Standardizationof EnzymeAssays
Hans Ulrich Bergmeyer
Now
a few words
regarding
each
of these
five
Additional Keyphrases
enzyme assay methods, criteria
#{149} international
nomenclature,
units,
standard
temperature
#{149}
isoenzymes
#{149} variables
affecting samples
#{149} reference
standards
Standardization
of methods
requires
an enormous amount of work, organization,
and the good
will and cooperation
of many people.
The work
on standardization
has to be done in such a manner
that all laboratories
are willing to follow the decisions of these standardizing
commissions.
Therefore, the methods
must be selected
carefully
in
order to make them acceptable
to most laboratories. Also, one should only choose those methods
that have already been proven and that fulfill the
strict requirements
necessary for modern analytical
methods.
This is the first step in the standardization of methods.
A second part of the work is the effort of international
commissions
to create definite terms and
units in clinical chemistry-for
enzyme nomenclature, enzyme activity,
weight, volume,
and concentration
(units)-and
to fix an international
standard
temperature.
The third part will be a great deal of experimental
work for the determination
of optimal
measurement
conditions for the selected methods.
Having obtained
these results, we get the standardized
conditions
for measurement,
but,
as
Figure 1 shows, it is not enough for the completely
standardized
enzymatic
assay
method.
Other
factors are to be considered:
the stability
of the
compound
to be assayed in the sample as well as
the sampling
itself, and on the other hand, the
availability
of stable and defined reference
standards. Only with all these factors considered
do we
get a standardized
enzymatic
assay method (1).
From the Dept. of Biochemistry,
University of Wuerzburg and
Boehringer
Mannheim
GmbH,
8132 Tutzing/Obb.,
Bahnhofstrasse 5, Mannheim
31, Germany.
Presented
at the International
Seminar and Workshop
on
Enzymology,
Chicago, Ill., May 2 1-24, 1972.
parameters.
Selection
of Methods
Routine methods should
the following requirements.
be selected according
The method
to
#{149}
must be sufficiently
sensitive
#{149}
must be highly specific
#{149}
must give reproducible
and correct results
#{149}
must be sufficiently simple for practical work.
Methods
should be selected
according
to these
requirements.
A few explanations
of this subject
may follow.
Sensitivity.
In general, enzymatic
methods
and
those for measurement
of enzyme
activities
are
sufficiently
sensitive, as shown (2) in Table 1.
Specificity.
This requirement
is primarily
of importance for metabolite
assays, but in a certain sense
also for enzyme activity measurements.
“Enzymes
are the most specific reagents known today” (Otto
Warburg)-this
statement
is valid in spite of the
fact that a few enzymes can react with several chemically related substances.
In such cases, however,
one can achieve
specificity
by combining
these
nonspecific
enzymes with absolutely
specific ones.
An example is the determination
of glucose wherein
Fig. 1. Standardization
of enzyme assays
CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972 1305
Table 1. Sensitivity of Enzymatic Methods
Method
Limit of detection
(approx. vaIues)
Limit of readings
Metabolites
uv-test, endpoint
A = 0.002
fluorometry, end- 0.01 scale units
point
catalytic
test
enzymatic re.cy.
cling
10
10
mol/liter
mol/liter
A/5 mm = 0.002 10-s mol/liter
0.01 scale units!
10’s mol/ liter
5mm
Enzyme Activities
uv-test
A/5
fluorometric test
0.01 scale units/
5 mm
7 h/5 mm = 1
manometric test
radioisotope
test
mm
0.002
=
500 cpm; incubation time, 30 mm
0.1 U/liter
0.001 U/liter
10 U/liter
0.0001 U/liter
#{149}
Related to liter of the sample.
the nonspecific
hexokinase
is coupled
with the
glucose-6-phosphate-specific
glucose-6-phosphate
dehydrogenase.
Because
the indicator
enzyme,
glucose-6-phosphate
dehydrogenase
(EC 1.1.1.49),
is specific, the overall reaction becomes specific, too.
With respect to enzyme activities there are other
problems.
In particular
the activity
of hydrolases
is often measured
with artificial
substrates,
for
methodological
reasons.
For example,
we determine protease
activity
with benzoyl-i-arginineethylester
as a substrate.
This measured
activity
does not have an unquestioned
correlation
to the
physiological
role of the enzyme. Other substrates
are also converted
with similar velocity, an exception being the physiological
substrates,
the proteins. We can thus establish
an excellent method,
but it remains the task, together with the clinicians,
to correlate
the values obtained
with disorders
of
protein metabolism.
The same is true for esterases.
Precision
and accuracy.
In most cases the volume
measurement
of reagents
and samples is the chief
determinant
of precision. Therefore,
one preferably
should use a premixed reagent in order to decrease
pipetting
steps. It has been demonstrated
(3) with
measurements
of enzyme activities
that the precision in the series is far better when a reaction mixture is used than when each pipetting
step is done
individually.
The coefficient of variation is reduced
to less than half.
Other main sources of error in assays of enzyme
activities are inadequate
setting and control of temperature, and, in coupled tests, a false ratio of indicator (auxiliary)
enzyme to measured
enzyme.
For
example,
if in the reaction
step A
of which
B is catalyzed by an enzyme, the activity
is to be measured,
the indicator
reaction
B 4- C should
be arranged
A
-
so that
B 4- C the
only part
1306 CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972
first
of a
minute is allowed to attain a steady state in which
both reactions
proceed with equal velocity;
in this
case only a negligible
amount
of B accumulates.
Therefore,
the indicator
reaction indicates
at any
moment of the overall reaction how fast B is produced by the enzyme to be measured.
In order to
assure this according to Michaelis-Menten
kinetics
the ratio of
(vm/Km)E.:
(Vm/Km)E,
has to be at least 1: 100. The error of different
ratios has already been calculated
(4) (Table 2).
This requirement
results from the fact that in
the steady state small amounts of B (in relation to
Km) cause the indicator
reaction to run relatively
slowly, so that the effective velocity is far below
Vmax.
Therefore,
one has to measure
with
a
relatively large amount of indicator enzyme.
In general,
enzymatic
methods
for the determination
of metabolites
have a range of error
(standard
deviation)
of about 1% to 3%, whereas
those for the determination
of enzyme activities are
about 3 to 5%.
Correct results. These depend on the specificity of
the enzymes used, and on their activity and purity.
These three variables
cannot be separated
from
each other, because they influence each other. It is
obvious
that a certain
amount
of activity
is
necessary to bring the reaction to completion.
Nonspecificity
or impurity
(that is to say, contamination with other enzymes)
results in several compounds
in the same sample
being acted
upon
simultaneously.
By using high-quality
enzymes, one
can assure correct results by recovery measurements
in the same cuvet; that is, by adding a definite
amount of the pure substance
to be measured,
one
can check the system for absence of interferences.
Simplicity.
This requirement
for the method
must not interfere in any way with any of the other
three requisites.
If this is assured, simplicity
should
then be sought for two reasons:
to save time in
routine work and to decrease the number of steps
and so decrease the number of possible sources of
error. Mechanization
of the procedure
is one such
simplification.
Not in every case are enzyme reactions
of zero
order (linear conversion
curve).
In the case of
nonlinear
conversion
curves,
the calculation
of
Table 2. Determination of Enzyme Activity
in a Coupled Test: Percentage Error at
Different Activity Ratios of E and E1
(vmx)E
/(K,,)EI
1:10
1:100
1:1000
1:
Error
-23%
-4%
-0.7%
0%
enzyme activities
is always based on the tangents
of these curves at t = 0. The initial velocity (vo) is
therefore
used for definition
of enzyme units. It is
therefore
always recommended
that the curves be
drawn with an automatic
recorder and that v0 be
ascertained
graphically
from nonlinear
curves.
Two-point
measurements
are allowed only with
linear conversion
rates. With respect to simplicity,
methods
with zero-order
kinetics
are therefore
preferable.
All of these criteria should be considered,
when
methods
have to be selected. Because it is almost
impossible
to fulfill all optimum
requirements
simultaneously,
certain
compromises
have to be
made.
International
Nomenclature,
Units,
Standardized
Considering
the fact that the maintenance
of
temperatures
that are below room temperature
is
not a technical problem with modern thermostats,
I suggest that the physico-chemical
standard
temperature
of 25#{176}C
be used to avoid the aforementioned disadvantages.
The reasons are as follows:
#{149}
Preheating
time for all reagents
and experimental
materials
in cuvets,
especially
plastic
cuvets with low thermal conductivity,
is shorter.
#{149}
Temperature
drop of preheated
solutions during pipetting
and of preheated
cuvets during handling is smaller.
#{149}
Growth
of contaminating
bacteria
is minimized in the solutions used throughout
the day.
#{149}
Hydrogen
ion concentrations
for all commonly
used buffer mixtures
are quoted for 25#{176}C.
Temperature
For measurements
in clinical chemistry,
various
The proposals
for international
units for enauthors
have suggested
the use of temperature
zymes, weight,
volume,
and concentrations
preconversion factors instead of fixing one temperature
sented by Dybkaer
and Jorgensen
several years
as a standard
temperature.
Such an approach
is
ago will not be discussed
here. It should be mennot correct for several reasons.
tioned that one should perhaps
speak of enzyme
Human body fluids in many cases contain sevactivity
instead of enzyme amount.
Furthermore,
eral isoen zymes
(e.g., lactate
dehydrogenase).
it is suggested
that a switch-over
from the fiftyThe reaction,
catalyzed
by each of them may have
year-old term “unit” to “katal” be made only when
a different
temperature
coefficient.
This assumes
internationally
standardized
methods
are availparticular
importance
in the many diseases in which
able, not sooner. One should not confuse hospital
the isoenzyme pattern varies considerably.
lAThen test-kits
are used, measurement
at other
personnel
any more than necessary;
that means
that there should be only one more change to a
temperatures
than indicated
in their instructions,
new method together with new terminology.
and then applying
a conversion
factor,
leads to
Standardized
temperature
(5). At present in clinfalse results.
The optimum
substrate
concentraical chemistry,
temperatures
of 25#{176},
30#{176},
32#{176},
35#{176}, tion is temperature-dependent.
For example,
in
37#{176}C,
and others are used in enzymatic
methods.
the case of lactate
dehydrogenase
(EC 1.1.1.27),
With the use of a standarized
temperature,
all
optimal pyruvate
concentration
varies by a factor
kinetic,
methodologic,
and diagnostic
aspects
of
of 10 for measurements
at 25#{176}
or 37#{176}C.
enzymes should be properly
rechecked.
There are
Test-kits
in general contain buffers that are ina number
of advantages
in conducting
activity
tended for use at a certain pH and a definite temmeasurements
at higher temperatures
rather than
perature.
The effect of temperature
change on the
at lower temperatures.
These include the greater
pH of several buffers differs-i.e.,
the p11-variation
differences
in absorbance
per time unit, and thereof buffers between 25#{176}
and 37#{176}C
may be as much as
fore greater sensitivity.
This makes possible either
0.35 pH unit. This influence on the reaction rate of
more analyses per hour or smaller sample volumes.
an enzyme-catalyzed
reaction is negligible only in
The decisive disadvantage
of a higher temperature
special
cases (when the pH-dependence
of the
such as 37#{176}C
is the possibility
of alterations
in the
rate is slight). One can calculate the dependence
of
protein structure
or even irreversible
inactivation
pH on temperature
by means of the dissociation
of certain enzymes during temperature
equilibraconstants
of the buffer components.
We have meation in the assay, a thermal
lability of substrates
sured it for the buffers most often applied in enzyme
(e.g.,
p-nitrophenyl
phosphate,
creatine
phosmeasurements
[aminotransferases
(EC 2.6.1.1 and
phate),
of the cofactors
(e.g., nicotinamide
ad2.6.1.2), creatine kinase (EC 2.7.3.2), and alkaline
enine dinucleotide),
and of auxiliary and indicator
phosphatase
(EC 3.1.3.1)].
Figure 2 shows that
enzymes. It is also well known that even the transthe largest deviation
is obtained
with Tris [trisfer of a warmed
solution to the cuvet in a pipet
(hydroxymethyl)aminomethane]
buffer, the smallkept at room temperature
results in a significant
est with phosphate
buffer.
decrease in the temperature
of the reaction
mixAddition
of compounds
needed as substrates
or
ture. The best conditions for measurements
at 37#{176}C substituents
in the enzyme reaction
may or may
would be to work in a room that is at the same temnot lead to a more or less “temperature
stability”
perature;
unfortunately,
this is somewhat
troubleof pH. This depends on the buffer efficiency and
some.
the dissociation
constants
of the added
cornCLINICAL CHEMISTRY, Vol. 18, No. 11, 1972 1307
8.0
TRIS
PHOSPHATE
7.5
--
+ 1 M L-AIa
A..#{149}+25OmM
LAsp
pH
7.0
‘j.,.
30
25
35
sEDIA
Mg2
Glucose
40
9.5
pH
DEA
9.0
15
20
Fig. 2. Temperature-pH
25
30
35
relationships
40
45
for various
so
#{176}c
buffers
TRIS = tris(hydroxymethyl)aminomethane,
0.1 mol/liter; TRA
=
triethanolamine,
0.1 mol/liter; DE.4 = diethariolamine,
1
mol/liter,
pH = 0.020/#{176}C
(upper curve) and 0.1 mol/liter,
L
pH = 0.018/#{176}C
(tower curve).
Calibration
buffers:
phosphate
buffer (50 mmol/liter;
pH, 6.86; 25#{176}C),
borate buffer (50 mmol/
liter; pH 9.18; 25#{176}C).
Temperature measured with NBS standardized thermometer
pounds. Thus, one cannot calculate
the temperature effect on pH of these buffers. One has to measure and adjust the pH at 37#{176}C,
for example, with
an instrument
calibrated
with a standard
buffer at
37#{176}C.
If used at all, temperature
conversion
factors
should be valid only within narrow limits (e.g., between 25#{176}
and 30#{176}C),
and optimum
conditions
must be established
for measurements
at both temperatures.
Optimum
Conditions
for Measurements
First, one has to decide on the definition
of the
term “optimum
conditions.”
It is suggested
that
the term “optimizing”
should be used for the establishment
of optimum
conditions
as far as they are
practicable
(6), and if they allow for the best possible discrimination
of results
(as for a-hydroxybutyrate
dehydrogenase,
EC 1.1.1.27, LD1); this
1308 CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972
means that in some cases, one cannot apply such a
high substrate
concentration
that the enzyme is saturated, because the highly concentrated
substrate
solution may be oversaturated
or too viscous; in the
case mentioned
before, the differences in activity of
the lactate dehydrogenase
isoenzymes
1 and 5 under
optimal substrate
concentration
become too small
to give results that are useful for the physician.
So
we come from “optimum”
to “optimized
conditions,” which are to be standardized.
Optimized
and standardized
conditions
include
a lot of variables.
At constant
temperature
they
include substrate
concentration,
pH, ionic strength,
type of buffer, and concentration
of cofactors.
In the widely used transaminase
(aminotransferase)
determinations
(two-substrate
reactions)
we have six variables that must be adjusted to each
other. In the case of determination
of lactate dehydrogenase
activity
with pyruvate
as substrate
and nicotinamide
adenine
dinucleotide
as coenzyme,
not considering
any other factors, there
are five variables.
To determine
optimum
conditions one has to vary one condition
while keeping
the others constant.
Moreover,
the standardization
is not limited to a
single lactate dehydrogenase
isoenzyme.
There are
five of these isoenzymes
in each organ. For each
isoenzyme
the optimum
condition
for measurement has to be determined.
We (7) did this in 1960
for human serum from healthy subjects,
and from
patients
with heart infarction
or with hepatitis.
The results are shown in Figure 3. As a first step,
we determined
an optimal pyruvate
concentration
at different
pH values
(dashed
curves,
lower
abscissa).
For the three groups of serum in the
pH range 7.3 to 7.5, optimal concentration
is 5 X
10
mol/liter
(perpendicular
line). In the second
step we have established
for this substrate
concentration
the optimal
pH for each of the three
serum groups (heavy curves, upper abscissa).
One
can see (open arrows) that at constant
temperature in phosphate
buffer (50 mmol/liter)
the isoenzyme complex in liver and heart differ considerably in relation
to pH and substrate
concentration.
This makes it clear that if we standardize
the
measurements
of gross, overall lactate
dehydrogenase activity,
we have to make a compromise
insofar
as we have to establish
a standardized
method for a variety
of applications
that cannot
be foreseen;
namely, for measurements
in serum
after myocardial
infarction,
for measurement
in
serum in cases of liver diseases, and for measurement in serum in cases of diseases
of skeletal
muscles. Our standardized
conditions
can oniy be
“mean value conditions”
with regard to optimal
conditions
for each isoenzyme.
Having selected the methods
and standardized
for optimized
conditions,
and after having
con-
pH
60
&5
7.0
7.5
8.0
U
S
U
S
C
E
0
102
PA pyruvate
25#{176}c
Fig. 3. Determination of optimum conditions for measurements of lactate dehydrogenase activity in serum.
Phosphate buffer (50 mmol/liter, 25#{176}C)
Section A: Normal serum. Section B: Serum in hepatitis. Section
C: Serum after myocardial
infarction.
Black arrow: optimal
pyruvate concentration
at pH 7.3 to 7.5; white arrows: optimal
pH at 5 X 10 mol of pyruvate per liter. Left hand and bottom
co-ordinates (broken and thin-lined curves): Dependence of the
activity on the pyruvate concentration with constant pH (shown
on each curve). Right and upper co-ordinates (thick-lined curves
0-0-0): Dependence of the activity on the pH with constant
pyruvate (0.3 mmol/liter; optimum activity)
sidered standard
temperature
and internationally
established
nomenclature,
we now have standardized conditions for measurements.
However, we still
have to consider samples and reference standards
in order to attain
standardized
enzymatic
assay
methods.
Samples
We all know that for blood sugar determinations, blood cannot be stored indefinitely.
We also
know that anticoagulants
added to the syringe for
blood withdrawal
may cause errors in the measurement
of enzyme
activities.
Furthermore,
it is
clear that one cannot assay enzyme activities
in a
serum that was in the mail for several days during
warm weather.
All this leads to the fact that one has to recognize
in general three points:
#{149}
Sampling
#{149}
Sample stability
#{149}
Sample contents
affecting
measurements
Sampling.
One can, of course,
measure
in a
sample only what it really contains.
It is nonsense to complain that a specific method for blood
alcohol determination
shows too much ethanol in
samples from a painter,
who worked for 8 h with
varnish dissolved in ethyl acetate and inhaled the
fumes.
The esterases
of his body have released
ethanol.
Not so striking,
but similary important,
are mistakes
such as drawing blood for the determination
of enzyme activities
after considerable
stress, in which for example
the creatine
kinase
activity increases strongly.
If at all possible, blood samples should be drawn
and stored with use of disposable
equipment
that
can be thrown away after use. Each dishwashing
might cause errors because of incomplete
cleaning
or residual detergents,
which could inhibit enzymes
[E. Bernt, personal
communication]
(Table 3).
In the course of preliminary
treatment
(separation of plasma
or serum)
hemolysis
must
be
avoided.
The blood cells contain
some of the
enzymes
to be measured
in serum or plasma in
high concentration,
such as malate- (EC 1.1.1.37)
and lactate
dehydrogenase.
Hemolysis
is a frequent
source of error. Thrombocytes
may also
release enzymes into the serum, even though only
on a relatively
small scale. Nevertheless,
the question is whether to perform the assay in serum or in
plasma.
Sample stability. In some cases samples can be
stabilized,
but these are still exceptions,
e.g.,
prostatic
phosphatase
and creatine kinase (Table
4). The stability
of enzymes in the sample during
storage is different,
as may be seen from Table 5.
Generally,
it would be best to measure within a few
hours after sampling,
but even at normal
room
temperature
most of the enzymes do not lose more
than 10% of their activity.
Sample
contents
affecting
measurements.
The
possible
interference
by anticoagulants
has already been mentioned.
More severe, because
in
most cases not readily recognized,
may be interference by drugs or other therapy.
It is well known,
that ascorbic acid or uric acid can cause false results
in blood sugar determinations
with the glucose
oxidase (EC 1.1.3.4) method.
We had established
an excellent enzymatic
color test for uric acid, but
it was affected by many pharmaceuticals.
Methods
for enzyme assays should also be checked in every
case as to the possible effects of interfering
substances in the sample. Consider,
for example,
the
usefulness
of values that would be obtained
in the
assay of xanthine
oxidase (EC 1.2.3.2) activity
in
serum of allopurinol-treated
patients
if one neglected the fact that this compound
inhibits
this
enzyme. Another example: therapy with a variety
of pharmaceuticals
for heart
and
metabolic
diseases causes disturbance
of the determination
of
activity
of lactate dehydrogenase
with lactate as
substrate
and diaphorase
(lipoamide
dehydrogenase, EC 1.6.4.3) as indicator
enzyme
(8; see
also 9, 10). Information
on the influence
of
therapeutic
agents should be included in every instruction
sheet for the assay of enzyme activities.
This is not only useful, but necessary.
CLINICAL CHEMISTRY,
Vol. 18, No. 11, 1972 1309
Table 3. Influence of Detergents on Enzyme Activities
Detergent
Deutsche Hydrier.
Werke
Texapon T
Concentration
In the assay
g/100 ml
No Inhibition
aspartate
0.06
Inhibition
aminotransferase
alanine aminotransferase
malate dehydrogenase
45%
glutamate dehydrogenase
100%
0.08
peroxidase 60%
lactate
aspartate aminotransferase
Trinon NJ
alkaline
dehydrogenase
phosphatase
50%
17%
creatine kinase 30%
acid phosphatase 37%
Bie u. Berntsen/
Kopenhagen
0.09
alanine aminotransferase
lactate dehydrogenase
aldolase
Q.9
0.08
alanine aminotransferase
aspartate amino-
creatine
alkaline phosphatase 24%
acid phosphatase 10%
choline esterase 25%
transferase
Ferak/Berlin
kinase
lactate dehydrogenase
Ultravon JF
Ciba
0.2
lactate dehydrogenase
16%
a.hydroxybutyrate dehyd rogen ase
aspartate aminotransferase
alanine aminotransferase
creatine kinase
alkaline
phosphatase
glutamate dehydrogenase
leucine aminopeptidase
RBS25
0.006
aspartate
aminotransferase
none
alanine aminotransferase
aldolase
hexokinase
glucose-6-phosphate dehydrogenase
Roth/Karlsruhe
alkaline
phosphatase
glutamate dehydrogenase
peroxidase
and 26 other enzymes
Table 4. Stabilization and
Reactivation of Creatine Kinase
Activity,
Sample
after 1
%
2
Serum at 2#{176}C
32#{176} 16#{176}
Serum + cysteineb at 2#{176}C 100
100
Serum + cysteine#{176}
at 22#{176}C100
50
a
3 days
12#{176}
16#{176}
0
May be reactivated by cysteine to 100%.
Three mg of cysteine per milliliter of serum.
enzyme,
but there
is no guarantee
that
this
absolute
content
of enzyme
activity
will be recovered correctly.
By applying a false method, the
activities
of standards
are determined
wrongly,
too. So the reference
material
has to be assayed
by the same standardized
method as that for which
serve as a standard.
It is therefore
necessary,
first
to standardize
the methods
and then to set up
appropriate
reference
standards.
Additional
Reference
Standards
It is self-evident
that reference standards
should
be stable and well defined. The problems involved
in the preparation
and definition
of standards
for
the assay of metabolites
can be overcome.
The
exact definition
of enzyme standards,
however,
is
more complicated.
It is possible
to weigh in a
definite amount
of a crystallized
and well-defined
1310 CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972
Remarks
Standardization
and optimization
of methods
for enzyme
assays is vitally
important,
because
enzyme activity
is highly dependent
on temperature, pH, buffer, ionic strength,
and substrate
concentration
(if the enzyme is not saturated
with
substrate).
Measurements
of coupled reactions
are
subject to special requirements
for the auxiliary
and indicator
reaction. One has to try to convert a
two-substrate
reaction to a one-substrate
reaction
Table 5. Stability of Enzymes in Serum under Various Storage Conditions
(<10% of Original Activity Lost during Specified Time)
Enzyme
Acid phosphatase
Alkaline phosphatase
Aldolase
a-Amylase
Cholinesterase
Creatine kinase, activated
y-Glutamyl transpeptidase
Glutamic dehydrogenase
Aspartate aminotransferase
Alan me aminotransferase
Leucine arylamidase
Lactate dehydrogenase
+25#{176}C
(rm.
temp.)
4 hours#{176}
2-3 daysc
2 days
1 month
1 week
2 days
2 days
1 day
3 days
2 days
1 week
1 week
0#{176}
to +4#{176}C
(refrigersted)
3 days
2-3 days
2 days
7 months
1 week
1 week
1 week
2 days
1 week
1 week
1 week
1-3 days6
References
-25#{176}C
(frozen)
(3)
(4)
3 days
1 month
unstable4
2 months
1 week
1 month
1 month
1 day
1 month
unstable4
1 week
1-3 days6
()
(1)
()
(4)
(1)
()
(1)
()
(I)
()
At pH 5-6.
#{176}
b
With added citrate or acetate.
Activity may increase.
Enzyme does not tolerate thawing well.
#{149}
Depending on isoenzyme pattern in the serum. (See ref. 5).
A
References
1. Amador, E., and Wacker, W. E. C., In Methods of Biochemical
Analysis,
XIII, D. Glick, Ed. lnterscience Publishers, New York,
N.Y., 1965 pp 274-276.
2. Methoden der Enzymatischen
Ed.. 2nd ed., Verlag Chemie,
136-138.
3. Ibid.,
Analyse, 1, H. U.
Weinheim/Bergstr,
Bergmeyer,
1970, pp
5. Hoffmeister,
8, 613 (1970).
H., and Junge, B., Z. Kim. Chem. Kim. Bioche,n.
6. Henry, R. J., Clinical Chemistry, Principles
Harper & Row, New York, N.Y., 1965.
p 818.
of zero order by developing
experimental
conditions that will avoid errors in the establishment
of
the initial velocity.
All this is in contrast
to the determination
of
metabolites.
If one can cope with all this, one also
should be able to use clear terms, and mathematically correct writing of formulas and dimensions.
What can be omitted from method standardization is the definition
of normal,
transitory,
and
pathological
values. This is not a matter
of the
method itself, but of the interpretation
of the results obtained with this method. Moreover,
it may
not be practical to establish normal values that are
supposedly
valid for the whole world, since there
might be significant
differences
between different
groups of people.
References
1. Bergmeyer,
H. U., The
standardization
of
methods.
Relazioni
VI Congresso
Internationale
Clinica,
Rome, 1966, p 175.
4. BMC Laboratories, N.Y.C. (“Wherever contradictions
arose
in the literature, we chose the shortest time allowance and confirmed it in practical studies,” BMC).
enzymological
di Patologia
and Technics,
2. Michal,
G., Enzymatic
Analysis,
Methodicum
I, G. Thieme
Verlag, Stuttgart,
1972, p 1110.
3. Engelhardt,
untersuchungen.
A., and
Aerztl.
Houben-Weyl,
Noetges,
A., Fehlerquellen
Lab. 16, 42 (1970).
bei Enzym-
4. Bergmeyer,
H. U., Grundlagen
der enzymatischen
Analyse,
In Methoden der Enzymatischen
Analyse,
2nd ed., H. U. Bergmeyer, Ed. Verlag Chemie,
Weinheim
1970, p 115.
5. German
Society
of Clin. Chem.,
Festlegung
einer einheitlichen
Messtemperatur
f#{252}r
Enzymaktivitatsbestimmungen
in
der klinischen
Chemie.
Z. Kim. Chem. Kim. Biochem. 9, 464
(1971),
6. Recommendations
of the German
Society
for Clinical
Chemistry. Standardization
of methods
for the estimation
of enzyme
activity
in biological
fluids. Z. Kim. Chem. Kim. Biochem. 8,
659 (1970). (In English).
7. Bergmeyer,
H. U., Bernt,
E., and Hess, B., Lactic
dehydrogenase.
In Methods
in Enzymatic
Analysis,
H. U. Bergmeyer,
Ed. Academic
Press, New York, N.Y., 1965, p 737.
8. Haug,
H.,
von Pharmaka
9. Elking,
laboratory
Dorloechter,
G., and
auf AutoAnalyzer-Tests.
Hermann,
G.,
Diagnostik
M. P., Kabat,
H. F., Drug
test values. Amer. J. Hosp.
induced
Pharm.
Der Einfluss
5, 85 (1972).
modifications
25, 485 (1968).
of
10. Christian,
D. G., Drug interference
with laboratory
blood
chemistry
determinations.
Amer. J. Clin. Pathol. 54, 118 (1970).
CLINICAL CHEMISTRY, Vol. 18, No. 11, 1972 1311