Alteration of the Canine Metabolome After a 3-Week Supplementation of Cannabidiol (CBD) Containing Treats: An Exploratory Study of Healthy Animals

Subjects and Diets

Sixteen dogs (eight male, eight female, 9 months to 4 years of age, 18.2 ± 3.4 kg BW) of various mixed breeds were received from a local shelter for inclusion in this study. Additionally, the shelter was informed of and gave consent for the use of the dogs for research purposes before their arrival. Prior to beginning the experiment, each dog had a complete blood count (CBC) and serum chemistry analysis (IDEXX Laboratories, Inc., Westbrook, Maine) performed, along with physical evaluation by the attending veterinarian and a fecal examination to rule out any underlying disease that might preclude enrollment. Dogs were excluded if they demonstrated serious behavioral issues, such as human aggression that would endanger research personnel, were severely emaciated, classified as a body condition score <3.5 on a 9-point scale (where one is emaciated and 9 is obese), or if their initial evaluations revealed an underlying disease that required more than routine treatments (such as heartworm positive dogs).

Dogs were individually housed in 1.2 × 1.8 m cages within one of two dog kennels at the LMU DeBusk Veterinary Teaching Center. They were stratified by treatment and sex and evenly distributed between the two kennels. Dogs were fed Purina Pro Plan EN Gastroenteric Fiber Balance Dry Dog Food (Nestle Purina, Inc., St. Louis, MO) to meet the daily metabolizable energy requirements of neutered adult dogs at maintenance, calculated as (70 ^* BW^0.75) ^* 1.6 and split into two meals per day. Body weight and body condition score (5-point scale) were assessed weekly for the adjustment of diets. Dogs arrived from the shelter and were started on the study diet more than 37 days prior to starting treatments and 58 days before collecting samples for this study.

Experimental Design and Treatments

These dogs were participating in a concurrent study evaluating the impact of CBD on canine voluntary activity (20) with treatments consisting of 0 (placebo treats; CON) or 75.6 ± 5.86 mg CBD/d (CBD). Dogs were blocked by baseline activity before being stratified by age, weight, and sex and randomly assigned to treatments within each block. The CBD was a constituent of a proprietary industrial hemp extract (AgTech Scientific, Paris, KY) that was incorporated into treats and administered in the form of 2 treats daily, each containing half the daily dose. Both CON and CBD treats were composed of the following ingredients: chicken, chicken liver, Asian carp, catfish, and in the case of the CBD treats, industrial hemp extract. Cannabidiol was the primary constituent of the industrial hemp extract; however, trace THC was present in the CBD treatment (2.9 ± 0.22 mg THC/d). Based on the mean BW of dogs included in the study and analysis of the treats, mean dose of CBD ± SD was 4.5 ± 0.77 mg CBD/kg BW/d. Treats were offered solely as a reward upon kennel re-entry following twice-daily exercise within 30 min of meals.

Blood Sample Collection

After 21 d of treatment administration, ~6 mL of blood was collected via cephalic catheter or jugular venipuncture ~2 h after the final treat administration. The selection of this time point was based on previous work demonstrating the half-life of elimination of CBD to be between 1 and 4 h after oral administration (21–23). Blood samples were collected into tubes containing sodium heparin and were immediately centrifuged at 1,645 × g for 10 min. Plasma was collected after centrifugation then stored at −20°C (<12 h) before long-term storage at −80°C.

CIL/LC-MS-Based Untargeted Metabolomics Analysis

Untargeted metabolomic profiling was done using a CIL/LC-MS-based technique with an Agilent 1100 LC system (Palo Alto, CA) connected to a Bruker Impact HD quadrupole time-of-flight (QTOF) MS (Billerica, MA). This technique uses a differential isotope labeling (¹²C and ¹³C-labeling) to separate metabolites based on chemical groups followed by LC-MS analysis (19). Detailed information regarding sample preparation, labeling, normalization, LC-UV and LC-MS setup, and metabolite quantification have been reported elsewhere (24, 25). Typical coefficient of variation for this high-performance chemical isotope labeling LC-MS method for metabolome analysis is in the range of 5–10% for individual metabolites (26–28). In this study, amine/phenol- and carbonyl-containing metabolites were analyzed. A total of 19 LC-MS data files were generated (three quality control samples, eight CBD samples, and eight CON samples).

Metabolite Data Processing

Raw data processing on the 19 LC-MS data files was performed using ISOMS Pro 1.0 according to procedures described by Mung and Li (25). Peak pairs whose mean (sample) / mean (blank) was ≤ 4.0 were filtered out. Peak pairs with no data present in at least 80% of the samples were filtered out. The final metabolite-intensity table was generated using IsoMS-Quant (29).

Metabolite Identification

A two-tier identification approach was used to perform metabolite identification. In tier 1, peak pairs were searched against a chemical isotope-labeled metabolite library (CIL Library) based on accurate mass and retention time (29). The CIL Library contains 1,213 experimental entries, including 711 amines/phenols and 90 carbonyls. In tier 2, a linked identity library (LI Library) was used for the identification of the remaining peak pairs. The LI Library includes over 2,000 human endogenous metabolites from 68 metabolic pathways, providing high-confidence putative identification results based on accurate mass and predicted retention time matches (30).

Glucose Metabolism

The increase in glucose, an isomer of glucosamine, 2′-deamino-2′-hydroxy-6′-dehydroparomamine, and an isomer of 2-deoxy-schyllo-inosamine may suggest that glucose metabolism was altered by CBD. The endocannabinoid system—by which CBD and other cannabinoids exert physiological effects—plays a well-established role in glucose and energy metabolism, marking it as a target for the treatment of metabolic diseases like type 2 diabetes (59). Cannabinoids like CBD and THC have been suggested to reduce hyperglycemia and increase insulin production in rodents (60, 61), but this has yet to be investigated in a canine model.

Glucosamine is an amino sugar that is readily synthesized in the body from glucose and glutamine. It is an essential component of mucopolysaccharides that are incorporated into connective tissue, mucous secretions, skin, tendons, ligaments, and cartilage. Additionally, it helps regulate the synthesis of collagen in cartilage (62–64). Because of its high concentration in joint tissues, glucosamine is commonly used as a dietary supplement in humans, horses, and dogs as a support for joint health and function and to relieve symptoms of osteoarthritis, though there is little scientific evidence supporting these effects (65). Both 2-deoxy-scyllo-inosamine and 2′-deamino-2′-hydroxy-6′-dehydroparomamine are intermediates in the biosynthesis of aminoglycoside antibiotics, like kanamycin, from glucose in Streptomyces bacterial species (66, 67). However, since these metabolites are not known to be generated in mammalian systems, the biological significance of this is unclear.

The decrease in gamma-aminobutyric acid (GABA), glyceraldehyde, and an isomer of glyceraldehyde may also indicate that CBD altered glucose metabolism. Best known as the primary inhibitory neurotransmitter in the central nervous system, GABA is also produced by insulin-producing β cells of the pancreas and immune cells (68, 69). In the pancreas, GABA inhibits glucagon secretion from neighboring α cells and modulates glucose homeostasis (68, 70, 71). This action of GABA in the pancreas has highlighted its potential as a target for diabetes treatment (72). It has also been shown to regulate cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells and is thought to exert anti-inflammatory effects (73–75).

Glyceraldehyde, a triose monosaccharide, is an intermediate in glycolysis in its phosphorylated form (GAP). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the enzyme that catalyzes the conversion of GAP into 1,3-bisphosphoglycerate, is a major regulator of carbon flux in the body (76). It is also known to play a role in other cellular functions such as redox sensing, membrane fusion, iron homeostasis, and cell death (77). The decrease in glyceraldehyde may lend support to the suspected anti-obesity and anti-diabetic effects of CBD; however, the increase in glucose and decrease in GABA would appear to be incongruous with this potential effect. These results highlight a relatively unexplored avenue of CBD research that warrants further investigation.

Hydroxycinnamic Acid Derivatives

The increase in plasma isoferulic acid (IFA), trans-2,3-dihydroxycinnamate, and vanillic acid may suggest that CBD altered the metabolism of hydroxycinnamic acid derivatives. Isoferulic acid is a naturally occurring hydroxycinnamic acid derivative commonly found in Lobelia and Cimicifuga species. It is an isomer of ferulic acid, a phenolic compound that is a component of lignin, which is commonly found in cell walls of plants, including C. sativa (78). Ferulic acid has also been isolated from hemp seed meal, a byproduct of hemp oil processing (79). Both ferulic acid and IFA have been reported to have anti-inflammatory, anti-viral, anti-oxidative, and anti-diabetic properties and are commonly used as ingredients in herbal medicines in Japan and China (80, 81). Isoferulic acid has been shown to reduce plasma glucose in diabetic rats and to inhibit IL-8 production in mice (82, 83). Additionally, IFA has been suggested to act as an anti-glycation compound. Protein glycation is a non-enzymatic reaction associated with oxidative stress and reactive oxygen species (ROS) production; it is thought to be a contributor to age-related diseases (84). In several studies, IFA protected against fructose- and glucose-mediated glycation and inhibited ROS production in vitro (80, 85). In humans, IFA has been shown to be an intermediate in the metabolism of plant-derived phenolic compounds like caffeic acid (86), with humans obtaining the majority from dietary consumption.

Trans-2,3-dihydroxycinnamate is a derivative of cinnamic acid, which is an intermediate in the biosynthesis of lignin, flavonoids, and other secondary metabolites produced by plants like C. sativa (87). Cinnamic acid and its derivatives, like trans-2,3-dihydroxycinnamate, possess antioxidizing activity (88). Vanillic acid is a dihydroxybenzoic acid derivative commonly used as a flavoring agent. It is also an intermediate in the synthesis of vanillin from ferulic acid and a phenolic compound that, like IFA, is a component of lignin present in the secondary cell wall of plants, including C. sativa (89, 90). Like other lignin-associated aromatic acids, vanillic acid has been reported to exert antimicrobial properties (91). If the increase in these metabolites is a result of CBD supplementation, it is possible that these compounds may contribute to the suspected anti-microbial, anti-inflammatory, and antioxidative effects of hemp. However, it is unlikely that the changes in these compounds are solely due to increased dietary consumption as the treats used in this study provided a small quantity of industrial hemp extract. Conversely, it may be possible that changes in the metabolism of these hydroxycinnamic acid derivatives occurred as a result of CBD; additional work is warranted to further investigate these potential effects.

Vitamin and Nucleotide Metabolism

Increased concentrations of pyrimidodiazepine, 4-amino-4-deoxychorismate, 7-carboxy-7-carbaguanine, 2-formylglutarate, and ascorbate may indicate an alteration of vitamin and nucleotide metabolism. Pyrimidodiazepine is a derivative of uracil and a substrate for pyrimidodiazepine synthase, an enzyme that can contribute to glutathione synthesis. Uracil derivatives like pyrimidodiazepine are also thought to possess antimicrobial and antioxidant properties (92). 4-amino-4-deoxychorismate is a precursor for para-aminobenzoic acid (pABA) biosynthesis, which is a precursor for folic acid (vitamin B9) synthesis in plants and microorganisms (93, 94). While vitamin B9 is an essential cofactor that facilitates methyl transfers, mammals do not possess the enzymes to produce folic acid and instead rely on dietary consumption of the vitamin (93). 7-carboxy-7-carbaguanine is pyrrolopyrimidine that, like 4-amino-4-deoxychorismate, is involved in the biosynthesis of vitamin B9 (95). 2-formylglutarate is an intermediate in nicotinamide metabolism in several bacterial species [(96, 97)]. Ascorbate, or vitamin C, can be synthesized in dogs from glucose or ingested in the diet (98, 99). Vitamin C serves as a cofactor in several essential reactions, including collagen synthesis and wound healing, as well as an antioxidant. If the increase in ascorbate was due to CBD supplementation, this may contribute to the suspected antioxidative effects of CBD.

Decreased concentrations of 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate and (Z)-3-peroxyaminoacrylate may also support an effect of CBD on vitamin and nucleotide metabolism. 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate is an intermediate in the pyridoxine (vitamin B6) degradation pathway (100). Vitamin B6 is an essential cofactor in several enzymatic reactions, including the synthesis of glutathione, an important antioxidant (101). (Z)-3-peroxyaminoacrylate is an intermediate in bacterial pyrimidine degradation pathway known as the Rut pathway. However, this pathway and intermediate are not known to play a role in mammalian pyrimidine metabolism (102).

Since the relative concentrations of pyridoxine, uracil, and folate were not affected by treatment, and since several of these metabolites are not known to be generated in mammalian systems, the biological significance of these changes is unclear. However, increased pyrimidodiazepine and ascorbate, along with decreased 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate, may indicate an influence of CBD on antioxidant status. Further studies are needed to determine the roles of these metabolites and the potential effects of CBD on these pathways.

We thank Lincoln Memorial University students K. Athey, H. Barnhart, L. Calvin, K. Dubois, J. Gauldin, S. Swears, M. Kight, M. Mendoza, J. Steen, S. Swears, and K. Williams for their assistance in caring for dogs and facilitating data collection.