Table I. Concentrations apply to the complete reaction mixtu–
re and are specified at 37 °C, where conversion rates and
Michaelis eonstants are higher, to ensure that maximal ASAT
catalytic activity will be supported at both 37 °c and 30 °c.
A buffer adjusted to pH 7.8 0 (30 °C) may well be used. The
catalytic activity concentration of the rnalate and lactate
dehydrogenase have been
de,ter~ined
as d.5scribed in the IFCC
ASAT Reference Method at 30 C and 37 C. The average tempe–
rature conversion factors are found to be 1,50 for Malate
dehydrogenase and 1.35 for Lactate dehydrogenase (17). The
values in Table I earrespond to those obtained at 37 °c.
Stage A. Incubation-activation reagent A.
volume fraction in: incubation-reac–
tivation stage : 0.91
complete reaction
mixture
0.833
Tris(hydroxymethyl}aminomethane
buffer pH 7.65
L-Aspartate
80 mmol/L
240 mmol/L
Exa.mple of
volume
(pL}
NADH
180 JllilOl/L
500 JlL
Pyridoxal-5#-phosphate
Malate dehydrogenase (600 U/L)
>
Lactate dehydrogenase (900 U/L)
>
Add Sample.
100
~umol./L
10 Jlkat/L
15 )lkat/L
Volume fraction in reagent A:
Volume fraction in complete mixture
0.0909
50
pL
0.0833
Mix carefully. During an incubation period of
300 s the Apo-ASAT enzyme proteins become satu–
rated with pyridoxal-5'-phosphate, the LDH-NADH
reaction with sample pyruvate goes to comple–
tion, and the reaction temperature has to be
reached.
Stage B. Substrate start with reagent B:
j2-oxoglutarate.
jVolume fraction:
0.0833
2-0xoglutarate
12 :mmol/L
Mix carefully. After a lag-phase of up to 60 s a
eonstant rate is achieved that is monitored for
the minimum time and
n~mber
of data points to
obtain an acceptable precision around the upper
reference limit.
50
pL
44
Klinisk kemi
i
Norden 2: suppl, 1990
