Biochemical and haematological changes in synthetic cannabinoids and marijuana users; a retrospective evaluationAbstract
Toxic and hazardeus substances have negative effects on both the environment and human health. In this study, we aimed to determine the relationship between use of synthetic cannabinoids and complete blood count parameters, neutrophil/ lymphocyte ratio (NLR), platelet/eosinophil ratio (PER) and platelet/lymphocyte ratio (PLR). 104 patients aged 18-65 years were evaluated retrospectively. Patients were divided into 3 groups; in control group (Group 1) no metabolites detected in urine samples, Group2 positive for bonsai and Group3 positive for marijuana metabolites. Neutrophils, NLR and PER were higher, while %lymphocyte and %eosinophil were lower in Group 2 than Group 1 (p<0.05). The NLR of Group 3 were higher whereas %lymphocyte were lower compared to Group 1(p<0.05). The %eosinophil were lower, in contrast PER was higher in Group 2 compared to Group 3 (p<0.05). The findings of our study showed that bonsai consuming decreased lymphocyte percentage, eosinophil percentage while increasing neutrophil percentage, NLR and PER.We also found that marijuana consumption increased neutrophil percentage and NLR, and reduced lymphocyte percentage values. We believe that further research is needed to fully elucidate the effects of cannabinoid use on blood parameters.
Keywords:
Synthetic cannabinoids; Marijuana; Hematological parameters; Biochemical parameters; Neutrophil lymphocyte ratio; Platelet eosinophil ratio.
INTRODUCTION
Marijuana (Cannabis), obtained from Cannabis sativa, is a substance used for various purposes due to its medicinal and psychoactive properties. The primary active constituent of cannabis is ∆9-tetrahydrocannabinol (THC). THC is derived from monocarboxylic acid (tetrahydrocannabinolic acid) in Cannabis sativa. Containing 21 carbon atoms in tricyclic structure, THC has high lipid solubility. This characteristic contributes to the prolongation of half-life (Kwong et al., 2013; Barrales-Cureño et al., 2020).
However the bioavailability changes based on the route of administration, the most commonly way to intake Marijuana is inhalation. THC is metabolized mainly in the liver. In oral intake, bioavailability is lower due to first pass elimination. It is converted to 11-hydroxy-THC (active metabolite) and some inactive metabolites in the liver, which are excreted via urine and partly, bile (Gonçalves et al., 2019; Uzbay, 2006).
Different synthetic cannabinoids are named variously e.g., Bonsai, K2 (Spice), and Bombay Blue. Bonzai is a cannabinoid subgroup with aminoalkylidol structure that has clinical effects similar to THC (Seely et al., 2011). While producing Bonzai, synthetic cannabinoid is primarily dissolved in an organic solvent such as acetone or ethanol. The dissolved substance is then sprayed onto the herbal material and dried (Kevser, 2017). Since mini tree leaves called "bonzai" were originally used in production, these kinds of materials were called “Bonzai”.
Both types of cannabinoids (bonzai and marijuana) have many effects such as tachycardia, hypertension, vascular tone change, changes in perception of external stimuli, impaired cognitive functions and perception of the environment which, are exerted via CB1 and CB2 receptors. The severity of the effect varies with dose, the route of administration, metabolic properties and weight of the individuals (Koltai et al., 2019; Gonçalves et al., 2019). Although CB1 receptors are concentrated mainly in the central nervous system, especially in the cerebral cortex, hippocampus, and striatum, they are also found in the peripheral nervous system affecting various organs such as the lung, liver, and kidney. CB2 receptors are mainly found in immune cells, but also in the brainstem and spinal cord. The protein content of CB1 and CB2 receptors is 44% similar. Both receptors are G-protein coupled receptor types.
Although not fully understood, there are also other receptors in the central nervous system and endothelial cells called non-CB1 and non-CB2 (Koltai et al., 2019). Limited data are available on the pharmacodynamics and pharmacokinetics of synthetic cannabinoids.
While most bonzai-derived drugs are known to be potent CB1 agonists, the mechanisms underlying their mode of action have not been precisely identified yet, and the CB and nonCB receptor interactions in the brain and peripheral tissue have not been fully explained (Seely et al., 2012). Bonsai and its metabolites reportedly have greater affinity for cannabinoid receptors than THC (Seely et al., 2012; Seely et al., 2011). Gurney et al. stated that cannabinoid derivatives reduce prolactin and luteinizing hormone (LH) levels via the CB1 receptor but do not affect dopamine levels (Gurney et al., 2014). Another study suggested that cannabinoid receptors have a regulatory effect on thyroid hormones (Ameri, 1999).
When the effects of cannabinoids on the inflammation system are examined, it is thought that the major effect is immune suppression by stimulating Apoptosis with CB2 receptors, suppression of cytokines and chemokine production (Artuç, Doğan, Demirci, 2014). Since CB2 receptors are present in the spleen and thymus, they may also play a role in hematopoietic development. This hypothesis is supported by the expression of CB2 mRNAs in macrophages, mast cells, B lymphoids, T-lymphoids, and erythroid cells. In addition, effects of anandamide, an endocannabinoid derivative, as growth stimulators for hematopoietic cells (Valk et al., 1997). The expression of CB2 receptors in leukocytes was examined with anti-CB2 antibodies. CB2 receptor expression is reportedly most common in B lymphocytes, followed by natural killer (NK) cells and T lymphocytes. These receptors were found to be particularly effective in B lymphocyte differentiation (Carayon et al., 1998; Alshaarawy, 2019).
In a study examining the effect of anandamide on platelets, an increase in platelet concentration and activation after cannabinoid exposure was found (Grambow et al., 2016). In addition to their primary function in hemostasis, platelets are also effective in the innate and acquired immune system (Voudoukis, Karmiris, Koutroubakis, 2014). This is thought to be accomplished by secretion of pro-inflammatory agents from the intracellular granules when activated. Therefore, the proinflammatory effects of the cannabinoid derivative, anandamide, may be monitored by the PLR. In addition, synthetic cannabinoids are known to have proinflammatory effects, affect complete blood count (CBC) and indirectly calculated NLR (Guzel et al., 2017). Neutrophil count increases and lymphocyte count decreases during inflammation. NLR enables the evaluation of both parameters together and information about both immune pathways can be obtained (Elbistanli et al., 2017). In addition, there are studies showing that NLR is used as an indicator of subclinical inflammation in the evaluation of disease processes in coronary artery disease and cancer (Duffy et al., 2006; Halazun et al., 2009).
In recent years, studies on the effects of cannabinoids on the environment and human health have been increasing. However, the preparation, content and exposure amount of cannabinoids cannot be standardized, different opinions stand out on the effects of cannabinoids on the inflammation system. In our study, we investigated the changes between cannabinoid use and the NLR, PLR, PER which are seen as potential inflammatory markers.
MATERIAL AND METHODS
This retrospective study included 104 patients who were admitted to Sakarya University Faculty of Medicine Training and Research Hospital between July 2017-2019. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Creatinine, Sodium (Na), Potassium (K) and CBC parameters (White Blood Cell (WBC), Red Blood Cell (RBC), Hemoglobin (Hb), Hematocrit (Hct), Platelet number (PLT), Mean Erythrocyte Volume (MCV), Mean Platelet Volume (MPV), Erythrocyte Distribution Width (RDW) and Platelet Distribution Width(PDW), Mean Erythrocyte Hemoglobin (MCH), Mean Erythrocyte Hemoglobin Concentration (MCHC), Neutrophil%, Neutrophil, Lymphocyte, Lymphocyte%, Monocyte, Monocyte%, Eosinophil, Eosinophil%, Basophil, Basophil%) levels in blood samples were analyzed along with spot urine toxic substance levels. In addition, NLR, PER and PLR were calculated. Participants were divided into three groups based on the level of cannabinoid metabolites found in spot urine samples: Those testing negative for any toxic substances were included Group 1 (Control group). Group 2 comprised of those with urine bonsai metabolite levels >20 ng/mL, and Group 3 included those with >50 ng/mL marijuana metabolites. The used cutoff values were determined by Substance Use and Mental Health Services (SAMHSA) for immune methods (50 ng/mL for marijuana and 20 ng/mL for synthetic cannabinoids) (Clinical Drug Testing in Primary Care, 2012). Participants who were not between the ages of 18-65 years, whose serum ALT, AST, Urea, Creatinine, Na, K, and CBC parameters and spot urine toxic substances were not determined within the same day and samples that did not meet the criteria of the Working Principles of Medical Laboratories that Perform Illicit Drug and Substance Analysis in Urine Samples and Medical Laboratories in Substance Addiction Diagnosis and Treatment Centers were excluded from the study. Ethics committee approval was obtained from the Non-Interventional Ethics Committee of the Faculty of Medicine of Sakarya University with the decision number 104 on 02.10.2019. CBC parameters were measured by CELL-DYN 3700 CD-3700SL with LED
Flow Cell method (Abbott Diagnostics Diver, Abbott Laboratories Abbott Park IL, 60064, USA). Biochemical analytes were analyzed using the spectrophotometric method in an Olympus AU5800 (Beckman Coulter, Inc. Brea, CA92821 U.S.A.) and a Beckman AU680 Coulter autoanalyzer (Beckman Coulter, Inc. Brea, CA92821 U.S.A.). Urine toxic substances levels were determined by Homogen Enzyme Immunoassay method with Olympus AU480 autoanalyzer (Beckman Coulter, Inc. Brea, CA92821 U.S.A.). At all stages from sample collection to sample analysis, the "Working Principles of Medical Laboratories that Perform Illicit Drug and Substance Analysis in Urine Samples and Medical Laboratories in Substance Addiction Diagnosis and Treatment Centers (2016)" guidelines were taken into consideration.
Statistical Analysis
Statistical analysis of the data was performed with SPSS 20.0 (SPSS Inc., Chicago, IL, United States). Mean and standard deviations of the measurands within each group were calculated. The normality of distribution was determined by Shapiro-Wilk test. One-way analysis of variance (Oneway ANOVA) and Post-hoc Tukey test were used to compare the groups with normally distributed. Kruskal Wallis Analysis and Mann Whitney U test were performed for non-normally distributed parameters. The relationship between the parameters was determined by the Spearman correlation test and the Pearson correlation test. p <0.05 was considered significant.
RESULTS AND DISCUSSION
The mean ages of individuals in Groups 1 (n=38), 2 (n= 25) and 3 (n=41) were 37,3±26,8, 35,2±26,8 and 31,7±20,6 years, respectively (Table I).
We found that neutrophil count, NLR and PER were higher, while lymphocyte percentage, eosinophil count and eosinophil percentage were lower in Group 2 than Group 1 (p <0.05). The neutrophil count and NLR values of Group 3 were higher whereas lymphocyte percentage and lymphocyte count were lower compared to Group 1 (p <0.05). The neutrophil count and NLR were higher, and lymphocyte% and lymphocyte count were lower in patients testing positive for marijuana when compared to the control group. Finally, eosinophil count and eosinophil % were lower and PER was higher in Group 2 compared to Group 3. The complete comparison of CBC and biochemical parameters are presented in Table II.
Association of Oxidative Stress biomarkers with self-declared medications class of methotrexate users from a private clinic. Ijuí RS, 2021 - 2022 n=100
Correlation analysis was performed to determine the relationship between the parameters across all three groups: Bonsai usage positively correlated with neutrophil%, neutrophil, NLR and PER and negatively correlated with lymphocyte%, eosinophil%, red blood cell count, eosinophil count and lymphocyte count (p <0.05). Marijuana use positively correlated with neutrophil%, NLR and PLR, and negatively correlated with lymphocyte%, monocyte% and lymphocyte count (p <0.05) (Table III).
Marijuana and synthetic cannabinoid consumption are important health problems throughout the world. Several studies have been conducted on the clinical presentation, treatment, and blood parameter changes of patients abusing these drugs and numerous studies are still ongoing. In this study, we aimed to investigate the effects of two different cannabis species (marijuana and bonsai) on biochemical, hematological, and thyroid hormone parameters. As a result of our retrospective study, we found that the use of marijuana and bonsai had different effects on various CBC parameters which could also be used as inflammation markers (NLR, PLR, PER).
In studies investigating the relationship between synthetic cannabinoid use and hematological parameters, cannabinoids were found to have proinflammatory effects as shown by some CBC parameters, especially the neutrophil-lymphocyte ratio (Guzel et al., 2017). In case of inflammation, neutrophil count increases while lymphocyte count decreases. Therefore, increased NLR, which is considered an indicator of subclinical inflammation, has been the subject of numerous studies (Elbistanli et al., 2017; Halazun et al., 2009; Soder et al., 2020).
In two separate studies, (Duffy et al., 2006; Halazun et al., 2009) NLR was considered a suitable predictor in evaluating coronary syndrome and hepatocellular cancer progression. There are various studies which report an increase in NLR in schizophrenia and bipolar disorder (Çakır et al., 2015; Semiz et al., 2014). In our study, NLR was significantly higher in both marijuana and bonsai users compared to the control group, but there was no significant difference between the two. This finding suggests that marijuana and synthetic cannabinoids may trigger the inflammatory process. Although it is an easily obtainable parameter, NLR levels are may also be elevated in many other diseases or by the abuse of numerous drugs (Cıcek et al., 2018; Çakır et al., 2015; Semiz et al., 2014). Therefore, we believe that more comprehensive and detailed studies are needed to determine the relationship between NLR, marijuana and synthetic cannabinoid consumption.
Güzel et al. (2017) compared the sub-parameters of whole blood counts between synthetic cannabinoid users and a control group to find that MPV and lymphocyte ratios were significantly lower in synthetic cannabinoid users, whereas MPV, monocyte and neutrophil counts were higher. A statistically significant difference was found between the groups in terms of NLR, while no significant difference was reported for PLR. In our study, akin to the results of Güzel et al. neutrophil% and NLR were higher in bonsai users than the control group, and PLR values was similar. However, we found that eosinophil% and eosinophil counts were lower in bonsai-using patients than both the control and marijuana-positive groups. This may be due to the differences in the characteristics and gender distribution of the patient groups in the studies, as well as the different amounts of chemicals contained in synthetic cannabinoids. In their study comparing marijuana-user patients (24 individuals) with a control group (16 individuals), Oseni et al. (2006) found a 12.5% decrease in lymphocyte levels of marijuana users. Similarly, in our study, the number of lymphocytes and lymphocyte% in the marijuana positive group and the percentage of lymphocytes in the bonsai-positive group were lower than the control group. Neutrophils and NLR were increased in both bonsai-positive and marijuana-positive groups.
Valk et al. (1997) stated that CB2 receptors may play a role in hematopoietic development. These researchers found that CB2 mRNA was expressed in macrophages, mast cells, B lymphoid, T-lymphoid, and erythroid cells. Anandamide, an endocannabinoid, acts as a growth stimulator for hematopoietic cells.
Similarly, Alshaarawy (2019) stated that the WBC count increased after cannabinoid use, especially in the level of neutrophils. However, it was stated that this increase may also be due to the increase in susceptibility to infections after cannabis use.
Carayon et al. (1998) also examined the expression of CB2 receptors in leukocytes, showing that the amount of cannabinoid receptors are increased in lymphocytes. Considering this information, the decrease in lymphocyte number and rates in our study may be the due to the effects of marijuana and bonsai on CB2 receptors. It may also be considered that the lymphocyte number and ratio may be decreased to compensate for the increased neutrophil ratio. As a matter of fact, it is known that lymphopenia may be accompanied by neutrophilia in inflammation (Elbistanli et al., 2017).
In our study, there was no statistically significant difference in eosinophil levels between the marijuana-positive and control groups. However, the eosinophil percentage and count of the bonsai-positive group were lower than that of the marijuana-positive and control groups. Unlike our study, Oseni et al. (2006) found an increase in eosinophil counts in 4.2% of marijuana users compared to non-marijuana users. In their study comparing marijuana users and control individuals, Eledo et al. (2015), found an increase in eosinophil counts in the marijuana group. They reported that this difference may be due to individual response, as well as contamination with fungal spores during cannabis preparation. Oka et al. (2004) found that eosinophils had CB2 receptors and examined the effect of endocannabinoids such as Anandamide and 2-Arachidonoylglycerol (2-AG) on them. They indicated that 2-AG induces the migration of eosinophils with a full agonistic effect on the CB2 receptor, which may be closely related to inflammation. They also found that anandamide, which is also endocannabinoid, does not affect eosinophil migration as opposed to 2-AG, which is a partial agonist of CB2. In our study, the decrease in eosinophil levels in bonsai consumers supports the idea that synthetic cannabinoids affect eosinophil count via CB2 receptors. It should also be noted that the additives contained in the commercially available synthetic cannabinoid forms may cause such effects.
The results of our study showed that the use of bonsai increased neutrophils, NLR and PER while decreasing eosinophil%, lymphocyte and eosinophil counts. It also reveals that the use of Marijuana increases neutrophil count and NLR while decreasing lymphocyte% and lymphocyte count. We think that these changes may occur by acting on the CB2 receptors in various blood cells of synthetic cannabinoids.
THC and synthetic cannabinoid consumption are an increasingly important health problem. It is now known that there are over a hundred distinct types of synthetic cannabinoids, and very few of them can be detected in laboratories. There are different studies on the effects of THC and synthetic cannabinoids on receptor levels. We may conclude that the conflicting results across various studies are due to the different study populations, differences in dose and metabolism between individuals, inability to standardize the chemicals contained in commercially available synthetic cannabinoids and the use of different synthetic cannabinoids that cannot be detected in the laboratory. Therefore, further research is needed in this field both in vitro and in large populations to fully elucidate the mechanisms and effects of synthetic cannabinoid use on blood parameters.
DATA AVAILABILITY STATEMENT
Data available from the corresponding author upon reasonable request.
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Associate Editor:
Luis Salazar
