2.11. Inosine monophosphate (IMP) measurement
Five different samples were used for the measurements. Differences
between groups were evaluated using a two-sample t test. High-speed
liquid chromatography was used for measuring IMP. We used a Shodex
Asahipak GS-320 HG column (φ7.6 mm × 300 mm, Showa Denko) at a
temperature of 30 °C. An 875-UV (JASCO) detector was used, and the
detection wavelength was 260 nm. As the mobile phase, we used
200 mM sodium dihydrogen phosphate (pH2.9) filtered through a
membrane filter (pore diameter 0.20 μm); its flow rate was 0.6ml/min.
A portion of each sample, 2.0 ± 0.3 g, was homogenized in 5 ml of
chilled 10% perchloric acid. Then, the solution was centrifuged at
16,000 g using a centrifuge (CF15R, Hitachi Koki). The supernatant
was decanted; 5ml of 10% perchloric acid was then added to the residue
and mixed well. This mixture was centrifuged for collecting the
supernatant. The supernatant and the 10% perchloric acid were mixed
to obtain a 25-ml supernatant diluent. One milliliter of the diluent was
neutralized using 10 M KOH and 1 M KOH, and the neutralized diluent
was centrifuged at 14,000 g. The supernatant was decanted, and 1 ml
of Milli-Q was added to the residue and mixed well; the mixture was
then centrifuged at 14,000 g, and the supernatant was collected. The
supernatant was centrifuged again and decanted, and then, Milli-Q
was added to the decanted supernatant to prepare 10 ml of test liquid.
The test liquid was filtered through a syringe filter unit (pore diameter
0.20 μm), and 20 μl of the test liquid was injected into the sample for
measurement. An autosampler (Model 09, System Instruments) was
used for injection.