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IFCC 1986/
1: Enzymes
, III. IFCC Method for
alanine aminotransferase
J. Clin. Chem. Clin. Biochem.
Vol.24, 1986, pp. 481-495
© 1986 Walter de Gruyter & Co.
Berlin New York
OFFPRINT
481
IFCC 1986!1
INTERNATIONAL FEDERATION OF CLINICAL CHEMISTRY (IFCC)1),2)
Scientific Committee, Analytical Section
Expert Panel on Enzymes3)
Approved Recommendation (1985) on IFCC Methods
for the Measurement of Catalytic Concentration of Enzymes
Part 3. IFCC Method for Alanine Aminotransferase
(L-Alanine: 2-Oxoglutarate Aminotransferase, EC 2.6.1.2)
Prepared for publication4) by
H. U. Bergmeyer, M. Harder
and
R. Rej
This paper is based on a Provisional Recommenda-
tion (1) which has been revised to take account of
the comments received, and has been accepted by the
Council of the International Federation of Clinical
Chemistry by maol ballot in June 1985.
This paper forms part of a series of recommendations
on measurements of catalytic concentrations of en-
zymes. Others deal with:
Part 1. General Conditions, (2)
Part 2. Method for Aspartate Aminotransferase, (3)
Part 4. Method for y-Glutamyltransferase, (4)
Part 5. Method for Alkaline Phosphatase, (5)
Part 6. Reference Materials for Enzyme Measure-
ments,
Part 7. Method for Creatine Kinase
Contents
1. Introduction
2. Principle
1) The exclusive 0 for all languages and countries is vested in
The International Federation of Clinical Chemistry.
2)
IFCC Sections reprinted in
J. Clin
.
Chem. Clin. Biochem
.
are listed in the Cumulative
Index, which appeared in connection with the contents of
this journal Volume
23, 1985.
Since 1986 have been printed in J. Clin. Chem. Clin. Bio-
chem.
IFCC
1986
/1 Vol. 24
,481-495
IFCC
1986
/2 Vol. 24,497 - 510
3. Optimal Conditions for Measurement
4. Instrumentation and Equipment
5. Reagents
6. Purity of Reagents
7. Preparation of Solutions
8. Stability of Solutions
9. Specimen Procurement, Transportation and Stor-
age
10. Measurement
11. Calculations
12. Analytical Variability
13. Reference Values
14. References
Appendix A: Description of Pertinent Factors in Ob-
taining Optimal Conditions for Mea-
surement
Appendix B: Reagent Specifications
Appendix C: A Comparison of the Catalytic Activi-
ties Calculated for Various Combina-
tions of the Two Substrates for Alanine
Aminotransferase
3)
4)
Expert Panel Members: R. Rej (US) (Chairman), M. Herder
(DK), M. Mathieu (FR), L. M. Shaw (US), J. H. Stremme
(NO).
Reprint requests
and
inquiries
may be addressed to:
Dr. Robert Rej
Wadsworth Center for Laboratories and Research
New York State Department of Health
Albany, NY 12201 (U.S.A.)
or to
Dr. Mogens Herder
Department of Clinical Chemistry
Odense University Hospital
DK-5000 Odense C.
Received for publication 1986-02-27
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
482 IFCC
1986
%
1: Enzymes
, III. IFCC Method
for alanine aminotransferase
1. Introduction
The principles applied in the selection of the condi-
tions of measurement are those stated in previous
publications by this expert panel (2). Serum has been
used as the source of enzyme. The selected concentra-
tions of substrates and indicator enzyme have been
calculated from rate equations applicable to the pri-
mary and indicator reactions and have been con-
firmed experimentally.
2. Principle
The recommended method for the measurement of
alanine aminotransferase activity in serum is based
on the principles outlined by
Wroblewski (6).
Modifi-
cations include optimization of substrate concentra-
tions, replacement of phosphate with tris(hydroxyme-
thyl)aminomethane as buffer and addition of pyri-
doxal phosphate.
The reaction catalyzed by alanine aminotransferase
(I) and the indicator reaction (II) are as follows:
L-Alanine +
2-Oxoglutarate
Pyruvate
4
Alanine
amino-
transfe
_ rase
Pyruvate +
L-Glutamate
Lactate
dehydrogenase')
(I)
(II)
+ NADH + H+ + NAD+
The equilibrium of the indicator reaction (II) lies far
to the right (7); therefore, the equilibrium of reaction
(I) is not relevant because pyruvate is continuously
consumed in the indicator reaction.
The catalytic activity
of alanine aminotransferase is
determined by measurements of the rate of NADH
oxidation
in reaction (II).
3. Optimal Conditions for Measurement
Optimized6) reaction conditions (2) for the assay of
alanine aminotransferase in serum from healthy
individuals and from patients with myocardial infarc-
tion or liver disease are shown in table 1.
Apoenzyme of alanine aminotransferase is reconsti-
tuted with pyridoxal phosphate prior to measure-
ment. During the period of preincubation the concen-
s) L-Lactate: NAD oxidoreductase EC 1.1.1.27
6) Optimized
reaction conditions
are defined
as those condi-
tions which are most
favorable for both
the kinetic reactions
and the technical aspects
of the
measurement
(
i. e., optimum
conditions do not necessarily provide maximum
activity).
L-Lactate
Tab. 1.
Optimized conditions for measurement.
Temperature
30,0 °C
pH (30 °C)
7,3
Tris(hydroxymethyl)aminomethane
100
L-Alanine
500
2-Oxoglutarate
15
Pyridoxal phosphate
0,10
NADH
0,18
Lactate dehydrogenase
20
Volume fraction of sample
0,083
mmol x 1-^
mmol x 1-^
mmol x 1-
mmol x 1-'
mmol x 1-'
µkat x 1-'
(1: 12)
Note: In the above, the unit mmol x 1_t implies the kind of
quantity substance concentration and the unit µkat x l1 the
catalytic concentration. Measurement of the catalytic concen-
tration of lactate dehydrogenase is described in Appendix B.
The concentrations apply to the complete reaction mixture
except for small increases due to the presence of substances
and enzymes in the specimen.
tration of pyridoxal phosphate in the reaction is 109
.tmol x 1-1 and the serum volume fraction is 1: 11
(0,0909). In addition, at this stages reactions of
NADH with substances in serum are allowed to reach
completion prior to the initiation of the alanine ami-
notransferase reaction by addition of 2-oxoglutarate.
4. Instrumentation and Equipment
Spectrometer suitable for accurate measurements at
339 nm with constant temperature cuvette compart-
ment. The specifications for the equipment (e. g. spec-
tral band width, light path, accuracy of temperature
control) should meet those of previous recommenda-
tions (2).
5. Reagents
1. Tris(hydroxymethyl)aminomethane,
C4H11N03,
Mr 121,14
2. L-Alanine and D-Alanine, free acids, C3H7N02,
Mr 89,1
3. 2-Oxoglutaric acid, C5H605, Mr 146,10
4. 13-Nicotinamide-adenine-dinucleotide, reduced
form, disodium salt, C21H27N7O14P2Na7, Mr 709,4,
5. L-Lactate: NAD oxidoreductase, EC 1.1.1.27,
Lactate dehydrogenase from pig skeletal muscle,
specific catalytic activity higher than 9 kat x kg
(30 °C), in glycerol, Reagent 8
6. Pyridoxal-5-phosphoric acid monohydrate, Pyri-
doxal phosphate C$H1006NP H2O,
Mr 265,2
7. Hydrochloric acid, HC1, Mr 36,47 (1 mol x 1-1)
8. Glycerol, C3Hg03, Mr 92,10 (volume fraction 0,5
in aqueous solution).
9. Sodium chloride, NaCl, Mr 58,45
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
IFCC 1986/1: Enzymes, III. IFCC Method for alanine aminotransferase 483
6. Purity of Reagents
The specifications regarding purity as given in 1. c.
(2) must be assured. 2-Oxoglutarate must be free of
any pyruvate and lactate. The purity of the pyridoxal
phosphate is estimated from the measurement of its
absorbance at 388 nm and at 25 °C in a solution of
NaOH, 0,1 mol x 1-1, (8). The purity must be higher
than 0.995 based on molar lineic absorbance of fully
ionized pyridoxal phosphate of 650 m2 x mol-1. The
contamination of lactate dehydrogenase with alanine
aminotransferase should not exeed 5 x 10-5 (kat/
kat). The relative contamination of lactate dehydro-
genase with glutamate dehydrogenase7) should not
exceed 3 x 10-5 (kat/kat). (Details in Appendix B).
IV. Reduced nicotinamide-adenine dinucleotide ((3-
NADH, 11,3 mmol x 1-1):
Dissolve 16,1 mg NADH, disodium salt or an amount
equivalent to this as determined by water of hydra-
tion in 2,0 ml of reagent II.
V. Lactate dehydrogenase (catalytic concentration
2,52 x 101, mkat x 1-1)8):
Mix the enzyme solution in glycerol according to
its catalytic concentration and adjust with glycerol,
volume fraction 0,5 (reagent 8) to give the above
indicated catalytic concentration.
VI. Reagent mixture for overall alanine aminotrans-
ferase reaction:
7. Preparation of Solutions .
To prevent the growth of micro-organisms in the
solutions, sterilized containers should be used. All
solutions should be prepared in calibrated flasks with
fresh deionized and distilled water of reagent-grade.
The pH of solutions must be adjusted at 30 °C (2)
with standardized reference buffers (e. g. IUPAC or
NBS).
1. Tris/L-alanine (Tris 110 mmol x 1-1, L-alanine
630 mmol x 1-1, pH 7,3, approx. chloride 100
mmol x 1-1):
Dissolve 1,33 g tris(hydroxymethyl)aminomethane
and 5.61 g L-alanine, free acid in 80 ml water, adjust
pH to 7.3 with HCl, 1 mol x 1-1 at 30 °C (approx.
10 ml), then adjust temperature to the calibration
temperature of the flask and make up to 100 ml with
water.
II. Tris/Hydrochloric acid buffer (Tris 110 mmol x
1-1, pH 7,3, chloride approx. 80 mmol x 1-1):
Dissolve 2,66 g tris(hydroxymethyl)aminomethane in
160 ml water, adjust pH to 7,3 with HCl, 1 mmol
x 1-1, then adjust temperature to the calibration
temperature of the flask and make up to 200 ml with
water.
III. Pyridoxal phosphate (6,3 mmol x 1-1):
Dissolve 16,7 mg pyridoxal phosphate in reagent II
and make up to 10,0 ml with reagent II.
Mix 100 ml tris/L-alanine solution (I) with 2 ml pyri-
doxal phosphate solution (III), 2 ml NADH-solution
(IV) and 1 ml enzyme solution (V).
VII. Reagent mixture for individual sample blank:
Dissolve 5,61 g D-alanine, free acid, in 50 ml tris/
hydrochloric acid buffer (II). Adjust pH to 7,3 at
30 °C and make up to 100 ml with reagent II.
Mix these 100 ml with 2 ml pyridoxal phosphate
solution (III), 2 ml NADH-solution (IV) and 1 ml
enzyme solution (V).
VIII. Tris/2-oxoglutarate (Tris 110 mmol x 1-1, 2-
oxoglutarate 180 mmol x 1-1) pH 7,3:
Dissolve 1,32 g Tris(hydroxymethyl)aminomethane
and 2,63 g 2-oxoglutaric acid in 80 ml of water. Adjust
pH to 7,3 at 30 C. Then adjust the temperature to
the calibration temperature of the flask and make up
to 100 ml with water.
IX. Sodium chloride (154 mmol x 1-1):
Dissolve 0,9 g sodium chloride in 100 ml water.
8. Stability of Solutions
All solutions should be stoppered and stored in a
refrigerator at 0 to 4 °C. Prepare the reagent mixtures
(solutions VI and VII) fresh each day. Prepare the
pyridoxal phosphate, the NADH, and the 2-oxoglu-
tarate (solutions III, IV and VIII) every two weeks.
7) L-Glutamate: NAD(P) oxidoreductase (deaminating). EC 8) Measurement of catalytic concentrations as described in
1.4.1.3 Appendix B.
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
484 IFCC 1986/
1: Enzymes
, III. IFCC Method
for alanine aminotransferase
The tris/L-alanine and tris/HC1 buffers and the indi-
cator enzyme (solutions I, II and V) are stable for at
least 6 months if bacterial contamination is pre-
vented.
Deterioration of these last three solutions due to
bacterial contamination can be prevented
by addition
of 8 mmol x 1-' sodium azide
(
8). Store solutions
III, IV, VI and VII
in dark bottles.
9. Specimen Procurement
,
Transportation and Stor-
age
Collect blood with minimal venous stasis. The use
of serum from freshly-collected unhaemolyzed blood
without visible haemolysis is preferred. Store serum
protected from light. Plasma is not recommended (cf.
Appendix A).
Alanine aminotransferase in serum remains stable at
+ 4 °C for a minimum of 3 days. Freezing of serum
is not recommended (10).
Tab. 2. Composition of the four
reaction
mixtures (A, B, C
and D) for the reaction
rate measurements
that consti-
tute one alanine aminotransferase
rate
measurement.
Kind of reaction rate
Sample
Reaction
Mixture
(Solution No.)
(A) Overall reaction
Serum
VI
(B) Reagent blank for
Solution IX
VI
overall reaction
(C) Overall sample blank
Serum
VII
(D) Reagent blank for
Solution IX
VII
sample blank
10. Measurement
10.1 Measurement conditions
Wavelength:
339 nm (± I nm)
Light path:
10,0 + 0,01 mm
Final volume of reaction mixture: 2,40 x
10-3 1
Temperature:
30,0 + 0,05 °C (thermostated cuvette
compartment)
10.2. Handling of solutions
Before pipetting, the temperature of reagent solutions
and of specimen must be brought to the calibration
temperature of the pipettes.
During the preincubation period the solution in the
cuvettes must attain a temperature of 30 °C before
initiation of the reaction by addition of solution VIII.
10.3. Subprocedures that constitute one
measurement
Four different reaction mixtures are necessary,
table 2. The starting solution is VIII for all four
reactions. All measurements are made against a cu-
vette containing water. The timing of the individual
steps of the measurements must be identical for the
overall reaction (A) and the various blank reactions
(B, C and D). This is described in table 3.
10.4 Measurement interval
A lag phase of up to 90 seconds may occur. Thereafter
the values of
(AA/At)
of the overall alanine amino-
transferase reaction are constant over a period of at
least 300 seconds for sera with catalytic concentra-
tions of alanine aminotransferase up to about 4 .tmol
Tab. 3. Analytical system for measurement of the overall alanine aminotransferase reaction rate.
Pipette
Volume
into the cuvettes:
(ml)
Reagent Mixture VI
2,000
Serum
0,200
Substance or catalytic concentrations in final complete reaction mixture
Tris(hydroxymethyl)aminomethane 100 mmol x 1-'
L-Alanine 500 mmol x 1-1
NADH 0,18 mmol x 1_1
Pyridoxal phosphate 0,10 mmol x 1-'
Lactate dehydrogenase 20 µkat x 1-1
Volume fraction 0,0833 (1:12) (1 + 11)
Mix and monitor the absorbance in a spectrophotometer at 30 °C for at least 600 s to ensure that a constant
AA/At
is attained
for each sample. During this preliminary period of incubation alanine aminotransferase is saturated with pyridoxal phosphate
(100 ttmol x 1-1) and NADH is oxidized due to the presence of pyruvate and other endogenous substrates in serum.
2-Oxoglutarate solution VIII 0,200
2-Oxoglutarate 15 mmol x 1-1
Mix and monitor change in absorbance for 90 s to eliminate any error due to the lag phase. Then monitor change in absorbance
continuously for at least 300 s.
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
IFCC 1986/1: Enzymes, III. IFCC Method
for alanine aminotransferase
x 1-1. If the value of
(AA/At)
is greater than 0,0025
s-1 or decreases during monitoring, then dilute the
sample 5 to 10 fold with sodium chloride, 154 mmol
x 1-1 (Solution IX) and repeat the measurement.
The period of observation of the blank rates should
be the same as for the overall reaction.
10.5 Corrections for blank reactions
Calculate the mean
(AA/At),
s-1 for the overall ala-
nine aminotransferase reaction
,
the individual sample
blank reaction
,
and the two corresponding reagent
blank reactions
.
The corrected
(A/A/At),
s-1 for ala-
nine aminotransferase is:
(AA/At
)
corrected
=
[(AA/At)A - (AA/At
)$]
- [(AA/At)c - (AA/At)
D],
5-1.
The subscripts, A, B, C and D indicate the composi-
tion of reaction mixtures referred to in table 2.
The corrected value of
(AA/At),
s-1 is used in the
following calculations. It corresponds to the initial
rate of conversion (mol x s-1) catalyzed by alanine
aminotransferase.
11. Calculations
From the following equation the catalytic concentra-
tion, b, is given by
b= V
x (AA/
At),
kat x 1-1
exlxv
where V is the reaction volume (1), v is the sample
volume (1), t is the reaction time (s), e is the molar
lineic absorbance (m2 • mol-1), and 1 is the pathlength
of cuvette (mm).
14. References
1. Bergmeyer, H. U. & Herder, M. (1980)
IFCC Methods for the Measurement of Catalytic Concen-
tration of Enzymes. Part 3. IFCC Method for Aaanine
Aminotransferase.
J. Clin. Chem. Clin. Biochem. 18, 521-534; Clin. Chim.
Acta
105,
147F-172F.
2. Bowers, Jr, G. N., Bergmeyer, H. U., Herder, M. & Moss,
D. W. (1979)
Approved Recommendation on IFCC Methods for the
Measurement of Catalytic Concentration of Enzymes.
Part 1. General Considerations Concerning the Determina-
tion of of the Catalytic Concentration of an Enzyme in the
Blood Serum or Plasma of Man.
Clin. Chim. Acta 98, 163F-174F; J. Clin. Chem. Clin.
Biochem. (1980)
18,
89-95.
3. Bergmeyer, H. U., Herder, M. & Rej, R. (1986)
485
The value for
the molar lineic
absorbance of NADH
under the
conditions
of this IFCC method is (11, 12):
E30 nm = 630 m2 x mol - 1
The calculation of the catalytic concentration b, for
measurements performed at max, at 339 nm, be-
comes:
2,4 x 10
b = x (AA/
At), kat x l'
630x10x0
,
2x10_3
= 1,905 x 10 x (AA/At), kat x 1-
Example: Measurement
at 339 nm,
(AA/At)
corrected
= 0,030/
60 s = 0
,0005 s-1
b= 1,905 x 10_3 x 0,0005 kat x 1-1
= 1,905 x 10_3 x 5 x 10_4 = 9,52 x 10-^ kat
x 1-1
= 0,95 µkat x 1-1
12. Analytical Variability
A within-run relative standard deviation of less than
0,02 was obtained at catalytic concentration of 0,50
tkat x 1-1 (n = 20). In a study of between-labora-
tories variation using lyophilized human serum (13),
relative standard deviations were 0,065 and 0,035 for
isolated between-laboratories variation and average
within-laboratory variation, respectively. The cata-
lytic concentration was 0,40 µkat x 1-1, the number
of participating laboratories was 5.
13. Reference Values
Preliminary reference interval limits are 0,08 to 0,58
µkat x 1-1 for healthy, young adults.
Approved Recommendation (1985) on IFCC Methods for
the Measurement of Catalytic Concentration of Enzymes.
Part 2. IFCC Method for Aspartate Aminotransferase.
J. Clin. Chem. Clin. Biochem. 24, 497 - 510.
4. Shaw, L. M., Stremme, J. H., London, J. L. & Theodorsen,
L. (1983)
IFCC Methods for the Measurement of Catalytic Concen-
trations of Enzymes. Part 4. IFCC Method for 7-Glutamyl-
transferase.
J. Clin. Chem. Clin. Biochem. 21, 633-646; Clin. Chim.
Acta
135,
315F-338F.
5. Tietz, N. W., Rinker, A. D. & Shaw, L. M. (1983)
IFCC Methods for the Measurement of Catalytic Concen-
tration of Enzymes, Part 5. IFCC Method for Alkaline
Phosphatase.
J. Clin. Chem. Clin. Biochem. 21, 731-748; Clin. Chim.
Acta
135,
339F-367F.
J. Clin
.
Chem
.
Clin. Biochem
. / Vol. 24,
1986 / No. 7
486
IFCC 1986/1: Enzymes, III. IFCC Method for alanine aminotransferase
6. Wroblewski, F. & La Due, J. S. (1956)
Serum Glutamic Pyruvic Transaminase in Cardiac and He-
patic Disease.
Proc. Soc. Exp. Biol. Med.
91, 569-571,
7. Hakala, M. T., Glaid, A. J. & Schwert, G. W. (1956)
Lactic Dehydrogenase II. Variation of Kinetic and Equili-
brium Constants with Temperature.
J. Biol. Chem. 221, 191-209.
8. Peterson, E. A. & Sober, H. A. (1954)
Preparation of Crystalline Phosphorylated Derivatives of
Vitamin B6.
J. Amer. Chem. Soc. 76, 169-175.
9. Rej, R. & Vanderlinde, R. E. (1975)
Azide as a Preservative in Assays of Aspartate Aminotrans-
ferase Activity.
Clin. Chem. 21, 158-161.
APPROVED RECOMMENDATION (1985)
10. Cuccherini, B., Nussbaum, S. J., Seeff, L. B., Lukacs, L. &
Zimmermann, H. J. (1983)
Stability of aspartate aminotransferase and alanine amino-
transferase activities.
J. Lab. Clin. Med.
102,
370 - 376.
It. Horecker, B. L. & Kornberg, A. (1948)
The Extinction Coefficients of the Reduced Band of Pyri-
dine Nucleotides.
J. Biol. Chem.
175,
385-390.
12. Ziegenhorn, J., Senn, M. & Bucher, T. (1976)
Molar Absorptivities of (3-NADH and 13-NADPH.
Clin. Chem. 22, 151-160.
13. Bowers, G. N., Alvarez, R., Cali, J. P., Eberhardt, K. R.,
Reeder, D. J., Schaffer, R., Uriano, G. A., Elser, R., Ewen,
L. M., McComb, R. B., Rej, R. & Shaw, L. M. (1983)
The measurement of the catalytic (activity) concentration
of seven enzymes in NBS human serum SRM 909.
National Bureau of Standards Special Publication 260-83.
U. S. Government Printing Office, Washington: 1983.
IFCC Method for Alanine Aminotransferase
Appendix A
Description of pertinent factors in obtaining optimal conditions for measurements
1. Introduction
Optimum pH and substrate concentrations as well as
optimum conditions for the indicator reaction have
been investigated by several authors (1- 5) using
different buffers at different temperatures. However,
efforts to obtain optimized and standardized condi-
tions for measuring the catalytic activity of alanine
aminotransferase in human sera have been intensified
during the present decade (6 -10).
In the following, confirmed data for these complex
relationships at 30 °C are considered (8-10). The
sources of alanine aminotransferase employed were
pools of human sera from patients with heart or
liver diseases respectively, according to a previous
recommendation by this panel (11). These are re-
ferred to as "heart enzyme" and "liver enzyme".
For some experiments serum from individual patients
were used. Pyridoxal-5-phosphate was added to the
reagent mixture (Reagent VI and VII) to ensure satur-
ation of the apo-enzyme with this co-enzyme.
2. Buffer
Catalytic activity of an enzyme and optimal concen-
trations of the substrates are dependent in the type
of buffer, ionic strength and pH. Similarly, optimal
pH is dependent on the type of buffer and ionic
strength. The pK of the buffer should be such that
adequate buffering capacity is available at the opti-
mal pH. Optimum catalytic activity is found in the
interval between pH 7,1 and 7,9 depending on the
type of buffer and substrate concentration. Therefore
only a few buffers merit consideration (11). Phos-
phate was excluded because it inhibits the recombina-
tion of the apo-enzyme with pyridoxal phosphate
(fig. 1). The buffers TES and HEPES described by
Good (12)
would satisfy the requirements with respect
to pK but they were also excluded, since they possess
no advantages over triethanolamine and especially
tris(hydroxymethyl)aminomethane. The usefulness of
the last two mentioned buffers has been carefully
investigated (8, 10). In Tris buffer, the catalytic activ-
ity of alanine aminotransferase was 3% ('liver en-
zyme') to 10% ('heart enzyme') higher than in tri-
ethanolamine buffer, and the sample blank was 14%
and 28% resp. lower. Therefore, Tris buffer is recom-
mended.
The optimal pH for alanine aminotransferase in
human serum lies close to pH 7,3 (fig. 2) although
the dependence of catalytic activity on pH close to
this value is of minor importance. Therefore, pH 7,3
is recommended. The fact that pH 7,3 is not close to
the pK (30 °C) 7,94 of Tris is overcome by recom-
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
IFCC 1986/1: Enzymes, III. IFCC Method for alanine aminotransferase
ZO 40 60 SO
Inorganic phosphate,
Substance concentration Em mol • 1-11
Fig. 1. The effect of inorganic phosphate on the recombination
of pyridoxal phosphate and apo-enzyme of alanine ami-
notransferase. The source of enzyme was pooled human
serum.
mending a buffer concentration of 100 mmol x 1-1.
The buffering capacity of Tris at this pH has been
examined both theoretically and experimentally using
patient sera. The complete oxidation of NADH in
the assay results in an increase in the concentration
by 0,18 mmol x 1-1 and a pH change of less than
0,01 pH unit. Addition of an acidified patient speci-
men (pH 5,62) resulted in no significant change in
the assay pH.
3. Effects
of pyridoxal phosphate
The dissociation of pyridoxal phosphate from and
the recombination with the apo-enzyme of alanine
aminotransferase is different from that of aspartate
aminotransferase. In contrast to the latter enzyme,
alanine aminotransferase is unstable after removal of
the pyridoxal phosphate. It is, however, difficult to
dissociate pyridoxal phosphate from the enzyme e. g.
by dialysis, since the binding is strong (10). Neverthe-
less, increased catalytic activities of alanine amino-
transferase result after supplementation of serum
with pyridoxal phosphate; the average fractional in-
crease of the catalytic activity in serum from healthy
individuals in 1,2-fold. The activation effect is smaller
than for aspartate aminotransferase where the aver-
age increase is 1,4-fold.
The effects of pyridoxal phosphate concentration on
alanine aminotransferase were investigated in serum
of patients with heart and liver disease, at catalytic
concentrations of 1500 nkat x 1-1 and 2500 nkat x
1-1, (fig. 3). Phosphate inhibits the recombination of
7,3
pH
7,0
7,6
200 500 800
1-Alanine,Substance concentration [mmotxl-11
Fig. 2. Confirmation of reaction conditions by response surface
optimization:
The sources of enzyme were pools of human serum. 2-
Oxoglutarate and L-alanine were varied as shown; L-
alanine was at 500 mmol x 1-1 (a), and 2-oxoglutarate
was at 15 mmol x 1-' (b). Areas of constant catalytic
activity are shown as a percentage of maximum catalytic
concentration.
(Reproduced by permission from the A1aAT study
group of the American Association of Clinical Chem-
istry).
the apo-enzyme with pyridoxal phosphate in all buf-
fers investigated (fig. 1) (8, 10). In Tris buffer contain-
ing the reagents except for 2-oxoglutarate necessary
for the assay of the final catalytic activity, it is pos-
sible at 30 °C to reach complete saturation within
480 s to 600 s (8 to 10 min) at a final concentration
of 115 µmol/l (fig. 3). The activation of individual
sera is shown in figure 4. The pyridoxal phosphate
concentration in the preincubation mixture should be
109 µmol x 1-1 (fig. 4).
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
488 IFCC 1986/
1: Enzymes
, III. IFCC Method
for alanine aminotransferase
"fiver enzyme"
1 _.
200 400 600 800 200 400 600 800
Period of incubation with pyridoxat phosphate prior to measurement Esl
Fig. 3. The effect of the period of incubation with pyridoxal phosphate (• 40 µmol x 1-'; o 80 µmol x 1-'; x 160 µmol x
1-1, substance concentration in final reaction mixture) on the measured catalytic concentration of alanine aminotransferase.
The source of the enzymes were pools of human sera from patients with heart and liver diseases (`heart enzyme' and
`liver enzyme').
The ordinate values are the ratio of the alanine aminotransferase concentration measured with pyridoxal phosphate
compared to the enzyme concentration of the sample measured without pyridoxal phosphate (= ratio 1,0).
Pyrid000l-5-phosphate,
Substance concentration ( µmol • I ' )
Fig. 4. The effects of pyridoxal phosphate (0-200 smol x
L1), during 600 s of preincubation on the measured
catalytic concentration of alanine aminotransferase.
The sources of the enzymes were sera from 14 patients
with heart or liver diseases. The ordinate values are the
fraction of the alanine aminotransferase concentration
measured with pyridoxal phosphate compared to the
enzyme concentration of the sample measured without
pyridoxal phosphate (= ratio 1,0).
4. Effects of other substances
Chloride, added as sodium chloride or hydrochloric
acid, inhibits the catalytic activity of the enzyme
significantly in concentrations of more than 100
mmol x 1-1 at optimum pH (10), but this concentra-
tion is not reached in the assay system described here.
A slight but significant activation of alanine amino-
transferase by hydrogen carbonate was found (10).
Sodium hydrogen carbonate concentrations between
10 and 20 mmol x 1-1 activate alanine aminotrans-
ferase in these sera about 3%. The effect is diminished
to zero at a sodium hydrogen carbonate concentra-
tion of 80 mmol x 1-1. More than 80 mmol x 1-1
inhibits the `heart enzyme', and more than 25 mmol
x 1-1 inhibits the `liver enzyme'. The slight activation
does not justify inclusion of hydrogen carbonate.
5. Kind of specimen and volume fraction of sample
((p)
Plasma obtained by addition to whole blood of 9,2
g/1 sodium heparin may cause turbidity of the reagent
mixture and should not be used. The method is in-
tended for serum.
At an infinite dilution of the sample the potential side
reactions that will results in departure from absolute
specificity of the method will be reduced to a mini-
mum. However, at a certain minimal value of the
volume fraction of sample (cp) the measurement signal
will approach the noise level of the instrumentation
used. The volume fraction of sample (cp) must be a
compromise between these two opposite effects.
A slight dependence of catalytic concentration on
volume fraction may exist only for the `liver enzyme'
(fig. 5) (10). The cause of this effect may be the
presence of enzyme inhibitor(s) in the serum. A sam-
ple volume fraction of 0,0833 for sera with a catalytic
concentration of approximately 300 nkat x 1-1 re-
sults in
(AA/At)
of about 0,010 (60 s)-1 at wave-
lengths of 334 or 339 nm.
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
IFCC 1986/
1: Enzymes
, III. IFCC Method
for alanine aminotransferase
"heart enzyme"
0,167 0,125 0,100 0,083 0,071
Volume
traction `P
Fig. 5. Relationship between the catalytic concentration of al-
anine aminotransferase and the volume fraction of
serum (cp). The sources of the enzymes were pools of
human sera from patients with heart and liver diseases
(`heart enzyme' and `liver enzyme').
6. Substrate concentrations
In Tris buffer (100 mmol x 1-1; pH 7,5) optimum
substance concentrations of 2-oxoglutarate and L-
alanine (fig. 6) have previously been determined em-
pirically for the enzyme in serum from patients with
liver and heart diseases (9).
However, the selection of optimum substrate concen-
trations can be made less empirical by calculations
based on rate equation for the reaction catalyzed by
alanine aminotransferase (9, 10).
The reaction catalyzed by alanine aminotransferase
follows a two substrate, ping-pong bi-bi mechanism
(13, 14). The rate equation for this mechanism is less
complex than for an ordered or random mechanism
(14, 15). Values of Km determined (9, 10) in this assay
system under the reaction conditions selected for this
method are shown in table 1. Km (not Ks) is used for
the calculation to reaction rate (16). Km values of
table 1 were estimated from the procedures by
Flo-
rini & Vestling (17).
The catalytic concentration of
the serum samples from patients with liver and heart
diseases were measured at 30 'C, in Tris buffer, 100
mmol x 1-1; pH 7,5; pyridoxal phosphate 100.tmol
x 1-1.
The highest possible (theoretical) value for the cata-
lytic activity of the primary reaction in the system
described here, Vpr;m, is reached if for both substrates,
L-alanine and 2-oxoglutarate, [S] >> Km. If one tol-
erates a certain deviation of the measured catalytic
activity, v; from V, it is possible to calculate a distinct
substrate concentration (9, 10) that ensures the par-
ticular v, be obtained, since [S] _ (v;
x Km)/(V - v;)
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
b
{
2000
oy
a=
o-
0
o .o
o
a '=
0
0
u
000
3 6 9 12 15 18 21 24
2-Oxoglutarate. Substance concentration fmmolxl ]
ver enzyme
"heart enzyme"
100 200 300 400 500 600 700 800
a l. - Alanine
,
Substance concentration
(
mmol • 1-1
l
489
Fig. 6. Dependence of the catalytic concentration of alanine
aminotransferase on substance concentrations
of L-al-
anine and 2-oxoglutarate
.
The sources of enzyme were
pools of human sera from patients with heart and liver
diseases
(`
heart enzyme
'
and `liver enzyme').
Tab. 1.
Michaelis
and inhibitor constants (mmol x 1-1).
Parameter Source of enzyme in pool of serum
Heart Liver
(K0)5 (L-alanine
)*
26,5
± 2,3
21,9
± 2,5
(K0)5' (2-oxoglutarate
)*
0,73
± 0,
03
0,69
± 0,02
(Kr)2-oxoglutarate
**
334
- 387
347
- 442
(KI)gtutamate
**
26,5
- 27.2
13,2
- 14.2
* For Km values: mean -i- 2 SD (mmol x 1-1)
** For K1 values: range of three measurements (mmol x l-')
= F X Km. This expression has been used for the
calculation of the substrate concentrations. For two-
substrate enzyme reactions no uniquely optimum
condition with respect to substrate concentrations
exists. Instead, there is an infinite number of concen-
tration pairs (9, 10) whose interrelationship can be
expressed by a hyperbola for each particular value
of F.
Alanine aminotransferase is subject to substrate and
product inhibition. From all possibilities of inhibition
which might occur theoretically (13) one can for
practical purposes neglect all but
490 IFCC 1986/1: Enzymes, III. IFCC Method for alanine aminotransferase
(a) the competitive inhibition by 2-oxoglutarate (S2)
against L-alanine (S1) on the pyridoxal phosphate
form of the enzyme, and
(b) the competitive inhibition by L-glutamate (P2)
against (S1). In addition, the alanine aminotransferase
reaction is inhibited noncompetitively by pyruvate
(P1) against L-alanine (13, 18). However, this inhibi-
tory effect is small, because pyruvate is removed by
the indicator reaction and the steady state concentra-
tion of Ps thus is small. If the activity of the primary
enzyme is low and the reaction period short, the
accumulation of L-glutamate will be minimal. Within
a reaction time of 300 s and a serum containing 5 µkat
x 1-1 of alanine aminotransferase the concentration
of L-glutamate will be aprox. 0.125 mmol x 1-1.
This is less than I% of the
K1
value for L-glutamate.
Thus, for practical purposes the only inhibition by 2-
oxoglutarate into account, the interaction between
[S1] and [S2] for a given fraction of the maximum
velocity is expressed by a parabola (fig. 7).
Three points have to be considered in the choice of
the pair of substrate concentrations:
a) The concentration of each should be the lowest
possible
(
most economical),
b) The concentrations should be such that a slight
deviation of one of them from the normal value does
not alter the other too much
(
lowest variability),
c) The concentrations should be most practical (with
respect to e. g. light absorbance at the wavelength of
measurement
,
solubility
,
and inhibitory effects).
Therefore
,
the substance concentration of 2-oxoglu-
tarate was set to be lower than 20 mmol x 1-1 taking
its light absorbance into account
.
With 15 mmol x
1-1 for 2-oxoglutarate the substance concentration
for L-
alanine is calculated by the relationship
In the formula above, no consideration is given to
the known product inhibition by L-glutamate and
pyruvate. These effects can be neglected for reaction
periods of up to 300 s and in a coupled reaction
where the steady state concentration of pyruvate is
small (10).
50 100
2-OxogIutarate,
Substance
concentration t mmol - I -' 1
Fig. 7.
Interrelationships
(
calculated
)
of substance concentra-
tions of substrates according to
[S1] =
(Km)s
, x 1 +
1 (Km)S2
F [S
2]
X + [S2]
(K
)
[S2]
m
s,
v
K
[S
] =
,
`
F =
K1
i
;
I (Km)s2 V- vi
F [S2]
in which S1 is L-alanine; S2 is 2-oxoglutarate;
K1
is
inhibitor constant for competitive inhibition by 2-
oxoglutarate; (Km)s, is the
Michaelis
constant with
respect to L-alanine; (Km)s2 is the
Michaelis
constant
with respect to 2-oxoglutarate. F = 10,11 corre-
sponding to v; = 0,91 x V. The fraction is the highest
practical fraction obtainable. Numerical values for
the kinetic parameters are chosen of table 1.
The dotted and broken lines indicate the 95% confi-
dence interval (± S. D.). The results of an experiment
(using serum from a patient with liver disease) is shown
as three points. This assesses the validity of the equa-
tion. Vi = 0,91 V was used. Abbreviations used are:
[S2], substance concentration of 2-oxoglutarate; [I], sub-
stance concentration of competitive inhibitor; (Km),,,
Michaelis
constant of alanine aminotransferase with
respect to L-alanine; (Km)s2,Michaelis
constant of ala-
nine aminotransferase with respect to 2-oxoglutarate;
K1, inhibitor constant of alanine aminotransferase with
respect to 2-oxoglutarate, (numerical values see table 1,
page 489).
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
IFCC 1986/
1: Enzymes
, III. IFCC
Method for alanine aminotransferase
15
Tab. 2. Selected pairs of substrate concentrations for the measurement of L-alanine-aminotransferase. The limits denote a 95%
confidence interval. The resulting initial reaction rate vi will be 0,91 x V.
Kind of substrate Source of enzyme in serum
"Liver"
Substrate concentration
,
mmol x 1-1
Lower limit
L-Alanine, S,
390
2-Oxoglutarate, S2
-
Recommended concentrations:
Mean
"Heart"
Substrate concentration, mmol x 1-1
Upper limit Lower limit Mean
430 461 522 550
15
L-alanine: 500 mmol x 1-1 (Corresponds to v1 = 0,916 x V for liver enzyme
2-oxoglutarate: 15 mmol x 1-1 and vi = 0,906 x V for heart enzyme.)
The results of calculations are summarized in table 2.
The recommended pair of substrate concentrations is
indicated by arrows in figure 7. The chosen values
are located between the parabolas for the two en-
zymes. This is a compromise. Calculating the corre-
sponding value of F, the result is vi = 0,916 x V for
the `liver enzyme' and vi = 0,906 x V for the `heart
enzyme'. That means that, in practice, with the indi-
cated pair of substrate concentrations the reaction
rate of vi = 0,91 x V is measured irrespective of
whether the main part of alanine aminotransferase in
serum originates from liver or heart.
7. Indicator reaction
The indicator enzyme is lactate dehydrogenase which
is also necessary to achieve complete reduction of
endogenous pyruvate of the serum specimen by
NADH.
The relationship between the time lag needed to re-
duce various concentrations of pyruvate added with
the sample, and the catalytic concentration of lactate
dehydrogenase is similar to that in the assay system
for aspartate aminotransferase (11). A catalytic con-
centration of lactate dehydrogenase of 0,6 µkat x
1-1 will allow for complete reduction, within 10 min
(600 s), of pyruvate concentrations higher than ten
times the mean concentration in serum.
The catalytic concentration, b, of lactate dehydroge-
nase needed as indicator enzyme is shown in figure 8.
It can be calculated more precisely, (9, 10) from the
kinetic parameters that are applicable to this assay
Upper li
572
to make the term 1 + (^P )n, x [S3] much greater
IN
than (Km)s3, so that
(Veff.)ind. =
1 +
(Km)Pi
+
(Km)S3
[P1]
[S3]
Vnd. X
[S3]
(Veff.)ind. =
(Km)P,
X [
S3] + (Km)s3
[P1]
Vind.
(Km)P,
[P1]
In steady state [P1] is constant, therefore 1 + (Km)/
[P1] is also constant. Vind, represents k x [E] accord-
ing to
Michaelis-Menten;
thus this rate of the indi-
cator reaction is constant (i. e. the progress is linear)
and reflects the rate of the reaction.
2
000 I
1 500
E
E
systems. The effective velocity of the indicator reac-
tion depends on the steady state concentration [P1]
of one of the two products of the primary reaction,
that is, pyruvate. The second substrate of this two-
substrate reaction is NADH. It has been shown (10)
that the commonly used substance concentrations [S3]
of NADH in dehydrogenase reactions are sufficient
10 000 20 000
Indicator enzyme, Catalytic concentration n kat . 1
491
T
30000
Fig. 8. The dependence of the catalytic concentration of ala-
nine aminotransferase on the catalytic concentration of
lactate dehydrogenase in the reaction mixture.
1 000 -I
500 -I
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
492
IFCC 1986/
1: Enzymes
, III. IFCC Method
for alanine aminotransferase
To achieve a uniform reaction rate for the coupled
reaction in the steady state
(Veff.)
ind.
must equal
(vi)p
rim,
which is 0,91 x
Vpri
Therefore
nd.
= 1 + (Km)
p
, x 0,91 x
Vprim.'
1
in which [Pi
]
is the unknown steady state concentra-
tion of pyruvate and (Km
)
p, is the
Michaelis
constant
of lactate dehydrogenase for pyruvate.
(Km)pl is 0.16 mmol x 1-1 (10). The value [Pi] is
determined by the acceptable duration of the lag
phase of the coupled reaction
.
To keep this short,
[Pt] was set to 0,003 mmol x 1-1, corresponding to
a lag phase during which A becomes 0,021 at 339
nm. This corresponds to a period of approx
.
90 s for
sera with normal alanine aminotransferase and 7 s for
sera containing the highest catalytic concentrations
measurable with this method.
V ind.
= 1
+
0,16 X 0,91 x Vrim_ = 49,44 x
Vprim.
0,005
If the catalytic concentration of alanine aminotrans-
ferase in the assay system is limited to b = 400 nkat
x 1-1 (corresponding to b = 4,800 nkat x 1-1 in the
serum sample),
Vprim, = 0,4 p.mol x s-1 x 1-1 and
Vind. = 49,44 x 0,4 = 19,78.tmol x s-1 x 1-1.
The suggested catalytic concentration of the indicator
enzyme lactate dehydrogenase is b = 20 pkat x 1-1.
8. References
1. Henley, K. S. & Pollard, H. M. (1955)
A New Method for the Determination of Glutamic Oxal-
acetic and Glutamic Pyruvic Transaminase in Plasma.
J. Lab. Clin. Med. 46, 785-789.
2. Wroblewski, F. & La Due, J. S. (1956)
Serum Glutamic Pyruvic Transaminase in Cardiac and He-
patic Disease.
Proc. Soc. Exp. Biol. Med. 91, 569 - 571.
3. Laursen, T. & Hansen, P. F. (1958)
A Fluorimetric Method for Measuring the Activity in
Serum of the Enzyme Glutamic Pyruvic Transaminase.
Scand. J. Clin. Lab. Invest.
10,
53 - 58.
4. Bergmeyer, H. U. & Bernt, E. (1963)
Glutamate-Pyruvate Transaminase, Optimum Method, In:
Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.)
1st ed. pp. 850-851, Academic Press, New York.
5. Schmidt, E. & Schmidt, F. W. (1968)
Optimale Bedingungen zur Bestimmung der Transaminasen
and der Kreatin-Phosphokinase im Serum and ihre Konse-
quenzen fur die Diagnostik.
Z. Analyt. Chem. 243, 398-414.
The concentration of NADH (0,18 mmol x 1-1) is
sufficient to support the optimal reaction rate (fig. 9),
as well as to ensure the reduction during the preincu-
bation period of concentrations of pyruvate likely to
occur in the majority of sera.
Excessive oxidation of NADH during the preincuba-
tion period due to exceptionally high concentrations
of endogenous pyruvate may occur. This can result
in a decrease in absorbance during preincubation of
more than 0,600 thus reducing the concentration of
NADH to less than 100 µmol x 1-1. If this occurs,
and also if the absorbance at the end of the period
of preincubation is less than 1,000, the serum speci-
men must be diluted with sodium chloride, 154 mmol
x 1-1, and the measurement repeated.
2 00
1500
00
50
0,05 0,10 0,15 0,20 0,25
NAOH, Substance concentration ( mmot • I-' I
Fig. 9
.
The dependence of the catalytic concentration of alan-
ine aminotransferase on the concentration
of NADH.
6. Deutsche Ges. Klin. Chem.: Empfehlungen der Deutschen
Gesellschaft fur Klinische Chemie, Standardisierung von
Methoden zur Bestimmung von Enzymaktivitaten in biolo-
gischen Fhissigkeiten. Experimentelle Begriindung der opti-
mierten Standard-Bedingungen
J. Clin. Chem. Clin. Biochem. (1972),
10,
182-192.
7. Scandinavian Committee on Enzymes: `Recommended
Methods for the Determination for Enzymes in Blood'
(1974)
Scand. J. Clin. Lab. Invest.
33,
291-306.
8. Commission Enzymologie (1978) Societe Francaise de Bio-
logic Clinique. Recommandation pour la determination
dans le serum humain de la concentration catalytique de
l'alanine aminotransferase a 30 °C
Ann. Biol. Clin. 36, 457-462.
9. Bergmeyer, H. U. (1977)
Evaluation of Optimum Conditions of Two-Substrate En-
zyme Reactions.
J. Clin. Chem. Clin. Biochem.
15,
405-410.
J. Clin
.
Chem
.
Clin. Biochem
. / Vol. 24,
1986 / No. 7
IFCC 1986/
1: Enzymes
, III. IFCC Method
for alanine aminotransferase
10. Bergmeyer, H. U., Scheibe, P. & Wahlefeld, A. W. (1978)
Optimization of Methods for Aspartate Aminotransferase
and Alanine Aminotransferase.
Clin. Chem. 24, 58 - 73.
11. IFCC Expert Panel on Enzymes (1976)
Provisional Recommendations on IFCC Methods for the
Measurement of Catalytic Concentrations of Enzymes.
Clin. Chim. Acta 70, F19-F42; J. Clin. Chem. Clin. Bio-
chem.
15
(1977) 39-51; Clin. Chem.
23 (1977)
887-899.
12. Good, N. E., Winget, G. D., Winter, W., Conolly, T. N.,
Izawa, S. & Singh, R. M. M. (1966)
Hydrogen Ion Buffers for Biological Research.
Biochemistry 5, 467-477.
13. Henson, C: P. & Cleland, W. W. (1964)
Kinetic Studies of Glutamic Oxalacetic Transaminase Iso-
enzymes.
Biochemistry 3, 338 - 345.
APPROVED RECOMMENDATION (1985)
493
14. Velick, S. F. & Vavra, J. (1962)
A Kinetic and Equilibrium Analysis of the Glutamic Oxal-
acetate Transaminase Mechanism.
J. Biol. Chem. 237, 2109-2122.
15. Cleland, W. W. (1963)
The Kinetics of Enzyme-Catalyzed Reactions with Two or
More Substrates or Products. I. Nomenclature and Rate
Equations.
Biochim. Biophys. Acta 67, 104-137.
16. Enzyme Nomenclature (1972) p. 29, Elsevier, Amsterdam.
17. Florin, J. R. & Vestling, C. S. (1957)
Graphical Determination of the Dissociation Constants for
Two-Substrates Enzyme Systems.
Biochim. Biophys. Acta 25, 575-578.
18. Saier, M. H. & Jenkins, W. T. (1967)
Alanine Aminotransferase, I. Purification and Properties.
J. Biol. Chem. 242, 91-100.
IFCC Method for Alanine Aminotransferase
Appendix B
Reagent specifications
1. Measurement of the catalytic concentration of the
reagent enzyme
I.I. Catalytic concentration of the reagent
enzyme
The measurement conditions (temperature, pH, type
of buffer, substance concentrations of buffers, ions,
substrates and cofactors) correspond to those applied
for the assay of alanine aminotransferase in this
IFCC method.
Conditions for measurement of lactate dehydrogenase (EC
1.1.1.27)
Temperature
30,0 °C
pH (30 °C)
7,3
Tris (hydroxymethyl)
aminomethane
100
L-Alanine
500
Pyridoxal phosphate
0,10
NADH
0,18
Pyruvate
3,0
Volume fraction of sample
0,083
1.3. Reagents
1.2. Preparation of samples from stock re-
agent enzyme
Dilute the reagent enzyme from stock solution no. V,
lactate dehydrogenase from skeletal muscle (e. g. pig
skeletal muscle), specific catalytic concentration
higher than 9000 nkat x mg-1. Prepare two samples
from the stock solution with catalytic concentrations
of lactate dehydrogenase of approx. 5 and 10 µkat
x 1-1. For making dilutions use sodium chloride,
154 mmol x 1-1 containing albumin 10 g x 1-1.
mmol x 1-'
mmol x 1-1
mmol x 1-1
mmol x 1-1
mmol x 1-1
(1:12)
(1 + 11)
In making up the reagents for this measurement,
proceed as described for alanine aminotransferase in
the main except for the exclusion of lactate dehydro-
genase from solution VI. Also replace reagent solu-
tion VIII 2-oxoglutarate with sodium pyruvate, 36
mmol x 1-1 and use this solution as starting reagent.
1.4. Measurement procedures
Use the same equipment as for the measurement
of alanine aminotransferase. Determine the catalytic
J. Clin. Chem. Clin. Biochem. / Vol. 24, 1986 / No. 7
494
IFCC 1986/1: Enzymes, III. IFCC Method
for alanine aminotransferase
reaction in the two samples of the diluted stock
solution and in the reagent blank, in which the sample
is solution IX, sodium chloride.
Analytical system
Pipette
Vol-
into the
ume
cuvettes
(ml)
Reagent
mixture VI
(without
lactate
dehydro-
genase)
2,000
Sample
0,200
Substance or catalytic concentrations
in final complete reaction mixture
Tris
(hydroxy-
methyl)
aminomethane 100 mmol x 1-1
L-Alanine 500 mmol x 1-1
NADH 0,18 mmol x 1-1
Pyridoxal 0,10 mmol x 1-1
phosphate
Volume fraction 0,0833
Mix, and ensure temperature equilibrium at 30 °C, then add:
Sodium 0,200 Sodium 3,0 mmol x 1-1
pyruvate pyruvate
36 mmol x 1-1 (VIII)
Register the change in absorbance immediately after adding
pyruvate and mixing. Use the change in absorbance
(AA/At)
in the time period of 20 - 60 s corrected for the reagent blank
rate when calculation the catalytic concentration, b1, of lactate
dehydrogenase in the sample:
b1 = 1,905 x 10-3 x
(AA/At),
kat x I-
2. Measurement of contaminant enzymes in the re-
agent enzyme
Two potential contaminant enzymes of the reagent
enzyme lactate dehydrogenase are glutamate dehy-
drogenase (NAD(P+)), (EC 1.4.1.3) and alanine ami-
notransferase (EC 2.6.1.2).
2.1. L-Alanine aminotransferase (EC 2.6.1.2):
The method is identical to the method described in
the main document, therefore the rate of conversion
rate of the reagent blank (B) for the overall reaction
can be used in calculating the catalytic concentration
of alanine aminotransferase of the reaction mixture,
b2
b2 =
1,905 x 10-3
x (AA/At)
x 0,083, kat x 1-1
The relative contamination of the reagent enzyme,
lactate dehydrogenase is the ratio between the cata-
lytic concentration of alanine aminotransferase of the
reaction mixture, b2, and the catalytic concentration
of lactate dehydrogenase, 20 tkat x 1-1, i. e. b2/(20
x 10-6). This should not exceed 5 x 10-5 (kat/kat).
2.2. Glutamate dehydrogenase
(EC 1.4.1.3):
Temperature 30,0 °C
pH (30 °C) 7,3
Tris(hydroxymethyl)aminomethane 100 mmol x 1-
NADH 0,18 mmol x 1-1
Lactate dehydrogenase 20 µkat x 1-1
NH4 100 mmol x 1-1
Volume fraction of sample (sol IX) 0,0833 (1:12)
2-Oxoglutarate 7 mmol x 1-1
Preincubate at 30 °C the above reagent mixture (total volume
2,2 ml) containing all reagents except for 2-oxoglutarate. Start
the reaction by adding 2-oxoglutarate, sot. VIII, 0,2 ml. Ob-
serve that a lag-phase of 120 s may occur. Correct the observed
rate of conversion with the rate of conversion of a mixture
containing all reagents except for NW. The catalytic concen-
tration of glutamate dehydrogenase of the reaction mixture is:
bz =
1,905 x 10-3
x (AA/At)
x 0,0833, kat x 1-1.
The ratio between the catalytic concentration of glu-
tamate dehydrogenase, b3, and the concentration of
lactate dehydrogenase of the reaction mixture, 20
.tkat x 1-1, is the relative contamination of the
reagent enzyme. It should not exceed 3 x 10-5 (kat/
kat).
3. Method
for checking the presence of L-alanine in
D-alanine
The specimen blank activity measurement requires
use of D-alanine rather than the active L-alanine.
Although use of this reagent provides for measure-
ment of an ideal specimen blank reaction, a small
contamination of this reagent with L-alanine will
result in an inappropriate correction. This contamina-
tion may be assessed by the following protocol:
A solution is prepared in aqueous glycerol (volume
fraction 0,5 - reagent B) that contains alanine ami-
notransferase of porcine skeletal muscle at a catalytic
concentration higher than 1,6 mkat x 1-1. The cata-
lytic activity of this solution may be assessed by
determining the overall alanine aminotransferase re-
action using dilutions of this solution with reagent B.
Follow subprocedures C and D outlined in Method
Section 10.3 using the above enzyme solution in sub-
procedure C. Immediately upon addition of solution
VIII in these measurements, record the absorbance
at 339 nm. Monitor change in absorbance until rate
of change for both reaction mixtures becomes equal
(this may take several hours and is dependent upon
the degree of contamination with L-alanine). Record
absorbance at 339 nm for both mixtures. Subtract
final absorbance from initial absorbance for each
mixture (An ria1- Afinal). Subtract the absorbance
change for mixture D from that found for mixture C
(AA = AAc - AAD).
This change in absorbance is
due to L-alanine. The concentration of L-alanine is
calculated:
J. Clin. Chem
. Clin. Biochem
. / Vol. 24, 1986 / No. 7
IFCC 1986/
1: Enzymes,
III. IFCC
Method for alanine aminotransferase
[L-alanine] = Ale x l
where e and l are defined as above (Section 11). For
measurements using 10 mm path length cuvettes the'
calculation is:
L-Alanine (mol x 1-1)
= AA x
1,587 x 10-4
Using the above procedure a typical preparation of
D-alanine was found to contain a fractional portion
APPROVED RECOMMENDATION (1985)
495
of L-alanine of 2,6 x 10-s; the concentration of L-
alanine in reaction mixture C is thus 0,013 mmol x
1-1. At the upper limit of linearity of the procedure
(ca 4 µkat x 1-1) this concentration supports no
true alanine aminotransferase activity. At catalytic
concentrations of 25 pkat x 1_1 the true alanine
aminotransferase measured in the reagent blank
measurement is < 20 nkat x 1-1.
IFCC Method for Alanine Aminotransferase
Appendix C
A Comparison of the Catalytic Activities
Calculated
for Various
Combinations
of the Two Substrates for
Alanine Aminotransferase
Appendix A describes how the pair of substrate con-
centrations selected will yield 0,91 of the theoretical,
maximal velocity obtainable under the defined condi-
tions of temperature, buffer, pH, cofactors etc. It has
been demon: :ted also that a constant fraction of
the maxii_:__' :c -y may be obtained for other com-
binations of su >strate concentrations. Such pairs of
substrates lie on a parabola, see figure 8, Appendix A.
For the purpose of comparison, v/ V has been calcu-
lated from the rate equation (page 490 of Appendix
A) and using the numerical values of the kinetic
constants described in table I of Appendix A for dif-
ferent substrate pair combinations. It is thus possible
from the two tables to find pairs of substrate that
will yield the identical initial velocity or alternatively
to find out the change in vi that will result from
the increase or decrease in one or both substrate
concentrations. It should be stressed that the values
of the tables are valid only for the system under
consideration i. e. alanine aminotransferase in human
serum obtained from patients with liver (tab. 1) and
heart (tab. 2) diseases. It is valid also only for mea-
surements performed under conditions of this method
except for the variation in substrate concentrations.
Tab. 1.
Nomogram showing the concentration of L-alanine [S11
as a function of 2-oxoglutarate
[
S2], and the fractional,
maximal velocity
[v1/l]
of alanine aminotransferase*).
v;/V
0,87
0,88
0,89
0,90
0,91
0,92
0,93
0,94
0,95
[S21
5
-
-
-
-
- -
- -
6
646
-
-
- -
-
-
-
7
438
590
-
-
-
-
-
-
8
354
446
598
- -
-
-
-
9
308
375
477
650
-
-
-
-
10
279
333
411
533
751
-
- -
11
260
306
370
465
622
- -
-
12
245
286
341
421 545
766
-
-
13
235
272
321
390
493 667
-
-
14
226
260
305
367
457
602
- -
15
220
251
293
349
430
555
776
-
16
214
244
283
335
408
520 709
-
17
210
238
275
324
392
492
658
-
18
206
233
268
315
378
471
620
898
19
203
229
263
307 367
453
589
834
20
200
226
258
300
357
438
564
784
(1268)
*) The values refer to human serum containing alanine amino-
transferase from patients with liver diseases. The equation
from which the nomogram was constructed and the values
of the kinetic constants are described in Appendix A (p 490
and tab. 1). If no concentration of [S1] is given, the values
calculated are above the limit of solubility of S1.
Tab. 2.
Nomogram showing the concentration of L-alanine [S1]
is a function of 2-oxoglutarate
[
S2], and the fractional,
maximal velocity
[v,/ V] of
alanine aminotransferase*).
v;/V 0,87
0,88
0,89
0,90
0,91
0,92 0,93
0,94 0,95
[S
21
5
-
-
-
-
-
-
6
971
-
-
- -
-
7
598
842
-
-
-
-
-
-
8
466
601
837
- - - -
-
9
398
492
639
905
- - -
-
10
356
530
538
715
-
-
-
-
11
329
390
477 610
839
-
-
-
12
309
363
436
545
719
- -
-
13
294
342
407
500
642
891
- -
14
283 327
385
467
589
791
- -
15
274
315 368
442
550
721
-
-
16
267
305
355
423 520
670
934
-
17
261
297
344
407
496
630
858
-
18
256
290
335
394
477
600
802
(1195)
19
251
285
328
384
461 575
757
(1098)
20
248
280
321
375
448
554
721
(1023)
*) The values refer to human serum containing alanine amino-
transferase from patients with heart diseases. The equation
from which the nomogram was constructed and the values
of the kinetic constants are described in Appendix A (p 490
and tab. 2). If no concentration of [S1 is given, the values
calculated are above the limit of solubility of S1.
J. Clin. Chem. Clin. Biochem. / Vol. 24,1986 / No. 7
