is flat
between
pH 7.0 and 7.4 (Figure 3). Final absorbance
values
increase
with
increasing
concentration
of
triethanolamine
buffer
and become
optimum
at a
concentration
of 85 mmol/liter
in the working
reagent (Figure 4).
CLINICAL
CHEMISTRY,
Vol. 20, No.3,
1974
333
0.7
-
13A/
/16 mm.
Pool.d
s.nm,
Table 1. Inorganic Phosphate Determinations in
10 Sera Containing Abnormal Activities of
5’-Nucteotidase (5’-N) and Alkaline
Phosphatase (ALP)a
Phosphorus values by
0.3
-
0.2
-
0.1
5nig/dl
A---A-----L
stondcrd
Pool.d urum
/_.___......-_.
-
I
I
6.8
7.0
7.2
Manual
molybdenumblue method
(FiskeSubbaRow)
Enzymatic
method
(CentrlfiChem)
Inorganic phosphate, as mg P/dl
1.6
1.6
2.7
2.7
3.2
3.3
3.3
7.4
pH
Fig. 3. Dependence of absorbance on pH in triethanolamine buffer for an inorganic phosphate standard (5 mg
phosphorus/dI) and two pooled sera
ALP#{212}
U/liter.
305
246
3.4
4.6
4.8
4.4
5.0
5.1
5.3
5.0
5.2
6.3
6.3
5’-N’
6.2
6.3
24
19
495
8
594
352
310
368
1002
231
31
76
88
64
344
16
68
248
AMP concn in the working reagent: 1.69 mol/liter.
Normal range: 6-hO U/liter.
Normal range: 5-15U/liter.
A/.
0.7
-
0.6
-
0.5
-
0.4
-
/mm.
#{149}
Pooi.d
sarum
0.7
-
tA/
/16 ml,,,
__A
L
/,_.__.#{149}
S
5mg/di
standard
Pootad urum
0.2
-
.-
UfUI7
0.’
II
7
I
43
55
I
0.3
-
0.2
-
0.1
-
Sn,g/dl standard
170
BUFFER COC. (mind/I)
Fig. 4. Dependence of absorbance on buffer concentration for an inorganic phosphate standard (5 mg phospho-
rus/dI) and two pooled sera
#{149}__-..e..-__p
I
I
3.5
10.1
16.5
MQCI5(mind/I)
Fig. 5. Dependence
of absorbance
on the concentration
of magnesium
for an inorganic phosphate
standard (5 mg
phosphorus/dl) and two pooled sera
Magnesium
chloride.
Magnesium
ion is necessary
to activate
the enzyme system. With a concentration
of 10.1 mmol of magnesium
chloride
per liter in the
working
reagent
the highest
absorbance
is obtained
(Figure 5).
Adenosine
5’-monophosphate
(AMP). AMP is necessary to activate
the enzyme
phosphorylase.
However, because
AMP is hydrolyzed
by 5’-nucleotidase
(5’-ribonucleotide
phosphohydrolase,
EC
3.1.3.5)
and alkaline
phosphatase
(orthophosphoric
monoester phosphohydrolase,
EC 3.1.3.1) to yield inorganic
phosphate,
a high concentration
of AMP should be
avoided.
As shown in Table 1, AMP at a concentration of 1.69 ,zmol/liter
in the working reagent
is not
hydrolyzed
by sera with abnormal
activities
of 5’nucleotidase
and alkaline
phosphatase.
When these
sera were analyzed
for inorganic
phosphate
with an
AMP concentration
of 7 ,zmol/liter
or greater
in the
working reagent,
absorbance
continuously
increased,
334
CLINICAL CHEMISTRY, Vol. 20, No.3.1974
owing to hydrolysis
of AMP. When the concentration
of AMP is less than 0.85 cmol/1iter,
reaction
time is
16 mm, but the final absorbance
values are lower.
Therefore
we chose an AMP concentration
of 1.69
,zmol/liter,
to eliminate
interference
from the enzymes 5’-nucleotidase
and alkaline
phosphatase
and
to obtain maximum
sensitivity.
Glycogen.
Absorbance
is maximum
with a concentration
of 0.423 g of glycogen
per deciliter
in the
working reagent (Figure 6).
NADP.
With
a concentration
of 1.01 mmol of
NADP per liter in the working
reagent,
absorbance
is maximum
(Figure 7).
Enzymes.
Reaction
rate increases
as phosphorylase
activity
is increased.
However,
the cost per test also
Activity
0.7
-
0.6
-
hA/
Table 2. Effect of Phosphorylase Activity on the
Time of Reaction and Cost of Reagents per Test
ii6 miii.
of
phosphorylas.
In
working reagent,
U/mIa
Posted annum
0.5
Reaction
time,
2.5
1.7
1.0
miii
(Cents)
Cost/test
10
16
22
29
23
20
#{149}
One unit willform 1.0 moI of glucose-i-phosphate
glycogenand orthophosphate
perminuteatpH 6.8.
from
0.4
-
0.3
-
0.2
-
mg/dS standard
Posted uris,,
#{149}_.
0.I
Table 3. Recovery of Inorganic Phosphate Added
to Serum and Urine
Inorganic phosphate,
expressed as P
In serum
in urine
Added
Found
Expected
Recovery,
025
I
I
I
051
LOl
151
NADP
W
(mind/I)
Fig. 7. Dependence of absorbance on NADP concentration for an inorganic phosphate standard (5 mg phosphorus/dI) and two pooled sera
mg/dI
2.86
2.86
5.22
8.86
2.18
7.72
2.18
5.45
4.76
22.20
9.10
5.22
4.87
10.54
10.58
7.30
7.40
10.67
13.70
10.67
13.62
92
99
95
100
102
31.38
31.30
101
5.04
Table 4. Precision of the Method (on the
CentrifiChem)
Serum pool
Within-run
Lyophiliz.d
Day-to-day
0.7
-
0.6
-
0.5
-
0.4
-
0.2
SD
5.04
0.04
CV, %
0.83
Pooled serum
4___A-________4
5mg/s
#{149}
Pealed serum
___-S
-
standard
ref. serum
Within-run
Day-to-day
4.96
2.48
0.10
2.01
0.07
2.82
2.52
0.08
0.I
I
mg P/di
Mean
#{149}‘-.
I
03#{128}
09
PHOSPHOGLUCOMUTASE (U/nW)
3.17
Fig.8. Dependence of absorbance on phosphoglucomutase activity
for an inorganic phosphate standard (5 mg
phosphorus/dI) and two pooled sera
0.7
-
0.6
-
0.5
-
0.4
-
0.3
-
4_
0.2
-
‘-
hA/
116mm.
0.7
mis.
Pooled serum
0.6
-
0 5
#{149}
Pooled serum
#{149}_-e
04
Smg/di
__.4-8---._
standard
Pooled serum
0.I
I
0.2
-
5mg/dl standard
-4
#{149}
Pooled serum
-
I
0.332 0.423 0304
GLYCOGEN
3.0
(g/dI)
phorus/di) and two pooled sera
2). We selected
6.0
50
GPO
Fig. 6. Dependence
of absorbance on glycogen concentration for an inorganic phosphate standard (5 mg phos-
(Table
.
0.1
0.I1I
increases
0.3
a phosphorylase
of 1.7 U/ml for use in the working reagent.
an activity
of 0.9 U/ml for phosphoglucomu(Figure 8), and 3.0 U/ml for glucose-6-phos-
activity
With
tase
phate dehydrogenase
(Figure 9) in the working
agent, absorbance is maximum.
re-
(U/mi)
Fig. 9. Dependence of absorbance on glucose-6-phosdehydrogenase activity for an inorganic phosphate
standard (5 mg phosphorus/dI) and two pooled sera
phate
Recovery
and
precision.
Inorganic
phosphate
added to serum and urine was excellently accounted
for analytically
(Table
3). Within-run
precision
(Table 4) was determined
by simultaneously
performing 24 determinations
on a serum pool and lyophilized
commercial
reference
serum (“Versatol
CLINICAL CHEMISTRY, Vol. 20, No. 3, 1974
335
Table 5. Inorganic
Phosphate
in Serum of
Hyperbilirubinemic Subjects, as Determined by
the Present and by the Manual
Phosphomolybdenum Blue Methods
Present method
Biiirubin,
mg/di
Manual method
mg P/dI
4.6
4.8
5.5
5.3
5.6
5.1
5.5
6.1
69
17.2
3.8
4.6
6.8
4.8
3.8
4.4
6.8
5.0
fants by a manual
phosphomolybdenum
blue method and by the proposed
enzymatic
method.
The results (Table 5) indicate
no interference
from bilirubin.
The proposed
enzymatic
method,
which
avoids
deproteinization
of the sample,
is a rapid, reliable,
and accurate
micromethod
for the automated
estimation of inorganic
phosphate
in serum and urine.
This
Corp.
work
was
supported
by
a
grant
from
Union
Carbide
References
1. Fiske, C. H., and SubbaRow,
tion of phosphorus.J.
Biol. Chern.
AA”).
Day-to-day
precision
was measured
during
seven days, with use of the same serum pool and lyophilized
reference serum (Table 4).
Comparison
with other methods.
The enzymatic
method
was compared
to the molybdenum-blue
method
as used with the Technicon
SMA 12/60. For
81 sera, a least-squares
fit of the data produced
the
equation
y = 0.14 + 0.97x, with a correlation
coefficient (r) of 0.969. The enzymatic
method
was also
compared
to a manual
method
(Fiske.-SubbaRow).
For 87 plasmas,
a least-squares
fit of the data produced the equation
y = 0.05 + 0.99x (r = 0.990). For
25 urines, a least-squares
fit of the data produced
the
equation
y = 0.32 + 1.03x (r = 0.968). Results obtained
by the enzymatic
method
compare
favorably
with those for both the automated
and the manual
methods.
For a test to be useful.in
a hospital
for children,
it
must be free of bilirubin
interference.
To determine
the effect of bilirubin
on inorganic
phosphate
values,
we compared
samples
from hyperbilirubinemic
in-
336
CLINICAL
CHEMISTRY,
Vol. 20, No. 3, 1974
2. Dryer, R. L., Tammes,
tion of phosphates
with
Chem. 225, 177 (1957).
Y., The colorimetric
66,375(1925).
determina-
A. R., and Routh, J. I., The
N-phenyl-p-phenylenediamine.
determina-
J. Biol.
3. Bayinski,
E., Foa, P., and Zak, B., Determination
of phosphate:
Study
of liable
organic
phosphate
interference.
Clin.
Chim. Acta 15, 155(1967).
4. Kraml,
M., A semi-automated
determination
of phospholipids
Clin. Chim. Acta 13,442(1966).
5. Yee,
analysis
H. Y., A simplified
method
for automated
of serum and urine. Gun. Chem. 14, 898 (1968).
6. Parekh,
A. C., and Jung, D. H., Serum
determination
using p-phenylenediamine
Clin. Chim. Acta 27,373(1970).
7.
Drewes, P. A., Direct colorimetric
in serum and urine. Clin. Chim. Acta
8.
phosphorus
inorganic
phosphorus
as a reducing
agent.
determination
of phosphorus
39,81(1972).
Daly,
J. A., and Ertingshausen,
G., Direct method
for deterinorganic
phosphate
in serum with the “CentrifiChem.”
Clin. Chem. 18, 263 (1972).
mining
9. Pesce,
M. A., and Bodourian,
S. H., Bilirubin
interference
with ultraviolet
determination
of inorganic
phosphate
in the
“CentrifiChem.”
Clin. Chem. 19,436(1973).
Letter to the Editor.
10. Fawaz, B. N., Roth, L., and Fawaz,
G., The enzymatic
estimation of inorganic phosphate.
Biochem. Z. 344, 212 (1966).
11. Schulz,
D. W., Passonneau,
J. V., and Lowry, 0. H., An enzymic method
for the measurement
of inorganic
phosphate.
Anal.
Biochem.
19,300(1967).