Effect of non-alcohol beer on anxiety: Relationship of 5-HIAA

Abstract

Beer contains hop (Humulus lupulus L.) as flavoring and preservative. This cannabaceae plant, which possesses sedative and hypnotic properties, is present in non-alcoholic beer as well. Likewise regular beer, non-alcoholic beer includes the essential amino acid lysine with relaxing effects, which are related to the inhibition of serotonin receptors. It is known that the main neuroendocrine signal from stress is the release of the hormone cortisol, alongside with the altered levels of the hormone melatonin and the neurotransmitter serotonin. In this study we aimed to analyze the possible anxiolytic effect of non-alcoholic beer on neuroendocrine levels of cortisol, melatonin and serotonin, in a population under stress. To achieve this goal, a healthy student population was enrolled under the stressful conditions evoked by official academic exams. They consumed 1 non-alcoholic beer (330 mL) at dinner time for 14 nights. Melatonin, serotonin and cortisol levels were measured by quantifying their early morning urinary metabolites collected weekly. The consumption of non-acoholic beer reduced significantly the nocturnal serotonin levels whereas there were no substantial changes on melatonin and cortisol levels. However and in line with this, Anxiety/State parameters underwent a significant decreased after consumption of non-alcoholic beer at dinner time regarding to control values. These results point to the anxiolytic response of non-alcoholic beer is performed through the serotoninergic pathway. All in all, consumption of non-alcoholic beer might be an additional tool to deal with serotonin-related stress profile.

Full text

ISSN 18197124, Neurochemical Journal, 2015, Vol. 9, No. 2, pp. 149–152. © Pleiades Publishing, Ltd., 2015.
149
12
INTRODUCTION
Hops (
Humulus lupulus
, fam. Cannabaceae) are a
perennial dioecious herb whose crops have an eco
nomical importance for the brewery industry. For cen
turies, this plant has provided flavor and aroma to beer
[1] and has been considered as a sedative [2]. The
importance of the female plants (the only ones culti
vated for brewery) lies in the lupulin glands, which are
developed at the base of the bracteoles [3], and contain
resins and essential oils required for the flavor and
aroma of beer [4].
The sedative activity of hops lies mainly in its bitter
acids: the
γ
acids (or humulones) which are very sen
sitive to oxidation [5] and generate 2methyl3buten
2ol [6], also the polyphenol xanthohumol [7] and the
terpene myrcenol [8]. The main mechanism of action
of hops is by increasing the activity of neurotransmit
1
The article is published in the original.
2
Corresponding author; address: Laboratory of Health Education,
Experimental Sciences Education Area, University of Extrema
dura, Excellence Campus Hidranatura, av. Elvas s/n 06006
Badajoz, Spain; email: jcubero@unex.es.
ter
γ
aminobutyric acid (GABA) through modulation
of brain GABA A receptors [9–11], thus inhibiting the
central nervous system (CNS). Also hops modulate
serotonin central nervous system receptor [12] and
may act through modification levels of serotonin [13]
generated by chronic social defeat stress in rats [14].
Also lysine is one of the most important essential
amino acids in hops and in nonalcoholic beer, with a
concentration of 12 mg/100 mL [15, 16] which signif
icantly reduce chronic anxiety in human assay [17].
Llysine acts like a partial 5HT4 antagonist and inhib
its serotoninmediated intestinal pathologies in rats
[18] and also reduces the anxiety and lessens stress in
humans [19].
It has been reported that hops may be beneficial
both in animal and human populations as an aid to
sleep, among others health disorders, like obesity and
antiinflammatory or antithrombotic effects. In addi
tion to the nonalcoholic beer consumption in people
with sleep problems, the sedative action associated
with the components of hops and the amino acid
lysine has been used to correct temporary sleep latency
Effect of NonAlcohol Beer on Anxiety: Relationship of 5HIAA
1
L. Franco
a
, C. Galán
a
, R. Bravo
a
, I. Bejarano
a
, E. PeñasLledo
b
, A. B. Rodríguez
a
,
C. Barriga
a
, and J. Cubero
a
,
c
,2
a
Neuroimmunophysiology and Chrononutrition Research Group, Department of Physiology,
University of Extremadura, Badajoz, Spain
b
Department of Pharmacology, University of Extremadura, Badajoz, Spain
c
Health Education Laboratory, Experimental Science Education Area, University of Extremadura,
Campus of Excellence: Hidranatura, Badajoz, Spain
Received November 5, 2014
Abstract
—Beer contains hop (
Humulus lupulus
L.) as flavoring and preservative. This cannabaceae plant,
which possesses sedative and hypnotic properties, is present in nonalcoholic beer as well. Likewise regular
beer, nonalcoholic beer includes the essential amino acid lysine with relaxing effects, which are related to
the inhibition of serotonin receptors. It is known that the main neuroendocrine signal from stress is the release
of the hormone cortisol, alongside with the altered levels of the hormone melatonin and the neurotransmitter
serotonin. In this study we aimed to analyze the possible anxiolytic effect of nonalcoholic beer on neuroen
docrine levels of cortisol, melatonin and serotonin, in a population under stress. To achieve this goal, a
healthy student population was enrolled under the stressful conditions evoked by official academic exams.
They consumed 1 nonalcoholic beer (330 mL) at dinner time for 14 nights. Melatonin, serotonin and cor
tisol levels were measured by quantifying their early morning urinary metabolites collected weekly. The con
sumption of nonacoholic beer reduced significantly the nocturnal serotonin levels whereas there were no
substantial changes on melatonin and cortisol levels. However and in line with this, Anxiety/State parameters
underwent a significant decreased after consumption of nonalcoholic beer at dinner time regarding to con
trol values. These results point to the anxiolytic response of nonalcoholic beer is performed through the sero
toninergic pathway. All in all, consumption of nonalcoholic beer might be an additional tool to deal with
serotoninrelated stress profile.
Keywords: nonalcohol beer, hops, lysine, serotonin, anxiety
DOI:
10.1134/S181971241502004X
CLINICAL
NEUROCHEMISTRY

150
NEUROCHEMICAL JOURNAL Vol. 9 No. 2 2015
FRANCO et al.
and sleep fragmentation disorders in human popula
tions [20].
Induced sleep is a physiological process in which
molecules, like the neurotransmitter GABA, the
indolamines serotonin and melatonin, or some hor
mones like cortisol among other molecules, are
involved. Therefore, our aim was to analyze the possi
ble anxiolytic effect of nonalcoholic beer on the neu
roendocrine levels of cortisol, melatonin and seroto
nin, in a population under stress.
MATERIALS AND METHODS
Subject
The research was carried out on a population of
16 university students who volunteered to do it. The
students who participated in the research were healthy,
they were not under or overweight and they were not
taking any medication, stimulant or drink consump
tion that might have influenced the outcome of the
research. Table 1 shows the anthropometric character
istics of the sample population. This population con
sisted of 10 women and 6 men (
n
= 16). This project
was approved by the Bioethics Committee of the Uni
versity of Extremadura.
The subjects enrolled to participate in our research,
were stressed as the experiment was carried out during
their official university exam period in February 2013.
This stress is observed by increasing the variable Anxi
ety/State (A/E) in the StateTrait Anxiety Inventory,
both groups of participants (males and females) were
higher than the 50 percentile obtained from the scale
published by Spielberg et al. [21].
Experimental Design
We chose a longitudinal and crossed intervention
model in which each subject was his own control. The
experimental period was 3 weeks; the first 7 days were
used for the control, when participants did not have
any beer intake (control week). During the following
14 days (2 weeks), the participants ingested 330 mL of
nonalcohol beer (Mahou Laiker Without
®
) during
their dinner. The StateTrait Anxiety Inventory was
completed by all the participants at the end of each
one of the three weeks that the trial lasted. Hormone
and neurotransmitter levels were measured through
the excretion of metabolites in urines collected in the
morning (09:00 a.m.) at the end of each week).
StateTrait Anxiety Inventory (STAI)
This questionnaire consists of two sections, each
containing 20 questions. The questions on the first
section are about Anxiety/State (A/S), conceptual
ized as a transitory, subjective, emotional state or con
dition of the human organism, of tension and appre
hension, and hyperactivity of the autonomic nervous
system. The second section asks questions about Anx
iety/Trait (A/T), a relatively stable characteristic of
individuals with a tendency to perceive situations as
threatening. The time for the test is 20 minutes and the
population is spread by sex [21].
Analytical Methods
To determine the hormones levels: cortisol, 6sulf
atoximelatonin (aMT6S) and neurotransmitter levels:
5hidroxi3indolacetic acid (5HIAA), a competitive
enzyme immune essay method E.L.I.S.A. kit (DRG
©
)
was used, by measuring the absorbance in a Nano
Quant InfiniteN200 150 modular reader, with wave
length a 450 nm absorbance. All the analytical deter
minations were performed by duplicate.
Statistical Treatment of Data
For the resolution, statistical data were obtained by
GraphPad Prism
®
software v.5.50 for Windows
®
envi
ronment. A descriptive study was conducted where the
Mean ( ) ± Standard Dsviation (SD) were calculated.
These are formulas commonly used for the type of data
handled. For the inferential study, a paired Tstudent
test was performed. Due to the fact that the size of the
sample was variable, we applied the Kolmogorov–
Smirnov test for normality of the data. Having deter
mined that the population was in the Normal, the
results were analyzed by
t
Student test comparing two
groups. Significant values were determined as those
with
p
value <0.05.
RESULTS
In order to study the effects of nonalcoholic beer
on Anxiety/State the volunteers consumed a dose of
330 mL at dinner time for 14 nights. The results indi
cate that nonalcoholic beer consumption signifi
cantly reduced (
p
< 0.05) the Anxiety/State from the
24 ± 4.24 (50th percentile) to 18.13 ± 2.9 (Table 2). To
support these results with physiological parameters,
cortisol, melatonin and serotonin earlymorning lev
els were measured. Cortisol levels (Table 3) decreased
moderately (153.74 ± 0.96
μ
g/mL) compared to its
control (156.58 ± 1.10
μ
g/mL). On the other hand,
nonsubstantial changes were observed in urinary
x
Table 1.
Anthropometric characteristics of a population of
students under exams stress
Age (years) 20.09 ± 2.23
Weight (kg) 67.80 ± 15.86
Height (m) 1.72 ± 0.11
BMI 23.13 ± 3.03
Each value represents the mean ± SD of participants.
n
= 16.

NEUROCHEMICAL JOURNAL Vol. 9 No. 2 2015
EFFECT OF NONALCOHOL BEER ON ANXIETY: RELATIONSHIP OF 5HIAA 151
aMT6S values (Table 3), (91.35 ± 15.89
μ
g/mL) ver
sus its control (98.11 ± 12.68
μ
g/L).
The last but not the least, the most relevant result
was the significant decrease (
p
< 0.05) in early morning
5HIAA (5.55 ± 0.95 mg/L) compared to its control
(8.8 ± 0.5 mg/L) (Table 3). This substantial decrease
manifests a drop in nocturnal serotonin levels after
nonalcoholic beer consumption.
DISCUSSION
Stress activates the hypothalamicpituitaryadre
nal (HPA) axis, a process that is characterized by a rise
in the systemic adrenocorticotrophic hormone
(ACTH) followed by the release of glucocorticoids.
The increase of glucocorticoids is necessary to supply
the energy and to induce the arousal state against
stressful situations. Also the hyperactivity of the HPA
axis is implicated in the pathogenesis of depression.
This physiological and psychological abnormality
generates disturbances in the serotonin (5HT) neu
rotransmission exhibiting an increase (striatum, fron
tal, cortex) or a decrease (amygdala, lateral septum) in
the release of 5HT [22]. In addition, the interesting
assays of Keeney et al. [14], have demonstrated that
the hippocampal serotonin release, measured via
microdialysis, generated chronic social stress in rats.
The importance of this limbic structure, the hippoc
ampus, is that it is a brain region involved in the co
ordination of behavioral and physiological response to
stress, and because it is densely innervated by seroton
ergic nerve terminals [14].
The most peculiar issue to deal with in this research
regards the different central nervous system (CNS)
5HT recapture and 5HT metabolism in the periph
ery. Measurements of urinary metabolites of 5HT
cannot directly assess the CNS. It has been known for
decades that the majority of urinary 5HIAA seems to
come from peripheral sources such as gastrointestinal
enterochromaffin cells that contain large amounts of
5HT [23].
Regarding to the decreased urinary levels of
5HIAA after the consumption of nonalcoholic beer
to explain that it could be generated by the anxiolytic
effect of hops [6–8] and lysine [18], it has been irrefut
ably proved by the sedative effect of nonalcoholic
beer on a stressed population [20, 24, 25], because
abnormal urinary levels of 5HIAA have been associ
ated to generalized anxiety disorder (GAD) [26, 27]
and the severity of the Hamilton Anxiety Rating Scale
[28]. In particular, increased 5HIAA levels were cor
related with the Hamilton somatic subscale (items 7
to 13) with items related to somatic signs of anxiety:
tension, intellectual difficulties, somaticmuscular
symptoms, somatic sensory symptoms, and genitouri
nary symptoms.
This reduction in the 5HIAA levels could be asso
ciated with the inhibition of the action in the
monoamine oxidase (MAO), as it occurs in any anxi
olytic medicine. Unfortunately, there is no scientific
evidence in the bibliography about a possible effect of
hops and lysine contained in beer, over the IMAO.
This hypothesis would be an interesting and future line
of investigation.
Also, in humans, a 3month randomized double
blind study tested if lysine fortification of wheat
reduced anxiety and stress in poor Syrian communi
ties. In the lysinefortified group, lysine significantly
reduced chronic anxiety measured by the trait anxiety
inventory in males. Lysine also reduced the plasma
cortisol response to the blood drawing caused by stress
in females, like was sympathetic arousal in males mea
sured by skin conductance. These results suggest that
some stress responses in economically weak popula
Table 2.
Influence of nonalcoholic beer consumption on anxiety levels of a population of students under exams stress
Control Nonalcoholic beer treatment
Anxiety/State 24 ± 4.24 18.13 ± 2.9*
Each value represents the mean ± SD of participants. Participants completed both tests at 10.00 a.m. before the beginning of the trial
(control score) and one day following its termination (14 days). Up to 10 min was allowed for completion of these paper and pencil surveys.
*
p
< 0.05 respect to the control. The results were analyzed by
t
Student test comparing two groups.
n
= 16.
Table 3.
Influence of nonalcoholic beer consumption on neuromolecules levels on cortisol, 6sulfatoxymelatonin
(aMT6S) and 5hidroxyindolacetic acid (5HIAA) of a population of students under exams stress
Control Nonalcoholic beer treatment
Cortisol 1.56 ± 1.10 1.53 ± 0.96
aMT6S 98.11 ± 12.68 91.35 ± 15.89
5HIAA 8.8 ± 0.5 5.55 ± 0.95*
Each value represents the mean ± SD of participants. *
p
< 0.05 respect to the control. The results were analyzed by
t
Student test com
paring two groups.
n
= 16.

152
NEUROCHEMICAL JOURNAL Vol. 9 No. 2 2015
FRANCO et al.
tions consuming cerealbased diets can be improved
with lysine fortification [17]. And lysine dietary defi
cits in experimental animals (prolonged) increases
stressinduced anxiety, which is reverted by prolonged
lysine treatment that has been shown to have anxi
olytic effects in normally fed stressed rats. Llysine
acts like a partial 5HT4 receptor antagonist, and
inhibits serotoninmediated intestinal pathologies and
anxiety in rats. Agonists of 5HT4 receptor increase
propulsive mobility in the mesenteric plexus and
antagonists prevent it, and modulate visceral pain and
anxiety in humans and animals [18, 29, 30]. Thus,
lysine has been proposed as a potential therapeutic
tool in the treatment of stressrelated intestinal disor
ders, in which there is a diagnosis of 5HT sensitiza
tion and concurrent anxiety disorder.
In conclusion, stress has been shown to modulate
the expression and binding of 5HT and 5HT level,
rate of synthesis, release, uptake, and reception in a
variable manner with a type of stressor in brain
regional differences, and in particular in the peripheral
regions, mainly in gastrointestinal tract (GIT) with the
5HT4 receptor by anxiety. We conclude therefore that
the population enrolled with stress, after drinking
nonalcoholic beer for 14 nights, improves anxiolytic
response through via serotonergic.
ACKNOWLEDGMENTS
This research has been funded by The Government
of Extremadura (Fondos FEDER), PIIAcción
VIIUEX 2010 and the Centro de Información
Cerveza y Salud (CICS).
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