Absence of InsP6 and presence of inositol phosphate
phosphatase in human plasma. All the extracts were resolved by PAGE
and visualized with toluidine blue staining. (a)
EDTA was added to 20 ml of commercial human plasma from male (MP)
and female (FP); InsP6 was added to the spiked aliquot
(InsP6; 2 nmol). The samples were then acidified and
subjected to TiO2 enrichment. (b) Plasma
from healthy anonymous donors (D1 to D4) was prepared as described
in Material and methods, with spiking (InsP6; 1 nmol),
and subjected to TiO2 extraction. (c) 4
nmol of InsP6 was added to 1 ml of human plasma and
incubated at 37°C for the indicated time before acidification
and extraction of inositol phosphates with the TiO2
procedure. Standards: InsP6 (4 nmol); InsP5 (6
nmol of Ins(1,3,4,5,6)P5); InsP4 (5 nmol of
Ins(1,4,5,6)P4); InsP3 (20 nmol of
Ins(1,4,5)P3). (d) 4 nmol of
InsP6 was added to 1 ml of a different source of
human plasma (HP) and bovine plasma (BP) before incubation at
37°C for the indicated time, followed by acidification of the
samples and TiO2 extraction. The gels presented are
representative of experiments performed two to four times.