1,4-dihydroxynonene mercapturic acid, the major end metabolite of exogenous 4-hydroxy-2-nonenal, is a physiological component of rat and human urine

Article abstract of DOI:10.1021/tx970139w

In the present study 1,4-dihydroxynonene mercapturic acid (DHN-MA), previously shown to be the major urinary metabolite of 4-hydroxy-2-nonenal (HNE) administered to the rat, was characterized and determined to be a normal constituent of rat and human urine. DHN-MA was excreted as a mixture of at least two stereoisomers as determined by ion trap LC-MS/MS/MS after solid-phase extraction and HPLC purification. The 24-h urinary excretion of this compound was about 10 ng and 5 μg for rat and human, respectively. This end metabolite of the lipid peroxidation product HNE could represent a specific and noninvasive biomarker.

Full text of DOI:10.1021/tx970139w

130
Chem. Res. Toxicol. 1998, 11, 130-135
1,4-Dih yd r oxyn on en e Mer ca p tu r ic Acid , th e Ma jor En d
Meta bolite of Exogen ou s 4-Hyd r oxy-2-n on en a l, Is a
P h ysiologica l Com p on en t of Ra t a n d Hu m a n Ur in e
J acques Alary,* Laurent Debrauwer, Yvette Fernandez, J ean-Pierre Cravedi,
Dinesh Rao, and Georges Bories
Laboratoire des Xe´nobiotiques, INRA, BP 3, 31931 Toulouse Cedex, France
Received August 11, 1997
In the present study 1,4-dihydroxynonene mercapturic acid (DHN-MA), previously shown
to be the major urinary metabolite of 4-hydroxy-2-nonenal (HNE) administered to the rat,
was characterized and determined to be a normal constituent of rat and human urine. DHN-
MA was excreted as a mixture of at least two stereoisomers as determined by ion trap LC-
MS/MS/MS after solid-phase extraction and HPLC purification. The 24-h urinary excretion
of this compound was about 10 ng and 5 µg for rat and human, respectively. This end metabolite
of the lipid peroxidation product HNE could represent a specific and noninvasive biomarker.
lifetime of free HNE represents a serious drawback for
its use as a biomarker.
In tr od u ction
Lipid peroxidation of lipoproteins, cellular and organite
membranes by reactive oxygen species, is thought to play
a role in the pathogenesis of several diseases including
atherosclerosis, ischemia/reperfusion injury, adult res-
piratory distress syndrome, and several inflammatory
diseases (1, 2). At this time, whether lipid peroxidation
is a cause or a consequence of these diseases remains
equivocal, but it seems likely that this phenomenon can
at least contribute to tissue injury. Lipid peroxidation
results in the formation of reactive aldehydes, and one,
4-hydroxy-2-nonenal (HNE),1 is a major compound pro-
duced in vivo by the peroxidation of ω-6 polyunsaturated
fatty acids.
Recently, 1,4-dihydroxynonene mercapturic acid (DHN-
MA) (Figure 1) was shown to be the major urinary
metabolite of exogenous HNE in the rat, accompanied
to a lesser extent by the corresponding aldehyde, acid,
and lactone mercapturic acids (17). The stability of DHN-
MA in urine appears appropriate for a urinary biomarker
of lipid peroxidation. To determine if this phenomenon
represents a permanent physiological process, the pres-
ence in the urine of DHN-MA generated in situ was
examined. The present paper reports the identification
and quantitation of DHN-MA in urine obtained from rats
kept in normal physiological conditions and from healthy
human volunteers.
The expired hydrocarbons ethane and pentane, as well
as malondialdehyde, have been widely used in the past
decade as an index of lipid peroxidation. However HNE
is now considered by numerous authors as a reliable
index of both the extent (3, 4) and consequences (5) of
lipid peroxidation. As a matter of fact, methods have
been developed to determine the concentration of free
HNE in the plasma of rats (6) and healthy human
volunteers (4, 7-9). However, owing to its high reactivity
toward -SH groups (10, 11) and amino groups (12, 13),
HNE readily reacts at physiological pH via Michael
addition with endogenous compounds such as amino
acids and proteins. Conjugation with glutathione, oc-
curring either spontaneously (11) or through catalysis by
glutathione transferases (14, 15), has been shown to
represent a major metabolic pathway in several cell types
(16). Moreover, the aldehyde group of HNE can also be
either oxidized by aldehyde dehydrogenase to 4-hydroxy-
nonenoic acid which is metabolized by hepatocytes through
â-oxidation (16) or reduced by alcohol dehydrogenase to
1,4-dihydroxynonene (16). The resulting short biological
Exp er im en ta l P r oced u r es
Ch em ica ls. Sodium azide, sodium borohydride, and N-
acetyl-L-cysteine were purchased from Aldrich (Saint Quentin
Fallavier, France). All chemicals and solvents for the prepara-
tion of buffers and HPLC eluents were the highest grade
commercially available from Merck (Nogent-sur-Marne, France)
or Carlo Erba (Rueil Malmaison, France). Ultrapure water from
Milli-Q system (Millipore, Saint Quentin en Yvelines, France)
was used for HPLC eluent preparation.
Ra d ioa ctivity Deter m in a tion . The samples were directly
counted in a model 4330 Packard Tricarb scintillation counter
(Packard Instrument Co., Downers Grove, IL) with Ultimagold
(Packard) as the scintillation cocktail.
Ur in e Collection . Urine was collected for 24 h from seven
healthy male volunteers (nonsmokers, 40-60 years old) who had
not taken any drugs during the previous week. Six male Wistar
rats (200-250 g) were placed in individual metabolism cages
for 24-h urine collection. The animals were allowed free access
to tap water and commercial chow. Urine samples were
collected in vials containing either 1 or 100 mg of sodium azide
for the rat and human samples, respectively. The volume of
urine was measured to quantitate DHN-MA excreted in 24 h.
The urine samples were stored at -20 °C until analysis.
HP LC P u r ifica tion of DHN-MA Sta n d a r d : System 1.
Semipreparative HPLC was used for the purification of syn-
thesized DHN-MA. The system consisted of a Philips 4100
* To whom correspondence should be addressed. Tel: 33 (0)561 28
5383. Fax: 33 (0)561 28 5244.
1
Abbreviations: DHN-MA, 1,4-dihydroxynonene mercapturic acid;
HNE, 4-hydroxy-2-nonenal; DHN, 1,4-dihydroxynonane.
S0893-228x(97)00139-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/28/1998

1,4-Dihydroxynonene Mercapturic Acid in Urine
Chem. Res. Toxicol., Vol. 11, No. 2, 1998 131
F igu r e 1. Structure and full scan positive electrospray mass spectrum of DHN-MA (MW 321).
apparatus (Pye Unicam, Cambridge, U.K.) equipped with an
Ultrabase ODS column (5 µm, 250 × 7.5 mm) (SFCC, Eragny,
France) thermostated at 35 °C and connected to a Radiomatic
Flo-one A-500 instrument (Radiomatic, La-Queue-Lez-Yvelines,
France) with Flo-scint II as scintillation cocktail or to a Gilson
model 202 fraction collector (Gilson France, Villiers-le-Bel,
France). Three mobile phases were used. Mobile phase A
contained 2.5% acetonitrile and 97.5% of 0.1% (v/v) acetic acid.
Mobile phase B consisted of 20% acetonitrile and 80% of 1%
(v/v) acetic acid. Mobile phase C contained 70% acetonitrile and
30% of 1% (v/v) acetic acid. Solvents were delivered at a flow
rate of 2 mL/min as follows: 0-4 min, 100% A; 4-5 min, linear
gradient from 100% A to 100% B; 5-45 min, linear gradient
from 100% B to 100% C; 45-60 min, 100% C.
HP LC Sep a r a tion of Ur in a r y DHN-MA: System 2. The
system consisted of a model 420 Kontron pump (Kontron
Instruments, Saint Quentin en Yvelines, France) connected to
a Gilson 202 fraction collector. HPLC separations were per-
formed with a Spherisorb ODS 2 RP column (5 µm, 250 × 4.6
mm) from Shandon (Eragny, France) protected by a Spherisorb
precolumn (5 µm, 10 × 4.6 mm). Eluent A contained 90% 20
mM ammonium acetate adjusted to pH 4.5 with acetic acid and
10% acetonitrile. Eluent B consisted of 80% of 1% (v/v) acetic
acid and 20% acetonitrile. Solvents were delivered at a flow
rate of 1 mL/min as follows: 0-20 min, 100% A; 20-60 min,
100% B. The separation was carried out at ambient tempera-
ture (20-22 °C), and the fractions were collected at a rate of 2
fractions/min.
determined spectrophotometrically at 223 nm (ꢀwater ) 13 750
M-1 cm-1). The yield of HNE recovery was about 80%.
The aqueous solution of HNE (100 µmol) was added to 2 mL
of 0.2 M phosphate buffer (pH 8.5) and 1 mL of a 10% (w/v)
aqueous solution of NaHCO3 containing 100 µmol of N-acetyl-
L-cysteine, then placed at 37 °C, and stirred for 24 h. The
reaction mixture was brought to pH 7.0 and loaded on a 1-g
LC-C18 Supelco cartridge (Saint Quentin Fallavier, France)
preconditioned with 5 mL of methanol and 10 mL of distilled
water. The cartridge was washed with 20 mL of distilled water,
dried under nitrogen, and eluted with 10 mL of methanol. The
eluate was evaporated to dryness, the residue was dissolved in
10 mL of 0.05 M sodium hydroxide solution, and sodium
borohydride (400 µmol) was added. The mixture was incubated
for 2 h at 37 °C, then brought to pH 2 by dropwise addition of
1 M H3PO4 (to remove the excess of borohydride) adjusted to
pH 6.0, and loaded on a 1-g LC-C18 cartridge as described above.
After evaporation of the methanol eluate, the residue was taken
up with 3 mL of acetonitrile-water (1:2). Purification of DHN-
MA was carried out using HPLC system 1. In order to
determine the retention time of DHN-MA,
a mixture of
[3H]-labeled and unlabeled (1 mg) DHN-MA was injected and
radioactivity determined by on-line scintillation counting. Then
two 1.5-mL aliquots of the acetonitrile-water sample were
injected, and the effluent containing DHN-MA was collected,
based on the retention time of [3H]DHN-MA (20-22 min) and
UV absorbance at 205 nm. The combined eluents were evapo-
rated to dryness under vacuum. To remove traces of water, 2
mL of absolute ethanol was added and the mixture evaporated
to dryness at 45 °C under vacuum. The product was stored
overnight in a vacuum dessicator (P2O5) and weighed. The
reaction yield was about 15%. The product was analyzed by
proton nuclear magnetic resonance in CD3OD using a Bruker
ARX 400 spectrometer. Proton spectrum (all δ, ppm, from
tetramethylsilane): 0.92 (t, CH3-CH2-), 0.33-1.9 (m, CH3-CH2-
CH2-CH2-CH2-CHOH-CH-CH2-CH2OH), 2.00 (s, CH3-CO-
NH-), 2.87 (m, -S-CH-), 3.30 (m, -S-CH2-), 3.68 (m, -CHOH-),
Syn th esis of DHN-MA. HNE diethyl acetal was synthe-
sized as described by Esterbauer et al. (18). J ust prior to use,
racemic HNE was prepared from the diethyl acetal derivative
(30 mg) by 1 mM HCl hydrolysis (1 h, 25 °C). The aqueous
solution was vortex-extracted (2 min) five times with 10 mL of
dichloromethane. The organic phase was filtered on a phase-
separation filter (1PS Whatman). The filtrate was evaporated
under vacuum without heating and the liquid residue resolu-
bilized in 10 mL of distilled water. HNE concentration was

132 Chem. Res. Toxicol., Vol. 11, No. 2, 1998
Alary et al.
ESI-MS as well as LC-ESI-MS analyses were carried out
on a Finnigan LCQ quadrupole ion trap mass spectrometer
(Thermo Quest, les Ulis, France) equipped with a Finnigan
electrospray ionization source. For infusion studies, the sample
solution [typically 10 ng/µL in acetonitrile-water-acetic acid
(49:49:2, v/v/v)] was infused into the ESI source at a flow rate
of 3 µL/min, using an electrospray needle voltage of 5.2 kV and
a heated capillary temperature of 200 °C. On-line LC-MS
analyses were achieved using MS/MS/MS experiments in the
trap. The m/z 322 (MH+) to 304 and m/z 304 to 164 transitions
were monitored in the MS2 and MS3 steps, respectively. The
electrospray needle voltage was 5.4 kV, and the heated capillary
temperature was 220 °C. Helium was used as damping as well
as collision gas for MSn experiments. All analyses were
performed under automatic gain control conditions. The LC
system consisted of a Thermo Separation P2000 (Thermo Quest,
les Ulis, France) LC pump equipped with a Rheodyne 7725I
injector. The LC column was an Ultrabase C18 (5 µm, 250 × 2
mm) (Life Sciences International, Eragny, France). The flow
rate was 0.2 mL/min without postcolumn splitting into the ESI
source. The mobile phases consisted of acetonitrile-0.5% acetic
acid mixtures, 10:90 for A and 30:70 for B. The following
gradient was used: 0-3 min, 100% A; 3-13 min, linear gradient
from 100% A to 100% B; 13-25 min, 100% B. When the LC-
MS/MS/MS analyses were concerned, the LC effluent was
directed into waste during 8 min at the beginning of the run
using a programmable divert valve in order to discard the
endogenous urine compounds. Quantitation was based on peak
area and was achieved using external standard calibration.
F igu r e 2. HPLC radiochromatogram (system 2) of synthesized
[4-3H]DHN-MA.
3.75 (m, -CH2OH), 4.57 (dd, -NH-CH-COOH). DHN-MA was
obtained with a purity of 98%.
The fast atom bombardment mass spectrometry (FAB-MS)
spectrum exhibited an intense ion (MH-) at m/z 320. By positive
mode electrospray ionization mass spectrometry (ESI-MS), the
product gave a quasi molecular ion (MH+) at m/z 322 and a
daughter ion at m/z 304, corresponding to loss of water (Figure
1). With LC coupled to MS/MS/MS, synthetic DHN-MA showed
the presence of two unresolved compounds with the same
fragmentation pattern consistent with the presence of two
stereoisomers.
Syn th esis of [3H]DHN-MA. [4C-3H]DHN-MA was synthe-
sized from [4C-3H]HNE diethyl acetal (19) as described for
unlabeled DHN-MA. The compound was purified using HPLC
system 1. HPLC-purified [3H]DHN-MA showed
a specific
Resu lts
activity of 180 GBq/mmol. The determination of exchange of
tritium from DHN-MA (10 mM phosphate buffer, pH 7.4, 72 h,
37 °C) amounted to 0.1% (0.08-0.15, n ) 3). This value will be
considered as nonsignificant. Radio HPLC chromatogram of
[3H]DHN-MA using system 2 revealed the presence of two
partially resolved radioactive products (Figure 2). After collec-
tion, these two compounds were characterized by ESI-MS
(positive mode). They exhibited the same quasi molecular ion
(MH+) at m/z 324, corresponding to the two stereoisomers.
Extr a ction a n d P u r ifica tion of DHN-MA fr om Ur in e. A
10-mL aliquot of filtered (0.45 µm) urine (pH 6-7) was diluted
1:1 with 10 mL of distilled water and spiked with 30 µL of
methanol containing [3H]DHN-MA (39 000 dpm, 1.1 ng) as
tracer. The sample was applied to a 1-g LC-C18 cartridge
(Supelco) and passed through by gravity. The cartridge was
washed with 20 mL of distilled water, dried under a nitrogen
stream, and eluted with 5 mL of methanol. The eluate was
evaporated to dryness under vacuum at 45 °C. The residue was
added to 400 µL of eluent A (HPLC system 2). The suspension
was vortexed for 2 min and the vial rinsed with 100 µL of eluent
A. The extract and wash were combined and filtered on a 0.45-
µm Ultrafree MC filtration unit (Millipore) prior to HPLC
injection. Separation of DHN-MA was performed using HPLC
system 2. The radioactive fractions were collected, combined,
and then evaporated to dryness at 45 °C under vacuum. The
residue was taken up with 250 µL of 0.5% (v/v) acetic acid-
acetonitrile (90:10), and a 100-µL aliquot was analyzed by LC-
MS/MS/MS.
Id en tifica tion of DHN-MA by LC-MS/MS/MS. An
LC-MS/MS/MS-based method was developed for the
unequivocal identification of DHN-MA. The positive
electrospray ionization produces an intense MH+ ion at
m/z 322. When subjected to MS/MS experiments by
collisional activation in the trap, the selected m/z 322
parent ion decomposes mainly into the m/z 304 ion,
corresponding to a loss of water from the MH+ species,
and to a lower extent into the m/z 164 fragment ion,
which corresponds to the N-acetylcysteine moiety of
DHN-MA. An additional isolation/fragmentation se-
quence was performed on the m/z 304 fragment ion
produced by decomposition of the m/z 322 precursor ion.
This resulted in the formation of an intense m/z 164 ion
as a product of the m/z 304 fragmentation in the MS/
MS/MS experiment. Indeed, compared to LC-MS/MS
based on the selective detection of m/z 322 to 304 and
m/z 322 to 164 transitions, the use of LC-MS/MS/MS in
the quadrupole ion trap instrument greatly improved the
selectivity of the mass spectrometric detection without
affecting the sensitivity, owing to the high transmission
efficiency of ion trap tandem mass spectrometry (20).
Id en t ifica t ion of DHN-MA in R a t a n d Hu m a n
Ur in e. Figure 3a illustrates the chromatogram obtained
from a SPE plus HPLC-purified urine sample. In an
attempt to overcome the HPLC purification step, some
experiments were carried out by direct injection of the
crude preconcentrated SPE methanol eluate. Figure 3b
shows that even in the absence of any purification step,
the background noise remains low and does not hamper
the determination of the peak area. This result convinc-
ingly demonstrates the high selectivity of the LC-MS/
MS/MS method.
For experiments carried out without previous HPLC purifica-
tion, the crude solid-phase extraction (SPE) methanol eluate was
evaporated to dryness, the residue was dissolved in 500 µL of
the acetic acid-acetonitrile eluent mentioned above, and a 100-
µL aliquot was injected.
Ma ss Sp ectr om etr y. FAB mass spectra were obtained on
a
Nermag R-10-10-H single quadrupole instrument (Quad
Service, Poissy, France) fitted with a M-scan (Ascott, U.K.) FAB
gun. Xenon gas was used for bombardment at an acceleration
voltage of 8 kV. Typically a 0.5-µL aliquot of the sample (1 µg/
µL in methanol) was mixed with ca. 1 µL of Magic Bullet
(dithiothreitol-dithioerythritol, 5:1) used as the matrix for
analyses which were performed in the negative ion mode.
The linearity of the mass spectrometric response has
been assessed between 1 and 100 ng of injected DHN-
MA. The detection limit of the system ca. 0.1 ng (i.e.,

1,4-Dihydroxynonene Mercapturic Acid in Urine
Chem. Res. Toxicol., Vol. 11, No. 2, 1998 133
F igu r e 3. Typical LC-ESI-MS/MS/MS (positive mode) chromatogram of N-acetylcysteine ion (MH+, m/z 164) from DHN-MA
fragmentation following injection of DHN-MA purified from rat urine by SPE plus HPLC (a) or SPE (b).
300 fmol) was reached for standard DHN-MA injected
onto the LC column. However, the limit of quantitation
with external standard calibration was estimated to be
about 2 ng of injected DHN-MA, owing to the important
run-to-run variations of the results with the lower
amounts injected.
DHN-MA. The standard deviation of the measured peak
area was found to be 8.4% (n ) 6).
Qu a n t it a t ion of DHN-MA in R a t a n d Hu m a n
Ur in e. The use of [3H]DHN-MA as a tracer allowed
quantification of the recovery at each step of the proposed
method. To determine the extraction yield, the radioac-
tivity not retained on the C18 cartridge was counted after
concentrating the combined aqueous phase and wash.
The repeatability of the LC-MS/MS/MS method was
evaluated by replicate injections of 5 ng of standard

134 Chem. Res. Toxicol., Vol. 11, No. 2, 1998
Alary et al.
The results were confirmed by comparing the radioactiv-
ity found in the methanol eluate with that of the spiked
dose. Separate measurements revealed that the yield of
SPE for urinary DHN-MA was 97 ( 1% (mean ( SD, n
) 6).
isomers, all the chromatograms from rat and human
urine show that DHN-MA generated in situ is excreted
in the urine as a mixture of at least two stereoisomers.
This result is in accordance with that of a recent HNE
metabolism study which showed that DHN-MA was
excreted in rat urine as a pair of diastereoisomers (23).
Indeed, other authors (24) showed that HNE formed in
vivo was a racemic mixture of 4R and 4S isomers.
Assuming that all the amino acids constitutive of glu-
tathione have an L configuration, Michael addition of
glutathione at the double bond leads to an additional
chiral center at the C3 position. Accordingly, excreted
DHN-MA would be composed theoretically of four isomers
providing that a metabolic stereoselective process is not
involved during both the formation and degradation of
endogenous DHN-MA. Considering that the synthesized
DHN-MA standard also shows the presence of two
isomers with the same parent ion at m/z 322 and a
similar fragmentation pattern leading to a unique ion
with m/z 164, it appears that the recorded peak area
corresponds to the total urinary DHN-MA.
The current results demonstrate for the first time that
DHN-MA is a physiological component of rat and human
urine and that in nonpathological conditions the forma-
tion of this end product of lipid peroxidation is relatively
low. The low levels of excreted DHN-MA are likely a
direct consequence of the low level of lipid peroxidation
occurring under physiological conditions.
Additionally, two recent HNE metabolic studies carried
out in the rat showed that DHN-MA is the major urinary
metabolite of HNE. However, DHN-MA accounted for
only 13% of the HNE dose following iv injection (17) and
3.5% after ip injection (23). Thus in these two cases only
a small fraction of the HNE dose is excreted in urine as
DHN-MA.
For the determination of HPLC recovery, the radioac-
tive fractions were collected and combined and the
corresponding radioactivity was compared with that of
the eluent A filtrate. HPLC yield was 97 ( 0.5% (mean
( SD, n ) 6). The major losses of DHN-MA accounting
for 7% of the dose originated from the resuspension of
the residue in eluent A and the subsequent filtration.
Indeed the results showed that urinary DHN-MA was
recovered with a final yield of 88.2 ( 4% (mean ( SD, n
) 13). All the values given below have been corrected
to take into account the individual recovery yield.
The mean value for DHN-MA excreted during a 24-h
period by the six rats was 11.07 ( 8.5 ng with values
ranging from 6 to 27 ng. For the seven healthy human
volunteers, the corresponding mean value was 4.64 (
2.72 µg with a range of 2-9 µg. These excreted amounts
corresponded to DHN-MA concentrations of about 0.8 and
2.7 ng/mL in rat and human urine, respectively.
Discu ssion
A preliminary study (unpublished results) character-
izing urinary DHN-MA under physiological conditions,
based upon Raney nickel desulfurization of HPLC prepu-
rified DHN-MA into 1,4-dihydroxynonane (DHN) and
subsequent GC/MS SIM mode analysis, allowed identi-
fication of this compound and revealed that the urinary
level of DHN-MA was extremely low. However this
method suffered from both a poor DHN recovery and a
lack of reproducibility. Moreover, the total ionization
current background in the molecular mass region of the
DHN main fragments was too high to ensure sufficient
specificity and sensitivity.
In order to characterize and quantify urinary DHN-
MA under normal physiological conditions, a more sensi-
tive and specific method had to be developed. In order
to satisfy these requirements, an LC-MS/MS/MS-based
method which allows the direct determination of unmodi-
fied DHN-MA was investigated. The method showed a
high yield of recovery, as DHN-MA is a nonvolatile,
unreactive, and stable compound which remains un-
changed along the whole purification step. An additional
advantage of the method is that neither derivatization
nor liquid/liquid extraction is required, all steps known
to decrease the yield of recovery especially at the nano-
gram level.
It is assumed that HNE occurring in the body origi-
nates from continuous production by basal endogenous
lipid peroxidation (25). Therefore, and based on previous
data (17, 23), it might be expected that the presence of
DHN-MA in urine results from this generation of HNE.
To test the validity of DHN-MA as a biomarker of lipid
peroxidation in vivo, studies are in progress using rats
in which lipid peroxidation is experimentally enhanced.
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