Do mammals make all their own inositol hexakisphosphate?

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Inositol hexakisphosphate (InsP6) is a crucial signaling molecule.
• Accurate quantification of InsP6 in biological samples is essential for understanding its cellular roles.
• Previous methods for InsP6 measurement may lack specificity or sensitivity.

Purpose of the Study:

• To characterize a highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6).
• To quantify InsP6 levels in various biological samples using the developed assay.
• To evaluate the endogenous synthesis of InsP6 in mammalian cells and its presence in human bodily fluids.

Main Methods:

• Development and characterization of a mass assay involving [32P]ATP phosphorylation of InsP6 using a recombinant kinase.
• Separation and quantification of 32P-labeled products via High-Performance Liquid Chromatography (HPLC) with a [3H]InsP7 internal standard.
• Analysis of InsP6 concentrations in rat tissues, Dictyostelium discoideum, HeLa cells, human blood (white cells, serum, plasma), and urine.

Main Results:

• The assay demonstrated high specificity and sensitivity for InsP6 quantification.
• InsP6 concentrations varied across tissues, with Dictyostelium discoideum showing significantly higher levels (352 µM) than rat tissues (10-20 µM).
• HeLa cells synthesized InsP6 endogenously (1.952 ± 0.117 nmol/dish), independent of serum content. InsP6 was detected in human white blood cells but not in serum, plasma, or urine (<1-5 nM).

Conclusions:

• Mammalian cells possess the capability to synthesize their own InsP6.
• The absence of detectable InsP6 in human serum, plasma, and urine suggests that previous dietary studies may have misidentified the analyte.
• Claims regarding health benefits from dietary or topical InsP6 application, based on extracellular levels, require reassessment due to the lack of endogenous detection in these fluids.