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Endocannabinoid Analysis Service

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Overview of Endocannabinoid

In the intricate tapestry of biological signaling, the endocannabinoid system emerges as a critical network, governing both central and peripheral processes. This elaborate network, comprising G protein-coupled cannabinoid type 1 and type 2 receptors (CB1 and CB2), along with their endogenous ligands—the endocannabinoids—and the enzymes that govern their synthesis and degradation, operates both centrally and peripherally within the body. Research has focused extensively on well-studied endocannabinoids such as anandamide (arachidonoyl ethanolamine) and 2-arachidonoylglycerol. These compounds have been scrutinized in various disease contexts, including obesity, thereby highlighting their crucial roles and potential as diagnostic or prognostic biomarkers. Therefore, Protheragen conducts the reliable quantification of these endocannabinoids in samples, which is of utmost importance given their relevance in conditions like obesity.

At Protheragen, the analysis of endocannabinoids has been pivotal in identifying key targets for the development of anti-obesity therapeutics. These targets, such as cannabinoid receptors and metabolic enzymes, are crucial in regulating appetite and energy balance. The subsequent anti-obesity therapy development focuses on creating drugs that modulate these targets effectively, while preclinical studies rigorously test these therapeutics for safety and efficacy in animal models, laying the groundwork for potential therapy development.

Unveiling the Secrets of the Endocannabinoid System for Healthier Living

At Protheragen, our researchers are dedicated to providing a comprehensive and specialized analysis of endocannabinoids. To ensure the precise quantification of trace endocannabinoids, we have developed a meticulous process for handling tissues.

Tissue Homogenization

We typically homogenize tissues in organic solvents, precipitate proteins, and extract lipids. Tissues are frozen in liquid nitrogen or cold 2-methylbutane and kept at -80°C for analysis. Before homogenization, frozen tissues are quickly weighed without thawing. To minimize postmortem metabolite changes, lipids are extracted before thawing. Homogenization methods include glass or electric homogenizers. This process is done quickly on ice to prevent degradation, and an internal standard is added for quantification.

Internal Standards for Quantification

The construction of a standard curve (calibration curve) is a crucial step to ensure the accuracy, reproducibility, and reliability of our analytical methods. We use the internal standard approach, which involves adding a constant amount of nonendogenous or nonanalyte molecules to each sample before homogenization. These internal standards must behave similarly to the analytes throughout the extraction procedures and achieve a recovery rate of at least 90%. For mass spectrometry (MS)-based analysis, we use stable-isotope labeled internal standards due to their distinct atomic mass units compared to analytes. In chromatography, ultraviolet (UV) or fluorescence-based analyses, we use internal standards with different carbon chain lengths.

Protein Precipitation

Before conducting liquid chromatography-mass spectrometry (LC-MS/MS) analysis of endocannabinoids, we typically perform a protein precipitation step. Acetone is the most commonly used water-miscible solvent in this step, although other solvents like acetonitrile or methanol are also effective for precipitating proteins. The supernatant is collected and subjected to further lipid extraction.

Lipid Extraction and Purification

Due to the lipophilic nature of endocannabinoids, we extract them from biological samples using water-immiscible solvents like chloroform or a mixture of ethyl acetate and hexane. Repeated extraction steps are conducted to increase the lipid yield. High levels of matrix components interfere with the analysis, necessitating further purification. This is achieved through thin-layer chromatography (TLC) or solid-phase extraction (SPE).

  • Lipid extracts in chloroform/methanol are spotted onto a TLC plate and developed using various solvent systems. The spots containing endocannabinoids are scraped off and re-extracted with organic solvents for further analysis.
  • SPE is more widely used for high-throughput analysis and is fully automated.

Separation and Quantification of Endocannabinoids

We use liquid gas-mass spectrometry (GC-MS) to separate and identify endocannabinoid metabolites from biological samples. Nonpolar capillary columns and helium as the carrier gas are standard, with ionization modes including electron impact (EI), positive chemical ionization (PICI), and negative chemical ionization (NICI). For quantification, we use selected ion monitoring (SIM) and match the mass spectrum of the analyte to a standard. To improve sensitivity, we perform derivatization using N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) before GC-MS analysis. This modifies the hydroxyl groups of anandamide and 2-arachidonoylglycerol, creating TMS derivatives that are dissolved in hexane for detection. Additionally, we use LC with techniques like high-performance liquid chromatography (HPLC)-UV/fluorescence and LC-MS/MS for direct analysis of endocannabinoids using soft ionization like electrospray ionization (ESI) and selected reaction monitoring (SRM) to enhance selectivity and sensitivity.

Workflow

Workflow of endocannabinoid analysis. (Protheragen)

Applications

  • Our service can be used to identify key targets for anti-obesity therapeutics, such as cannabinoid receptors and metabolic enzymes, which regulate appetite and energy balance.
  • Our service can be used to develop and test anti-obesity drugs that modulate the endocannabinoid system, with preclinical studies conducted in animal models to ensure safety and efficacy.
  • Our service can be used to investigate the role of the endocannabinoid system in obesity-related metabolic disorders, such as insulin resistance and dyslipidemia, by providing detailed endocannabinoid profiles.

Advantages

  • We develop a meticulous process for handling tissues, including tissue homogenization, lipid extraction, and purification, to ensure accurate results.
  • We utilize advanced techniques such as GC-MS, LC-MS/MS, and HPLC-UV/fluorescence for the separation, identification, and quantification of endocannabinoids, ensuring high sensitivity and selectivity.
  • We offer customized solutions tailored to the specific needs of clients, addressing the unique challenges of each project.

Publication Data

DOI: 10.1371/journal.pone.0008792

Journal: PloS one

Published: 2010

IF: 2.9

Result: The study examined the connection between the endocannabinoid system and severe obesity by analyzing plasma levels of anandamide and related N-acylethanolamines (NAEs) alongside genotyping for the FAAH 385 C→A (P129T) mutation in 96 severely obese individuals (BMI ≥40 kg/m2) and 48 normal-weight individuals (BMI ≤26 kg/m2). Key findings revealed that carriers of the FAAH 385 A mutant allele exhibited significantly higher plasma levels of anandamide and NAEs compared to those with the wild-type FAAH genotype, even after controlling for BMI, suggesting that the mutant allele directly influences endocannabinoid system activation in severe obesity. This discovery offers novel biomarkers for obesity and potential endocannabinoid system-based therapeutic strategies.

Frequently Asked Questions

  1. How does the endocannabinoid system affect obesity?

    The endocannabinoid system plays a crucial role in regulating food consumption, fat accumulation, insulin responsiveness, and inflammation—factors that are key to the onset and progression of obesity.

  2. What functions in the body do endocannabinoids regulate?
    • Appetite
    • Fertility
    • Immune functions
    • Learning
    • Mood
    • Memory
    • Pain
    • Sleep
    • Body temperature.

Protheragen stands out due to our comprehensive Obesity Biomarker Identification and Analysis, meticulous processing, advanced instrumentation, use of internal standards, expertise in obesity research, high-throughput capability, derivatization for enhanced sensitivity, and potential for customized solutions. If you want to conduct a comprehensive and specialized analysis of endocannabinoids, ensuring precise quantification and identification of key targets for anti-obesity therapeutics, contact us!

Reference

  1. Sipe, J.C.; et al. Biomarkers of endocannabinoid system activation in severe obesity. PloS one. 2010, 5(1): e8792. (CC BY)

All of our services and products are intended for preclinical research use only and cannot be used to diagnose, treat or manage patients.

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