Beta-carboline alkaloids and essential tremor: exploring the environmental determinants of one of the most prevalent neurological diseases.

Abstract

Essential tremor (ET) is among the most prevalent neurological diseases, yet its etiology is not well understood. Susceptibility genotypes undoubtedly underlie many ET cases, although no genes have been identified thus far. Environmental factors are also likely to contribute to the etiology of ET. Harmane (1-methyl-9H-pyrido[3,4-beta]indole) is a potent, tremor-producing beta-carboline alkaloid, and emerging literature has provided initial links between this neurotoxin and ET. In this report, we review this literature. Two studies, both in New York, have demonstrated higher blood harmane levels in ET cases than controls and, in one study, especially high levels in familial ET cases. Replication studies of populations outside of New York and studies of brain harmane levels in ET have yet to be undertaken. A small number of studies have explored several of the biological correlates of exposure to harmane in ET patients. Studies of the mechanisms of this putative elevation of harmane in ET have explored the role of increased dietary consumption, finding weak evidence of increased exogenous intake in male ET cases, and other studies have found initial evidence that the elevated harmane in ET might be due to a hereditarily reduced capacity to metabolize harmane to harmine (7-methoxy-1-methyl-9H-pyrido[3,4-beta]-indole). Studies of harmane and its possible association with ET have been intriguing. Additional studies are needed to establish more definitively whether these toxic exposures are associated with ET and are of etiological importance.

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TheScientificWorldJOURNAL (2010) 10, 1783–1794
ISSN 1537-744X; DOI 10.1100/tsw.2010.159


*Corresponding author.
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Published by TheScientificWorld; www.thescientificworld.com


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β-Carboline Alkaloids and Essential
Tremor: Exploring the Environmental
Determinants of One of the Most Prevalent
Neurological Diseases
Elan D. Louis1,2,3,4,* and Wei Zheng5
1GH Sergievsky Center, 2Department of Neurology, 3Taub Institute for Research on
Alzheimer’s Disease and the Aging Brain, College of Physicians and Surgeons,
Columbia University, New York; 4Department of Epidemiology, Mailman School of
Public Health, Columbia University, New York; 5School of Health Sciences, Purdue
University, West Lafayette, IN
E-mail: EDL2@columbia.edu; wzheng@purdue.edu
Received May 24, 2010; Revised July 15, 2010; Accepted July 15, 2010; Published September 1, 2010
Essential tremor (ET) is among the most prevalent neurological diseases, yet its
etiology is not well understood. Susceptibility genotypes undoubtedly underlie many
ET cases, although no genes have been identified thus far. Environmental factors are
also likely to contribute to the etiology of ET. Harmane (1-methyl-9H-pyrido[3,4-
β]indole) is a potent, tremor-producing β-carboline alkaloid, and emerging literature
has provided initial links between this neurotoxin and ET. In this report, we review this
literature. Two studies, both in New York, have demonstrated higher blood harmane
levels in ET cases than controls and, in one study, especially high levels in familial ET
cases. Replication studies of populations outside of New York and studies of brain
harmane levels in ET have yet to be undertaken. A small number of studies have
explored several of the biological correlates of exposure to harmane in ET patients.
Studies of the mechanisms of this putative elevation of harmane in ET have explored
the role of increased dietary consumption, finding weak evidence of increased
exogenous intake in male ET cases, and other studies have found initial evidence that
the elevated harmane in ET might be due to a hereditarily reduced capacity to
metabolize harmane to harmine (7-methoxy-1-methyl-9H-pyrido[3,4-β]-indole). Studies
of harmane and its possible association with ET have been intriguing. Additional
studies are needed to establish more definitively whether these toxic exposures are
associated with ET and are of etiological importance.
KEYWORDS: essential tremor, environmental epidemiology, etiology, toxin, harmane

INTRODUCTION
Essential tremor (ET) is a chronic neurological disease that is usually progressive. Its hallmark clinical
feature is a 4- to 12-Hz kinetic tremor (i.e., tremor that occurs during volitional movements) of the

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arms[1], which may manifest during a broad range of daily tasks (e.g., eating, writing, and shaving) (Fig.
1). Across affected individuals, this tremor may range in severity from mild and functionally
inconsequential to severe and disabling[2,3,4,5]. In addition to the classical kinetic tremor of the arms,
patients may exhibit a variety of tremors that differ by location (e.g., head, voice, and jaw) and condition
(postural, intentional, and rest)[1,6,7,8]. Aside from tremor, other motor manifestations have been
documented; perhaps most notable of these is a gait that has been described as mildly ataxic[9,10]. In
addition, there is growing recognition of the presence of mild cognitive changes (especially executive
dysfunction)[11,12,13,14,15,16]. In epidemiological studies, ET has been shown to be associated with
increased risks of Alzheimer’s disease[17,18] and Parkinson’s disease[19]. Although traditionally viewed
as a disease of morbidity rather than mortality, in the single, prospective, population-based study that
enrolled a control group, there was a 45% increased risk of mortality[20], despite the absence of such a
finding in one earlier retrospective study using historical controls[21].

FIGURE 1. The effects of tremor are apparent in an ET patient’s attempt to
draw an Archimedes spiral.
ET is one of the most common neurological diseases; in a meta-analysis of the 28 population-based
prevalence studies, the pooled prevalence (all ages) = 0.9%[22]. A commonly cited value is that the
population prevalence is 4.0% after age 39 years[23]. The prevalence increases markedly with age,
especially with advanced age (i.e., 4.6% among persons aged 65 years and older, and as high as 21.7% in
persons aged 95 and older)[22].
The underlying pathogenesis of ET is poorly understood, although recent postmortem studies indicate
the presence of degenerative changes in the cerebellum in the majority of cases, including Purkinje cell
loss in excess of that seen in similarly aged controls[24,25].

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ETIOLOGY OF ET
The common presence, in clinic samples, of ET families with multiple affected individuals over several
generations has led to the view that genetic susceptibility is of importance in disease etiology[26].
Although no genes have yet been identified, linkage to regions on chromosome 2p[27], 3q[28], and
6p[29] indicate that genetic factors are indeed of importance in this disease. A commonly cited value is
that 50% of ET cases occur on a familial basis[30,31].
Although widely considered to have a large genetic component, it is likely that environmental factors also
contribute to the etiology of ET[32]. The high preponderance of ET cases without affected relatives (>50% of
ET cases in some series)[31,33,34,35,36,37,38] and the results from concordance studies (60% concordance in
monozygotic twins in one study and 63% in another)[39,40] are among the observations that have been used to
support the notion that nongenetic factors are important in this disease[32,41]. The existence of intrafamilial
differences in age of onset and severity of tremor[42,43] also suggests that environmental (or perhaps other
genetic) factors may be serving as modifiers of underlying susceptibility genotypes.
Environmental factors are also thought to play a substantial role in other neurological disorders
(Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis)[44,45,46,47,48,49,50,51,52,
53,54,55], so that it is not inconceivable that they could play a role in ET. Using the commonly cited value
of 50% for the percentage of cases that occur on a familial basis[30,31] and given its population prevalence
of 4.0% after age 39 years[23], this then suggests that approximately 2.0% of the population aged 40 years
has a nonfamilial form of ET[23,32]. Despite the apparent extent of the problem, the environmental
correlates for this tremor are just beginning to be explored. One possible link has been established with the
exposure to β-carboline alkaloids. The purpose of this article is to critically review the published literature
on the links between β-carboline alkaloids and ET.
β-CARBOLINES AS POSSIBLE ENVIRONMENTAL TOXINS IN ET
Rationale for Their Study in ET
The β-carboline alkaloids are a group of naturally occurring, tremor-producing chemicals that include
harmane, harmine, harmaline, and several others[32,56,57,58]. Structurally, the β-carboline alkaloids are
heterocyclic amines, comprised of a combination of five- and six-ringed (i.e., cyclic) carbon structures,
containing an amine group[59,60] (Fig. 2). There is considerable structural similarity with 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is used to produce one of the main toxin-induced
animal models for Parkinson’s disease[61,62,63]. Like MPTP, the β-carboline alkaloids are highly
neurotoxic, and the administration of β-carboline alkaloids to a wide variety of laboratory animals
produces an intense and generalized action tremor that resembles ET[32,64]. Indeed, β-carboline alkaloid
administration is currently viewed as the main animal model for ET and new pharmacotherapies are
tested using exposed animals[65,66,67,68,69]. The tremor that is produced by β-carboline alkaloids
shares several features with ET, including its primary clinical features and drug-response
characteristics[69,70,71,72,73,74], and underlying brain changes (destructive changes in the cerebellum,
including Purkinje cell loss, which has now been documented in ET and in the β-carboline alkaloid model
of tremor)[25,64,69,70,75,76,77,78,79]. Human volunteers exposed to intravenously administered
harmine exhibit neurological signs, including an acute, coarse tremor[80,81].
Harmane (1-methyl-9H-pyrido[3,4-β]indole) is a potent, tremor-producing β-carboline alkaloid[56],
capable of inducing tremor in laboratory animals within 3.1 min of subcutaneous injection[57]. Due to its
high lipid solubility, harmane accumulates in brain tissue, with brain concentrations in laboratory animals
6.5 times those injected into peripheral tissues[57]. Furthermore, harmane is one of the most abundant of all
dietary heterocyclic amines and human exposure to harmane through diet is greater than that of other
heterocyclic amines[82,83], making it a β-carboline alkaloid of particularly high interest in epidemiological
studies of humans[32].

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FIGURE 2. Chemical structures of harmane (1-methyl-9H-pyrido[3,4-β]indole) and
harmine (7-methoxy-1-methyl-9H-pyrido[3,4-β]-indole).
β-Carboline alkaloids are produced by the body endogenously[84,85], although exogenous sources
are likely to be more important; one study estimated that dietary sources were 50 times greater than these
endogenous sources[86]. β-Carboline alkaloids may be found in particularly high ng/g concentrations in
animal protein (i.e., meat) and cooking results in additional increases in concentrations[83,87,88,89,90].
β-Carboline alkaloids are also present in varying concentrations in a variety of plant-derived
foods/substances, including coffee, ethanol, and tobacco[32,91]. The demonstrated tremor-inducing
property of the β-carboline alkaloids, along with their presence in the food supply, provides the rationale
for an in-depth scrutiny of their potential role in the etiology of ET, the most common tremor disorder.
Are Levels Elevated in ET?
Hypothesizing that β-carboline alkaloids might be an environmental exposure of etiological importance in
ET, Louis and colleagues began to study their blood concentrations in ET cases in 2000[60,92].
Concentrating their efforts on harmane, which is one of the most abundant of all dietary heterocyclic
amines[82,83], and harmine (7-methoxy-1-methyl-9H-pyrido[3,4-β]-indole), a probable metabolic
breakdown product of harmane[60], they demonstrated that blood harmane concentration was elevated in
100 ET cases at the Neurological Institute of New York compared to 100 matched control subjects
(median blood harmane [cases] = 5.21 vs. 2.28 g–10/mL [controls], p = 0.005)[93]. Blood harmine
concentrations were also marginally, but not significantly, elevated in ET cases (median blood harmine
[cases] = 1.60 vs. 0.65 g–10/mL [controls], p = 0.19). A larger replicate sample of 150 ET cases and 135
matched controls was assembled over the ensuing 5 years (2002–2007)[94]; while overall concentrations
were lower in that sample, the case-control difference persisted (median blood harmane [cases] = 2.61 vs.
1.82 g–10/mL [controls], p = 0.016)[94]. Among ET cases, there was no correlation between blood
harmane concentration and tremor severity (total tremor score, Pearson's r = −0.01, p = 0.87), yet in a
small subgroup of 24 ET cases with very severe tremor (total tremor score ≥25), blood harmane
concentrations were very high. In linear regression analysis in which log blood harmane concentration
was the outcome variable, the ordinal independent variable was coded as follows: ET with high total
tremor score (2), remainder of ET cases (1), and controls (0), beta = 0.16, p = 0.03, indicating a
significant trend (i.e., as one progressed from controls to ET cases and finally to ET cases with severe
tremor, blood harmane concentration progressively increased)[94].
These two studies provide preliminary evidence for elevated blood harmane concentrations in ET.
There is, however, a need to replicate these data at other centers with additional ET case samples.
Are Levels Elevated in the ET Brain?
Levels of harmane have been shown to be elevated in the blood in two studies to date. This result is
intriguing; however, the brain is the presumptive target organ for harmane-induced effects in patients with

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ET. Are brain levels of harmane elevated in ET? Data are indirect and very limited and will be reviewed
below.
Magnetic resonance spectroscopic imaging studies[95,96] have demonstrated reductions in the N-
acetyl aspartate to creatine ratio in the cerebellum of ET cases compared with controls. N-Acetyl aspartate
is an amino acid that is present in the cytosol of neurons and is a marker of neuronal integrity. Reductions
in N-acetyl aspartate (often expressed as a ratio to brain creatine) are a marker of neuronal damage and
probable neuronal loss[97,98]. These reductions in the N-acetyl aspartate to creatine ratio in the ET
cerebellum are consistent with postmortem studies that have documented degenerative changes, including
Purkinje cell loss, in the ET cerebellum[79,99]. Hypothesizing that blood harmane concentration in ET
cases would be inversely correlated with cerebellar NAA/tCR, a neuroimaging measure of neuronal
dysfunction or degeneration, Louis et al.[100] used magnetic resonance spectroscopic imaging to assess
12 ET cases in a pilot study. A priori, the neuroanatomic structure of interest was the cerebellar cortex.
Secondary regions were the central cerebellar white matter, cerebellar vermis, thalamus, and basal
ganglia. Blood concentrations of harmane and another neurotoxin, lead, were also assessed. In a linear
regression model that adjusted for age and gender, log blood harmane concentration was a predictor of
cerebellar NAA/tCR (p = 0.009) such that every 1 g–10/mL unit increase in log blood harmane
concentration was associated with a 0.41 unit decrease in cerebellar NAA/tCR. The association between
blood harmane concentration and brain NAA/tCR only occurred in the cerebellar cortex; it was not
observed in any of the secondary brain regions of interest. Furthermore, the association was specific to
harmane and not lead[100]. These results provide preliminary support for the view that increased blood
harmane concentration in ET is associated with cerebellar neuronal damage, consistent with the animal
study data that harmane and other β-carboline alkaloids produce cerebellar damage[64,69,70,75,
76,77,78]. Of course, this pilot study did not directly measure brain harmane concentration in ET cases,
and studies of human postmortem tissue are needed in order to test the association between brain harmane
levels and ET directly.
Other Possible Biological Correlates of Elevated Harmane in ET
In a number of studies of ET patients, additional biological correlates of elevated harmane have been
examined in an attempt to explore the wider biological effects of this toxin. In the first study[101], the
association between elevated blood harmane and impaired olfaction was explored. Olfactory dysfunction
occurs in a variety of diseases, including patients with cerebellar diseases[102,103], and there is evidence
that the cerebellum itself may play a role in central olfactory processing[102,103,104,105]. Olfactory
dysfunction has also been reported in patients with ET in some studies[106]. As noted above, the
pathophysiology of ET is not well understood, although recent postmortem studies indicate the presence
of degenerative changes in the cerebellum[24,25]. β-Carboline alkaloids have been shown to produce
marked cerebellar damage in exposed laboratory animals in several studies[70,77]. To further test the
model that harmane, through cerebellar toxicity, leads to ET, Louis et al.[101] hypothesized that they
would find a correlation between blood harmane concentrations and smell test scores in ET cases (i.e.,
higher blood harmane concentration would be associated with greater olfactory dysfunction) and
furthermore, that such a correlation would not be detected among a group of controls. In that study of 83
ET cases and 69 controls, they indeed found that higher blood harmane concentration was correlated with
lower smell test scores in ET cases (rho = –0.46, p < 0.001); however, in controls, higher blood harmane
concentration was not correlated with lower smell test scores (rho = 0.12, p = 0.32)[101].
In a second study, Louis et al.[107] examined associations between harmane and cancer. Many of the
heterocyclic amines are also mutagens and are linked with several types of cancer (especially colon and
prostate)[59,108,109]. Harmane itself is not mutagenic, yet it exerts comutagenic activity both in bacterial
and mammalian cells[110,111]. Harmane is a comutagen and a high blood concentration could
conceivably predispose an individual to cancer. As noted above, blood harmane concentrations also seem
to be elevated in initial studies of ET patients[93,94]. Louis et al.[107], therefore, tested the hypothesis