CLINICAL CHEMISTRY, Vol.

31/2, 185-190 (1985)

Effectsof Temperatureon Measurementof AlkalinePhosphataseActivity

William H. Copeland, Daniel A. Nealon, and Robert Rej1
We examined the effects of temperature
on the activity and
steady-state
kinetic properties of alkaline phosphatase
(EC
3.1.3.1). Purified isoenzymes
from human liver, intestine,
and placenta were used, as was human serum, and the
enzyme from porcine kidney. Phosphatase
activity was estimated by two different assay techniques. For all isoenzymes,
apparent Michaelis constants for the substrate 4-nitrophenyl
phosphate
decreased
with increased temperature;
!
at
37 #{176}C
was typically half that determined
at 25 #{176}C.
All enzymes of human origin exhibited similar linear Arrhenius
relationships
over the range examined, 20-37 #{176}C
(E5 of 3036 kJ mol).
The porcine kidney enzyme obeyed an Arrhenius relationship
that was slightly, but significantly, different
from the isoenzymes of human origin. Temperature
relationships based upon Arrhenius behavior and individual activity
measurements
are presented. For human alkaline phosphatases, they differed by no more than 10%.
Keyphrases:
enzyme assay
kinetic analysis
enzyme kinetics
characteristics of isoenzymes
Arrhenius relationships
quality control

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

What assay temperature

is most suitable for measurement of enzyme activities has been the subject of considerable discussion,
with substantial
differences in opinion
evident (1-8). Because of this lack of consensus regarding a
standard temperature,
the use of temperature-conversion
factors in the measurement
of clinically useful enzymes has
been proposed (9, 10) and observed in practice through
interlaboratory
surveys (11, 12). However, in many cases
correction factors are applied without regard for the possible
isoenzyme heterogeneity
of the specimen, even though some
isoenzymes may respond differently to changes in temperature (13-15). Thus the validity of using temperature-correction factors has been questioned in at least some applications (16-18).
The case of alkaline phosphatase
lorthophosphoric-monoester phosphohydrolase
(alkaline
optimum),. EC 3.1.3.1]
presents particular analytical problems for accurate estimation of total enzyme activity. Not only may its isoenzymic
forms in the human differ considerably in certain properties,
but also the enzymes used in quality-control
or calibration
fluids often are of nonhuman
origin and may be very
Wadsworth Center for Laboratories and Research, New York
State Department of Health, Albany, NY 12201.
Address
correspondence to this author.
Portions of this paper were included in presentations at the 35th
national meeting of the AACC, New York, July 1983 (abstract: Clin
Chem 29. 1158, 1983) or at the 11th International Symposium on
Enzymology, Rome, February 1984.
Received October 9, 1984; accepted October 30, 1984.

Materials and Methods

Alkaline phosphatase
was determined
by the AACC/
IFCC reference method. We followed the published protocols
(21, 22) exactly except for altering temperature
as will be
described. Individual
specimen-blank
reactions were not
determined. The magnitude
of the reagent blank was assessed at each temperature;
at most, it was equivalent to
<0.4 U of alkaline phosphatase activity per liter of specimen
at 37#{176}C,
and it was not deducted from overall reaction rates.
Enzyme activity was also determined
according to the
general method of Bowers et al. (23), with use of the
following final concentrations,
per liter: 800 mmol of 2amino-2-methyl-1-propanol
(2A2M1P), 1 mmol of Mg, and
15 mmol of 4-mtrophenyl
phosphate. The pH at 30 #{176}C
was
10.4 and the volume fraction of specimen was 0.0196.
For both assay techniques,
we continuously
monitored
production of 4-nitrophenol
at 405 nm with a Cary Model
219 spectrophotometer
(Varian Instruments,
Palo Alto, CA
94303) interfaced with an Apple H Plus inicrocomputer,
used for directly estimating
the reaction rates. Spectral
band width was set at 1.0 nm. Temperature
was controlled
with a Varian Peltier-controlled
thermocuvette
and verified
with a Model 45 CU cuvette thermometer
(Yellow Springs
Instruments,
Yellow Springs, OH 45387) calibrated with a
gallium melting-point
cell (SRM 1968; U.S. National Bureau of Standards, Washington,
DC 20234). For both assay
procedures, reactions were initiated by adding specimen.
Units (U) of activity are tmol min1, calculated with use of
the molar absorptivity of 4-nitrophenol,
1845 m2 mol (21,
22). Steady-state
kinetic parameters
were calculated both
by direct iterative-fit procedures (24, 25) and by the usual
reciprocal methods. Parameters
obtained from reciprocal,
.

CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985

185

Arrhenius, and regression plots were calculated according to

a robust linear regression technique based on the algorithm
of Paulson and Nicklin (26). Activation energies and thermodynamic quantities
were estimated
as previously described (27).
Human liver and intestinal alkaline phosphatases
were
prepared by the procedure of Sussman et al. (28) through the
stage of Sephadex G-200 chromatography.
Alkaline phosphatase (B Grade) from human placenta was from Calbiochem-Behring,
San Diego, CA 92112. Porcine-kidney
alkaline phosphatase
was from Miles Laboratories,
Elkhart, IN
46515. The purified phosphatases
were added to matrixes as
described previously (29) and stored in 5.0-mL aliquots at
temperatures
below -20 #{176}C.
Patients serum specimens,
selected to cover the normal and abnormal ranges of alkaline phosphatase
activity, were obtained from a hospital
clinical laboratory, stored at 4#{176}C,
and assayed within 24 h of
collection.
2A2M1P was lot number 1545, from Research Organics,
Inc., Cleveland, OH 44125. We screened this material for
chelating inhibitors
of phosphatase
activity, using both
catalytic activity (22) and metal binding procedures (30),
and found it acceptable by both criteria. Magnesium acetate
and zinc sulfate were from J. T. Baker Chemical Co.,
Phillipsburg,
NJ 08865. N-Hydroxyethylethylenediaminetriacetic acid was from Aldrich Chemical Co., Milwaukee,
WI 53233. 4-Nitrophenyl
phosphate-the
disodium salt,
hexahydrate-was
from Sigma Chemical Co., St. Louis, MO
63178.

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

six different estimation

techniques.
The Km value determined by any method agreed within 5% of the average Km
value reported in Table 1. The variation of apparent Km
with temperature
was log-linear with respect to the reciprocal of the absolute temperature.
Apparent enthalpy changes
for substrate binding (.H8), calculated from the slopes of
such plots, are also presented in Table 1.
Arrhenius plots in Figure 3 show the effects of temperature (20-37 C) on the activity of the various alkaline
phosphatase isoenzymes, as measured by both assay methods. Also shown in Figure 3 is the Arrhenius relationship
determined for patients sera over this temperature
range.
Mean and 1 SD are shown for individually
determined
values. Thermodynamic
parameters
calculated from these
Arrhenius
relationships,
activation enthalpy (H
c), and
activation energies (Fa) are shown in Table 2. Also shown in
Table 2 are the activation energies (Ea) calculated by using
determined
values of Vmj, rather than measurements
of
initial velocity. Estimates of each thermodynamic
parameter are similar despite the differences in enzyme source. In
addition, the average values for H37
and Ea obtained for
29 individual sera agree well with those obtained with the
semipurifled alkaline phosphatase from the various human
sources. These sera varied in enzyme activity from about 25
to 550 U/L at 30 #{176}C.
Table 2 also shows temperature
coefficients, calculated from Arrhenius relationships
in percent increase in activity per degree Celsius.
Table 3 gives temperature/activity
ratios calculated from
the Arrhenius
relationships
for the different sources of
alkaline phosphatase
and for the patients sera. Direct
comparison of alkaline phosphatase activities of 29 patients
sera measured at 25, 30, and 37 #{176}C
are shown in Figure 4.
The slopes of the robust regression lines obtained for these
measurements,
given in the legend to Figure 4, agree well
with those calculated by the Arrhemus relationships
for the
purified isoenzymes and the patients sera shown in Table 3.
.

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

CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985

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

Method of Bowers at al. (23)

(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

enthalpy change for substrate binding, in kJ . mol1

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(

Fig. 4. Comparison of alkaline phosphatase measurements at 25, 30,

and 37#{176}C
Patients sera(n = 29) were assayed in quadruplicate at each temperature by (A)
the MCC/IFCC procedure(21, 22) and (B) the procedureof Bowerset al. (23).
Data points representmeanvalues. Robustlinear-regressionestimatesare: (A)
U
=
0.790(U37.,)) - 3.0 (r2 = 0.996);
=
1.242(U25..) + 2.1 (r2 =
0.998).(5) U5, c = 0.789(U37c) -4.6 (r2 = 0.997); U5, ., = 1.242(U25 c) + 4.9

(r2

isoenzymes and

human serum alkaline phosphatase

Enzyme activity was determinedby (A) the AACC/IFCC procedure(21, 22) and
(B)the procedureof Bowerset al. (23). Arrheniusslopeswerecalculatedfor each
preparation of alkalinephosphataseand for each of the individualpatients sera.
For the patients sera, the shaded area representsthe 1 SD range about the
meanvalue

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.

used in analytical measurements,

25-37 #{176}C
(Figures 1 and
2, Table 1). For all isoenzymes we examined, the determined
Km decreased with increased
temperature;
the difference
was approximately
twofold for measurements
at 25 and at
37 C. The values for the Michaelis constants
when 4-

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

Method of Bowers et al. (23)

(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

Values for enthalpy change. E,,,and E, are in kJ#{149}

mol. All values, except E,,, were calculated by using initial velocity measurements. Data for E,, were
determinedby using the dependenceof apparent V,. with temperature.Temperaturecoefficientsare given in terms of averageincreasein activity per #{176}C
over the
range 25-37 C (see text). Relative standard deviations for replicate data were <8% for all measurements.

Table 3. Relationships of Alkaline Phosphatase Activities at Various Temperaturesa

AACC/IFCC method

(21, 22)

Method of Bowers et al. (23)

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

CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985

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

CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985

relationships:

compare the slopes we obtained (Figure 4)

with the factors presented in Table 3. Particularly
notable is
the close agreement between the temperature
relationships
for any single isoenzyme for the two methods studied (Table
3). Heerspink
et al. (10), using the French-recommended
procedure (8), found temperature
relationships
in very close
agreement to those given for serum in Table 3. These data
indicate that temperature
relationships
for alkaline phosphatase probably are identical among assay techniques that
are comparable in basic principles (e.g., identical substrates
and nucleophilic
acceptor) but that may differ in some
details. Furthermore,
Heerspink
et al. (10) found significantly different factors for the procedure of the German
Society (3), indicating that more profound changes in assay
technique will not allow use of factors derived for different
procedures.
It is well known that the responses of enzymes in qualitycontrol sera and calibrators to changes in assay conditions
differ from those of patients sera, and this is particularly
true for alkaline phosphatases
(19, 20, 45). Differences in
behavior of alkaline phosphatases
of quality-control
fluids
in response to changes in temperature
have been described
(10, 19). Separate reports from our laboratory (20, 46) have
suggested that porcine-kidney
phosphatase has characteristics resembling those of the enzyme from human liver and
human serum. Thus we chose this nonhuman
enzyme for
the present study, and our results show that the activity of
this enzyme does in fact differ from those of human origin
when temperature
is varied (Figure 3, Tables 2 and 3).
Changes in activity with temperature
were slightly but
significantly
lower than those found for any isoenzyme of
human origin. This observation
was not expected on the
basis of our earlier studies. Perhaps differences in specimen
matrix or in degree of enzyme purity may account for this
observation. Alternatively,
the differences between the response of this nonhuman enzyme and that of human liver or
serum origin are slight [<5% difference between 30 and
37 C (Table 3)1, so that the similarities
of these two
enzymes [e.g., in apparent Km (Table 1)1 may outweigh
differences with respect to temperature.
The use of factors to interrelate
enzyme activities obtained at various temperatures
has met with some skepticism (16-18).
A major argument
against applying such
factors to patients sera is the probability that there is a
mixture of isoenzymes in the sera, each differently affected
by temperature
(13-15). Yet there is a renewed interest in
the application of conversion factors in clinical enzymology
(47). We found nearly identical temperature
relationships
for human serum and human liver alkaline phosphatase in
each of the two assay techniques used here. Intestinal and
placental isoenzymes of human origin obeyed similar but
not identical relationships.
These differences may contribute
to the somewhat wide variability
in the Arrhenius
slopes
found for specimens of patients serum, relative SD about
11% (Figure 3). If factors derived from the response of
human liver isoenzyme, or the average response of human
serum, are applied to specimens containing predominantly
intestinal
or placental isoenzyme, the error introduced is
slight, about 5%. However, this may exceed the relative
imprecision of the analytical
procedure; a relative SD of
<2% has been demonstrated
for the AACC/IFCC method
(48). The error is greater
if such factors are applied to
porcine kidney alkaline phosphatase-an
enzyme
that is
otherwise similar to that of human serum.
Our data are thus equivocal regarding the routine application of such factors for the case of alkaline phosphatase.
However, we present clear evidence that, for any single
isoenzyme, essentially identical temperature
relationships

are obeyed in two assay techniques

that differ substantially
in their final assay conditions. It appears likely that the
relationships
presented
in Table 3 are valid for other
measurement
techniques in which 4-nitrophenyl
phosphate
and 2A2M1P are used. The close agreement of factors found
for human serum with a third assay procedure (10) further
supports this view.
We are grateful for the excellent assistance of Penelope Purzycki,
David Stalter, and Gertrude Troxell. We also thank Drs. G. N.
Bowers, Jr., R. B. McComb, and N. W. Tietz for their helpful
comments.

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