Plasma protein binding of TTX was determined
using an equilibrium dialysis unit (Sanplatec corp.,
Tokyo, Japan). Approximately 25mLblood/kg
body weight was collected from a puffer fish on
the day of study. The blood was centrifuged at
1200g for 10 min and the plasma was collected. The
dialysis membrane (MWCO 3500, Spectrum
Laboratories, CA, USA) was rinsed twice in MilliQ
water, soaked in modified Hank’s balanced salt
solution (pH 7.4) for 30 min and set into the dialysis
unit to separate compartments of a dialysis chamber.
A mixture of 50 mL TTX solution (020 ng)
and 900 mL puffer fish plasma was put into one side
of the chamber (termed as the plasma sample), and
950 mL of buffer was put into the counter side of the
chamber (termed as the buffer sample). Plasma
protein binding of TTX was assessed after incubation
for 24 h at 20 1C. The plasma and buffer
samples were collected from each side of the