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Original Article

Assessment of early morning serum cortisol levels

in adult male patients with alcohol-related
disorders

Barun K. Chakrabarty a, Karan Sud b, Prosenjit Ganguli c,*, S.A. Khan d

a
Classified Specialist (Pathology), 151 Base Hospital, Guwahati, Assam, India
b
Graded Specialist (Psychiatry), 151 Base Hospital, Guwahati, Assam, India
c
Senior Advisor (Path & Micro), Command Hospital (Eastern Command), Kolkata, India
d
Classified Specialist (Psychiatry), Command Hospital (Eastern Command), Kolkata, India

article info abstract

Article history: Background: Alcohol-related disorders are a major health problem among Indian male
Received 20 August 2018 professionals because of the unique nature of socioeconomic and demographic conditions.
Accepted 5 March 2020 Various studies have highlighted the association between alcohol-related disorders and
Available online 26 November 2020 hypothalamic-pituitary-adrenal (HPA) axis dysfunction, but the evidence accrued so far is
inconclusive. In our study, we have assessed early morning serum total cortisol concen-
Keywords: tration among Indian adult male population affected with alcohol-related disorder.
Serum cortisol Methods: A case-based cross-sectional study in which all consecutive patients admitted in
Alcohol-related disorder the psychiatry ward of a tertiary care hospital with diagnosis of ‘alcohol-related disorders’,
Enzyme-linked fluorescent assay who were meeting all the inclusion criteria, and who had none of the exclusion criteria
were part of the study. Diseased controls and healthy controls were chosen by applying
strict inclusion and exclusion criteria. Serum early morning (0400 h) total cortisol levels
were estimated using automated quantitative enzyme-linked fluorescent assay technique.
Results: 98 psychiatric patients and 50 healthy controls were evaluated. Out of these 98
patients 66 patients were diagnosed cases of alcohol-related disorder. Morning serum total
cortisol levels in patients with alcohol-related disorders was found to be significantly
different from healthy controls.
Conclusion: Our study suggests that alcohol-related disorders are associated with chronic
changes in HPA axis and significant alteration of early morning serum total cortisol levels
were demonstrated in this group of patients.
© 2020 Director General, Armed Forces Medical Services. Published by Elsevier, a division of
RELX India Pvt. Ltd. All rights reserved.

* Corresponding author.
E-mail address: prosenjitganguli@hotmail.com (P. Ganguli).
https://doi.org/10.1016/j.mjafi.2020.03.001
0377-1237/© 2020 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd. All rights
reserved.
48 m e d i c a l j o u r n a l a r m e d f o r c e s i n d i a 7 8 ( 2 0 2 2 ) 4 7 e5 3
Introduction
The conceptualization and extent of alcohol-related disorders
have evolved substantially over time. Two main diagnostic
classification systems for this group of disorders established
by the World Health Organization and the American Psychi-
atric Association are the International Classification of Dis-
eases (ICD) and Diagnostic and Statistical Manual of Mental
Disorders (DSM), respectively. The present classification of
ICD-10 distinguishes between harmful use of alcohol and
dependence which is broadly similar to the previous version
of the DSM namely, the DSM-IV (text revision). The most
recent edition of the DSM series, the DSM-5, was published in
May 2013 and is widely deployed in the United States by cli-
nicians and researchers alike. The DSM-5 brought about a
paradigm shift by incorporating two separate DSM-IV termi-
nologies, that is, alcohol abuse and alcohol dependence under
a single disorder by renaming them as alcohol use disorder
(AUD). Under the DSMe5, the diagnostic threshold was also
modified, whereby anyone meeting any two of the 11 criteria
during the 12-month period would receive a diagnosis of
AUD.1 According to the DSM-5, alcohol-related disorders
comprise AUD, intoxication, withdrawal, other alcohol-
induced disorders, and other unspecified alcohol-related dis-
orders. The DSM-5 Task Force showed substantial interest in
establishing biomarkers, but due to unavailability of appro-
priate diagnostic tests, it recommended for the requirement of
continued research in this area.2,3
Recent research highlighted the effects of alcohol on cortisol
levels in the human body.4 Cortisol secreted from the adrenal
gland, which is a part of the hypothalamic-pituitary-adrenal
(HPA) axis, acts as a stress hormone. High stress-induced
cortisol release can result in the temporary increase in heart
rate and blood pressure with simultaneous cessation of various
metabolic physical processes related to digestion, reproduction,
developmental, and immune function aiming to conserve energy
against the stress response but may result in damage to the
human body.5 AUD represents a multidimensional psychiatric
abnormality where alcohol intoxication and withdrawal, func-
tion as two distinct activators of the HPA axis to increase circu-
latory levels of cortisol. But the repeated activation of the HPA
axis by chronic exposure to alcohol appears to produce neuro-
endocrine tolerance resulting in altered glucocorticoid response
to alcohol.6 Although the mechanisms underlying these endo-
crine disturbances are not completely understood, recent
research has suggested that the alcohol affects the central ner-
vous system and the corticotropin-releasing factor neurons of
HPA axis, resulting in alteration of cortisol levels by the feedback
mechanism.7 However, themajor limiting factors that determine
the relation of chronic alcohol exposure with cortisol levels is the
coexisting effects of other psychiatric anomalies (e.g., depres-
sion, mania, and so on) and other chronic illness (e.g., liver dis-
ease, malnutrition, and so on).
Cortisol levels in the human body maintains the circadian
rhythm. In most people, cortisol levels are highest when they
get up in the morning and lowest around midnight hours.
There are various methods of measurement of cortisol levels,
and the level can be measured from serum/plasma, saliva, or
from urine of the patient.8
In our study, we have measured the early morning serum
total cortisol levels by automated quantitative enzyme-linked
fluorescent assay (ELFA) technique in patients diagnosed with
alcohol-related disorder admitted in the psychiatric ward of a
multispecialty hospital and compared the measured levels
with both healthy controls and psychiatric nonalcoholics.
Materials and methods
This study was conducted in the department of laboratory
medicine in collaboration with the department of psychia-
try of a 699-bedded hospital for a period of one month in
January 2018. It is a single-centre case-based cross-sectional
study in a tertiary care hospital located in North eastern
India. The sample size was calculated on the basis of a study
by Mendelson et al,9 using mean value of morning serum
cortisol levels in alcoholics and non-alcoholics after period
of cessation of drinking, with an alpha cut-off of 5% and
setting the study power at 90%, it was determined as 58.
This was determined using the minimum number of study
subjects per group required for the conducted study using
the standard formula for sample size calculation in a
comparative study.10 The sample size of the study was 148
study subjects comprising 66 patients with alcohol-related
disorder, 32 nonalcoholic diseased controls, and 50 healthy
controls.
Study subjects
We have followed universal sampling technique for our study.
During the period of the study, of one month, all patients
attending out patient department (OPD) who had been diag-
nosed as having alcohol-related disorders as per the DSM-5
guidelines and admitted to the psychiatric ward were
enrolled for the study following inclusion and exclusion
criteria. The participants were interviewed and examined by a
competent psychiatrist, and the diagnosis was arrived at by a
detailed alcohol history. All the patients evaluated were
ambulatory during the period of study, were under appro-
priate treatment, had access to all ward recreational facilities,
and were not segregated from other patients. These subjects
were maintained on a well-balanced diet along with daily
multivitamin supplements. The patients had no history of
alcohol intake after admission. From the study population, the
target sample group was derived by applying the following
strict exclusion criteria. All female patients, patients with
malignancy, patients on hormonal therapy, nonambulatory/
bedridden patients, patients with history of recent trauma or
surgery within the last one month, and patients with a history
of alcohol intake within the last 48 h were excluded from the
study. Healthy controls were taken from caregivers accom-
panying the psychiatric patients residing within the ward, and
the diseased controls were taken from nonalcoholic psychi-
atric patients. Both for the patients and controls, meals were
provided at 0900, 1330, and 2000h, and room lights were
turned off between 21:00 and 23:00 h before the day of the test.
Institutional ethical committee approval was obtained, and
written informed consent was obtained from all participants.
 m e d i c a l j o u r n a l a r m e d f o r c e s i n d i a 7 8 ( 2 0 2 2 ) 4 7 e5 3 49

Procedure

Early morning (0400hrs) fasting sample of 05 mL of venous

blood was collected from each participant in gel vacutainer.
Controls refrained from alcohol consumption for at least 24 h
before testing. Collected blood serum was then separated and
processed within a maximum of six hours of collection, and
the blood samples were processed as per recommendations of
the kit manufacturer. The serum total cortisol level was esti-
mated using automated quantitative estimation by enzyme-

linked fluorescent assay technique (ELFA) on BIOMERIEUX
Vidas table topped immune auto analyzer equipment. Proper
calibration procedure using the calibrator provided within the
kit was performed. Tests were performed using proper quality
control measures. The measurement range of the used VIDAS
Cortisol S kit was from 2 to 650 ng/mL. The normal reference
range for early morning serum total cortisol used was Fig. 1 e Box plots for early morning serum total cortisol
54.94e287.56 ng/mL which was obtained from the kit values of studied population.
literature.

Statistical analysis
All data were tabulated into MS Excel with Windows 10
operating system and analyzed using Statistical Package for
the Social Sciences (SPSS Inc., USA) software, version 20. Pa-
tient groupespecific mean (Xm) and standard deviation (Sx) of
serum cortisol levels were calculated separately. Multiple
comparison analysis was performed using analysis of
variance and post hoc analysis to see the significance of dif-
ferences among the studied groups for a level of significance
p < 0.05. The Pearson's correlation coefficient was used for
analyzing the correlation between age and early morning
serum total cortisol.
Results
A total of 98 psychiatric patients were admitted to the psy-
chiatric ward during the study period, and 50 healthy controls
were evaluated. Of 98 patients, 66 patients were diagnosed as
cases with alcohol-related disorder. The nonalcoholic disease
control group comprised 12 patients with mood disorder, 04
patients with psychotic disorder, 10 patients with anxiety
disorder, and 06 patients with other psychiatric illnesses. The
average age and the serum total cortisol of the studied groups
are shown in Table 1. There was a statistically significant
difference observed in the early morning serum cortisol level
among the studied groups (p ¼ 0.001). Box plots of three
studied groups of serum cortisol values are depicted in Fig. 1.
The observed values of serum cortisol were found to be low in
patients with alcohol-related disorder than in ‘healthy control
population’ which was statistically significant (p ¼ 0.001).
Although the cortisol level was also low in the diseased con-
trol, this difference was not statistically significant (p ¼ 0.105)
from healthy controls. No statistically significant correlation
was found between the age of the persons and early morning
serum total cortisol concentrations in any of the studied
groups.
Discussion
Alcohol-related disorders are a known health problem,
especially among the professional male population. The dis-
order may exhibit several clinical, hematological, biochem-
ical, and hormonal changes. There is growing consciousness
of the widespread metabolic effect that forms the basis of
various laboratory tests for alcohol abuse.11 Recent research
has demonstrated that alcoholism affects hormonal axis but
to what extent, is yet to be discovered.12 Alcohol-related dis-
order changes are seen in HPA axis and extrahypothalamic
glucocorticoid pathways as a part of the compensatory con-
sequences, and it is postulated that these effects may play a
role in compulsive alcohol intake motivation.13 In patients
with AUD, alcohol was found to produce acute and chronic
effects on the hypothalamic, extrahypothalamic limbic-
striatal, and prefrontal stress pathways which may influence
alcoholic behaviour.14,15

Table 1 e Characteristics of serum total cortisol levels in the study population.

Study groups Number of Age p Serum total cortisol level p
participants (mean ± SD) value (mean ± SD) value
Patients with alcohol-related 66 34.98 ± 6.24 0.000 145.79 ± 40.72 0.001
disorder
Nonalcoholic psychiatric disorder 32 32.78 ± 5.79 156.10 ± 44.03
Healthy controls 50 28.02 ± 5.8 175.56 ± 42.52

SD, standard deviation.

50 m e d i c a l j o u r n a l a r m e d f o r c e s i n d i a 7 8 ( 2 0 2 2 ) 4 7 e5 3
Glucocorticoids are synthesized in the adrenal cortex, and
cortisol is the main glucocorticoid among them. The amount
of circulating cortisol is normally subject to the circadian
rhythm depending on a negative feedback mechanism con-
trolling the release of adrenocorticotropic hormone. Its
rhythm is suggested to be further modulated by various
other factors, including sleep cycle, age, ethnicity, gender,
body mass index, socioeconomic status, chronic illness, and
menstrual cycle, but data available are conflicting. If we
demonstrate normal 24-h blood cortisol secretion curve in
human being, the secretion reaches at the highest level in
early morning at the time of awakening. Subsequently the
secretion level reaches a nadir in the first half of sleep cycle.
Minor upsurges can be appreciated in relation to meals
during the middle of the day and early evening. Cortisol
predominantly circulates in the blood in carrier protein
corticosteroid-binding globulin or transcortin and albumin-
bound form (90%).8,16 Since 1950s, many studies have
shown a relationship between the body cortisol level and
alcoholism. However, most of the previous studies were
limited to short-term alcohol consumption effects. In 1966,
the first study carried out by Mendelson et al.9 showed that
during active drinking, long-term alcohol ingestion increases
cortisol levels in both alcoholics and in nonalcoholics, but
the cortisol levels were found to be higher in alcoholics than
in nonalcoholics. Furthermore, in the study, cortisol levels
were also suggested as a potential marker for alcohol with-
drawal. In another study, overnight urinary cortisol levels
were found to be increased with increased amounts of
alcohol ingestion.17 Stalder et al. 18 showed in their study
that alcoholics had higher hair cortisol concentrations than
abstinent alcoholics or nonalcoholics. However, a study
carried out by Bernardy et al.19 and Blaine et al.11 indicated
that up to four weeks there was a decreased adrenocortical
response to behavioural stress in abstinent alcoholics and
higher stress cortisol values were seen in patients with se-
vere withdrawal. Several other studies have indicated
attenuated cortisol response in chronic alcoholics.20 In our
study, we found that during abstinence in the hospital ward
admission period and while under management, the early
morning serum cortisol levels in patients with alcohol-
related disorder were significantly lower than in the
healthy controls, suggesting altered HPA function in patients
with alcohol-related disorder. It has been hypothesized that
reduced cortisol responses in alcoholics may be suggestive of
greater intrinsic or acquired tolerance to alcohol.19 We may
speculate that the lower early morning serum cortisol level is
due to lower activation of the HPA axis observed in patients
with alcohol-related disorder and may represent a useful
diagnostic information of inclination to alcohol misuse,
although direct confirmation for this theory is required. This
finding may be associated with other findings of hypores-
ponsiveness to physical and psychological stress in patients
with alcohol-related disorder.11,21 The prestress baseline
cortisol values also suggest the alteration of a normal
circadian rhythm in the patients with alcohol-related dis-
order. Patients with alcohol-related disorder are exhausted
in the morning without their alcohol and have likely altered
circadian rhythm in such a way that they need alcohol to
boost the level of easiness they used to achieve without it.
Studies revealed that alteration of normal usual upsurging
pattern of cortisol secretion is reestablished if strict absti-
nence is maintained.22 Therefore measured attenuated value
in our study can be used in follow-up of patients. It also in-
dicates the chances of relapse. However, it should also be
remembered that HPA regulation and stress response may
not completely recover even after the diurnal pattern be-
comes normal.23 Scant, but allusive, evidence indicates that
a hyporesponsive HPA axis and resultant alteration of
cortisol levels correlates with the severity of the underlying
addictive practice.24
Regarding methodological issues in this study, the human
body cortisol may be sampled from either blood, urine, or
saliva with each sample source having definite advantages and
disadvantages, thereby focusing on different views of HPA
functioning. The serum or plasma level of cortisol determines
the intensity of cortisol secretion and its tissue effects,
whereas urine cortisol indicates the function of the cortex of
the adrenal gland. Studies exhibited that there is a good cor-
relation between saliva cortisol and serum cortisol levels but
saliva cortisol level is difficult to be evaluated.25 Most studies
have used blood sampling for its definite advantages.8 There-
fore, in our study, we had used serum samples. Serum/plasma
cortisol levels can be measured by chromatographic or
immunoassay methods. Chromatographic methods have a
specific advantage of accuracy that they are suitable only for
experimental/research studies. On the contrary, immuno-
assay which is the most frequently used technique for serum
cortisol assessment has its inherent limitations and proce-
dural advantages. In this study, we have used nonisotopic
quantitative automated enzyme-linked fluorescent assay
technique. Measurement of cortisol blood levels can be per-
formed by total cortisol assay or free cortisol assay. Methods
for serum-free cortisol assay are limited by the inherent
complexity, and they are time-consuming, expensive proced-
ures. In our study, we have performed the total serum cortisol
level assay which is more accurate and established.26
Based on several studies, it is hypothesized that in-
terventions that normalize the high tonic and blunted phasic
response of cortisol by decreasing the tonic level as well as
enhancing the phasic response to alcohol may effectively
address the AUD management.27 Our study highlighted the
altered biological response of the HPA axis which may be
useful in the objective identification of alcohol-related disor-
ders and also indicates the possible therapeutic implications
of the study results.
In studies, cortisol dynamics demonstrated different fre-
quency and basal secretion in women than in men. Various
studies showed conflicting results of age on cortisol level
except on extreme ages.28 Our study could not demonstrate
statistically significant age effects on early morning serum
cortisol concentrations.
Many previous studies have tried to develop a diagnostic
system incorporating a test panel for alcoholism.29 Until now
most of the assessment of alcoholism is solely based on
clinical parameters evaluated either by an interview format or
a questionnaire method. There are several limitations of these
methods leading to long drawn management difficulties and
loss of effective manpower. The controversy over the effec-
tiveness of incorporation of diagnostic tests is principally
Table 2 e Traditional alcohol biomarkers.
Biomarkers
Gamma-glutamyltransferase
(GGT)
Aspartate aminotransferase
Sensitivity Specificity Detection limits Current and future Limitations
possible use
37e95% 18e93% Minimum >5e6 drinks of alcohol Screening of alcoholism; 1.Low specificity
intake required for detection; Chronic alcohol abuse 2.Analysis of samples must be executed on
After consumption maximum time of the same day
positivity 6e8 weeks in absence of liver 3.Not suitable for monitoring of abstinence
abnormality 4.Performs poorly in people younger (<30 years)
and in women
5.More strongly associated with obesity than
alcohol
25e60% 47e68% AST/ALT return to normal after Chronic alcohol abuse 1.1.Low specificity

m e d i c a l j o u r n a l a r m e d f o r c e s i n d i a 7 8 ( 2 0 2 2 ) 4 7 e5 3
(AST) termination or reduction of alcohol 2.2.Analysis of samples must be executed on the
Alanine aminotransferase 15e40% 50e57% consumption for 2e4 weeks same day
(ALT) 3.3.Performs poorly in people younger (<30 years)
and older (>70 years)
4.4.Not suitable for monitoring of abstinence
Mean corpuscular 40e50% 80e90% MCV return to normal after termination or Heavy alcohol usea; 1.1.Low specificity
volume (MCV) reduction of alcohol consumption for Screening of alcoholism 2.2.Analysis of samples must be executed on
8e16 weeks; the same day
Minimum >5e6 drinks of alcohol intake 3.3.Not suitable for monitoring of abstinence
required for detection
Carbohydrate-deficient 46e90% 70e100% MCV return to normal after termination or Heavy alcohol usea; 1.1.Specific but not very sensitive
transferrin (CDT) reduction of alcohol consumption for Current heavy drinking and 2.2.Moderate or episodic drinking show normal
2e3 weeks; relapse detection range CDT levels.
Minimum >5e6 drinks of alcohol intake 3.False-positive results may occur due to rare
required for detection genetic variations
4. Difficult to measure accurately.
5. Yet ongoing standardization
6. Analysis of samples must be
executed on the same day
CDT, MCV, and GGT in 88% 95% Chronic excessive drinking
combination
a
More than 60 g per day (4e5 standard drinks).

51
52 m e d i c a l j o u r n a l a r m e d f o r c e s i n d i a 7 8 ( 2 0 2 2 ) 4 7 e5 3
related to test panel protocols. With the introduction of the
DSM-5, the spectrum of alcohol-related disorders has
expanded which also necessitates the requirement of
extended research on the diagnostic tests.
Traditional biomarkers that are related with hazardous
drinking are gamma-glutamyltransferase, aspartate amino-
transferase, alanine aminotransferase, mean corpuscular
volume, and carbohydrate-deficient transferrin. There are
various limitations and shortcomings noted for currently used
traditional biomarkers of alcohol-related disorders (Table
2).30,31 Recent research revealed several potential biochem-
ical markers for a more accurate reflection of alcohol-related
disorders than traditional markers (e.g.. ethyl glucuronide,
ethyl sulphate, acetaldehyde, acetaldehyde adducts, and
antiadduct antibodies, fatty acid ethyl esters, phosphatidyle-
thanol, b-hexosaminidase, total serum sialic acid, plasma
sialic-acid index of apolipoprotein J, cholesteryl ester transfer
protein, 5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid,
salsolinol, dolichol, circulating cytokines, proteomic tech-
niques). Most of these markers are validated only in research
laboratories and are yet to be implemented in a clinical
setting. There is also a growing consensus to develop panels of
biomolecules rather than a single marker to improve the
sensitivity and specificity of diagnosis.32 Our present findings
can add to this growing body of evidence.
The strength of our study is that though it is performed in
the Northeast region of the country, it is based on the studied
male population which is economically productive and has a
pan-India profile. The study population is considered rela-
tively healthier than the average general population, resulting
in the representation of an ideal alcohol-related disorder
population without severe comorbid illnesses which can
affect serum cortisol levels. We have assessed serum total
cortisol by the automated ELFA method, which is the most
commonly assessed, economical, standardized, and readily
available parameter in our country.33
Our study had certain limitations. The sample size was
small, and the studied groups were not age matched, that
is, the mean age difference was statistically significant
(p ¼ 0.00). Therefore age-adjusted values and larger sample
size would have provided a better comparison. Further,
extensive studies with more stringent inclusion and exclusion
criteria and increased sample size can improve our strength of
data and help to provide more conclusive results of diagnostic
values.
To conclude, our study suggests that in Indian men,
alcohol-related disorders are associated with significant
alteration of early morning serum total cortisol levels,
possibly due to chronic changes in the functional HPA axis.
Therefore, it is important to study the cortisol levels by the
most readily available, standardized, and economical labora-
tory diagnostic technique in a pan-Indian populationebased
reference method which could be developed as a potential
tool for diagnosing and treating patients with alcohol-related
disorders. To promote this concept, we have determined a
single-point assessment of early morning serum total cortisol
levels using the automated ELFA method with intent to use it
in the Indian male patients with alcohol-related disorders in
future.
Ethical approval
All procedures performed in this study were in accordance
with the ethical standards of the institutional research com-
mittee and with the 1964 Helsinki declaration and its later
amendments or comparable ethical standards.
Declaration of Competing Interest
All authors have none to declare.
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