Table I. Concentrations apply to the complete reaction mixtu–
re and are specified at 37 °C, where conversion rates and Mi–
chaelis eonstants are higher, to ensure that maximal ALAT ca–
talytic activity will be supported at both 37 °C and 30 °c .
A buffer adjusted to pH 7.30 (30 °C) may well be used. The
catalytic activity concentration of lactate deh ydrogenase has
been determined as described in the IFCC ALAT
Reference Me–
thod at 30 °C and 37 °C. The average temperature conversion
factor 37 °c/ 30 °c was found to be 1.35 (17). The value in
.
.
o
Table I earresponds to that
obta~ned
at 37 C.
Stage A Incubation-activation reagent A.
Volume fraction in:
incubation-reactivation stage
0.91
complete reaction mixture
0.833
Example of
volume {pL)
Tris(hydroxymethyl)aminomethane
100 mmol/L
buffer pH 7.15
L-alanine
500 mmol/L
500
pL
NADH
180 )llilOl/L
Pyridoxal-5'-phosphate
100 jllilOl/L
Lactate dehydrogenase {1700 U/L)
28 Jlkat/L
Add sample
50
JIL
Volume fraction in reagent A
0.0909
Volume fraction in complete mixture
0.0833
Mix carefully. During an incubation period of
300 s the Apo-ALAT enzyme proteins become sa tu-
rated with pyridoxal-5'-phosphate, the LDH-NADH
reaction value to sample pyruvate goes to c om-
pletion, and the reaction temperature has to be
reached.
stage B. Substrate start with reagent B:
2-oxoglutarate.
Volume fraction:
0.083
50
JIL
2-0xoglutarate
12 mmol/L
Mix carefully. Af ter a lag-phase of up to 60 s a
eonstant rate is achieved that is monitored for
the minimum time and number of data points to
obtain an acceptable precision arou nd the upper
reference limit.
60
Klinisk kemi
i
Norden 2: supp/, 1990
