Liquid chromatography/electrospray ionisation mass spectrometric tracking of 4-hydroxy-2(E)-nonenal biotransformations by mouse colon epithelial cells using [1,2-13C2]-4-hydroxy-2(E)-nonenal as stable isotope tracer

Article abstract of DOI:10.1002/rcm.5033

4-Hydroxy-2(E)-nonenal (HNE), a product of lipid peroxidation, has been extensively studied in several areas, including metabolism with radio-isotopes and quantification in various matrices with deuterium-labelled HNE as standard. The aim of this work was to evaluate the relevance of ¹³C-labelled HNE in biotransformation studies to discriminate metabolites from endogens by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). ¹³C-Labelled HNE was synthesised inimproved overall yield (20%), with the incorporation of two labels in the molecule. Immortalisedmouse colon epithelial cells were incubated with 2:3 molar amounts of HNE/¹³C-HNE in order to gain information on the detection of metabolites in complex media. Our results demonstrated that the stable isotope m/z values determined by mass spectrometry were relevant in distinguishing metabolites from endogens, and that metabolite structures could be deduced. Six conjugate metabolites and 4-hydroxy-2(E)-nonenoic acid were identified, together with an incompletely identified metabolite. Stable-isotope-labelled HNE has already been used for quantification purposes. However, this is the first report on the use of ¹³C-labelled HNE as a tracer for in vitro metabolism. ¹³C-Labelled HNE could also be of benefit for in vivo studies. Copyright

Full text of DOI:10.1002/rcm.5033

Research Article
Received: 31 January 2011
Revised: 22 March 2011
Accepted: 28 March 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2011, 25, 2675–2681
(wileyonlinelibrary.com) DOI: 10.1002/rcm.5033
Liquid chromatography/electrospray ionisation mass
spectrometric tracking of 4‐hydroxy‐2(E)‐nonenal
biotransformations by mouse colon epithelial cells using
[1,2‐13C2]‐4‐hydroxy‐2(E)‐nonenal as stable isotope tracer†
*
I. Jouanin , M. Baradat, M. Gieules, S. Taché, F. H. F. Pierre, F. Guéraud and L. Debrauwer
INRA, UMR1331, Toxalim, Research Center in Food Toxicology, F‐31027 Toulouse, France
4‐Hydroxy‐2(E)‐nonenal (HNE), a product of lipid peroxidation, has been extensively studied in several areas,
including metabolism with radio‐isotopes and quantification in various matrices with deuterium‐labelled HNE as
standard. The aim of this work was to evaluate the relevance of 13C‐labelled HNE in biotransformation studies
to discriminate metabolites from endogens by liquid chromatography/electrospray ionisation mass spectrometry
(LC/ESI‐MS). 13C‐Labelled HNE was synthesised in improved overall yield (20%), with the incorporation of two labels
in the molecule. Immortalised mouse colon epithelial cells were incubated with 2:3 molar amounts of HNE/13C‐HNE in
order to gain information on the detection of metabolites in complex media. Our results demonstrated that the stable
isotope m/z values determined by mass spectrometry were relevant in distinguishing metabolites from endogens, and
that metabolite structures could be deduced. Six conjugate metabolites and 4‐hydroxy‐2(E)‐nonenoic acid were
identified, together with an incompletely identified metabolite. Stable‐isotope‐labelled HNE has already been used
for quantification purposes. However, this is the first report on the use of 13C‐labelled HNE as a tracer for in vitro
metabolism. 13C‐Labelled HNE could also be of benefit for in vivo studies. Copyright © 2011 John Wiley & Sons, Ltd.
The γ‐hydroxy‐α,β‐unsaturated aldehyde 4‐hydroxy‐2(E)‐
nonenal (HNE) is one of the major aldehydes among the
breakdown products formed during the peroxidation of ω6
fatty acids. These polyunsaturated fatty acids (PUFAs) are
part of the cell membrane constituents and their peroxidation
to reactive aldehydes can alter cell integrity. The cytotoxicity
of HNE has been shown and its possible involvement in the
development of pathologies related to oxidative stress and
aging has been investigated.[1,2] HNE has also been detected
in food.[3–5] Therefore, HNE can have both endogenous and
exogenous origin.
An enhanced consumption of haem‐rich food, red and
processed meat, is associated with an increased risk of colon
cancer.[6,7] Haem is able to induce the peroxidation of PUFAs
in vitro. Haemin, a model molecule for haem, increases
precarcinogenic lesions in the colon of rats fed with haemin‐
rich and calcium‐poor diet.[8] Therefore, there could be a link
between the toxic aldehydes formed by lipid peroxidation
and colon cancer.
mouse colon epithelial cells bearing or not a mutation on the
Apc gene, as a model to study and compare the biological
effects of food components and HNE in terms of cytotoxicity.[9]
We have also used this model to compare the biotransforma-
tion capacities of the two cell lines in a quantitative way by
monitoring HNE consumption and metabolite formation,
using a radio‐isotope (manuscript in preparation).
In metabolism studies, the identification of metabolites by
mass spectrometry (MS) can be hindered by interfering
endogens coming from the matrices and co‐eluting with the
metabolites of interest. In this case, the use of radio‐isotopes is
the method of choice for the selective and efficient detection
and quantification of metabolites by liquid chromatography
(LC)/radio‐isotope counting. However, since the radio‐
isotope is used in small amount, identification in LC/MS
is not possible. Stable isotopes can overcome this problem
since they can be used in quantitative amounts compared
with the naturally abundant isotope, thus providing ions of
characteristic m/z values for selective detection by MS.
13C/12C isotopic ratio analyses by isotope ratio mass
spectrometry (IRMS) and conventional MS are used in various
fields.[10] The applications of stable‐isotope‐labelled drugs and
toxic compounds in metabolism studies have been recently
reviewed.[11] In disposition studies, deuterium‐labelled and
13C‐labelled glucose have been used simultaneously to study
kinetics after ingestion, using gas chromatography (GC)/MS
after derivatisation, and gas chromatography/combustion/
isotope ratio mass spectrometry (GC/C/IRMS).[12] Fatty acid
fluxes have been traced after the ingestion of a 13C‐triglyceride
using GC/C/IRMS.[13] Jian et al. reported on the LC/MS
The mutation of the Apc gene is known to be involved in
early stages of colon cancer. We have used immortalised
* Correspondence to: I. Jouanin, INRA, UMR1331, Toxalim,
Research Center in Food Toxicology, F‐31027 Toulouse,
France.
E‐mail: ijouanin@toulouse.inra.fr
†
Presented at the 6th Congress of the French Society of
Stable Isotopes (Société Française des Isotopes Stables,
SFIS) held 26–29 October 2010 in Toulouse, France.
Rapid Commun. Mass Spectrom. 2011, 25, 2675–2681
Copyright © 2011 John Wiley & Sons, Ltd.

I. Jouanin et al.
[1,2‐13C2]‐4‐Hydroxy‐2(E)‐nonenal (13C‐HNE)
quantification of a glutathione conjugate of 4‐oxo‐2(E)‐
nonenal obtained from cells.[14] To our knowledge, the
metabolism of lipid peroxidation products has not been
studied with a 13C stable isotope tracer by LC/MS. Never-
theless, syntheses of stable‐isotope‐labelled HNE have been
reported. HNE with one 13C atom has been synthesized to
study its in vitro interaction with proteins by 13C‐nuclear
magnetic resonance (13C‐NMR).[15] Deuterated HNE has been
validated as internal standard for quantification by MS.[16–18]
However, we reasoned that this molecule was not relevant for
metabolism studies because the deuterium could be cleaved
by biotransformations occurring at the methyl‐deuterated
terminus.[19] We recently reported on the synthesis of HNE
with two 13C labels.[20]
13C‐HNE was prepared according to our published method,[20]
with modifications. Ethyl [1,2‐13C2]‐2‐bromoacetate was
converted into ethyl [1,2‐13C2]‐4‐hydroxy‐2(E)‐nonenoate by
reacting consecutively with 4‐chlorothiophenol, hydrogen
peroxide and n‐heptanal as described.[21] The remaining steps
to 13C‐HNE were unchanged and the overall yield was
improved to 20% (Fig. 1).
4‐Hydroxy‐2(E)‐nonenal (HNE)
HNE was prepared from ethyl 2‐bromoacetate by the same
method as for 13C‐HNE.
Mixtures containing 2:3 molar amounts of HNE/13C‐HNE
were prepared for cell experimentation and were checked by
NMR (1H, CDCl3).
The aim of this work was to assess the use of a 13C stable
isotope tracer included into HNE to distinguish HNE‐specific
metabolites from endogens produced by mouse colon
epithelial cells, using LC/electrospray ionisation (ESI)‐MS.
We will first discuss the 13C‐HNE synthesis, and then the mass
spectrometry results obtained from cell incubation media.
Mouse colon epithelial cells experimentation
Mouse colon epithelial cells (Apc+/+) were established as
previously described.[22] Briefly, the cell line was established
from the colon of F1 progeny obtained after pairing a
heterozygous female Immortomouse from Charles River
Laboratories (Wilmington, MA, USA) with a male C57BL/
6 J mouse from Charles River Laboratories.
EXPERIMENTAL
Chemicals
The use of an Immortomouse makes it possible to obtain
cell lines C57BL/6 J‐Apc+/+ that express the heat‐labile SV40
large T antigen (AgT tsA58) under the control of an interferon
γ (INFγ)‐inducible promoter. At 33 °C and with INFγ (Becton
Dickinson, Le Pont de Claix, France) in the medium, the
temperature‐sensitive SV40 large T antigen is active and
drives cell proliferation. At 37 °C the temperature‐sensitive
mutation yields an inactive protein, and cells behave as non‐
proliferating colonic epithelial cells. The cells were cultured
at 33 °C in Dulbecco’s modified Eagle’s medium (DMEM;
Invitrogen, Cergy‐Pontoise, France) supplemented with 10%
Ethyl 2‐bromoacetate was purchased from Aldrich (Saint
Quentin Fallavier, France). Solvents, 4‐chlorothiophenol,
hydrogen peroxide (30% in water), n‐heptanal and diisobutyl-
aluminium hydride (DIBAL: 1 M in n‐hexane), were purchased
from Acros Organics (Geel, Belgium). n‐Heptanal was distilled
before use. Ethyl [1,2‐13C2]‐2‐bromoacetate (99% atom 13C) was
purchased from Eurisotop (Saint Aubin, France).
Caution: HNE is cytotoxic and ethyl 2‐bromoacetate is
carcinogenic. They should be handled with gloves in a
well‐ventilated fume cupboard.
O
S
O
1) 4-chloro-thiophenol
2) H2O2
heptanal
OEt
piperidine
*
*
Cl
O
O
PPh3 / NaHCO3
*
TBHP
*
OEt
OEt
Br
*
*
C5H11
C5H11
OEt
*
SeO2 cat.
*
heptanal
O
O
NaBH4
OH
OH
*
*
O
OEt
*
C5H11
C5H11
OTHP
*
(HNE)
O
PTSA
DHP
*
PPTS cat.
OTHP
MnO2
OTHP
DIBALH
OEt
*
*
*
OH
C5H11
*
O
C5H11
C5H11
*
O
Figure 1. Synthetic scheme for 13C‐HNE preparation. Asterisks indicate 13C labels.
wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 2675–2681

13C‐Labelled HNE for LC/MS analyses of mouse colon cells metabolites
Table 1. LC/MS identified Apc +/+ metabolites of HNE
LC
ESI‐MS
ESI‐MS2
ESI‐MS2
Metabolites
Retention time
(min)
product ions m/z
(precursor ion m/z)
product ions m/z
(precursor ion m/z)
m/z
16
276/278
464/466
462/464
318/320
171/173
460/462
476/478
316/318
189, 120 (276)
191, 120 (278)
HNE‐cysteine
19.1/21.1
19.9/22.0/24.4
32.2
446, 272, 254, 335 (464)
306, 444, 272, 254, (462)
162, 171, 189 (318)
448, 272, 254, 337 (466)
306, 446, 272, 254, (464)
162, 173, 191 (320)
DHN‐glutathione
HNE‐glutathione
HNE‐MA
33.5
153, 127, 71 (171)
155, 128, 72 (173)
HNA
33.9/34.5
37.2/38.1/39.0
39.8
306, 442, 272, 254, 288 (460)
254, 272, 458 (476)
306, 444, 272, 254, 288 (462)
254, 272, 460 (478)
HNA‐lactone‐glutathione
?
162, 187 (316)
162, 189 (318)
HNA‐lactone‐MA
OH
O
OH
O
NH2
S
m/z 306
S
HO
m/z 189
m/z 120
OH
H
N
N
O
O
H
H2N
O
O
HO
O
HNE-glutathione
HNE-cysteine
O
O
m/z 446
OH
OH
NH2
m/z 272
S
N
NH2
S
m/z 306
HO
H
HO
H
N
N
O
O
N
H
O
O
O
O
H
HO
m/z 335
O
O
HO
HNA-lactone-glutathione
DHN-glutathione
OH
O
O
O
S
m/z 162
OH
S
m/z 162
m/z 189
m/z 187
OH
N
N
H
O
H
O
O
O
HNA-lactone-MA
HNE-MA
m/z 71
OH
m/z 153
OH
O
m/z 127
HNA
Figure 2. Structures of HNE metabolites by mouse colonocytes. Arrows
indicate MS2 fragments.
Rapid Commun. Mass Spectrom. 2011, 25, 2675–2681 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

I. Jouanin et al.
16,0
100
50
foetal calf sera (Sigma‐Aldrich, Saint Quentin Fallavier, France),
2% penicillin/streptomycin, 2% glutamine (Invitrogen), and
10U/mL INFγ. The experiments were performed at 37 °C,
and without INFγ, to inhibit the SV40 transgene and limit
proliferation. For treatment, the Apc +/+ colonocytes were
incubated with 40 μM 13C‐HNE/HNE (3:2 molar amount)
in fresh DMEM supplemented with 2% glutamine, at +37 °C
for 60min. The supernatants were then removed, concentrated
on a C18 Sep‐pak cartridge (200 mg, Waters, Guyancourt,
France), stored with 1% 2,6‐di‐tert‐butyl‐4‐hydroxytoluene
(BHT) or acetic acid, and analysed.
m/z 276
m/z 466
m/z 464
m/z 462
m/z 460
m/z 171
m/z 476
a
0
21,1
19,1
100
b
c
50
0
22,0
24,4
24,4
100
19,1
50
0
22,0
100
34,5
33,9
LC/ESI‐MS
d
e
f
50
20,0
The cell culture supernatants were analysed by LC/ESI‐MS
using a Thermo Separation Products P1500 LC pump (Thermo
Fisher, Les Ulis, France) fitted with an ODS Spherisorb column
(5 μm, 250 × 4.6 mm; Waters, Guyancourt, France) with the
following mobile phases: A: MeCN/water/AcOH (2.5:97.5:0.1);
B: MeCN/water/AcOH (60:40:0.1) and gradient:0 min
(15%B); 10 min (25%B); 20 min (26%B); 40–45 min (65%B).
The flow rate was 1 mL/min with 1/5 post‐column splitting.
The liquid chromatograph was coupled to a Finnigan LCQ
quadrupole ion trap mass spectrometer (Thermo Fisher) fitted
with an ESI source operating in the negative ion mode.
Typical ionisation and ion transfer conditions were as follows:
electrospray needle voltage, 4.5 kV; heated transfer capillary
temperature, 220 °C; heated transfer capillary voltage, –20 V;
tube lens offset voltage, 10 V. The MS2 experiments were
carried out under automatic gain control using helium as the
collision gas. The tandem mass spectrometric (MS/MS) ion
excitation conditions (isolation width, excitation voltage,
excitation time) were adjusted to obtain maximum sensitivity
and structural information for each compound of interest.
0
34,5
33,8
100
50
0
33,5
100
50
0
38,1
39,0
100
g
37,2
50
0
0
5
10
15
20
25
30
35
40
45
Time (min)
Figure 3. Extracted mass chromatograms for ions at (a) m/z
276, (b) m/z 466, (c) m/z 464, (d) m/z 462, (e) m/z 460, (f) m/z
171, and (g) m/z 476, corresponding, respectively, to HNE‐
cysteine, to glutathione‐conjugated metabolites of HNE, to
4‐hydroxy‐2‐nonenoic acid, and to an unidentified metabolite.
RESULTS AND DISCUSSION
the HNE metabolites detected by ESI‐MS and analysed by
MS/MS in the ion trap. The structures of the various
metabolites identified together with their corresponding
proposed fragmentation pathways are reported in Fig. 2.
Typical LC/MS mass chromatograms are reported in Fig. 3.
For a compound eluting at 16 min (Fig. 3(a)), the corre-
sponding negative ESI‐MS spectrum (Fig. 4(a)) shows two ions
at m/z 276/278 displaying the characteristic isotopic pattern of
the incubated 12C/13C‐HNE mixture, which could correspond
to HNE‐cysteine.[23] The occurrence of product ions at m/z 189
and 120 corresponding to [HNE–S]– and [cysteine–H]–,
respectively, in the MS2 spectrum acquired from the m/z 276
precursor ion (Fig. 4(b)) was also consistent with this
compound being the HNE‐cysteine conjugate. This assign-
ment was further confirmed by the fragmentation of the m/z
278 ion, which yielded the [cysteine–H]– ion at m/z 120 whereas
the [HNE–S]– ion was shifted to m/z 191 (Fig. 4(c)). Based on
these data, the compound eluted at 16 min was identified as
HNE‐cysteine.[19] The absence of HNE‐cysteine from HNE
incubation with DMEM without the cells was checked.
Synthesis
In previous work, we reported on the synthesis of 13C‐HNE
with full characterisation (IR, NMR, HRMS) of the inter-
mediates and the final product.[20] The synthesis was first
tested with unlabelled HNE, then with the stable‐isotope‐
labelled compound. However, the overall yield was rather low
(4%). To improve this yield we used a synthetic method[21]
which enabled the introduction of the three functionalities of
HNE (alcohol, alkene, aldehyde) in a more efficient way. We
thus prepared directly the 13C‐labelled 4‐hydroxynonenoate
ester starting with the same stable‐isotope‐labelled precursor
(ethyl [1,2‐13C2]‐2‐bromoacetate) (Fig. 1). The yield was thus
improved to 20%.
LC/ESI‐MS
After incubation of the cells with HNE/13C‐HNE (2:3 molar
mixture), the supernatants were analysed by LC/ESI‐MS in
the negative ion mode.
The total ion current reflected the complexity of the
supernatants. Along the LC elution, the ions corresponding
to the metabolites were tracked using their characteristic
12C/13C patterns. The results are summarised in Table 1,
indicating the LC retention times, and the m/z values of
Other metabolites eluted in the 19–34 min retention time
range and displaying the characteristic M/M + 2 pattern were
detected as smaller peaks (see Table 1). Peaks at 19.1 and
21.1 min (Figs. 3(b) and 3(c)) both exhibited [M–H]– ions at
m/z 464/466, consistent with DHN‐glutathione conjugate
isomers. The [M–H]– ions of peaks at 19.9, 22.0 and 24.4 min
wileyonlinelibrary.com/journal/rcm Copyright © 2011 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2011, 25, 2675–2681

13C‐Labelled HNE for LC/MS analyses of mouse colon cells metabolites
278
10
8
a
276
6
4
2
279
338
314
336
312
231
171
191
0
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
189
10
8
6
4
2
0
b
c
276
120
171
119
188
275
260 280
80
100
120
140
160
180
m/z
200
220
240
191
10
8
6
4
120
120
278
277
2
190
171173
119
0
80
100
140
160
180
m/z
200
220
240
260
280
Figure 4. Quadrupole ion trap mass spectra obtained from HNE‐cysteine: (a)
negative ion ESI mass spectrum, (b) MS/MS spectrum of the m/z 276 precursor
ion (HNE‐cysteine), and (c) MS/MS spectrum of the m/z 278 precursor ion
([13C2]‐HNE‐cysteine).
were all detected at m/z 462/464 (Figs. 3(c) and 3(d)),
carboxylic acid form. The carboxylic acid moiety easily under-
goes cyclisation to the lactone (HNA‐lactone‐glutathione).
An additional small peak, partially coeluting with HNA‐
lactone‐glutathione, was detected thanks to the use of the
stable isotope labelling. This peak eluted at 33.5 min (Fig. 3(f))
and yielded [M–H]– ions at m/z 171/173. The MS/MS spectra
acquired from both the 12C‐ and the 13C‐labelled metabolites
(Table 1) displayed product ions corresponding to [M–H–
H2O]– (m/z 153 shifted to m/z 155) and [M–H–CO2]– (m/z 127
shifted to m/z 128), with the latter indicating that one labelled
carbon atom was involved in the CO2 elimination. A weaker
product ion is also present at m/z 71 (shifted to m/z 72),
resulting from cleavage of the C5–C6 bond of the hydro-
carbon chain, giving rise to a C4H7O‐ (12C313CH7O) ion
(Fig. 2). This characteristic fragmentation allowed us to
identify this metabolite as 4‐hydroxy‐2‐nonenoic acid (HNA).
Additional metabolites eluting at 32.2 and 39.8 min
showed [M–H]– ions which were characteristic of the
mercapturic acid (MA) conjugates of HNE (m/z 318/320)
and HNA‐lactone (m/z 316/318),[31,32] respectively, although
their retention times had been expected to be shorter on the
basis of previous work.[23] The corresponding MS2 analyses
confirmed these identifications, with the occurrence of the
diagnostic m/z 162 product ion, corresponding to [MA–H]–,
as presented in Fig. 2. In spite of reports demonstrating the
occurrence of HNE‐CysGly as a HNE‐glutathione metabolite
detected by LC/ESI‐MS (m/z 335 in the MS and a MS²
product ion at m/z 177 in positive ion mode),[33] this
compound was not detected in our experiments.
matching HNE‐glutathione conjugate isomers.[19] The [M–H]–
ions of peaks at 33.9 and 34.5 min (Figs. 3(d) and 3(e)) at m/z
460/462 were characteristic of the HNA‐lactone‐glutathione
conjugate.[23]
The MS/MS spectra acquired from the [M–H]– ions were in
agreement with the proposed structures, as indicated in
Table 1, showing the occurrence of the m/z 306 and /or 272
product ions, diagnostic of a glutathione moiety (Fig. 2).
These assignments were further confirmed by MS/MS
experiments performed on the [M–H]– ions selected from
the 13C‐labelled HNE metabolites, as reported in Table 1,
showing that none of the m/z values of the product ions were
shifted except for those of the [M–H–H2O]– ions. Note that
the DHN‐glutathione conjugate did not lead to the formation
of the m/z 306 product ion (cleavage of the thioether bond on
the DHN side of the conjugated metabolite, see Fig. 2) but to
the m/z 272 ion corresponding to cleavage of the glutathione
thioether bond. In the case of DHN‐glutathione, cleavage
of the glutamyl residue of glutathione leading to the for-
mation of the m/z 335 product ion (shifted to m/z 337 for the
13C‐labelled metabolite) was also observed whereas this
fragmentation process did not occur for the two other
glutathione conjugates (Table 1).
Those glutathione conjugated metabolites are known
as major biotransformation products of HNE in vivo and
in vitro[24–30] and have already been identified by comparison
with authentic synthesised standards. They result from
Michael‐type reaction of the glutathione and cysteine thiol
group on the electrophilic carbon C3 of HNE. The aldehyde
function of the resulting HNE‐glutathione can be reduced to the
corresponding alcohol (DHN‐glutathione) or oxidised to the
In addition to these metabolites formed by classical
xenobiotic metabolism processes, minor unidentified peaks
were detected later in the gradient elution, at retention times
Rapid Commun. Mass Spectrom. 2011, 25, 2675–2681 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

I. Jouanin et al.
478
10
8
a
b
476
462
6
4
479
500
341
2
273
258
512
320
231
300
300
558
550
0
150
200
250
350
m/z
400
450
500
600
476
10
8
6
4
458
2
272
254
347
210
436
0
140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480
m/z
478
10
8
c
460
6
4
272
254
2
477
459
210
349
179
0
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480
m/z
Figure 5. Quadrupole ion trap mass spectra obtained from the unknown
metabolite: (a) negative ion ESI mass spectrum, (b) MS/MS spectrum of the m/z
476 precursor ion (HNE metabolite), and (c) MS/MS spectrum of the m/z 478
precursor ion ([13C2]‐HNE metabolite).
36 and 39 min (Fig. 3(g)). These peaks exhibited [M–H]– ions
CONCLUSIONS
at m/z 476/478 displaying the characteristic isotopic ratio of
the incubated HNE/13C2HNE mixture (Fig. 5(a)). The
corresponding MS/MS spectra of the two peaks were
identical and showed a major product ion at m/z 458 (shifted
to m/z 460 in the MS/MS spectrum of the m/z 478 precursor
ion), together with product ions at m/z 272 and 254 (Figs. 5(b)
and 5(c)). The occurrence of these two latter ions was
indicative of glutathione conjugates as discussed before.
The occurrence of the minor [M–H–129]– product ion
corresponding to the loss of a glutamyl residue (m/z 347/
349) reinforced this conclusion. Thus, for these metabolites,
we first hypothesised isomers of a glutathione conjugate of
4‐oxo‐2(E)‐nonenoic acid (ONA‐SG), although a shorter
retention time would be expected from such a compound.
We successfully synthesised ONA‐SG but the resulting MS2
spectrum was different from that of the metabolite (data not
shown). The oxidation of the glutathione sulphur atom of
HNA‐lactone‐glutathione into a sulfoxide group may also
lead to [M–H]– ions at m/z 476/478[34] but the occurrence of
the m/z 458/460 (loss of water) product ions in the MS2
spectra rather suggests the presence of an hydroxyl group.
Thus, this metabolite could correspond to a hydroxylated
form of HNA‐lactone‐glutathione. Hydroxylation at C9
has been reported in the metabolism of HNE by rat as
urinary metabolites. However, these structures are expected
to be more polar.[35] The lactone ring may also constitute a
hydroxylation site. However, there was not sufficient
information available in the MS/MS spectra to allow the
precise location of the hydroxylation position of this
metabolite.
Understanding how HNE, a toxic lipid peroxidation product,
is biotransformed by colonocytes is of crucial importance for
mechanistic insight on early stages of colon carcinogenesis.
In this work, our aim was to test a chemical tool, namely
[1,2‐13C2]‐4‐hydroxy‐2(E)‐nonenal, in order to track metabo-
lites in mouse colonocytes culture media by revealing
characteristic 13C/12C m/z patterns in LC/ESI‐MS analyses.
13C was chosen as stable isotope tracer because the labels
could not be lost during metabolism. LC/MS analyses and
MS2 experiments proved to be relevant for tracking seven
metabolites and an additional incompletely identified com-
pound. Those metabolites, resulting from Michael‐type
additions and oxidation/reduction reactions, demonstrate
the involvement of detoxification pathways in colonocytes.
To our knowledge, this is the first report on the use of 13C‐
labelled HNE as a tracer for LC/MS analyses in the field of
metabolism studies, and this provides an interesting alter-
native to the use of radioactive compounds. 13C‐HNE should
be especially well suited for the LC/MS analyses of in vivo
experimentation samples, by providing identification of
metabolites from complex mixtures.
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