Where Does Nε-Trimethyllysine for the Carnitine Biosynthesis in Mammals Come from?
Balestrieri ML (2014) Where Does Ne-Trimethyllysine for the Carnitine Biosynthesis in Mammals Come
from? PLoS ONE 9(1): e84589. doi:10.1371/journal.pone.0084589
e Where Does N -Trimethyllysine for the Carnitine Biosynthesis in Mammals Come from?
Luigi Servillo 0
Alfonso Giovane 0
Domenico Cautela 0
Domenico Castaldo 0
Maria Luisa Balestrieri 0
Annalisa Pastore, National Institute for Medical Research, Medical Research Council, London, United Kingdom
0 1 Department of Biochemistry, Biophysics and General Pathology, Second University of Naples , Naples , Italy , 2 Stazione Sperimentale per le Industrie delle Essenze e dei Derivati dagli Agrumi (SSEA), Reggio Calabria, Italy, 3 Dipartimento di Ingegneria Industriale e ProdAl scarl, Universita` degli Studi di Salerno , Fisciano (SA) , Italy
Ne-trimethyllysine (TML) is a non-protein amino acid which takes part in the biosynthesis of carnitine. In mammals, the breakdown of endogenous proteins containing TML residues is recognized as starting point for the carnitine biosynthesis. Here, we document that one of the main sources of TML could be the vegetables which represent an important part of daily alimentation for most mammals. A HPLC-ESI-MS/MS method, which we previously developed for the analysis of NGmethylarginines, was utilized to quantitate TML in numerous vegetables. We report that TML, believed to be rather rare in plants as free amino acid, is, instead, ubiquitous in them and at not negligible levels. The occurrence of TML has been also confirmed in some vegetables by a HPLC method with fluorescence detection. Our results establish that TML can be introduced as free amino acid in conspicuous amounts from vegetables. The current opinion is that mammals utilize the breakdown of their endogenous proteins containing TML residues as starting point for carnitine biosynthesis. However, our finding raises the question of whether a tortuous and energy expensive route as the one of TML formation from the breakdown of endogenous proteins is really preferred when the substance is so easily available in vegetable foods. On the basis of this result, it must be taken into account that in mammals TML might be mainly introduced by diet. However, when the alimentary intake becomes insufficient, as during starvation, it might be supplied by endogenous protein breakdown.
Ne-trimethyllysine (TML) is a non-protein amino acid which
has an important role as metabolic intermediate being recognized
as the precursor of carnitine, a metabolite essential for the fatty
acid transport and utilization in mitochondria. Carnitine is
biosynthesized through a sequence of four enzyme catalyzed
reactions. In the first reaction, catalyzed by the Ne-trimethyllysine
hydroxylase, TML is converted into 3-hydroxy-TML (HTML).
Then, a specific aldolase cleaves HTML into glycine and
4-Ntrimethylaminobutyraldehyde, which is successively oxidized to
4N-trimethylaminobutyrate. Finally, the hydroxylation of the
3-hydroxy-4-N-trimethylaminobutyrate, which is better known as carnitine . In some animals,
especially carnivores, carnitine is largely introduced with the diet
and a recent study, which stimulated a considerable debate in the
scientific community, suggested the possibility that the intestinal
metabolism of exogenous carnitine could promote atherosclerosis
in humans . However, it is recognized that mammals synthesize
carnitine endogenously and, to date, no biosynthetic route
alternative to that reported above is known. The biosynthesis of
TML has been a puzzling biochemical problem for long time. In
fact, attempts aimed to demonstrate in higher organisms its
formation from lysine methylation have been unsuccessful so far.
Actually, the presence of an enzyme which methylates lysine by
using S-adenosylmethionine as a methyl donor and forming
Netrimethyllysine was reported only in the mould Neurospora crassa .
Since then, neither other reports in the scientific literature
describing a similar enzyme in some other organism nor more
information on the Neurospora crassa enzyme nor its cloning has
To date, the generally accepted view is that the metabolic
source of TML is the hydrolysis of proteins which contain TML as
a post-translational modification of some lysine residues. It is well
known, in fact, that in mammals proteins such as calmodulin,
histones, cytochrome c, and myosin contain TML residues [4,5].
Therefore, it is conceivable that free TML may be released in the
course of the protein turnover and, then, utilized for carnitine
production, although a direct evidence of this formation route has
never been reported. Indeed, only experiments, conducted on
exogenous proteins, chemically radiolabeled in vitro by the
incorporation of [3H]methyl groups into lysine residues and then
perfused into animal livers, showed that free TML was released
. On the other hand, it would have been difficult to imagine
another origin for TML, as this amino acid is believed to be rather
rare in its free form. Actually, in the natural world, besides
animals, there are few other sources known to contain free TML.
As matter of fact, the occurrence at fair level of TML, as a free
amino acid, has been reported in seaweeds, especially those of the
Laminaria genus. For this reason, TML has also the trivial name
of laminine [9,10].
Recently, we showed for the first time the occurrence of
NGmethylated derivatives of arginine in the most important
vegetables utilized for human nutrition . Intriguingly, also
these compounds are believed to be formed in human organism by
degradation of proteins containing NG-methylated arginine
residues arising from post-translational modifications . In the
course of this study, we developed a rapid HPLC method to
separate, before quantification by ESI-MS/MS,
NG-methylarginines in short times without need of derivatization. During the
analysis of numerous extracts of vegetables important in human
nutrition, we invariably noticed in the chromatogram the presence
of a peak much more retained and intense than those of arginine
NG-methylated derivatives. Here, we report the identification of
this peak and show that it corresponds to TML, which, therefore,
appears to be ubiquitous, not at negligible levels, in all the vegetal
Materials and Methods
Ne-trimethyllysine, NG-monomethylarginine, homoarginine,
formic acid, ammonium formate, and the 0.1% solution of formic
acid in water used for the LC-ESI-MS analyses were from
SigmaAldrich (Milan, Italy). The standard mixture of L-amino acids,
containing Ala, Arg, Asp, Glu, His, Ileu, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Tyr, Val, and Cys at 2.5 mM concentration in 0.01 M
HCl, was from Pierce. AccQ FLuor reagent was from Waters
(Milan, Italy). Milli-Q water was used for all the preparations of
solutions and standards.
Fruits, leafy vegetables, potato (tuber), flours of Gramineae and
Leguminosae were purchased in various local supermarkets. Four
different lots of 1 kg of each vegetal source were analyzed at
various times. Samples of alfalfa (Medicago sativa) and nettle (Urtica
dioica) plants were supplied by the city botanical gardens. All
vegetables used are reported in Table 1.
Sample preparations for TML determinations by
Citrus fruit juices were prepared by hand squeezing the fruits.
Juices were previously centrifuged at 120006 g for 15 min and
then the supernatants were diluted 1:1 with a 0.2% solution of
formic acid in water. As for flours and leafy vegetables, the samples
were homogenized in a mixer with formic acid 0.2% v/v in 1:3
(w/w) ratio, whereas, for fruits, the edible part (200 g) was
homogenized in a mixer with formic acid 0.2% v/v in 1:1 (w/w)
ratio. The homogenates were kept for 2 h under constant agitation
and then centrifuged at 180006 g for 30 min at 4uC. The
supernatants were finally frozen and kept at 220uC until used for
Determination of TML by LC with ESI-MS/MS detection
The LC-ESI-MS analyses were performed with HPLC Agilent
1100 series equipped with on line degasser and automatic injector
coupled on-line with an Agilent LC-MSD SL quadrupole ion trap.
The MS acquisition was performed by ESI in positive ion mode
with nitrogen as the nebulising and drying gas under the following
conditions: nebulizer pressure, 30 psi; drying temperature, 350uC;
drying gas, 7 L/min. The Ion Charge Control (ICC) was applied
with target set at 30000 and maximum accumulation time at
20 ms. The measurements were performed from the peak area of
the Extracted Ion Chromatogram (EIC). The quantification was
achieved by comparison with the calibration curves obtained with
standard solutions. The standard stock solution of TML was
prepared at a concentration of 200 mg/L and additional
Ranges were obtained by analyzing four lots of each sample in duplicate.
calibration levels (25, 5, 2, 1 and 0.1 mg/L) were prepared by
serial dilution with water containing 0.1% formic acid and stored
at 4uC. The calibration curve was built using these standard
solutions. The response of the MS/MS detection follows a linear
calibration curve between 0.1 and 25 mg/L with a correlation
coefficient of 0.99. The optimization of the instrumental
parameters for the analyses of TML was performed by continuous
infusion of 5 mM standard solution in 0.1% formic acid. The mass
cut-off and the fragmentation amplitude were optimised in order
to obtain the most efficient MS/MS transitions from the positively
charged precursor ion [M+H+] to the fragment ions. The more
intense 189.2R130 MS/MS transition was used for TML
quantification. The 189.2R84 and m/z 189.2R60 MS/MS
transitions were utilized to confirm TML identification.
Successively, volumes of 1020 mL of standard solutions or samples were
analyzed by HPLC-ESI-MS/MS by using the silica column
SupelcosilTM LC-Si 3.3 cm64.6 mm i.d., 3 mm particle size. The
elution was performed isocratically at a flow rate of 100 mL/min
by utilizing 75% of Sol. A (0.1% formic acid in water) and 25% of
Sol. B (100 mM ammonium formate in water titrated to pH 4.5
with formic acid).
The retention times and peak areas of the monitored fragment
ions were determined by the Agilent software Chemstation version
4.2. Recovery of TML from the various matrices was determined
by analyzing portions (10 g) of homogenized samples. Portions
were spiked with a know amount of TML in formic acid 0.1%.
The standard solutions added to the samples were 2 mmol/L.
Samples of the same homogenized matrix without the addition of
TML were also analyzed. The recovery of TML was obtained
separately. Percentage recoveries were based on the difference
between the total amount in the spiked samples versus that in the
unspiked samples. Reproducibility was assesses by the
determination of recovery of six individual samples. The limit of detection of
TML was assessed by utilizing only the standard solutions in
formic acid 0.1%. The limit of detection was determined as the
concentration of TML which gave a peak height three times that
of the background noise.
Determination of TML and amino acids by LC with
TML quantification in the samples was also performed by
reverse-phase HPLC employing a Waters instrument mod. 2690
equipped with the fluorescence detector mod. 474. The juice or
extract samples of about 10 mL were centrifuged at 120006 g at
4uC for 10 min. Then, 5 mL of supernatant was filtered and
loaded on a column (561 cm) filled with Bio-Rad AG
50WX8(H+) resin. After loading, the column was washed with five volumes
of Milli-Q water and, then, the amino acids were one-step eluted
with 10 mL of 15% ammonia solution in water. In order to
exhaustively remove ammonia which heavily interfered with TML
determination, after drying the eluate in a rotavapor, the residue
was dissolved in 2 mL of NaOH 0.01 M in water and then
vacuum dried again. The residue was finally dissolved in 2 mL of
0.01 M HCl in water and the solution filtered through a 0.45 mm
filter. The derivatization of TML in the samples was accomplished
with AccQ (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate),
which is usually employed in the quality control of fruit juices for
the determination of the free amino acids by HPLC with
fluorescence detection . AccQ-Ne-trimethyllysine was
fluorimetrically detected by excitation at 350 nm and emission at
395 nm. AccQ-Ne-trimethyllysine was identified on the basis of
the retention time and quantified by comparison of the sample
peak area with the calibration curve.
Determination of TML in vegetable sources
The quantification of TML in the vegetal matrices was
performed by the same chromatographic procedure that was
advantageously employed to analyze NG-methylated arginine
derivatives in vegetables . This procedure has proven to be
suitable for polar substance analysis with ESI-MS/MS detection
and does not require sample derivatization. The chromatography
was performed with a short silica column (SupelcosilTM LC-Si),
employing isocratic elution conditions. When analyzing the
content of methylarginines in vegetables, multiple reaction
monitoring was used for the analyte quantification. The transition
utilized for NG-monomethylarginine (NMMA) MS2 quantification
was 189.2R74. Incidentally, the m/z of protonated TML is 189.2
too. In all vegetable samples, this lucky coincidence allowed the
observation of an intense peak in the total ion chromatogram
(TIC), emerging at a retention time (r.t.) of about 15 min, much
more retained than that of NMMA. As an example, the MS2 total
ion chromatogram (TIC) by isolating at m/z 189.2 from a sample
of sweet pepper berry extract is reported (Figure 1, panel A). The
MS2 fragmentation pattern of the peak at r.t. 15 min (Figure 1,
panel A) shows only three fragments at m/z 130, 84, 60 (Figure 1
panel C) which are typical of Ne-trimethyllysine MS2
fragmentation , as also confirmed by comparison with the fragmentation
pattern of the TML standard solution. The smaller peak at r.t.
6.0 min (Figure 1, panel A) corresponds to
NG-monomethylarginine. Anyway, besides the identity of the MS2 fragmentation
pattern with that of TML standard solution, the identification of
the unknown peak was also confirmed by the identity of its
retention time with that of an authentic TML standard solution
(Figure 1, panel B). Moreover, when changing the
chromatographic conditions by varying the ammonium formate
concentration in the eluent, it was found that the retention time of TML on
the LC-Si column strongly varied depending on the concentration
of ammonium formate in the eluent, as also observed for
methylarginines . Therefore, in order to further confirm the
peak identity, various eluent composition changes were tried. In
each case, the retention times of the standard TML peak and that
of the unknown compound varied in the same way. However,
besides NMMA, TML is also isobaric with homoarginine (HAG),
a substance also reported to occur in some vegetables such as grass
pea (Lathyrus sativus)  and lentils . In order to exclude the
possibility to mistake homoarginine for TML, we run HAG
standard solution in the same chromatographic conditions. We
found that HAG shows a much shorter retention time (about
5.5 min) than TML, thus the two compounds are completely
resolved in the chromatographic conditions we utilized (Figure 1,
panel B). This is important as the fragments at m/z 130, 84 and 60
present in MS/MS fragmentation pattern of TML also occur in
the MS/MS fragmentation pattern of homoarginine, besides other
fragments (Figure 1, panel E). Therefore, a complete
chromatographic resolution is mandatory for a reliable attribution and
quantification when both compounds are present in the same
sample. Our results show that TML is present in all the vegetables
analyzed (Table 1), as demonstrated for each vegetable source by
the comparison of retention times and MS2 fragmentation patterns
with the authentic standard. In particular, the highest
concentration was found in sweet pepper fruit. It is interesting to note that
fruit juices, largely utilized in human nutrition, such those of
orange and grapefruit, also contain levels of TML in the same
range of the content of some essential amino acids, such as lysine
and leucine . Also, it is of interest the observation that one
of the highest level of TML was found in leaves of alfalfa, a plant
of paramount importance for the cattle feeding.
Evaluation of the percentage recoveries
In order to check the performance of the analytical procedure,
the percentage recoveries, the reproducibility and the limits of
detection for TML were assessed. Recoveries of TML, determined
as reported in Materials and Methods section, were in each case
higher than 95% for all analyzed samples. The reproducibility,
assessed by calculating the standard deviation of six recoveries,
resulted less than 5%. The detection limit, determined as that
concentration of TML which gave a peak height three times that
of the background noise, was 10 nmol/L.
The presence of TML in the vegetable samples was further
confirmed by an independent analytical approach, based on
HPLC with fluorescence detection, commonly used to analyze
amino acids in food sources . This procedure involves the
purification of the vegetal extract by a passage on a short column
of AG50WX8 (H+ form). Moreover, this procedure also allowed
to have an estimation of the matrix effect in the analyses of some
samples by comparing the results obtained by HPLC-ESI-MS/MS
with and without the purification passage on the AG50WX8
column (see Materials and Methods). In all cases, the samples
without the passage on column showed values from 2% to 15%
lower than the purified samples. An important aspect of TML
analysis by HPLC with fluorescence detection consists in the
sample treatment after the passage on the AG50WX8 column.
Generally, for amino acid analysis, the column is eluted with a
15% ammonia solution. Afterwards, the eluted solution is vacuum
dried and the residue is suspended in a suitable solvent and
derivatized. However, we found that in this way a broad peak, due
to residual ammonia presence, heavily interfered with
determination of derivatized TML, as both substances emerged in the same
time range. Instead, the treatment with a diluted solution of
sodium hydroxide, as described in Materials and Methods,
exhaustively removed ammonia from the dried samples and
completely eliminated such interference (Figure 2). Results
obtained by HPLC-ESI-MS/MS and by HPLC with fluorescence
detection were also compared for tomato berry, alfalfa leaves and
sweet pepper berry extracts (Figure 2). The HPLC analyses
confirmed the presence of TML in these vegetables at levels
comparable within +/210% to those obtained by
In this paper we reported the occurrence of TML in a consistent
number of vegetables and vegetable derived products. Specifically,
in the samples examined, free TML levels were in the same range
as other free amino acids . This result is of noticeable
interest as the presence of free TML in vegetables, excepting
seaweeds, was practically ignored so far. To date, only one study
reports unequivocal identification and quantification of free TML
in a plant extract . On the contrary, several studies report in
some plants the occurrence of proteins containing the TML
residue in their primary structure as a post-translational
modification of lysine residues. Among these proteins, there are
calmodulin from Papaver somniferum and Euphorbia lathyris [24,25],
and cytochrome c from wheat germ, which has been found to
contain two TML residues . Another important protein, owing
to its essential role in carbon dioxide fixation in the vegetal cell
chloroplasts, is the ribulose-1,5-bisphosphate carboxylase
oxygenase (RuBisCo) which, in some plant species, has been found to
contain TML in the N-terminal region of its large subunit [27,28].
Since the biosynthetic pathway for TML formation, as free amino
acid, is still unexplored in plants, it cannot be ruled out that the
free TML also in plants origins from the breakdown of TML
residue containing proteins, as it is supposed for mammals. The
role of free TML in all plants we examined can be at the present
only hypothesized. It may be supposed that, at least in part, TML
is utilized for carnitine biosynthesis, as it happens for mammals. In
fact, it has been recently demonstrated that in Arabidopsis thaliana
the carnitine biosynthetic pathway shares similar features with the
pathway of mammals and fungi . An other possible role of
TML could be that to be part of the pool of osmolytes, as it is
hypothesized for seaweeds , and thus it could play a stress
protective function in the vegetal cells. Anyway, whatever the
TML role in plants may be, an important question arises, as a
consequence of our results, about the true source of TML in
The current opinion is that mammals utilize the breakdown of
their endogenous proteins containing TML residues as starting
point for carnitine biosynthesis (Figure 3). But is it conceivable to
resort to such tortuous and energy expensive route when the
substance is so easily available from vegetable foods? This
consideration even better applies to herbivorous mammals for
which the intake of free TML through foods should be higher than
for other mammals. From this point of view, it is of interest our
finding that alfalfa leaves, one of the most important forage grass
for the cattle feeding, resulted one of the richest source of free
TML among the vegetables examined (Table 1). In this respect,
besides the consideration that the amount of TML from vegetables
assumed by the herbivores seems sufficient for their carnitine
biosynthesis, it is also important to point out that rat and guinea
pigs orally supplemented with free TML have an increased rate of
carnitine biosynthesis and excretion . Conversely, dietary
proteins (casein, soy proteins, and wheat gluten) were found to be
poor source of TML . In light of such evidence, our hypothesis
seems founded on a firm ground as it has already been proved that
animals orally supplemented with free TML showed increased
rates of carnitine biosynthesis and excretion [29,31].
On the other hand, also for humans with the particular dietary
habit to eat only vegetable foods (vegetarians or vegans), it is
hardly conceivable that only endogenous TML can suit the needs
for their carnitine biosynthesis. Indeed, it has been shown that
vegetarian adults have slightly reduced plasma carnitine levels
compared to omnivorous individuals [32,33]. However, serum
carnitine levels are more markedly depressed in vegetarian
children  and in premature infants who do not receive a
Figure 3. Pathways for the carnitine formation. Left side: The biochemical pathway, according to the view generally accepted, entails, as the
metabolic source of TML in mammals, the hydrolysis of proteins which contain TML as a post-translational modification of some lysine residues. Right
side: The vegetal food intake, as suggested in this paper, directly provides mammals with the TML to be converted into carnitine. Circles depict amino
acids. The black filled circle indicates lysine. AdoMet: S-adenosylmethionine. Both protein and AdoMet syntheses require ATP (indicated in
dietary source of carnitine . However, this could be ascribed to
immaturity of the biosynthetic conversion machinery of TML into
carnitine. Lastly, a warning that could be relevant to vegans is that
the assumption of free TML through vegetables consumption may
represent a risk factor for those subjects affected by dysregulation
of the carnitine biosynthesis due to a mutation of the
Netrimethyllysine hydroxylase gene .
In conclusion, although it cannot be excluded that a certain
amount of TML might be produced by the hydrolysis of
endogenous proteins containing such residue as a posttranslational
modification, it does not appear likely that this route might be the
preferred one when the substance is so easily available through a
vegetable containing diet (Figure 3). Conversely, when the dietary
intake become insufficient, it cannot be ruled out that the
breakdown of endogenous proteins could become the prime
source of TML. From this point of view, it seems reasonable to
suggest that in mammals TML utilized for carnitine biosynthesis
might be mainly taken from diet (for herbivore animals this should
be even more likely). However, when the alimentary intake fails, as
in case of starvation, TML might be produced from endogenous
protein breakdown, similarly to the essential amino acids when
their intake becomes insufficient.
Conceived and designed the experiments: LS D. Castaldo MLB.
Performed the experiments: LS AG D. Castaldo D. Cautela MLB.
Analyzed the data: LS D. Castaldo MLB. Contributed reagents/materials/
analysis tools: LS D. Castaldo MLB. Wrote the paper: LS D. Castaldo
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