Nutrition 31 (2015) 13171323

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Nutrition
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Review

TMAO: A small molecule of great expectations

Marcin Ufnal M.D., Ph.D. a, *, Anna Zadlo M.Sc. b, Ryszard Ostaszewski Ph.D. b
a
Department of Experimental Physiology and Pathophysiology, Laboratory of Centre for Preclinical Research, Medical University of Warsaw,
Warsaw, Poland
b
Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Trimethylamine N-oxide (TMAO) is a small organic compound whose concentration in blood
Received 18 February 2015 increases after ingesting dietary L-carnitine and phosphatidylcholine. Recent clinical studies show a
Accepted 10 May 2015 positive correlation between elevated plasma levels of TMAO and an increased risk for major
adverse cardiovascular events dened as death, myocardial infarction, or stroke. Several experi-
Keywords: mental studies suggest a possible contribution of TMAO to the etiology of cardiovascular diseases
Trimethylamine N-oxide
by affecting lipid and hormonal homeostasis. On the other hand, TMAO-rich seafood, which is an
TMA
important source of protein and vitamins in the Mediterranean diet, has been considered benecial
Circulatory system
Diabetes for the circulatory system. Although in humans TMAO is known mainly as a waste product of
Diet choline metabolism, a number of studies suggest an involvement of TMAO in important biological
Osmolyte functions in numerous organisms, ranging from bacteria to mammals. For example, cells use TMAO
Cancer to maintain cell volume under conditions of osmotic and hydrostatic pressure stresses. In this
Oxidative stress article, we reviewed well-established chemical and biological properties of TMAO and dietary
sources of TMAO, as well as looked at the studies suggesting possible involvement of TMAO in the
etiology of cardiovascular and other diseases, such as kidney failure, diabetes, and cancer.
2015 Elsevier Inc. All rights reserved.

Introduction
In Charles Dickens novel Great Expectations, Pip, a poor
orphan, faces the turning point in his life when he receives an
unexpected fortune. This raises great expectations in Pip that go
beyond the said fortune. Unfortunately, as Pip lives a new life, his
great expectations are ruined. Likewise, in a world in which more
people die annually from cardiovascular diseases (CVD) than
from any other cause, a newly discovered marker raises great
expectations in both patients and doctors. Recently, an associa-
tion between an elevated fasting plasma trimethylamine N-oxide
(TMAO) and an increased risk for major adverse cardiovascular
events has been identied. New research suggests that TMAO
affects lipid and hormonal homeostasis and thereby possibly
contributes to the development of CVD. TMAO is a metabolite of
phosphatidylcholine and L-carnitine, both abundant in red meat.
A.Z. and R.O. wrote the paragraph Chemical and physical properties of
TMAO . and prepared illustrations 1 to 4. M.U. wrote the remaining para-
graphs and prepared illustration 5. All the authors have approved the nal
version of the manuscript.
* Corresponding author. Tel.: 48 (0) 22 116 6113; fax: 48 (0) 22116 6201.
E-mail address: mufnal@wum.edu.pl (M. Ufnal).
http://dx.doi.org/10.1016/j.nut.2015.05.006
0899-9007/ 2015 Elsevier Inc. All rights reserved.
For this reason, TMAO has been proposed to constitute a link
between diet and CVD. On the other hand, it is well established
that TMAO plays a protective role in cell homeostasis in
numerous animal species. For example, cells use TMAO to
maintain cell volume under conditions of osmotic and hydro-
static pressure stresses.
Is TMAO the long sought-after link between unhealthy diet
and CVD, or do we, like Pip, rely too much on what we do not
have yet? In what follows here, we review studies on chemical
and biological properties of TMAO and its possible role in the
development of CVD and other diseases.
Chemical and physical properties of TMAO, interactions
with proteins
TMAO is an organic compound belonging to the class of amine
oxides with the formula (CH3)3 NO (Fig. 1). It occurs in the form
of a colorless solid and is usually encountered as a dihydrate.
Chemically, it is obtained in a straightforward procedure starting
from trimethylamine (TMA), (Fig. 2).
TMAO is capable of affecting the structure and activity of
many biologically important compounds. For example, it has
1318 M. Ufnal et al. / Nutrition 31 (2015) 13171323
Fig. 1. Molecular structure of TMAO. TMAO is an amphiphile consisting of a small
hydrophilic NO group and a bulky hydrophobic three-methyl group. Compared
with other amphiphiles, the hydrophilic part is highly polar. TMAO, trimethyl-
amine-N-oxide.
been well established that TMAO is an important stabilizer of the
protein-folded state and nucleic acids [1,2]. Numerous thermo-
dynamic studies on the effects of TMAO on proteins have
conrmed that it counteracts the effects of protein denaturants,
heat, and pressure [3]. Several mechanisms have been proposed
to explain the folding propensity of TMAO [4,5], however, they
are still not fully understood (Figs. 3 and 4).
It has been speculated that TMAO affects the stability of
proteins dissolved in water by modifying the water structure and
hydrogen bond strength. Several studies have pointed to the
signicance of a strong interaction between the hydrophilic
group of TMAO and water for TMAO osmolytic activity. The
implication from these ndings is that TMAO stabilizes proteins
by modifying protein polarity and activity of water. It would
indirectly affect the equilibrium between the unfolded and fol-
ded conformations of proteins [6,7]. However, several molecular
dynamics simulations in aqueous solutions of TMAO in which no
signicant alteration of water structure was found negated this
mechanism [8,9]. Other studies have suggested the preferential
solvation model [10], in which water and urea tend to solvate
TMAO rather than the protein residues. Hydrogen bonds
between TMAOwater and TMAOurea have the largest energies
in that system. In line with this model, a decrease in the number
of peptidewater hydrogen bonds [11] and dehydration of a
Fig. 2. Tertiary amines have been found to react directly with molecular oxygen under high oxygen pressure [91] or with peroxides [92] to give the corresponding N-oxide in
high yields. The applications of amine N-oxides are very wide in organic and organometallic chemistry, including in oxidation reactions, decarbonylation of metal carbonyl
derivatives, liberation of organic ligands from the metal complexes, application as a ligand in coordination chemistry. TMAO may also be obtained by biosynthesis [93]. TMAO,
trimethylamine-N-oxide.
carbon nanotube [12] were observed. However, the mechanism
requires removal of urea from the protein solvation shell, which
is inconsistent with the observation that TMAO is not interfered
by the presence of a competing osmolyte [13]. Other studies,
based on thermodynamic measurements, have suggested that
stabilizing effects could be induced by direct interaction of TMAO
with the protein backbone and/or TMAO with the side chains of
proteins [14]. The oxygen atom on TMAO can favorably interact
as a hydrogen bond acceptor, while three methyl groups can take
part in hydrophobic interactions with the side chains of proteins.
In contrast, entropically unfavorable interactions can occur in
particular with the backbone amides. If such unfavorable in-
teractions prevail, TMAO is depleted from the protein surface.
The resulting concentration gradient in TMAO may lead to
osmotic pressure favoring the folded conformation. TMAO,
which is a stabilizing osmolyte, is preferentially excluded from
the protein backbone because water reacts favorably with
backbone polar groups, whereas destabilizing osmolytes, such as
urea or guanidine hydrochloride, are preferentially accumulated
in the vicinity of protein backbone because they exhibit a
stronger propensity to interact with the backbone than water [9].
The priority interaction of destabilizers with the unfolded
ensemble of the protein provides the thermodynamic driving
force for unfolding. It is generally accepted that TMAO is pref-
erentially excluded from the protein surface [15,16]. Based on
computer modeling studies and the experimental osmotic
pressure measurements, it has been proposed that interaction of
cosolvents with protein surfaces preferentially excludes TMAO
because of repulsive self-interaction in the solvation shell [17].
TMAO: A waste product?
TMAO is a common compound found in animals, but is also
present in plants and fungi [1820]. The origin of TMAO may be
exogenous and endogenous. In humans, most TMAO seems to
come from the oxygenation of TMA, a TMAO precursor produced
by gut ora [21]. In contrast, in marine animals TMAO may be
synthetized endogenously [22]; however, the mechanisms
involved are not clear [23]. The concentration of TMAO in marine
animals signicantly exceeds its concentration in other organ-
isms [24]. Some bacteria reduce TMAO to TMA, which is
responsible for the characteristic smell of decaying seafood.
Interestingly, the TMA-to-TMAO ratio has been used as a marker
of freshness in the shing industry [25]. In humans, TMAO is
known mostly as a waste product of choline metabolism.
Nevertheless, research provides evidence for involvement of
TMAO in important biological functions in numerous organisms,
ranging from bacteria to mammals [24] (Fig. 5).
 M. Ufnal et al. / Nutrition 31 (2015) 13171323
Fig. 3. Schematic illustration of the postulated interaction between TMAO and
three water molecules. At the oxygen atom, three lone pairs are located in one of
the resonant molecular structures. Therefore, TMAO can accept up to three strong
hydrogen bonds at its hydrophilic head [17]. TMAO, trimethylamine-N-oxide.
TMAO and bacteria
TMAO has been found to serve as an electron acceptor in the
anaerobic metabolism of various bacteria living mostly in the
three environments: the marine environment (e.g., Alteromonas
and Vibrio), the brackish pond (non-sulfur photosynthetic bacte-
ria), and animal intestines (Enterobacteriaceae) [26]. In the Enter-
obacteriaceae, a normal part of the human gut ora, anaerobic
respiration with TMAO has been shown to support oxidative
phosphorylation [26]. On the other hand, TMAO has been found to
inhibit growth of other bacteria, such as Staphylococcus aureus
[27], which is an important pathogen in humans and animals.
Substantial evidence exists that TMAO represents a signicant
nutrient for marine bacteria [28] and may play an important role
in the global carbon and nitrogen cycles [29].
Fig. 4. Model of TMAO and urea effects on protein folding. TMAO, a stabilizing osmolyte, does not bind to proteins. It is preferentially excluded from the proteins hydration
layer. By contrast, destabilizers such as urea generally bind to proteins, causing them to unfold. TMAO, trimethylamine-N-oxide.
1319
Mammalian gut ora enzymes catalyze production of several
methylamines, including TMA, dimethylamine (DMA), and
monomethylamine, from dietary choline and lecithin. Some of
these methylamines are absorbed into the bloodstream and
eventually excreted in an unchanged or oxidized form (TMAO) in
urine or sweat, whereas methylamines remaining in the gut may
serve as substrates for the formation of nitrosamines by various
bacteria [30,31].
TMAO as an osmolyte
The ability to control cell volume is a pivotal survival capa-
bility for single- and multiple-cell organisms. Osmolytes are
small compounds used by cells to maintain cell volume. They
play a key role in the adaptation of cells to osmotic and hydro-
static stress. Different organisms use different osmolytes
depending on their enzymatic competence and the availability of
substrates [18,23].
The function of osmolytes has been elegantly described
elsewhere [18,24,32]. In short, there are several types of osmo-
lytes used by cells to regulate their volume, including amino
acids and derivatives, polyols and sugars, methylamines, and
urea. In contrast to salt ions, most osmolytes do not perturb
protein structures. Moreover, some osmolytes, such as TMAO,
seem to stabilize macromolecules by neutralizing perturbations
caused by changes in osmolarity, hydrostatic pressure, or urea
[23,24,33].
Osmotic stress usually results from water loss due to eva-
poration or attraction of water by hyperosmotic aqueous sur-
roundings. These apply to animals living in hot or saline habitats,
or both. The role of osmolytes, and TMAO in particular, has also
been recognized in deep-sea animals that are exposed to high
hydrostatic pressure. It has been suggested that deep-sea shes
use TMAO to counteract the protein-destabilizing effects of
osmotic and hydrostatic pressures [23,24].
Humans modify their environment to suit their needs, but
their tissues are also exposed to osmotic and hydrostatic
stresses. For example, cells in the kidney medulla operate in the
environment with the osmolarity three to ve times higher
than the osmolarity of plasma. To produce concentrated urine,
1320 M. Ufnal et al. / Nutrition 31 (2015) 13171323
Fig. 5. Biological and chemical properties of TMAO and their postulated effects on
homeostasis of organisms. TMAO, trimethylamine-N-oxide.
kidneys accumulate sodium and urea to establish high osmotic
gradient in the medullary region. It has been reported that
kidneys accumulate TMAO, which may serve as an osmotic
agent and protect kidney cell proteins from perturbations
caused by urea [33,34].
Moreover, cells in the gastrointestinal tract are subjected to
an extreme osmotic stress as the osmolarity of diet may vary
from nearly zero (e.g., tap water) to several hundred mOsm/kg
(e.g., potato chips or other highly salted foods). Osmotic stress
also affects the cells of patients suffering from waterelectrolyte
balance disturbances or diabetes. Finally, due to frequent
changes in blood pressure, hydrostatic stress affects the cells of
the heart, arteries, and veins, the latter especially in patients
suffering from venous valve insufciency. Intriguingly, to our
knowledge, except for a limited research on the role of TMAO in
the kidneys [24], the role of TMAO as an osmolyte in other
human organs has not yet been investigated.
Metabolism of TMAO
It seems that, in contrast to some marine animals that may
synthetize it endogenously, TMAO in terrestrial mammals
comes from exogenous sources [18,21,22,35]. TMAO concen-
tration in blood increases after ingestion of dietary choline
and L-carnitine [30,36]. Choline is acquired from the diet
and endogenous synthesis; however, the latter source is not
sufcient to meet human requirements. The average human
diet contains about 500 mg of free choline. Dietary sources rich
in choline include red meat, sh, poultry, and eggs. Other food
sources of choline are whole grains, soy, and vegetables such as
cauliower and cabbage [37]. Furthermore, numerous dietary
supplements marketed as antidementia drugs or products
improving heart and liver function contain phosphatidylcholine
in high concentration. It appears that there is a threshold
concentration of choline that must be ingested to be converted
to TMAO [35,36,38]. Choline is absorbed from the small intes-
tine via mediated transport, which is half saturated at choline
concentrations in the intestines of 200 to 300 mM [3941]. If
choline-rich food is ingested, and the concentration of choline
in the small intestine exceeds the transport capacity, choline
reaches the large bowel. It is metabolized in the large bowel by
intestinal bacteria producing TMA and DMA [42,43].
Another important source of TMAO in humans is L-carnitine
[44]. Similar to choline, L-carnitine is acquired from the
diet and biosynthesis [45]. It is found at high levels in red
meat, poultry, some dairy products [46,47], and various
over-the-counter dietary supplements. In contrast to choline,
L-carnitine is scarce in sh and eggs [46]. Carnitine in the large
bowel seems to undergo a similar bacterial processing to
choline to form TMA.
Most of TMA derived from bacterial metabolism of choline
and L-carnitine is absorbed into the bloodstream and then TMA
is rapidly oxidized to TMAO by avin-containing mono-
oxygenase-3 (FMO3), a hepatic enzyme [30,43,48]. Treatment
with broad-spectrum antibiotics has been found to reduce
TMAO concentration in the blood of humans and laboratory
animals, which seems to conrm that gut bacteria are the key
source of TMAO in the blood [21,43,49]. However, evidence
exists that dietary TMAO and TMA also may be signicant
sources of TMAO in humans. In this context, seafood, an
important source of proteins and vitamins in many diets around
the world, is rich in TMAO, TMA, and DMA [25,50,51]. It has
been demonstrated that there is an increased urinary excretion
of TMAO in the Swedish but not British population, which
seems to be associated with higher consumption of sh by the
Swedish population [52]. Similarly, high urinary concentrations
of TMAO were reported in the Japanese population as compared
with that of North America [53].
Moreover, high urine TMAO concentration has been associ-
ated with urinary nitrogen excretion and a high-protein diet
[54]. This may result from the fact that a high-protein diet may
modify gut microbiota or affect the absorption of choline and
carnitine in the small intestines. However, another possible
explanation is that in humans on a high-protein diet, TMAO is
synthetized to dispose the excess of amine groups formed in the
metabolism of amino acids, as it seems to be the case in some
marine animals.
In humans, DMA, TMA, and TMAO are excreted mainly in
urine, as well as in sweat and exhaled air [51,5557].
TMAO at the bench and bedside
So far, clinicians have focused mainly on the role of TMAO in
sh odor syndrome. However, recently TMAO has attracted large
attention after the publication of several papers suggesting that
it may be an important diagnostic marker in CVD [21,43,49].
TMAO blood level
The concentration of TMAO in human blood plasma is in the
range of 3 mmol/L in healthy individuals to 40 mmol/L in patients
with renal failure [21,49,58]. TMAO plasma concentration has
been reported at w0.6 mmol/L in rats [59] and <5 mmol/L in mice
[21,43]. These variations may be attributed to several factors,
including amount of dietary choline, carnitine, TMA, and TMAO,
as well as the action of the gut microbiota or the activity of FMO3.
Interestingly, it was found that a 2-wk infusion of TMAO,
which increased blood TMAO levels 100-fold, from 0.6 to
60 mmol/L, did not produce any apparent toxic effects in rats [59].
This nding may be less surprising if we look at TMAO levels in
the blood in deep-sea animals, ranging from 100 to 300 mmol/L
[34]. Unfortunately, little is known about the concentration of
 M. Ufnal et al. / Nutrition 31 (2015) 13171323
TMAO in tissues other than blood, and there is no data on
intracellular TMAO level in mammals.
TMAO in cardiovascular diseases
Accumulating evidence suggests a possible role of an elevated
concentration of plasma TMAO as a new marker of an increased
cardiovascular risk in humans [21,49,60,61]. TMAO, as a metab-
olite of phosphatidylcholine and L-carnitine, is abundant in red
meat, egg yolks, and other products that are considered to be
atherogenic, and has been proposed to constitute a link between
such diet and CVD [21]. Clinical studies suggest a positive
correlation between high blood plasma TMAO levels and an
increased cardiovascular risk, independent of traditional risk
factors, such as diabetes or kidney failure [21,60]. However, it
must be stressed that low glomerular ltration rate along with
other risk factors, such as hypertension and diabetes mellitus,
were common in the top quartile of TMAO blood levels in
populations evaluated in the studies [49,60].
Several experimental studies have found evidence of TMAO
affecting lipid and hormonal homeostasis, which may suggest a
contribution of TMAO to the development of CVD. One study
found that TMAO may modulate cholesterol and sterol meta-
bolism, thus leading to the development of atherosclerosis [21].
The study demonstrated that TMAO and its dietary precursors,
choline and carnitine, suppress reverse cholesterol transport
through gut microbiota-dependent mechanisms [21]. TMAO also
has been found to affect the hemodynamic effects of chronically
infused angiotensin II [59], a pivotal hormone in the circulatory
system homeostasis. Finally, TMAO has been repeatedly shown
to affect protein folding [2,46]. This may exert a signicant
effect on the afnity of receptors as well as on the activity of
enzymes and hormones involved in the control of the circulatory
system homeostasis.
Several lines of evidence suggest that TMAO may play a
protective role in cardiovascular system by reducing deleterious
effects of oxidative stress. For example, it has been found that
oral L-carnitine supplementation signicantly increases plasma
TMA and TMAO levels and decreases markers of vascular injury
and oxidative stress such as soluble forms of intracellular
adhesion molecule-1, vascular cell adhesion molecule-1, and
malondialdehyde [62]. Furthermore, treatment with TMAO has
been found to inhibit the negative effects of oxidative stress in
streptozotocin diabetic rats [63] and human neuroblastoma cells
[64]; however, the mechanisms are not clear. TMAO has also
been found to act as an electron acceptor in bacteria [26].
Therefore, it may neutralize electrons that leaked from the
mitochondrial electron transport chain reducing the formation
of reactive oxygen species.
Fish odor syndrome
Trimethylaminuria, or sh odor syndrome, is a rare, auto-
somal recessive disease. Its name comes from the characteristic
odor of decaying sh and seafood for which TMA is responsible.
Trimethylaminuria is characterized by FMO3 deciency, which
results in accumulation of TMA in the body and its excretion in
urine, breath, and sweat. Mutations in the FMO3 gene have been
shown to underlie trimethylaminuria [65]. However, one study
reported that a reduction in FMO3 activity and increased
excretion of TMA are present in a considerable percentage of
healthy individuals without the classical features of trimethyla-
minuria [66]. This may suggest some yet unidentied factors
playing a role in determining the presence of the symptoms.
1321
Kidney failure
Several studies have found increased accumulation of TMA
and TMAO to be characteristic in patients with end-stage renal
disease [51,58]. This may result from low glomerular ltration
rate and low TMAO plasma clearance. However, increased
plasma and urine TMAO levels also may be a consequence of the
release of TMAO from the renal medulla due to kidney ischemic
damage [67,68].
Diabetes
Some evidence suggests that TMAO may affect glucose
metabolism, and that high plasma TMAO levels may be associ-
ated with diabetes mellitus [69,70]. It has been found that
signicantly higher median plasma TMAO concentrations are
present in patients with diabetes than in the individuals without
diabetes [71]. On the other hand, it has been found that patients
with diabetes who were treated with metformin showed
decreased glucose but increased plasma TMAO compared with
untreated individuals [72]. Other studies suggest a low diag-
nostic value of TMAO measurements in obese patients with
diabetes due to a high variability of plasma TMAO concentrations
[73]. To our knowledge, the level of plasma TMAO in diabetic
laboratory animals has not yet been reported. However, it has
been found that the hepatic tissues of diabetic mice have
lowered levels of TMAO and choline [74]. Interestingly, it has
been demonstrated that treatment with TMAO counteracted
endoplasmic reticulum stress by promoting normal protein
folding in diabetic rats, suggesting a potentially benecial effect
of TMAO in diabetes [63].
Cancer
Recently, plasma TMAO levels have been shown to be posi-
tively associated with colorectal cancer (CRC) [75]. Furthermore,
experimental studies suggest that TMAO may serve as a useful
urinary biomarker for gastric tumorogenesis in a mouse model
[76]. However, a causative link between high TMAO concentra-
tion and cancer is unknown. It has been found that red meat
consumption is associated with an increased risk for CRC [77].
Therefore, an increased concentration of plasma TMAO, a red
meatderived metabolite, is likely to be present in patients with
CRC. Based on this information, it is possible that plasma TMAO
levels may be a lurking variable rather than a causative link
between consumption of red meat and CRC.
High-plasma TMAO levels reect high intestinal concentra-
tions of TMA and DMA, which are both produced in the large
bowel by bacteria [30]. In this context, TMA and DMA can be
nitrosated [78] and nitrosated amines may act as potent carcin-
ogens [79]. Interestingly, the production of promutagenic nitro-
sated amines occurs in both the upper and lower gastrointestinal
tract and does not require the presence of bacteria [80], whereas
production of TMAO requires bacterial processing [49]. Further
evidence against a causal association between TMAO and cancer
is provided by studies indicating a protective effect of TMAO
in cancerogenesis by correcting the folding defect of mutant
protein [81,82].
TMAO as a therapeutic agent
Several studies point to the benecial effects exerted by
TMAO and to its pharmacotherapeutic potential. For example,
TMAO has been shown to lower the critical concentration of
1322 M. Ufnal et al. / Nutrition 31 (2015) 13171323
tubulin needed for tubulin assembly by modulating binding of
protein kinase A-phosphorylated tau protein to tubulin,
which may have a therapeutic implication in Alzheimers disease
[83,84]. Furthermore, TMAO has been found to protect
epidermolysis bullosa simplex keratinocytes from heat
stress-induced keratin aggregation, which implies the thera-
peutic potential of TMAO in epidermolysis bullosa and other
keratinopathies [85]. Other potential therapeutic applications of
TMAO include corneal dystrophies [86], cataracts [87], glaucoma
[88], asthma [89], or MachadoJoseph disease/spinocerebellar
ataxia-3 [90].
Perspectives
Elevated plasma TMAO is associated with an increased risk for
major adverse cardiovascular events in humans and several
experimental studies suggest a possible involvement of TMAO in
the etiology of CVD. On the other hand, it is well established that
TMAO performs important, protective functions in numerous
organisms, ranging from bacteria to sh. For instance, it main-
tains cell volume, protecting cells from osmotic and hydrostatic
damage. It is possible that, although TMAO plays an important
role in lower animals, in humans it is merely a remnant of
the evolution of the osmolyte system. However, it cannot be
excluded that an accumulation of TMAO in humans in the course
of CVD and other diseases also may reect the adaptation of cells
to disturbances in waterelectrolyte balance, increased blood
pressure, or other disturbing factors. Further research, including
studies on intracellular concentration of TMAO in mammals, as
well as the effects of TMAO on the activity of enzymes and
hormones, is needed to establish the role of TMAO in health and
disease in humans.
Acknowledgments
The authors acknowledge Dr. T. Zera for his critical comments
on the manuscript.
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