Do mammals make all their own inositol hexakisphosphate?

A highly specific and sensitive mass assay for inositol hexakis-phosphate (InsP₆) was characterized. This centres around phosphorylating InsP₆ with [³²P]ATP using a recombinant InsP₆ kinase from Giardia lambia, followed by HPLC of the ³²P-labelled products with an internal [³H]InsP₇ standard. This assay was used to quantify InsP₆ levels in a variety of biological samples. Concentrations of InsP₆ in rat tissues varied from 10-20 μM (assuming 64%of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352 ± 11 μM InsP₆. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP₆ levels per mg of protein similar to rat tissues. This amounted to 1.952 ± 0.117 nmol InsP₆ per culture dish, despite the cells being grown in serum shown to contain no detectable (less than 20 pmol per dish) InsP₆. These results demonstrate that mammalian cells synthesize all their own InsP₆. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP₆ was detected (<1 nM InsP₆). Human urine was also examined, and also contained no detectable (<5 nM) InsP₆. These results suggest that dietary studies purporting to measure InsP₆ at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP₆ in the diet or applying it topically can produce health benefits by increasing extracellular InsP₆ levels may require reassessment.