Saliva-Based Endocannabinoid Quantification: A Research Review

By: Mike Robinson, Global Cannabinoid Research Center, Santa Barbara, California, USA  © 3/16/2024

Abstract:

The evolving legal landscape surrounding medicinal cannabinoid-based products underscores the growing interest in the endogenous cannabinoid (endocannabinoid) system. Despite its potential, the field of endocannabinoid research, significantly beyond blood matrices, remains nascent. This study reviews current research and pioneers developing and validating a method for simultaneously quantifying key endocannabinoids and hormones in human plasma and saliva. Utilizing liquid-liquid extraction and ultra-high performance liquid chromatography coupled with tandem mass spectrometry, we achieve a significant milestone in endocannabinoid research, opening new avenues for understanding the system's role in stress responses and peripheral tissue functions.

Introduction:

The discovery of the endocannabinoid system (ECS) followed the isolation of THC and the identification of cannabinoid receptors, CB1 and CB2. Endocannabinoids such as arachidonoyl-ethanolamide (AEA) and 2-arachidonoylglycerol (2AG) bind to these receptors, influencing numerous physiological processes.

However, human studies on endocannabinoids have been minimal. This study addresses the gap by developing methods to quantify endocannabinoids in saliva and plasma, exploring their role in stress responses and the potential influence of sex hormones on endocannabinoid levels (1).

Therefore, we aim to expose the pros and cons of the Saliva-Based Endocannabinoid Study on humans and animals with Endocannabinoid Systems and its potential in future medical applications.

Method Development and Validation:

In the reviewed study entitled Simultaneous quantification of endocannabinoids, oleoylethanolamide and steroid hormones in human plasma and saliva, researchers focused on AEA, 2AG, oleoylethanolamide (OEA), cortisol, and progesterone, using liquid-liquid extraction and ultra-high performance liquid chromatography-tandem mass spectrometry for quantification (1). Our method demonstrated high linearity (R2 = 0.98–0.99 for plasma and R2 = 0.99 for saliva) across a 1–20 ng/mL concentration range. This robust methodology lays the groundwork for extensive research into the endocannabinoid system's dynamics in non-blood matrices.

Results & Discussion:

The study found ethyl acetate:cyclohexane extraction superior for endocannabinoid recovery, highlighting the evolution of extraction methods for accurate quantification. The sensitivity to acute physiological changes, such as exercise-induced, was validated in saliva samples, suggesting a novel approach for future endocannabinoid research (1).

This method's success in detecting the acute reactivity of salivary endocannabinoids and cortisol to exercise underscores the saliva system's potential for noninvasive research applications.

The study needed an in-depth overview of saliva-based studies (2).

Noninvasive Sampling

Saliva collection is a noninvasive, stress-free method that can be performed without professional medical staff (2). This makes it ideal for frequent sampling in diverse settings, including at home or in clinical environments. The ease of collection facilitates longitudinal studies, where multiple samples over time are required to monitor changes in the ECS.

Reflecting Physiological Changes

Salivary endocannabinoids are considered to more acutely reflect the physiological status of the ECS than blood samples. This is because saliva, not a blood product, bypasses the metabolic processes in the liver (2), providing a more direct insight into peripheral endocannabinoid activity.

Saliva can quickly reflect changes in endocannabinoid levels in response to stress, exercise, and dietary intake, making it a valuable tool for studying the immediate effects of these factors on the ECS (3).

Saliva Composition and Endocannabinoid Measurement

Saliva contains many biological molecules, including hormones, enzymes, and, importantly, lipid-based compounds like endocannabinoids (3). The presence of endocannabinoid receptors and enzymes involved in the synthesis and degradation of endocannabinoids in salivary glands suggests that saliva contains not only endocannabinoids transported from blood but also those locally synthesized. This dual origin of salivary endocannabinoids offers a comprehensive view of systemic and local ECS activities (4).

Challenges and Solutions in Saliva-Based Measurements

One primary challenge in measuring endocannabinoids in saliva is the low concentration of these molecules, which demands susceptible analytical techniques (5). The development of ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) addresses this challenge by providing the sensitivity and specificity needed for accurate quantification of endocannabinoids in saliva.

Applications in Research and Medicine

Using saliva to measure endocannabinoids has significant implications for research and clinical practice. Research allows for the exploration of the ECS's role in stress, addiction, obesity, and other conditions without the need for invasive blood draws. Saliva-based endocannabinoid measurements could lead to developing diagnostic tools for ECS-related dysregulations and monitoring therapeutic interventions targeting ECS (5).

Key Saliva-Based Endocannabinoid Studies

Response to Exercise: Some studies have investigated the acute reaction of salivary endocannabinoids to physical exercise. These studies often focus on how exercise-induced stress and subsequent recovery influence endocannabinoid levels, potentially mediating the stress-reducing effects of exercise.

Obesity and Metabolic Health: Research has also explored the relationship between salivary endocannabinoids and metabolic health indicators, finding, for instance, that individuals with obesity may exhibit different levels of salivary endocannabinoids compared to those of a healthy weight. This ECS response may suggest a potential role of endocannabinoids in energy balance and metabolism (5).

Stress and Emotional Responses: Some research has examined how salivary endocannabinoids correlate with stress and emotional states. Given the endocannabinoid system's role in mood regulation and stress response, these studies aim to uncover how salivary endocannabinoid levels might reflect emotional states or stress levels (6).

Challenges and Considerations

Despite the potential, there are challenges and considerations in saliva-based endocannabinoid research:

Variability: Saliva composition can vary significantly among individuals and under different conditions, which may influence endocannabinoid levels and their interpretation(1,2,3,4,5,6).

Sensitivity and Specificity: Developing sensitive and specific analytical methods for detecting endocannabinoids in saliva is crucial, as concentrations may be lower than in blood(5).

Standardization: Standardization of collection, storage, and analysis protocols is needed to ensure consistent and reliable measurement of endocannabinoids in saliva(1).

Implications for Future Research:

The methodology developed in this study offers a promising avenue for noninvasive, comprehensive research into the endocannabinoid system. It encourages further exploration of the ECS's role in various physiological and pathological states, potentially leading to novel therapeutic targets and strategies for managing stress, obesity, and other conditions influenced by endocannabinoids.

Conclusion:

This groundbreaking study presents a validated method for quantifying endocannabinoids and related hormones in human plasma and saliva. It enhances our understanding of the endocannabinoid system's role in stress response and peripheral tissues and sets the stage for future explorations into the ECS.

Expanding the research into the pros and cons of Saliva Research shall contribute significantly to the broader comprehension and therapeutic exploitation of the endocannabinoid system by providing a reliable and sensitive tool for quantifying endocannabinoids.

With adequate measurements of endocannabinoids, further research may allow for determining the tone of the Endocannabinoid System (ECS) or measurement of all endocannabinoids and receptor availability.

Mike Robinson, Global Cannabinoid Research Center, Santa Barbara, California, USA  © 3/16/2024

References:

1.) Luke J. Ney, Kim L. Felmingham, Raimondo Bruno, Allison Matthews, David S. Nichols, Simultaneous quantification of endocannabinoids, oleoylethanolamide and steroid hormones in human plasma and saliva: Journal of Chromatography B, Volume 1152, 2020, 122252, ISSN 1570-0232, https://doi.org/10.1016/j.jchromb.2020.122252. https://www.sciencedirect.com/science/article/pii/S1570023220302208 Volume 1152, 1 September 2020

2.) Pittman TW, Decsi DB, Punyadeera C, Henry CS. Saliva-based microfluidic point-of-care diagnostic. Theranostics. 2023 Jan 31;13(3):1091-1108. doi: 10.7150/thno.78872. PMID: 36793864; PMCID: PMC9925318.

3.) Hillard, C. Circulating Endocannabinoids: From Whence Do They Come and Where are They Going?. Neuropsychopharmacol. 43, 155–172 (2018). https://doi.org/10.1038/npp.2017.130

4.) Dipanjan Chanda, Dietbert Neumann, Jan F.C. Glatz,The endocannabinoid system: Overview of an emerging multi-faceted therapeutic target" Prostaglandins, Leukotrienes and Essential Fatty Acids,Volume 140, 2019, Pages 51-56, ISSN 0952-3278, https://doi.org/10.1016/j.plefa.2018.11.016. https://www.sciencedirect.com/science/article/pii/S0952327818301765

5.) Matias I, Gatta-Cherifi B, Tabarin A, Clark S, Leste-Lasserre T, Marsicano G, Piazza PV, Cota D. Endocannabinoids measurement in human saliva as potential biomarker of obesity. PLoS One. 2012;7(7):e42399. doi: 10.1371/journal.pone.0042399. Epub 2012 Jul 31. PMID: 22860123; PMCID: PMC3409167.

6.) Luke Ney, Caleb Stone, David Nichols, Kim Felmingham, Raimondo Bruno, Allison Matthews, Endocannabinoid reactivity to acute stress: Investigation of the relationship between salivary and plasma levels, Biological Psychology, Volume 159, 2021, 108022, ISSN 0301-0511, https://doi.org/10.1016/j.biopsycho.2021.108022. https://www.sciencedirect.com/science/article/pii/S0301051121000132