2.6 Start of Reaction with Serum (Activated ALAT)
Certain analyzers in routine use today cannot perform the re–
commended two-stage method. It is possible to activate sample
ALAT by incubation of serum samples at an excess concentra–
tion of P-5'-P in the reagent TRIS buffer, e.g. 250 Fmol/L
for 10 minutes at room temperature prior to analysis (7 - 9,
o
17
20). Other authors recommend 20 minutes at 30 C at a
concentration of 400pmol/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 inta tubes, released from a stirring rod.
The activated sample is then added to the complete ALAT reac–
tian mixture. Achievement of eonstant reaction rates with
same 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 at around 405 nrn.
2.7 Minimum NADH Concentration
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 BOpmol / 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 nrn below 1.000 at the
start of rnanitaring of reaction rate indicates that the NADH
concentration is too low. (l).
Klinisk kemi
i
Norden 2: suppl, 1990
63
