Methods
Analysis of ECs and related compounds in plasma: artifactual isomerization and ex vivo enzymatic generation of 2-MGs[S]

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The analysis of peripheral endocannabinoids (ECs) is a good biomarker of the EC system. Their concentrations, from clinical studies, strongly depend on sample collection and time processing conditions taking place in clinical and laboratory settings. The analysis of 2-monoacylglycerols (MGs) (i.e., 2-arachidonoylglycerol or 2-oleoylglycerol) is a particularly challenging issue because of their ex vivo formation and chemical isomerization that occur after blood sample collection. We provide evidence that their ex vivo formation can be minimized by adding Orlistat, an enzymatic lipase inhibitor, to plasma. Taking into consideration the low cost of Orlistat, we recommend its addition to plasma collecting tubes while maintaining sample cold chain until storage. We have validated a method for the determination of the EC profile of a range of MGs and N-acylethanolamides in plasma that preserves the original isomer ratio of MGs. Nevertheless, the chemical isomerization of 2-MGs can only be avoided by an immediate processing and analysis of samples due to their instability during conservation. We believe that this new methodology can aid in the harmonization of the measurement of ECs and related compounds in clinical samples.

2-arachidonoylglycerol
2-oleoylglycerol
validation
Orlistat
human
endocannabinoids
2-monoacylglycerol

Cited by (0)

This work was supported by DIUE (Department of Innovation, Universities and Enterprise) de la Generalitat de Catalunya 2009 (grant SGR 718) and CIBEROBN (Spanish Biomedical Research Centre in Physiopathology of Obesity and Nutrition, CB06/03, CIBEROBN is an initiative of ISCIII).

    Abbreviations

    AEA

    N-arachidonoyl ethanolamide

    AG

    arachidonoylglycerol

    Am. Ac.

    ammonium acetate

    CB

    cannabinoid

    CV

    coefficient of variation

    DAG

    diacylglycerol

    DAGL

    sn-1-diacylglycerol lipase

    DEA

    N-docosatetraenoyl ethanolamide

    DGLEA

    N-dihomo-γ-linolenoyl ethanolamide

    DHEA

    N-docosahexaenoyl ethanolamide

    EC

    endocannabinoid

    EPEA

    N-eicosapentaenoyl ethanolamide

    ERC

    endocannabinoid-related compound

    ISTD

    internal standard

    LEA

    N-linoleoyl ethanolamide

    α-LEA

    N-α-linolenoyl ethanolamide

    LG

    linoleoylglycerol

    LLOQ

    lower limit of quantification

    LOD

    limit of detection

    MG

    monoacylglycerol

    MRM

    multiple reaction monitoring

    NAE

    N-acylethanolamide

    OEA

    N-oleoyl ethanolamide

    OG

    oleoylglycerol

    PEA

    N-palmitoyl ethanolamide

    POEA

    N-palmitoleoyl ethanolamide

    QC

    quality control

    QC-H

    quality control-high

    QC-L

    quality control-low

    QC-M

    quality control-mid

    RF

    response factor

    SA

    surrogated analyte

    SEA

    N-stearoyl ethanolamide

    TBME

    tert-butyl-methyl-ether

[S]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one table.