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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Am J Addict. 2020 Jun 2;30(1):88–91. doi: 10.1111/ajad.13064

Relationship between cotinine levels and peripheral endogenous concentrations of oxytocin, β-endorphin and orexin in individuals with both alcohol and nicotine use disorders

Carolina L Haass-Koffler 1,2,3, Roberta Perciballi 2,4, Zoe E Brown 2, Mary R Lee 3, William H Zywiak 2,5, Jonathan Kurtis 6, Robert M Swift 1,7, Lorenzo Leggio 2,3,8
PMCID: PMC7944578  NIHMSID: NIHMS1671307  PMID: 32488890

Abstract

Background and Objectives

In this secondary analysis of a pilot clinical trial with individuals with alcohol and nicotine use disorders, we investigate the relationship between serum concentrations of oxytocin, β-endorphin, melatonin, α-melanocyte-stimulating hormone, substance P and orexin, with objective biomarkers (salivary cotinine and serum γ-glutamyl transferase (GGT)) as well as with self-reported smoking and alcohol drinking.

Results

We found significant positive correlations for cotinine and oxytocin (p=0.002), β-endorphin (p=0.008) and orexin (p<0.001), but not for either GGT, or self-reported smoking or alcohol drinking.

Conclusions and Scientific Significance

These preliminary results suggest a relationship between cotinine and oxytocin, β-endorphin and orexin which opens new potential hypotheses on the potential role of these endocrine pathways in tobacco smokers.

BACKGROUND

Tobacco smoking is highly prevalent among individuals with alcohol use disorder (AUD). The interactions between the reinforcing effects of nicotine and alcohol have been studied both in humans and in animal models.

Several preclinical studies have suggested a relationship between alcohol-[1] and smoking-[2] related behaviors and a variety of endogenous peptides. These peptides may act as both neurotransmitters (short-lived, local response) and hormones (longer, distant target cell), including oxytocin, β-endorphin, melatonin, α-melanocyte-stimulating hormone (α-MSH), substance P and orexin. While there is a paucity of studies on the relationship between various peptides and co-use of alcohol and tobacco, relationships among peptides and the use of each single substance have been investigated. Oxytocin administration may reduce cravings associated with alcohol and smoking relapse, and indeed oxytocin has received attention as a potential treatment for addictive disorders, including alcohol and nicotine[3, 4]. Deficits in the endogenous peptide β-endorphin may affect alcohol consumption in humans and animals[5, 6]. The melanocortin family may be an important regulator of the rewarding component in the addiction process. Substance P is crucial for the neurochemical response to stress-induced situations[7]. Substance P’s neuronal signaling plays a role in opioid response, suggesting interactions in pathways contributing to addiction-dependent behavior[8]. There is a relationship between orexin plasma concentration and intensity of tobacco craving in nicotine-dependent individuals[9], also for a recent review see[10].

Although previous studies demonstrate potential relationships between peptides and either alcohol- or smoking-related behaviors, research on the endogenous levels of these peptides in individuals with both alcohol and nicotine use disorders is lacking. Most of the previous research has focused on subjective self-reported outcomes, rather than on objective biomarkers of smoking or alcohol drinking. As such, the goal of this study was to investigate the relationship between the peripheral levels of these peptides and smoking- and alcohol-related biomarkers in patients with alcohol and nicotine use disorder. We further investigated the relationship between these peptide levels and self-reported measures of alcohol consumption and smoking. This study was exploratory in its nature and, the hypotheses developed within this study are meant to serve as a guide for more focused future studies.

METHODS

Setting of the Parent study

This secondary analysis was developed using data from a pilot clinical study (International Clinical Trial Registry: ISRCTN62137064) conducted at the Brown University and Roger Williams Medical Center, Providence, RI; both centers Institutional Review Boards approved the study. All participants signed a written informed consent, had heavy use of alcohol (men ≥5 standard drink units (SDUs), and women ≥4 SDUs, daily) and ≥10 cigarettes a day, during the 90-day period before screening. Although blood alcohol levels were zero at the time of blood draw, smokers were not nicotine deprived[11]. To ensure that participants were not intoxicated at the time of the study visits, we required participants having a BrAC of 0.00 g/dL prior to partaking in all study sessions.

Blood and Saliva Samples Collection, Storage and Analysis

Only blood samples collected at baseline were used for this secondary analysis. Blood samples were centrifuged, serum was extracted, and serum aliquots were stored at −80°C. Peptides were measured using a multiplexed, competitive format immune-assay (Millipore). Saliva samples for cotinine, a biomarker of nicotine use[12], were collected (anytime between 12:00–17:00 hour), stored at −80°C and analyzed using enzyme competitive immunoassay (Salimetrics). Blood samples for γ-glutamyl transferase (GGT) were processed by East Side Laboratory using standard autoanalyzer procedures for clinical samples. The parent study was started in 2010 and completed in 2012. The multiplexed immune-assay for oxytocin, β-endorphin, melatonin, α-MSH, substance P and orexin was run in 2011. Saliva cotinine samples were processed, and data was generated in 2012.

Data Analytic Strategy

Distributional characteristics of outcome measures were examined to evaluate similarities to the normal distribution. Normal distribution was assessed using skewness and kurtosis values between −2 and +2. Serum peptide was used to evaluate potential correlations with serum GGT, salivary cotinine, cigarettes per day and heavy drinking days, via regression analysis using Pearson’s correlation coefficient.

Results were checked for collinearities, statistical tests were two-sided and to control for multiple comparison (six peptides analyzed), significance was accepted with a conservative p-value of <0.0083. Cotinine, GGT and self-reported drinking and smoking were considered independent tests and did not require corrections for multiple comparison. Statistical Package for the Social Sciences (SPSSv.24) was used to conduct the analysis and GraphPad Prism (v.7) to generate the figures.

RESULTS

Description of the samples

Out of the 30 participants in the parent study, paired blood and saliva samples for 19 subjects were available for this analysis. There were no significant differences in the demographics: age (47.5±9.1 years), gender (46%), race (36.84 white%), and baseline characteristics: Heavy Drinking Days (9.1±5.0), Alcohol Dependence Scale (12.9±7.8), Cigarettes per day (22.3±8.5) and Fagerström Test for Nicotine Dependence, (7.0±.5) between the main sample of the parent study and the sub-group of this analysis (p’s>0.05).

Relationship between endogenous serum levels of the peptides and salivary cotinine and self-reported smoking

There was a significant positive correlation between salivary cotinine concentrations and the following peptides: oxytocin (r16=0.667, p=0.002, Figure 1A), β-endorphin (r16=0.604, p=0.008, Figure 1B) and orexin (r14=0.742, p<0.001, Figure 1C). There were no significant correlations between salivary cotinine concentrations and melatonin, α-MSH and substance P. Furthermore, there were no significant correlations between self-reported cigarettes per day and any of the peptides analyzed.

Figure 1 – Significant and positive correlations between saliva continine concentrations and serum levels of oxytocin, β-endorphin and orexin in smoker with AUD.

Figure 1 –

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Results are expressed as M ± SEMs. Significance was accepted as a pcorrected-value of < 0.0083.

Relationship between endogenous serum levels of the peptides and serum GGT levels and self-reported drinking

There were no significant correlations between any of the peptides analyzed here and either serum GGT levels or self-reported heavy drinking days.

CONCLUSIONS

This exploratory analysis revealed significant positive correlations between salivary cotinine levels and the peripheral endogenous concentrations of oxytocin, β-endorphin and orexin in individuals with both alcohol and nicotine use disorders. The peripheral concentrations of these peptides did not correlate with GGT levels, nor did they correlate with either self-reported alcohol drinking or tobacco smoking.

The positive correlation that we found between β-endorphin and cotinine is consistent with the observed effect of nicotine in increasing β-endorphin and in the reduction of anxiety and tension in smokers. Beta-endorphins are known to be involved in nicotine-related stress responses, as they are released in the peripheral circulation by nicotine stimulation. Nicotine induced neuroadaptation in the brain oxytocin system and chronic nicotine administration upregulates oxytocin receptor binding in the amygdala, the brain structure involved in stress regulation. In rodents, oxytocin increases nicotine intake and reduces stress. Nicotine increases appetite via increasing orexin. Preclinical data showed that nicotine increases ingestive behaviors by acting on pre-pro-orexin mRNA production, with consequent large distribution within the hypothalamus[13].

The positive correlations between cotinine and orexin, β-endorphin and oxytocin are supported by findings that these peptides can influence smoking behaviors. Nicotine is metabolized in the liver by cytochromes P450 and about 70–80% of metabolites are converted to cotinine which is later excreted in the urine. Cotinine represents the most reliable and objective biomarker of smoking-related behavior. The liver enzymes are not biomarkers specific for alcohol use, as elevated GGT levels may also rise as a result of obesity, diabetes, and hypertriglyceridemia. One may speculate that somehow these hormones are more related to smoking markers than alcohol markers, at least in individuals with both alcohol and nicotine use disorders, a hypothesis that should be studied in prospective a priori human studies and in preclinical models.

A limitation of this study is that the blood samples reflect the peripheral circulating levels of these hormones, therefore, no conclusion may be drawn on the potential role of these hormones at the central level. It is also important to note that, while hormones were measured in the blood, saliva was used to measure cotinine. However, we note that minimal protein binding in blood and water solubility increases concentration of cotinine in saliva by 15% to 40%, salivary cotinine can distinguish differences between active and passive smoking [14], and is just as accurate as both blood[15] and urinary[16] cotinine.

Bed-to-bench translational efforts are needed to shed light on potential mechanisms that can exemplify how the endocrine pathways analyzed here may relate to the co-use of alcohol and tobacco smoking in addictive disorders. This work represents an exploratory analysis, which on its own strongly limits the overall conclusions that can be drawn from these tentative and preliminary results. Rather, the goal of this report is to highlight data that may guide future hypothesis-driven studies on the role of these peptides in individuals with both alcohol and nicotine use disorders.

ACKNOWLEDGMENTS

Dr. Haass-Koffler is supported by the National Institute on Alcohol Abuse and Alcoholism (K01 AA023867; R01 AA026589; R01 AA027760; R21 AA027614) and by the National Institute of General Medical Sciences (NIGMS), Center of Biomedical Research Excellence (COBRE, P20 GM130414). Drs. Lee and Leggio are supported by the National Institute on Drug Abuse Intramural Research Program and the National Institute on Alcohol Abuse and Alcoholism Division of Intramural Clinical and Biological Research (ZIA AA000218, Clinical Psychoneuroendocrinology and Neuropsychopharmacology Section; PI: Leggio). Dr. Kurtis is supported by NIAID (R21 AI131047, R01 HD092301, R01 AI127699, and R0 1AI110699)

The parent study was funded by the National Institute on Alcohol Abuse and Alcoholism (R03 AA020169; PI: Leggio) and by a grant from the ABMRF/The Foundation for Alcohol Research (PI: Leggio). The authors also thank Bianca Persaud for her input in the analysis. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. The authors thank William P Koffler for his contribution to the editing of the manuscript.

Footnotes

DECLARATION OF INTERESTS

The authors report no biomedical financial interests or potential conflicts of interest.

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