The association of plasma acyl ghrelin level with alcohol craving in early abstinent alcohol dependent patients.

BACKGROUND: Craving plays an important role in maintenance of alcohol dependence. Earlier studies have analyzed the role of ghrelin in craving and their results have been heterogenous. Acyl ghrelin is its more active form as it crosses the blood brain barrier. Hence we aimed to examine the relationship between plasma acyl ghrelin and craving in Indian patients having alcohol dependence syndrome.
METHODS: The present study was a hospital-based prospective study. A total of 60 drug-naive patients of alcohol dependence and 30 healthy controls were included. After taking informed consent fasting blood samples were collected from them on day 1 and tested for plasma acyl ghrelin level. Fasting blood samples were repeated in all cases on day 14. During this time, we also assessed the patients' cravings by obsessive compulsive drinking scale, and alcohol craving questionnaire; and withdrawal by clinical institute withdrawal assessment for alcohol scale. These scales were repeated on day 14. Data analysis was done by SPSS version 25.0.
RESULTS: Plasma concentrations of acyl ghrelin increased significantly during early abstinence in patients from day 1 to day 14 (P < 0.0001). Pearson correlation test revealed a trend of positive correlation between plasma concentration of acyl ghrelin on day 14 and severity of craving on day 1.
CONCLUSION: Our results suggest the plasma concentration of acyl ghrelin may be a predictor of severity of alcohol craving during early abstinence. Anti-craving drugs acting on acyl ghrelin level in brain may open an innovative avenue for optimum treatment of alcohol dependence.

Introduction

Ghrelin and its active form

Ghrelin is mainly synthesized and secreted by the entero-endocrine cells of the stomach and intestine as a precursor protein, preproghrelin.[12] This preproghrelin undergoes proteolytic cleavage to form a 28-amino-acid peptide hormone ghrelin. Then post translational acetylation of ghrelin is required for it to become functional as acyl ghrelin, which is the more active form.[3] Only this acetylated form of ghrelin is able to cross the blood—brain barrier to activate central growth-hormone secretagogue receptors (GHS-R1A) in the mesolimbic reward system[4]

Prevalence of alcohol use and craving

Alcohol use is an important public health problem worldwide. Alcohol contributes substantially to the global burden of disease with 4% mortality and 4-5% of disability adjusted life years (DALY) which accounts for 36.4% of all neuropsychiatric DALY.[56] Craving or pathological appetite means an uncontrollable desire to take a substance; a desire which, if not satisfied, provokes physical and psychological suffering. Craving plays an important role in the development and maintenance of alcohol dependence and in relapse after periods of abstinence.[7] Craving is capable of altering the will of the alcoholic patient forcing him to take alcohol in -spite of the related damage.[8]

Ghrelin and alcoholism

Few studies have shown the extension of ghrelin signals to brain regions outside the hypothalamus such as in mesolimbic dopaminergic pathways, suggesting that ghrelin may be involved in the development and maintenance of addictive disorders, such as alcohol addiction.[9] Indeed, there is growing evidence that central ghrelin-signaling plays a significant role in the regulation of alcohol intake.[1011] One pre-clinical study has shown that administering ghrelin in the ventral tegmental area (VTA) of rats increased their voluntary alcohol intake.[10] One human study has shown a link between ghrelin function and alcoholism, by showing polymorphisms in the genes responsible for encoding pro-ghrelin and ghrelin receptors to be associated with heavy alcohol consumption.[12]

Ghrelin and craving

Recently few clinical studies have investigated the relationship between plasma ghrelin levels and alcohol craving which showed a positive correlation between these two variables with increasing ghrelin plasma levels in early abstinence.[711] But there were few studies showing contradictory findings, like no relationship between plasma ghrelin levels and alcohol craving, also a statistical trend for a negative correlation between alcohol craving and plasma levels of total ghrelin in Lesch Type 1 alcoholics.[1314]

Knowledge gaps and need of the study

The heterogeneous results of aforementioned clinical studies may be due to differences in sample compositions. Few studies included intoxicated patients, whereas others included those in withdrawal. The literature was not conclusive regarding abstinence. Also these studies have analyzed the preprohormone ghrelin instead of acetylated ghrelin which is the active form; called acyl ghrelin that can cross the blood brain barrier. Only a few studies found a positive correlation between plasma acylated ghrelin and alcohol craving.[315] To the best of our knowledge, there is no study in Indian population which compared plasma concentration of acylated ghrelin in alcohol dependent patients and its relationships to severity of craving and withdrawal in early abstinence; which is the period of second stage of rehabilitation during recovery having withdrawal symptoms and craving hence more prone to relapse.[16]

So considering the heterogeneity of results in previous studies, lack of studies on active form of ghrelin in Indian population we aimed to estimate the levels of plasma acylated ghrelin in patients of alcohol dependence syndrome and compare them with matched healthy controls and to compare changes on day 1 and day 14 of detoxification in those patients. We also aimed to estimate plasma acylated ghrelin level and its relationship with craving and withdrawal among those patients in their early abstinence.

Methods

Subject and methods

This was a hospital based prospective study. The sample was drawn with computer based simple random allocation from the inpatient department of Addiction Psychiatry of a tertiary care psychiatric hospital, located in eastern part of India from May 2019 to February 2020.

Mode of selection of cases

After informed consent, 60 male patients diagnosed with alcohol dependence syndrome as active dependence, continuous and episodic use or in uncomplicated withdrawal state, as per ICD-10 (DCR) (WHO 1993) criteria abstinent for more than 72 h in the age group 18-60 years were included.[17] Patients with the severity of Alcohol Dependence Questionnaire (SADQ) score more than 20 were included.[18] Sample size was calculated with the help of statistician using the G Power 3.1.9.2 software dated April. 2019 with the input parameters like effect size 0.8, alpha error probability 0.05 and power 0.95, allocation ratio 2 (2:1). Due to paucity of female patient with alcohol dependence attending this hospital and one previous study findings of gender differences in plasma ghrelin level we included only male patients in our study.[19] Patients with present or past history of any major physical or psychiatric illness other than alcohol dependence were excluded. Patients having any other co-morbid substance dependence except nicotine and caffeine and those who were on any psychotropic medication in the 4 weeks preceding the study were excluded.

Mode of selection of controls

Control group comprised of 30 males without any psychiatric or major medical illness, which had GHQ12 score less than 3. They were demographically matched regarding age, marital status, education and occupation with the study group. Those who used substances like alcohol were excluded except usage of nicotine or caffeine. Informed consent was taken. As duration of study was short with sample collection period was less than six months we decided to enroll only 30 controls.

Operational procedure

The study was initiated after obtaining the permission from the Institutional Ethics Committee. Then clinically diagnosed patients of alcohol dependence syndrome as per ICD-10 (DCR) criteria who fulfilled the inclusion and exclusion criteria were taken up for the study after admission which ensure abstinence. Controls were recruited from the attendants of few outdoor patients who were not their first- or second-degree relatives. After obtaining written informed consent necessary sociodemographic and clinical information were collected by using structured sociodemographic and clinical information sheet from both patients and controls. Then on the first day Alcohol Craving Questionnaire-Revised version (ACQ-R) and Obsessive Compulsive Drinking Scale (OCDS) were administered to measure the different parameters of alcohol craving in the patient group.[2021] Clinical Institute Withdrawal Assessment for Alcohol scale (CIWA-Ar) was applied to know the withdrawal state severity of the patients.[22] Patients were detoxified with Benzodizapine and psychological management like motivational enhancement therapy and supportive psychotherapy was offered uniformly to all patients based on existing institutional policies. Detoxification was started on the day of admission. Till our knowledge Benzodiazepine does not have any effect on acyl ghrelin level, so considered for detoxification. Scales like OCDS, ACQ-R, CIWA-Ar were repeated on day 14 also. Healthy controls were rated only on GHQ-12.[23] Blood (10 ml) were drawn on next day morning (day 1) before breakfast from both cases and controls; after they had fasted overnight and under standardized conditions (sitting patient; from cubital vein). Same was repeated from cases on day 14 also. After collection of blood, it was allowed to clot in a serum separator tube at room temperature. It was centrifuged for approximately 15 min. The serum was aliquoted and stored at -20°C for analyzing later. ELISA kits were used for detection of plasma acyl ghrelin concentration. The kit had 96-well plates pre-coated with secondary antibody. The measured level was reported by a Biochemist and recorded.

Description of tools

Sociodemographic and clinical data sheet

A semi-structured pro-forma for recording demographic details like age, sex, marital status, education, occupation, income, were prepared. Also clinical data of alcohol intake such as duration, age of regular intake, last intake were prepared. It also included history of medical and psychiatric illness, treatment history, details of physical examination, mental status examination. Finally, diagnosis of the patient was according to the International Classification of Disease – 10th revision (Diagnostic Criteria for Research) (WHO 1993).

Severity of alcohol dependence questionnaire (SAD-Q)

It is a 20 item clinical screening tool designed to measure the presence and level of alcohol dependence. Each item is scored on a 4-point scale, giving a possible range of 0 to 60. A score of over 30 indicates severe alcohol dependence.

Alcohol Craving Questionnaire-Revised version (ACQ-R)

The ACQ-R is a thirty-item, self-report measure that includes questions for assessing an individual's desires and intentions to use alcohol, compulsivity and expectancy. The first factor contained 21 items and was labeled 'urge and intention to drink alcohol' and the second factors, containing nine items indicating positive and negative reinforcement about drinking at that specific moment. The responses range from 1 = 'strongly disagree' to 7 = 'strongly agree'.

Obsessive Compulsive Drinking Scale (OCDS)

It is a 14-item self-rating instrument which measures the severity and improvement during treatment of alcohol dependence and two subscale scores. The first 10 questions of the scale are divided into an obsessionality subset (questions 1-5) and a compulsivity subset (questions 6-10). The items, forming the subscales like drinking obsession, loss of control, alcohol consumption and social interference. All the items are rated from 0 (being the minimum) to 4 (being the maximum).

The Clinical Institute Withdrawal Assessment for Alcohol, (CIWA-Ar)

It is a ten item scale used in the assessment and management of alcohol withdrawal. Each item on the scale is scored independently, and the summation of the scores yields an aggregate value that correlates to the severity of alcohol withdrawal, with ranges of scores. Mild alcohol withdrawal being defined as a score less than or equal to 15, scores of 16–20 usually mean moderate alcohol withdrawal and, scores over 20 usually mean severe alcohol withdrawal.

General Health Questionnaire-12 (GHQ-12)

The 12- item screening questionnaire was used to see the general health and screen out any psychiatric morbidity in healthy controls. Each item was assessed on a four-point scale (less than usual, no more than usual, rather more than usual, much more than usual).

Data analysis

Data was tabulated and analyzed using Statistical Package for Social Sciences (SPSS for Windows, Version 25.0. Chicago, SPSS Inc.). The descriptive statistics used means and standard deviation for the unpaired data. Comparison was done between two groups with these measures of dispersion and variance. Group differences of continuous variables were measured by using independent Student's t test. Group differences of categorical variables were computed by using Chi-square test. Comparisons of variables between cases and controls were done using independent Student's t test and paired t test. Correlation between the clinical and biochemical variables was done with Pearson correlation test. A value of P < 0.05 was taken as significant in all aspect.

Results

Table 1 compares the sociodemographic variables between the patients and control group. Both the groups were comparable except occupation and marital status where unemployment was significantly more in patient group (P = 0.015). Most of the patients were married in comparison to control group (P = 0.001). There was no significant difference in the serum levels of acyl ghrelin between patients and control group on day 1 as shown in Table 2. Table 3 compares the serum level of acyl ghrelin levels in patient group on day 1 and day 14. There was significant rise in acyl ghrelin level day 14 (P = 0.001). Table 4 shows correlation between the clinical variables and biochemical parameters in the patients. There was significant negative correlation between OCDS score on day 1 and acyl-ghrelin level on day 1. There was significant positive correlation between ACQ score on day 1 and acyl-ghrelin level on day 14.

Table 1

Comparison of sociodemographic variables between patients and controls

Variables		Cases n=60 (%)	Controls n=30 (%)	t/X2	df	P
Age (in years)		35.63±7.99	34.13±6.24	0.89(t)	88	0.371
Education	Educated	54 (90.0%)	27 (90.0%)	0.000 (X2 )	1	1.000
	Uneducated	6 (10.0%)	3 (10.0%)
Occupation	Employed	42 (70.0%)	28 (93.3%)	6.300 (X2 )	1	0.015
	Unemployed	18 (30.0%)	2 (6.7%)
Marital Status	Married	45 (75%)	7 (23.3%)	21.885 (X2 )	1	0.000
	Unmarried	15 (25%)	23 (76.7%)

X 2=Pearson Chi-square test, t=Independent t-test

Table 2

Comparison of plasma acyl ghrelin levels between patients and controls at baseline (n=90)

Biochemical Parameter	Cases (n=60) Mean±SD	Controls (n=30) Mean±SD	t	P
Acyl Ghrelin day 1	41.12±19.35	44.34±36.93	-0.543	0.588

t=Independent t-test

Table 3

Comparison of plasma acyl ghrelin levels in patients over time (n=60)

Variables	Groups	Mean±SD	t	P
Acyl ghrelin Day 1	Cases (n=60)	41.12±19.35	-7.133	<0.001***
Acyl ghrelin day14	Cases (n=60)	88.92±52.36

t=Paired t-test

Table 4

Correlation between clinical variables and plasma acyl ghrelin levels in patient group (n=60)

Variables	AcylGhrelin day 1	AcylGhrelin day 14
OCDS (day1)	–.321*	–0.026
OCDS (day14)	–0.019	–0.138
ACQ-R (day1)	0.216	0.303*
ACQ-R (day14)	0.092	–0.016
CIWA-Ar (day1)	–0.075	–0.032
CIWA-Ar (day14)	–0.138	–0.065
SAD-Q (day1)	–.019	–.064

*P ≤ 0.05 (Pearson correlation test was used); OCDS = Obsessive Compulsive Drinking Scale; ACQ-R = Alcohol Craving Questionnaire-Revised version; CIWA-Ar = Clinical Institute Withdrawal Assessment for Alcohol; SAD-Q = Severity of alcohol dependence questionnaire

Discussion

Sociodemographic and clinical characteristics

The mean age of the alcohol dependent patients and healthy controls were (35.63 ± 7.99) years and (34.13 ± 6.246) years, respectively, which were previously matched. This finding was consistent with previous Indian study.[24] In our study greater percentage of educated patients (90%) reflects increased awareness and better identification of the illness, and thus seeking medical help. The findings of major percentage of employed patients of alcohol dependence (70%) in our study were consistent with previous epidemiological study.[23] This might reflect better financial security which motivates them for seeking medical help. However some studies found negative associations between alcohol consumption and employment.[25] So this area needs further research. Like previous Indian study majority of the patients in this study were also married (75%) as marriage before middle age is common in Indian population.[24] Marriage being a phase of transition in life, which might play the role of stressor, hence becoming an aggravating factor for alcohol use.[26] Table 1.

The mean baseline score of severity of alcohol craving and withdrawal measured by OCDS, ACQ-R and CIWA-Ar were on the higher side. This might be due to the fact that the cases selected were inpatients in a tertiary level institute; where patients who had longer duration and severe level of alcohol dependence get referred for expert management.

Comparison of acyl ghrelin level between patients and controls at baseline and its changes over time

There was no statistically significant difference between the serum levels of acyl ghrelin between the two groups at baseline. Previous studies by Calissendorff J et al. 2005 and Leggio L et al. 2013 have shown acute decrease in blood ghrelin level after administration of alcohol.[2728] But in our study alcohol dependent patients with their last intake more than 72 h were included. As the cases were all inpatient subjects, they were all in a protected environment and hence surely abstinent after admission. So in this abstinence phase where there was no exposure to alcohol their serum level of acyl ghrelin remain almost same as in controls which was decreased in previous two studies [Table 2].

There was a significant increase in the fasting plasma acyl ghrelin level in patients from baseline through day 14. Thus the level of acyl ghrelin increased during early abstinence. This finding supports the idea of affect of long term alcohol ingestion and ghrelin secretion and its activation to acyl ghrelin which in turn affect its plasma level. Such findings were similarly replicated in an earlier study where they found increase in plasma acyl ghrelin level between day 1 and day 14 of abstinence among 61 alcohol dependent inpatients[15] [Table 3].

Correlation between clinical variables and acyl ghrelin level in patient group

The present study tried to analyze the relationship between acyl ghrelin level with baseline severity of alcohol dependence (SAD-Q), craving (OCDS, ACQ-R) and alcohol withdrawal (CIWA-Ar). Similar analysis was done on day 14 during their early abstinence except for SAD-Q. There was a weak negative correlation between the plasma concentration of acyl ghrelin level and OCDS score at day 1. Though there was significant inverse relationship between them but strength of their correlation on day 1 was weak as this depends on sample size too. In a previous study there was a negative correlation between alcohol craving and plasma level of total ghrelin in Lesch type 1 alcoholics on day1.[14] There was also a positive correlation between plasma concentration of acyl ghrelin on day 14 and ACQ on day 1, which suggested that patients with higher level of craving on day 1 had higher acyl ghrelin levels on day 14. This result was in concordance with an earlier study finding.[315] This study didn't find any significant correlation of acyl ghrelin level with severity of alcohol dependence and withdrawal symptoms [Table 4].

Limitation of the study and future direction

The limitations of the study were a small sample size drawn from a specific segment of population by purposive sampling method and only including male patients. Hence a higher number of patients including both the genders would have made the results more generalizable. The study used self-report measures of craving which might have been influenced by the individual reporting. So we need to apply objective measures to increase sensitivity and specificity of the measurement. Again long term effect on craving and relapse prevention has not been studied as the objective was limited to early abstinent period only. Hence a longitudinal assessment for longer time period is recommended.

Conclusion

The plasma concentration of acyl ghrelin level increases during early abstinence period in alcohol dependent patients. Also the study shows the trend that plasma concentrations of acyl ghrelin positively affect alcohol craving during this period. Hence plasma acyl ghrelin level may be a predictor of alcohol craving. So present study adds to the growing literature on a relationship between the ghrelin system and craving in alcohol dependent patients. Hence craving mechanism can be better understood by studying the changes in ghrelin system in abstinent period. Anticraving drugs acting on acyl ghrelin level or its receptors in brain may open a new and innovative avenue for the treatment of alcohol dependence.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.