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dissimilar to those of human serum (19,20). Recent recommendations (21) from the American Association for Clinical
Chemistry (AACC) and (22) the International
Federation of
Clinical Chemistry (IFCC) propose similar reference procedures for measurement
of alkaline phosphatase
activity at
30 #{176}C.
Because these procedures probably will be used as
reference techniques
for routine measurements
made at
different temperatures
and under various modified assay
conditions, we examined the effects of temperature
on the
measurement
and steady-state kinetic properties of alkaline
phosphatase
isoenzymes from several human sources and
from porcine kidney, the latter representative
of a nonhuman enzyme used in quality-control
fluids. Because the
proposed reference method includes features presently uncommon to routine alkaline phosphatase
methods, such as
addition of zinc(ll) and metal chelator and use of lower
concentrations
of buffer, we examined the effects of temperature, using both the reference assay (21,22) and one based
upon a routine procedure (23).
Additional
185
Results
The effects of temperature
on the affinities of the alkaline
phosphatase
isoenzymes for 4-nitrophenyl
phosphate were
measured at three temperatures
commonly used for the
assay of the enzyme: 25, 30, and 37 #{176}C.
Representative
Hanes plots are shown in Figure 1 for results obtained by
the AACC/IFCC reference method (21,22) and in Figure 2
on using the method of Bowers et al. (23). For all phosphatases we examined, the results show that as the temperature increased, the apparent Km values for 4-nitrophenyl
phosphate decreased. Table 1 summarizes the average apparent Km values obtained on analyzing the original data by
E
>
6/
4-NITROPHENYLPHOSPHATE
(mol L
0)
Fig. 2. Hanes plots of initial velocity of alkaline phosphatases with 4nitrophenyl phosphate as the substrate being vaned
Isoenzymeswere: A, humanlter; B, humanintestine;C, humanplacenta;and D,
porcinekidney.Enzymeactivitywas determinedby the procedureof Bowerset
al. (.
Curves(topto bottom)are for 25. 30, and 37 C
E
>
3
5
7
-I
4-NITROPHENYLPHOSPHATE
(mol L
10)
Fig. 1. Hanes plots of initial velocity of alkaline phosphatases with 4nitroptienyl phosphate as the varied substrate
lsoenzymeswere: A, humanliver;B. humanintestine;C, humanplacenta;and 0.
porcinekidney. Enzymeactivitywas determinedby the AACC/IFCC procedure
(21, 2. Curves (top to bottom)are for 25, 30, arid 37#{176}C
186
Discussion
Evidently there are significant changes in apparent Michaelis constants for the human and porcine isoenzymes of
alkaline phosphatases
in the range of temperatures
usually
Table 1. Effect of Temperature on the Apparent Km (mmol L1) of Alkaline Phosphatase Isoenzymes
for 4-Nitrophenyl Phosphate
.
AACC/IFCC method
(21, 22)
K,,,
K,,,
-pp
Enzyme source
Human
Human
Human
Porcine
liver
intestine
placenta
kidney
aparent
25 C
1.05
1.02
30 C
0.86
0.79
0.61
0.74
0.63
1.11
37#{176}C
0.58
0.53
0.43
0.47
H
-38.7
-41.4
-24.4
-54.5
-pp
25 C
30 C
37#{176}C
1.22
1.28
0.62
1.01
1.03
0.93
0.53
0.67
0.76
0.61
0.39
H
-30.8
-47.6
-28.9
-39.6
0.54
37 3230
25
20
C 37
3230
25
20
B
I.,
CV
0
PATIENT
SERA
(n29(4)10i420
PATIENTSERA
(n 19) 4120
LIVER
4030
460
LIVER
4180
INTESTINE
3370
INTESTINE
PORCINE KIDNEY
PLACENTA
4340
3330
32
PORCINE
KIDNEY
3930
PLACENTA
4)50
3470
34
32
r
Fig. 3. Arrhenius
relationships
UL
(K
3,4
Ol
of alkaline phosphatase
(30#{176}C(
(r2
isoenzymes and
0.996)
nitrophenyl
phosphate is the substrate
(Table 1) are in
substantial agreement with those found by other authors for
either the human or corresponding
nonhuman isoenzymes
(31-35). There are, however, significant
differences in reported steady-state
kinetic properties of alkaline phosphatase, owing to differences in purification
technique and
choice of assay conditions (35). Our data for 30 #{176}C
compare
particularly well with the recent report of Duncan et al. (35),
who used that temperature
and a similar assay system to
examine human alkaline phosphatase
reference materials.
Table 2. Apparent Enthalpy Change (H437 c) Energies of Activation (E5 and Ea), and Temperature
Coefficients for Alkaline Phosphatases from Various Sources
AACC/IFCC method
Enzyme source
Patients serum
Human liver
Human intestine
Human placenta
Porcine kidney
Ht37 .
31.6
31.1
27.1
33.5
25.1
E
34.2
33.7
29.7
36.1
27.7
(21, 22)
Temp. coeff., %
5.9
28.1
30.5
30.9
25.3
5.8
4.9
6.3
4.5
31.7
32.2
30.3
31.9
26.3
E
34.3
34.8
32.8
34.5
28.9
Temp. coeff., %
28.9
25.6
33.3
23.2
5.9
6.0
5.6
5.9
4.7
(21, 22)
Enzymesource
25-. 30#{176}C
37-. 30#{176}C
25-. 37#{176}C
25-. 30#{176}C
Patients serum
1.255
0.736
1.705
1.256
Human liver
1.251
0.740
1.691
1.260
Human intestine
1.218
0.767
1.589
1.244
Human placenta
1.271
0.724
1.756
1.258
Porcine kidney
1.202
0.780
1.541
1.212
Relationships are shown as factors for conversion of numerical results between the two temperaturesindicated.
37-. 30C
0.736
0.732
0.745
0.734
0.772
25-. 37C
1.707
1.720
1.670
1.714
1.569
187
unit per degree Celsius (40). We did not attempt to vary the
The inverse relation between apparent Km and temperature resulted in negative enthalpies of substrate binding
pH of the assay mixture to compensate for this change.
(MI,,, Table 1). This effect was unanticipated,
because posiIt has been suggested (41) that Arrhenius
plots where
tive MI,, values and increased Km values with increased
V
is used are more meaningful
than those utilizing
temperature
are more commonly observed for other eninitial velocities, such as in Figure 3. We determined
the
zymes (15, 16, 27, 36, 37). Hiwada and Wachsmuth
(38)
relationships,
using V
data. The results, given as Ea in
examined the effects of temperature
on the steady-state
Table 2, did not differ greatly from the relationships
deterkinetic behavior of porcine-kidney
alkaline phosphatase and
mined using initial velocities (E8 in Table 2). Our primary
found a small, but consistent, increase in Km for 4-nitrogoal was to determine the effects of temperature
on the
phenyl phosphate with increased temperature
in the range
measurement
of alkaline
phosphatase
activity, so it is
25 to 37 #{176}C.
This dependency
was more substantial
for
appropriate that all other calculations are based upon initial
changes in temperature
below 25 #{176}C.
In that study tris(hyvelocity data.
droxymethyl)methylaniine
buffer was used as the nucleoFor all calculations, we utilized a value for molar absorpphilic phosphate acceptor at 50 xmnolfL and pH 9.5. Appartivity of 1845 m2 mol,
the value obtained for 4-nitroent Km depends upon the nature and concentration
of the
phenol under the conditions of the AACC/IFCC assay (21,
acceptor molecule and the pH of assay, so differences in
22). An identical value was reported by Bowers et al. (42) for
assay conditions may account for our contradictory
results.
solutions of the compound in NaOH at 30#{176}C.
They found an
However, we found decreased apparent Km for the two
increased value for molar absorptivity of solutions containassays used (Table 1, Figures 1 and 2) where 2A2M1P
ing 2A2M1P at 1 moJiL, a medium similar to that used in
concentration
differed by nearly threefold. Although both
the assay of Bowers et al. (23). These authors suggest the
use of a molar absorptivity value approximately
3% greater
assay techniques
support nearly maximal rates of transphosphorylation
activity, that would not be the case at
than the one we used here. Because of the uncertainty
of
acceptor concentrations
at 50 mmoIJL. Because differences
estimates of molar absorptivity
of 4-nitrophenol
solutions
in degree of purification
and preparation
technique influcontaining 2A2M1P at a concentration
of about 1 molIL, we
chose to use a single, well-characterized
value for all meaence steady-state kinetic behavior for this enzyme (35), this
surements.
The molar absorptivity
of 4-nitrophenol
also
factor cannot be excluded as a source of the observed
increases with temperature;
the observed change from 30#{176}C
differences between our data and those of Hiwada and
to either 25 or 37 C is <0.5% (42, 43). Thus estimates of Ea
Wachsmuth (38). The observed change in apparent Km was
and Arrhenius slopes are systematically
overestimated
by
roughly equivalent for the two assay techniques for a given
approximately
this amount. We did not correct for this
phosphatase
isoenzyme (Table 1). Changes in apparent Km
variation because it is far less than the analytical error of
were also similar for each phosphatase
studied, except for
the human placental isoenzyme. For it, the decrease was
the estimates shown (Table 2, Figure 3). In addition, on a
less pronounced with increased temperature,
hence lower
practical basis, conversion factors such as those provided in
Table 3 should be calculated
without such a correction,
MI,, values were observed. At any given temperature
the
because they should also compensate for this effect as well as
observed Km were generally higher in the Bowers et al. (23)
changes in the enzyme activity. Estimates
of apparent
method, reflecting the competition ascribable to the greater
Michaelis constants (Table 1) are independent of the molar
concentrations
of 2A2M1P used in that technique as compared with the AACC/IFCC procedure (21,22).
absorptivity of 4-nitrophenol.
The effects of temperature
on alkaline phosphatase activiIf one assumes a simple Michaehs-Menten
model, the
ratio of substrate concentration
[SI to measured Km at 37 #{176}Cty have been examined by others (10,33,34,38,40,44)
and
is theoretically preferable analytically to that at 25 or 30#{176}C. reviewed by McComb et al. (19). In that review they provide
temperature
coefficients normalized as the increase in enThe ratios of [SI/Km and the percentage
of V
for the
zyme activity per degree Celsius. We therefore also provide
human liver isoenzyme as measured by the AACC/IFCC
this information in Table 2. Activity is not linearly related
procedure (21, 22) are 15.2 (93.8% of V,,,) at 25 #{176}C,
18.6
to assay temperature,
so we present this coefficient solely for
(94.9%) at 30 C, and 27.6 (96.5%) at 37 C. However, on a
the purpose of comparison.
Our results (Tables 2 and 3)
practical basis, measured initial velocities were linear durshow that the effect of temperature
on alkaline phosphatase
ing the interval they were measured-usually
the first 2
activity is less diverse than that summarized elsewhere (19),
miii of the reaction-and
we saw no difference in the
where increases from 4% (human serum) to 15% (porcine
linearity of the catalyzed reaction among the temperatures
kidney) were noted. Differences in assay techniques probaexamined. Furthermore,
as shown in Figure 3, the Arrhenibly contribute to the disparity in the latter case.
us relationships
were linear over the range 20-37 #{176}C,
indiArrhenius relationships
have been described for several
cating that the changes in Km did not substantially
affect
alkaline phosphatases
(34,38,44). In general, linear Arrhemeasured velocities.
nius relationships
have been characterized
by others, alThe decrease in apparent Km with increased temperature
though a break in the Arrhenius
plot at about 25#{176}C
has
may be related to the considerable
hydrophobic nature of
been reported for the porcine kidney enzyme (38). Most
the enzyme; at increased temperatures,
hydrophobic interactions increase and the enzyme may assume a more
other studies do not give values for activity below 25 C, so
they neither confirm nor refute that finding. Our data
favorable state. Significant decreases in Km with increases
(Figure 3) show linear Arrhenius relationships for the range
in temperature
have recently been demonstrated
(39) for
another membrane-associated
enzyme, asialomucin-sialyl20 to 37 C, including that for porcine kidney phosphatase.
Our data (Figure 3, Table 3) indicate that human alkaline
transferase (EC 2.4.99.1). That report also describes differphosphatases
are similarly affected by changes in temperaences in the temperature
effects between solubilized and
ture, differences in the response of isoenzymes from any two
membrane-associated
preparations.
Differences in lipid content among purified preparations
may also at least partly
sources being less than 10% (Table 3) over the range 25explain interlaboratory
discrepancies
regarding the effects
37 C. There is excellent agreement between human liver
phosphatase
and that of serum. Direct comparison of meaof temperature
on alkaline phosphatase.
The pH optima of alkaline phosphatases are influenced by
sured activity of serum phosphatase
at any two temperatures (Figure 4) confirmed data obtained from Arrhenius
temperature,
but only slightly, typically less than 0.03 pH
188
relationships:
References
1. Haeckel R, H#{216}rder
M, Zender R. IFCC report on the survey of
opinions concerning preferred incubation temperatures
for measurement of enzymes (and possibly other components) in clinical
chemistry. J Clin Chem Clin Bischem 20, 947-958 (1982).
2. Bergmeyer HU. Standardization
of the reaction temperature for
the determination
of enzyme activity. Z Kim Chem Kim Biochem
11, 39-45 (1973).
3. Recommendations
of the German Society for Clinical Chemistry:
Standardization
of methods for the estimation of enzyme activities
in biological fluids. J Clin Chem Clin Bwchem 15, 255-260 (1977).
4. Duggan PF. Activities of enzymes in plasma should be measured
at 37 C. Clin Chem 25, 348-352 (1979).
5. Bergmeyer HU, Bowers GN Jr. Another discussion on measuring temperature. In Clinical Enzymology Symposia, 4, A Burlina, L
Galzigna, Eds, Piccin Medical Books, Padua, Italy, 1984, pp 271-
276.
6. NCCLS Proposed Position Paper (PPC-1O), Enzyme Assay Conditions: Choice of Standard Reaction Temperature for Assay Activity
Values of Enzymes in Human Sera. National
Committee for Clinical Laboratory Standards, Villanova, PA, 1979.
7. Bowers GN Jr, Bergmeyer HU, H#{248}rder
M, Moss DW. Approved
recommendation (1978) on IFCC methods for the measurement
of
catalytic concentration of enzymes. Part I. General considerations
concerning the determination
of the catalytic concentration
of an
enzyme in the blood serum or plasma of man. Clin Chim Acta 98,
163F-174F (1979).
8. Mathieu M, Aubry C, Arthur M, et al. Societe Francaise
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