European Heart Journal - Cardiovascular Pharmacotherapy Advance Access published August 8, 2016
European Heart Journal – Cardiovascular Pharmacotherapy (2016) 2, 1–8
doi:10.1093/ehjcvp/pvv037
REVIEW
Melatonin has multiorgan effects
Lionel H. Opie* and Sandrine Lecour
Department of Medicine, Hatter Cardiovascular Institute for Research in Africa, University of Cape Town, Cape Town 7925, South Africa
Received 13 July 2015; revised 27 August 2015; accepted 15 September 2015
Melatonin, widely used to counter transatlantic travel jet lag and insomnia, is synthesized in the suprachiasmatic nucleus of the anterior pituitary
gland. Its release into the circulation is stimulated by the onset of darkness, followed by a progressive decrease in blood levels with the onset of
dawn. Melatonin administration can maintain the quality of sleep and help to counteract age-induced cognitive decline. Melatonin can also limit
the severity of a variety of cardiovascular and cerebrovascular diseases, diabetes, and cancer.
Keywords
Melatonin † Sleep † Cardioprotection † Anticancer † Day–night cycle † Multiorgan
Introduction
Melatonin is widely used to counter transatlantic travel jet lag and
insomnia. Melatonin is less well known for its multiple and widespread physiological and pharmacological effects. The multiorgan
targets of melatonin, as here reviewed and as shown in Figure 1,
have important implications for optimal health at a time when
cardiovascular diseases (CVDs) are the main causes of death and
disease in the developed world and soon will be so in the developing
world.1 One preventative approach to CVD is adherence to the
traditional Mediterranean diet.2 Here, we propose that the unique
properties of melatonin, a naturally occurring hormone best known
for counting jet lag also provides cardiovascular protection and limits cancer. The well-known diurnal pattern of variation of blood
melatonin levels has the nocturnal rise that promotes sleep followed
by a gradual fall after midnight until low levels in the early morning.3
Melatonin is also found in red wine or even in grape juice in which it
has cardioprotective effects.4 – 7 As reviewed below, a wide range of
other health benefits are now been attributed to melatonin. These
multiple mechanisms are the topic of this review.
Melatonin synthesis physiological
properties
Melatonin (N-acetyl-5-methoxytryptamine) plays a major role in
regulating the circadian light-sensitive biological clocks that promote
sleep when darkness prevails or help waking up with the morning
light. Melatonin is synthesized primarily in the pineal gland and its secretion into the blood stream is regulated by the environmental
light/dark cycle via the suprachiasmatic nucleus. The initial event is
that very sensitive ocular photoreceptors stimulate the formation
of melanopsin within a subset of photosensitive retinal ganglion
cells,8 which in turn send signals to the pineal gland from which release of melatonin, synthesised from serotonin, is either stimulated
or inhibited.9 Physiological levels of melatonin vary from 5 to
200 pg/mL function according to the time of the day.3
The exact site in the brain where melatonin acts to induce sleep is
not yet established. A clue comes from the mechanisms underlying
insomnia, a recent proposal being that there are abnormalities in the
sleep patterns in the ventromedial prefrontal cortex prefrontal
lobes during episodes of rapid eye movement in sleep.10 That may
be where melatonin acts centrally.
Experimentally, melatonin is a powerful antioxidant molecule,11,12 with also proven antihypertensive and lipid lowering
effects.13,14 Melatonin even at low concentrations confers protection against myocardial ischaemic –reperfusion injury (IRI) by activation of protective downstream signalling pathways.15 Protective
mechanisms include the following pathways: Janus kinase 2/signal
transducers and activators of transcription 3, and nuclear factor erythroid 2-related factor 2 (Nrf2).16 In human, leucocytes
melatonin protected from free radicals acting on the MT1/MT2
receptors.17 In patients undergoing coronary artery bypass, the protective dose was 10 mg as shown by increased levels of Nrf2
activity.18
Regarding possible effects anti-inflammatory effects, the data
give no clear clinical message as yet. Thus, larger trials are required
to establish that melatonin reduces inflammatory markers. Experimentally, melatonin reduced the proinflammatory marker IL-1b
(P , 0.01) but not the levels of TNF-a and IL-6.19
Melatonin molecular effects
The pathway whereby melatonin crosses the cell membrane involves
components of the SLC2/GLUT family of glucose transporters.20
* Corresponding author. Tel: +27 21 406 6358, Fax: +27 21 447 8789, Email: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2015. For permissions please email: [email protected]
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L.H. Opie et al.
Melatonin attenuated glucose-induced tumour progression in mice
with prostate cancer and prolonged their lifespan.20 Melatonin also
experimentally restored glucose transporter-4 loss in adipocytes.21
Melatonin is capable of influencing in development of diabetic complications by neutralizing the unnecessary production of reactive
oxygen species (ROS), protection of beta cells. These observations
suggest the potential use of melanin, with few or no serious side effects, to inhibit of the development of adverse effects of hyperglycaemia in prediabetes and diabetes.21
Melatonin pretreatment attenuated cerebral ischaemic–reperfusion mitochondrial injury by activation of the Silent information
regulator 1 (SIRT1) which is a histone deacetylase that gives highly
effective protection against IRI.22 The melatonin-induced upregulation of SIRT1 was also associated with an increase in the antiapoptotic factor, Bcl2, and a reduction in the pro-apoptotic factor
Bax. In adult mice subject to IRI, melatonin gave cerebral protection
by reducing infarct volume and brain oedema with increased neurological scores.23
Widespread health benefits
of melatonin
Among the remarkable protective effects of melatonin are protection from cancer by acting on glucose transporters,20 lessening of
insulin resistance associated in experimental type 2 diabetes,21 followed by prevention of mitochondrial dysfunction in experimental
ischaemic stroke and anti-apoptotic effects.22,24 These remarkable
properties will now be reviewed. Adequate sleep is required for
normal daytime cognitive functions.25 Melatonin has a hypnotic/
sedative effect on cognition when given orally. This is explained by
the role of melatonin in circadian rhythm regulation, an effect
mediated by stimulation of the melatonin receptors MT1 and
MT2. Binding of melatonin to the brain MT1 receptor acts via the
G-coupled protein pathway that enhances binding of gammaaminobutyric acid (GABA) to the GABAA receptor. This results in
a high-dose anaesthetic effect.26
Melatonin induces sleep even when several circadian rhythms are
out of phase with each other and with the external environment.
Melatonin is over-the-counter in the USA while in most European
countries, it is available as a prescription drug (example: Circadin).
Melatonin acts both by inducing sleep and by restoring the inherent
sleep rhythm that is related to the rise and fall of blood melatonin
levels.3 Melatonin therapy also helps to restore these human circadian rhythms resulting in better cognition and less day time fatigue.25
Furthermore, melatonin can improve sleep duration even in totally
blind subjects.26
Direct proof of an effect on disordered sleep rhythm comes from
studies on the Angleman syndrome a rare neurodevelopmental disorder which is associated with an abnormality of chromosome
15q11-q13.27 In those with this sleep disorder, the night-time serum
melatonin levels were lower than those of the controls. Serum
melatonin levels were altered (peak levels in affected patients 60
vs. 108 ng/L in controls; P , 0.01), whereas the peak melatonin nocturnal levels in controls were normal.27 In children with the 24-h
motor activity of this syndrome, such activity decreased during
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Figure 1 The multiple health benefits of melatonin on multiple organs.
3
Melatonin has multiorgan effects
the total sleep period following melatonin treatment, and an increase in the duration of the total sleep period.28
Melatonin and cardioprotection
Melatonin and cerebroprotection
Experimentally, melatonin has anti-apoptotic effects in transient
brain ischaemia. It strongly promotes the potentially lethal opening
of the mPTP in isolated brain mitochondria from striatal neurons
after middle cerebral artery occlusion in mice.34 In early experimental stroke, whether caused by embolism or haemorrhage, there is a
defined period after the insult has occurred within which the cerebral cells take to die. The mechanism may involve neuronal oxidative
stress, which suggests that therapy should be directed towards
blocking the generation of such stress.35 An early timing of such
therapy would be essential. In cerebral artery occlusion in monkeys
30 min to 4 h of ischaemia caused multiple, non-confluent deep infarcts with prominent grey mater necrosis.36 Such brain cells are
threatened by death and could be rescued by timely reperfusion
within 4 h in mice.37 Disruption of the blood –brain barrier permeability decreased by 53% (P , 0.001).Transplantation of melatoninsecreting cells protected against experimental stroke.38 In the
development of stroke, melatonin decreased inflammation as a
contributory mechanism.39 Exogenous melatonin treatment and
transplantation of melatonin-secreting cells both have supporting
laboratory data.38
In searching for a therapy against the adverse cellular effects of an
early stroke, melatonin had experimental anti-apoptotic effects in
Melatonin, obesity, and diabetes
Melatonin supplementation may act to prevent the adverse health
consequences of obesity. In a double-blind, placebo-controlled trial,
44 obese women were randomly given placebo or 6 mg melatonin
with a low-calorie diet for 40 days, melatonin decreased serum
TNF-a (P ¼ 0.02) and IL-6 (P ¼ 0.03).41 The proposed mechanism
was two-fold, anti-inflammatory, and decreased oxidative stress.
Melatonin also has direct anti-diabetic effects, hence potentially
relevant to the therapy of type 2 diabetes.42 Experimentally, one
mechanism proposed is that melatonin can neutralize the production of ROS.43 The Gaq receptor and the phosphatidylinositol
3-kinase signalling cascade mediate melatonin effects in pancreatic
cells.15 The prediabetic rat is a model of diet-induced obesity, in
which chronic melatonin consumption (4 mg/kg/day) reduced the
gain of body weight together with reduced visceral adiposity, serum
insulin and triglyceride, besides protecting the heart against IRI.15
The molecular mechanisms involved PKB/Akt and extracellular
signal-regulated kinase (ERK)42/44 activation. As might be expected, immunoreactive melatonin was appropriately found in pancreatic islet cells.44
Melatonin has anti-cancer properties
Recently, the benefits of melatonin in the treatment of cancer, both
in vitro and in vivo, have been reported and carefully analysed by the
US Department of Health, showing the importance of the concept
of the topic. As the value of melatonin for cancer therapy has been
disputed. Wang et al.45 performed a systematic review of randomized controlled trials of melatonin in solid tumour cancer patients
and observed its effect on tumour remission, 1-year survival, and
side effects due to radiochemotherapy. The patients had a variety
of solid malignancies of the lung, breast, liver, gastrointestinal tract,
head, and neck. In such patients, there were more remissions (16.5
vs. 32.6%, P , 0.00001), 1-year survival rate was better (28.4 vs.
52.2%, P ¼ 0.001), and there was less thrombocytopenia (RR ¼
0.13; P , 0.00001).
The US Department of Health analysis of this major prospective
study was that the overall trial quality of this trial was acceptable,
though somewhat impaired in that blinding or use of placebo was not
present in any of the trials (https://nccih.nih.gov/health/melatonin).
Also, loss to follow-up was unknown in most trials. Despite these
defects, there was clear benefit with melatonin in the therapy of
these cancers. In the specific case of advanced prostate carcinoma
in patients, lower urinary levels of a melatonin metabolite were associated with an increased risk for cancer.45 In 761 patients, there
were more remissions (16.5 vs. 32.6%, P , 0.00001); 1-year survival
rate was better (28.4 vs. 52.2%, P ¼ 0.001), and there was less
thrombocytopenia (RR ¼ 0.13; P , 0.00001). Regarding the basic
mechanisms, members of the SLC2/GLUT family of glucose transporters have a central role. Melatonin interacts at the same location
in GLUT1 as does glucose. Thus, there is a facilitated transport of
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Melatonin invokes sleep, thus raising the hypothesis that melatoninenhanced sleep could decrease the elevated blood pressure associated with insomnia. Insomnia associated with physiological hyperarousal was associated with an increased risk of hypertension.
Of note, chronic insomnia with symptoms lasting 6 months or
more was associated with an increased risk of hypertension.28 In
16 hypertensive patients treated with beta-blockers, 3 weeks of
nightly melatonin 2.5 mg improved sleep quality besides increasing
total sleep time (+36 min; P ¼ 0.046).29 Add-on prolonged-release
melatonin (PRM) was used for antihypertensive therapy.30 The quality of sleep and behaviour following awakening improved substantially with PRM compared with placebo (P , 0.0001).
In 16 hypertensive patients already treated with beta-blockers,
3 weeks of nightly melatonin (2.5 mg) improved total sleeping time
(P ¼ 0.046) and the onset of sleep (P ¼ 0.001), without apparent tolerance and without rebound sleep disturbance when melatonin was
withdrawal,31 thus suggesting that melatonin is implicated in cardioprotection in IRI. The protective effects of melatonin in the heart after
experimental myocardial infarction (MI) include inhibition of the
opening mitochondrial permeability transition pore (mPTP) that potentially mediates lethal damage.32 Reperfusion ventricular arrhythmias in spontaneously hypertensive rats were also inhibited.33
After experimental MI, melatonin synthesis in the pineal gland increased substantially.3 One day thereafter, synthesis of pineal melatonin increased by 4.3 times (P , 0.001), which was associated with
the increased concentration of melatonin in the plasma (P , 0.001)
and left ventricle (P ¼ 0.01). The molecular mechanism of such benefits may include decreased formation of free radicals during oxidative stress and reduced superoxide generation during reperfusion.
transient brain ischaemia. It strongly promoted the potentially lethal
opening of the mPTP in isolated brain mitochondria from striatal
neurons after middle cerebral artery occlusion in mice.40
Theoretically, the mPTP could be blocked with expected salvage
of threatened brain tissue, yet firm data are presently lacking. The
logical next step would be to gather data from a clinical trial.
4
How do these basic studies relate
to cancer in humans?
An Icelandic population study evaluated the prospective association
between levels of the primary urinary metabolite of melatonin,
aMT6s, and the risk of prostatic carcinoma in 928 men.51 Lower levels of aMT6s were associated with an increased risk for advanced
prostatic carcinoma. There was a four-fold increased risk for advanced cancer compared with men with aMT6 levels above the median (hazard ratio: 4.04). In women in five prospective case–control
studies, there was an inverse relationship between carcinoma and
levels of urinary aMT6.51 Women with the highest values of
aMT6s also had a lower risk of breast cancer.
The blood melatonin levels in women might have implications for
the occurrence of hormonal-dependent breast cancer. Melatonin
has potential oncostatic activity so that the link between night
work and breast cancer risk was studied in the Nurses’ Health Study
studied over 14 years.52 Among 78 562 women, without cancer at
baseline, 2441 developed invasive breast cancer at follow-up. The
relative risk was 1.08 [95% confidence interval (CI), 0.99 – 1.18].
The risk was further increased among women who worked 30 or
more years on the night shift (RR ¼ 1.36; 95% CI ¼ 1.04 to 1.78).
An indirect speculation, based on this study, is that sustained insomnia could be a cancer risk.
Exposure of xenograft-bearing rats to dim light at night (LAN)
completely suppressed elimination of the normal pattern of
increased blood melatonin levels at night.53 The result was unsuppressed high rates of tumour metabolism and growth. The authors
propose that this mechanism may help to explain why women
working rotating night shifts have higher risks of breast and other
cancers.
Melatonin and mood disorders
The biological melanin clock is especially important in the aetiology
of seasonal affective disorder (SAD) in which psychological depression, for which bright light is a very effective treatment.53 Forty winter depressives were treated for a week with bright light (4.300 lx
for 30 –45 min shortly after awakening) or dawn simulation (gradually increasing light during the last 30 min of sleep achieving
100 lx before alarm beep, with the dawn simulator placed closer
to the open eyes for a further 15 min: 250 lx). Dawn simulation
was similarly effective to bright light in the treatment of winter depression. Patients with more severe depression tended to report
greater improvement with bright light; in such cases, this would outweigh the non-clinical advantages of dawn simulation.
In persons with SAD depressive symptoms in the evening, these
are related to abnormal patterns of plasma melatonin which are too
high at night. In a 22-day design, 22 patients suffering from a major
depression with a seasonal pattern of SAD were given light treatment (10 000 lx) for 2 weeks on workdays.54 Therapy to advance
the onset of blood melatonin rise improved sleep, alertness, and
mood
Melatonin and ageing
The sleep-wake cycle changes with age, with prominent sleep phase
delay during youth and reduced circadian strength in older persons.55 Aging is one of the major contributors to accelerated morbidity and mortality with an established increasing incidence of
CVD, hypertension, and stroke. In a cohort of elderly Japanese persons, the HEIJO-KYO cohort, urinary melatonin excretion was significantly associated with decreased night-time systolic blood
pressure (SBP), independently of age, gender, body mass index, current smoking status, diabetes, and daytime physical activity.56 Specifically, an increase in the urinary melatonin excretion from 4.2 to
10.5 mg (25th to 75th percentile) went with a 2.0 mmHg decrease
in night-time SBP. Thus, melatonin secretion was inversely associated with night-time blood pressure in that elderly population
not reviving antihypertensive drug therapy.
In a study on over 1000 elderly persons (mean age, 72 years),
melatonin secretion was significantly and inversely associated with
white blood cell (WBC) and platelet counts which were taken as
markers of inflammation. Melatonin secretion was inversely associated with WBC and platelet counts in this elderly population.57
Regarding mechanisms for this clinical benefit, animal data suggest
that growth hormone and melatonin can help to prevent cardiac
aging,58 as well as reducing the nocturnal blood pressure.59
Melatonin and for early cognitive decline
Cognitive impairment inevitably increases with ageing. But when
does age-related cognitive decline begin? In healthy educated adults
even in their 20s and 30s, certain limited aspects of such decline had
already started.60 Hypertension predisposes to early cognitive decline. The deterioration in mild cognitive declined could be countered by fast-release melatonin 3–9 mg given at bedtime for up to
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melatonin across the cell membrane. More specifically, lower urinary levels of a melatonin metabolite were associated with an increased risk for advanced prostate carcinoma. 45 Furthermore,
there was an inverse association between the incidence of breast
cancer and high levels of the urinary melatonin metabolite,
6-sulfatoxymelatonin (aMT6s) as found in in five prospective
case –control studies.46
At a molecular level, melatonin reduced the uptake of glucose and
modified the expression of GLUT1 transporter in prostate cancer
cells.47 Melatonin stimulated transmembrane glucose transport via
insulin receptor substrate-1/phosphatidylinositol 3-kinase pathway
in murine skeletal muscle cells.48 Furthermore, experimentally, glucose supplementation promoted prostate cancer progression in
TRAMP mice (TRansgenic Adenocarcinoma of mouse Prostate)
with decreased expression of Toll-like receptors 4 and Toll-like receptors 5 occurred as the prostate tumour progressed. In contrast,
normal prostate tissue from wild-type mice showed strong expression of receptors 4 and 5.48 The authors proposed that decreased
expression of receptors TLR4 and TLR5 may contribute to prostatic
tumorigenesis.
Sirtuin 1 (Sirt1) is a NAD+-dependent histone deacetylase that is
a direct intracellular target of melatonin.49 The TRAMP, oral administration of melatonin, at human-achievable doses, significantly inhibited prostate cancer tumour growth as shown by decreases prostate
and genitourinary weight and by mRNA and protein levels of proliferation markers.49 Melatonin uptake through the glucose transporter is proposed to be new target for melatonin inhibition of cancer.50
L.H. Opie et al.
5
Melatonin has multiorgan effects
Melatonin in other conditions
Alzheimer’s disease
Poor sleep quality is a major aspect ofAD, as recently suggested.63
Endogenous melatonin levels would have already fallen at the preclinical stages of AD so that added melatonin therapy is logical. In
a 6-month trial in patients with mild to moderate AD given melatonin in the PRM preparation, sleep efficiency improved in the melatonin group vs. placebo after 24 weeks of treatment (P ¼ 0.017).64
experimental evidence is that melatonin decreases the severity of induced hepatic fibrosis.69
Sleep quality and potentially
carcinogenic urban lighting
As melatonin promotes sleep, and as breast cancer is related to
poor sleeping patterns, it follows that melatonin use could be associated with decreased breast cancer. What are the data? Tumour
growth in nude rats bearing human breast cancer xenografts could
be indirectly linked as tumour growth was accelerated by exposing
the rats to LAN.70 Their study established that artificial LAN, even at
minimal levels, accelerated human breast cancer xenograft growth.
Are the cancer links to city lighting applicable for both genders, in
women for breast cancer and for men prostate cancer? An Icelandic
study evaluated the prospective association between the urinary
melatonin metabolite (aMT6) levels and risk of prostatic carcinoma
in 111 men.50 Lower levels of aMT6 were associated with an increased risk for advanced prostatic carcinoma, with a four-fold increased risk for advanced cancer compared with men with aMT6
levels above the median (hazard ratio: 4.04; 95% CI, 1.26 – 12.98).
Thus, there are links between hormonally induced cancers and
the anti-cancer activity of melatonin in both men and women.
What is the implication for current city dwellers? They pay a
health price for the easy access to LAN, namely perturbations in
melatonin rhythm with implication for the risk of hormonally related
cancers. The cause is a disorganized circadian system called chronodisruption.71 Blue light, particularly beneficial during the daytime, is
more disruptive at night, and induces the strongest melatonin inhibition. The remedy would be the development of lighting systems for
large cities that preserve the natural melatonin rhythm, thereby reducing the health risks induced by chronodisruption.
Pregnancy
Melatonin, produced in the ovary and placenta, acts on the foetus.65
Furthermore, at the time of parturition, the maternal melatonin
rhythm is increased to induce uterine contractility. When ova
were collected for in vitro fertilization-embryo transfer, melatonin
treatment improved implantation and pregnancy rates. Melatonin
projected male gametes from oxidative damage and preserved their
viability. Melatonin preserves the integrity of the ovum prior to and
at the time of ovulation. Furthermore, when ova were collected for
in vitro fertilization-embryo transfer, treatment with melatonin improved implantation and pregnancy rates.66
Renal and hepatic disease
In animal models of chronic kidney disease (CKD) involving experimental hypertension, diabetes mellitus, and models of nephrotoxicity, melatonin attenuated the CKD. 67 Melatonin reduced the
oxidative burden, inflammatory changes, and apoptosis. In haemodialysed patients, melatonin attenuated sleep disturbances. Experimentally, melatonin-activated p38, ERK, and nuclear factor-kB in
human dental pulp stem cells.68 The authors suggest that combined
treatment of grafted human dental pulp stem cells and melatonin
could be a viable future approach for the treatment of human liver
cirrhosis which at present has no adequate therapy. More direct
Melatonin in clinical trials for
patients with coronary heart
disease
Recently, a relevant experimental study found beneficial effects of
melatonin in countering oxidative stress in IRI.18 Clinically, Nrf2, limited oxidative stress that occurred during and after coronary artery
bypass grafting (CABG). Thirty volunteers undergoing CABG were
randomized to receive 10 mg oral melatonin before sleeping at night
for 1 month before surgery. In those randomized to melatonin, cellular damages resulting from CABG surgery via theNrf2 pathway were attenuated (melatonin group given Nrf2 compared with
placebo: 15.2 + 4.6 pmol/L, 0.28 + 0.01 vs. 1.1 + 0.59 pmol/L,
0.20 + 0.07, P , 0.05).
There are several clinical trials underway. The background for
these clinical studies is that experimentally induced MI, as studied
in rodents, influenced the synthesis, concentration, and receptor expression of endogenous melatonin.72,73 In response to experimental
MI, melatonin synthesis in the pineal gland increased rapidly, suggesting an important role for endogenous melatonin in post-MI cardioprotection.74 The day/night pattern of changes in melatonin blood
levels,3 may help to explain the timing of human infarcts.74
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3 years.61 Those treated with melatonin added to their prior medication, often donepezil, performed better in the Mini-Mental State
Examination and in the Alzheimer’s disease (AD) Assessment Scale.
Furthermore, depression scores decreased in melatonin-treated patients, while the quality of sleep and wakefulness also improved. But
when does age-related cognitive decline begin? Controversially, the
decline in healthy educated adults could start even when they were
only in their 20s and 30s.60 When could melatonin be started to
counter this age-related adverse effect? Sensitive testing with a complex video game (StarCraft 2), using a dataset of 3305 players,
showed that age-related slowing began early and not late in life.61
An overt decline of cognitive capacities, such as reasoning,
memory, and semantic fluency, could be detected in middle age
(age 45– 49).62 In that study of cognitive decline on 5198 men and
2192 women, aged 45 –70 over 10 years, all cognitive scores, except
vocabulary, declined in all five age categories (age 45 – 49, 50 – 54,
55–59, 60 –64, and 65–70 at baseline), with faster decline in older
people. From the point of view of maintaining full cognition and
lessening the inevitable rate of decline in middle age, the implication
is that is that melatonin could beneficially be started even before
middle age, yet there are no trials as yet to prove this point.
6
Summary
Melatonin is a hormone with multiple sites of impact, with its major
effect in regulating the physiological light – darkness cycle and the
subjective states of wakefulness and sleep. Melatonin is synthesized
in the suprachiasmatic nucleus of the anterior pituitary gland.
Physiologically, the initial stimulatory event to it release is the onset
of darkness light acting on very sensitive ocular photoreceptors. The
pathways of melatonin synthesis and its physiological properties are
reviewed. The melatonin-induced hormonal changes are many, including upregulation of SIRT1 with an associated increase in the antiapoptotic factor, Bcl2. The normal sleep rhythm depends on a 24 h
pattern of rise and fall of circulating melatonin levels with the rhythm
of its action on the brain. As normal sleep is an imperative component of mental and cardiovascular health, the pattern of secretion of
melatonin has now been widely studied and is here reviewed in
countering the cognitive decline of ageing, maintaining the quality
of sleep, sustaining mental health prevention and prevention of
some aspects of cardiovascular and cerebrovascular diseases, diabetes, and cancer. Currently there are large clinical trials underway
to test the efficacy of melatonin in patients with acute coronary
heart disease.
Conflict of interest: none declared.
References
1. Nieuwlaat R, Schwalm JD, Khatib R, Yusuf S. Why are we failing to implement
effective therapies in cardiovascular disease? Eur Heart J 2013;34:1262 – 1269.
2. Turati F, Dilis V, Rossi M, Lagiou P, Benetou V, Katsoulis M, Naska A,
Trichopoulos D, La Vecchia C, Trichopoulou A. Glycemic load and coronary heart
disease in a Mediterranean population: the EPIC Greek cohort study. Nutr Metab
Cardiovasc Dis 2015;25:336 –332.
3. Hickie IB, Rogers NL. Novel melatonin-based therapies: potential advances in the
treatment of major depression. Lancet 2011;378:621 –631.
4. Demrow HS, Slane PR, Folts JD. Administration of wine and grape juice inhibits in
vivo platelet activity and thrombosis in stenosed canine coronary arteries. Circulation 1995;91:1182 – 1188.
5. González-Flores D, Gamero E, Garrido M, Ramı́rez R, Moreno D, Delgado J,
Valdés E, Barriga C, Rodrı́guez AB, Paredes SD. Urinary 6-sulfatoxymelatonin
and total antioxidant capacity increase after the intake of a grape juice cv. Tempranillo stabilized with HHP. Food Funct 2012;3:34– 39.
6. Opie LH, Lecour S. The red wine hypothesis: from concepts to protective signalling
molecules. Eur Heart J 2007;28:1683 –1693.
7. Lamont KT, Somers S, Lacerda L, Opie LH, Lecour S. Is red wine a SAFE sip away
from cardioprotection? Mechanisms involved in resveratrol- and melatonininduced cardioprotection. J Pineal Res 2011;50:374–380.
8. Gooley JJ, Lu J, Fischer D, Saper CB. A broad role for melanopsin in nonvisual
photoreception. J Neurosci 2003;23:7093 –7106.
9. Cahill GM, Grace MS, Besharse JC. Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock. Cell Mol
Neurobiol 1991;11:529 –560.
10. Chang CH, Chen MC, Qiu MH, Lu J. Ventromedial prefrontal cortex regulates
depressive-like behavior and rapid eye movement sleep in the rat. Neuropharmacology 2014;86:125 –132.
11. Reiter RJ, Tan DX. Melatonin: a novel protective agent against oxidative injury of
the ischemic/reperfused heart. Cardiovasc Res 2003;58:10 –19.
12. Yang Y, Sun Y, Yi W, Li Y. A review of melatonin as a suitable antioxidant against
myocardial ischemia-reperfusion injury and clinical heart diseases. J Pineal Res 2014;
57:357 – 366.
13. Simko F, Paulis L. Melatonin as a potential antihypertensive treatment. J Pineal Res
2007;42:319 –322.
14. Koziróg M, Poliwczak AR, Duchnowicz P, Koter-Michalak M, Sikora J, Broncel M.
Melatonin treatment improves blood pressure, lipid profile, and parameters of oxidative stress in patients with metabolic syndrome. J Pineal Res 2011;50:261 –266.
15. Nduhirabandi F, Du Toit EF, Blackhurst D, Marais D, Lochner A. Chronic melatonin
consumption prevents obesity-related metabolic abnormalities and protects the
heart against myocardial ischemia and reperfusion injury in a prediabetic model
of diet-induced obesity. J Pineal Res 2011;50:171 –182.
16. Yang Y, Sun Y, Yi W, Fan C, Xin Z, Jiang S, Di S, Qu Y, Reiter RJ, Yi D. A review of
melatonin as a suitable antioxidant against myocardial ischemia-reperfusion injury
and clinical heart diseases. J Pineal Res 2014;7:357–366.
17. Espino J, Rodrı́guez AB, Pariente JA. The inhibition of TNF-a-induced leucocyte
apoptosis by melatonin involves membrane receptor MT1/MT2 interaction.
J Pineal Res 2013;54:442 – 452.
18. Haghjooy Javanmard S, Ziaei A, Ziaei S, Ziaei E, Mirmohammad-Sadeghi M. The effect of preoperative melatonin on nuclear erythroid 2-related factor 2 activation in
patients undergoing coronary artery bypass grafting surgery. Oxid Med Cell Longev
2013;2013:676829.
19. Alamili M, Bendtzen K, Lykkesfeldt J, Rosenberg J, Gögenur I. Melatonin suppresses
markers of inflammation and oxidative damage in a human daytime endotoxemia
model. J Crit Care 2014;184.e9 –184.
Downloaded from http://ehjcvp.oxfordjournals.org/ by guest on August 25, 2016
Hypothetically ROS generated during myocardial IRI could influence
ischaemia-modified albumin (IMA) levels as studied in in 27 patients
with ST-segment elevation MI (STEMI) undergoing primary angioplasty.18 Circulating IMA was negatively correlated to melatonin in
these patients.
The MARIA trial is testing the hypothesis that in patients with
acute myocardial infarction (AMI) melatonin will confer cardioprotection against IRI.75 If successful, the finding would support the use
of melatonin in therapy of IRI of the heart. The background is that
intra-platelet melatonin levels predict angiographic no-reflow after
primary percutaneous intervention in patients with STEMI.75
In planning trials of melatonin in AMI, platelet reactivity could be a
useful marker of beneficial effects as studied in 494 STEMI patients
with a median follow-up of 2.3 years of whom 58 suffered from a
cardiovascular death. High levels of High on-treatment Platelet Reactivity and of C-reactive protein identified a subgroup of patients at
higher risk of cardiovascular death.76
In the MARIA trials the acronym stands for: Melatonin as an
Adjunct in patients with acute myocaRdial Infarction undergoing
primary Angioplasty.75 In the first MARIA study circulating IMA correlated negatively to melatonin in STEMI patients, leading the
authors to suggest that melatonin might exert a beneficial effect
as a radical scavenger in human myocardial ischaemia – reperfusion.75 Low intra-platelet melatonin concentrations in 180 consecutive patients with a first STEMI predicted angiographic no-reflow
after primary percutaneous coronary intervention (pPCI) in patients with STEMI.76 Decreased levels of melatonin in serum predicted adverse left ventricular remodelling after AMI. 77 These
ongoing studies on patients with AMI in the MARIA trials test the
hypothesis that melatonin will confer cardioprotection against IRI.
In Denmark, the IMPACT trial has started. Owing to its relatively
non-toxic profile, melatonin is an easily implementable drug in the
clinical setting, and melatonin has the potential to reduce morbidity
in patients with AMI.78 The IMPACT trial aims to evaluate the preventative effect of intracoronary and systemic melatonin on the severity of ischaemic –reperfusion injuries following pPCI in early AMI.
The primary outcome measures are: death, sustained ventricular arrhythmias, resuscitation from cardiac arrest, cardiogenic shock,
heart failure, major bleedings, stroke, need for revascularization, recurrent ischaemia, re-infarctions, and re-hospitalization; MRI of the
heart focusing on quantification infarct size, area at risk and myocardial salvage index, secondary outcome measures: serum creatinine
kinase Myocardial Band (CK-MB) 96 h after PCI; clinical events
within 90 days of PCI; high-sensitive Troponin T or Troponin I.
L.H. Opie et al.
Melatonin has multiorgan effects
45. Wang YM, Jin BZ, Ai F, Duan CH, Lu YZ, Dong TF, Fu QL. The efficacy and safety of
melatonin in concurrent chemotherapy or radiotherapy for solid tumors: a
meta-analysis of randomized controlled trials. Cancer Chemother Pharmacol 2012;
69:1213 –1220.
46. Basler M, Jetter A, Fink D, Seifert B, Kullak-Ublick GA, Trojan A. Urinary excretion
of melatonin and association with breast cancer: meta-analysis and review of the
literature. Breast Care (Basel) 2014;9:182 –187.
47. Hevia D, González-Menéndez P, Quiros-González I, Miar A, Rodrı́guez-Garcı́a A,
Tan DX, Reiter RJ, Mayo JC, Sainz RM. Melatonin uptake through glucose transporters: a new target for melatonin inhibition of cancer. J Pineal Res 2015;58:234 –225.
48. Han JH, Park JH, Kim BY, Chang SN, Kim TH, Park JH, Kim D. Decreased expression
of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. J Vet Sci 2015;16:
281 –287.
49. Jung-Hynes B, Schmit TL, Reagan-Shaw SR, Siddiqui IA, Mukhtar H, Ahmad N. Melatonin, a novel Sirt1 inhibitor, imparts antiproliferative effects against prostate cancer in vitro in culture and in vivo in TRAMP model. J Pineal Res 2011;50:140 – 149.
50. Sigurdardottir LG, Markt SC, Rider JR, Haneuse S, Fall K, Schernhammer ES,
Tamimi RM, Flynn-Evans E, Batista JL, Launer L, Harris T, Aspelund T,
Stampfer MJ, Gudnason V, Czeisler CA, Lockley SW, Valdimarsdottir UA,
Mucci LA. Urinary melatonin levels, sleep disruption, and risk of prostate cancer
in elderly men. Eur Urol 2015;67:191 – 194.
51. Basler M, Jetter A, Fink D, Haneuse S, Fall K, Schernhammer ES, Tamimi RM,
Flynn-Evans E, Batista JL, Launer L, Harris T, Aspelund T, Stampfer MJ,
Gudnason V, Czeisler CA, Lockley SW, Valdimarsdottir UA, Mucci LA,
Trojan A. Urinary excretion of melatonin and association with breast cancer:
meta-analysis and review of the literature. Breast Care (Basel) 2014;9:182 –187.
52. Schernhammer ES, Laden F, Speizer FE, Willett WC, Hunter DJ, Kawachi I,
Colditz GA. Rotating night shifts and risk of breast cancer in women participating
in the nurses’ health study. J Natl Cancer Inst 2001;93:1563 –1568.
53. Blask DE, Dauchy RT, Brainard GC, Hanifin JP. Circadian stage-dependent inhibition of human breast cancer metabolism and growth by the nocturnal melatonin
signal: consequences of its disruption by light at night in rats and women. Integr
Cancer Ther 2009;8:347 –353.
54. Meesters Y, Dekker V, Schlangen LJ, Bos EH, Ruiter MJ. Low-intensity blueenriched white light (750 lux) and standard bright light (10,000 lux) are equally effective in treating SAD. A randomized controlled study. BMC Psychiatry 2011;11:17.
55. Robillard R, Naismith SL, Smith KL, Rogers NL, White D, Terpening Z, Ip TK,
Hermens DF, Whitwell B, Scott EM, Hickie IB. Sleep-wake cycle in young and older
persons with a lifetime history of mood disorders. PLoS ONE 2014;9:e8776.
56. Obayashi K, Saeki K, Tone N, Kurumatani N. Relationship between melatonin secretion and nighttime blood pressure in elderly individuals with and without antihypertensive treatment: a cross-sectional study of the HEIJO-KYO cohort.
Hypertens Res 2014;37:908 –913.
57. Obayashi K, Saeki K, Kurumatani N. Higher melatonin secretion is associated with
lower leukocyte and platelet counts in the general elderly population: the HEIJOKYO cohort. J Pineal Res 2015;58:227 – 233.
58. Paredes SD, Forman KA, Garcı́a C, Vara E, Escames G, Tresguerres JA. Protective
actions of melatonin and growth hormone on the aged cardiovascular system.
Horm Mol Biol Clin Investig 2014;18:79– 88.
59. Reiter RJ, Tan DX, Korkmaz A. The circadian melatonin rhythm and its modulation:
possibleimpact on hypertension. J Hypertens Suppl 2009;27:S17 –S20.
60. Cardinali DP, Vigo DE, Olivar N, Vidal MF, Furio AM, Brusco LI. Therapeutic application of melatonin in mild cognitive impairment. Am J Neurodegener Dis 2012;1:
280 –291.
61. Thompson JJ, JBlair MR, Henrey AJ. Over the hill at 24: persistent age-related
cognitive-motor decline in reaction times in an ecologically valid video game task
begins in early adulthood. PLoS ONE 2014;9:e94215.
62. Singh-Manoux A, Kivimaki M, Glymour MM, Elbaz A, Berr C, Ebmeier KP, Ferrie JE,
Dugravot A. Timing of onset of cognitive decline: results from Whitehall II prospective cohort study. BMJ 2012;344:d7622.
63. Wade AG, Farmer M, Harari G, Fund N, Laudon M, Nir T, Frydman-Marom A,
Zisapel N. Add-on prolonged-release melatonin for cognitive function and sleep
in mild to moderate Alzheimer’s disease: a 6-month, randomized, placebocontrolled, multicenter trial. Clin Interv Aging 2014;9:947–961.
64. Xu J, Wang LL, Dammer EB, Li CB, Chen SD, Wang G. Melatonin for sleep disorders and cognition in dementia: a meta-analysis of randomized controlled trials.
Am J Alzheimers Dis Other Demen 2015. pii: 1533317514568005.
65. Voiculescu SE, Zygouropoulos N, Zahiu CD, Zagrean AM. Role of melatonin in embryo fetal development. J Med Life 201;7:488 –492.
66. Reiter RJ, Rosales-Corral SA, Manchester LC, Tan DX. Peripheral reproductive
organ health and melatonin: ready for prime time. Int J Mol Sci 2013;14:
7231 – 7272.
Downloaded from http://ehjcvp.oxfordjournals.org/ by guest on August 25, 2016
20. Hevia D, González-Menéndez P, Quiros-González I, Miar A, Rodrı́guez-Garcı́a A,
Tan DX, Reiter RJ, Mayo JC, Sainz RM. Melatonin uptake through glucose transporters: a new target for melatonin inhibition of cancer. J Pineal Res 2015;58:234–250.
21. Zephy D, Ahmad J. Type 2 diabetes mellitus: role of melatonin and oxidative stress.
Diabetes Metab Syndr 2015;9:127 – 131.
22. Yu L, Sun Y, Cheng L, Jin Z, Yang Y, Zhai M, Pei H, Wang X, Zhang H, Meng Q,
Zhang Y, Yu S, Duan W. Melatonin receptor-mediated protection against myocardial ischemia/reperfusion injury: role of SIRT1. J Pineal Res 2014;57:228 –238.
23. Yang Y, Jiang S, Dong Y, Fan C, Jiang S, Zhao L, Di S, Xin Z, Wang B, Wu G, Li X, Li Z,
Gao X, Dong Y, Qu Y. Melatonin prevents cell death and mitochondrial dysfunction
via a SIRT1-dependent mechanism during ischemic-stroke in mice. J Pineal Res 2015;
58:61–70.
24. Andrabi SA, Sayeed I, Siemen D, Wolf G, Horn TF. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for antiapoptotic effects of melatonin. FASEB J 2004;18:869 –887.
25. Amirian I, Andersen LT, Rosenberg J, Gögenur I. Working night shifts affects surgeons’ biological rhythm. Am J Surg 2015;210:389 – 395.
26. Roth T, Nir T, Zisapel N. Prolonged release melatonin for improving sleep in totally
blind subjects: a pilot placebo-controlled multicenter trial. Nat Sci Sleep 2015;7:
13 –23.
27. Takaesu Y, Komada Y, Inoue Y. Melatonin profile and its relation to circadian
rhythm sleep disorders in Angelman syndrome patients. Sleep Med 2012;13:
1164 –1170.
28. Li Y, Vgontzas AN, Fernandez-Mendoza J, Bixler EO, Sun Y, Zhou J, Ren R, Li T,
Tang X. Insomnia with physiological hyperarousal is associated with hypertension.
Hypertension 2015;65:644 –650.
29. Scheer FA, Morris CJ, Garcia JI, Smales C, Kelly EE, Marks J, Malhotra A, Shea SA.
Repeated melatonin supplementation improves sleep in hypertensive patients treated with beta-blockers: a randomized controlled trial. Sleep 2012;35:1395 –1402.
30. Lemoine P, Wade AG, Katz A, Nir T, Zisapel N. Efficacy and safety of prolongedrelease melatonin for insomnia in middle-aged and elderly patients with hypertension: a combined analysis of controlled clinical trials. Integr Blood Press Control 2012;
5:9– 17.
31. Scheer FA, Morris CJ, Garcia JI. Repeated melatonin supplementation improves
sleep in hypertensive patients treated with beta-blockers: a randomized controlled
trial. Sleep 2012;35:1395 – 1402.
32. Petrosillo G, Colantuono G, Moro N, Ruggiero FM, Tiravanti E, Di Venosa N,
Fiore T, Paradies G. Melatonin protects against heart ischemia-reperfusion injury
by inhibiting mitochondrial permeability transition pore opening. Am J Physiol Heart
Circ Physiol 2009;297:H1487 –H1489.
33. Diez ER, Renna NF, Prado NJ, Lembo C, Ponce Zumino AZ, Vazquez-Prieto M,
Miatello RM. Melatonin, given at the time of reperfusion, prevents ventricular arrhythmias in isolated hearts from fructose-fed rats and spontaneously hypertensive
rats. J Pineal Res 2013;55:166 –173.
34. Lecour S. Multiple protective pathways against reperfusion injury: a SAFE path
without Aktion? J Mol Cell Cardiol 2009;46:607 –609.
35. Manzanero S, Santro T, Arumugam TV. Neuronal oxidative stress in acute ischemic
stroke: sources and contribution to cell injury. Neurochem Int 2013;62:712 –718.
36. DeGirolami U, Crowell RM, Marcoux FW. Selective necrosis and total necrosis in
focal cerebral ischemia. Neuropathologic observations on experimental middle
cerebral artery occlusion in the macaque monkey. J Neuropathol Exp Neurol 1984;
43:57–71.
37. Chen HY, Chen TY, Lee MY, Chen ST, Hsu YS, Kuo YL, Chang GL, Wu TS, Lee EJ.
Melatonin decreases neurovascular oxidative/nitrosative damage and protects
against early increases in the blood-brain barrier permeability after transient focal
cerebral ischemia in mice. J Pineal Res 2006;41:175 –182.
38. Shinozuka K, Staples M, Borlongan CV. Melatonin-based therapeutics for neuroprotection in stroke. Int J Mol Sci 2013;14:8924 – 8947.
39. Esposito E, Cuzzocrea S. Antiinflammatory activity of melatonin in central nervous
system. Curr Neuropharmacol 2010;8:228 –242.
40. Andrabi SA, Sayeed I, Siemen D, Wolf G, Horn TF. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for antiapoptotic effects of melatonin. FASEB J 2004;18:869 –871.
41. Mesri Alamdari N, Mahdavi R, Roshanravan N, Lotfi Yaghin N, Ostadrahimi AR,
Faramarzi E. A double-blind, placebo-controlled trial related to the effects of melatonin on oxidative stress and inflammatory parameters of obese women. Horm
Metab Res 2015;4:504 – 508.
42. Korkmaz A, Ma S, Topal T, Rosales-Corral S, Tan DX, Reiter RJ. Glucose: a vital
toxin and potential utility of melatonin in protecting against the diabetic state. Ml
Cell Endocrinol 2012;349:128–137.
43. Zephy D, Ahmad J. Type 2 diabetes mellitus: role of melatonin and oxidative stress.
Diabetes Metab Syndr 2015;9:127 – 1346.
44. Söderquist F, Hellström PM, Cunningham JL. Human gastroenteropancreatic expression of melatonin and its receptors MT1 and MT2. PLoS ONE 2015;10:
e0120195.
7
8
67. Hrenak J, Paulis L, Repova K, Aziriova S, Nagtegaal EJ, Reiter RJ, Simko F. Melatonin
and renal protection: novel perspectives from animal experiments and human studies (review). Curr Pharm Des 2015;21:936 –949.
68. Cho YA, Noh K, Jue SS, Lee SY, Kim EC. Melatonin promotes hepatic differentiation
of human dental pulp stem cells: clinical implications for the prevention of liver
fibrosis. J Pineal Res 2015;58:127 –135.
69. Hong RT, Xu JM, Mei Q. Melatonin ameliorates experimental hepatic
fibrosis induced by carbon tetrachloride in rats. World J Gastroenterol 2009;15:
1452– 1458.
70. Blask DE, Brainard GC, Dauchy RT, Hanifin JP, Davidson LK, Krause JA, Sauer LA,
Rivera-Bermudez MA, Dubocovich ML, Jasser SA, Lynch DT, Rollag MD, Zalatan F.
Melatonin-depleted blood from premenopausal women exposed to light at night
stimulates growth of human breast cancer xenografts in nude rats. Cancer Res
2005;65:11174 – 11184.
71. Reiter RJ, Rosales-Corral S, Coto-Montes A, Boga JA, Tan DX, Davis JM,
Konturek PC, Konturek SJ, Brzozowski T. The photoperiod, circadian regulation
and chronodisruption: the requisite interplay between the suprachiasmatic nuclei
and the pineal and gut melatonin. J Physiol Pharmacol 2011;62:269 –274.
72. Sallinen P, Mänttäri S, Leskinen H, Ilves M, Vakkuri O, Ruskoaho H, Saarela S. The
effect of myocardial infarction on the synthesis, concentration and receptor expression of endogenous melatonin. J Pineal Res 2007;42:254 –256.
L.H. Opie et al.
73. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, Samimi-Fard S,
Kaski JC, Reiter RJ. Light/dark patterns of soluble vascular cell adhesion molecule-1
in relation to melatonin in patients with ST-segment elevation myocardial infarction. J Pineal Res 2008;44:65 –69.
74. Sallinen P, Mänttäri S, Leskinen H, Ilves M, Vakkuri O, Ruskoaho H, Saarela S. The
effect of myocardial infarction on the synthesis, concentration and receptor expression of endogenous melatonin. J Pineal Res 2007;42:254 –260.
75. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, Kaski JC,
Reiter RJ, Jimenez-Sosa A. A unicenter, randomized, double-blind, parallel-group,
placebo-controlled study of l: study design and rationale. Contemp Clin Trials 2007;
28:532–539.
76. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Saiz MM, Aldea-Perona A, de
la Torre JM, Garcia-Camarero T, Consuegra-Sanchez L; MARIA Study Investigators.
MARIA Study Investigators. Cardioprotection with melatonin in the acute myocardial infarction: awaiting results of MARIA trial? Int J Cardiol 2015;182:54 –55.
77. Dogan NB, Ozpelit E, Akdeniz S, Bilgin M, Baris N. Simple clinical risk score for noreflow prediction in patients undergoing primary percutaneous coronary intervention with acute STEMI. Pak J Med Sci 2015;31:576–581.
78. Dominguez-Rodriguez A, Abreu-Gonzalez P, Arroyo-Ucar E, Reiter RJ. Decreased
level of melatonin in serum predicts left ventricular remodelling after acute myocardial infarction. J Pineal Res 2012;53:319 –323.
Downloaded from http://ehjcvp.oxfordjournals.org/ by guest on August 25, 2016