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 ASAT Reference Method. The
o
stancard 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 ASAT in serum lies between pH
7. 6 and 8.
O
(l, l
O) •
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, the same
o
reaction system, adjusted to 7.80 at 30 C, will change to a
o
pH of 7.65
±
0.02 at 37
c.
However, due to the broad pH op-
timum described in the IFCC ASAT Reference Method (1, 10),
this shift from optimum pH does not decrease the ASAT cataly–
tic activity concentration by more than about l%. Consequent–
ly, for routine use the same TRIS-HCl buffer adjusted to pH
o
7.80 (30
C) will support the reaction with no significant
loss at either reaction temperature.
The ECCLS standard Procedure invalves two stages (Table l).
In the first, the sample is incubated with reagent A, which
contains pyridoxal-5'-phosphate but not 2-oxoglutarate. Apo–
ASAT
is reconstituted during this phase, and the LDH-cataly–
zed NADH reaction with sample pyruvate reaches completion.
In the seeond stage, the ASAT-catalyzed reaction is initiated
by addition of 2-oxoglutarate (reagent B).
Klinisk kemi
i
Norden 2: supp/, 1990
43
