Novel α-Oxoamide Advanced-Glycation Endproducts within the N6-Carboxymethyl Lysine and N6-Carboxyethyl Lysine Reaction Cascades

Article abstract of DOI:10.1021/acs.jafc.7b05813

The highly reactive α-dicarbonyl compounds glyoxal and methylglyoxal are major precursors of posttranslational protein modifications in vivo. Model incubations of N²-t-Boc-lysine and either glyoxal or methylglyoxal were used to further elucidate the underlying mechanisms of the N⁶-carboxymethyl lysine and N⁶-carboxyethyl lysine reaction cascades. After independent synthesis of the authentic reference standards, we were able to detect N⁶-glyoxylyl lysine and N⁶-pyruvoyl lysine for the first time by HPLC-MS² analyses. These two novel amide advanced-glycation endproducts were exclusively formed under aerated conditions, suggesting that they were potent markers for oxidative stress. Analogous to the well-known Strecker degradation pathway, leading from amino acids to Strecker acids, the oxidation of an enaminol intermediate is suggested to be the key mechanistic step. A highly sensitive workup for the determination of AGEs in tissues was developed. In support of our hypothesis, the levels of N⁶-glyoxylyl lysine and N⁶-pyruvoyl lysine in rat livers indeed correlated with liver cirrhosis and aging.

Full text of DOI:10.1021/acs.jafc.7b05813

Article
pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Novel α‑Oxoamide Advanced-Glycation Endproducts within the
N6‑Carboxymethyl Lysine and N6‑Carboxyethyl Lysine Reaction
Cascades
Tim Baldensperger,† Tobias Jost,† Alexander Zipprich,‡ and Marcus A. Glomb*,†
†Institute of Chemistry, Food Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 2, 06120 Halle/Saale,
Germany
‡Department of Internal Medicine I, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Strasse 40, 06120 Halle/Saale,
Germany
ABSTRACT: The highly reactive α-dicarbonyl compounds glyoxal and methylglyoxal are major precursors of posttranslational
protein modifications in vivo. Model incubations of N2-t-Boc-lysine and either glyoxal or methylglyoxal were used to further
elucidate the underlying mechanisms of the N6-carboxymethyl lysine and N6-carboxyethyl lysine reaction cascades. After
independent synthesis of the authentic reference standards, we were able to detect N6-glyoxylyl lysine and N6-pyruvoyl lysine for
the first time by HPLC-MS2 analyses. These two novel amide advanced-glycation endproducts were exclusively formed under
aerated conditions, suggesting that they were potent markers for oxidative stress. Analogous to the well-known Strecker
degradation pathway, leading from amino acids to Strecker acids, the oxidation of an enaminol intermediate is suggested to be
the key mechanistic step. A highly sensitive workup for the determination of AGEs in tissues was developed. In support of our
hypothesis, the levels of N6-glyoxylyl lysine and N6-pyruvoyl lysine in rat livers indeed correlated with liver cirrhosis and aging.
KEYWORDS: Maillard reaction, amide advanced-glycation endproducts, methylglyoxal, glyoxal, oxidative stress, aging
to fragment by oxidative α-dicarbonyl cleavage and amine-
induced β-dicarbonyl cleavage, resulting in mixtures of short-
chained carbonyls, carboxylic acids, and their corresponding
amide AGEs.14,15 On the other hand, two of the quantitatively
most relevant AGEs, CML and N6-carboxyethyl lysine (CEL),
are formed nonoxidatively by isomerization after a reaction
between protein-bound lysines and the short-chained dicar-
bonyl structures GO and MGO, respectively. Alternative
products formed in the complex reaction cascades include
monovalent AGEs with an amide structure, like N6-glycoloyl
lysine (GALA) and N6-lactoyl lysine, and cross-linking
compounds, like the glyoxal lysine amide (GOLA) and the
glyoxal lysine dimer (GOLD).9
It is important to understand that in contrast to all the other
structures identified so far within this mechanistic scheme, only
CML can be formed by additional oxidative pathways. A main
route was shown to proceed via the direct oxidative
fragmentation of the Amadori product of lysine and glucose,
although the exact mechanism is still lacking. In addition,
multiple minor reaction pathways have been published, mainly
related to the formation of glyoxal or glycolaldehyde from
higher sugars via, for example, the metal-catalyzed autoxidation
of glucose or the amine-catalyzed single-electron transfers of
pyrazinium-radical cations within the Namiki pathway.16
Consequently, CML has been used as a marker of oxidative
stress in diabetes, for example, which is generally characterized
INTRODUCTION
■
Louis-Camille Maillard described the nonenzymatic browning
reaction of reducing sugars and amines upon heating in 1912.
Already at this early stage, he postulated the reaction’s great
importance in the physiology and pathology of humans on the
basis of the wide availability of carbohydrates and amines.1
Starting in the 1940s, Maillard-reaction research focused not
only on the formation of colors, aromas, and tastes in food2 but
also on the negative side-effects, like the formation of
foodborne toxins, such as acrylamide.3 In addition, Maillard
reactions are widely accepted as an important source of reactive
intermediates that lead to advanced-glycation endproducts
(AGEs) in vivo. AGEs are stable posttranslational protein
modifications and were correlated with the development and
progression of various chronic and age-related pathologies. As
an example, the levels of the most abundant AGE, N6-
carboxymethyl lysine (CML), increased with the severity of
diabetes4 and rose in an almost linear fashion with age in
human lens proteins.5
Because of the progress in analytical methods, the under-
standing of Maillard-reaction mechanisms has strongly
improved in recent years. Highly reactive α-dicarbonyl
compounds, like glyoxal (GO), methylglyoxal (MGO), and
deoxyglucosones, have been identified as key intermediates and
quantitated in order to understand the chemistry of AGE
formation in vitro and in vivo.6−10 These precursors are
transformed to the stable endproducts mainly by three different
mechanisms under physiological conditions:9 oxidative α-
dicarbonyl cleavage,11 amine-induced β-dicarbonyl cleavage,12
and isomerization.13 Rather-long-chained dicarbonyl structures,
for example, 1-deoxyglucosone and 2,3-diketogulonic acid, tend
Received: December 11, 2017
Revised: January 29, 2018
Accepted: February 6, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.7b05813
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
purified by column chromatography on a silica gel 60 using hexane−
acetone (3:1). Fractions containing 5 (TLC: Rf 0.22 in hexane−
acetone (3:1), ninhydrin detection) were collected, concentrated in
vacuo, and dried under a high vacuum to afford compound 5 as a
yellow viscous oil (841 mg, 50%). As described by Katayama, the N,N-
dimethyl hydrazone protective group was removed by silica-gel
purification.22 1H NMR (400 MHz, MeOH-d4): δ (ppm) = 1.44 (s,
9H), 1.46 (s, 9H), 1.56 (m, 2H), 1.63 (m, 2H), 1.74 (m, 2H), 2.38 (s,
by increases in both carbonyls and oxidative stress, accelerating
the formation of late-stage complications.17 However, because
of the multiple routes leading to CML formation, the value of
CML as a suitable chemical parameter for oxidative stress has to
be challenged. The present work therefore successfully aimed
to identify two novel AGEs specific for oxidation within the
isomerization-reaction cascade and test their applicability in
vivo.
3
3
3H), 3.24 (t, J = 7.0 Hz, 2H), 3.93 (t, J = 5.4 Hz, 1H). 13C NMR
(100 MHz, MeOH-d4): δ (ppm) = 24.2, 24.8, 28.3, 28.7, 29.7, 32.3,
39.9, 55.7, 157.8, 162.9, 172.3, 197.6. HR-MS: m/z 373.2333 (found);
m/z 373.2338 (calcd for C18H33O6N2 [M + H]+).
MATERIALS AND METHODS
■
Chemicals. All chemicals of the highest quality available were
provided by Sigma-Aldrich (Munich/Steinheim, Germany), Fluka
(Taufkirchen, Germany), Merck (Darmstadt, Germany), Roth
(Karlsruhe, Germany) and Sigma (Taufkirchen, Germany), unless
otherwise indicated. The NMR solvents were purchased from ARMAR
Chemicals (Leipzig/Doettingen, Germany).
N6-Pyruvoyl Lysine. Compound 5 (590 mg, 1.59 mmol) was
dissolved in acetone and 6 M HCl (10 mL each). After being stirred
for 30 min, the mixture was diluted with 100 mL of water and
concentrated to a volume of approximately 20 mL under a vacuum.
After being washed with 20 mL of EtOAc, the aqueous phase was
separated and evaporated to dryness. The crude product was purified
by column chromatography on a Lichroprep RP C18 using water−
methanol (9:1). Fractions with positive ninhydrin detections (TLC: Rf
0.19 in water−butanol−acetic acid (8:1:1)) were collected, evaporated
to dryness, and lyophilized to afford N6-pyruvoyl lysine as an orange
The standard reference substances CML,7 GALA,13 CEL,18 and N6-
lactoyl lysine12 as well as the Amadori product of glucose and N2-t-
Boc-lysine,7 N2-t-Boc-lysine t-butyl ester (1),19 glyoxylic acid diethyl
acetal (2),20 and pyruvic acid N,N-dimethyl hydrazone (4)21 were
synthesized according to the literature.
1
N2-t-Boc-N6-(2,2-diethoxy acetyl) Lysine t-Butyl Ester (3).
Compound 2 (647 mg, 4.37 mmol) and hydroxybenzotriazole
(HOBt, 590 mg, 4.37 mmol) were dissolved in 10 mL of dry THF
under an argon atmosphere at 0 °C. After 10 min, 745 mg (4.8 mmol)
of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) was
added. A solution of 1.319 g (4.37 mmol) of 1 in 5 mL of dry THF
was added dropwise after 20 min. The reaction mixture was warmed to
room temperature and stirred for 16 h. The solvent was evaporated
under a vacuum, and the residue was dissolved in 10 mL of EtOAc and
washed with a saturated NaHCO3 solution and brine (10 mL each).
The organic layer was dried over Na2SO4, and the solvents were
evaporated. The crude product was purified by column chromatog-
raphy on a silica gel 60 using hexane−acetone (3:1). Fractions
containing 3 (TLC: Rf 0.35 in hexane−acetone (3:1), ninhydrin
detection) were collected, concentrated in vacuo and dried under a
high vacuum to afford compound 3 as a yellow viscous oil (633 mg,
amorphic material (302 mg, 88%). H NMR (400 MHz, D2O): δ
(ppm) = 1.42 (m, 2H), 1.61 (m, 2H), 1.90 (m, 2H), 2.43 (s, 3H), 3.26
3
(t, J = 7.0 Hz, 2H), 3.82 (m, 1H). 13C NMR (100 MHz, D2O): δ
(ppm) = 22.7, 25.3, 28.7, 30.8, 39.7, 55.1, 163.1, 174.7, 199.1. HR-MS:
m/z 217.1186 (found); m/z 217.1188 (calcd for C9H17O4N2 [M +
H]+).
Aerated Incubations. Mixtures containing N2-t-Boc-lysine (40
mM), phosphate buffer (0.1 M, pH 7.4), and either GO, MGO,
ascorbic acid, the Amadori product, maltose, pyruvic acid, or glyoxylic
acid (40 mM) were incubated in screw-cap vials. Incubations were
done at 37 °C in a shaker for 7 days. Aliquots of 100 μL were collected
each day and instantly stored at −20 °C until the analyses.
AGEs were analyzed by HPLC-MS2 after the deprotection of the
N2-t-Boc group. Pyruvic and glyoxylic acid formation were analyzed by
GC-MS after silylation. Each sample was prepared at least three times.
Deaerated Incubations. The incubations were modified by using
a phosphate buffer with 1 mM diethylenetriaminepentaacetic acid. The
buffer was degassed with helium before the samples were placed in 0.7
mL screw-cap vials without air and incubated under an argon
atmosphere.
1
3
34%). H NMR (400 MHz, MeOH-d4): δ (ppm) = 1.23 (t, J = 7.1
Hz, 6H), 1.44 (s, 9H), 1.46 (s, 9H), 1.38 (m, 2H), 1.56 (m, 2H), 1.75
(m, 2H), 3.22 (t, 3J = 7.0 Hz, 2H), 3.61 (m, 4H), 3.93 (t, 3J = 5.0 Hz,
1H), 4.79 (s, 1H). 13C NMR (100 MHz, MeOH-d4): δ (ppm) = 15.4,
24.3, 28.3, 28.8, 30.0, 32.7, 39.8, 55.8, 63.4, 99.7, 158.2, 170.4, 173.8.
HR-MS: m/z 433.2900 (found); m/z 433.2908 (calcd for C21H41O7N2
[M + H]+).
pH Incubations. Aerated incubations were performed as described
above, but the pH of the phosphate buffer was adjusted to 4.5 or 9.6
using 1 M HCl or NaOH.
N6-Glyoxylyl Lysine. Compound 3 (178 mg, 0.41 mmol) was
dissolved in acetone and 6 M HCl (10 mL each). After being stirred
for 30 min, the mixture was diluted with 100 mL of water and
concentrated to a volume of approximately 20 mL under a vacuum.
After being washed with 20 mL of EtOAc, the aqueous phase was
separated and evaporated to dryness. The crude product was purified
by column chromatography on a Lichroprep RP C18 using water−
methanol (9:1). Fractions with positive ninhydrin detections (TLC: Rf
0.14 in water−butanol−acetic acid (8:1:1)) were collected, evaporated,
and lyophilized to afford N6-glyoxylyl lysine as an amorphic material
(75 mg, 90%). 1H NMR (400 MHz, D2O): δ (ppm) = 1.34 (m, 2H),
1.48 (m, 2H), 1.85 (m, 2H), 3.13 (t, 3J = 7.0 Hz, 2H), 3.96 (t, 3J = 6.5
Hz, 1H), 5.15 (s, 1H). 13C NMR (100 MHz, D2O): δ (ppm) = 21.4,
27.7, 29.3, 38.5, 52.7, 86.7, 172.0, 172.1. HR-MS: m/z 203.1028
(found); m/z 203.1026 (calcd for C8H15O4N2 [M + H]+).
Housing of Animals and Induction of Cirrhosis by Carbon
Tetrachloride (CCl4). Male Wistar rats were used for all the
experiments. The control and cirrhotic rats were bred in the Center of
Medical Basic Research (ZMG), Medical Faculty, University of Halle,
and the old rats were purchased from Janvier Laboratories (Le Genest-
Saint-Isle, France). The rats were housed in standard cages in a climate
room with 12 h light and dark phases and free access to food. The
cirrhotic rats underwent inhalation exposure to CCl4 three times a
week. Phenobarbital (0.35 g/L) was added to the drinking water as
described previously.23 The treatment was given for 12 weeks. The
livers were isolated 7−10 days after the last dose of CCl4. The
principles for the care and use of animals from the American
Physiological Society guide were followed. All animal experiments
were approved by the local animal committee (42502-2-1123 MLU,
Landesverwaltungsamt Sachsen-Anhalt, Germany).
Tissue Collection. The rats were anaesthetized with 150 mg/kg
bodyweight narcoren (Merial, Lyon, France). In deep narcosis, the
animals were killed by exsanguination, the livers were dissected after
perfusion with Krebs-Ringer, and the samples were immediately snap-
frozen. For the sirius-red staining, the livers were fixed in formaline
(Histofix Roth, Karlsruhe, Germany) and processed routinely. For the
Western blots, the livers were treated with a protein lysis buffer
(RIPA) for the protein isolation. The primary antibodies were for
vinculin (SC-5573, Santa Cruz Biotechnology, Dallas, Texas), α-SMA
(ab5694), and TGF-β (ab66043, both abcam, Cambridge, UK). The
N2-t-Boc-N6-pyruvoyl Lysine t-Butyl Ester (5). Compound 4 (590
mg, 4.54 mmol) and HOBt (613 mg, 4.54 mmol) were dissolved in 10
mL of dry THF under an argon atmosphere at 0 °C. After 10 min, 775
mg (5.0 mmol) of EDC was added. A solution of 1.369 g (4.54 mmol)
of 1 in 5 mL of dry THF was added dropwise after 20 min. The
reaction mixture was warmed to room temperature and stirred for 16
h. The solvent was evaporated under a vacuum, and the residue was
dissolved in 10 mL of EtOAc and washed with saturated NaHCO3
solution and brine (10 mL each). The organic layer was dried over
Na2SO4, and solvents were evaporated. The crude product was
B
DOI: 10.1021/acs.jafc.7b05813
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Journal of Agricultural and Food Chemistry
Article
Table 1. Mass-Spectrometric Parameters for AGE Quantitation
a
b
b
precursor
product ion 1
product ion 2
CE
product ion 3
retention time
(min)
m/z
DP
(V)
m/z
CE
CXP
(V)
m/z
(amu)
CXP
(V)
m/z
CE
CXP
(V)
AGE
(amu)
(amu)
(eV)
(eV)
(amu)
(eV)
N6-carboxymethyl lysine
N6-glycoloyl lysine
12.3
12.2
11.7
205.1
205.2
221.1
42
40
40
130.2
142.1
203.3
17
20
15
9
84.1
84.1
30
36
25
14
14
12
56.1
56.2
59
64
19
9
8
11
13
N6-glyoxylyl lysine
140.1
157.3
12
hydrate
N6-carboxyethyl lysine
N6-lactoyl lysine
N6-pyruvoyl lysine
N6-glycoloyl lysine-d1
N6-lactoyl lysine-d1
17.8
16.2
19.5
12.2
16.2
219.1
219.2
217.0
206.2
220.2
54
40
55
40
40
130.1
156.2
154.0
143.1
157.2
18
20
19
20
20
11
8
84.1
84.1
84.1
84.1
84.1
33
35
32
36
35
7
9
56.1
173.1
171.0
160.1
174.1
59
17
15
64
17
8
8
12
11
8
6
12
8
14
9
8
a
b
MRM transition used for quantitation. MRM transition used for confirmation.
Western-blot signals were quantified using a Fusion-Fx-7 imager with
BD-Software (Peqlab, Erlangen, Germany).
spectrometer. Nitrogen was used as the sheath and auxiliary gas. To
measure the analytes, the scheduled-multiple-reaction-monitoring
(sMRM) mode of HPLC-MS2 was used. The optimized parameters
for the mass spectrometry are given in Table 1. The quantitation was
based on the standard-addition method with known amounts of the
pure authentic reference compounds. The values for the limits of
detection (LOD) and limits of quantitation (LOQ) for the respective
matrices are shown in Table 2.
Protein Workup. The protein was worked up by a modified
procedure from Pogo et al.24 Minced rat liver (250 mg) was weighed
in a 14 mL round-bottom cell-culture tube (Greiner Bio-One,
Kremsmuenster, Austria). Cooled (4 °C) lysis buffer (2 mL, 0.32 M
sucrose, 3 mM MgC12) was added, and the sample was lysed at 6500
rpm by an Ultra-Turrax (IKA, Staufen, Germany) for 2 min on ice.
The lysate was cleared by 20 μm CellTrics filtration (Sysmex,
Norderstedt, Germany) and centrifugation at 10 000 RCF for 15 min.
The supernatant was separated, and 50% trichloroacetic acid (TCA)
was added to a final concentration of 10%. The precipitate was washed
twice with 5% TCA and isopropanol. After the precipitate was dried
under a high vacuum, 3 mg of protein were dissolved in 1 mL of
phosphate-buffered saline (PBS, pH 7.4, 150 mM NaCl, 10 mM
phosphate) and homogenized by a MM 400 mixer mill (Retsch, Haan,
Germany). After the addition of 200 μL of a NaBD4 solution (15 mg/
mL in 0.01 M NaOH), reduction was allowed to occur for 1 h at room
temperature; then, the excess NaBD4 was destroyed by 100 μL of 1 M
HCl, and the solution was neutralized by 100 μL of 1 M NaOH. The
samples were evaporated in a vacuum concentrator (Savant-Speed-Vac
Plus SC 110 A combined with a Vapor Trap RVT 400, Thermo Fisher
Scientific, Bremen, Germany), dissolved in water, and lyophilized. The
lyophilizate was dissolved in 1 mL of PBS, and aliquots were used for
the acid and enzymatic hydrolyses.
Table 2. LODs and LOQs of AGEs in Incubations and
Protein Hydrolysates
incubation
LOD LOQ
protein hydrolysate
LOD
(mmol/mol (mmol/mol (μmol/mol
LOQ
(μmol/mol
leucine)
analyte
lysine)
lysine)
leucine)
N6-
0.040
0.121
0.777
2.590
carboxymethyl
lysine
N6-glycoloyl
0.003
0.130
0.019
0.008
0.391
0.058
0.250
0.834
nd
lysine
a
N6-glyoxylyl
nd
lysine hydrate
N6-carboxyethyl
0.413
1.375
lysine
N6-lactoyl lysine
0.003
0.009
0.011
0.028
0.071
nd
0.238
nd
Acid Hydrolysis. The acid hydrolysis was performed as described
by Glomb and Pfahler.13 Aliquots of the protein solution (400 μL)
were dried, and 800 μL of 6 M HCl was added. The solution was
heated for 20 h at 110 °C under an argon atmosphere. The volatiles
were removed in a vacuum concentrator, and the residue was dissolved
in 492 μL of 0.05 M HCl. Prior to their injection into the HPLC-MS2
system, the samples were filtered through 0.45 μm cellulose acetate
Costar SpinX filters (Corning Inc., Corning, NY).
N6-pyruvoyl
lysine
N6-glycoloyl
nd
nd
nd
nd
0.144
0.043
0.481
0.144
lysine-d1
N6-lactoyl lysine-
d1
a
nd = not determined.
Enzymatic Hydrolysis. The enzymatic hydrolysis followed a
slightly modified protocol by Smuda et al.5 Pronase E (0.3 U, two
additions), leucine aminopeptidase (1 U), and carboxypeptidase Y
(0.95 U) were added to 500 μL aliquots of the protein solution. The
enzymes were applied stepwise, and the incubations with each were for
24 h at 37 °C in a shaker incubator. A small crystal of thymol was
added with the first digestion step. Once the total digestion procedure
was completed, the reaction mixtures were filtered through 3000 Da
molecular weight cutoff filters (VWR International, Radnor, PA).
For each sample, the efficiency of the acid hydrolysis was assigned
to 100%, and the efficiency of the enzymatic hydrolysis was calculated
by HPLC-MS2 analysis of acid-stable CEL.
Model Incubations. Prior to the injection into the HPLC-MS2
system, 100 μL of 6 M HCl was added to 100 μL aliquots of the
incubation solutions; the samples were kept at room temperature for
30 min and diluted on a scale of 1:10 with water.
Chromatographic separations were performed on a stainless-steel
column (VYDAC 218TP54, 250 × 4.6 mm, RP18, 5 μm, Hesperia,
CA) using a flow rate of 1 mL/min and a column temperature of 25
°C. The samples (10 μL) were injected at 2% B and run isocratically
for 5 min. The gradient was changed to 100% B within 15 min (held
for 10 min), and then the gradient was changed to 2% B within 5 min,
at which it was held for 15 min. The eluents were ultrapure water (A)
and a mixture of methanol and demineralized water (7:3, v/v; B), both
of which were with 1.2 mL/L HFBA. sMRM mode was used for the
mass-spectrometric detection.
Analytical HPLC-MS2. A Jasco PU-2080 Plus quaternary gradient
pump with a degasser and a Jasco AS-2057 Plus autosampler (Jasco,
Gross-Umstadt, Germany) were used. The mass analyses were
performed using an Applied Biosystems API 4000 quadrupole
instrument (Applied Biosystems, Foster City, CA) equipped with an
API source that used an electrospray-ionization (ESI) interface. The
HPLC system was connected directly to the probe of the mass
Protein Hydrolysates. Chromatographic separations were per-
formed on a stainless steel column (XSelect HSS T3, 250 × 3.0
mm, RP18, 5 μm, Waters, Milford, MA) using a flow rate of 0.7 mL/
min and a column temperature of 25 °C. The samples (10 μL) were
C
DOI: 10.1021/acs.jafc.7b05813
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 1. Synthesis of N6-glyoxylyl lysine and N6-pyruvoyl lysine.
injected at 2% B and run isocratically for 2 min. The gradient was
changed to 14% B within 10 min (held for 0 min); 87% B within 22
min (held for 0 min); and 100% B within 0.5 min, at which it was held
for 7 min. Then, the gradient was changed to 2% B within 2.5 min, at
which it was held for 8 min. The eluents and mode were the same as
described above.
Ninhydrin Assay. The ninhydrin assay was performed as described
by Smuda et al.5 After the complete workup, the contents of the amino
acid modifications were standardized to that of L-leucine. A calibration
of L-leucine, concentrated between 5 and 100 μM, was performed and
referenced to the diluted samples. Each sample was prepared four
times.
Analytical GC-MS. The GC-MS method was established by Smuda
et al.12 The samples were analyzed on a Thermo Finnigan Trace GC
Ultra coupled to a Thermo Finnigan Trace DSQ (both Thermo Fisher
Scientific GmbH, Bremen, Germany). The GC column was a HP-5
(30 m × 0.32 mm, 0.25 μm film thickness; Agilent Technologies, Palo
Alto, CA), with an injector at 220 °C, a split ratio of 1:30, and a
transfer line at 250 °C. The oven-temperature program was as follows:
100 °C (0 min), 7.5 °C/min to 200 °C (0 min), and 50 °C/min to
320 °C (10 min). Helium 5.0 was used as the carrier gas in constant-
flow mode (linear velocity of 28 cm/s, flow of 1 mL/min). Mass
spectra were obtained with EI at 70 eV (source: 210 °C) in selected-
ion-monitoring mode: glyoxylic acid (m/z 221.0/193.0/73.0) and
pyruvic acid (m/z 217.0/189.0/73.0). The retention times were as
follows: glyoxylic acid was tR= 5.85 min, and pyruvic acid was tR= 4.00
min. Aliquots of the incubations (100 μL) were dried in vacuo, the
residues were dissolved in 100 μL of pyridine, and 100 μL of N,O-
bis(trimethylsilyl)-acetamide with 5% trimethylchlorosilane was added.
The samples were kept 4 h at 70 °C prior to injection into the GC-MS
system.
High-Resolution Mass Determination (HR-MS). Positive-ion
high-resolution ESI mass spectra were obtained from an Orbitrap Elite
mass spectrometer (Thermo Fisher Scientific, Bremen, Germany)
equipped with an HESI electrospray ion source (spray voltage of 4 kV,
capillary temperature of 275 °C, source heater temperature of 40 °C,
FTMS resolution >30.000). Nitrogen was used as the sheath and
auxiliary gas. The sample solutions were introduced continuously via a
500 μL Hamilton syringe pump with a flow rate of 5 μL/min. The data
were evaluated by the Xcalibur software 2.7 SP1.
MHz for 13C. SiMe4 was used as the reference for calibrating the
chemical shift.
Statistical Analysis. Analyses were performed in triplicate for each
model incubation and resulted in coefficients of variation less than 5%
for the AGE concentrations. All significance tests for the AGEs were
performed by two-sample t-tests. Nonparametric tests (Kurskal−
Wallis or Mann−Whitney) were used to detect differences between
the groups for the markers of fibrosis.
RESULTS AND DISCUSSION
■
Syntheses of Authentic Reference Standards. N6-
glyoxylyl lysine and N6-pyruvoyl lysine were synthesized in an
amide-coupling reaction catalyzed by EDC and HOBt (Figure
1). First, derivatives of glyoxylic acid and pyruvic acid with
protected carbonyl functions were synthesized in order to
increase their solubilities and prevent side reactions. Ethyl
diethoxyacetate was treated with NaOH to yield glyoxylic acid
diethoxy acetal, 2, which was coupled with 1 to give the fully
protected amide 3. Pyruvic acid was protected as a N,N-
dimethyl hydrazone, 4, and coupled with 1 to give the
protected amide 5. The N,N-dimethyl hydrazone group had
already been cleaved off during the silica gel chromatography,
as described in literature.22 Finally, amides 3 and 5 were
deprotected by treatments with 3 M HCl to give N6-glyoxylyl
lysine and N6-pyruvoyl lysine.
Both of the target compounds as well as the intermediates 2,
3, 4, and 5 were unequivocally verified by nuclear-magnetic-
resonance (NMR) experiments and accurate mass determi-
nations via HR-MS. Furthermore, the chemical shifts at 5.15
1
ppm in the H NMR experiments and 86.7 ppm in the 13C
NMR experiments for the terminal aldehyde function showed
that N6-glyoxylyl lysine existed mainly as a hydrate. In contrast,
the keto function of N6-pyruvoyl lysine had a chemical shift of
199.1 ppm in the 13C NMR experiments, indicating its free
carbonyl moiety.
Formation of N6-Glyoxylyl Lysine and N6-Pyruvoyl
Lysine. The HPLC-MS2 screenings of the aerated incubations
of N2-t-Boc-lysine, phosphate buffer and either glyoxal (GO),
methylglyoxal (MGO), ascorbic acid, maltose, or the Amadori
product of glucose and N2-t-Boc-lysine under physiological
conditions indicated the exclusive formation of N6-glyoxylyl
Nuclear-Magnetic-Resonance Spectroscopy (NMR). NMR
spectra were recorded either on a Varian VXR 400 spectrometer
1
operating at 400 MHz for H and 100 MHz for 13C or on a Varian
1
Unity Inova 500 instrument operating at 500 MHz for H and 125
D
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Article
lysine by GO and the formation of N6-pyruvoyl lysine by MGO
after 7 days at 37 °C. The time course of AGE formation was
recorded by collecting aliquots every 24 h (Figure 2). After a 7
oxidative α-dicarbonyl cleavage or amine-induced β-dicarbonyl
cleavage of any putative reaction side-products resulting from,
for example, aldol condensation. In addition, incubations of N2-
t-Boc-lysine with glyoxylic acid or pyruvic acid showed no
formation of the corresponding amide AGEs, ruling out any
mechanism of direct amide formation between the carboxylic
acid function and the amine under the present conditions. The
results therefore strongly pointed toward an isomerization-
based reaction as the only possible source of N6-glyoxylyl lysine
and N6-pyruvoyl lysine, which must include an oxidative step by
definition. Consequently, the two novel amide AGEs should be
alternative products of the established N6-carboxymethyl lysine
(CML)- and N6-carboxyethyl lysine (CEL)-reaction cascades,
which are based on isomerization (Figure 3).9 The reaction
starts with formation of the imine at one of the carbonyl
functions (the keto function in the case of MGO, I). Hydration
of the other carbonyl group allows the formation of an
enamine, which rearranges to give the respective carboxyalkyl
endproduct. If alternatively the isomerization starts from the
hemiaminal intermediate (in the case of MGO at the aldehyde
function, II) the respective α-hydroxyamide endproducts result
via an enaminol intermediate. Typically, the formation of the
carboxyalkyl structure is favored by a factor of 5−30. Indeed, in
the above model incubation, both structural classes were
detected, reaching 0.73 mol % N6-glycoloyl lysine (glycolic acid
lysine amide, GALA) and 11.24 mol % CML in the case of GO,
and 0.03 mol % N6-lactoyl lysine and 0.12 mol % CEL in the
case of MGO (Figure 2, data not shown for CML and CEL).
This opened up the hypothesis that the novel α-oxoamide
AGEs, N6-glyoxylyl lysine and N6-pyruvoyl lysine, might stem
from the direct oxidation of GALA and N6-lactoyl lysine,
respectively. However, this notion was undermined by an
aerated incubation of the authentic α-hydroxyamide references,
where no oxidation products were detected after 7 days.
Influence of Oxygen and pH. Comparative incubations
under aerated and deaerated conditions confirmed the
nonoxidative pathway of CML, CEL, GALA, and N6-lactoyl
lysine formation, as there were almost no changes in their
concentrations (Figure 4, aerated and deaerated 113 and 118
mmol/mol lysine, respectively, for CML; aerated and deaerated
both 1.2 mmol/mol lysine for CEL). On the other hand, N6-
glyoxylyl lysine and N6-pyruvoyl lysine were formed exclusively
under aeration. This again stressed the need for an oxidation
Figure 2. Formation of AGEs in incubations of N2-t-Boc-lysine with
glyoxal (A) or methylglyoxal (B) at 37 °C and pH 7.4 under aeration.
6
6
6
●
○
N -Glycoloyl lysine ( ), N -glyoxylyl lysine ( ), N -lactoyl lysine
6
■
□
( ), and N -pyruvoyl lysine ( ).
day incubation, the molecular yield was 0.48 mol % N6-
glyoxylyl lysine and 0.01 mol % N6-pyruvoyl lysine. Taking the
short carbon chain of the precursors into account, a formation
mechanism based on direct fragmentation of the educts was
highly unlikely. Nevertheless, aliquots of the incubations were
analyzed by GC-MS in order to determine the corresponding
glyoxylic and pyruvic acids, which would be the main
byproducts of any dicarbonyl-cleavage reaction.14 No acid
formation was monitored. This unequivocally excluded the
formation of N6-glyoxylyl lysine and N6-pyruvoyl lysine by the
Figure 3. N6-carboxymethyl lysine and N6-carboxyethyl lysine isomerization reaction cascades.
E
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Figure 4. Effects of oxygen on the formation of AGEs in aerated
(closed bars) and deaerated (open bars) incubations of N2-t-Boc-lysine
with glyoxal (A) or methylglyoxal (B) for 7 days at 37 °C and pH 7.4.
Figure 5. Effects of pH 4.5 (closed bars), 7.4 (open bars), and 9.6
(hatched bars) on the formation of AGEs in incubations of N2-t-Boc-
lysine with glyoxal (A) or methylglyoxal (B) for 7 days at 37 °C.
step within the isomerization-reaction scheme. The most likely
electron-rich candidate is the enaminol (Figure 3), which
should be readily oxidized to give the novel α-oxoamide AGEs.
In fact, Hofmann et al. described a similar enaminol
intermediate in their explanation of the alternative formation
of acids in the general Strecker degradation of amino acids. The
nonoxidative degradation of the enaminol led to Strecker
aldehydes, whereas in the presence of oxygen, the ratio
significantly shifted from 4:1 to almost 1:2 toward the Strecker
acids. However, because their setup was more related to food
processing at high temperatures, they measured 0.83 mol %
phenylacetaldehyde and 1.22 mol % phenylacetic acid in
aerated incubations of glyoxal and phenylalanine after 60 min.
In full support of our results, a direct oxidation of Strecker
aldehydes to Strecker acids was ruled out by isotope-labeling
experiments that pointed strongly toward the enaminol as the
Strecker acid precursor.25
The CML- and CEL-reaction cascades were strongly
dependent on pH values (Figure 5). In general, hardly any
AGEs are formed under acidic conditions, because the N6-
amino function of lysine is protonated. This leads to a loss of
nucleophilicity and prevents the necessary formation of Schiff
base adducts. In contrast, the N6-amino function is sufficiently
deprotonated at pH 7.4 to allow an attack at the carbonyl
carbon and initiate the formation of AGEs. A factor of about
100 was found between the yields of CML and CEL regardless
of whether the reaction proceeded at pH 7.4 or 9.6. This must
be attributed to the different character of the carbonyl function
initiating the reaction. Whereas for CML, the N6-amino group
of the lysine attacks an aldehyde function of GO, CEL
formation requires an attack at the much-less-reactive ketone
moiety of MGO. On the other hand, the reason for the
pronounced difference in the concentrations of GALA and N6-
lactoyl lysine and of N6-glyoxylyl lysine and N6-pyruvoyl lysine
at pH 7.4 versus 9.6 by a factor of approximately 4 is less
obvious. In diluted aqueous solutions, dicarbonyl structures
exist predominantly in their hydrated forms; that is, GO almost
exclusively exists as the dihydrate,26 and 56 and 44% of MGO
exist in the mono- and dihydrated forms, respectively.27
Although this does not change as a result of changes in the
pH, the rate of enolization required for the rearrangement step
does. This implies that the general course of the reaction is
triggered mainly by two aspects, the stability of the endproducts
and the rate of isomerization. Obviously in the case of CML
and CEL, the high thermodynamic stabilities of the resulting
carboxyalkyls are the main driving forces, resulting in almost no
differences, whereas in the case of the α-hydroxyamides and α-
oxoamides, pH-related changes in the kinetics of rearrangement
prevail.
Oxidative-Stress and Aging Markers. Analyses of the
AGEs in the model incubations were done after the acid
hydrolysis of the BOC protection groups. As is apparent from
the NMR data, there is an equilibrium between the carbonyl
and hydrate forms. Consequently, HPLC separation led to very
broad peaks with poor signal-to-noise ratios for the novel α-
oxoamide AGEs. This was especially pronounced for N6-
glyoxylyl lysine (LOD, 0.130 mmol/mol lysine; LOQ, 0.391
mmol/mol lysine) but still satisfactory for quantitation.
However, the signals for the native α-oxoamide AGEs were
completely lost in the analyses of the liver-tissue samples
because of the inevitable load of the matrix. In the first attempt
to access the power of the novel AGEs as parameters of
oxidative stress and aging, the soluble proteins of the rat livers
were extracted by a lysis protocol. Because of their chemical
natures, amide AGEs have to be released by enzymatic
hydrolysis. The efficiency of this step was referenced to that
of acid-stable CEL by a parallel acid-hydrolysis workup for each
sample and was typically around 70−80%. Acid hydrolysis must
include a reduction step with sodium borohydride to prevent
any artifact formation, especially for CML. In the present study,
the use of sodium borodeuteride allowed the differentiation of
F
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N6-glyoxylyl lysine, in the form of N6-glycoloyl lysine-d1, from
any parallel-formed GALA and of N6-pyruvoyl lysine, in the
form of N6-lactoyl lysine-d1, from N6-lactoyl lysine. This also
eliminated the chromatographic issues with the native α-
oxoamides and led to very sharp peaks and significantly reduced
background noise. As a result, the sensitivity was improved by a
factor of about 200 for N6-glyoxylyl lysine (LOD, 0.144 μmol/
mol leucine; LOQ, 0.481 μmol/mol leucine) and a factor of
about 50 for N6-pyruvoyl lysine (LOD, 0.043 μmol/mol
leucine; LOQ, 0.144 μmol/mol leucine). The calculated
concentrations of N6-glyoxylyl lysine and N6-pyruvoyl lysine
were corrected by the subtraction of the natural-isotope peaks
(10.16% of GALA and 11.17% of N6-lactoyl lysine).
formation mechanism was already highlighted by the above N2-
t-Boc-lysine model incubations. This implies that both of the
parameters are indeed specific chemical markers for oxidative
stress in vivo (Figure 6).
To test for the biological significance of the novel α-
oxoamides in aging, a rat model was chosen, and the AGEs
were analyzed in the soluble-protein fraction of the liver.
Inflammatory processes and oxidative stress in this organ were
triggered by the induction of cirrhosis with tetrachloromethane.
As shown in Table 3, the mean levels of all the AGEs
a
Table 3. AGEs in Soluble Proteins from Rat-Liver Lysates
AGE (μmol/mol
3 month old
(healthy)
3 month old
(cirrhosis)
22 month old
(healthy)
leucine)
N6-carboxymethyl
lysine
5.22 3.34
5.40 3.83
10.07 7.01**
N6-glycoloyl lysine
N6-glyoxylyl lysine
0.73 0.07
0.37 0.03
11.73 2.32
0.70 0.09
0.57 0.16*
8.34 2.52
1.36 0.32**
0.58 0.03*
17.27 4.35**
N6-carboxyethyl
lysine
N6-lactoyl lysine
0.28 0.06
0.11 0.04
0.34 0.09
0.55 0.06**
0.27 0.07*
N6-pyruvoyl lysine
0.23 0.05*
markers of fibrosis (%)
sirius-red staining
TGF-β
0.49 0.14
100 26
100 31
23.16 2.67*
947 661*
1995 1057*
1.70 0.40*
478 143*
453 192*
Figure 6. Correlation of GO-specific AGEs (A) and MGO-specific
AGEs (B) in rat livers with cirrhosis. Black lines are median values.
smooth muscle antigen
a
Means
standard deviations, n = 5. Significant differences were
determined via the t-test or Mann−Whitney test by comparison with
the AGE levels and markers of fibrosis in 3 month old healthy rats. *p
< 0.05, **p < 0.001.
The hypothesis of oxidative stress leading to α-oxoamide
formation was supported by sirius-red staining and Western
blotting of transforming growth factor β (TGF-β) and α
smooth-muscle actin (α-SMA, Table 3). Histological sirius-red
staining of collagen is an established test for fibrosis, which is
associated with increased oxidative stress.29 Whereas collagen
was nearly absent in the healthy, young rat livers (0.49%),
fibrosis increased slightly with age (1.70%) and by a factor of
about 50 in the cirrhotic animals (23.16%). Western blotting of
TGF-β and α-SMA was another method used to access fibrosis-
induced tissue changes. Additionally, TGF-β signaling and
expression as well as its activation from the latent complex are
increased by reactive oxygen species, and TGF-β itself in turn
induces the production of reactive oxygen species.30 The
intensity of both markers was referenced as 100% for the
young- and healthy-rat-liver proteins. In the 22 month old rats,
the levels of TGF-β (478%) and α-SMA (453%) were nearly 5-
times higher than they were in the young rats. The induction of
fibrosis under oxidative stress was strongly accelerated in the
cirrhotic rats, in which the TGF-β levels elevated to 947%, and
the α-SMA levels elevated to 1995%. These results were thus in
line with the significantly higher amounts of α-oxoamides found
in the old and cirrhotic rat livers.
approximately doubled when 3 month and 22 month old rats
were compared, showing p values <5% for N6-glyoxyly lysine
and N6-pyruvoyl lysine and <0.1% for all the other
modifications. It is important to understand that GALA and
N6-glyoxyly lysine are selective parameters for GO, whereas N6-
lactoyl lysine and N6-pyruvoyl lysine are formed exclusively
from MGO. On the other hand, CML can result not only from
GO but also from many other precursors as glycolaldehyde or
Amadori products. Taking the higher reactivity of GO and the
various pathways leading to CML into account, it is quite
surprising that the average CML levels in the aged rat livers
(10.07 μmol/mol leucine) were lower than the CEL levels
(17.27 μmol/mol leucine). A similar observation was found in
the renal glomeruli of rats, in which CML was quantitated at
269 μmol/mol lysine, and CEL was quantitated at 329 μmol/
mol lysine.28
When cirrhosis was induced, it was obvious that N6-glyoxyly
lysine and N6-pyruvoyl lysine were influenced not only by age
but also by oxidative stress. Whereas all the other AGE
parameters showed virtually no changes compared with the age-
matched healthy control group, the two α-oxoamides increased
significantly (p < 5%). The prerequisite oxidation step in their
To decouple the influences of aging and oxidative stress on
the formation of α-oxoamides, the ratios between GALA and
G
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N6-glyoxylyl lysine and between N6-lactoyl lysine and N6-
pyruvoyl lysine were calculated. Whereas the N6-glyoxylyl/
GALA and N6-pyruvoyl/N6-lactoyl lysine ratios remained on
average constant at around 0.5 in the young and aged rat livers,
the ratios in the cirrhotic livers increased to 0.8 for N6-
glyoxylyl/GALA and 0.7 for N6-pyruvoyl/N6-lactoyl lysine.
These changes in the ratios may thus be valuable markers of
liver cirrhosis in future research. Further investigations have to
validate the present preliminary results and open up the
applicability of these novel markers to other pathologies with
inflammatory oxidative stress.
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under physiological conditions. Glycoconjugate J. 2016, 33, 499−512.
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T. Protein modification by a Maillard reaction intermediate
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(11) Davídek, T.; Robert, F.; Devaud, S.; Vera, F. A.; Blank, I. Sugar
fragmentation in the Maillard reaction cascade. Formation of short-
chain carboxylic acids by a new oxidative α-dicarbonyl cleavage
pathway. J. Agric. Food Chem. 2006, 54, 6677−6684.
(12) Smuda, M.; Voigt, M.; Glomb, M. A. Degradation of 1-deoxy-D-
erythro-hexo-2,3-diulose in the presence of lysine leads to formation of
carboxylic acid amides. J. Agric. Food Chem. 2010, 58, 6458−6464.
(13) Glomb, M. A.; Pfahler, C. Amides are novel protein
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: marcus.glomb@chemie.uni-halle.de, Fax: ++049-345-
5527341.
ORCID
Tim Baldensperger: 0000-0002-9653-3156
Marcus A. Glomb: 0000-0001-8826-0808
Funding
Funding was supported by the Deutsche Forschungsgemein-
schaft (DFG, Germany) Research Training Group 2155,
ProMoAge.
(15) Smuda, M.; Glomb, M. A. Maillard degradation pathways of
vitamin C. Angew. Chem., Int. Ed. 2013, 52, 4887−4891.
(16) Thorpe, S. R.; Baynes, J. W. CML. A brief history. Int. Congr.
Ser. 2002, 1245, 91−99.
Notes
(17) Nowotny, K.; Jung, T.; Hohn, A.; Weber, D.; Grune, T.
̈
The authors declare no competing financial interest.
Advanced glycation end products and oxidative stress in type 2
diabetes mellitus. Biomolecules 2015, 5, 194−222.
ACKNOWLEDGMENTS
(18) Fujioka, M.; Tanaka, M. Enzymic and Chemical Synthesis of e-
N-(L-Propionyl-2)-L-Lysine. Eur. J. Biochem. 1978, 90, 297−300.
(19) Gellett, A. M.; Huber, P. W.; Higgins, P. J. Synthesis of the
unnatural amino acid Nα-Nε-(ferrocene-1-acetyl)-l-lysine. A novel
organometallic nuclease. J. Organomet. Chem. 2008, 693, 2959−2962.
(20) Pinto, A.; Conti, P.; Grazioso, G.; Tamborini, L.; Madsen, U.;
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■
We thank Dr. D. Strohl from the Institute of Organic
̈
Chemistry, Halle, Germany, for recording the NMR spectra
and Dr. A. Frolov from the Leibniz Institute of Plant
Biochemistry, Halle, Germany, for performing the accurate
mass determination.
ABBREVIATIONS USED
■
AGEs, advanced-glycation endproducts; GO, glyoxal; MGO,
methylglyoxal; CML, N6-carboxymethyl lysine; CEL, N6-
carboxyethyl lysine; GALA, N6-glycoloyl lysine; EDC, 1-ethyl-
3-(3-(dimethylamino)propyl)carbodiimide; HOBt, hydroxy-
benzotriazole; TCA, trichloroacetic acid; PBS, phosphate-
buffered saline; HFBA, heptafluorobutyric acid; sMRM,
scheduled multiple-reaction monitoring; TGF-β, transforming
growth factor β; α-SMA, α smooth-muscle actin
(22) Katayama, H.; Utsumi, T.; Ozawa, C.; Nakahara, Y.; Hojo, H.;
Nakahara, Y. Pyruvoyl, a novel amino protecting group on the solid
phase peptide synthesis and the peptide condensation reaction.
Tetrahedron Lett. 2009, 50, 818−821.
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Groszmann, R. J. Nitric oxide and vascular remodeling modulate
hepatic arterial vascular resistance in the isolated perfused cirrhotic rat
liver. J. Hepatol. 2008, 49, 739−745.
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