Anal Bioanal Chem (2012) 402:2727–2735 DOI 10.1007/s00216-012-5729-9
ORIGINAL PAPER
Peripheral endocannabinoid microdialysis: in vitro characterization and proof-of-concept in human subjects Alexander A. Zoerner & Christin Rakers & Stefan Engeli & Sandor Batkai & Marcus May & Jens Jordan & Dimitrios Tsikas
Received: 11 November 2011 / Revised: 2 January 2012 / Accepted: 9 January 2012 / Published online: 4 February 2012 # Springer-Verlag 2012
Abstract In vivo endocannabinoid (EC) microdialysis has only seldom been performed, mostly in rodent brain tissue. Low solubility in aqueous media, adsorption to surfaces, and instability with co-present human serum albumin (HSA) are the major obstacles in EC microdialysis. The addition of hydroxypropyl-ß-cyclodextrine (HPCD) to the perfusion fluid has been previously described to facilitate lipid microdialysis, but the general biophysical properties of HPCD, especially with respect to peripheral EC microdialysis, have not been described before. We report on the characterization of EC microdialysis using an in vitro system using Ringer’s solution with 10% HPCD as the perfusion fluid and with fatty acid-free HSA as the matrix fluid. The endocannabinoids anandamide (AEA) and 2arachidonoyl glycerol (2AG) were measured using LCMS/MS. AEA was stable in the perfusion and matrix fluids, whereas 2AG was only stable in the perfusion fluid. In the matrix fluid, 2AG underwent rapid isomerization to 1arachidonoyl glycerol. A relative recovery of 3.5% for AEA was found with 10% HPCD in the perfusion fluid and a flow rate of 1 μL/min. For 2AG, a similar relative recovery of 3.5% was estimated. Since 2AG was found unstable in the matrix fluid, a reliable calculation of the relative recovery rates was not possible. Delivery and recovery experiments revealed unequal inward and outward EC transport across the microdialysis membrane. Contrary Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-5729-9) contains supplementary material, which is available to authorized users. A. A. Zoerner (*) : C. Rakers : S. Engeli : S. Batkai : M. May : J. Jordan : D. Tsikas Institute for Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany e-mail:
[email protected]
to usual microdialysis findings, we observed increasing recovery rates for AEA with increasing flow rates. Long equilibration times of several hours were necessary to obtain constant relative recovery rates. In a proof-of-concept study in humans, we collected AEA from subcutaneous abdominal adipose tissue employing the described methodology. Our study suggests that the microdialysis technique is not suitable for the exact quantification of tissue EC concentrations, but it allows for their rough estimation. Keywords Microdialysis . Endocannabinoids . Anandamide . 2-Arachidonoyl glycerol . LC-MS/MS . Hydroxypropyl-ß-cyclodextrine
Introduction Microdialysis has been increasingly used over the last two decades to study the interstitial compartment in the central nervous system and in peripheral tissues [1]. The technique involves perfusion of a suitable aqueous fluid through a microdialysis catheter. The microdialysis catheter comprises a flexible tube with a semi-permeable membrane that can be introduced into the tissue of interest. Through the semipermeable membrane, the perfusate and the interstitial fluid equilibrate. Thus, analyte concentrations in microdialysates correspond to in vivo concentrations in the interstitial space. Endocannabionoids (ECs), notably anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG), are lipid mediators involved in a number of physiological processes [2]. For example, ECs and their two receptors CB1 and CB2 are studied in conjunction with obesity [3], psychiatric disorders [4], pain [5], inflammation [6], and asthma [7]. Microdialysis is a promising technique
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to study the peripheral physiological functions of ECs, for example in muscle and adipose tissue. EC microdialysis has previously been performed mainly in rodent brain tissue [8–10]. In human subjects, anandamide microdialysis has been described in a case report on a patient undergoing brain surgery following an ischemic stroke [11]. However, microdialysis of lipophilic substances such as AEA and 2AG is associated with a number of difficulties such as low solubility, low relative recovery, and interfering adsorption effects. To overcome these obstacles, cyclodextrines can be added to the perfusion fluid [12]. Indeed, hydroxypropyl-ß-cyclodextrine (HPCD) has been reported to increase relative EC recovery in microdialysis [9, 10]. Yet, neither HPCD’s biophysical properties nor important microdialysis parameters, such as the required equilibration times, observed relative recoveries, and calibration procedures, have been sufficiently described in the literature. We evaluated EC microdialysis in an in vitro system with HPCD at a concentration of 10% (w/v) added to the aqueous perfusion fluid. We used Ringer’s solution with fatty acidfree human serum albumin (HSA) at 35 g/L as the artificial interstitial fluid. Finally, we demonstrate for the first time the feasibility of peripheral anandamide microdialysis in human abdominal adipose tissue.
Materials and methods Chemicals Anandamide (AEA), tetradeutero-anandamide (d4-AEA), 2AG, and pentadeutero-2AG (d5-2AG) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Sterile lactate-free Ringer’s solution was obtained from B. Braun (Melsungen, Germany), HPCD was acquired from Acros Organics (Geel, Belgium, 97% purity) for in vitro experiments, and Cavitron W7 HP5 (in compliance with Ph.Eur. 6, Hydroxypropylbetadex), from ISP Fine Chemicals (Assonet, MA, USA), was used for in vivo studies. Albumin (HSA, fatty acid-free) was purchased from Sigma-Aldrich (Steinheim, Germany). Instrumentation and analytical procedures CMA 60 microdialysis catheters (CMA/Microdialysis, Stockholm, Sweden) with polyaryl ether sulphone membranes and a 20-kDa cutoff were used. In a custom-made in vitro microdialysis system (University of HalleWittenberg, Germany), approximately 15 mL of the matrix fluid was constantly kept at 37 °C under permanent stirring. The matrix fluid was used as the artificial interstitial fluid and contained 35 g/L HSA in Ringer’s solution, unless
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otherwise noted. A CMA 200 microdialysis pump was connected to the microdialysis catheters. The perfusion fluid consisted of Ringer’s solution with 10% (w/v) HPCD, if not otherwise stated. AEA and 2AG were quantified by LC-MS/MS using a XEVO TQ MS tandem quadrupole mass spectrometer connected to an ACQUITY UPLC system (Waters, Boston, MA, USA) as previously described [13]. Briefly, the internal standards, d4-AEA and d5-2AG, were added to the microdialysis samples at concentrations of 66 and 85 nM, respectively, before solvent extraction with toluene. For in vivo microdialysis samples, d4-AEA was used at a concentration of 4.8 nM. After toluene evaporation to dryness, the samples were reconstituted in the LC-MS/MS mobile phase which consisted of 25% water and 75% methanol, both containing 2 mM ammonium acetate. A flat gradient from 75% methanol to 79% methanol was used with a flow rate of 0.5 mL/ min; chromatographic separation took place on an ACQUITY BEH C18 column (100-mm length, 2.1-mm i.d., 1.7μm particle size). Quantitative measurements were carried out in the selected reaction monitoring and positive electrospray ionization modes. The following transitions were used: m/z 348 → m/z 62 (d0-AEA), m/z 352 → m/z 66 (d4AEA), m/z379→m/z287 (d0-2AG), and m/z384→m/z287 (d5-2AG). No HPCD was found in toluene extracts. The limit of quantitation (LOQ) for AEA extracted from microdialysis samples (Ringer’s solution containing 10% (w/v) HPCD, 240 μL sample volume) with toluene was determined to be 50 pM. At this concentration, accuracy and imprecision were determined to be within the commonly accepted ranges of 100±20% and
