1.1 ECCLS Measurement Procedure:
o
o
Substrate start, 37 C and 30 C
The component concentrations in the final reaction mixture
are the same as those of the
IFCC ALAT Reference Method
(2).
o
The standard reaction conditions have been defined at 37 C in
Table l below. The reaction system performs equally well at
o
o
30
C. Optimum pH (30 C) for ALAT in serum lies in the in-
terval 7.1 to 7.9, depending on the type of buffer with an
optimum for TRIS-HCl buffer at 7.3 (2, 10 b).
The pH of the TRIS-HCl buffer decreases 0.15 for an increase
o
o
in temperature from 30 C to 37
C (9). Consequently,
o
the
same reaction system, adjusted to 7.30 at 30 C will change
o
to a pH of 7.15
0.02 at 37
c.
However, due to the broad pH
optimum described in the IFCC ALAT Reference Method (2, lOb)
this shift from optimum pH does not decrease the ALAT
cata~.y
tic activity concentration by more than about 1%. Consequent–
ly, for routine use, the same TRIS-HCl buffer adjusted to pH
o
7.30 (30
C) will support the reaction with no significant
loss at either reaction temperature.
The ECCLS standard Procedure involves two stages (Table l).
In the first, the sample is incubated with
reagent A,
which
contains pyridoxal-5'-phosphate but not 2-oxoglutarate. Apo–
ALAT is reconstituted during this phase, and the LDH-cataly–
zed NADH reaction with sample pyruvate reaches completion.
In the seeond stage, the ALAT catalyzed reaction is initiated
by addition of 2-oxoglutarate
(reagent B).
Klinisk kemi
i
Norden 2: supp/, 1990
59
