HNE Michael adducts to histidine and histidine-containing peptides as biomarkers of lipid-derived carbonyl stress in urines: LC-MS/MS profiling in Zucker obese rats

Article abstract of DOI:10.1021/ac7016184

A new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach, based on the precursor ion scanning technique using a triple-stage quadrupole, has been developed to detect free and protein-bound histidine (His) residues modified by reactive carbonyl species (RCS) generated by lipid peroxidation. This approach has been applied to urines from Zucker obese rats, a nondiabetic animal model characterized by obesity and hyperlipidemia, where RCS formation plays a key role in the development of renal and cardiac dysfunction. The immonium ion of His at m/z 110 was used as a specific product ion of His-containing peptides to generate precursor ion spectra, followed by MS ² acquisitions of each precursor ion of interest for structural characterization. By this approach, three novel adducts, which are excreted in free form only, have been identified, two of them originating from the conjugation of 4-hydroxy-fraras-2-nonenal (HNE) to His, followed by reduction/oxidation of the aldehyde: His-1,4-dihydroxynonane (His-DHN), His-4-hydroxynonanoic acid (His-HNA), and carnosine-HNE, this last recognized in previous in vitro studies as a new potential biomarker of carbonyl stress. No free His-HNE was found in urines, which was detected only in protein hydrolysates. The same LC-MS/MS method, working in multiple reaction monitoring (MRM) mode, has been developed, validated, and applied to quantitatively profile in Zucker urines both conventional (1,4-dihydroxynonane mercapturic acid, DHN-MA) and the newly identified adducts, except His-HNA. The analytes were separated on a C12 reversed-phase column by gradient elution from 100% A (water containing 5 mM nonafluoropentanoic acid) to 80% B (acetonitrile) in 24 min at a flow rate of 0.2 mL/min and analyzed for quantification in MRM mode by applying the following precursor-to-production transitions m/z 322.2 → 164.1 + 130.1 (DHN-MA), m/z 314.7 → 268.2 + 110.1 (His-DHN), m/z 312.2 → 110.1 + 156.0 (His-HNE), m/z 383.1 → 266.2 + 110.1 (CAR-HNE), m/z 319.2 → 301.6 + 156.5 (H-Tyr-His-OH, internal standard). Precision and accuracy data, as well as the lower limits of quantification in urine, were highly satisfactory (from 0.01 nmol/mL for CAR-HNE, His-DHN, His-HNE, to 0.075 nmol/mL for DHN-MA). The method, applied to evaluate for the first time the advanced lipoxidation end products profile in urine from obese Zucker rats, an animal model for the metabolic syndrome, has proved to be suitable and sensitive enough for testing in vivo the carbonyl quenching ability of newly developed RCS sequestering agents.

Full text of DOI:10.1021/ac7016184

Anal. Chem. 2007, 79, 9174-9184
HNE Michael Adducts to Histidine and
Histidine-Containing Peptides as Biomarkers of
Lipid-Derived Carbonyl Stress in Urines: LC-MS/
MS Profiling in Zucker Obese Rats
Marica Orioli, Giancarlo Aldini, Maria Carmela Benfatto, Roberto Maffei Facino, and Marina Carini*
Istituto di Chimica Farmaceutica e Tossicologica “Pietro Pratesi”, Faculty of Pharmacy, University of Milan, Via Mangiagalli
25, I-20133 Milan, Italy
A new liquid chromatography-tandem mass spectromet-
ric (LC-MS/MS) approach, based on the precursor ion
scanning technique using a triple-stage quadrupole, has
been developed to detect free and protein-bound histidine
(His) residues modified by reactive carbonyl species
(RCS) generated by lipid peroxidation. This approach has
been applied to urines from Zucker obese rats, a nondia-
betic animal model characterized by obesity and hyper-
lipidemia, where RCS formation plays a key role in the
development of renal and cardiac dysfunction. The im-
monium ion of His at m/z 110 was used as a specific
product ion of His-containing peptides to generate precur-
sor ion spectra, followed by MS2 acquisitions of each
precursor ion of interest for structural characterization.
By this approach, three novel adducts, which are excreted
in free form only, have been identified, two of them
originating from the conjugation of 4-hydroxy-trans-2-
nonenal (HNE) to His, followed by reduction/oxidation
of the aldehyde: His-1,4-dihydroxynonane (His-DHN),
His-4-hydroxynonanoic acid (His-HNA), and carnosine-
HNE, this last recognized in previous in vitro studies as
a new potential biomarker of carbonyl stress. No free His-
HNE was found in urines, which was detected only in
protein hydrolysates. The same LC-MS/MS method,
working in multiple reaction monitoring (MRM) mode,
has been developed, validated, and applied to quantita-
tively profile in Zucker urines both conventional (1,4-
dihydroxynonane mercapturic acid, DHN-MA) and the
newly identified adducts, except His-HNA. The analytes
were separated on a C12 reversed-phase column by
gradient elution from 100% A (water containing 5 mM
nonafluoropentanoic acid) to 80% B (acetonitrile) in 24
min at a flow rate of 0.2 mL/min and analyzed for
quantification in MRM mode by applying the following
precursor-to-product ion transitions m/z 322.2 f 164.1
+ 130.1 (DHN-MA), m/z 314.7 f 268.2 + 110.1 (His-
DHN), m/z 312.2 f 110.1 + 156.0 (His-HNE), m/z
383.1 f 266.2 + 110.1 (CAR-HNE), m/z 319.2 f
301.6 + 156.5 (H-Tyr-His-OH, internal standard).
Precision and accuracy data, as well as the lower limits
of quantification in urine, were highly satisfactory (from
0.01 nmol/mL for CAR-HNE, His-DHN, His-HNE, to
0.075 nmol/mL for DHN-MA). The method, applied to
evaluate for the first time the advanced lipoxidation end
products profile in urine from obese Zucker rats, an
animal model for the metabolic syndrome, has proved to
be suitable and sensitive enough for testing in vivo the
carbonyl quenching ability of newly developed RCS se-
questering agents.
Reactive carbonyl species (RCS) generated by lipid peroxida-
tion, such as 4-hydroxy-trans-2-nonenal (HNE), 2-propenal (ac-
rolein, ACR), malondialdehyde (MDA), and glyoxal (GO), leading
to covalently modified proteins, may contribute to oxidative tissue
damage. They are known to be involved in the pathogenesis of
several cardiovascular (atherosclerosis, cerebral or heart is-
chemia-reperfusion injury) and neurodegenerative (Alzheimer’s,
Parkinson’s) diseases, as well as in diabetes and cancer.1-3
Most of the biological effects of intermediate lipid-derived RCS
are attributed to their capacity to react with the nucleophilic sites
of peptides and proteins (Cys, His, or Lys residues) forming
advanced lipoxidation end products (ALEs), such as MDA-Lys
(Schiff base adduct), HNE-Lys (Michael adduct),4 HNE-Lys
ꢀ
(pyrrole derivative),5 FDP-Lys [N -(3-formyl-3,4-dehydropiperi-
ꢀ
dino)lysine],6 MP-Lys [N -(3-methylpyridinium)lysine],7 levuglan-
din adducts (pyrrole derivatives),8 CMC [S-(carboxymethyl)-
(1) Aldini, G.; Dalle-Donne, I.; Colombo, R.; Maffei Facino, R.; Milzani, A.; Carini,
M. ChemMedChem 2006, 1, 1045-1058.
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Modifications to Cellular Dysfunction and Diseases; Dalle-Donne, I., Scaloni,
A., Butterfield, A., Eds.; John Wiley & Sons Inc.: Hoboken, NJ, 2006; pp
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* To
whom
correspondence
should
be
addressed.
E-mail:
(8) Davies, S. S.; Amarnath, V.; Roberts, L. J., II. Chem. Phys. Lipids 2004,
128, 85-99.
marina.carini@unimi.it. Phone: + 39-02-50319298. Fax: +39-02-50319359.
9174 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007
10.1021/ac7016184 CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/03/2007

ꢀ
cysteine],9 CML [N -(carboxymethyl)lysine],10 and GOLD (GO-
already established order of reactivity of the nucleophilic amino
acids (Cys . His > Lys) toward R,â-unsaturated aldehydes,22 as
demonstrated for a number of peptides, proteins, and enzymes
by mass spectrometry.23 Structural information on the nature of
the HNE modification of histidine side chain was first reported
by Uchida and Stadtman.24 Reduction of the aldehyde group of
the primary Michael addition product with sodium borohydride
converts it to the hydroxy derivative that is stable to strong acid
hydrolysis. This approach formed the basis of methods for the
identification and quantification of the HNE-histidine Michael
adduct of proteins by conventional amino acid analytical tech-
niques. Immunohistochemistry using monoclonal antibodies to
HNE-histidine conjugates has been widely applied to detect
HNE-histidine protein adducts in human astrocytomas,25 in the
ischemic rat heart after transplantation,26 in the target organ of a
ferric nitrilotriacetate-induced renal carcinogenesis model,27 in
ischemia/reperfused rat heart,28 and in serum of type 2 diabetic
patients.29
On the contrary, very few studies report free His modified by
RCS as a tool to confirm involvement of carbonyl stress in different
pathological conditions. Increased levels of the His-HNE adduct
have been found in the middle frontal gyrus of AD patients
compared with controls, but no details on the analytical method
used (LC-MS/MS) are reported.30 Other endogenous histidine-
containing dipeptides have been more recently recognized as
detoxifying agents against cytotoxic HNE and ACR: carnosine
Lys dimer).11,12 In particular, CML (formed by reaction of GO with
Lys residues) is now considered as a general marker of carbonylic
stress and long-term damage to proteins in aging, Alzheimer’s
disease (AD), atherosclerosis, and diabetes.13 Antisera for quan-
titative immunoassays of protein-bound ALEs in body fluids and
tissues, although widely used, are questionable, because they yield
only semiquantitative results. Hence, more specific and sensitive
analytical approaches, mainly based on LC, GC/MS, and LC-
MS/MS methodologies, have been developed for their quantitative
determination in proteins hydrolysates. Free CMC or CMC
released from proteins by hydrolysis can be easily determined
by reversed-phase HPLC with fluorescence detection after pre-
column derivatization with o-phthaldialdehyde.9 CML has been
estimated in modified skin collagen, plasma, or urines by reversed-
phase HPLC method with o-phthalaldehyde precolumn derivati-
zation14 or by isotope dilution, selected ion monitoring gas
chromatography/mass spectrometry (SIM-GC/MS).15,16 CML (in
free and protein-bound form after hydrolysis) is now determined
by isotope dilution LC-MS/MS.17 This method can be accurately
applied to all the biological matrices (plasma, tissues, and urine).
The mercapturic acid conjugate of 1,4-dihydroxynonane (DHN-
MA)18 is another well-established and highly stable biochemical
biomarker for lipid peroxidation and carbonylation. This adduct,
representing the main urinary end metabolite of HNE following
conjugation with GSH, has been shown to be a physiological
component of rat and human urine.19 DHN-MA is generally
determined by LC-MS/MS.20 More recently, an enzyme immu-
noassay has been developed and validated for its determination
in urines.21
(â-alanyl-
tidine, HCAR), and anserine (â-alanyl-
L
-histidine, CAR), homocarnosine (γ-amino-butyryl-his-
L
-1-methylhistidine, ANS).31-33
These compounds are widely distributed in vertebrates and
particularly abundant in excitable tissues such as nervous system
and skeletal muscles,34,35 only CAR and HCAR being normally
present in human tissues. In particular, the Michael adduct of
HNE with CAR (CAR-HNE) has been identified as a novel, early,
specific, and stable marker of lipid peroxidation in those biological
districts where CAR is specifically located, and an LC-MS/MS
Hence, the mainly used in vivo biomarkers for carbonylation
are RCS-modified Cys and Lys residues. On the contrary, little
attention has been devoted to covalently modified His as a possible
diagnostic tool. This is quite surprising, in view of the following
evidence: (a) the well-documented reactivity of His residues
toward several RCS (mainly HNE, ACR, and MDA);1-3 (b) the
(22) Doorn, J. A.; Petersen, D. R. Chem.sBiol. Interact. 2003, 143-144, 93-
100.
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W. Biochemistry 1995, 34, 3702-3709.
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305.
(24) Uchida, K.; Stadtman, E. R Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 4544-
4548.
(11) Wells-Knecht, K. J.; Brinkmann, E.; Thorpe, S. R.; Baynes, J. W. J. Org.
Chem. 1995, 60, 6246-6247.
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Soc., Perkin Trans. 1 1995, 1, 2817-2818.
(13) Girone`s, X.; Guimera`, A.; Cruz-Sa´nchez, C. Z.; Ortega, A.; Sasaki, N.; Makita,
Z.; Lafuente, J. V.; Kalaria, R.; Cruz-Sa´nchez, F. F. Free Radical Biol. Med.
2004, 36, 1241-1247.
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Busch, A. E.; Dingermann, T.; Somoza, V.; Baynes, J. W.; Huber, J.
Diabetologia 2005, 48, 1645-1653.
(25) Zarkovic, K.; Juric, G.; Waeg, G.; Kolenc, D.; Zarkovic, N. Biofactors 2005,
24, 33-40.
(26) Renner, A.; Sagstetter, M. R.; Harms, H.; Lange, V.; Go¨tz, M. E.; Elert, O.
J. Heart Lung Transplant. 2005, 24, 730-736.
(27) Ozeki, M.; Miyagawa-Hayashino, A.; Akatsuka, S.; Shirase, T.; Lee, W. H.;
Uchida, K.; Toyokuni, S. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci.
2005, 827, 119-126.
(28) Chen, J.; Henderson, G. I.; Freeman, G. L. J. Mol. Cell. Cardiol. 2001, 33,
1919-1927.
(15) Alderson, N. L.; Chachich, M. E.; Youssef, N. N.; Beatti, R. J.; Nachtigal,
M.; Thorpe, S. R.; Baynes, J. W. Kidney Int. 2003, 63, 2123-2133.
(16) Petrovicˇ, R.; Futas, J.; Chandoga, J.; Jakus, V. Biomed. Chromatogr. 2005,
19, 649-654.
(17) Teerlink, T.; Barto, R.; ten Brink, H. J.; Schalkwijk, C. G. Clin. Chem. 2004,
50, 1222-1228.
(29) Toyokuni, S.; Yamada, S.; Kashima, M.; Ihara, Y.; Yamada, Y.; Tanaka,
T.; Hiai, H.; Seino, Y.; Uchida, K. Antioxid. Redox Signaling 2000, 2, 681-
685.
(30) Cutler, R. G.; Kelly, J.; Storie, K.; Pedersen, W. A.; Tammara, A.; Hatanpaa,
K.; Troncoso, J. C.; Mattson, M. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
2070-2075.
(18) Peiro, G.; Alary, J.; Cravedi, J. P.; Rathahao, E.; Steghens, J. P.; Gue´raud, F.
Biofactors 2005, 24, 89-96.
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Biophys. Res. Commun. 2002, 298, 699-706.
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Chem. Res. Toxicol. 1998, 11, 130-135.
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Analytical Chemistry, Vol. 79, No. 23, December 1, 2007 9175

method for its quantification in biological matrices (rat skeletal
muscle) has been developed.36
system. NFPA (nonafluoropentanoic acid) was purchased from
Sigma-Aldrich (Milan, Italy). Carnosine (â-alanyl- -histidine) and
L
In view of these findings, the first aim of this work was to
develop an LC-MS/MS approach, based on the precursor ion
scanning mode, to detect all free and protein-bound His species
modified by RCS. This technique is a valuable tool for the rapid
confirmation of targeted compounds or for the nontargeted
detection of compounds bearing a common moiety. It has been
widely used in drug discovery and development37 and in vitro and
in vivo drug metabolism studies,38-40 although it has never been
applied to screen a biological matrix for the presence of covalently
modified peptides. The only application in this field has been
reported by Fenaille et al.,41 which used the precursor ion scanning
technique to identify peptides containing hexanal-modified Lys
residues within an unfractionated tryptic digest of hexanal-
modified apomyoglobin (in vitro studies).
We used the immonium ion of histidine (m/z 110) as a well-
known and specific fragment ion of His-containing peptides36 to
generate precursor ion spectra, with the final aim to identify in
urines from Zucker obese rats covalently modified His residues.
The Zucker fa/fa rat has been chosen as a nondiabetic animal
model characterized by obesity and hyperlipidemia, where ALEs
formation plays a key role in the development of renal and cardiac
dysfunction.15 The involvement of lipid peroxidation in renal injury
in obese Zucker rats has been recently demonstrated by the heavy
deposition of HNE adducts, using a specific rabbit polyclonal
antibody to HNE adducts.42
Furthermore, although a wide number of papers have been
published on determination of ALEs in biological fluids, none of
them tried to simultaneously quantitate the most important
biomarkers of carbonylic stress in urines from Zucker rats. In
order to fill this gap, and to define for the first time the urinary
profile of both conventional ALEs (DHN-MA) and the newly
identified adducts to histidine, the second aim of this work was
to develop an LC-MS/MS methodology in multiple reaction
monitoring (MRM) for their quantitation. This approach was done
with the final goal to use the obese Zucker rat as a pharmacologi-
cal tool to evaluate, through the determination of a series of highly
stable biochemical markers for carbonylation, the protective effects
of a long-term administration of newly developed RCS sequester-
ing agents.
the internal standard (IS) H-Tyr-His-OH were a generous gift
from Flamma S.p.A. (Chignolo d’Isola, Bergamo, Italy). HNE was
prepared from synthesized 4-hydroxy-non-2-enal diethylacetal as
previously described36,43 and quantitated by UV spectroscopy (λmax
224 nm; ꢀ 13.75 × 104 cm-1
M
-1). CAR-HNE and DHN-MA
adducts were synthesized according to the published methods.36,44
His-HNE was prepared as described for CAR-HNE, using
histidine instead of carnosine,36 and His-DHN by NaBH4 reduc-
tion of His-HNE.45 At the end of the incubation periods, sample
aliquots relative to CAR-HNE and His-HNE reaction mixtures
were directly analyzed by HPLC for determination of HNE
consumption, according to the method previously described,35 and
by ESI-MS (infusion) to confirm adduct formation. The final
concentrations of CAR-HNE, His-HNE, and His-DHN were
calculated by considering that CAR and His form a 1:1 molar ratio
adduct with HNE and by calculating the residual amount of HNE
(0.9% for CAR; 1.0% for His). His-HNA was prepared by treatment
of His-HNE with sulfamic acid and sodium chlorite as previously
described.46
LC-MS/MS Instrument and Conditions. The HPLC system
(Surveyor, ThermoFinnigan Italia, Milan, Italy), equipped with a
quaternary pump, a Surveyor UV-vis diode array programmable
detector 6000 LP, a vacuum degasser, a thermostatted column
compartment, and a Surveyor autosampler (200 vials capacity),
was used for solvent and sample delivery. Separations were
performed on a C12 Phenomenex Sinergy polar-RP column (150
mm × 2 mm i.d.; particle size 4 µm) (Chemtek Analytica, Anzola
Emilia, Italy) protected by a polar-RP guard column (4 mm × 2
mm i.d.; 4 µm) kept at 25 °C and equipped with an on-line sample
preconcentration C18 cartridge (Opti-lynx, El-Chimie SrL, Cinisello
Balsamo, Milan, Italy). Gradient elution was employed from 100%
water containing 5 mM nonafluoropentanoic acid (A) to 80%
acetonitrile (B) in 24 min at a flow rate of 0.2 mL/min (injection
volume 50 µL) followed by a 6 min isocratic elution. The
composition of the eluent was then restored to 100% A within 2
min, and the system was reequilibrated for 6 min. The samples
rack was maintained at 4 °C.
A TSQ Quantum triple-quadrupole mass spectrometer (Ther-
moFinnigan Italia, Milan, Italy) with electrospray ionization (ESI)
source was used for mass detection and analysis. Mass spectro-
metric analyses were performed in positive ion mode. ESI interface
parameters were set as follows: middle position; capillary tem-
perature 270 °C; spray voltage 4.0 kV. Nitrogen was used as
nebulizing gas at the following pressure: sheath gas 30 psi;
auxiliary gas 5 a.u. MS conditions and tuning were performed by
mixing through a T-connection the water-diluted stock solutions
of analytes (flow rate 10 µL/min), with the mobile phase
maintained at a flow rate of 0.2 mL/min: the intensities of the
[M + H]+ ions were monitored and adjusted to the maximum by
using the Quantum Tune Master software.
EXPERIMENTAL SECTION
Chemicals and Reagents. All chemicals and reagents were
of analytical grade and purchased from Sigma-Aldrich Chemical
Co. (Milan, Italy). HPLC-grade and analytical-grade organic
solvents were also purchased from Sigma-Aldrich (Milan, Italy).
HPLC-grade water was prepared with a Milli-Q water purification
(36) Orioli, M.; Aldini, G.; Beretta, G.; Maffei Facino, R.; Carini, M. J. Chromatogr.,
B: Anal. Technol. Biomed. Life Sci. 2005, 827, 109-118.
(37) Papac, D. I.; Shahrokh, Z. Pharm. Res. 2001, 18, 131-145.
(38) Zhang, J. Y.; Wang, Y.; Dudkowski, C.; Yang, D.; Chang, M.; Yuan, J.;
Paulson, S. K.; Breau, A. P. J. Mass Spectrom. 2000, 35, 1259-1270.
(39) Kostiainen, R.; Kotiaho, T.; Kuuranne, T.; Auriola, S. J. Mass Spectrom. 2003,
38, 357-372.
(43) Aldini, G.; Granata, P.; Orioli, M.; Santaniello, E.; M. Carini M. J. Mass
Spectrom. 2003, 38, 1160-1168.
(40) Liu, D. Q.; Hop, C. E. J. Pharm. Biomed. Anal. 2005, 37, 1-18.
(41) Fenaille, F.; Tabet, J. C.; Guy, P. A. Rapid Commun. Mass Spectrom. 2004,
18, 67-76.
(44) Ahmed, M. U.; Thorpe, S. R.; Baynes, J. W. J. Biol. Chem. 1986, 261, 4889-
4894.
(45) Alary, J.; Bravais, F.; Cravedi, J. P.; Debrauwer, L.; Rao, D.; Bories, G. Chem.
(42) Dominguez, J. H.; Wu, P.; Hawes, J. W.; Deeg, M.; Walsh, J.; Packer, S. C.;
Nagase, M.; Temm, C.; Goss, E.; Peterson, R. Kidney Int. 2006, 69, 1969-
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39, 1417-1428.
9176 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Spectra acquisition for qualitative analysis was based on a
survey precursor ion scan for m/z 110.2. The Q1 quadrupole was
scanned from m/z 50 to 400 in 1 s (scan time) with resolution of
0.70 m/z, and the precursor ions were fragmented in Q2 using
collision potentials from 20 to 40 V (20 V). Finally, Q3 was set to
transmit only ions at m/z 110.2 with resolution 0.70 m/z. This
scan mode was followed by an enhanced resolution experiment
for the ions of interest and then by MS2 acquisitions. MS2 spectra
were acquired using the optimized collision energy (35 V).
Ionization efficiency in the presence of the mobile phase
modifiers was monitored in MRM mode at 2.00 kV multiplier
voltage, by selecting for each analyte the MRM transitions of [M
+ H]+ precursor ions f product ions and optimizing the relative
collision energies by the Quantum Tune Master software. The
selected MRM transitions were also used for quantitative analysis.
The parameters influencing these transitions were optimized as
follows: argon gas pressure in the collision Q2, 1.5 mbar; peak
full width at half-maximum (fwhm), 0.70 m/z at Q1 and Q3; scan
width for all MRM channels, 1 m/z; scan rate (dwell time), 0.2
s/scan. Data processing was performed by the Xcalibur 2.0 version
software.
in samples (ultrafiltered and hydrolyzed) used for qualitative
analysis in precursor ion scanning mode.
Preparation of Standards and Quality Control Samples.
Stock solutions of 1 µmol/mL DHN-MA, His-DHN, His-HNE,
CAR-HNE, and the internal standard stock solution (IS; 0.1 µmol/
mL) were prepared in PBS and stored at 4 °C for 1 week. Stock
solutions were diluted further with PBS, and the solutions of each
analyte except His-HNE (separate working solutions) were mixed
together to obtain working solutions. The 1 nmol/mL working
solutions were analyzed by LC-MS/MS to ensure that the
concentrations of the original solutions were within the limits of
the maximum established error (e3%). Urine samples from 4 week
old LN rats were used as blank urine to prepare positive control
urine samples. This urine was checked by the applied analytical
procedure to ensure it did not contain the selected ALEs above
the limit of detection. Calibration samples for adducts in free form
were prepared by spiking blank urine samples with each working
solution to provide the following final concentrations: 0.075, 0.10,
0.5, 1.0, 2.5, 5.0, 10.0 nmol/mL for DHN-MA, 0.01, 0.05, 0.10,
0.50, 1.00, 2.50, 5.00 nmol/mL for His-DHN and CAR-HNE. IS
was added at the concentration of 1 nmol/mL.
The SEQUEST database (Bioworks version 3.2; Thermo
Electron San Jose, CA) was used for peptide peak assignments.
Animals, Sample Collection, and Preparation. Male Zucker
obese (fa/fa) rats (ZO n ) 5; body mass 696.1 g ( 13.8) and lean
(LN) littermates (Fa/fa) (n ) 5; body mass 454.5 ( 22.8 g)
were purchased at 14 weeks of age from Charles River Italia spa
(Calco, Como, Italy). The animals were housed individually in
metabolic cages with a light-dark cycle of 12 h each and with
free access to food and water. Additional LN rats (n ) 5; body
mass 82.4 ( 4.7 g) were purchased at 4 weeks of age and used
as controls. ZO rats develop with age a significant proteinuria,
mainly due to albumin leakage.15 The animals were maintained
in compliance with the policy on animal care expressed in
National Research Council guidelines (NRC 1985). Urines from
each animal were collected daily at alternate days for 3 days
starting from the 24th week of age. One milliliter of a 360 mM
BHT ethanolic solution was added to the urine collection
tubes that were placed in a unit maintained at 0 °C during
collection. The total volume of urine collected in each day was
recorded, and 5 mL aliquots were frozen and stored at -80 °C
until analysis.
Renal disease was assessed by measurements of 24 h urinary
albumin and total urinary protein, using the Sentinel 17600 and
17620 commercial kits (Sentinel, Milan, Italy). Urine samples were
centrifuged at 18 000 rpm for 10 min; 200 µL of the supernatants
was added with 10 µL of internal standard (1 µM dissolved in
mobile phase) and immediately ultrafiltered by centrifugation at
15 000 rpm in a Microfuge through a 3 kDa molecular mass cutoff
membrane (YM-3000, Millipore, Milan, Italy). The filtrate was
transferred to the autosampler vial insert (50 µL injected) for
quantitative analysis. For determination of protein-bound adducts
(His-HNE), urine aliquots corresponding to 3.0 mg of protein
were subjected to hydrolysis (6 N HCl for 20 h at 110 °C in a
heating block) as described by Teerlink et al.17 The residue of
the final digest was taken up in 100 µL of mobile phase containing
the internal standard, filtered through 0.2 µm nylon filters, and
the filtrate was injected (80 µL). The internal standard was omitted
Quality control (QC) samples at four concentrations, 0.075, 0.5,
2.5, 10.0 nmol/mL for DHN-MA, 0.01, 0.10, 1.00, 5.00 nmol/mL
for His-DHN and CAR-HNE, were prepared in the same way,
using blank urine spiked with independently prepared stock
standard solutions. Each calibration and QC sample was processed
as described in sample preparation.
Calibration and QC samples for determination of protein-bound
His-HNE were prepared by spiking protein hydrolysates (3 mg
of BSA) with the His-HNE working solutions (0.01, 0.05, 0.10,
0.50, 1.00, 2.50 e 5.00 nmol/mL).
Method Validation. Calibration standards were prepared and
analyzed in duplicate in three independent runs. The calibration
curves were constructed by weighted (1/x2) least-square linear
regression analysis of the peak area ratios of each analyte to the
IS against nominal analyte concentration. The lower limit of
quantification (LLOQ) was determined as the lowest concentration
with values for precision and accuracy within (20% and a signal-
to-noise (S/N) ratio of the peak areas g10.
Intra- and interday precisions and accuracies of the method
were determined by assaying five replicates of each of the QC
samples (four levels in the low, intermediate, and high concentra-
tion range) in three separate analytical runs. Precision and
accuracy were determined by calculating the coefficient of varia-
tion (CV%) and the relative error (RE%).
The absolute recovery of the free analytes after ultrafiltration
was determined by comparing the mass spectrometric response
of ultrafiltered urine standards to that of ultrafiltered urine blanks
spiked with a corresponding set of concentrations (containing 100%
of the theoretical concentration) over the entire calibration range.
The overall absolute recovery was measured as the ratio of the
slopes of the two calibration curves and expressed as percentage.
The specificity of the assay was evaluated by comparison of
LC-MS/MS chromatograms of the analytes at the LLOQ to those
of blank urine samples in triplicate.
The stability of all the analytes in stock solutions (4 weeks at
4 °C) and in processed samples, including the resident time (24
h) in the autosampler (4 °C), were determined in triplicate. The
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007 9177

mean values of the triplicate samples were compared with those
of the initial condition or those of freshly prepared QC samples,
and the percentage deviation was calculated. In addition, for His-
DHN and His-HNE, three replicates of urine were used for
stability studies at two concentration levels (0.05 and 2.50 nmol/
mL) in different conditions: at laboratory room temperature
(25 °C), storage temperature (-20 °C), and after freeze-thaw
cycles. The stability of His-HNE under hydrolytic conditions was
established only indirectly, by adding known amounts of His-
HNE (10 nmol/mL) to a BSA solution (3 mg in 100 µL of
ammonium acetate buffer pH 5.5). The samples, after protein
precipitation, were submitted to acidic hydrolysis and treated as
described above. Percentage recoveries were calculated by
comparing the mass spectrometric response of BSA spiked with
His-HNE hydrolyzed samples to that of hydrolyzed samples
spiked with the corresponding concentration (containing 100% of
the theoretical concentration).
mation Figure S-1) indicate that NFPA significantly suppressed,
in respect to HCOOH, the extracted ion current for all the analytes
(monitored in MRM mode), except for DHN-MA, showing a poor
ionization efficiency in all experimental conditions. Anyway, this
suppressing effect was less than that induced by HFBA: a 40%
and a 15% increase was in fact observed for DHN-MA and CAR-
HNE, respectively. Overall, these results led us to replace HFBA
with NFPA as the ion-pairing agent.
Infusion experiments were performed in order to obtain the
full mass spectra of DHN-MA, CAR-HNE, and IS, showing the
[M + H]+ at m/z 322, 383, and 319, respectively, and to establish
the optimal fragmentation reactions for MRM.
Under the optimized LC and MS conditions, no interferences
from endogenous compounds were found in 4 week old LN urines,
and the retention times for the analytes were 24.80 min (DHN-
MA), 27.84 min (CAR-HNE), and 23.16 min (IS). Preliminary
LC-MS/MS profiles, obtained by spiking LN urines with known
amounts of the analytes (data not shown), indicate that they can
be selectively detected and quantitated, even if they are simulta-
neously present, by the following precursor-product ion combina-
tions:
RESULTS AND DISCUSSION
Optimization of LC and MS/MS Conditions. In previous
studies36 we have developed a sensitive and selective method for
the simultaneous determination in rat skeletal muscle of the
Michael adducts between HNE and glutathione (GS-HNE) and
the endogenous histidine-containing dipeptides carnosine and
anserine (CAR-HNE, ANS-HNE), using a gradient elution with
HFBA as ion-pairing agent and H-Tyr-His-OH as internal
standard for quantitative analysis. H-Tyr-His-OH was maintained
as IS as an alternative approach to isotope-labeled analytes because
they are not readily and commercially available. In addition, it
was found to fulfill some basic criteria: it is not an endogenous
dipeptide, it is stable and matches the chromatographic retention,
recovery, matrix effects and ionization properties with all the
analytes.36,45,47 The same chromatographic conditions were not
suited for separation of IS, CAR-HNE, and DHN-MA in the
presence of a C18 preconcentration cartridge (used to increase
sensitivity), since IS coelutes near the void volume. In addition,
the UV-DAD profile of blank urines (data not shown) indicates
that the bulk of endogenous interfering material eluted within 9
and 11 min and coeluted with all the other analytes. Because these
conditions caused loss of sensitivity by reduced ionization as a
result of ion suppression, the gradient elution was suitably
modified, by increasing the polarity of solvent A, the chromato-
graphic run, and by replacing HFBA (0.1%) with NFPA (5 mM;
0.08%),17 to increase retention on the reversed phase of very
hydrophilic compounds (IS) and to shift beyond the matrix
components the more lipophilic analytes (DHN-MA, CAR-
HNE).
m/z 322.2 f 130.1 + 164.1
m/z 383.1 f 110.1 + 266.2
m/z 319.2 f 156.5 + 301.6
(collision energy 15 eV)
DHN-MA
(collision energy 40 eV)
CAR-HNE
(collision energy 25 eV)
H-Tyr-His-OH (IS)
Precursor Ion Monitoring for Qualitative Analysis of
Zucker Rat Urines. The first step was to apply to urine from ZO
rats the precursor ion scanning technique, a valuable tool for the
rapid confirmation of targeted compounds or for the nontargeted
detection of compounds bearing a common moiety. We used the
immonium ion of histidine (m/z 110) as a well-known and specific
fragment ion of His-containing peptides36 to generate precursor
ion spectra in order to screen ZO urines for the presence of
covalently modified His residues, both in free (analysis of
ultrafiltrates) and protein-bound (analysis of protein hydrolysates)
form. The precursor ion mass spectra were evaluated in order to
identify the m/z of the precursor ions. Figure 1 reports the
bidimensional plot (m/z values against retention times) of urine
ultrafiltrates from 4 week old LN (panel a), 6 month old LN (panel
b), and ZO (panel c) rats. This latter only contains three distinct
[M + H]+ signals at m/z 328, 314, and 383, with retention times
of 16.71 min, 25.23, and 27.84 min, respectively, two of them being
detectable also in urines from 6 month old LN rats. Retention
times and molecular weights were confirmed by the extracted
ion currents corresponding to the identified precursor ions
(Figure 2).
NFPA was already successfully employed as ion-pairing agent
for analysis of CML in human plasma protein by tandem mass
spectrometry.17 Hence, we studied the effect of NFPA on the
ionization efficiency of all the other adducts and compared it to
that of HFBA, previously used for CAR-HNE,36 and to that of
HCOOH, used for DHN-MA.48,49 The results (Supporting Infor-
Each [M + H]+ ion of interest was then selected for MS2
acquisitions obtained by applying the optimized collision energy
for each species, in order to obtain structural characterization
(Figure 3). In detail, the MS2 of the [M + H]+ at m/z 383 (Figure
3a) contains two main product ions at m/z 266.2 and 110.2. The
first is diagnostic for HNE-modified His residues (immonium
ion),36,50,51 and the second (His immonium ion) is due to a retro-
(47) Liang, Y.; Xie, L.; Liu, X. D.; Lu, T.; Wang, G. J.; Hu, Y. Z. J. Pharm. Biomed.
Anal. 2005, 39, 1031-1035.
(48) Rathahao, E.; Peiro, G.; Martins, N.; Alary, J.; Gue´raud, F.; Debrauwer, L.
Anal. Bioanal. Chem. 2005, 381, 1532-1539.
(49) Peiro, G.; Alary, J.; Cravedi, J. P.; Rathahao, E.; Steghens, J. P.; Gue´raud, F.
Biofactors 2005, 24, 89-96.
9178 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Figure 3. LC-MS/MS analysis of urine ultrafiltrates from ZO rats:
MS2 spectra of the precursor ions at (a) m/z 383.1; (b) m/z 314.7; (c)
m/z 328.2.
Figure 1. LC-ESI-MS/MS analysis of urine ultrafiltrates (free
adducts). Data were acquired in precursor ion scanning mode
(product ion m/z 110) and reported as two-dimensional plots (m/z
values against retention times): (a) 4 week old LN; (b) 6 month old
LN; (c) ZO rats.
227, corresponding to the molecular mass of the peptide moiety
(carnosine). All these data (MW, retention time, and MS2) allowed
assigning the peak with retention time of 27.8 min to the structure
of CAR-HNE, previously identified in rat skeletal muscle as a
marker of carbonylation36 but never before identified in urines.
The same approach was applied to the other two peaks
recognized by the precursor ion scan experiments. The MS2
spectrum of the [M + H]+ species at m/z 314 (Figure 3b) is
dominated, besides the common ion at m/z 110.2, by a main
product ion at m/z 268.2 (+2 Da with respect to CAR-HNE),
corresponding to the immonium ion of 1,4-dihydroxynonane-
modified His residue. Hence, this adduct was preliminarily
identified as His-1,4-dihydroxynonane (His-DHN), arising by
HNE Michael adduction to His followed by metabolic conversion
of the aldehyde into the alcohol function.
Finally, the MS2 spectrum relative to the [M + H]+ species at
m/z 328.2 (Figure 3c) is characterized by the product ion at m/z
282.1 [M + H - HCOOH]+, the typical immonium ion of HNE-
modified His residues increased by 16 Da (m/z 266.2 in CAR-
HNE adduct). This, indicating aldehyde metabolic oxidation to
the corresponding carboxylic derivative, allowed assigning to the
[M + H]+ adduct at m/z 328.2 the structure of His-4-hydrox-
ynonanoic acid (His-HNA).
Figure 2. Extracted ion chromatograms (SICs) of the identified
precursor ions in urines ultrafiltrates from ZO rats: (a) m/z 328.2; (b)
m/z 314.7; (c) m/z 383.1.
Protein hydrolysates from LN and ZO urine were analyzed by
the same methodology to recognize in the biological matrix
Michael reaction from the ion at m/z 266. The same retro-Michael
reaction occurs also on the [M + H]+ at m/z 383 (neutral loss of
the HNE moiety, 156 Da) and formation of the product ion at m/z
(50) Bolgar, M. S.; Gaskell, S. J. Anal. Chem. 1996, 68, 2325-2330.
(51) Fenaille, F.; Guy, P. A.; Tabet, J. C. J. Am. Soc. Mass Spectrom. 2003, 14,
215-226.
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007 9179

Figure 5. LC-MS/MS analysis of protein hydrolysates from ZO
rats: MS2 spectra of the precursor ions at m/z 269.4 (a) and m/z
312.2 (b).
which was confirmed by comparison with the retention time of
the authentic standard treated under the same hydrolytic condi-
tions. These results seem to indicate protein carnosinylation, a
hypothesis already postulated by Hipkiss et al. who, using model
systems in vitro, demonstrated that carnosine, by reacting with
protein carbonyls with generation of “carnosinylated“ polypeptides,
may intracellularly suppress the deleterious effects of protein
carbonyls.52 It has been speculated that carnosinylation may
facilitate inactivation/disposal of carbonylated proteins through
the formation of inert lipofuscin, proteolysis via the proteasome
system, and exocytosis following interaction with receptors.53,54
Because this proposal pointing to a hitherto unrecognized mech-
anism by which cells normally defend themselves against protein
carbonyls has never been supported by experimental data in vivo,
our preliminary results, if confirmed on a larger sample of animals
and/or other experimental models, may represent the first
evidence for protein carnosinylation.
The MS2 spectrum of the [M + H]+ species at m/z 312.2
(Figure 5b), 2 Da over that of the His-DHN adduct (dehydro-
genation), contains the diagnostic product ion at m/z 266,
corresponding, as previously described for CAR-HNE, to the
immonium ion of HNE-modified His. Another significant product
ion, although of lower relative abundance, is that at m/z 156 [His
+ H]+, arising from the [M + H]+ by neutral loss of the HNE
moiety (typical retro-Michael reaction). All this information
indicates for the precursor ion at m/z 312.2 the structure of the
His-HNE Michael adduct.
Figure 4. Protein-bound covalently modified His residues: LC-
ESI-MS/MS analysis of protein hydrolysates from lean and Zucker
obese rats. Panels a and b report the total ion current (TIC) profiles
of the precursor ion scanning of m/z 110 in LN and ZO urine,
respectively; panels c and d show the SICs of the identified precursor
ions in protein hydrolysates from ZO rats at m/z 269.4 (#) and 312.2
(/).
protein-bound covalently modified His residues. Figure 4a (LN)
and Figure 4b (ZO) report the total ion current (TIC) profiles of
the precursor ions which give a product ion of m/z 110. The
abundant peak in lean (retention time 21.24 min; [M + H]+ at
m/z 255), which almost disappeared in Zucker urine, was not
considered as a potential biomarker of carbonylation, because its
MS2 spectrum (data not shown) indicates Val-His (Bioworks
analysis). Two chromatographic peaks were significantly elevated
in ZO rats (retention times 23.41 and 26.12 min). The correspond-
ing mass spectra indicate the m/z of the precursor ions at 269
and 312, respectively, further confirmed by the extracted ion traces
reported in Figure 4, parts c and d.
The MS2 spectrum of the [M + H]+ at m/z 269.4 contains
several significant product ions only at 25 eV collision energy
(Figure 5a): the ion at m/z 227.3 [M + H - CH2dCO]+ typical
of N-acetylated derivatives, and the ions at m/z 209.2/210.2, 156.0,
and 110.2, which are characteristic of the fragmentation pattern
of carnosine.36 Hence, MW and fragmentation pattern indicate for
the peak eluting at 23.41 min the structure of N-acetylcarnosine,
Identity of all the adducts (free and protein-bound) was finally
supported by chemical synthesis and by the match of the
(52) Hipkiss, A. R.; Brownson, C.; Bertani, M. F.; Ruiz, E.; Ferro, A. Ann. N.Y.
Acad. Sci. 2002, 959, 285-294.
(53) Hipkiss, A. R.; Brownson, C.; Carrier, M. J. Biogerontology 2000, 1, 217-
223.
(54) Quinn, P. J.; Boldyrev, A. A. Biochemistry (Moscow, Russ. Fed.) 2000, 65,
771-778.
9180 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Standard curves for the analytes constructed on different
working days showed good linearity over the entire calibration
ranges (0.075-10 nmol/mL for DHN-MA; 0.01-5.00 nmol/mL
for all the other analytes), with coefficients of correlation (r2)
greater than 0.998. The equations for the calibration lines were
y ) 0.3808((0.005480)x - 0.05222((0.02384)
DHN-MA
y ) 2.963((0.01589)x - 0.02089((0.03204)
His-DHN
y ) 1.433((0.02425)x + 0.00367((0.00452)
His-HNE
y ) 1.435((0.02115)x - 0.01559((0.04265)
CAR-HNE
The LLOQs were 0.075 nmol/mL for DHN-MA, 0.01 nmol/mL
for His-DHN, His-HNE, and CAR-HNE; the lower LOQ found
for DHN-MA with respect to that described by Rathahao et al.48
may be explained on the basis of the decreased ionization
efficiency induced by NFPA with respect to HCOOH.
Representative MRMs traces relative to all the adducts and IS
(1 nmol/mL) obtained from blank urine spiked with LLOQ
concentrations (Figure 6) indicate that they can be selectively
detected and quantitated, even if they are simultaneously present,
by the above-reported precursor-product ion combinations. The
specificity of the method was demonstrated by the absence of
interfering peaks in the mass and time ranges for the ALEs and
IS in individual urine samples from five animals (triplicate injection;
free analytes) or in reconstituted protein matrix (protein-bound
His-HNE).
The intra- and interassay precision and accuracy of the method
were determined on QC samples prepared separately from
calibration standards, by analyzing five replicates at four concen-
tration levels, and the data are reported in Supporting Information
Tables S-1 and S-2. The intraday precision (CV%) was less than
3.98% (e8.72% at the LLOQs), and accuracy ranged from -4.40%
to +11.00% of nominal concentrations (within (16% at the LLOQs);
the interday CV values were less than 3.84% (e9.22% at the
LLOQs), and accuracy was in the range of -3.79% to +6.51%
(within (10.25% at the LLOQs).
The extraction recoveries for free adducts were determined
by comparing the peak area ratio of ultrafiltered urine standards
at three concentration levels (low, intermediate, and high) to those
of post-ultrafiltered blank urines spiked with the corresponding
concentrations. The mean extraction recovery was satisfactory,
being 96.5%, 92.7%, and 101.5% for DHN-MA, His-DHN, and
CAR-HNE, respectively. Recovery of His-HNE after the hydro-
lytic procedure was only 55%, and studies are in progress to
optimize this step.
Figure 6. LC-MS/MS profiles (MRM traces) of blank (bold lines)
and spiked (thin lines) urine with (a) IS (1 nmol/mL) and the analytes
at LLOQs: (b) DHN-MA (0.075 nmol/mL), (c) His-DHN, (d) His-
HNE, and (e) CAR-HNE (0.01 nmol/mL).
chromatographic and mass spectrometric properties. Presently,
the His-HNA adduct is available in minimal amount (due to the
low yields of reaction), just enough for structure confirmation,
but not sufficient to develop and validate a quantitative method.
Hence, further infusion experiments were performed to evalu-
ate the ionization efficiency of His-DHN and His-HNE (results
in Supporting Information Figure S-1) and to optimize their
fragmentation reactions for MRM analysis. The following precur-
sor-product ion combinations were used for quantitative analysis:
m/z 314.7 f 268.2 + 110.1
m/z 312.2 f 110.1 + 156.0
(collision energy 26 eV)
His-DHN
(collision energy 30 eV)
His-HNE
Method Validation. The LC-MS/MS approach was first
applied to urine from 1 month old LN rats to ensure their use as
a blank matrix for calibration: none of the analytes were detected
in the MRM mode (bold lines in Figure 6). A full validation
procedure (specificity, linearity, LOD, LOQ, intra- and interday
precision and accuracy, recovery, and stability) was then per-
formed on urine from young LN rats (free adducts) or on a
reconstituted protein matrix (protein-bound adducts).
The high stability of DHN-MA and CAR-HNE in aqueous
solutions, urines, or tissue extracts at room and lower tempera-
tures has already been widely documented.36,49 We therefore
checked their stability in QC samples and in the spiked urine
ultrafiltrates in the experimental conditions used. The stability was
guaranteed for at least 24 h at 4 °C, and no significant differences
(t test) were found between freshly prepared urine ultrafiltrates
and ultrafiltrates prepared from urine stored in liquid N2 (data
Analytical Chemistry, Vol. 79, No. 23, December 1, 2007 9181

vivo, has never been described in ZO rats but substantially
confirms previous findings in different experimental models and
conditions.48,49
Also the urinary levels of protein-bound adducts (His-HNE)
were significantly elevated in ZO rats, to reach values (when
expressed as total daily excretion tacking into account the protein
urinary content) greater than 2 µmol.
Table 1. Renal Function and Urinary Levels of ALEs in
Lean and Zucker Obese Ratsa
lean
Zucker
urinary volume (mL/24 h)
14.5 ( 1.1
20.9 ( 2.4c
total protein
mg/mL
mg/24 h
6.45 ( 1.20
93.52 ( 17.4
30.7 ( 6.0c
641.6 ( 125.4c
albumin
mg/mL
mg/24 h
CONCLUSIONS
2.75 ( 1.4
27.2 ( 2.8c
39.9 ( 20.3
568.5 ( 58.5c
The LC-MS/MS approach developed in the first part of this
study, based on the precursor ion scanning technique, was applied
for the first time for the rapid detection and identification in
biological matrices of new biomarkers of carbonylation in obese
Zucker rats, an experimental model where carbonyl stress plays
a key role in the development of renal and cardiac dysfunction.15
Our interest was focused to recognize covalent modifications of
His and His-containing peptides by RCS (especially in free form),
in view of the lack of such information. Although His-HNE
protein adducts have been immunohistochemically detected in
several biological systems, experimental animal models and in
humans,25-29 no studies report free His modified by RCS as a tool
to detect involvement of carbonyl stress in different pathological
conditions. Our approach allowed identifying for the first time in
urine from ZO rats two novel adducts, both originating from the
conjugation of HNE to His, which are excreted in free form only:
His-DHN, arising from the subsequent reduction of the aldehyde
by a member of aldo-keto reductase superfamily, and His-HNA,
arising from the conjugation of HNE followed by the oxidation of
the aldehyde by aldehyde dehydrogenase.56 No free His-HNE
was found in urines, but it was detected and quantified only in
protein hydrolysates. Very likely albumin, which accounts for more
than 90% of total excreted proteins, is the target protein for HNE.
This assumption is supported by the already demonstrated
reactivity of some His residues on human serum albumin (HSA),
namely, His242 (located in a fatty acid and drug binding cavity in
HSA subdomain IIa), according to Szapacs et al.,57 and His146,
according to Aldini et al.58 Studies are in progress to identify His
residues modified by HNE in albumin isolated from ZO rat urine,
using the LC-MS/MS approach recently developed to character-
ize the covalent modifications of HSA by HNE.58
Presently, we do not know (for the lack of a suitable standard)
the stability of His-HNE bound to proteins during the hydrolytic
step, but the high levels found seem to indicate it to be stable
enough. This is substantiated by the fact that HNE has been
shown to react with His to form a stable Michael addition-type
adduct in the cyclic hemiacetal structure, which is stabilized
toward retro-Michael reaction in acidic conditions. Stability of the
adduct, readily isolable, is due to the poor leaving group ability
of imidazole.55 Even considering the 50% loss during the experi-
mental procedure (determined only indirectly, working on His-
HNE added to a reconstituted protein mixture), the results of the
quantitative analysis indicate a massive formation of HNE-protein
adducts also in obese animals.
DHN-MA
nmol/mL
nmol/24 h
1.116 ( 0.058
16.182 ( 0.841
2.868 ( 0.174c
59.941 ( 3.637c
His-DHN
nmol/mL
nmol/24 h
0.061 ( 0.005
0.884 ( 0.073
0.184 ( 0.015c
3.846 ( 0.314c
CAR-HNE
nmol/mL
nmol/24 h
0.053 ( 0.008
0.768 ( 0.116
0.097 ( 0.008b
2.027 ( 0.167c
His-HNE (protein-bound)
nmol/mg protein
nmol/24 h
0.112 ( 0.009
10.474 ( 0. 842
3.160 ( 0.628b
2027.4 ( 402.9c
a Urinary levels were determined in every 24 h urine fraction (n )
3) of five animals. Values are the mean ( SD relative to five animals.
b p < 0.005 vs lean (t test). c p < 0.001 vs lean (t test).
not shown). Although His-DHN has been shown to be highly
stable under conditions required to release HNE-histidine
Michael adduct from proteins (strong acid hydrolysis),24,55 no
stability data in urines are available from the literature, or for His-
HNE under the same conditions. Stability data for His-HNE and
His-DHN, summarized in Supporting Information Table S-3,
indicated that the analytes was stable for at least the length of
time under different conditions listed, all the values ranging from
92.54% to 102.01% of the initial value, except for the second freeze-
thaw cycle, where a 21-28% decrease was observed for both the
analytes. Stock solutions, when stored in PBS at a nominal
temperature of 4 °C, were stable for at least 4 weeks.
Renal Function and Profiling of ALEs in Zucker Rats. Total
urinary protein and albumin were measured to evaluate renal
function. Urinary volumes were 14.5 ( 1.1 mL/24 h and 20.9 (
2.4 mL/24 h, respectively, in LN and ZO (p < 0.001 between
groups). A 10-fold increase in urinary albumin was observed in
ZO versus LN rats, which was paralleled by a similar increase in
total urinary protein (Table 1).
The HPLC-MS/MS method was then applied to profile the
ALEs content in rat urines. The results, summarized in Table 1,
indicate that all the adducts found in free form can be determined
in lean animals. As reasonably expected, DHN-MA, the main
indicator of HNE formation in vivo, represents the most abundant
excretion product. When the data are expressed as nmol/mL
urine, a significant increase (2-3-fold; p < 0.005 and p < 0.001)
in the urinary excretion of all the adducts was observed in Zucker
rats. If we consider the urinary volumes and the total daily
excretion, these increments become much more consistent,
ranging from 2- to 4-fold increase for all the adducts. The pattern
of DHN-MA excretion, the main indicator of HNE formation in
(56) Alary, J.; Fernandez, Y.; Debrauwer, L.; Perdu, E.; Gueraud, F. Chem. Res.
Toxicol. 2003, 16, 320-327.
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2006, 45, 10521-10528.
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2003, 278, 5044-5051.
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Facino, R.; Carini, M. J. Mass Spectrom. 2006, 41, 1149-1161.
9182 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Several studies on HNE metabolism in vitro (using rat hepatic
subcellular fractions or hepatocytes) and in vivo demonstrated
that HNE undergo phase I metabolism (reduction/oxidation of
the aldehydes), with formation of 1,4-dihydroxy-trans-nonene
(DHN) and 4-hydroxy-trans-2-nonenoic acid (HNA), and phase II
conjugation (GSH) and subsequent mercapturic acid formation.
This latter already has been recognized for a long time as the
mostefficientdetoxificationsystemtowardelectrophiliccompounds,45,56,59-63
DHN-MA being the most important excretion product. The high
urinary levels of DHN-MA found in LN and ZO rats confirm that
the GSH conjugative pathway plays a key role also in this animal
model.
The results of this study unequivocally indicate that HNE, in
addition to GSH-dependent metabolism, undergoes histidine-
dependent metabolism, following the same detoxification pathways
involving reductive/oxidative metabolic conversion. This in turn
implies that His-HNE, as HNE itself or its conjugation product
with GSH, is a good substrate for both dehydrogenases and
reductases.
The same MS/MS approach in precursor ion mode allowed
also identifying for the first time in urine the Michael adduct
between HNE and the endogenous dipeptide carnosine. This
confirms our previous in vitro studies performed on rat skeletal
muscle,36 i.e., that CAR-HNE is a stable and reliable biomarker
of lipid peroxidation and carbonylation in vivo. Unlike His-HNE,
CAR-HNE does not undergo further metabolism: no CAR-DHN
or CAR-HNA was in fact detected in LN or ZO rat urines, thus
to indicate that CAR-HNE is a poor substrate for aldehyde
dehydrogenases/reductases.
In this context, it must be stressed that this new histidine-
dependent detoxification pathway of HNE could be particularly
relevant in those pathological conditions (included those induced
by xenobiotics) involving a massive depletion of the endogenous
pool of GSH, which represents the first line of defense toward
HNE. This assumption has been already demonstrated in HNE-
spiked rat skeletal muscle.36 By LC-MS/MS monitoring of GSH-
HNE, CAR-HNE, and ANS-HNE adducts, conjugation with GSH
was confirmed to play a key role in HNE detoxification. Coincu-
bation with the alkylating agent N-ethylmaleimide (to mimic an
abrupt decay in GSH pool) resulted in a drastic fall of GSH-HNE
levels, with a concomitant significant elevation in the levels of both
CAR-HNE and ANS-HNE. Hence, inhibition of the thiol-
dependent pathway shifts HNE detoxification toward the histidine-
dependent pathway.
stress and inadequate antioxidant defenses in humans has been
accumulating over the past few years, and oxidative stress
biomarkers have been found to be elevated in several tissues,
including skeletal muscle, plasma, and erythrocytes.65 The occur-
rence of the alternative mechanism of detoxification of unsaturated
aldehyde via His residues in obese human subjects remains,
however, to be established.
Also the LC-MS/MS method developed in the second phase
of this study for the quantitative determination of HNE adducts
to His residues is novel since it permits, in a single chromato-
graphic run, the simultaneous determination of adducts that,
although structurally unrelated, have lipid peroxidation as a
common origin. Our method was not optimized to discriminate
between diastereomers (it is well-known, for example, that His-
HNE adduct, possessing three chiral centers in the cyclic
hemiacetal structure, gives rise to four diastereomers),55 because
the characterization of the possible configurational isomers of each
adduct and the knowledge of their relative amounts in vivo was
beyond the aim of this work. The method has been validated not
only for the newly identified His adducts but also for DHN-MA,
the most widely used biomarker of carbonyl stress in vivo. DHN-
MA, a physiological component of rat and human urine, is to be
considered as the main metabolic end product of HNE, and this
is confirmed by the high urinary levels found in both LN and ZO
rats, never reported before.
From an analytical point of view, the LC-MS/MS method
requires minimal sample manipulation, including a very simple
ultrafiltration step without further purification. This guarantees
high recovery rates for analytes bearing different chemical
structures and polarity, as well as high reproducibility, and
compensates the relatively long chromatographic run. Highly
satisfactory detection limits have been obtained for all the HNE
adducts to His (0.01 nmol/mL). By contrast, the LOQ for DHN-
MA was found to be slightly greater than that recently reported
by Rathahao et al.48 and Peiro et al.49 using an ion trap instead of
a triple-quadrupole MS system (performing quantification by MS3
acquisitions), and formic acid instead of NFPA as modifier, the
main factor responsible for the decreased ionization efficiency.
Also this apparent drawback is compensated by the high excretion
levels of DHN-MA and by the suitability of the method for the
simultaneous determination of more than one analyte.
From a biological point of view, it is important to stress that
this analytical method, specifically developed to profile in urines
His residues modified by RCS, allowed reporting for the first time
the excretion profile of some new ALEs (and DHN-MA) in ZO
rats. In previous studies carried out on this animal model, only
few markers of oxidative stress in plasma (lipid hydroperoxides
and 8-epi-PGF2R)66 and carbonyl stress in renal tissue (HNE
protein adducts, using a specific rabbit polyclonal antibody to HNE
adducts) have been reported.67
The decline of cellular thiol reserve, as a consequence of the
increased oxidative stress, has been shown to occur also in obese
Zucker rats, where a 40% decrease in hepatic GSH levels in respect
to the control lean animals was found.64 This could explain the
higher levels of HNE adducts to His and His-peptides in obese
animals than in the controls. Evidence of obesity-induced oxidative
Our results confirm HNE adduction to proteins in Zucker rats
and should represent a useful tool to better understand the
biochemical role of the newly identified biomarkers in different
physiopathological conditions.
(59) Esterbauer, H.; Zollner, H.; Lang, J. Biochem. J. 1985, 228, 363-373.
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Vermeulen, N. P. Xenobiotica 1996, 26, 1087-1100.
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Pharmacol. Physiol. 2004, 31, 506-511.
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Nagase, M.; Temm, C.; Goss, E.; Peterson, R. Kidney Int. 2006, 69, 1969-
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Analytical Chemistry, Vol. 79, No. 23, December 1, 2007 9183

Moreover, the results of this study show that ALEs excretion,
being negligible in young LN rats, becomes evident in adult LN
animals and significantly increased in the pathological model (ZO
rats). This from one side confirms, working on a simple biological
matrix (noninvasive withdrawal and easy to handle), that ALEs
formation takes place in physiological conditions and is strictly
associated with aging.2,68 On the other hand, these results clearly
indicate that the ZO rat represents an animal model where
carbonyl stress plays a key role in the development of long-term
complications of the metabolic syndrome. This makes the ZO rat
a highly suitable model for testing in vivo the carbonyl quenching
ability of newly developed RCS sequestering agents, by measuring
the urinary levels of the highly stable biomarkers considered in
this study.
ACKNOWLEDGMENT
This study was supported by COFIN2004 (Cofinanziamento
Programma Nazionale 2004, Ministero dell’Istruzione, dell’Universita`
e della Ricerca) and FIRST 2006-2007 (Fondo Interno Ricerca
Scientifica e Tecnologica, University of Milan).
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review July 31, 2007. Accepted September
27, 2007.
(68) Hamelin, M.; Borot-Laloi, C.; Friguet, B.; Bakala, H. Arch. Biochem. Biophys.
2003, 411, 215-222.
AC7016184
9184 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007