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Proposal of standard methods for the determination of enzyme

catalytic concentrations in serum and plasma at 37 °C I. Alkaline
phosphatase (orthophosphoric-monoester phosph....

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Schmidt et al.: Alkaline phosphatase Standard method 247

Eur. J. Clin. Chem. Clin. Biochem.

Vol. 30, 1992, pp. 247-256
© 1992 Walter de Gruyter & Co.
Berlin · New York

Deutsche Gesellschaft für Klinische Chemie

(German Society for Clinical Chemistry)

Proposal of Standard Methods for the Determination

of Enzyme Catalytic Concentrations
in Serum and Plasma at 37 °C
I. Alkaline phosphatase
(orthophosphoric-monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1)
Prepared for publication by
Working Group on Enzymes1),2)

This paper is one of four recommendations on measurements of catalytic concentrations of enzymes. Others
deal with:
II. Cholinesterase (this j. 30 (1992) 163-170)
III. Glutamate dehydrogenase
IV. Lactate dehydrogenase

Introduction
Alkaline phosphatases catalyse both the hydrolysis of
orthophosphate monoesters and the transfer of inor-
ganic phosphate. The degree of transphosphorylation
varies with isoenzyme distribution and depends on
the acceptor species. At least four human isoenzymes
are known: tissue non-specific (liver-bone-kidney type
with various posttranslational modifications), intes-
tinal, placental and placental-like (germ cell) alkaline
phosphatase, in addition to fetal forms that occur in
malignant disease. Hence, the measured catalytic con-
centrations and isoenzyme distributions vary accord-
ing to the buffer used, although all the methods use
4-nitrophenylphosphate as the most suitable substrate
for continuous monitoring.
In comparison with hydrogen carbonate buffer, or
with 2-amino-2-methyl-l-propanol, which is recom-
mended in the IFCC reference method (1) by the
American (2) and French (3) societies, diethanolam-
ine, which is selected in the recommended method of
the Scandinavian (4) and German (5) societies, clearly
enhances the activity of bone and liver phosphatase;
i.e. in diethanolamine both display a positive bias
versus isoenzymes present in sera of healthy people
and those of intestinal and placental origin (1). Both
aminoalcohols often contain inactivating impurities
as 5-amino-3-aza-2,2,5-trimethylhexanol (6) in 2-
amino-2-methyl-l-propanol, and monoethanolamine
(7) in diethanolamine. Therefore, we evaluated the
use of methylglucamine, a pure buffer substance with
moderate phosphate-acceptor properties, which has
been proposed by Chromy et al. (8) for alkaline phos-
phatase determination and recommended by the Ital-
ian society (9). We analysed sera from healthy adults,
children, pregnant women and patients with liver and
bone diseases. We tried to optimize all analytical
variables at 37 °C in order to establish a robust stand-
ard method for non-specific alkaline phosphatase.

*) Members: Ellen Schmidt (Hannover, chairholder), W. Ger-

hardt (Helsingborg), E. Henkel (Oldenburg/Hannover), R.
Klauke (Hannover), W. Liese (Magdeburg), K. Lorentz (Lü- Principle
beck), O. Sonntag (Hannover), W. Stein (Hamburg), G.
Weidemann (Nürnberg). Under assay conditions N-methyl-D-glucamine forms
2
) Received for publication: January 14, 1992. a phosphate ester, and the colourless 4-nitrophenyl-

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

248 Schmidt et al.: Alkaline phosphatase standard method

phosphate is converted to intensely yellow 4-nitro- 4. Hydrochloric acid, HC1, Mr 36,47 (2 mol/1).
phenoxide:
5. 4-Nitrophenylphosphate, disodium salt, hexahy-
drate, C6H4NNa2O6P · 6H2O, Mr 371,14.
4-Nitrophenyl- phosphatase 4-Nitrophenoxidc
phosphate + H2O * + Phosphate
Purity of reagents
4-Nitrophenyl- 4-Nitrophenoxide
Alkaline
phosphate + + Methylgluc- Specifications regarding the purity of reagents must
Methylglucamine amine phosphate conform to the criteria given in 1. c. (10) for the IFCC
method (1). Methylglucamine must be of highest
available purity (mass fraction above 0,99, single peak
Optimized Conditions for Measurement3) in gas chromatography as trifluoro and sililyl deriv-
Tab. 1. Concentrations in the assay.
atives on OV 1701 (0,2 mm χ 25m) fused silica
capillaries, temperature gradient 100-200°C, 4 °C/
N-Methyl-Z>-glucamine 500 mmol/1 60 s). Commercial products generally meet these cri-
pH (37 °C) 10,1
4-Nitrophenylphosphate 20 mmol/1 teria. The crystalline white powder is slightly hygro-
Magnesium acetate 0,5 mmol/1 scopic and should be kept under nitrogen, when it
Sodium chloride 110 mmol/1 may be stored for several years.
Volume fraction of sample 0,0179 (1 : 56)

Preparation of solutions
Technically most favourable measurement conditions,
which take account of the solubility, stability and Sterile containers should be used to prevent the
preincubation of the components, as well as adapta- growth of microorganisms. All solutions should be
bility to mechanized performance, are shown in prepared in calibrated flasks at the calibration tem-
table 2. perature with doubly distilled deionized water (sterile
with a conductivity below 2 μ8).
Tab. 2. Measurement conditions.
Temperature 37,0 ± 0,1 °C Solution I — activator/buffer
Wavelength (bandwidth) 405nm(^2nm)
Light path length 10,0 ± 0,01 mm Methylglucamine, 560 mmol/1; magnesium acetate,
Preincubation time To reach thermal equilibration
Starter substance 4-Nitrophenylphosphate 0,56 mmol/1, sodium chloride, 78,4 mmol/1, pH 10,6
Delay time 60s (20 °C)/10,1 (37 °C); hydrogen chloride, approx. 93
Measurement time 90s mmol/1.
Reagent blank Necessary
Sample blank Not necessary Dissolve 27,33 g of methylglucamine and 1,15 g of
sodium chloride in about 200 ml of water, adjust to
pH 10,5 with hydrochloric acid, 2 mol/1, (approx. 11,6
Instrumentation and Equipment ml) at 20 °C, add 30 mg of magnesium acetate tetra-
A spectrometer (preferably a recording instrument hydrate, dissolve completely and make up to 250 ml.
suitable for accurate measurement at 405 nm) with
constant temperature cuvette compartment is re- Solution II — substrate
quired. Specifications for the equipment (e. g. sample 4-Nitrophenylphosphate, 224 mmol/1.
and reagent handling, performance of the spectro-
meter, and temperature control) should meet those of Dissolve 4,16 g of disodium 4-nitrophenylphosphate
IFCC recommendations (10). hexahydrate and add water to a final volume of
exactly 50 ml.
Reagents
Solution III — diluent
1. N-Methyl-D-glucamine, C7H17NO5, MT 195,22.
Sodium chloride, 154 mmol/1.
2. Sodium chloride, NaCl, Mr 58,45.
Dissolve 0,9 g of sodium chloride in 100 ml of water.
3. Magnesium acetate, tetrahydrate, Mg(CH3COO)2
•4H 2 O, Mr 214,5.
Stability of solutions
3
) The decimal sign is a comma, as usual also in IFCC rec- Solution I should be kept in a tightly stoppered flask;
ommendations. it then has a shelf life of at least 2 months at 20 °C.

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

Schmidt et al.: Alkaline phosphatase standard method 249

Solutions exposed to air (250 ml with a surface of
38,5 cm2 in an open polypropylene bottle) show a
decrease of 0,02 pH-units per day at 4 °C. Solution
II should be prepared just before use and must be
used within 8 hours if stored at 20 — 25 °C or at the
most within 1 day after storage at 3 —5 °C.
Specimen Procurement, Stability and Storage
Blood should be collected by venipuncture with min-
imal stasis. Blood cells should be removed from serum
or plasma within two hours after collection. Serum is
the preferred specimen, and heparinized plasma is
acceptable, but plasma containing metal binding an-
ticoagulants such as ethylenediaminetetraacetic acid,
citrate, or oxalate must not be used.
Alkaline phosphatase in serum is stable for at least 2
days at 20 °C, 1 week at 4 °C, 3 months at -28 °C
or 6 months at —75 °C. After thawing, sera brought
to 4 °C do not lose activity within 2 days, but a few
refrigerated or frozen sera show slightly higher activ-
ities after warming to room temperature. Thus, fresh
serum specimens should be stored at 20 °C and when-
ever possible assayed within 4 hours after collection
(1).
Measurement procedure
The measurement consists of two subprocedures:
overall reaction and reagent blank reaction.
If the increase of absorbance is greater than 0,300 per
60 s, corresponding to 15 μkat/l (900 U/l), the sample
must be exactly diluted with solution III, and the
resulting ΔΑ/At multiplied accordingly.
If the increase of absorbance is smaller than 0,008 per
60 s, a sample fo 20 μΐ should be taken, and the
appropriate adjustment made in the calculation (see
below).
Reagent blank
The same procedure is followed when measuring the
reagent blank, except that the sample is replaced by
solution III (diluent). This change of absorbance must
be determined for each series of samples. The initial
absorbance may not exceed 0,500, and the increase
of absorbance must be less than 0,004 per 60 s at 405
nm. Otherwise, solution II (substrate) must be dis-
carded.
Correction for blank reaction
The reagent blank must be subtracted from the overall
reaction as follows:
(AA/At)overall - (AA/At)blank =
Calculation3)
Under the conditions of the assay, the molar absorp-
tion coefficient, ε, of 4-nitrophenoxide at 405 nm and
37°Cis 1875 ± 5,4m2/mol.
The light path length, /, is 0,01 m.
The total reaction volume, F, is 0,56 χ 10~3 1.
The sample volume, v, is 0,01 χ 10~ 3 1.
Let the increase in absorbance per second at 405 nm
be a.
a = (AA/At)corrected, s"1.

Overall reaction
Pipette successively into the cuvette Concentration in assay mixture

Solution I (activator-buffer) 500 μΐ N-Methylglucamine 500 mmol/1

Magnesium acetate 0,5 mmol/1
Sample 10 μΐ Volume fraction 0,0179 (1 : 56)

Mix thoroughly without removing any of the mixture from the Sodium 110 mmol/1
cuvette. Incubate at 37 °C until the mixture has attained this Chloride 171 mmol/1
temperature (at least 300 s).

Solution II (substrate) 50 μΐ 4-Nitrophenylphosphate 20 mmol/1

Mix and monitor the increase in absorbance after 60 s for up to

90 s with a recorder or an appropriate timer.
Note: The sodium chloride content comprises the sodium proportion of the substrate, chloride ions from buffer adjustment, and
added sodium chloride in solution I.

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

250 Schmidt et al.: Alkaline phosphatase standard method

From the following equation the catalytic concentra-

tion, b, is given by

b = a x
ε χ χ ν

0,56 χ ΙΟ
b =a x
1875 χ 0,01 χ 0,01 χ 10~3
s"1 x l
m χ mo!"1 χ m x l
2

56
ο =α χ χ 3
18,75 m χ ηιοΓ1

b = α χ 2,9867 kat/m3
b = a x 2987 μίαα/ΐ
Let the increase in absorbance per minute at 405 nm
be A. 40 60 80 100 120 140 160 180
1 Alkaline phosphatase
A = (AA/At)corrected, min" . (Recommended Method of the German Society for
Clinical Chemistry, 25 °C) [U/l]
, 56 mol 140
b — AΛ x x 3
18,75 m x min
b = A x 2987 U/l

If 20 μΐ of sample are used, the calculation factor

must be changed to 1520.

Note: l U corresponds to 16,67 nkat.

Multiplication by 60 converts μkat to U.

Analytical Variability
Performance data with the described measurement
procedure are given in table 3.

Tab. 3. Imprecision data from 12 manual determinations of

alkaline phosphatase.
Catalytic concentration Relative standard deviation
μkat/l 20 40 60 80
U/l intra-assay inter-assay
Alkaline phosphatase (IFCC method, 30 °C) [U/l]
1,27 76,2 0,0224 0,0427
2,05 123 0,0095 0,0264 Fig. 1. Comparison of alkaline phosphatase catalytic concen-
3,68 221 0,0080 0,0196 trations determined by
a) the Recommended Method of the German Society
for Clinical Chemistry at 25 °C (GSCC, abscissa) and
b) the IFCC Reference Method at 30 °C (IFCC, ab-
scissa) versus the proposed method at 37 °C (ordinate)
in 117 sera of healthy adults (see tab. 4).
Method Comparison and Reference Values
In order to compare results from the proposed method
at 37 °C with those of the IFCC Reference Method performed at 25 °C (5), 117 sera from healthy adults
at 30 °C (1) and those of the Recommended Method were investigated. Figure 1 shows the comparison of
of the German Society for Clinical Chemistry (GSCC) these values. Obviously, there is a close correlation

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

Schmidt et al.: Alkaline phosphatase standard method 251

between either the IFCC (r = 0,972, τ = 0,86) and Buffer and pH

the GSCC (r = 0,964, τ = 0,87) and the proposed
The selection of an appropriate buffer largely follows
method as shown in table 4.
the considerations outlined in the introduction to this
proposal and some specifications given by the Italian
Tab. 4. Comparison of results from 117 alkaline phosphatase society (9). Important advantages of methylglucamine
determinations by the proposed method (y) at 37 °C, over 2-amino-2-methyl-l-propanol are apparent from
the IFCC method at 30 °C (1) and the GSCC method table 6. These include the absence of inhibiting con-
(5) at 25 °C. Statistical analysis by rank correlation and
biometrical procedures (11). taminants, thus avoiding the addition of metal che-
lators, allowing the reaction to be started with sub-
Method (x) IFCC (1) GSCC (5) strate, and sustaining linear conversion rates during
Regression y = l,669x- 2,283 y - 0,872x- 1,896 extended measurement at 37 °C. These qualities are
equation evidenced by a linear temperature dependence, in
Linearity by yes yes contrast to 2-amino-2-methyl-l-propanol (fig. 2).
cusum test Compared with diethanolamine, a favourable pK
Significant deviation (with confidence intervals) value, lack of viscosity at optimal buffer concentra-
from 1 yes ( 1,591-1,754) yes ( 0,830-0,919) tion, and equal response to four human isoenzymes
(slope) are additional advantages.
fromO yes (-5,926-1,561) yes (-6,196-2,205)
(intercept)
Tab. 6. Properties of N-methyl-D-glucamine as selected buffer
substance in alkaline phosphatase measurement.

Since reference ranges for this method at 37 °C have State Crystalline white powder of very high
purity with a defined melting point
not been yet determined, preliminary 0,95-reference (128-131°C)
intervals were derived by assessing alkaline phospha- Purification Not necessary, but easy by recrystallis-
tase in sera with catalytic concentrations within the ation from water
reference limits of the Recommended Method of the Availability Common, multiple suppliers
German Society for Clinical Chemistry (GSCC) per- Inhibitors Not detected in commercially available
lots
formed at 25 °C (5). These data agree well with those
Metal ion control No need to add metal chelators to en-
reported previously by Franzini et al. (12). sure optimal zinc and magnesium con-
centration
Solubility High (583 g/1 at 20 °C in water)
Tab. 5. Preliminary 0,95-reference intervals for alkaline phos- pK value 9,63 (37 °C) with temperature depend-
phatase in 200 healthy adults at 37 °C versus corre- ency of -0,027 pH-units/ °C
sponding ranges at 25 °C. Phosphorylation Moderate, 0,75 that of diethanolamine
Concentration Optimal conditions for measurement
Popu- Number Proposed method (37 °C) GSCC method possible
lation of ((5); 25 °C)
samples μkat/l U/l U/l Isoenzyme bias Identical with that of 2-amino-2-
methyl-1 -propanol
Men 106 0,733-2,58 44-155 50-180 Initiation of reaction Serum or substrate start with identical
Women 94 0,624-2,42 37-145 45-170 results
Conversion rates Linear with time at 37 °C

The optimal buffer strength seems to be somewhat

Optimization of Conditions for Catalytic Activity
Measurements
Optimized reaction conditions for the determination
of alkaline phosphatase catalytic activity in human
sera were derived from results of univariate experi-
ments and response surface methodology quoted be-
low. These conditions consider both enzyme kinetics
and technical aspects of manual and mechanized per-
formance. They do not necessarily provide maximal
possible conversion rates, but the highest rates that
are consistent with robustness and transferability of
the method.
higher than proposed by Ceriotti et al. (9). Maximal
catalytic concentrations are observed between 450 and
525 mmol/1 in sera from healthy people and those
from patients, and these catalytic concentrations pre-
dominantly represent the placental, liver-bone or in-
testinal isoenzymes (fig. 3). Non-human control ma-
terial may display highest activities at buffer concen-
trations below 400 mmol/1. Between 400 and 500
mmol/1, blanks (with diluent or human serum albu-
min, 60 g/1) merely increase from 83 nkat/1 (5 U/l) to
112 nkat/1 (6,7 U/l). Hence, a substance concentration
of 500 mmol/1 is regarded as optimal.

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

252 Schmidt et al.: Alkaline phosphatase standard method

400 1,00
350
300
Λ
j? c 0,90
. 250 I8
ft
200 || 0,80
| c

150
"~ 0,70
I I I I I I I I I I I
9,5 10,0 10,5
pH
100

-
75
^^··— · — *" *
l l l ι ι ι l l ι ι l l l l l l l l l ι ι l l l l
20 25 30 35 40 I I I I I I I I I I I
Temperature [°C] 9,5 10,0 10,5
pH
Fig. 2. Temperature dependence of alkaline phosphatase activ-
ity in pool serum under the conditions of the assay Fig. 4. Variation of alkaline phosphatase activity (above) and
( ) and according to the IFCC Reference Method reagent blank (below) with pH. Conditions at 37 °C:
(actual , ideal · · -): Catalytic concentration calcu- Methylglucamine, 500 mmol/1, 4-nitrophenylphosphate,
lated from 60s (1), 60-240s (2), and 30-480s (3) 20 mmol/1, magnesium acetate, 0,5 mmol/1, and sodium
after start of the reaction. chloride, 110 mmol/1. The arrow indicates the selected
pH value.

} 1,00
1,00
.1
r!

50,90 0,90

200 300 400 500 600 700 800

N-Methyl-D-glucamine [mmol/l]

Fig. 3. Effect of methylglucamine concentration on alkaline

ί
0,80
phosphatase activity. Recommended assay conditions
were used, but the buffer concentration was varied. The 12 16 20 28 32 36
arrow indicates the selected condition.

Unexpectedly, a rise of pH influences the reagent 10

blank to a small extent, so that only reactions cata-
lysed by different sera with a rather small maximal
range between pH 10,0 and 10,3 need to be considered
(fig. 4). An optimum pH of 10,1 is chosen as a
convenient compromise based on the reaction of sera
Fig. 5. Effect of substrate concentration on alkaline phospha-
from healthy men. Higher conversion rates at pH 10,2
may occur with control material.
Substrate
The qualities of 4-nitrophenylphosphate as the sub-
strate of choice, which have been convincingly dem-
onstrated by Tietz et al. (1), likewise apply to this
method: a ready enzymatic hydrolysis, the high and
almost invariable absorbance of 4-nitrophenoxide at
16 20 24 28 32 36 40
4-Nitrophenylphosphate [mmol/l]
tase activity (above) and reagent blank (below). Apart
from 4-nitrophenylphosphate the recommended assay
conditions were used. The arrow indicates the selected
condition.
the reaction pH which enables its sensitive continuous
monitoring, and a minimal isoenzyme bias. Maximal
reaction velocity occurs in the broad range between
20 and 36 mmol/1 (fig. 5), but the reagent blank
doubles from 20 mmol/1 to 40 mmol/1. Therefore, 20

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

Schmidt et aL: Alkaline phosphatase standard method 253

mmol/1 is chosen to keep the detection limit low. When

methylglucamine is used as the buffer substance, the
linear measurement interval is not decreased by in- 28
creasing substrate concentrations; this is not the case 8-8
when the reaction is buffered with 2-amino-2-methyl- it
1-propanol.
c
ο 0,90

Wavelength
100 200 300
The reasons for wavelength selection, discussed by Sodium chloride [mmol/l]

Tietz et al. (1), also apply in the present choice of 405
nm. Compared with the IFCC method, which rec-
ommends 4-nitrophenylphosphate, 16 mmol/1 (1), the
slightly higher substrate concentration could favour
measurements at 410 nm, where its absorption is only
0,40 of that at 405 nm. However, this wavelength
should only be used if 405 nm cannot be selected with
the instrument to be used.
The molar absorption coefficient at 405 nm slightly
increases with temperature: 1850 ± 5,2m2/mol at
20 °C corresponds to 1875 ± 5,4m2/mol (n = 10) at
37 °C, a value close to 1867 ± 6 m2/mol in a similar
medium reported by Lewandrowski et al. (13).
Fig. 6. Influence of sodium chloride concentration on alkaline
phosphatase activity. Other conditions as in table 2. The
arrow indicates the selected condition.
Sodium chloride does not apparently contribute to
stability, as deduced from preincubation studies with
different concentrations. Enzyme activation is maxi-
mal between 100 and 250 mmol/1 (fig. 6) and varies
slightly within this range with different sera. The
selected concentration of 110 mmol/1 is above that of
Ceriotti et al. (9).
Volume fraction of the sample

Effectors Varying the sample volume fractions of 10 sera with

catalytic concentrations between 0,65 (40) and
Magnesium is essential both for alkaline phosphatase 7 !ikat/l (420 U/l) from 0,00362 to 0,0833 leads to
catalytic activity and stability, thus suggesting that identical results between 0,009 (1/111) and 0,0385
samples should be preincubated in the protective pres- (1/26) and yields 5 percent lower activities below and
ence of magnesium before starting the reaction with above this range. The selected fraction of 0,0179 per-
substrate. Under these conditions, maximal activity mits sufficient sensitivity and low imprecision (tab. 3)
and extended linear conversion rates are observed of this method.
between 0,5 mmol/1 and 2 mmol/1 (see below), whereas
in assays lacking magnesium (except for its presence
in the sample) about 0,95 of maximal velocity is Preincubation time and initiation of reaction
obtained. Alkaline phosphatase inactivation at 37 °C is still a
The addition of zinc(II) ions is unnecessary for the matter of discussion, although stability in the presence
following reasons. Firstly, proceeding from an average of activator and substrate has been reported (16). In
zinc concentration of 14 μηιοΐ/l in human serum, the confirmation of the protective effect of magnesium
assay contains about 0,25 μηιοΐ/ΐ, which is far above mentioned above, 20 sera with catalytic concentra-
the free zinc concentration of 3,16 fmol/1 in the IFCC tions within and 5 sera with catalytic concentrations
method (1), which arises from added zinc sulphate higher than the preliminary reference range were sta-
and the dissociation constant of zinc(II)-N-hydroxy- ble over a 15 minutes preincubation period with so-
ethylenediaminetriacetic acid (HEDTA) (14). Sec- lution I at 37 °C. This contradicts the results of Le-
ondly, analytical grade sodium chloride contains zinc wandrowski et al. (11), who observed a triphasic loss
5 — 50 mg/kg, so that buffer containing added sodium over 400 minutes in buffer devoid of magnesium.
chloride, 70 mmol/1, also contains at least 5 nmol/1 of Therefore, it is advisable to start the reaction with
zinc. Thirdly, the addition of the IFCC metal ion substrate. However, initiation with samples is possible
buffer (Mg-Zn-HEDTA) to the proposed buffer in- and yields identical results to those with substrate
stead of magnesium, 0,5 mmol/1, neither improves start.
enzyme stability nor enhances catalytic activity, which
has been confirmed by Ceriotti et al. (15). On the
Measurement interval
other hand, less than 12 μιηοΐ/ΐ of zinc is not delete-
rious, probably because zinc is complexed with vicinal With the proposed procedure, no lag phase exceeds
diol groups of methylglucamine. 45 s. Hence, a delay of 60 s is sufficient to guarantee

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

254 Schmidt et ah: Alkaline phosphatase standard method

linear conversion rates. Such rates are always ob-

tained for catalytic concentrations up to 15 μkat/l
(900 U/l) for at least 240 s. Although constant values
for ΔΑ/At are observed for samples between 15 |ikat/l
(900 U/l) and 22 μkat/l (1,32 kU/1) under the condi-
tions given in the measurement procedure, the result-
ing activities were 0,95 — 0,97 of those after fourfold
dilution.

Reagent blank and detection limit

Due to the reaction temperature of 37 °C, the spon-
taneous generation of 4-nitrophenoxide must always
be taken into account in calculating the true alkaline
phosphatase activity. Thus, the reagent blank defines
the detection limit, which was calculated to be 0,170
μkat/l (10,2 U/l) (in terms of mean plus 3 standard
deviations). Usually, the reagent blank of about 0,105
μkat/l (6,3 U/l) can be subtracted automatically in
modern analysers. 150 200 250 300 350
Alkaline phosphatase
(Recommended Method of the German Society for
Interferences and sample blank Clinical Chemistry, 25 °C) [U/l]

The deleterious effect of metal complexing anticoag-
ulants has been mentioned above. Haemolyzed spec-
imens should not be used. Lipaemic sera with tria-
cylglycerol concentrations above 12 mmol/1 must be
diluted appropriately. Icteric samples with more than
340 μηιοΐ/ΐ of bilirubin produce a loss in activity of
nearly 0,1, most possibly as a result of pigment pho-
tolysis during measurement. Interfering drugs have
been cited by Young (17).
Fig. 7. Comparison of alkaline phosphatase catalytic concen-
trations determined by the Recommended Method of
the German Society for Clinical Chemistry at 25 °C
(GSCC, abscissa) and the proposed method at 37 °C
(ordinate) in sera of 34 women between 30th and 39th
week of pregnancy (closed circles; r = 0,914, τ = 0,78,
y = 1,13x -f 11,4) and 6 children (open circles;
r = 0,595, τ = 1,00, y = 0,67x + 8,07) calculated ac-
cording to I.e. (11).
10,3

Dependence on isoenzyme composition

Human alkaline phosphatases promote diverse phos-
phorylation effects on amino alcohols, which makes
the choice of an appropriate buffer to some degree
arbitrary. Therefore, the enzyme's hydrolytic activity,
solely exerted in hydrogen carbonate, may serve as a
justified basis of critical buffer examination. On this
basis, methylglucamine and 2-amino-2-methyl-l-pro-
panol offer equal advantages in comparison with die-
thanolamine, and both of them are likewise suited for
alkaline phosphatase measurement without isoenzyme
bias as can be deduced from figure 7, which shows
results from sera with different predominant isoen-
zymes. 10,0
400 450 500
N-Methyl-D-glucamine [mmol/l]

Optimization by Response Surface Methodology
The selected conditions, examined with respect to pH
value, substrate and buffer concentrations and eval-
uated by multivariate analysis, are visualized as con-
Fig. 8. Alkaline phosphatase activity response surface for vary-
ing pH and methylglucamine concentration at 4-nitro-
phenylphosphate, 20 mmol/1, magnesium acetate, 0,5
mmol/1, and sodium chloride, 110 mmol/1. The ciphers
within the figure denote the fractional catalytic concen-
trations, the point symbolizes the selected combination.

Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4

Schmidt et al.: Alkaline phosphatase standard method
600
E 500
c 0,950
400
"Ö)
Q
300
200
10 20 30
4-Nitrophenylphosphate [mmol/l]
Fig. 9. Alkaline phosphatase activity response surface for vary-
ing 4-nitrophenylphosphate and methylglucamine con-
centrations. With the exception of these variables, rec-
ommended assay conditions were used. The ciphers
within the figure denote the fractional catalytic concen-
trations, the point symbolized the selected combination.
tour plots showing minimal changes of activity near
the reaction maximum. Response-surface data display
relatively flat maxima for concentrations of buffer
versus substrate (fig. 8) and for pH value versus buffer
concentration (fig. 9), whereas the maximum plateau
for pH value versus substrate concentration is some-
what narrower (fig. 10). In all cases the proposed
conditions always lie within the 0,99 area close to the
reaction maximum.
References
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10 12 14 16 18 20
4-Nitrophenylphosphate [mmol/l]
Fig. 10. Alkaline phosphatase activity response surface for
varying pH and 4-nitrophenylphosphate concentra-
tion. With the exception of these variables, recom-
mended assay conditions were used. The ciphers within
the figure denote the fractional catalytic concentra-
tions, the point symbolizes the selected combination.
Acknowledgement
The authors are indebted to E. Merck, Darmstadt (Germany)
for the preparation and kind supply of solutions used for
method comparison and for the establishment of preliminary
reference values, and they thank Barbara Gülschow, Renate
Henkel and Annette Hinrichs for their excellent technical as-
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Schmidt et al.: Alkaline phosphatase standard method
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Prof. Dr. K. Lorentz
Institut fur Klinische Chemie
Medizinische Universität Lübeck
Kronsforder Allee 71 -73
W-2400 Lübeck
Bundesrepublik Deutschland
Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 4