Is N,N-dimethylglycine N-oxide a choline and betaine metabolite?

Article abstract of DOI:10.1515/hsz-2016-0261

Choline metabolism is by oxidation to betaine, which is demethylated to N,N-dimethylglycine; dimethylglycine is oxidatively demethylated to sarcosine. This pathway is important for osmoregulation and as a source of methyl groups. We asked whether another metabolite was involved. We synthesized the N-oxide of dimethylglycine (DMGO) by oxidizing dimethylglycine with peracetic acid, and measured DMGO in human plasma and urine by HPLC-MS/MS with positive ion detection, using two chromatography procedures, based on ion exchange and HILIC separations. The molecular ion DMGOH⁺ (m/z=120) yielded four significant fragments (m/z=103, 102, 58 and 42). The suspected DMGO peak in human body fluids showed all these fragments, and co-chromatographed with added standard DMGO in both HPLC systems. Typical plasma concentrations of DMGO are under 1 μmol/l. They may be lower in metabolic syndrome patients. Urine concentrations are higher, and DMGO has a higher fractional clearance than dimethylglycine, betaine and choline. It was present in all of over 80 human urine and plasma samples assayed. Plasma DMGO concentrations correlate with plasma DMG concentrations, with betaine and choline concentrations, with the osmolyte myo-inositol, and strongly with urinary DMGO excretion. We conclude that DMGO is probably a normal human metabolite.

Full text of DOI:10.1515/hsz-2016-0261

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Biological Chemistry ’Just Accepted’ paper
ISSN (online) 1437-4315
DOI: 10.1515/hsz-2016-0261
Research Article
Is N,N- dimethylglycine N-oxide a choline and
betaine metabolite?
Michael Lever1,*, Christopher J. McEntyre1,2, Peter M. George2,3 and Stephen T. Chambers3
1Department of Chemistry, University of Canterbury, P.O. Box 4800, Christchurch 8140,
New Zealand
2Canterbury Health Laboratories, Christchurch 8140, New Zealand
3Department of Pathology, University of Otago, Christchurch 8140, New Zealand
*Corresponding author
e-mail: michael.lever@canterbury.ac.nz
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Dimethylglycine-N-oxide
Abstract
Choline metabolism is by oxidation to betaine, which is demethylated to
N,N-dimethylglycine; dimethylglycine is oxidatively demethylated to sarcosine. This pathway
is important for osmoregulation and as a source of methyl groups. We asked whether another
metabolite was involved. We synthesized the N-oxide of dimethylglycine (DMGO) by
oxidizing dimethylglycine with peracetic acid, and measured DMGO in human plasma and
urine by HPLC-MS/MS with positive ion detection, using two chromatography procedures,
based on ion exchange and HILIC separations. The molecular ion DMGOH+ (m/z = 120)
yielded four significant fragments (m/z = 103,102,58 and 42). The suspected DMGO peak in
human body fluids showed all these fragments, and co-chromatographed with added standard
DMGO in both HPLC systems. Typical plasma concentrations of DMGO are under 1 μmol/l.
They may be lower in metabolic syndrome patients. Urine concentrations are higher, and
DMGO has a higher fractional clearance than dimethylglycine, betaine and choline. It was
present in all of over 80 human urine and plasma samples assayed. Plasma DMGO
concentrations correlate with plasma DMG concentrations, with betaine and choline
concentrations, with the osmolyte myo-inositol, and strongly with urinary DMGO excretion.
We conclude that DMGO is probably a normal human metabolite.
Keywords: amine oxides; dimethylglycine metabolism; one-carbon metabolism;
osmoregulation; trimethylamine N-oxide.
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Dimethylglycine-N-oxide
Introduction
Choline is an essential nutrient (Zeisel, 2000, Ueland, 2011), with multiple roles. It is required
for the synthesis of the phospholipid component of cell membranes, and in mammals as a
component of circulating lipoproteins. An important role is as the main source of betaine, a
major osmolyte used by many organisms in most taxa, and specifically by most mammalian
tissues for cell volume regulation (Ueland, 2011, Lever and Slow, 2010, Day and Kempson,
2016). There are other roles for choline metabolites in cell volume regulation, and in
mammals in cellular signalling. The metabolism of choline and betaine has been often
described (Figure 1); choline and betaine are considered major sources of methyl groups, used
to remethylate homocysteine to methionine and hence replenish S-adenosylhmethionine
(SAM). Choline is oxidized to betaine by two mitochondrial steps (Figure 1), one methyl
group of betaine is transferred by cytosolic betaine-homocysteine methyltransferase, giving
N,N-dimethylglycine (DMG), which is further metabolized by mitochondrial dimethylglycine
dehydrogenase (DMGDH) to sarcosine and ultimately glycine, the methyl groups being
transferred to tetrahydrofolate (THF) to give 5,10-methylene-THF, and important
intermediate in one-carbon metabolism. All of the enzymes involved in this pathway are
subject to numerous controls, and in particular they are osmoregulated (Hoffmann et al. 2013)
which suggests that one-carbon metabolism and cell-volume regulation are intimately
interdependent.
Here, we show that another probable metabolite of DMG, N,N-dimethylglycine N-oxide
(DMGO) is present in human plasma and urine. This may be an alternative pathway for DMG
metabolism. If this is confirmed, the accepted metabolism outlined in the last paragraph will
need to be revised. Further, since DMGO is a carboxylated derivative of trimethylamine
N-oxide (TMAO), and TMAO is a metabolite that has received much recent attention as a risk
marker for human vascular disease (Tang et al., 2013, 2014, 2015, Wang et al., 2014), a
possible connection needs to be evaluated. An alternative interaction is that if DMG and
trimethylamine are oxidized by the same enzyme, tissue concentrations of DMG may affect
the oxidation of trimethylamine. Our initial observational data are strongly suggestive rather
than conclusive, and should initiate investigations into novel metabolic pathways. Since both
betaine and TMAO have been associated with vascular disease and diabetes (Tang et al.,
2013, 2014, 2015, Wang et al., 2014, Svingen et al., 2016,Walford et al., 2016, Ejaz et al.,
2016), the relevance of DMGO to human health should also be evaluated.
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Dimethylglycine-N-oxide
Results
Detection of N,N-dimethylglycine N-oxide (DMGO)
The mass, and the fragmentation patterns, of DMGO were characterized using three different
mass spectrometric systems. An Agilent single quadrupole mass spectrometer was used for
preliminary work, and to identify candidate HPLC systems for separating DMGO from other
plasma and urine components, by detecting a cation with m/z = 120 in acidified samples. The
limited sensitivity of this system meant that it was only suitable for quantitation in urine.
DMGO was initially quantified on an AB Sciex API4000 triple quadrupole mass spectrometer
in positive ion mode using the mass transitions m/z = 120.1 → 58.1 and 120.1 → 103.1. A
third mass transition for DMGO, 120.1 → 102.1, was also monitored in urine, but was below
the detection limits in plasma. For analysis, the decoupling potential was 41 V, the collision
energy was 27 V for the m/z = 120.1 → 58.1 (and 17 V for 103 and 102 fragments), and the
collision cell exit potential was 6 V. Samples were quantified using external aqueous
standards of DMGO with D3-N,N-dimethylglycine-N-oxide (D3-DMGO) used as an internal
standard. The mass transition for D3-DMGO was 123 → 61, the decoupling potential was 46
V, the collision energy was 29 V, and the collision cell exit potential was 4 V.
As sensitivity was found to be a problem for quantifying DMGO in some plasma samples, a
more sensitive tandem mass spectrometer (Agilent 6490) was also used. A fourth mass
transition for DMGO (120 → 42) was identified during compound optimization on this
instrument. For analysis, the collision energy was 32 V for the 120 → 58 mass transition,
56 V for the 120 → 42 mass transition, and 10 V for the 120 → 103 and 120 → 102 mass
transitions. The collision energy used for the internal standard mass transition (123 → 61)
was 32 V. The gas temperature was 220°C, the gas flow rate was 12 L/min, the sheath gas
temperature was 350°C, the sheath gas flow rate was 11 L/min, the capillary voltage was
3000 V, and the cell accelerator voltage was 5 V.
Presence of DMGO in human urine and plasma
Using a cation exchange column, an acetonitrile-based mobile phase, and detecting positive
ions in the mass spectrometer, chromatograms of extracts of a pool of human urine contained
a peak eluting at 7.66 minutes with m/z = 120 (Figure 2) which co-chromatographed with
added standard DMGO. These peaks gave all four prominent fragments with m/z = 58,
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Dimethylglycine-N-oxide
m/z = 42, m/z = 103 and m/z = 102 with relative intensities 10:6.1:1.6:0.7. The
chromatographic traces based on monitoring each of these four fragments showed a peak
corresponding to DMGO in the same place. The identity of this urine component with DMGO
was confirmed using a second chromatography system, an acetonitrile-based solvent on a
Diamond Hydride normal-phase column, again using positive ion detection. In this
chromatography system DMGO eluted earlier at 4.37 min.
DMGO peaks were observed in human plasma using both chromatography systems and all
four transitions (Figure 3). However, the DMGO peak obtained using the m/z = 120 → 102
mass transition lacked sufficient sensitivity for quantitation
The initial work was carried out on plasma and urine samples sent to the laboratory for
diagnostic testing, and on small pools of these. Two different mass spectrometers were used.
These samples were independent of the study samples on which quantitative results are
reported. The pools, with and without a known amount of added DMGO, were used as control
samples in the study sample analyses. Endogenous DMGO was always observed.
The candidate DMGO peak was found in all of 62 urine and 62 plasma samples from the
metabolic syndrome study group (Figure 4), with concordant results obtained by measuring
each of two fragments in positive ion mode. The same results were obtained using both the
ion exchange and HILIC chromatography systems (Figure 4). Concordant results (r2 > 0.98)
were also found using the m/z = 42 fragment, and for urine using the m/z = 102 fragment. The
results obtained in the small (n = 16) healthy normal subject group led to the same conclusion:
DMGO has been found to be present in every human urine or plasma sample that has been
assayed.
Performance characteristics of the provisional assays for DMGO
The provisional quantitative method used for the analysis of the available study samples was
based on quantifying the 120 → 58 mass transition on peaks eluting from the Diamond
Hydride column (HILIC chromatography). The less sensitive transition (m/z 120 → 103) was
also monitored for quantification and gave almost identical results; although the m/z 120 →
58 mass transition produced the largest peaks for DMGO, the baseline noise was greater than
for the m/z 120 → 103 mass transition, and the sensitivity was similar when using the 42 and
103 fragment ions; the limit of detection was below 0.015 μmol/l (signal to noise ≥3). The
imprecision for plasma (coefficients of variation, CV) was from 4.9% to 18% for a low
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Dimethylglycine-N-oxide
concentration of 0.05 μmol/l, and CVs 6% and lower at a concentration of 1 micromolar. The
imprecision (CV) was less than 4.1% when using the three strongest mass transitions to
measure DMGO in urine.
D3-DMGO was used as the internal standard. Recoveries were estimated by standard addition,
25 μmol/l for pooled human urine and 1 μmol/l for human plasma. They were 97 to 99%
(urine) and 84 to 91% (plasma). Linearity was assessed by adding, 25, 50, and 100 μmol/l of
DMGO to urine, and 3 replicates of each level of were measured by tandem mass
spectrometry, and 0.1, 0.25, 0.5, 1, 5, and 10 μmol/l were added to plasma; in all cases the
assay was linear (r2 ≥ 0.99) over the ranges tested.
Is N-hydroxy N-methylglycine (HMG) present?
Using the ion-exchange based chromatography system (as for DMGO) and detection with the
m/z = 106 → 60 and 106 → 42 transitions for detection, HMG eluted as a peak at 5.8 min.
There was no detectable peak corresponding to HMG at this place in plasma extracts. There
was a small peak in urine extracts that appeared to co-chromatograph with the crude HMG
standard material, this was estimated to correspond to a urine concentration considerably less
than 1 micromolar. Thus the presence of a trace of HMG could not be excluded, but if any is
present the concentration is very low.
Relationship of DMGO to other choline and betaine metabolites
Other plasma and urine metabolites were also assayed by LC-MS/MS as previously described
(Lever et al., 2014a,b, McEntyre et al., 2015).
The median plasma concentrations of DMGO and related metabolites are shown in Table 2;
usually, there is substantially less DMGO in plasma than DMG and urinary DMG excretion
was higher than DMGO excretion. The metabolic syndrome study population (n = 62)
appeared to have lower plasma concentrations than the healthy student group (n = 16);
however, urinary excretions were similar. As a result, calculated fractional clearances
(clearance of DMGO as a percentage of creatinine clearance) were significantly greater in the
metabolic syndrome group. Differences between the groups in the concentrations of related
metabolites (Table 2) were consistent with data reported in previous studies (Konstantinova
et al. 2008).
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Dimethylglycine-N-oxide
In samples from the metabolic syndrome study, the plasma concentrations of DMGO strongly
correlated with plasma DMG concentrations (Table 3), and also significantly with plasma
betaine and free choline, and with plasma concentrations of the osmolyte myo-inositol
(Table 3). No correlation was observed with plasma concentrations of TMAO. The urinary
excretion of DMGO was strongly correlated with plasma concentrations of DMGO
(Spearman’s r = +0.86, p<0.001), and also significantly with both the plasma concentrations
and urinary excretions of DMG.
The associations in the small student group (n = 16) were consistent with those found in the
metabolic syndrome population, but given the small sample size fewer correlations were
statistically significant (p<0.05).
Urine from patients with trimethylaminuria
DMGO was measured in urine samples from two patients with confirmed trimethylaminuria;
the DMGO concentrations were 11% and 5% of the median urine DMGO concentrations in
the metabolic syndrome patients.
Discussion
N,N-Dimethylglycine N-oxide (DMGO) is present in human plasma and urine
The proton NMR and elemental analysis data show that the white substance we synthesized
by the oxidation of N,N-dimethylglycine (DMG) is the N-oxide of DMG, and its cation has
the expected molecular mass, detected in three mass spectrometers. The fragmentation of the
observed molecular ion (DMGOH+) is consistent with this assignation; the m/z = 103 and
m/z = 102 fragments are analogous to the m/z = 59 and m/z = 58 fragments from
trimethylamine-N-oxide (TMAO, parent cation m/z = 76) (Wang et al., 2014) and represent
the elimination of a hydroxyl radical and a water molecule respectively after the rupture of the
relatively weak N-O bond. More energetic fragmentation gives an m/z = 58 fragment that is
almost certainly the same as the m/z = 58 fragment obtained from TMAOH+ and is the
predicted result of further decarboxylation. The m/z = 42 fragment is consistent with the
further loss of a methyl group, and was not observed when using the ABI4000 instrument.
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Dimethylglycine-N-oxide
DMGO was found in all the human plasma and urine samples that we have analyzed. Our
identification of DMGO is confirmed because the same concentration estimates are obtained
using two different chromatography systems, based on different separation principles
(ion exchange and HILIC) and with each of the four fragment ions of DMGOH+. Preliminary
data (not presented here) suggests that it could also be detected in negative ion mode after
separation on an anion exchange column.
Preliminary data on other normal subjects and on a population with diabetes (unpublished)
show that DMGO is present in plasma and urine of both genders. Older healthy subjects may
not have as high plasma DMGO concentration as reported here in a student population, but
the initial results on subjects with diabetes suggest that these are similar to the metabolic
syndrome subjects we report. We have found detectable DMGO in all human samples we
have analyzed so far and we conclude that DMGO is a normal component of human body
fluids.
DMGO shows an association with choline and betaine metabolism
The preliminary data presented here is only observational, but the strong correlations between
plasma concentrations of DMGO, dimethylglycine (DMG) and betaine suggest a metabolic
connection. This is plausible; DMG is a tertiary amine chemically related to trimethylamine
(one of the methyl protons of trimethylamine is replaced by a carboxyl group) and
trimethylamine is well-known to be efficiently metabolized to its amine oxide by a flavin
monooxygenase (FMO3) (Mackay et al., 2011). It could be reasonably expected that DMG
could be oxidized to DMGO by this, or a similar enzyme, and this needs to be experimentally
confirmed. Low concentrations of DMGO in the urine of patients with trimethylaminuria
(the result of a defect in FMO3) are consistent with trimethylamine and DMG being oxidized
by the same enzyme, but this is only circumstantial evidence.
DMGO shows an association with osmolytes
Plasma and urine DMGO concentrations of not only correlate with concentrations of the
osmolyte betaine, but plasma DMGO correlates with another major osmolyte, myo-inositol.
The reason for this association needs to be investigated: it may simply reflect that DMGO
oxidation is osmoregulated as are all other steps in betaine metabolism (Hoffmann et al.,
2013), or that the supply of DMG is osmoregulated.
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Dimethylglycine-N-oxide
What is the significance of DMGO?
The concentrations of DMGO are substantially lower than the concentrations of the presumed
precursor, DMG. The correlations suggest that it is a metabolic product of choline and betaine
metabolism, but the low concentrations suggest that it is only a minor metabolite. However,
the concentration appears to be lower in the metabolic syndrome group than in the student
group. This needs to be confirmed by further studies, since the two sets of specimens studied
here also differed in the age of the subjects, and in the way (and length of time) they had been
stored.
The preliminary observational data presented here does not provide information about the
functions of DMGO. Given its chemical structure and the correlation of plasma
concentrations with those of DMG, it is reasonable to presume that it is formed by the
oxidation of DMG, probably by a flavin or cytochrome P450 monooxygenase, but this needs
to be experimentally verified. The low concentrations of DMGO in FMO3 deficient patients
is circumstantial evidence that the enzyme responsible for the conversion of trimethylamine to
trimethylamine N-oxide (TMAO) is also responsible for DMG oxidation; if this is the case,
tissue DMG may inhibit the formation of TMAO, and thus provide another mechanism to
explain the association between TMAO metabolism and betaine metabolism (Obeid et al.,
2016).
An early study, using liver mitochondrial extracts, suggested that DMGO is not a substrate for
dimethylglycine dehydrogenase (Mackenzie and Frisell, 1958); this should be re-examined,
and the possibility that it is an inhibitor of this enzyme should also be investigated. It may be
reduced back to DMG in some tissues. The possibility that it could be decarboxylated to give
TMAO is interesting, given the recent attention to the significance of TMAO as a marker of
vascular disease risk (Tang et al., 2013, 2014, 2015), though we did not detect a correlation
between plasma TMAO and plasma DMGO that would support this. The strong correlation
between plasma DMGO and its urinary excretion, and its relatively high fractional clearance,
suggest that excretion is a major fate of DMGO, which would be a previously overlooked loss
of methyl groups; this is consistent with DMGO being a minor metabolite. However, its tissue
concentrations need to be measured and its transport characterized. We detected minimal, if
any, N-hydroxy N-methylglycine (the predicted demethylation product) in plasma or urine, so
demethylation is not likely to be a major pathway for DMGO metabolism, but though DMGO
is not likely to be a good substrate for dimethylglycine dehydrogenase or betaine
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Dimethylglycine-N-oxide
homocysteine methyltransferase, its potential as an inhibitor should be investigated. The
presence of DMGO also raises the question of whether tissue concentrations of DMG affect
the conversion of trimethylamine to TMAO.
Since the plasma concentrations and urinary excretions of related compounds such as
dimethylglycine (Svingen et al., 2013, 2015), betaine (Ueland, 2011, Lever and Slow, 2010,
Day and Kempson, 2016) and TMAO (Tang et al., 2013, 2014, 2015, Wasang et al., 2014)
have all been shown to be related to disease, the significance of DMGO as a risk marker, or
even a risk factor, need to be evaluated. For the present pilot study we used stored matched
human plasma and urine samples, and these were all from male subjects; we have preliminary
evidence that DMGO is present in the general human population, but systematic studies of
both the general population and populations with clinical conditions are needed. It would be
unwise to draw conclusions from differences between the two sets of samples reported here
because of the other differences between them (the ages and BMIs of the subjects, and the
differences in specimen storage). It has been recently shown that betaine metabolism is
closely associated with the development of diabetes (Svingen et al., 2016, Walford et al.,
2016, Ejaz et al., 2016), so the possibility that the differences we observed are real should be
investigated in more appropriate sample sets. It needs to be confirmed that other mammals
also make DMGO, so that DMGO can be included in future models of mammalian choline
and betaine metabolism.
The limitations of this study
This is a preliminary study and presents strong evidence that there is a novel metabolite,
N,N-dimethylglycine N-oxide, present in human plasma and urine. Although it is a plausible
presumption that it is formed by the oxidation of DMG, this needs to be confirmed
experimentally. The observational data is based on the analysis of specimens stored for
different times under different conditions. Although it is expected that DMGO would be as
stable in plasma as TMAO (Sweeley and Horning, 1957), this also needs to be confirmed, and
systematic studies of DMGO planned.
Conclusions
A probable new choline and betaine metabolite, N,N-dimethylglycine N-oxide (DMGO), has
been identified in human plasma and urine. A synthesis and assay methods for it are
presented. DMGO formation is probably a branch of the established pathway, but this needs
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Dimethylglycine-N-oxide
to be experimentally verified. Our observations raise many new questions that will need
experimental studies, and may lead to a revision of our understanding of the pathways for
mammalian choline and betaine metabolism.
Materials and methods
Materials
N,N-Dimethylglycine was purchased from Sigma-Aldrich (Sydney, Australia) and D3-N,N
dimethylglycine from CDN Isotopes (Quebec, Canada). Peracetic acid solution (32 wt.% in
dilute acetic acid) was purchased from Sigma-Aldrich. Other reagents were of analytical or
HPLC grade quality.
Synthesis of N,N-dimethylglycine N-oxide (DMGO)
DMGO has been previously synthesized by the oxidation of N,N-dimethylglycine with
hydrogen peroxide (Sweeley and Horning, 1957, Ikutani and Matsumura, 1968). Ikutani and
Matsumura (1968) used hydrogen peroxide in acetic acid, in which case the active oxidant is
peracetic acid. We obtained high yields using commercial peracetic acid.
N,N-dimethylglycine (DMG, 4.00 g, Sigma, free base) was dissolved in 25 ml of peracetic
acid (32 wt% in dilute acetic acid). The reaction mixture was refluxed for 2 hours. The excess
peracetic acid was removed on a rotary evaporator at 40°C. The reaction mixture was
acidified with sulfuric acid and then added to a column containing acidified Dowex 50WX8
(200-400 mesh, Sigma) strong cation exchange resin. The impurities were removed by
washing approximately three column volumes of water. When the eluent no longer tested
positive for peroxides (using a potassium iodide starch test), the DMGO product was eluted
off the column with aqueous ammonia solution (8% v/v). The ammonia and water were
removed on a rotary evaporator at 40°C. DMGO crystallized out of 2-propanol as a white
powder. The yield was 4.13 g (89%).
The DMGO was validated by LCMS and NMR spectroscopy. For NMR analysis,
approximately 15 mg of DMG or DMGO was added to 1 ml of D2O, and approximately
10 mg of 3-(trimethylsilyl)propionic-2,2,3,3-D4 acid sodium salt (TSP, Sigma) was added as
1
a reference standard. The TSP H signal was set to 0 ppm. A proton NMR spectrum was
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Dimethylglycine-N-oxide
obtained in 5 mm NMR tubes using a Varian INOVA 500 MHz NMR spectrometer with 8
scans. There was no detectable unreacted DMG present in the product by NMR, or by LC-MS
spectroscopy. The methyl group resonance shifted from 2.931 ppm in DMG to 3.485 ppm in
DMGO, and the CH2 resonance shifted from 3.731 ppm to 4.157 ppm. The peak area ratio of
the CH3 peak to the CH2 peak was 3:1 as expected.
DMG: 1H NMR (D2O) H 2.931 (3 H, s, CH3), 3.731 (2 H, s, CH2)
DMGO: 1H NMR (D2O) H 3.485 (3 H, s, CH3), 4.157 (2 H, s, CH2)
Elemental analysis (Microlab, Dunedin, NZ) showed that the product was anhydrous, and was
close to the theoretical composition: %C-40.35;40.27: %H-7.86;7.80: %N-11.82;11.75
(theoretical for anhydrous DMGO: C-40.33%: H-7.62%: N-11.76%).
A solution of D3-DMGO was prepared, for use as an internal standard in plasma and urine
assays, by scaling down this synthesis and starting with D3-DMG.
A crude sample of a possible metabolite of DMGO, N-hydroxy-N-methylglycine was also
prepared; 1.00 g (0.012 mol) of N-methylhydroxylamine hydrochloride, 10 g of sodium
bicarbonate, and 50 mL of dichloromethane were added to a 100 ml conical flask and stirred
for 3 hours. The reaction mixture was filtered, and 1.67 g (0.012 mol) bromoacetic acid
dissolved in dichloromethane was added. This mixture was stirred for 3 hours at room
temperature and then left to sit for 7 days. A syrup separated at the bottom of the reaction
mixture. Mass spectrometry confirmed that the main component of this had a mass of 106,
corresponding to the expected mass of N-methyl N-hydroxyglycine (HMG). Water (50 ml)
was added and the mixture shaken, and the aqueous layer was collected and dried under
vacuum. The N-methyl N-hydroxyglycine hydrobromide did not crystallize in 2-propanol, and
dried to a yellow syrup. Using LC-MS/MS, fragments were detected (positive ion mode) with
m/z 42.1and 60.2, consistent with the proposed product.
Mass spectrometry of DMGO
The mass of the molecular ion, and the fragmentation patterns, of DMGO were characterized
using three different mass spectrometric systems. An Agilent 6120 (Agilent Technologies
Australia, Mulgrave, VIC, Australia) single quadrupole mass spectrometer, connected to an
Agilent 1200 Series HPLC system, was used for preliminary work and identifying candidate
HPLC systems for separating DMGO from other plasma and urine components, with
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Dimethylglycine-N-oxide
detection by measuring the cation with m/z = 120, in acidified samples. The fragmentation of
this ion was investigated using two triple-quad mass spectrometers: an AB Sciex API4000
(Applied Biosystems, Mulgrave, VIC, Australia) triple quadrupole mass spectrometer in
positive ion mode and an Agilent 6490 (Agilent Technologies Australia, Mulgrave, VIC,
Australia) triple quadrupole mass spectrometer in positive ion mode.
Assays for DMGO in plasma and urine
An extraction solvent was made up containing 10 µmol/l D3-dimethylglycine N-oxide and
0.1% v/v trifluoroacetic acid (TFA) in 10% methanol and 90% acetonitrile. Fifty microliters
of plasma, urine, or aqueous standards were pipetted into 1 mL of extraction solvent. Samples
were vortexed and centrifuged at 13 000 g for 5 minutes, then transferred to HPLC vials and
capped for analysis.
Two different chromatography systems were used. The first system used a Cogent Diamond
Hydride silica column (100 × 2.1 mm, 4 µm, Microsolv Technologies, NJ, USA), and the
second system used an Epic strong cation exchange column (SCX, 125 × 3 mm, 3 µm, ES
Industries, NJ, USA). The two DMGO chromatography systems were developed using the
Agilent 6120 single-quad mass spectrometer for detection of the DMGOH+ ion (m/z = 120).
This was not sensitive enough for quantifying DMGO in plasma.
The chromatography system for the Cogent Diamond Hydride silica column was as follows:
Mobile phase solvent A contained 10 mmol/l formic acid, and 10 mmol/l ammonium formate
in 50% distilled water and 50% acetonitrile (v/v). Solvent B contained 7.5 mmol/l TFA and
15 mmol/l formic acid in acetonitrile. A gradient was used starting with 5% A and 95% B,
then to 25% A and 75% B at 3 minutes, and 100% A at 7.5 to 8.5 minutes, and then back to
starting conditions at 9 minutes. The run time was 12 minutes, the flow rate was 0.3 ml/min,
the injection volume was 10 µl, and the column temperature was 40°C.
The chromatography system used for the Epic SCX column was as follows: Solvent A
contained 10 mmol/l formic acid and 10 mmol/l ammonium formate in 50% distilled water
and 50% acetonitrile. Solvent B contained 7.5 mmol/l trifluoroacetic acid and 15 mmol/l
acetic acid in acetonitrile. The gradient started at 3% A and 97% B, going to 30% A and 70%
B at 4 minutes, then to 100% A from 7.5 to 8.5 minutes, and back to 3% A and 97% B at 9
minutes. The run time was 12 minutes. The injection volume was 10 µl, the flow rate was 0.3
ml/min, and the oven temperature was 40°C.
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Dimethylglycine-N-oxide
Biological samples
Human plasma and urine samples had been collected as baseline samples for two studies and
had been stored at -35°C for 10 years (first study) or -85°C for less than 2 years (second
study). Both studies had been approved by the Upper South B Regional Ethics Committee,
and all subjects gave written informed consent. The first study was of 16 healthy young adult
males, described elsewhere (Atkinson et al., 2008); the second (unpublished) study was of 62
overweight adult males with features of the metabolic syndrome. The entry criteria for the
unpublished study were: a waist measurement > 94 cm, or a BMI >28 and a body fat %
(impedence) > 25; hypertension or receiving medication for hypertension; dyslipidemia
(triglycerides >1.7; HDL <1.0; LDL >3.5 mmol/l). They are all male, aged 18 to 70 years.
Exclusion criteria were diabetes, diagnosed renal failure, a plasma creatinine > 130 µmol/l,
and having cancer, surgery or acute myocardial infarction 6 months prior to recruitment. The
characteristics of the study groups are summarized in Table 1.
Statistical analyses
Data was evaluated statistically using SigmaPlot vs 11.2 (Systat Software Inc, San Jose, CA).
Correlations were calculated as Spearman’s correlation coefficient
Acknowledgements
The study involving 16 healthy students was funded by the Heart Foundation of New
Zealand. The study of metabolic syndrome subjects was funded by DuPont Nutrition &
Health. The funding bodies had no influence on this study.
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Dimethylglycine-N-oxide
References
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Dimethylglycine-N-oxide
Tables and figures
Table 1
Characteristics of the study groups.
Students
Metabolic syndrome
Signif. difference (p)
Number
16
62
Age (years)
BMI
Pl creatinine (μmol/l)
Pl betaine (μmol/l)
Pl DMG (μmol/l)
25.0 (23.25-32.5)
23.8 (19.8-27.8)
75.3 (67.8-81.1)
40.9 (33.5-50.0)
2.51 (2.24-3.21)
57.0 (50.0-63.0)
33.4 (31.1-36.1)
79.1 (75.6-84.9)
32.6 (27.4-42.8)
2.82 (2.25-3.33)
<0.001
<0.001
0.058
0.018
0.421
Students: normal male subjects (n = 16) from previously reported study (Atkinson et al. 2008); Metabolic
syndrome: male subjects with symptoms of the metabolic syndrome (n = 62). All values are medians
(interquartile range). Pl: plasma; DMG: N,N-dimethylglycine. Significance of differences assessed by the Mann-
Whitney rank sum test.
Table 2
Concentrations and urinary excretions of dimethylglycine N-oxide and related
metabolites in normal subjects and metabolic syndrome subjects.
Students
Metabolic syndrome
Signif. difference (p)
Plasma concentrations (μmol/l)
DMGO
Betaine
DMG
0.57 (0.38-1.02)
40.9 (34.4-49.4)
2.5 (2.3-3.1)
0.13 (0.09-0.20)
32.6 (27.4-42.6)
2.8 (2.3-3.3)
<0.001
0.018
0.42
Free choline
myo-Inositol
TMAO
10.0 (9.4-12.5)
25.6 (21.9-27.7)
3.4 (1.7-4.7)
9.6 (8.0-11.3)
25.1 (22.6-29.0)
3.9 (2.7-9.9)
0.14
0.50
0.078
Urinary excretions (mmol/mol creatinine)
DMGO
Betaine
DMG
Free choline
myo-Inositol
TMAO
1.12 (0.91-1.65)
1.19 (0.95-1.63)
6.3 (4.6-10.3)
2.7 (2.0-3.8)
1.6 (1.3-2.0)
3.5 (2.9-5.6)
0.73
0.011
0.003
0.010
0.30
4.6 (3.6-6.7)
1.7 (1.1-2.1)
2.1 (1.6-3.1)
3.4 (2.6-4.0)
37.8 (23.8-72.0)
36.3 (27.2-86.9)
0.58
Fractional clearances (%)
DMGO
Betaine
15.0 (7.3-22.4)
0.80 (0.6-1.4)
4.5 (3.2-6.5)
1.6 (1.0-2.3)
1.0 (0.8-1.2)
75.4 (60.9-91.5)
1.6 (1.1-2.4)
7.2 (5.0-9.7)
1.3 (1.1-1.7)
1.1 (0.8-1.6)
79.1 (71.5-85.9)
<0.001
<0.001
0.007
0.29
0.18
0.11
DMG
Free choline
myo-Inositol
TMAO
88.8 (70.8-155.4)
Students: normal male subjects (n = 16) from previously reported study (Atkinson et al. 2008); Metabolic
syndrome: male subjects with symptoms of the metabolic syndrome (n = 62). All values are medians
(interquartile range). Pl: plasma; DMG: N,N-dimethylglycine; DMGO: N,N-dimethylglycine N-oxide; TMAO:
trimethylamine N-oxide. Significance of differences by Mann-Whitney rank sum test.
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Dimethylglycine-N-oxide
Table 3
Correlations of plasma concentration, and urinary excretion, of
dimethylglycine N-oxide with other choline metabolites and with osmolytes (significance in
brackets).
Plasma DMGO
Urinary excretion DMGO
Plasma components:
Betaine
Dimethylglycine
Free choline
myo-Inositol
TMAO
+0.41 (0.001)
+0.67 (<0.001)
+0.26 (0.039)
+0.31 (0.015)
+0.06 (0.62)
+0.42 (<0.001)
+0.66 (<0.001)
+0.15 (0.25)
+0.24 (0.066)
+0.06 (0.66)
Urinary excretions:
Betaine
Dimethylglycine
Free choline
myo-Inositol
TMAO
-0.15 (0.25)
+0.27 (0.035)
-0.03 (0.83)
-0.05 (0.71)
+0.08 (0.55)
-0.20 (0.11)
+0.29 (0.022)
-0.02 (0.89)
-0.06 (0.66)
+0.09 (0.49)
Population of overweight males with symptoms of the metabolic syndrome (n = 62). Urinary excretions as
mmole/mole creatinine. Spearman correlation coefficients. DMGO: N,N-dimethylglycine N-oxide; TMAO:
trimethylamine N-oxide. The correlation between plasma DMGO concentration and the urinary excretion of
DMGO was +0.86 (p<0.001). Significant (p<0.05) associations indicated in bold.
Figure 1
Metabolic pathway for mammalian choline oxidation in an idealized liver cell.
DMG: N,N-dimethylglycine; DMGO: N,N-dimethylglycine N-oxide; THF: tetrahydrofolate;
5,10-meTHF: 5,10 methylene tetrahydrofolate. Enzymes: CHDH: choline dehydrogenase;
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Dimethylglycine-N-oxide
BADH: betaine aldehyde dehydrogenase; BHMT: betaine homocysteine methyltransferase;
DMGDH: dimethylglycine dehydrogenase. Dashed line suggested additional pathway.
Figure 2
Co-chromatography of DMGO and a novel metabolite in human urine, using
mass detection in positive ion mode.
Black solid lines: human urine; dashed red lines: human urine with 25 μmol/l DMGO added.
Left: chromatograms from a Diamond Hydride HPLC column (HILIC chromatography);
right: chromatograms from an SCX column (cation exchange chromatography). Detection
based on the m/z = 120 to (from top) 103, 102, 58 and 42 transitions.
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Dimethylglycine-N-oxide
Figure 3
Co-chromatography of DMGO and a novel metabolite in human plasma, using
mass detection in positive ion mode.
Black solid lines: human plasma; red dashed lines: human plasma with 1 μmol/l DMGO
added. Left: chromatograms from a Diamond Hydride HPLC column (HILIC
chromatography); right: chromatograms from an SCX column (cation exchange
chromatography). Detection based on the m/z = 120 to (from top) 103, 102, 58 and 42
transitions.
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Dimethylglycine-N-oxide
Figure 4
Presence of DMGO in 62 plasma and urine samples.
Top: urine responses, bottom: plasma responses. Left: the same results are obtained using the
m/z 120→58 and 120→103 transitions; right: the same results are obtained using HILIC or
ion-exchange chromatography.
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