acid. (3), stannous chloride-hydrazine. (4), ferrous ammonium sulfate- thiourea. (5), p-phenylenediamine. (6), and p-methyl- aminophenol sulfate (7). Recently,.
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).
