Simultaneous measurement of anandamide and 2-arachidonoylglycerol by polymyxin B-selective adsorption and subsequent high-performance liquid chromatography analysis: increase in endogenous cannabinoids in the sera of patients with endotoxic shock.
Anandamide (ANA) and 2-arachidonoylglycerol (2-AG), two endogenous cannabinoids, can be generated by activated macrophages and platelets, respectively, in the context of endotoxic shock, and are proposed to play a crucial role in the induction of the shock-related hypotension. Taking advantage of our recently discovered function of polymyxin B (PMB) binding to ANA and 2-AG, we developed a new method for measuring ANA and 2-AG by applying PMB-immobilized beads to selectively adsorb them in biological fluids, instead of organic solvent extraction. The eluate from beads can be directly fractionated by reverse-phase high-performance liquid chromatography (HPLC), and the fractionations corresponding to authentic ANA and 2-AG are collected and derivatized with fluorogenic reagent and subsequently quantified by HPLC with fluorometric detection. The calibration graphs of ANA and 2-AG were linear over a range of 1 to 500 pmol/ml. The limits of detection for ANA and 2-AG were 20 and 50 fmol, respectively. Intraassay precision was 2.24-4.25 and 3.47-5.44%, and interassay was 4.05-6.14 and 4.92-7.28% for ANA and 2-AG, respectively. Using this method, we first determined a 4-fold and 3-fold higher level of ANA and 2-AG, respectively, in the sera of patients with endotoxic shock than in normal serum. This finding should help in elucidating the role of the endogenous cannabinoids in the hypotension of human endotoxic shock. This method is rapid, sensitive, and reliable for simultaneously quantifying ANA and 2-AG in biological fluids, and has potential for clinical usage.
Wang
Y
Y
Department of Laboratory and Molecular Medicine, Kagoshima University School of Medicine, Kagoshima, Japan.
Liu
Y
Y
Ito
Y
Y
Hashiguchi
T
T
Kitajima
I
I
Yamakuchi
M
M
Shimizu
H
H
Matsuo
S
S
Imaizumi
H
H
Maruyama
I
I
D000327:Adsorption; D000818:Animals; D001095:Arachidonic Acids / Q000097:blood*; D002138:Calibration; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D063388:Endocannabinoids; D005989:Glycerides / Q000097:blood*; D006801:Humans; D051379:Mice; D011112:Polymyxin B / Q000737:chemistry*; D053284:Polyunsaturated Alkamides; D015203:Reproducibility of Results; D012772:Shock, Septic / Q000097:blood*; D013050:Spectrometry, Fluorescence
D001095:Arachidonic Acids; D063388:Endocannabinoids; D005989:Glycerides; D053284:Polyunsaturated Alkamides; C094503:glyceryl 2-arachidonate; D011112:Polymyxin B; C078814:anandamide