Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS
- Flaminia Fanelli*,†,
- Valentina D. Di Lallo*,†,
- Ilaria Belluomo*,†,
- Rosaria De Iasio§,
- Margherita Baccini*,†,
- Elena Casadio*,†,
- Daniela Ibarra Gasparini*,†,
- Michelangelo Colavita*,†,
- Alessandra Gambineri*,†,
- Gabriele Grossi§,
- Valentina Vicennati*,†,
- Renato Pasquali*,† and
- Uberto Pagotto1,*,†
- *Endocrinology Unit, Department of Clinical Medicine, Alma Mater Studiorum, University of Bologna, Bologna, Italy; and
- †Endocrinology Unit and Centre of Applied Biomedical Research, Department of Clinical Medicine, Alma Mater Studiorum, University of Bologna, Bologna, Italy; and
- §Central Laboratory, S.Orsola-Malpighi Hospital, Bologna, Italy
Abstract
The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.
Footnotes
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↵1 To whom correspondence should be addressed. e-mail: uberto.pagotto{at}unibo.it
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- Abbreviations:
- 1AG
- 1-arachidonoyl-glycerol
- 2AG
- 2-arachidonoyl-glycerol
- 2D
- two-dimension
- AEA
- arachidonoyl-ethanolamide
- BES
- Binge Eating Scale
- BITE
- Bulimic Investigation test Edinburgh
- BMI
- body mass index
- CB
- cannabinoid receptor
- Chol/HDL
- cholesterol to HDL ratio
- DBP
- diastolic blood pressure
- EC
- endocannabinoid
- ECS
- endocannabinoid system
- ERC
- endocannabinoid related compounds
- EtOH
- ethanol
- FDA
- Food and Drug Administration
- IQ
- interquartile range
- IR
- ion ratio
- IS
- internal standard
- LLE
- liquid-liquid extraction
- LLOQ
- lower limit of quantitation
- LOD
- limit of detection
- MAG
- monoacyl-glycerols
- MeOH
- methanol
- MRM
- multiple reaction monitoring
- NAE
- N-acyl-ethanolamide
- OEA
- oleoyl-ethanolamide
- PEA
- palmitoyl-ethanolamide
- QC
- quality control
- RT
- room temperature
- S/N
- signal to noise ratio
- SBP
- systolic blood pressure
- SPE
- solid phase extraction
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This research was supported by grants from the European Union (REPROBESITY FPVII-223713 and NEUROFAST FPVII-KBBE-2009-3-245009) and the Progetto Regione-Università of the Emilia Romagna Region.
- Received October 12, 2011.
- Revision received December 13, 2011.
- Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
