Simultaneous ultra-high performance liquid chromathograpy-electrospray ionization-quadrupole-time of flight mass spectrometry quantification of endogenous anandamide and related N-acylethanolamides in bio-matrices.

We describe and validate a sensitive UHPLC-ESI-QTOF-MS method for the simultaneous quantification of seven endocannabinoids and non-endocannabinoids related N-acylethanolamides: N-arachidonoylethanolamide, N-palmitoylethanolamide, N-stearoylethanolamide, N-oleoylethanolamide, N-linoleoylethanolamide, N-α-linolenoylethanolamide and N-eicosapentaenoylethanolamide in several bio-matrices for the purpose of research and clinical application. We examined effects of different liquid-liquid and solid phase extraction on the recovery of endocannabinoids and N-acylethanolamides. Protein precipitation with cooled acetone and extraction with acetonitrile (1% v/v formic acid) using OASIS HLB cartridge gave better results. Separation was performed on a Waters Acquity UPLC HSST3 column using a 9min elution gradient coupled with high resolution mass spectrometry (QTOF/MS). The high sensitivity of the developed method allow its application on sample with low volumes or low levels of endocannabinoids and N-acylethanolamides and make the method suitable for routine measurement in human bio-matrices, such as plasma, serum (500µL), urine (1mL) and tissues (10-30mg). Its application in clinical research could contribute to unravel pathophysiological roles of these family of lipid mediators and disclose novel diagnostic and prognostic markers.

Simultaneous measurement of three N-acylethanolamides in human bio-matrices using ultra performance liquid chromatography-tandem mass spectrometry.

Endocannabinoids including N-acylethanolamides (NAEs) are a family of lipid-related signaling molecules implicated in many physiological and disease states which elicit their activities via the cannabinoid receptors. Anandamide (N-arachidonoylethanolamine, AEA) is the most characterized endocannabinoid and has been detected in many tissues and bio-fluids including human plasma and the central nervous system. The endocannabinoid-like NAEs, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are described as entourage compounds because they illicit similar physiological effects to AEA but have little or no affinity for cannabinoid receptors. As entourage compounds, levels of these NAEs can greatly influence the efficacy of AEA yet there are few studies which measure these compounds in bio-fluids. Here we describe a rapid, highly sensitive, specific and highly reproducible ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of AEA, OEA, and PEA in human bio-fluids including plasma, serum, breast milk, and amniotic fluids. This validated method using deuterated (AEA-d(8), OEA-d(2), and PEA-d(4)) internal standards, represents an improvement over previous analyses in terms of run time (4 min), limit of detection (0.9 fmol on column for AEA and PEA and 4.4 fmol on column for OEA), precision (relative standard deviations of peak areas: 3.1% (AEA), 2.9% (OEA), and 5.4% (PEA) for 133 fmol on column) and accuracy (95.1-104.9%). The sensitivity and precision of the validated method described here suggests that this method is suitable for the analysis of AEA, OEA, and PEA in clinical samples and may be utilized for the investigation of bio-matrices containing limited amounts of NAEs.

Lack of promoting effects of alpha-linolenic, linoleic or palmitic acid on urinary bladder carcinogenesis in rats.

Potential promoting effects of alpha-linolenic, linoleic and palmitic acids were investigated in a two-stage urinary bladder carcinogenesis model. In experiment 1, male F344 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks and then basal diet containing 10% alpha-linolenic, 10% linoleic or 10% palmitic acid along with 0.2% butylated hydroxyanisole (BHA) as an antioxidant for 24 weeks. The development of tumors in the urinary bladder was not increased by treatment with any of the fatty acids. In experiment 2, male F344 rats were given 10% alpha-linolenic, 10% linoleic or 10% palmitic acid along with 0.2% BHA in their diet for 8 weeks without prior BBN treatment. The administration of fatty acids was not associated with any increase in the 5-bromo-2'-deoxyuridine labeling index of the urinary bladder epithelium. Serum and/or urine fatty acid levels increased in the cases of alpha-linolenic and linoleic acid treatments, but not with palmitic acid. Under the present experimental conditions neither the two polyunsaturated nor the one saturated fatty acid exerted any promoting effect on urinary bladder carcinogenesis.

Qualitative and quantitative analysis of urinary lipids in the nephrotic syndrome.

A qualitative and quantitative analysis of urinary lipids in the nephrotic syndrome is presented. The following lipids were identified in the urine of patients with the nephrotic syndrome: free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phospholipids. Glass paper chromatography identified the cholesterol esters as palmitate, oleate, linoleate, and arachidonate, and identified the phospholipids as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Urinary lipid excretion was much greater in patients with the nephrotic syndrome than in patients with chronic renal disease and minimal proteinuria, or in patients with hyperlipidemia from other causes. Urinary lipid excretion varied widely among the 13 patients with the nephrotic syndrome studied, and no quantitative correlation with serum lipid levels was observed. However, qualitatively at least, the proportion of cholesterol esters excreted in the urine was similar to the proportion of these esters in plasma. A good correlation was found between lipid excretion and glomerular permeability. Furthermore, during steroid therapy urinary lipid excretion decreased concomitant with a decrease in proteinuria. All these observations support the idea that lipiduria in the nephrotic syndrome is related to protein loss and that most of the lipid in the urine enters the glomerular filtrate in the form of lipoproteins.