Metabolomics and integrated network analysis reveal roles of endocannabinoids and large neutral amino acid balance in the ayahuasca experience

This study (n=23) assessed the human metabolomics signature after consumption of ayahuasca and its connection with both the psychedelic-induced subjective effects and the plasma concentrations of ayahuasca alkaloids. Compared to baseline, the consumption of ayahuasca increased N-acyl-ethanolamine endocannabinoids, decreased 2-acyl-glycerol endocannabinoids, and altered several large-neutral amino acids (LNAAs). Enrichment analysis confirmed dysregulation in several pathways involved in neurotransmission such as serotonin and dopamine synthesis.

Abstract

“There has been a renewed interest in the potential use of psychedelics for the treatment of psychiatric conditions. Nevertheless, little is known about the mechanism of action and molecular pathways influenced by ayahuasca use in humans. Therefore, for the first time, our study aims to investigate the human metabolomics signature after consumption of a psychedelic, ayahuasca, and its connection with both the psychedelic-induced subjective effects and the plasma concentrations of ayahuasca alkaloids. Plasma samples of 23 individuals were collected both before and after ayahuasca consumption. Samples were analysed through targeted metabolomics and further integrated with subjective ratings of the ayahuasca experience (i.e., using the 5-Dimension Altered States of Consciousness Rating Scale [ASC]), and plasma ayahuasca-alkaloids using integrated network analysis. Metabolic pathways enrichment analysis using diffusion algorithms for specific KEGG modules was performed on the metabolic output. Compared to baseline, the consumption of ayahuasca increased N-acyl-ethanolamine endocannabinoids, decreased 2-acyl-glycerol endocannabinoids, and altered several large-neutral amino acids (LNAAs). Integrated network results indicated that most of the LNAAs were inversely associated with 9 out of the 11 subscales of the ASC, except for tryptophan which was positively associated. Several endocannabinoids and hexosylceramides were directly associated with the ayahuasca alkaloids. Enrichment analysis confirmed dysregulation in several pathways involved in neurotransmission such as serotonin and dopamine synthesis. In conclusion, a crosstalk between the circulating LNAAs and the subjective effects is suggested, which is independent of the alkaloid concentrations and provides insights into the specific metabolic fingerprint and mechanism of action underlying ayahuasca experiences.”

Authors: Francisco Madrid-Gambin, Alex Gomez-Gomez, Arnau Busquets-Garcia, Noemi Haro, Santiago Marco, Natasha L. Mason, Johannes T. Reckweg, Pablo Mallaroni, Lilian Kloft, Kim van Oorsouw, Stefan W. Toennes, Rafael de la Torre, Johannes G. Ramaekers & Oscar J. Pozo

Summary

A study was conducted to investigate the human metabolomics signature after consumption of ayahuasca and its connection with the subjective effects and the plasma concentrations of ayahuasca alkaloids.

1. Introduction

Ayahuasca is a psychedelic brew used by indigenous cultures in South America and is now being consumed by modern Brazilian religions.

Ayahuasca is traditionally made with the bark of Banisteriopsis caapi and the leaves of Psychotria viridis, which contain a synergistic combination of alkaloids responsible for the strong psychedelic effects.

Although there is a lack of biochemical knowledge on the human consumption of ayahuasca, it has a dual effect on human health and involves risks, such as serotonin syndrome and nonpsychotic mania.

The mechanism and molecular pathways involved in the impact of ayahuasca on healthy humans are yet to be investigated. The study of the metabolome may provide significant knowledge about which pathways ayahuasca may alter.

Several studies have been conducted on the ayahuasca beverage, but scarce metabolomics investigations have been conducted in humans. Previous studies in animal models have suggested that activation of 5-HT2A also modulates endocannabinoids.

Ayahuasca consumption produced a metabolomic signature, which was analyzed by an integrative network. The metabolites were connected to pathways, reactions, or relation networks that could explain the mechanism of action.

2.1. Subjects and study design

Twenty-three healthy volunteers, 14 males, 9 females, with an average age of 37.9 10.1 years, were enrolled in a within-subject observational study. They consumed ayahuasca individually, separated by a one-hour time window, and were assessed on consecutive testing days.

All participants were informed of all procedures, possible adverse reactions, legal rights and responsibilities, expected benefits, and their right to voluntary termination without consequences.

2.2. Psychological measures

Ayahuasca induces psychological effects that are measured by the validated 5-Dimension Altered States of Consciousness Rating Scale. The scale consists of 11 subscales, including the experience of unity, spiritual experience, blissful state, insightfulness, disembodiment, impaired control and cognition, and anxiety.

2.3. Ayahuasca brew alkaloids

The alkaloid concentrations of the ayahuasca brew used in the ceremony were determined using high performance LC – electrospray ionization – MS.

2.5. Metabolomics analysis

After protein precipitation, blood samples were analysed via six protocols, which assessed each family of compounds using previously reported methods. A total of 164 targeted biomarkers were determined by LC-MS/MS system.

2.6. Statistical analysis

The characteristics of the participants were described, and statistical analyses were performed using R software version 4.0.3. A principal component analysis was performed to detect outliers, and paired two-tailed Wilcoxon tests were used to evaluate potential pre-post consumption differences.

Ayahuasca consumption was predicted by multilevel-partial least squares discriminant analysis (mPLS-DA) using a repeated double cross-validation framework. A significant p-value lower than 0.05 was considered to indicate a good model.

2.7. The integrated pathway network construction

Regularized canonical correlation analyses were performed on all individuals to assess correlations between metabolomics, ayahuasca alkaloids, and the ayahuasca-induced subjective effects. A pathway network and corresponding clustered heat map were built from the rCCA outputs.

2.8. Enrichment analysis

Significant metabolites were mapped to metabolic pathways enrichment using the FELLA package and a KEGG knowledge graph was created. The diffusion method of the propagation algorithm was employed to score graph nodes and a Cytoscape network visualization was performed.

3. Results

Twenty-three individuals participated in the ayahuasca ceremony, and their plasma samples were analysed for the metabolomics set and the ayahuasca-alkaloids set.

3.1. Metabolic changes after ayahuasca consumption

The mPLS-DA resulted in a model with a classification rate of 100.0% and a permutation test p-value of 0.01. Thirty-one differential metabolites were extracted.

Most of the metabolites associated with ayahuasca intake were involved in either endocannabinoid or amino acid metabolism. The levels of certain steroid metabolites such as cortisone, 20-DHE and 20-DHE increased after ayahuasca intake.

3.2. Integrated pathway network analysis

The rCCA revealed five distinguishable clusters of metabolic markers, including endocannabinoids, lipids, and organic acids, which were correlated with subjective ratings of consciousness and ayahuasca alkaloids. However, some of these markers were not statistically connected with the plasma concentration of the ayahuasca alkaloids.

A positive relationship was observed between ayahuasca consumption and a set of metabolic markers, including tryptophan, 5HIAA, serotonin and several monoacylglycerol species. However, no relationship was observed between ayahuasca consumption and the cluster of alkaloids or the dose.

Several metabolic markers were found to be inversely correlated with ASC clusters. Tyrosine was found to be particularly associated with the experience of unity.

The integrative network graph of the ayahuasca-induced subjective effects shows that several metabolic groups are connected to the ayahuasca alkaloids arm, including the groups of amino acids, endocannabinoids, certain LPCs, and energy metabolism metabolites.

3.3. Metabolic pathway enrichment

The metabolic pathway enrichment analysis mapped 11 out of 31 significant metabolites with 243 items. The analysis suggested several interconnected pathways, including branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), melatonin biosynthesis from serotonin and tryptophan, and dopamine biosynthesis from tyrosine.

4. Discussion

Ayahuasca consumption alters serotonin metabolism, endocannabinoids levels and amino acids balance, which is in agreement with the MAO inhibition produced by -carbolines and the accumulation of serotonin at the nerve terminals leading to serotonin syndrome.

Ayahuasca modulates peripheral endocannabinoid levels by inhibiting the fatty acid amide hydrolase (FAAH) and activating 5-HT2A receptors, although its impact on peripheral 2-AG levels is unknown.

Our study focused on peripheral metabolites, but an important unresolved question is whether circulating metabolites are a biomarker for brain endocannabinoid signalling and how these are related to behaviour.

Studies showed that the levels of 2-AG and AEA in the brain are different from those in the blood, which could explain why there is no correlation between endocannabinoid levels and the subjective effects of ayahuasca. However, the endocannabinoid system may be involved in the therapeutic effects of ayahuasca.

Ayahuasca modified the plasmatic levels of LNAA, which might be key in the role of ayahuasca in ameliorating addiction. Ayahuasca also increased BCAAs, which may stimulate the mTOR signalling pathway, which leads to increased neuroplasticity.

Relative levels of tryptophan and tyrosine correlated with subjective effects, whereas levels of BCAAs and AAAs showed a negative correlation with subjective effects. Both amino acids are precursors of monoamine neurotransmitters. Tryptophan has been associated with the stimulation of serotonin and serotonergic activity, and a lower concentration of tyrosine points to an altered concentration of dopamine in the brain, which may be related to subjective effects of “unity”, “blissful state” and “insightfulness”.

Despite the strong association between LNAA concentrations and ayahuasca subjective effects, we found only a moderate association between peripheral LNAA and DMT concentrations.

The limitations of this study include the fact that we did not control for the set and setting characteristics of the ceremonies, and that the plasma alkaloid data do not represent the complete pharmacokinetic analysis.

5. Conclusions

To our knowledge, this report is the first to show the impact on the human metabolome produced by a psychedelic drug, ayahuasca. The study identified plasma biomarkers that can profile the neurophysiological effects, as well as the connected metabolic compounds that might underlie the different subjective effects.

CRediT authorship contribution statement

This paper presents the results of a study on the influence of social networks on psychological well-being.

Acknowledgments

FMG was supported by the Dutch Research Council, the Departament d′ Universitats, Recerca i Societat de la Informacio de la Generalitat de Catalunya, and the European Social Fund.