Kinetic Determination
Phosphatase
MARY F. MASSOD, B.S.,
MT
of
Serum
Alkaline
Activity
(ASCP), KENT R.
AND SANDRA L. MCGUIRE,
WERNER,
B.A.
Department of Clinical Chemistry, Brockton Hospital, Brockton, Massachusetts 02402
ABSTRACT
Massod, Mary F., Werner, Kent R., and McGuire, Sandra L.: Kinetic determination of serum alkaline phosphatase activity. Amer. J. Clin. Path. 54:
110-117, 1970. An automated multipoint spectrophotometric serum alkaline
phosphatase procedure based on the liberation of p-nitrophenol from p-nitrophenyl phosphate is presented. The enzyme activity is a linear function of
the absorbance change at 405 nm. and is proportional to the increase in
p-nitrophenol. T h e method employs 2-amino-2-methyl-l,3-propanediol, an
amino alcohol buffer that enhances alkaline phosphatase activity and resists
changes in pH under conditions of the assay. Zero-order rates of transphosphorylation and activities proportional to the concentration of enzyme are
observed. Analytical conditions have been optimized and normal data determined. T h e method is highly precise, having an error of less than 1%.
Multiple samples are monitored automatically, simultaneously, and accurately on a recording spectrophotometer in only 2 min. with a maximum of
0.2 ml. of serum. Sample requirements may be reduced tenfold to accommodate ultramicro serum volumes. In keeping with the recommendation of the
International Union of Biochemistry, values are expressed in International
Units as milliunits per milliliter. With the described kinetic alkaline phosphatase procedure, the simplicity, convenience, and accuracy of spectrophotometric enzymology is achieved with a minimum of time, effort, and technical
skill.
trophenyl phosphate as substrate. 2 In 1965,
Frajola and associates4 reported a kinetic
analysis based on the BLB colorimetric
technic.
Enhancement of alkaline phosphatase activity and pH stability have been observed
when amino alcohols have been employed
as buffers.1 Because of the simplicity and
convenience of spectrophotometric enzymology, it was deemed worthwhile to investigate the kinetic determination. T h e
Received October 1, 1969; accepted for publica.
, , . n
. , ,„
n
tion December 8, 1969.
amino alcohol, 2-amino-2-methyl-l,3-pro110
ALKALINE PHOSPHATASE of serum is generally classified as Type I of the phosphomonoesterases.0 Elevations are common in
many conditions, including disorders of
bone, liver, and kidney.5
Since 1930 numerous methods and a
variety of substrates have been used to measure this important enzyme. In 1946, Bessey
and associates3 described a serum alkaline
phosphatase (BLB) method utilizing p-ni-
July 1970
111
KINETIC ALKALINE PHOSPHATASE
panecliol, has been incorporated as buffer
reagent.
This study presents the modified spectrophotometric serum alkaline phosphatase
procedure. Optimal assay conditions and
normal data have been established. Multiple samples are monitored automatically
and simultaneously in only 2 min. with a
maximum of 0.2 ml. of serum. Microsample requirements may be scaled down to
ultramicro volumes. Values are expressed
in International Units 7 as milliunits (mU.)
per ml. With the proposed multipoint spectrophotometric assay, serum alkaline phosphatase activity may be determined accurately with a minimum of time, effort, and
technical skill.
Materials and Methods
Equipment
Spectrophotometer. Changes in absorbance are measured in a Gilford 2000 spectrophotometer equipped with attachments
for recording, scale expansion, positioning,
and temperature control.
Reagents
Buffered Solution of p-Nitrophenyl Phosphate, Sodium Salt (p-NPP, 0.0065 M),
2-Amino-2-Methyl-l,3-Propanediol
(AMP,
0.3 M, pH 25 C, 10.1) and Magnesium
Chloride (0.0005 M). First, 195 mg. p-NPP,
di(cyclohexylammonium) salt (Calbiochem
No. 487611), 3.1542 Gra. AMP (Eastman),
5 ml. 200 mg. per 100 ml. MgCl 2 -6H 2 0
solution, and approximately 50 ml. deionized water are placed in a 150 ml. beaker.
These are dissolved by mixing and the pH
is adjusted to 10.1 with 1 N HC1. This
solution is transferred quantitatively to a
100-mI. volumetric flask, and diluted to
volume with deionized water. The solution
is stored in 3-ml. aliquots in the freezer.
The substrate is stable for at least 2 months
when maintained at —20 C.
Principle
P-Nitrophenyl phosphate is used as the
substrate for the determination of alkaline
phosphatase activity. The substrate contains 2-amino-2-methyl-l,3-propanediol as
buffer at pH 10.1. Alkaline phosphatase
acts on the substrate and liberates p-nitrophenol, a yellow anion in the presence of
alkali. As the reaction proceeds, the increase in p-nitrophenol is monitored automatically in a recording spectrophotometer.
The level of alkaline phosphatase activity
is, therefore, a linear function of the increase in absorbancy at 405 nm.
Procedure
Micro
1. T u r n on water bath and pump device
for thermal equilibration at 25 C.
2. Allow the instrument to warm up for
at least half an hour prior to use.
3. Calibrate the recorder according to
the manufacturer's instructions.
4. Place 3.0 ml. AMP buffered substrate
in a 12 by 100-mm. tube and bring to 25 C.
in a water bath.
5. Add 0.2 ml. serum, stopper, and mix
by triple gentle inversion.
6. Transfer to a 3-ml., 1-cm. path length
cuvette.
7. Allow the recorder to plot the increase
in p-nitrophenol for 2 min. The alkaline
phosphatase forms a straight line.
Ultramicro
1. Calibrate the recorder.
2. Prepare assay sample with 3.0 ml.
substrate, 0.18 ml. 0.85% NaCl, and 0.02
ml. serum.
3. Allow the recorder to plot the increase
in p-nitrophenol for 8 min. and calculate
accordingly.
Calculations
One milliunit of alkaline phosphatase
activity is described as the number of mi-
112
A.J.C.P.—Vol. 54
MASSOD ET AL.
cromoles of p-nitrophenyl phosphate converted to p-nitrophenol per minute per ml.
at 25 C. with a 1-cm. light path and read
at 405 nm. Therefore, mU. per ml. equals:
(Absorbance change)(reaction total volume)(l,000) (temperature factor) (temperature coefficient)
(Extinction coefficient)(serum volume)(reaction time)
where reaction total volume = 3.2 ml.
1,000 = conversion factor for IU per 1.
to mU. per ml.
Temperature factor (BLB Units at 37 C.
to mU. at 25 C.) = 1.6
Temperature coefficient = assay temperature corrected to 25 C.
Extinction coefficient of p-nitrophenol at
405 nm. = 18.5
Serum volume = 0.2 ml.
Reaction time = 2 min.
Substituting, mU. per ml. =
(absorbance change)(3.2)(l,000)(1.6)/
(18.5)(0.2)(2.0) = (absorbance change)
(692)
Full-scale absorbance of 100 mU. = 100/
692 or 0.145
Recorder chart paper scale = 0 to 100 mU.
mU. per ml. = 2-min. reading—zero-min.
reading.
For the ultramicro analysis employing a
serum volume of 0.02 ml. and a reaction
1.500 -
§ 0.750 m
§
<
MINUTES
Fic. 1. The rate of catalysis of serum alkaline
phosphatase, shown as a function of time, is linear
and regresses through zero. The change in absorbance per minute per milliliter is 0.361.
time of 8 min., the full-scale absorbance
for 1,000 mU. equals 0.578 and the activity
is derived from the formula: mU. per ml. =
8 min. reading—zero-min. reading.
Results
The kinetic multipoint serum alkaline
phosphatase procedure yielded linear reaction rates in all instances in which the
absorbance change per minute was no
greater than 0.361 (Fig. 1). Activities were
proportional to the volumes of serum analyzed. The rate of catalysis of serum alkaline phosphatase was linear up through
500 mU. per ml. (Fig. 2). At higher activities linearity was obtained by dilution of
serum.
Optimum assay conditions of the prop o s e d automated spectrophotometric
method were evaluated using serum specimens with both normal and elevated alkaline phosphatase activities. The optimal
concentration of p-nitrophenyl phosphate
is 4.5 to 7.5 mM (Fig. 3), that of 2-amino-2methyl-l,3-propanediol is 250 mM or more
(Fig. 4), that of magnesium chloride is 0.25
to 0.50 mM (Fig. 5), and the optimum pH
ranges from 10.0 to 10.2 (Fig. 6). Straight
lines with identical slopes were obtained
when the log of activity was plotted against
the reciprocal of absolute temperature. Coefficients prepared from these data are
employed to standardize reaction temperatures to 25 C. (Table 1). Within-day repeatability of the described microdetermination as performed on 14 replicate
measurements on the same serum sample
July 1970
113
KINETIC ALKALINE PHOSPHATASE
1.500
W
O
FIG. 2. Serum alkaline
phosphatase activity,
shown as a function of
volume, is proportional
to the volume of serum
analyzed. Linearity is
maintained over a range
of 10 to 500 mU. per ml.
<
X
o
H
U
0.1
ML OF SERUM
with a mean activity of 71.2 ± 0.3 mU. per
ml. was 0.5%.
The microanalysis was used to compare
15 separate serum specimens for their alkaline phosphatase activities in International Units. Statistical evaluation of the
experimental and calculated values reveals
a standard deviation of 2.7 mU. and a correlation coefficient of 0.9997 (Table 2).
The micro and ultramicro technics were
compared using 12 separate serum specimens. The standard deviation and correla-
0.200 _,
FIG. 3. Activity of serum
alkaline phosphatase
shown as a function of
p-nitrophenylphosphate
concentration. Assays employing both normal
(•—•) and elevated
(O—O) serum specimens
yielded maximal activities
in the presence of 0.0045
to 0.0075 M of substrate.
••
«
0.100 .
0
'
1
0.0055
p-NPP SUBSTRATE MOLARITY
1
0.0110
114
A.J.CP.—Vol.
MASSOD ET AL.
54
0.220
§
FIG. 4. Activity of serum alkaline phosphatase shown as a function of 2-amino-2-methyl-l,3-propanediol concentrations. Tests of
both normal (•—•) and elevated
(O—O) serum specimens revealed
maximal activities with buffer levels of 0.25 M or more.
a
u
w
o
CO
o
<!
AMP BUFFER MOLARITY
tion coefficient for this study was 3.5 mU.
and 0.9997, respectively (Table 3).
Normal alkaline phosphatase activities of
57 apparently healthy adults, 31 men and
26 women, 17 to 57 years old, were determined spectrophotometrically. At the 95%
confidence limits the normal range was 17
to 81 mU.
Discussion
Results of this study indicate the sensitivity and flexibility of the recommended
0.300 w
o
<:
x
u
w
o
0.150 «
o
a<
•
0-
•
•
•
1
0.0005
MAGNESIUM CHLORIDE MOLARITY
•
1
0.0010
FIG. 5. Activity of serum
alkaline phosphatase
shown as a function of
magnesium chloride concentration. Analyses utilizing both normal (•—•)
and elevated (O—O)
serum specimens yielded
optimal magnesium chloride concentrations of
0.00025 M or more.
July 1970
Fie. 6. Activity of serum
alkaline phosphatase
shown as a function of
pH. Measurements employing both normal
(•—•) and elevated
(O—O) serum specimens
illustrate peak activities
in a pH range of 10.0 to
10.2.
115
KINETIC ALKALINE PHOSPHATASE
§ 0.060
SUBSTRATE pH
spectrophotometric serum alkaline phosphatase procedure. Analytical conditions
provide zero-order kinetics at all levels of
activity and reveal broad maximal plateaus
with optimal concentrations of substrate,
buffer, and magnesium chloride. The pH
optimal scale of 10.0 to 10.2 is acceptable
over a relatively wide range.
The original buffer of Bessey and associates was subject to pH changes from
atmospheric C 0 2 and sera with abnormal
pH levels. These problems are not encountered with our method because of the
stability and superiority of AMP as a buffer. Hemoglobin and bilirubin both absorb
at the same wavelength as p-nitrophenol.
Hence, blanks prepared from hemolyzed or
icteric sera for the BLB colorimetric analysis were falsely elevated. The absorbance
change of spectrophotometric methods is
not influenced by blank color. Consequently, the described kinetic alkaline
phosphatase assay is independent of the
blank chromogenic status.
Table 1. Temperature Coefficients of Multipoint Spectrophotometric Serum
Alkaline Phosphatase Procedure
Reaction
Temperature
(C)
Temperature
Coefficient
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
1.33
1.25
1.18
1.11
1.05
1.00
0.95
0.91
0.87
0.83
0.80
0.77
0.74
0.71
0.69
0.67
0.65
0.61
116
A.J.CP.—Vol.
MASSOD ET AL.
Table 2. Serum Alkaline Phosphatase Activities
as Obtained with the Micro
Spectrophotometric Assay
Spectrophotometric Serum Alkaline
Phosphatase Activities
Serum Experimental Calculated
DifferNo.
Result
Result*
ence
(Milliunits per ml.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
10
20
31
39
44
51
70
79
109
122
163
233
248
309
381
10
20
30
39
43
50
69
81
105
119
160
237
241
314
389
0
0
1
0
1
1
1
2
4
3
3
4
7
5
8
Mean difference = 2.7
Standard deviation of differences between data
from the two technics = 2.7 mU.
Correlation coefficient = 0.9997
toring of multiple samples is accomplished
simultaneously and rapidly. Data derived
at temperatures from 20 to 37 C. are corrected easily to 25 C. BLB results are standardized to International Units with simple conversion formulae. Calculated activities correlate closely with experimental
results.
With the proposed multipoint spectrophotometric kinetic procedure, large-scale
requests for serum alkaline phosphatase determinations may be fulfilled with speed,
simplicity, and accuracy.
Conclusions
The absorbance change based on the
liberation of p-nitrophenol from p-nitrophenyl phosphate buffered with 2-amino-2methyl-1,3-propanediol is the principle of
Table 3. Comparison of Micro and Ultramicro
Kinetic Serum Alkaline Phosphatase Assays 1
Serum
No.
* Calculated from the formula: mU./ml. = BLB
units/0.06.
The ultramicro adaptation manifests excellent agreement with the microassay and
requires only 0.02 ml. of serum, obtainable from a peripheral puncture. An ultramicro technic is an advantage when venipuncture is difficult or impossible. The
multipoint nature of the analysis allows observation of linearity while the reaction is
in progress. Thus, samples exhibiting nonlinear rates may be evaluated and adjusted
promptly.
The automated spectrophotometric technic is highly precise, having a within-day
repeatability of 0.5%. Unattended moni-
54
Kinetic Spectrophotometric Serum
Alkaline Phosphatase Method
Micro
Ultramicro
Difference
(Milliunits per ml.)
1
2
3
4
5
6
7
8
9
10
11
12
11
21
30
42
51
60
69
82
170
251
330
419
13
23
31
46
53
60
75
82
158
246
336
419
2
2
1
4
2
0
6
0
12
5
6
0
Mean difference = 3.3
Standard deviation of differences between results of both assays = 3.5 mU.
Correlation coefficient = 0.9997
July 1970
117
KINETIC ALKALINE PHOSPHATASE
the multipoint spectrophotometric serum
alkaline phosphatase procedure. The
method is sensitive, flexible, and precise,
revealing zero-order rates of transphosphorylation with absorbancy changes of 0.361
per min., activities proportional to enzyme
concentrations over a range of at least 10
to 500 mU. per ml., broad maximal plateaus of substrate components, and optimal
pH, and an error of less than 1%.
Multiple samples are monitored automatically and simultaneously on a recording spectrophotometer in only 2 min. with
a maximum of 0.2 ml. of serum. The versatility of the Gilford 2000 spectrophotometer allows ultramicro adaptations of the
technic with only 0.02 ml. of serum. Data
in milliunits per milliliter relate arbitrary
enzyme activity values to standard activities.
With the proposed spectrophotometric
kinetic assay, large volumes of alkaline
phosphatase determinations may be performed rapidly, conveniently, and accurately.
References
1. Babson, A. L., Greeley, S. J., Coleman, C. M.,
and Phillips, G. E.: Phenolphthalein monophosphate as a substrate for serum alkaline
phosphatase. Clin. Chem. 12: 482-490, 1966.
2. Bergmeyer, H. U.: Methods of Enzymatic Analysis. New York, Academic Press, 1965, pp. 32,
783-784.
3. Bessey, O. P., Lowry, O. H., and Brock, M. J.:
A method for the rapid determination of alkaline phosphatase with five cubic millimeters of
serum. J. Biol. Chem. 164: 321-329, 1946.
4. Frajola, W. J., Williams, R. D., and Austad,
R. A.: The kinetic spectrophotometric assay
for serum alkaline phosphatase. Amer. J. Clin.
Path. 43: 261-264, 1965.
5. Hawk, P. B., Oser, B. L., and Summerson, W. H.:
Practical Physiological Chemistry. 13th ed.
New York, McGraw-Hill Book Co., Inc., 1954,
pp. 637-638.
6. Henry, R. J.: Clinical Chemistry, Principles and
Technics. New York, Hoeber Medical Division,
Harper and Row, 1964, pp. 482-486.
7. International Union of Biochemistry. Report of
the Commission on Enzymes. Symposium Series, Vol. 20. New York, Pergamon Press, 1961,
pp. 8^0.
ERRATUM
In the article "An Evaluation of the Unitest System Based on Comparison
with Clinical Laboratory Methods" by John H. Glick, Jr., Ph.D., and Joseph W.
Timberlake, M.S. (53: 315-323, 1970), the senior author reports that the value
203A which appeared in Table 3 (page 318), row 1 of the third column, under
the heading "Cholesterol," should read 230A.