Chemistry Reference
In-Depth Information
of L-lactate to pyruvate with simultaneous reduction of nicotinamide adenine
dinucleotide (NAD
+
). One mole of NAD
+
is converted to one mole of NADH for
each mole (equivalent) of lactate present.
LDH
Pyruvate
NAD
þ
!
H
þ
L-lactate
þ
þ
NADH
þ
Hydrazine is used to trap the pyruvate (hydrazone) as it is formed, thus driving
the reaction to completion. The absorbance due to NADH is directly proportional to
the concentration of lactate and is measured using a two-
lter (340
383 nm) end
-
point technique. Reagents included NAD
+
(2.0
µ
M, tablet, wells 1, 2), dihydrazine
sulphate (3.7
M, liquid, wells 3, 4), LDH (40 U, liquid, sourced from rabbit, wells
5, 6) and tris buffer (0.24 mM, liquid, wells 7, 8). Hydrating, diluting and mixing
were automatically performed by the Dimension
®
System. Volumes of sample,
reagent 1, 2, 3, 4 and diluent volume were 4, 158, 20, 75, 20, and 197
µ
µ
L,
respectively. Experiments were carried out at 37
°
C. The assay range was
0.3
15.0 mmol/L and calibration scheme was based on 3 levels (n = 3). Calibrator
concentrations were 0, 8 and 15 mmol/L. The assigned coef
-
cients were
C
0
=
1.156 and C
1
= 0.0451. Two levels of a quality control material with known
lactic acid concentration were analysed without dilution.
The fructose assay was based on the enzymatic conversion of free fructose to
−
ʲ
cally converted to a product that reacted with
10-acetyl-3,7-dihydroxyphenoxazine (H
2
0
2
probe, OxiRed
™
) to generate a colour
(
-glucose, which was then speci
C and protected from light. The assay
buffer was allowed to warm up to room temperature (24
ʻ
= 570 nm) [
58
]. The kit was stored at
−
20
°
°
C) before use. All small
vials were brie
y centrifuged prior to opening. Phosphoglucose isomerase (EC
5.3.1.9, fructose converting enzyme) was only stable in ammonium sulphate
((NH
4
)
2
SO
4
) solution. A required amount (450
fl
L for each
well) was centrifuged for 5 min at 10,000 rpm. The supernatant was removed and
reconstituted with same volume of assay buffer. The enzyme mix was dissolved in
220
µ
l) for each assay (10
µ
L) were added into a 96-well
plate. Fructose standard solution (100 mM) was diluted to 1 mM by adding 10
µ
L assay buffer separately. The samples (50
µ
µ
L
of fructose standard to 990
L of assay buffer and mixed. The standard solution
(1 mM) was serially diluted 1:2 (v/v) in assay buffer to give standard solutions (500,
250, 125
µ
mol/L), which were added in a series of wells. Enough reagent was
mixed for the number of assays to be performed. For each well, reaction mix (50
µ
µ
L), OxiRed
™
Probe (2
L) contained assay buffer (36
µ
µ
L), enzyme mix (2
µ
L)
and fructose converting enzyme (10
µ
L). The solution was mixed by a vortex
mixer. Reaction mix (50
L) was added to each well containing fructose standard
and test samples. The resulting solution was mixed on the orbital plate shaker. The
reaction was incubated for 30 min at 37
µ
C and protected from light using Al foil.
Optical density (OD)
550 nm
was measured for the colorimetric assay in a microplate
reader. Due to the OxiRed
™
probe, glucose generates background, which was
subtracted by conducting a control without phosphoglucose isomerase in the
reaction. The background was corrected by subtracting the value from the zero-
°
