dIN.
CHEM. 29/5, 751-761 (1983)
A ReferenceMethodfor Measurementof AlkalinePhosphataseActivityin
HumanSerum1
Enzyme Working Group of the Subcommittee on Standards, American Association for Clinical Chemistry,
Study Group on Alkaline Phosphatase. Members: N. W. Tletz (Chairman), C A. Burtis, P. Duncan, K. Ervln,
C. J. Petitclerc, A. D. Rinker, D. Shuey, and E. A. Zygowlcz
We present an official AACC reference method for the
measurement of alkaline phosphatase, the culminationof
optimizationexperimentsconductedby a group of independent laboratories.The details of this method and evaluation
of factorsaffectingthe measurement are described.A metal
ion bufferhas been incorporatedthat maintainsoptimaland
constant concentrations
of zinc(II) and magnesium(Ii)
ions.
Final reaction conditionsare: pH (30 #{176}C),
10.40 ± 0.05; 2amino-2-methyl-1-propanol buffer,0.35 mol/L; 4-nitrophenyl
phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L;
zinc sulfate, 1.0 mmol/L;and N-(2-hydroxyethyl)ethylenediaminetriaceticacid, 2.0 mmol/L.
more than 50 years ago, the meaphosphatase (ALP, EC 3.1.3.1) activity
in serum as an aid in the diagnosis of hepatobiliary and
bone disease
enjoys continued
popularity.2
Because
measurement methods have proliferated (1,2), this enzyme has
long been cited as demonstrating the need for standardizing
enzyme
assays.
As a result, considerable effort has been
expended
at national and international levels to develop a
widely accepted optimal method for measuring ALP activity
Although
introduced
surement of alkaline
(3-7).
The optimized
method presented
in this report is the
result of active experimental
work carried out in several
laboratories, each following a pre-established
protocol.
Through
such a process, variations in methodology due to
nstrumentation,
reagent preparation, and patient population are readily evident, and the emerging method benefits
from repeated
testing in a variety
of clinical laboratory
settings.
Human
alkaline
phosphatase
consists
of at least five
tissue-specific
enzymes,
which can be separated
by electrophoresis at alkaline pH (2, 8, 9). Additional tissue-specific
enzymes
have been observed
in some pathological
conditions. The relative proportions
of the individual ALP enzymes found in serum of patients is related to the severity of
the lesion in the organ or tissue from which they originate
and the half-lives of the individual enzyme forms.’Thus, one
cannot predict the relative proportions of the different forms
of the enzyme in any one patient’s sample.
‘Optimization
experiments were performed in collaboration with
the Expert Panel on Enzymes of the International Federation of
Clinical Chemistry. Members: M. H#{216}rder
(Chairman), M. Mathieu,
R. Rej, L. M. Shaw, J. H. Strmme, and N. W. Tietz.
Direct reprint requests to N. W. Tietz, Department of Pathology,
University of Kentucky Medical Center, Lexington, KY 40536.
2Nonstandard
abbreviations:
ALP, alkaline
phosphatase;
2A2M1P, 2-amino-2-methyl-1-propanol;
HEDTA, N-(2-hydroxyethyl)ethylenediarninetriacetic
acid; IFCC, International Federation of
Clinical Chemistry; 4NPP, 4-nitrophenyl phosphate; 4NP, 4-nitrophenol; EDTA, ethylenediaminetetraacetic acid; DEA, diethanolamine; RSM, response surface methodology.
Received Jan. 31, 1983; accepted Feb. 1, 1983.
Because the catalytic
and physical properties of the
individual enzyme forms differ, we used various types of
specimens
in establishing
the method. Sera were selected to
include specimens
from patients with liver and bone diseases and from pregnant
women. In addition, serum pools,
sera from hospital patients with both normal and increased
alkaline
phosphatase
activity,
and purified preparations3
of
human
liver and bone alkaline phosphatase were used for
establishing the reaction conditions.
Principles
Alkaline phosphatases hydrolyze a large variety of organic monophosphate esters with the formation of an alcohol or
phenol and a phosphate ion. Phosphate groups are transferred from an enzyme-phosphate
complex to water and
other phosphate acceptors present in the reaction medium.’
Under the conditions of the optimized
method, alkaline
phosphatase
catalyzes
two transphosphorylation
reactions:
phosphate
+ H20
4-nitrophenoxide
(H20 serves as a phosphate acceptor)
(2) 4-Nitrophenyl phosphate + 2-amino-2-methyl-1-propanol (2A2M1P)
4-nitrophenoxide
+ 2A2M1Pphosphate
(1) 4-Nitrophenyl
+ phosphate
-+
-.
Under the pH conditions used in the procedure,
4-nitrophenyl phosphate is essentially colorless whereas the 4.nitrophenoxide
ion has an intense
yellow color [molar
absorptivity at 405 nm (bandpass 2 nm) under the reaction
conditions = 18450 ± 200 L x mor’ x cm’I. This allows
the rate of formation
of the 4-nitrophenoxide
ion to be
measured
with a spectrophotometer.
Guidelines
for the
determination
of the catalytic activity of enzymes require
that only methods be used that monitor the reaction rate
continuously
or at frequent
intervals (10).
This recommended
method is based on the principles first
proposed
by Bessey et al. (11). However, the pH and
concentrations of substrates are optimized, 2A2M1P replaces glycine as a buffer, and a metal-ion buffer is included
that is capable of maintaining optimal and constant concentrations of zinc(H) and magnesium(ll)
ions. Finally the
original one-point assay has been replaced by a technique
that monitors the progress of the reaction-rate curve continuously or at frequent intervals.
The kinetic mechanism for the alkaline phosphatase
reaction has not yet been clearly elucidated. Most studies
have been conducted
on alkaline phosphatase from Escherichia coli, and considerable
debate (12, 13) has ensued on
A purified liver enzyme preparation of human alkaline phosphatase was obtained by courtesy of C. J. Petitclerc, University of
Sherbrooke, Sherbrooke, Quebec, Canada.
Bone, liver, and placental ALP enzyme fractions purified from
human tissue sources were generously contributed to this study by
P. Duncan, Centers for Disease Control, Atlanta, GA.
CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983
751
the question of whether or not E. coli alkaline phosphatase
with its two equivalent active sites (one on each of two
structurally and functionally identical subunits) exhibits
“negative cooperativity.” Much less information
is available
on the kinetic mechanism of alkaline phosphatase from
human tissues. As a result, at this time it is not possible to
use an initial
velocity
kinetic
model to aid in the optimiza-
tion of a method for human alkaline
phosphatase
measurements.
Optimal Conditions for Measurement
Chemical Society Microchemical
specifications (tolerance is
± 0.3%). pH meters
must be calibrated
at 30 ± 1.0 #{176}C
by use
of standardized
reference buffers (e.g., NBS)4 with pH values
within 1 unit of the reaction measurement pH. Electrodes
used for pH measurement must have a ceramic junction and
a negligible sodium-ion error at pH values greater than
10.0.
activity
Reagents5
1. 2-Amino-2-methyl-1-propanol
(2A2M1P), C4H,1N0,
Mr 89.14.
2. 4-Nitrophenyl phosphate (4NPP), disodium salt, hexahydrate, Mr 371.2.
Optimized (14) and technically most favorable conditions
for the assay of alkaline phosphatase activity in serum are
presented
below. The concentrations apply to the complete
3. Magnesium
acetate,
Mg(C2H302)2
4H20, A.R grade,
reaction mixture (i.e., including sample).
M 214.5.
30 ± 0.1 #{176}C
Temperature
4. Zinc sulfate, ZnSO4- 7H20, AR grade, Mr 287.54.
pH (30 #{176}C)
10.40 ± 0.05
5. Hydrochloric
acid, HC1, Mr 36.47.
2-Amino-2-methyl-1-propanol
(2A2M1P)
0.35 mol/L
6. 4-Nitrophenol
(4NP), CGH5NO3, M 139.1.
16.0 mmol/L
4-Nitrophenyl phosphate
7. N-(2-Hydroxyethyl)ethylenedianirnetriacetic
acid, tn2.0 mmol/L
Magnesium acetate
sodium salt, dihydrate (HEDTA), C10H15N2Na3O71.0
mmolIL
Zinc sulfate
21120, Mr 380.24, purity : 99%.
N-(2-Hydroxyethyl)ethylenediaminetri8. Sodium hydroxide,
NaOH, AR grade, Mr 40.
2.0 mmolJL
acetic acid (HEDTA)
Volume fraction of sample
0.0196 (1:51)
Note: The dissociation constant for the chelate of zinc(II)Preparation of Solutions
HEDTA in aqueous solution is about 3.16 x i015 molJL
Prepare
all solutions in calibrated flasks with fresh rea(15). Therefore, it is assumed that most of the zinc(II) in the
gent-grade
water6 (17).
reaction mixture
is bound by HEDTA, with a very small
1. Metal-ion buffer stock solution-HEDTA,
1122 mmol/
proportion present as free zinc(ll) ions, which has experiL; ZnSO4, 56.1 mmol/L; Mg(C2H3O2,
1122 mmol/L: The
mentally been determined
to be optimal for the reaction.
order of addition of the individual reagents is extremely
Most magnesium(ll) binds to the remaining HEDTA, leavimportant,
to avoid precipitation.
Dissolve 4.266 g of
ing sufficient magnesium ions in solution to saturate the
HEDTA
in about 70 mL of water. Then add 1.613 g of
enzyme
[dissociation
constant
for magnesium(II)
and
ZnSO4 7H20 and allow it to dissolve completely. Next, add
HEDTA is about 1.66 x 10-6 molIL (15)]. This metal-ion
2.407 g of Mg(C2H302)2 4H0, dissolve completely by mixbuffering system controls the availability of zinc(H) and
ing, and dilute the solution to exactly 100 mL with water.
magnesium(H) ions for the alkaline phosphatase in the
This solution is stable for at least three months when stored
reaction mixture.
at 2-6#{176}C.
The interval during which the enzyme is exposed to the
2. 2A2M1P/metal
-ion
buffer-2-amino-2-methyl-1
-proreaction mixture
is minimized by initiating the reaction
panol, 393 mmol/L; HEDTA,
224 mmol/L;
Zn804,
1.12
with sample. This is also in accord with the IFCC recommmol/L; Mg(C2J1302)2, 224 mmol/L; pH30 -c 10.40 ± 0.05:
mendations (14) that pipetting steps be minimized.
If,
All 2A2M1P lots used for this procedure must be checked for
however, a system is not capable of this type of initiation,
suitability according to the protocol in Appendix B. Those
the reaction may also be measured by mixing the sample
preparations of 2A2M1P that we have obtained from Sigma
with buffer and initiating the reaction with substrate soluChemical
Co. (St. Louis, MO 63178) and 2A2M1P (Gold
tion (see Appendix
B). Results obtained with either method
Label) from Aldrich Chemical Co. (Milwaukee, WI 53201)
are comparable (see Appendix
A, X). However, preincubahave been found to be suitable for activity measurements.
tion of sample with buffer for more than 5 mm may lead to
Other sources may be satisfactory but have not been used
enzyme inactivation.
extensively.
Warm the 2A2M1P to approximately
37 #{176}C
until it is
Instrumentation and Equipment
completely liquefied. Mix 17.52 g of 2A2M1P with approximately 400 mL of reagent-grade water. Adjust the pH to
Spectrophotometers
suitable for accurate absorbance
10.40 at 30 #{176}C
by adding HC1, 1 moL’L. With stirring, slowly
measurements at the wavelength of 405 nm must be used.
add exactly 10.0 mL of metal-ion buffer stock solution
Specifications
for the equipment (e.g., sample and reagent
handling, temperature control, and overall photometric performance) should conform to previous recommendations (14,
16). Instruments must be capable of monitoring the linear
NBS Standard Reference Materials, NaHCO3 (SRM no. 191)
portion of the reaction-rate curve and should display both
and Na2CO3 (SRM no. 192), can be used as a 25 mmol/kg solution
the initial absorbance of the reaction mixture
and absorhaving a pH of 9.968 at 30#{176}C.
The slope of the pH meter can be
bance vs time during the measurement interval. The temadjusted with a 10 mmol/kg NBS borax buffer (SRM No. 187b)
perature of the reaction mixture in the cuvette must be
having a pH’of 9.143 at 30#{176}C.
following manufacturers generously contributed reagents
controlled at 30 ± 0.1 #{176}C.
Even within these limits, temperato this study: Sigma Chemical Co., St. Louis, MO 63178, 2A2M1P
ture deviations alone can cause a range of analytical error
and 4NPP; Aldrich Chemical Co., Milwaukee, WI 53201, 2A2M1P
as large as 1.0% (±0.5%), as experimentally
determined by
Gold Label; Calbiochem-Behring Corp., La Jolla, CA 92037, 4NPP;
the Study Group.
and J. T. Baker Chemical Co., Phillipsburg, NJ 08865, 4NPP.
All volumetric
glassware
used for the preparation of
Specifications are: microbiological content (colony-forming units
reagents
and for pipetting must meet U.S. National Bureau
per mL), 10; resistivity at 25 #{176}C
(megohm centimeter), 10; Si02
(mgfL), 0.05; particulate matter diameter, sO.2 i.m.
of Standards (NBS) Class-A specifications or American
.
‘
5
6
752
CLINICALCHEMISTRY,Vol.29, No.5, 1983
1 above) and readjust to pH31 -c 10.40 ± 0.05 by
adding HC1, 1 mol/L. Dilute to exactly 500 mL with reagentgrade water and store in a closed container to minimize
absorption of carbon dioxide. This buffer solution is stable
for at least three months if stored at 2-6 #{176}C
and protected
from CO2.
3. 4-Nitrophenyl
phosphate, 179.5 mrnol/L, in reagentgrade water: The 4-nitrophenyl phosphate reagent must be
of the highest possible purity and must meet the following
criteria: (a) enzymatic conversion of 4NPP to 4NP should
result in a hydrolysis of >98.0%; (b) the molar absorptivity
of 4NPP at 311 nm in NaOH, 10 mmol/L, at 25 #{176}C
should be
9867 ± 76 L x mol’ x cm; (c) the free 4NP content must
be less than 0.3 mmol per mole of 4NPP; and (d) inorganic
phosphate content must be less than 10 mmol per mole of
4NPP. Detailed procedures
for checking these conditions are
given by Bowers et al. (18).
Dissolve 666 mg of 4NPP (disodium salt, hexahydrate, Mr
371.2) in approximately 8 mL of reagent-grade water and
dilute to 10.0 mL. The 4NPP solution should be freshly
prepared each day, and it is stable for as long as 8 h at 2026#{176}C.
4. 4-Nitrophenol (4NP), 1 mmol/L, stock standard: The
crystalline
4-nitrophenol preparation selected for use must
meet the following specifications:
(a) color: colorless to
slightly yellow; (b) melting point: 113-114 #{176}C;
(c) water
content: <0.10 g per 100 g of 4NP; and (d) molar absorptivity at 401 nm in NaOH, 10 mmollL, at 24 #{176}C:
18 380 ± 90 L
x moY’ x cm
Acceptable
4NP can be prepared by
recrystallization
or sublimation techniques as described by
Bowers et al. (19), although many commercial preparations
already meet these criteria. 4NP is also available as a
clinical standard (SRM 938) from the National Bureau of
(solution
‘.
Standards,
Washington,
DC.
Heat about 400 mg of high-purity 4NP, in a 10-mL
beaker, in an oven at 50 #{176}C
for 24 h. Allow the 4NP to cool in
a desiccator for 2 h. To prevent water absorption, quickly
weigh and dissolve 139.1 mg of 4NP in reagent-grade water
and dilute to 1000 mL in a volumetric flask. Mix thoroughly. This solution is stable for at least three months if
protected
from light and evaporation.
5. 4-Nitrophenol,
0.04 mmol/L,
in 2A2MIP/metal-ion
buffer (solution 2): Using a Class A volumetric pipet, transfer exactly 10.00 mL of 4NP, 1 mmol/L (solution 4), to a 250mL volumetric flask. Dilute to the mark with 2A2M1P, 393
mmolfL (solution 2), and mix thoroughly. Measure the
absorbance of this buffered solution in a 10-mm cuvet at 405
mn at 30 ± 1 #{176}C
vs a buffer blank (prepared by diluting
10.00 mL of reagent-grade water to exactly 250 mL with
solution 2), using the same instrument and bandpass as for
the procedure [Note: the absorbance of 4NP is temperature
dependent (20)]. The 4NP solutions (solutions 4,5) should be
prepared at least twice and the absorbance measurements
made in triplicate. All readings should agree within 1.0% of
the mean.8 This net absorbance reading is used to confirm
the molar absorptivity of 4NP used for the calculation of
alkaline phosphatase activities (See Cakulations).
6. Alkaline buffered substrate solution for overall reaction:
Combine 10 volumes of solution 2 with one volume of
solution 3 to make an appropriate total volume. The absor-
‘
The relative
Na24NPP
can
factor should
molecular mass of various preparations
of
vary depending on the extent of hydration. This
be considered
when one is preparing the substrate
solution.
8With an Acta CIII spectrophotometer
(Beckman Instruments,
Fullerton, CA 92634) with a bandpass of 2 nm at 405 nm, we
observed an absorbance of 0.737 ± 0.00 1 for this solution.
bance of this solution at 405 nm should not exceed 0.5 A.
This solution may be used for up to five working days if
stored at 2-6#{176}C.
Alternatively, this reagent may be stored
frozen (-20 #{176}C)
in convenient aliquots and is stable for at
least three months if thawed only once.
7. Alkaline buffered solution for individual sample blank:
Combine 10 volumes of solution 2 with one volume of
reagent-grade water to make a total volume that is adequate for the intended use.
Specimen Procurement, Stability, and Storage
Collect blood with minimal venous stasis. Freshly collected serum, free of hemolysis, is the preferred specimen, but
heparinized plasma is acceptable. Complexing anticoagulants such as citrate, oxalate, and EDTA must be avoided
(2). Cell-free
serum should be obtained by centrifuging
clotted blood for 10 mm at a relative centrifugal force of
approximately 900 x g.
Increases in alkaline phosphatase
activity have been
observed
after storage of human serum but depend on
storage conditions. Activities of some sera may increase by
about 1 to 2% when kept at room temperature
up to 4 h after
venipuncture (21). However, activities increase significantly
after warming of previously refrigerated or frozen sera; the
rate of this increase is time- and temperature-dependent
(22-24) and may also be affected by the reaction conditions
(25). A similar effect is also observed in lyophilized samples
after reconstitution. This phenomenon has been attributed
to the dissociation of complexes formed between alkaline
phosphatase and lipoproteins at the lower temperatures (26)
or during lyophilization, thereby “reactivating” the enzyme
as it warms up or goes into solution. For these reasons,
freshly
collected
serum samples should be kept at room
temperature
and assayed as soon as possible, preferably
within 4 h after collection.
Procedure
1. Add 2.50 mL of buffered substrate solution (solution 6)
to the reaction cuvette and warm to 30 ± 0.1 #{176}C.
2. Initiate the reaction by adding 50 L of sample to the
cuvette and mix thoroughly.
3. Immediately
record the change in absorbance of the
reaction mixture at 405 ± 1 urn at 30 #{176}C
for up to 5 mm after
initiation of the reaction. Calculate the change in absorbance with time (&4/it) from the linear (16) portion of the
curve. Extending the reaction interval beyond the 5-mm
period may result in deviations from linearity for some
samples.
4. A reagent blank activity must be determined
for each
prepared batch of reagent mixture (solution 6) to account for
any possible spontaneous, nonenzymatic hydrolysis of the
substrate. Repeat steps 1-3 of the above procedure, substituting 50 L of reagent-grade water for sample. This blank
activity, if present, must be subtracted
from all alkaline
phosphatase
activity measurements
determined
with the
same batch of reagents. Generally, the observed reagent
blank activities under the stated measurement conditions
are less than 1 U/L and are thus insignificant.
5. A sample blank activity must be determined for each
sample by substituting solution 7 for solution 6 and following the same procedure as above. Sample blank reactions
may result in absorbances that are either increasing (+ LA!
t) or decreasing (- .A/t).
Under the stated measurement
conditions, sample blank reactions have been observed that
range from + 0.0008 A/mm (2.2 U!L) for a lipemic sample to
-0.0102 A/mm (28.1 U!L) for a severely icteric sample. Most
serum samples have blank activities corresponding to less
than ±0.0007 A/mm (1.9 U/L).
CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983
753
6. The values of LA/St for the overall alkaline phosphatase reaction are constant over a period of at least 5 mm for
sera with activities up to 800 UIL (0.289 A/mm) if accurate
absorbance readings up to 2.000 A can be made with the
spectrophotometer.
if the value of LA/mm exceeds 0.289 or
decreases during monitoring, the sample should be diluted
five- to 10-fold with 154 mmol/L sodium chloride and the
measurement repeated. The period of observation for the
blank rates should be the same as for the overall reaction.
Calculations
Catalytic
concentration
(U/L)
V
=
X
(LA/st)
rxlxv
V is reaction
volume
(L), e is the micromolar
absorptivity (L x mol’
x cm 1), 1 is pathlength of cuvette
(cm), v is sample volume (L), LA is change in absorbance,
and t is reaction interval (mm).
Where
For the described
LA
t
V
e
v
=
.Atest
=
1 mm
LA
method:
=
blank
reagent
2.55 x i0- L
18.45 x io- L
0.05 xl0-3 L
1 cm
=
=
=
U/L
-
X
-
moY’
LAsampie
blank
x cm1
=
2.55 x iO-3
(18.45 x 10-s) x (1) x (0.05 x 10-s)
=
2764 x (LA/mm)
s should be confirmed
A40
L
x 25 x iO
18.27 x 10 to 18.60 x
within this range, the
instrument
should be
X
(LA/mm)
in each individual laboratory as
and should be within the range of
iO3 L x .tmol’ x cm’. if it is not
spectrophotometric
accuracy of the
confirmed
and the measurement
repeated.
Analytical Variability
For nine separate laboratories, run-to-run imprecision for
samples having mean activities of 126 and 254 U/L showed
coefficients of variation from 0.7 to 2.6% (mean = 1.6%) and
0.4 to 2.6% (mean = 1.3%), respectively. Between-laboratory
imprecision for eight separate laboratories for samples with
activities of 48 and 226 U/L ranged from 4.9 to 7.1% and
from 2.7 to 5.3%, respectively.
A detailed report on these
transferability studies will be published in a separate communication.
Appendix A. Verification of Experimental
Conditions for the Measurement of Alkaline
Phosphatase
Optimum
conditions
for measuring
the catalytic
activity
of alkaline phosphatase in human sera have been investigated. Optimized reaction conditions are defined as those
conditions that are most favorable for both the enzyme
kinetic reactions and the technical aspects of the measurement (i.e., optimum conditions do not necessarily provide
maximum possible activity). In the following text, data
supporting the selection of these conditions are presented.
The sources of the alkaline phosphatase used were those
listed above, in the introduction.
I. Buffer Selection
Several buffers have been proposed for use in the measurement of alkaline phosphatase activity in serum, but the
three most commonly used are diethanolamine
(DEA), 2754
CLINICALCHEMISTRY,Vol. 29, No. 5, 1983
arnino-2-methyl-1-propanol
(2A2M1P),
and hydrogen
car-
bonate buffers.9 DEA and 2A2M1P have received strong
support because they function as phosphate acceptors, allowing transphosphorylation
to occur in addition to hydrolysis.’O
Hydrogen Carbonate Buffer
This
buffer offers the following advantages:
(a) it is
in very high purity
(A.it grade);
(b) maximum
alkaline phosphatase
activity is observed at relatively low
available
concentrations (-0.15 mol/L); (c) the pK (10.2) is close to the
pH optimum for alkaline phosphatase
activity; and (d) it is a
nonphosphorylating
buffer and thus allows measurement of
hydrolysis only (if this is desired) or it can be used in
conjunction with phosphate acceptors if greater sensitivity
is needed.
Hydrogen
carbonate buffer inhibits alkaline phosphatase
activity, which becomes more apparent as the concentration
of buffer increases. The degree of inhibition of some commercial control materials is even more pronounced than that
observed with patients’ sara. Inhibition can also be observed
by following the reaction-rate curve and noting deviations
from linearity with time. These deviations are most apparent at higher carbonate concentrations. The lower alkaline
phosphatase activity observed in the presence of hydrogen
carbonate buffer (as compared to 2A2M1P and DEA buffer)
is adequate for most purposes and instrumentation,
but can
be a liability when one is measuring the relatively low
activity of a tissue-specific enzyme after heat or chemical
inactivation of the other alkaline phosphatase enzymes. For
these reasons, hydrogen carbonate as a buffer for alkaline
phosphatase
activity measurements
was eliminated from
further consideration.
DEA and 2A2M1 P Buffer
DEA buffer has been recommended for use in the proposals by the German and Scandinavian
societies (3, 4), while
2A2M1P buffer has been recommended
by the French
society (7) and others (2,6). In spite of their popularity, these
two buffers have certain liabilities, which must be weighed
against their advantages:
A. Sensitivity.
Both buffers were primarily selected because of their ability to accept phosphate groups, resulting
in increased activities. This increased sensitivity allows the
use of small sample volumes. However, the significantly
increased sensitivity associated with use of DEA buffer may
be a liability, because the need for pipetting very small
sample volumes (e.g., 5 L) may result in increased imprecision. Alternatively,
if the sample size is kept larger, the
analytical
range is significantly
reduced. Therefore,
2A2M1P appears to be a good compromise between DEA
and hydrogen carbonate buffers inasmuch as its use results
in greater sensitivity than observed with hydrogen carbonate buffer but not in the extreme sensitivity associated with
DEA buffer.
B. Buffer concentration.
Maximum activities in DEA and
2A2M1P buffers are not obtained until concentrations
of
approximately 1.8 moJIL and 0.9 molIL, respectively, are
92-Amino-2-methyl-1,3-propanediol
buffer has also been suggested (27) but was found to have no advantages over preacreened lots of
2A2M1P.
‘#{176} has been suggested that the increased activity observed with
increases in concentration of these nucleophilic buffers is partly the
result of solvent and ionic strength effects (28). This has been shown
for systems containing nonphosphorylating buffers, in which addition of increasing amounts of sodium chloride resulted in increases
in the total reaction rate (28).
attained (6, 29). In the case of DEA, this concentration
results in an increase in viscosity of the solution, which may
cause measurement
errors. In addition, the previously mentioned solvent and iOnic strength effects will be observed,
and the effects of such high buffer concentrations on proteins, and especially on enzymes, have not been adequately
elucidated.
A concentration
of 1.0 mol/L for DEA (3, 4)
minimizes
but does not eliminate these concerns. In contrast, a system containing 2A2M1P buffer reaches 95t7
of
maximal
activity
(depending
on tissue-specific
enzyme
com-
position of sample) between 0.2 and 0.3 mol/L (Figure 1).
Further increases in activity at concentrations above 0.3
mol/L are minor and may be partly due to solvent and ionic
strength effects (28). Thus, in the case of 2A2M1P there
seems to be little justification
to use concentrations
ing 0.35 mol/L, a concentration
slightly
greater
exceedthan 0.3
mol/L, to protect against possible sample-to-sample
variations. Furthermore,
the use of these lower buffer concentrations
offers
the
advantage
of less
competition
between
substrate and buffer for their specific sites on the enzymes,
thus decreasing substrate requirements
as further discussed
in Section II, Substrate Selection. Increases in 2A2M1P
concentration
of 1 mollL increase the apparent
Km of
alkaline phosphatase
and 4NPP by a factor of 2 to 3 (30).
C. Buffer pK. The pK for DEA buffer at 30 #{176}C
is 8.7 (31),
which is 1.4 pH units away from the recommended reaction
pH for this buffer, 10.1. The pK of 2A2M1P buffer is 9.7 (32),
within 0.7 pH unit of the measurement
pH, making
2A2M1P buffer a better choice. More than adequate buffering capacity at a 2A2M1P buffer concentration
of 0.35 mol/L
for serum samples has been confirmed by the authors.
D. Buffer contaminants.
DEA preparations
have been
found to contain significant amounts of monoethanolamine,
a potent inhibitor of alkaline phosphatase.
The presence of
this inhibitor can readily be demonstrated by gas chromatography (6) and DEA lots must be screened for this
contaminant before use. It has also been demonstrated that
2A2M1P may contain inhibitors, possibly diamines (33) and
5-amino-3-aza-2,2,5-trimethylhexanol
(34), which inactivate the enzyme by binding zinc(II) ions. Adverse effects by
2A2M1P buffer can be avoided by (a) prescreening the
preparation with the procedure outlined in Appendix
B, (b)
prescreening for the presence of 5-amino-3-aza-2,2,5-trimethylhexanol
as described by Rej et al. (34); (C) use of a
metal-ion buffer containing zinc(Il), which will restore to the
enzyme any zinc(II) possibly bound to an inhibitor (see
Section IV, Optimization
of Magnesiumlil),
Zinc(JLI, and
HEDTA Concentrations);
and (d) initiating
the reaction
with sample, to avoid preincubation of sample in the buffer.
E. Bias of tissue-specific
alkaline phosphatase.
A method
for the measurement of total alkaline phosphatase activity
should have minimal bias toward any one individual tissuespecific alkaline phosphatase
enzyme. Data indicate that
reaction mixtures containing 2A2M1P buffer and 4NPP
show
a minimal
bias
toward
the tissue-specific
enzymes,
while reaction mixtures containing DEA buffer and 4NPP
demonstrate greater bias (35). These conclusions were based
on a comparison with a system containing hydrogen carbonate buffer and 4NPP, because there is no absolute reference
against which bias of tissue-specific enzymes can be measured.
Currently there appears to be no ideal buffer for alkaline
phosphatase
activity
measurements.
Based on the above
considerations, 2A2M1P buffer at a concentration of 0.35
mol/L appears to have advantages
over the other buffers
considered and was therefore selected.
II. Substrate Selection
4-Nitrophenyl
phosphate (sodium salt) was selected because (a) it is readily hydrolyzed by alkaline phosphatase;
(b) it is chromogenic,
with nearly maximum color of the
chromophore (4NP) obtained at the reaction pH, thus allowing continuous monitoring
of the reaction; (C) the molar
absorptivity of the hydrolysis product is high (about 18 450
L x mol’
x cm1), imparting
high sensitivity
to the
measurement
and thus allowing use of a small sample
volume and short incubation periods; and (d) the substrate
shows minimal bias towards the individual tissue-specific
enzymes normally found in human serum (29, 35). Maximum activities for alkaline phosphatase
in serum samples
were obtained in 2A2M1P buffer (0.35 mol/L) at a substrate
concentration of 16.0 mmollL (Figure 2). In the presence of
140
Liver
120
20-
Placental
Bone
l3O
Placental
l00
>
l:o
120H
>
C.,
0
0.20 040 0.60 0.80
2A2MIP
00
Concentration,
20 140
mol/L
.60
Fig. 1. Alkaline phosphatase activity vs buffer concentration for a serum
pool and serum samples containing a predominant tissue-specific
enzyme fraction
The serum pool was prepared by combining unselected serum specimens from
hospital patients.The liver and bone samples were obtained from patients with
liver disease and bone cancer, respectively, as determined by other clinical tests
and patientsymptoms. The placental sample was obtainedfrom a third-trimester
nonhospitalizedpregnant woman. The expanded scale for catalytic activity is
used to demonstrate more clearly small differences between buffer concentra-
tions
HOL
Pool
IllIli
6 8 10 12 14 16 1820
4NPP Concentration,
mmol/L
Fig. 2. Alkaline phosphatase activity vs substrate concentration for a
serum pool and serum samples containing a predominant tissuespecific enzyme fraction
The serumpooland serum samples were selectedas describedin the legendto
Fig. 1
CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983
755
2A2M1P, 0.9 molJL, some samples showed slight increases
in activity on use of substrate concentrations
exceeding 16
mmoIJL. This requirement
for a higher
tion is possibly ascribable
to competition
concentrated
buffer and the substrate
substrate
between
concentrathe highly
for enzyme catalytic
sites (30). However, with use of 2A2M1P, 0.35 mol/L, no
serum samples tested showed increases in activity with
4NPP concentrations
greater than 16.0 mmolIL. Use of
higher
concentrations
is not recommended
because
of the
associated higher initial absorbance readings at the measurement wavelength and the increased rate of spontaneous
hydrolysis. This optimal substrate concentration
has been
confirmed by using the multivanate
empirical optimization
technique of response surface methodology (see Section XI).
Some commercial 4NPP substrate preparations
have been
found to contain excessive amounts
of 4NP and other
contaminants
that result in high blank absorbances and (or)
decreased
alkaline
phosphatase
activities,
respectively.
Thus, it is extremely important
that 4NPP preparations
meet the purity criteria listed in the recommended
procedure.
III. pH Selection
The optimal pH was determined
to be 10.40 in the
presence of 2A2M1P, 0.35 mol/L, and 4NPP, 16 mmolIL, at
the reaction temperature
of 30 #{176}C.
This was established with
pooled sera, individual sera (Figure 3), and purified alkaline
phosphatase enzyme derived from liver, bone, and intestine.
However, this pH constitutes a compromise, because not all
patients’ sera demonstrate
a pH optimum
for alkaline
phosphatase
activity at 10.40 (e.g., samples with a high
proportion of placental alkaline phosphatase
show a higher
pH optimum). The pH optimum has been confirmed by use
of response surface methodology (see Section XI).
IV. Optimization of Magnesium(II),
HEDTA Concentrations
Alkaline
phosphatase
is an enzyme
Zinc(Il), and
that
requires
two
(Kznz) of the liver and bone enzymes for zinc(II) is approximately 4.6 x i0
mol!L (37) and for magnesium(II)
(KMg2+),
,29O
c
The dissociation
of magnesium(II)
ion from alkaline
phosis relatively
slow; association
is fast and dependent
on the magnesium(II)
concentration
(39). Therefore, sufficient magnesium(II)
ions must be added to the reaction
mixture
to ensure
fast and complete activation;
this is
phatase
particularly
important in the case of serum-initiated
reactions.
Both magnesium(II)
and zinc(II) ions form insoluble hydroxides at alkaline pH, especially above 10.0. This can
cause the development
of slight turbidity, which can simulate alkaline phosphatase
activity and can reduce the concentration of free magnesium(II)
and zinc(II) ions available
to the enzyme. For this reason, addition of excess zinc(II) to
the reaction mixture without a metal-ion buffer cannot be
used to overcome buffer inhibitions of alkaline phosphatase
activity. These problems
the use of a metal-ion
were
resolved
in the procedure
by
buffer that maintains
desirable
concentrations
of free magnesium(II)
and zinc(II) ions and
thus serves to protect against any fluctuations
in their
concentrations.
In addition, the buffer can potentially protect the enzyme against inhibition by the other trace metals
in the reaction mixture that reportedly
inhibit alkaline
phosphatase
activity (2, 40). HEDTA was selected as a
metal-ion buffer for this system because of its high affinity
for zinc(II) ions (15).
Methods commonly used in clinical laboratories
thus far
have not included exogenous zinc(II) in the reaction mixture. Our decision to do so is based on the following
considerations.
1. The zinc(II) component of the enzyme is essential for
its activity. Thus its optimal concentration
in the reaction
mixture
must be assured.
2. Some control specimens
are deficient
in zinc(II) because of the use of highly purified enzymes that are partly
stripped of zinc(H) during purification.
3. Zinc(II) bound by the metal-ion buffer serves as a
reservoir for replacement
of zinc(II) ions that have been
removed from the system by zinc(II) chelating contaminants
in 2A2M1P buffer. Reportedly, “titration” of 2A2M1P preparations with zinc(II) ions can completely remove the inhibitory effect of contaminants
in 2A2M1P (41). The metal-ion
buffer can also chelate other potentially inhibitory ions that
may be present as trace contaminants
in reagents or glassware.
Zinc(II), magnesium(II),
and HEDTA were optimized experimentally
and confirmed by computerized
response surface methodology (RSM) optimization (see Section XI) utilizing different combinations
of three concentrations
for each
of the components. These selected concentrations
were previously determined
to be near their respective
optimal
concentrations.
Table 1 presents results of enzyme activity
measurements
at each of these reagent concentrations.
In
each group of experiments,
the concentration
of one component was varied while the concentrations
of the other two
components were kept constant.
.
metals for maximal activity: zinc(II), which is essential for
catalysis, and magnesium(ll),
which is essential for stability
and maximum
activity
(36). The dissociation
constant
274
16 x 10 mol/L (38). In addition, the affinity of zinc(II) for
the magnesium(II)
site is approximately
10-fold that of the
affinity of magnesium(II)
for the same site. Therefore, an
excess concentration
of zinc(II) ion can displace magnesium(II) ions from the magnesium(II)
binding sites and cause
enzyme inhibition. An optimal Mg2 /Zn2 ratio is therefore
required; for rat placental enzyme, this ratio was found to be
500/1 (39).
Bone
25O’
Placental
l40
10.10 10.20 10.30 10.40 10.50 10.60
pH
V. Wavelength Selection
Fig. 3. Alkaline phosphatase activity vs pH for a serum pool and serum
samples containing predominantly a tissue-specific enzyme fraction
The serumpool and serumsampleswereselectedas describedin the legendto
Fig. 1
756
CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983
The reaction product 4NP absorbs maximally at about
402 nm, but at this wavelength the background absorbance
of the substrate 4NPP is significant (Figure 4). Thus use of
Table 1. Optimization of HEDTA, ZInc(II), and
Magnesium(II)a
Mg2, 2 mmol/L
Zn2, I mmol/L
HEDTA,
mmol/L
Mg2, 2 mmol/L
HEDTA, 2 mmol/I
HEDTA, 2 mmol/L
Zn2, 1 mmol/L
Acty,
Acty,
U/I
Mg2,
mmol/I
Acty,
U/L
220.6
1.5
0.5
1.0
227.0
2.0
2.5
221.6
1.5
207.1
2.5
a Sample used is from a pooled specimen of human serum.
mmol/L
U/L
1.5
217.0
218.8
2.0
225.2
225.2
217.0
VII. Sample Volume Fraction
Various sample volume fractions (ratios of sample volume
to volume of total reaction mixture) ranging from 0.0385 (1/
26) to 0.0139 (1/72) were used for alkaline phosphatase
activity measurements
on 19 individual patients’ sera. A
slight dilution effect in the form of an increase in activity
was observed up to a sample volume fraction of 0.0196 (1/
51), with no further increase in activity at greater dilutions
(paired t-test for dilutions 1/26 and 1/32 resulted in p <0.01
and for dilutions 1/32 and 1/51 p < 0.01, while dilutions 1/51
and 1/72 gave a value for p >0.99, n = 19). To maintain
adequate sensitivity while overcoming the observed dilution
effects, we selected a sample volume fraction of 0.0196 (1/51)
for the recommended
method.
VIII. Linearity of the Reaction Rate and Analytical
Range
080
Using a sample volume fraction of 0.0196 (1/51), a patient’s sample with above-normal
activity was diluted with
aqueous solutions of either sodium chloride, 154 mmolJL, or
bovine serum albumin, 30 g/L. A linear relation between
dilution and observed activity was observed up to at least
800 U/L with either diluent (Figure 5). As a result, the
analytical range for the recommended
method extends to
approximately
10 times the upper limit of the reference
interval.
-.
070
-
060
-
IX. Plasma vs Serum Specimens
050
Heparinized
plasma and serum samples were obtained
from a group of 10 healthy volunteers and alkaline phosphatase activity was determined on both types of samples by the
recommended method. No significant differences in activities (p > 0.99) were observed between results for plasma and
serum samples, in agreement with other investigators
(2).
040
030
-
020
-
0.10
-
X. Initiation of Reaction with Sample and Substrate
We initiated the alkaline phosphatase
reaction with either sample or substrate (solution 3), using specimens from
11 unselected hospitalized patients. The activities observed
4NPP.l6 mmol/L.
I
395
400
405
410
415
420 nm
Wove length
with the substrate-initiated
reactions were comparable (p>
0.99) with those obtained with the recommended
method if
we mixed the sample with the buffer (solution 2) and
preincubated
for 5 mm or less before initiating the reaction
Fig. 4. Spectral scan (absorbance vs wavelength) of 4NP, 0.040 mmol/
L, and 4NPP, 16 mmol/L, in the recommended reaction mixture
800
this wavelength
would limit the analytical
range of the
procedure. For this reason, use of 410 or 420 nm, at which
there is considerably
less absorption by 4NPP, has been
recommended.
Use of these wavelengths,
however, would
require measurement
of the reaction product (4NP) on the
slope of the absorption curve, increasing the possible error of
the measurement.
Use of 405 rim is a good compromise: it is
near the absorption peak yet results in a significant decrease in the initial absorbance
of the reaction mixture.
Selection of this wavelength also allows use of instruments
equipped with a mercury vapor lamp as light source.
(-)
600
0
-J
S..-
400
>.
I.-
I-
200
VI. Determination of Molar Absorptivity of 4-
Nitrophenol
The molar absorptivity of 4NP was determined under the
conditions of the recommended
method by workers in seven
different laboratories,
using the exact procedure described
under Preparation
of Solutions. 4NP was found to have a
molar absorptivity
of 18450 ± 200 L x mol’
x cm’ for
the specified reaction conditions.
0.2
0.4
0.6
0.8
1.0
SAMPLE FRACTION
Fig. 5. Alkaline phosphatase activity vs sample volume fraction for a
representative serum sample
Reactionconditionsare those of the recommended
method
CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983 757
25
with substrate.
However, preincubation
of sample with
buffer for longer than 5 mm may lead to enzyme inactivation with some 2A2M1P preparations.
Alkaline phosphatase
activities in samples from various
hospital patients and in commercial
controls were also
measured after preincubating
the samples with magnesium(II) ions (up to 1.0 mmolIL) before initiating the reaction.
No significant difference was observed between these activities and those obtained from the same samples on using the
recommended method with the metal-ion buffer.
To minimize inhibitions of alkaline phosphatase
activity
by potential inhibitors present in 2A2M1P preparations
and
by the highly alkaline pH conditions,
it is preferable to
minimize the interval during which the sample is in contact
with the buffer. Therefore, we recommend initiating
the
reaction with sample. Combining buffer and substrate into
one reagent also serves to decrease the number of pipetting
steps required for the activity measurement
and thus improves precision.
XI. Optimization of Substance Concentrations and
Reaction pH by Use of Computerized Response
Surface Methodology (RSM)11
As previously stated, the reaction mechanism for alkaline
phosphatase activity has not been clearly elucidated. Thus,
it is not yet possible to use an initial-velocity
kinetic model
to aid in optimizing reaction conditions. Therefore, we used
the empirical technique of response surface methodology,
which makes it possible to study optimal conditions for
enzyme reactions without knowledge of the kinetic mechanism for the enzyme under study (42). Contour plots of the
catalytic activity “surface” show that alkaline phosphatase
activity is maximal and changes little in the vicinity of the
proposed optimal reaction conditions, as evidenced by the
broad plateau of the plot. For example, examination
of the
contour plot in Figure 6 shows that the proposed optimal
concentrations
for zinc(II), 1 mmollL, and for magnesium(II), 2 mrnol!L (with HEDTA held constant at 2 mmol/
L), lie within the region of 99 to 100% of maximal activity
observed under the experimental
conditions and that total
activity changes little with changes in the concentration
of
the indicated reaction components.
Similarly, the plot in
Figure 7 shows the optimization
of HEDTA and magnesium(II) concentrations,
with zinc(II) kept constant at 1
mmoliL. Figure 8 indicates that the selected pH of 10.4 and
4NPP concentration
of 16.0 mmol/L (with 2A2M1P buffer
kept constant at 0.35 mol/L) lie in a very flat and optimal
region of the catalytic activity surface. If 2A2M1P is increased, small increases in catalytic activities occur (Figures
9 and 10). This is expected in view of the increased rate of
transphosphorylation
and perhaps
solvent
and ionic
strength effects. Figure 10 shows the relationship
between
2A2M1P and 4NPP concentrations.
Although the illustration suggests the use of a 4NPP concentration
slightly above
16 mmol/L, this was not borne out by other experimental
evidence, as indicated in Figure 3.
An independent
and more detailed evaluation
of the
reaction conditions based on response surface methodology
has been conducted by London, Tietz, Shaw, and Rinker and
will be published in a separate communication.
-j
0
E
E
z
0
I.I-
z
uJ
0
z
0
0
(I)
uJ
z
075
100
ZINC CONCENTRATION,
125
15
mmol/L
Fig. 6. Optimization of zinc(II) and magnesium(II) concentrations for a
serum pool by use of computerized response surface methodology
(RSM)
The serum pool was preparedby combiningunselected serum specimens from
hospital patients. All other reaction conditionsare those of the recommended
method. The x indicates the position on the surface for the recommended
conditions compared to the central point (optimum) of the surface, which is
marked by . The predictedactivitiesfor other reactionconditionsare indicated
by the percent figureson the individual contours
250
-J
.50
E
E
z
0
200
0
z
0
0
D
U,
uJ
I 50
200
HEDTA CONCENTRATION, mmolj
250
I 50
L
Fig. 7. Optimization of HEDTA and magnesium(II) concentrations for a
serum pool by use of computerized response surface methodology
(RSM)
See legendto Fig. 6
XII. Confirmation of the Usefulness of a Metal-Ion
Buffer
The benefits
“The
(Hospital
RSM contour plots were generated
using experimental
758
by J. W. London
of Pennsylvania,
Philadelphia,
PA),
data supplied to him from the authors.
of the University
CLINICAL
CHEMISTRY,
Vol. 29, No. 5, 1983
optimized method can
activities
so obtained with those by other methods in which a higher buffer
concentration
or a preincubation,
or both, are used. Expenbe demonstrated
of the recommended
by comparing
enzyme
8
8
-J
-J
5.-
-5.-
0
E
E
z
0
0
E
E
z
0
I-
16
z
I-.
z
IJJ
0
z
0
0
0
z
0
0
aa-
0
0
2
03
104
I4
t
I-
4
105
2
035
030
2A2MIP
CONCENTRATION,
040
4
mol/L
pH
Fig. 8. Optimization of pH and 4NPP concentration for a serum pool by
use of computerized response surface methodology
See legendto Fig.6
Fig. 10. Optimization of 2A2M1 P and 4NPP concentrationsfor a serum
pool by use of computerized response surface methodology
See legend to Fig.9
thus, buffers should be prescreened
and highly contaminated materials rejected. In experiments
1, 3, 5, 7, 9,
and 11 (columns B, C, D), where the reaction was initiated
with serum, nearly
equal activities
were observed, while
experiments
1, 9, 11 (column A) showed slight but significant decreases (p < 0.05) in activity with a contaminated
(not prescreened) buffer.
Initiation of reaction with substrate after a 10-mm preincubation [experiments 2, 4, 6, 8, 10, and 12 (columns A and
buffer;
Fig. 9. Optimization of 2A2M1 P concentration and pH for a serum pool
by use of computerized response surface methodology
All other reaction conditionsare those of the recommended method. The x
indicates the position on the surface for the recommended conditions. The
predicted activities for other reaction conditions are indicated by the percent
figureson the individual contours. The serum pool was prepared as described in
the legendto Fig. 6
B)] resulted in lower activities, probably owing to the effect
of small amounts of inhibitor that combined with zinc(II) or
magnesium(II)
ions (or both) from the enzyme during the
preincubation.
These inhibitions were not observed in experiments with the proposed metal-ion buffer (experiments
2, 4, 6, 8, 10, and 12; columns C and D). These experiments
clearly show the advantage of a system that is initiated with
sample as opposed to a system requiring preincubation
of
the sample in buffer.
The benefits of using a buffer concentration
of 0.35 vs 0.9
molIL are seen by comparing the relative inhibitions
observed in columns A and B. For example, the decrease in
activity for experiments
1 and 2(0.9 mol/L buffer) in column
A is 37% as opposed to the decrease of 18% in experiments
3
and 4 (0.35 molJL buffer) in column A. No inhibitions at all
were observed for the recommended method (columns C and
D). [Note: The slight increases in activities (p <0.01) observed at 0.9 as opposed to 0.35 molJL buffer with no
preincubation
of sample in buffer are due to slight increases
in transphosphorylation
at the higher buffer concentration.]
ments 1-12 (columns C and D) in Table 2 indicate that
equal enzyme activities (p >0.99) are observed with the
recommended method regardless of whether a contaminated
or prescreened
(see Appendix
B) 2A2M1P buffer is used.
This demonstrates
the protection that a metal-ion buffer
offers against the presence of metal-ion-binding
inhibitors in
2A2M1P buffer. Of course, these experiments do not exclude
the possibility that buffers containing higher concentrations
of inhibitors
could exceed the capacity of the metal-ion
In summary,
these experiments
demonstrate
that no
inhibitory effect is observed with the recommended method,
even if a buffer is used that contains some zinc(II) chelators.
As explained in the previous sections of this appendix, this
is attributable
to the combination of the use of a metal-ion
buffer, a functional test for the selection of2A2M1P buffer, a
serum-initiated
reaction, and use of a 2A2M1P buffer concentration of 0.35 mollL.
pH
2A2MIP
CONCENTRATION,mol/L
CLINICAL CHEMISTRY, Vol. 29, No. 5, 1983
759
Table 2. Effect of a Metal-ion Buffer on Alkaline Phosphatase Activities in Contaminated and
Prescreened 2A2M1 P Buffers Compared at Two Different Concentrations, with and without Sample
Preincubation a
Mg2,
2A2M1P
Experiment
concn,
no.
mol/L
Sample
Reaction system
Preincubatlon,
mm
HEDTA, 2 mmol/L: Zn2,
1 mmol/L Mq2, 2 mmol/L
I mmoIfL
Contaminated
2A2M1P
Prescreened
2A2MIP
Contaminated
2A2M1P
Prescreened
2A2M1P
A
B
C
0
Catalytic activities, U/L
Patient serum
1
2
3
0.90
0.35
4
Purified liver
isoenzyme
Patient serum
0
10
0
10
360
228
347
284
366
345
347
346
364
364
348
348
366
366
346
346
5
6
0.90
0
10
215
146
216
204
216
216
216
216
7
8
0.35
0
10
210
175
211
210
210
210
210
210
9
10
0.90
0
10
190
129
194
184
193
192
193
194
11
0.35
0
177
184
184
184
10
162
185
181
12
182
See text for discussion of data. All reaction mixtures contain 4NPP, 16 mmol.L. Other reaction components (2A2M1P. Mg2,
concentrations are indicated in the table.
Appendix B. Functional Test for Verification of
Suitability of 2A2M1 P Buffer
All chemicals used for preparation
of the solutions are the
as those described in the recommended method. However, the reagent substance
concentrations
are altered
slightly to permit convenient pipetting volumes. Prepare all
solutions in calibrated flasks with fresh reagent-grade
Wasame
1. Add 2.30 mL of Solution II to the reaction cuvette and
warm to 30 ± 0.1 #{176}C.
2. Add 50 L of sample to the cuvette and mix thoroughly. Incubate at 30 #{176}C
for exactly 10 mm.
3. Initiate the reaction by adding 200 iL of solution III,
which has been prewarmed to the reaction temperature.
4. Mix and monitor the absorbance of the reaction mixture at 405 ± 1 nm for as long as 5 mm after initiating
the
reaction,
5.
I. Metal-ion buffer stock solution-HEDTA,
L; ZnSO4, 55.4 mmolIL;
Mg(C2H302)2,
110.9
mmol/L:
order of addition
is extremely
important
to
avoid
precipitation.
reagents
Dissolve
110.9 mmoll
4.217
HEDTA) and their
Procedure
Preparation of Solutions
of the individual
Zn2,
The
g of
HEDTA in about 70 mL of water. Then add 1.593 g of
ZnSO4 7H2O and allow it to dissolve completely. Next add
2.379 g of Mg(C2H302)2 4H20, dissolve completely by mixing, and dilute the solution to exactly 100 mL with water.
This solution is stable for at least three months when stored
at 2-6 #{176}C.
to obtain
Repeat
the
.AIt
for the sample.
above
procedure
II and immediately
initiate
III, without
first allowing the
solution
tion
by adding
sample
to
the reaction
with solusample to incubate in the
buffer.
For pooled serum samples with normal and increased
alkaline phosphatase
activities, obtained from fasting individuals, observed values after the 10-mm incubation must
be 98% of the activities observed without preincubation.
References
1. Moss DW. International
and national views on methods for
alkaline phosphatase
activity measurements
in the clinical laboratory. In 2nd
Symp on Gun Enzymol, NW Tietz, A Weinstock,
and D Rodgerson, Eds., American
Association
for Clinical Chemistry, Washington,
DC, 1976, pp 41-50.
2. McComb RB, Bowers GN Jr, Posen S. Alkaline Phosphatase,
Plenum Press, New York, NY, 1979.
3. Empfehlungen
der Deutschen
Gesellschaft f#{252}r
Klinische
Chemie: Standardisierung
von Methoden
zur Bestimmung
von Enzymaktivitaten
in biologischen
FlUssigkeiten.
Z Kim Chem Kim
Biochem 10, 182-192 (1972).
4. Committee
on Enzymes of the Scandinavian
Society for Clinical
Chemistry
and Clinical Physiology,
Recommended
methods for the
determination
of four enzymes in blood. Scand J Clmn Lab Invest 33,
291 (1974).
mt
II. 2A2M1P/metal
panol, 388 mmolIL;
-ion
buffer-2-amino-2-niethyl-1
-proHEDTA,
222 mmol/L;
Zn.S04, 1.11
Mg(CJI3O7)2,
222 mmol/L; pH305
10.40 ± 0.05:
mmol/L;
Warm
the
2A2M1P to approximately
37 #{176}C
until it is
completely liquefied. Mix 17.29 g of 2A2M1P with approximately 400 mL of water. Adjust the pH to 10.40 ± 0.05 at
30 #{176}C
by adding HC1, 1 mol/L. With stirring, add exactly
10.0 mL of metal-ion buffer stock solution (solution I) and
readjust to pHo -c 10.40 ± 0.05 with 1 mollL HC1. Dilute to
exactly 500 mL with water and store in a closed container,
to minimize absorption of carbon dioxide. This buffer solution is stable for at least three months if stored at 2-6 #{176}C
and
protected
from carbon dioxide.
5. Draft
Proposal
(No. 8)
Gun Chem 19, 268-273
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ifi. 4-Nitrophenyl
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hexahydrate)
in approximately
8 mL of water and dilute to
10.0 mL. This solution should be freshly prepared.
760
CLINICAL
CHEMISTRY,
Vol. 29, No. 5, 1983
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