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performed as quickly as possible and then placed in the holder for
analysis. The reaction was monitored from 20 to 120 s after enzyme
addition, and the slope was calculated from 20 to 40 s using the
diode array software.
tified directly from the generated standard curve. A total of ten
fication of UCB, BMG1, BMG2, BDG and their isomers were based
on their lipophilicity and polarity, as well as the elution pat-
tern, chromatographic peak position and relative retention time
from previous reports [21–23]. The calibration curves for bilirubin
were used to determine the concentration of the mono- and di-
glucuronide species employing the gradient HPLC bilirubin method
described above. Quantification of UDPGA levels was determined
through the use of the constructed standard curve within the iso-
cratic HPLC method developed for UDGPA.
Bilirubin Glucuronide Formation: Bilirubin glucuronidation was
performed at 37 ◦C in a shaking water bath. All steps taken
were performed in the lowest light conditions possible; the glu-
curonide formed was found to be unstable to ambient lighting. The
following was added to an Eppendorf tube to achieve the final con-
centrations indicated, final volume 200 L: potassium phosphate
buffer (50 mM, pH 7.4), bilirubin (10 M), MgCl2·6H2O (0.88 mM),
rat liver microsomes (RLM, 100 g of protein/mL), alamethicin
(22 g/mL), and allowed to pre-incubated for 2 min. Addition of
UDPGA (3.5 mM), referred to as the zero-time point, initiated the
reaction. The mixture was allowed to shake at 37 ◦C for each of
the time course experiments. To each reaction 600 L of ice-cold
methanol containing 200 mM ascorbic acid was added to terminate
the enzymatic reaction, vortexed for 2 min, and then centrifuged at
12,000 rpm for 10 min. The supernatant was then analyzed by the
developed gradient HPLC protocol for separation and quantification
of UCB, BMG1, BMG2, and BDGs.
Inhibitor Assessment – Saturating NAD+ varying UDPG concentra-
tion: For each analysis, the cuvette was prepared in the same fashion
as outlined above. The final concentration of the components of
the mixture were 150 mM Gly-Gly, 0.1 unit/mL UDPGDH, 3 mM
NAD+ and varied concentrations of 0.1, 0.05, 0.025 and 0.02 mM of
UDPG, obtained from stock solution addition. Nanopure water was
used as a variable component to ensure that a final volume of 1 mL
was obtained. Inhibitor analysis of the four purines was performed
at two concentrations: 50 and 100 M for 6TP, 20 and 50 M for
6TX and 8OH-6TP, and 5 and 10 M for 6TU, obtained from their
corresponding stock solutions. Each assessment was performed in
triplicate. The average of the three were plotted and the slopes were
used to determined inhibition values.
Inhibitor Assessment − Saturating UDPG varying NAD+ concen-
tration: In an analogous protocol as described for NAD+ saturating
conditions (above), inhibitor analysis of UDPGDH was performed
under UDPG saturating conditions (0.6 mM) with varying concen-
trations of NAD+.
2.4. UDP-glucuronosyltransferase activity assay
Standard preparation: A bilirubin stock solution was prepared
by dissolving bilirubin in 100% dimethyl sulfoxide to yield a con-
centration of 2 mM, the stock solution was aliquoted, and stored
at −70 ◦C until use. A 25 mM UDPGA stock solution was prepared
by diluting 8 mg to 0.5 mL with nanopure water, and a 10 mg/mL
alamethicin solution was prepared by taking 5 mg and diluting to
500 L with methanol. Preparation of the 100 mM potassium dihy-
drogen phosphate buffer was done by dissolving 2.3 g of KH2PO4
into 80 mL of nanopure water, pH adjusted to 7.4 with 1 M HCl and
diluted to volume in a 100-mL volumetric flask.
Validation of Bilirubin Glucuronide Formation: Quantification of
UCB, BMG1&2 and BDGs were performed post the quenching of
UGT1A1, which was performed by immersing the Eppendorf tube
with the reaction mixture in a cold-water bath for two min. No
ascorbic acid was used, as the residual material would quench the
glucuronidase enzyme to be added. To this sample 0.1 mg/mL glu-
curonidase enzyme was added, inverted (x3), and then analyzed by
the HPLC protocol developed to quantify the levels of bilirubin and
BMG1&2 and BDGs for formation confirmation.
2.4.1. Chromatographic conditions – bilirubin and bilirubin
glucuronide
Bilirubin and its glucuronide were separated on a Discovery C18
analytical column, 4.5 mm x 100 mm, 5 M particle size with guard
column. A dual mobile phase was employed; the aqueous phase
consisted of an 8 mM imidazole & 2.5 mM tetrabutylammonium
hydrogen sulfate (TBAHS) buffer at a pH of 6.5 in nanopure water
and acetonitrile as the organic phase. A gradient elution profile
was employed for full separation at a flow rate of 0.5 mL/min, the
method begins at 10% acetonitrile and increases to 50% over 8 min,
held for 5.5 min, increased to 95% over 4.5 min, held for 10 min,
returned to 10% over 4 min and held at 10% for 2 min to allow
for column regeneration. The detection wavelength was 450 nm
with a sample injection volume of 5 L. The combined peak area
for bilirubin (sum of the three isomers) was plotted relative to the
concentration prepared for the generation of a working standard
curve.
Chromatographic Conditions – UDPGA: Chromatographic sepa-
rations were performed on a Discovery C18 analytical column,
4.5 mm × 100 mm, 5 m particle size (Supelco) with a guard col-
umn. UDPGA was separated under isocratic conditions, flow rate
at 0.5 mL/min using a mobile phase comprised of 40% methanol
and 60% buffer that was composed of 8 mM imidazole and 5 mM
TBAHS at a pH of 6.5. The detection wavelength was 262 nm with a
sample injection volume of 5 L. Various concentrations of UDGPA
were analyzed, and peak areas obtained were plotted relative to
said concentrations to generate a working standard curve.
Quantification of Bilirubin, Mono/Di-glucuronide, and UDPGA Lev-
els: Standard curves for both bilirubin and UDPGA were constructed
and used for the quantification of each species. Bilirubin was quan-
Inhibitor Assessment of Bilirubin Glucuronide Formation: Employ-
ing the same protocol delineated above for the formation of the
bilirubin glucuronide species, inhibitor assessment was performed.
To the Eppendorf tube, 6-thiopurine or 6-thiouric acid (50 and
75 M final concentrations) was added alongside a control (no
purine added) and allowed to pre-incubate for 2 min. Addition of
UDPGA initiated the reaction for each of the time course experi-
ments. The gradient HPLC method was employed for the 45-min
time course experiments for the quantification of the glucuronide
species. For experiments in which UDPGA was analyzed, the incu-
bation protocol for the formation was altered as follows: 300 L
final volume, 2.5 M of bilirubin, 260 M of UDPGA was employed
to start the reaction, no alamethicin, and the enzyme was quenched
with heat (87 ◦C). Quantification of UDPGA for the 1, 12, and 15-
h time course experiments was performed by the isocratic HPLC
method described above.
From
commercially
available
4,5-diamino-6-
hydroxypyrimidine, thiol installation about the C6 position
was accomplished under standard employed protocols with
Lawesson’s reagent [29] in a 43% yield. Following a Traube syn-
thesis protocol [30], 4,5-diamino-6-thiopyrimidine was heated
with urea in muffle furnace until the mixture underwent a
molting process. The reaction was worked up under acid-base
conditions followed by recrystallization to afford the desired
8OH-6-thiopurine in a 65% yield. The product matched reported