2.6 Start of Reaction with Serum {Activated ASAT)
Certain analyzers in routine use t oday cannot perform the re–
commended two-stage method. It is possible to activate sample
ASAT by incubation of serum sample s at an excess concentra-
tion of P-5'-P in the reagent TRIS buffer, e.g. 250 Jlmol/L
for lO minute s at room temperature prior to analys is ( 7
-
9,
o
17
-
20). Other authors recommend 20 minute s at 30
c
at a
concentration of 400 ymol/L of P-5'-P (18). Reactivation of
ALAT is fasterthan for ASAT (19). Thismay be done either by
adding the P-5'-P-TRIS solution to the sera or adding the
sufficient amount of P-5'-p in substance, e.g. included in
sample tubes, dosed into tubes, released from a stirring rod.
The activated sample is then added to the complete ASAT reac–
tian mixture. Achievement of eonstant reaction rates with
some sera may require 5 minutes or more (6
9). The NADH
consurning side reactions including that of LDH and pyruvate
do not start until sample has been added to the NADH contain–
ing reagent A. It must be realized that addition of P-5'-P to
sample increases the absorbance around 405 nm.
2.7 Minimum NADH Concentration for Measurement
During the incubation of sample with reagent A the endogenous
sample pyruvate-LDH reaction oxidizes NADH. At exceptionally
high sample pyruvate concentrations the remaining NADH
con–
centration in the complete reaction mixture may be decreased
to 80 pmol/L or less. If this occurs, the sample must be ap–
propriately diluted. At the conditions of the ECCLS Standard
procedure an absorbance value at 340 nm below 1.000 at the
start of monitoring of reaction rate indicates that the NADH
concentration is too low. (1).
Klinisk kem i
i
Norden 2: supp/, 1990
47
