Figure 4.

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.