Simultaneous analysis of saturated and unsaturated oxylipins in 'ex vivo' cultured peripheral blood mononuclear cells and neutrophils.

Oxylipins are a family of saturated and unsaturated fatty acids peroxidation products with bioactive properties. We have developed an improved method for the measurement of ex vivo oxylipin production by peripheral blood mononuclear cells (PBMCs) and neutrophils. We aimed to develop an analytical method to determine the production rates of polyunsaturated fatty acids (PUFAs), PUFA-oxylipin, and saturated-oxylipins by stimulated PBMCs and neutrophils based on solid phase extraction and HPLC-MS/MS technology. A UHPLC system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer was used to identify and quantify oxylipin production. For each oxylipin and PUFA their differential response was calculated with respect to a deuterated internal standard factor (ISF). To calculate oxylipin and PUFAs in the culture samples, the individual ISF was used for each oxylipin and PUFA with respect to the deuterated internal standard. PBMCs and neutrophils showed a different pattern of oxylipin production and fatty acid secretion. Lipopolysaccharide (LPS) did not stimulate oxylipin production or fatty acids secretion in PBMCs, whereas phorbol myristate acetate (PMA) stimulation increased the production rate of 5-HETE, 15-HETE, 15-HEPE, 17-DoHE, PGE2, AA, and DHA. LPS stimulation decreased 16-hydroxyl-palmitatte (16-OHPAL) production and DHA secretion in neutrophils, while PMA stimulation increased the production rate of AA and its derivate oxylipins, 5-HETE, 15-HETE, and PGE2. In conclusion, we have developed a new method to determine oxylipins derived from both saturated and unsaturated fatty acids in culture cell media. This method has enough sensitivity, and accuracy, to determine oxylipin production and fatty acid secretion by PBMCs and neutrophils.

Intake of myo-inositol hexaphosphate and urinary excretion of inositol phosphates in Wistar rats: Gavage vs. oral administration with sugar.

OBJECTIVE: To evaluate the urinary levels of inositol phosphates (InsPs) in rats that received different salts of myo-inositol hexaphosphate (InsP6) by gavage or by oral administration. METHODS: Thirty rats received AIN-76A diet (in which InsPs are undetectable) for 15 days. Then, 12 rats received InsP6 by gavage as a Na salt or a Ca/Mg salt; after 4 days, the Na or Ca/Mg InsP6 was administered with water containing 15 g/L sucrose and urine samples were collected. The other 18 rats received oral InsP6, in which 0.5 g of sugar was combined with InsP6 as a Na salt, a Ca/Mg salt, or a Na salt with CaCO3; daily urine samples were collected. Urine levels of InsPs were determined using a nonspecific method and a specific method (polyacrylamide gel electrophoresis, PAGE), and different InsPs were identified by mass spectroscopy (MS). RESULTS: After 15 days of the InsP6-free diet, the non-specific method detected no urinary InsPs, and MS detected only InsP2. After administration of Na-InsP6 by gavage, the non-specific method indicated more urinary InsPs than the amount of InsP6 determined by PAGE. MS indicated the presence of urinary InsP2, InsP3, InsP4, InsP5, and InsP6 in these rats, with notable variations among animals. Use of the same treatment to administer Ca/Mg-InsP6 led to a lower overall content of urinary InsPs and a lower level of InsP6. Oral administration of InsP6 as a sugar pill led to lower urinary levels of InsPs than administration of InsP6 by gavage, and administration as a Ca/Mg pill or a Ca/Mg pill with CaCO3 led to lower levels than administration as a Na pill. CONCLUSION: Administration of InsP6 to rats leads to the excretion of a mixture of different InsPs. Rats more effectively absorb InsP6 when supplied without dietary components that interfere with its uptake, such as the Ca ion and sugar.

Evaluation of inositol phosphates in urine after topical administration of myo-inositol hexaphosphate to female Wistar rats.

AIMS: Previous studies demonstrated a remarkable increase of urinary InsP6 by topical administration. However, the methodology used for InsP6 analysis was not specific. The aim of this paper is to measure urinary inositol phosphates InsPs using more advanced methodologies and to compare the results with those obtained by the non-specific method. MATERIALS AND METHODS: We fed 12 female rats with a diet without InsP6 for 16days. Then, we administered a topical InsP6 gel at high doses for 7days (50mgInsP6/day) or at low doses for 28days (20mgInsP6/day). We measured urine levels InsPs using a nonspecific method (based on the ability of InsPs to complex Al3+) and levels of InsP6 by a specific method (using polyacrylamide gel electrophoresis). Identification of different InsPs was performed by MS. KEY FINDINGS: At baseline, after dietary deprivation of InsP6, rats only excreted InsP2 in their urine, and there was no detectable InsP6 or other InsPs. Rats given the high dose treatment for 7days had abundant urinary InsP6, but also had other InsPs in their urine; cessation of InsP6 administration led to decreased levels of urinary InsPs. Rats given the low dose treatment for 28days had increasing levels of urinary InsPs over time. The maximum urinary InsP6 was at 21days, after which InsPs excretion decreased. SIGNIFICANCE: We conclude that the skin can absorb InsP6 from a topical gel, and that InsP6 is excreted in the urine, along with other InsPs (InsP5, InsP4, InsP3, and InsP2).