Extending the spectrum of α-dicarbonyl compounds in vivo

Article abstract of DOI:10.1074/jbc.M114.563593

Maillard α-dicarbonyl compounds are known as central intermediates in advanced glycation end product (AGE) formation. Glucose is the primary source of energy for the human body, whereas L-threo-ascorbic acid (vitamin C) is an essential nutrient, involved in a variety of enzymatic reactions. Thus, the Maillard degradation of glucose and ascorbic acid is of major importance in vivo. To understand the complex mechanistic pathways of AGE formation, it is crucial to extend the knowledge on plasma concentrations of reactive key α-dicarbonyl compounds (e.g. 1-deoxyglucosone). With the present work, we introduce a highly sensitive LC-MS/MS multimethod for human blood plasma based on derivatization with o-phenylenediamine under acidic conditions. The impact of workup and reaction conditions, particularly of pH, was thoroughly evaluated. A comprehensive validation provided the limit of detection, limit of quantitation, coefficients of variation, and recovery rates. The method includes the α-dicarbonyls 1-deoxyglucosone, 3-deoxyglucosone, glucosone, Lederer's glucosone, dehydroascorbic acid, 2,3-diketogulonic acid, 1-deoxypentosone, 3-deoxypentosone, 3,4-dideoxypentosone, pentosone, 1-deoxythreosone, 3-deoxythreosone, threosone, methylglyoxal, glyoxal; the α-keto-carboxylic acids pyruvic acid and glyoxylic acid; and the dicarboxylic acid oxalic acid. The method was then applied to the analyses of 15 healthy subjects and 24 uremic patients undergoing hemodialysis. The comparison of the results revealed a clear shift in the product spectrum. Inmost cases, the plasma levels of target analytes were significantly higher. Thus, this is the first time that a complete spectrum of α-dicarbonyl compounds relevant in vivo has been established. The results provide further insights into the chemistry of AGE formation and will be helpful to find specific markers to differentiate between the various precursors of glycation.

Full text of DOI:10.1074/jbc.M114.563593

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 41, pp. 28676–28688, October 10, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Extending the Spectrum of ␣-Dicarbonyl Compounds in Vivo*
Received for publication, March 7, 2014, and in revised form, August 25, 2014 Published, JBC Papers in Press, August 27, 2014, DOI 10.1074/jbc.M114.563593
Christian Henning‡, Kristin Liehr‡, Matthias Girndt§, Christof Ulrich§, and Marcus A. Glomb‡1
From the ‡Institute of Chemistry-Food Chemistry and the §Department of Internal Medicine II, Martin-Luther-University
Halle-Wittenberg, Kurt-Mothes-Strasse 2, 06120 Halle/Saale, Germany
Background: ␣-Dicarbonyls are central intermediates in the formation of advanced glycation end products (AGEs).
Results: A quantitation method for the complete spectrum relevant in vivo was established.
Conclusion: Non-enzymatic chemistry of glucose and L-ascorbic acid as precursors and ␣-dicarbonyl intermediates play an
important role in vivo.
Significance: Knowledge of plasma levels of ␣-dicarbonyls is crucial to understand the complex pathways of AGE formation in
vivo.
end products (AGEs).2 There are multiple sources and mecha-
nisms of AGE formation in vivo, involving oxidative and non-
oxidative chemistry of reducing sugars, Schiff bases, Amadori
adducts, ascorbic acid and metabolic intermediates (4, 5).
Because they are correlated to the severity of diabetic and ure-
mic complications their clinical relevance was established
(6–8).
Maillard ␣-dicarbonyl compounds are known as central inter-
mediates in advanced glycation end product (AGE) formation.
Glucose is the primary source of energy for the human body,
whereas L-threo-ascorbic acid (vitamin C) is an essential nutrient,
involved in a variety of enzymatic reactions. Thus, the Maillard
degradation of glucose and ascorbic acid is of major importance in
vivo. To understand the complex mechanistic pathways of AGE
formation, it is crucial to extend the knowledge on plasma concen-
trations of reactive key ␣-dicarbonyl compounds (e.g. 1-deoxyglu-
cosone). With the present work, we introduce a highly sensitive
LC-MS/MS multimethod for human blood plasma based on
derivatization with o-phenylenediamine under acidic conditions.
The impact of workup and reaction conditions, particularly of pH,
was thoroughly evaluated. A comprehensive validation provided
the limit of detection, limit of quantitation, coefficients of varia-
tion, and recovery rates. The method includes the ␣-dicarbonyls
1-deoxyglucosone, 3-deoxyglucosone, glucosone, Lederer’s glu-
cosone, dehydroascorbic acid, 2,3-diketogulonic acid, 1-deoxypen-
tosone, 3-deoxypentosone, 3,4-dideoxypentosone, pentosone, 1-de-
oxythreosone, 3-deoxythreosone, threosone, methylglyoxal, glyoxal;
the ␣-keto-carboxylic acids pyruvic acid and glyoxylic acid; and the
dicarboxylic acid oxalic acid. The method was then applied to the ana-
lyses of 15 healthy subjects and 24 uremic patients undergoing hemo-
dialysis. The comparison of the results revealed a clear shift in the
product spectrum. In most cases, the plasma levels of target analytes
were significantly higher. Thus, this is the first time that a complete
spectrum of ␣-dicarbonyl compounds relevant in vivo has been estab-
lished. The results provide further insights into the chemistry of AGE
formation and will be helpful to find specific markers to differentiate
between the various precursors of glycation.
AGE concentrations in vivo are markedly amplified in diabe-
tes but also in non-diabetic uremia with a significant loss of
renal clearance (9). Thus, besides substrate concentration, car-
bonyl stress, as described by Baynes et al. (10), is an alternative
explanation for the increase of chemical protein modifications
in various diseases. Carbonyl stress is caused by a generalized
increase in the concentration of reactive carbonyl precursors of
AGEs. Among those, ␣-dicarbonyl compounds (␣-DCs) play a
very prominent role. Their relevance as key intermediates in
AGE formation was extensively investigated in in vitro model
systems (11–15).
Endogenous formation is supposed to be the predominant
source of ␣-DCs in human circulation (16). However, informa-
tion on the physiological importance of exogenous dicarbonyl
intake is scarce and subject to recent investigations (17–19).
Regarding this topic, foods and peritoneal dialysis fluids are
considered to release considerable amounts of reactive ␣-DCs
(20–24).
For a comprehensive understanding of the complex formation
pathways of AGEs in vivo, the analytical assessment of the
complete ␣-DC spectrum is crucial. Nemet and Monnier (25)
examined the L-threo-ascorbic acid (ASA, vitamin C) degradation
products dehydroascorbic acid (DHA), 2,3-diketogulonic acid
2 The abbreviations used are: AGE, advanced glycation end products; ␣-DC, ␣-dicar-
bonylcompound;ASA,L-threo-ascorbicacid;DHA,dehydroascorbicacid;2,3-DKG,
2,3-diketogulonicacid(L-threo-2,3-hexodiulosonicacid);3-DT,3-deoxythresosone
(4-hydroxy-2-oxobutanal); GO, glyoxal; MGO, methylglyoxal (2-oxopropanal);
3-DG,3-deoxyglucosone(3-deoxy-D-erythro-hexos-2-ulose);OPD,o-phenylenedi-
amine; threosone, 3,4-dihydroxy-2-oxobutanal; 1-DG, 1-deoxyglucosone (1-de-
oxy-D-erythro-hexo-2,3-diulose); DHA, dehydroascorbic acid; DHAP, dihydroxyac-
etone phosphate; G3P, glyceraldehyde 3-phosphate; 1-DP, 1-deoxypentosone;
3-DP, 3-deoxypentosone; 3,4-DDP, 3,4-dideoxypentosone (3,4-dideoxypentos-2-
ulose);1-DT,1-deoxythresosone(1-hydroxy-2,3-butanedione);Q,quinoxaline;HD
patients, patients undergoing hemodialysis; 3-DGal, 3-deoxygalactosone; EtOAc,
ethyl acetate; 3,4-DGE, 3,4-dideoxyglucosone-3-ene; MeOH, methanol; CID, colli-
sion-induced dissociation; glucosone, D-arabino-hexos-2-ulose; Lederer’s glu-
cosone, N6-(3,6-dideoxyhexos-2-ulos-6-yl)-L-lysine; pentosone, pentos-2-ulose.
Advanced glycation of proteins has been investigated in
aging, diabetes, and nutrition (1–3). The Maillard reaction, a
non-enzymatic process, is initiated when proteins are exposed
to glucose or other carbohydrates. Through a series of reac-
tions, it eventually yields the irreversible advanced glycation
* This work was supported by German Federal Ministry of Education and
Research (BMBF) Project 13N11798.
1 To whom correspondence should be addressed. Fax: 49-345-55-27341;
E-mail: marcus.glomb@chemie.uni-halle.de.
28676 JOURNAL OF BIOLOGICAL CHEMISTRY
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␣-Dicarbonyl Compounds in Vivo
(2,3-DKG), threosone, 3-deoxythresosone(3-DT), andxylosonein ARMAR Chemicals (Leipzig/Doettingen, Germany). The qui-
human lens. In human blood plasma, however, only glyoxal (GO), noxaline derivatives of the ␣-DCs will be marked hereafter with
methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and DHA have the suffix “-Q”. 1-DG-Q, 3-DG-Q, glucosone-Q, Lederer’s glu-
been described in detail so far (26–33). The implemented analyt- cosone-Q, 1-DP-Q, 3-DP-Q, 3,4-DDP-Q, pentosone-Q, 1-DT-Q,
ical methods vary not only in the inevitable derivatization proce- 3-DT-Q, threosone-Q, pyruvic acid-Q, glyoxylic acid-Q, oxalic
dure but also in the chromatographic technique used. The com- acid-Q, and 3-DG were synthesized according to our previous
mon alternative approach for the quantitation of DHA is the work (37–39). The identities of target compounds were verified by
measurement of the difference of ASA before and after a reduction nuclear magnetic resonance (NMR) experiments.
step (34, 35). Consequently, concentrations of published plasma
Written informed consent was obtained from all patients.
levels of healthy subjects differ in a wide range and are thus not The study was approved by the Ethics Committee of the Med-
comparable(e.g. forGOfrom220to1150pmol/ml, forMGOfrom ical Faculty of the Martin Luther University Halle-Wittenberg.
120 to 650 pmol/ml, and for DHA from 550 to 6800 pmol/ml Blood samples were obtained from 15 healthy subjects with
(mean values)).
normal renal function and 24 non-diabetic patients undergoing
Glomb and Tschirnich compared common derivatization hemodialysis using EDTA as an anticoagulant (2 mg/ml whole
approaches (36). According to them, the use of aromatic o-di- blood). In HD patients, samples were obtained predialysis
amines (e.g. o-phenylenediamine (OPD)) is prerequisite for the before the midweek treatment session. Hemodialysis was per-
analysis of highly reactive ␣-DCs, such as 1-deoxyglucosone formed three times weekly for 4–5 h using polysulfone dialyz-
(1-DG). The detection of these short lived intermediates is lim- ers. All patients were treated with bicarbonate hemodialysis
ited by the rate of condensation of the reagent with the carbonyl (acid concentrate type 257, 8.4% sodium bicarbonate type 200,
moiety. However, these trapping reagents impose high oxida- MTN Neubrandenburg GmbH, Neubrandenburg, Germany)
tive stress on the system investigated and could lead to artifact with ultrapure water quality (by reverse osmosis and sterile fil-
formation, especially of ␣-DCs that originate from oxidative ters). Plasma was derived by centrifugation (3000 ϫ g, 10 min,
pathways (e.g. D-arabino-hexos-2-ulose (glucosone)).
4 °C) within 20 min of collection and immediately subjected to
The work group of Thornalley (31) developed a reliable the assay procedure described below. HbA1c, creatinine, and
method for the detection of MGO as 6,7-dimethoxy-2-meth- C-reactive protein were measured by routine methods at the
ylquinoxaline in human plasma. They stressed the importance central laboratory of Martin-Luther-University Clinical Cen-
of pH control to avoid the degradation of dihydroxyacetone ter, Halle (Saale, Germany).
phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to
2-(2Ј(R),3Ј(R),4Ј-Trihydroxybutyl)quinoxaline (3-DGal-Q)—
MGO, both central intermediates in several metabolic path- 3-Deoxy-D-threo-hexos-2-ulose (3-deoxygalactosone; 3-DGal)
ways in living organisms. was synthesized according to the literature (40) with the excep-
Although many methods for the detection of a few selected tion of the 3-DGal bis(benzoyl hydrazone) cleavage. Here,
␣-DCs were described already, a comprehensive method instead of benzaldehyde, sodium nitrite was used, following the
including all relevant compounds in vivo has been missing up to procedure of Henseke and Bauer (41). Purification of crude
now. The particular analytical challenge in this regard is the 3-DGal-Q was achieved by preparative high performance liquid
very diverging polarity of the target substances but also the chromatography with ultraviolet detection (HPLC-UV). NMR
baseline separation of isomeric pairs with identical molecular results were in line with those of Hellwig et al. (40).
masses (e.g. 1-/3-deoxypentosone (1-/3-DP)).
3-(D-erythro-Glycerol-1-yl)-quinoxaline-2-carboxylic Acid
We developed and validated a highly sensitive LC-MS/MS mul- o-Aminoanilide (DHA Precursor-Q)—DHA (250 mg; 1.44 mmol)
timethod suitable for routine analysis based on the derivatization was suspended in methanol (25 ml), and OPD was added (308.5
withOPD, including15␣-dicarbonylcompoundsandtwo␣-keto- mg; 2.85 mmol). The reaction mixture was heated for 2 h at
carboxylic acids. Both substance categories are hereafter referred 40 °C and allowed to cool. The precipitated solid was isolated by
to as ␣-DCs for reasons of simplicity. In detail, the method covers filtration; washed with water, ethanol, and ether; and dried
1-DG, 3-DG, glucosone, N6-(3,6-dideoxyhexos-2-ulos-6-yl)-L- (yield: 282.6 mg, 76%). Recrystallization from ethanol gave yel-
lysine (Lederer’s glucosone), DHA, 2,3-DKG, 4,5-dihydroxy-2,3- low needles.
pentanedione (1-DP), 4,5-dihydroxy-2-oxopentanal (3-DP),
1H NMR (500 MHz, DMSO-d6): ␦ (ppm) ϭ 3.44–3.55 (m,
3,4-dideoxypentosone (3,4-DDP), pentos-2-ulose (pentosone), 1-de- 2H), 4.03 (m, 1H), 5.42 (m, 1H), 6.63 (m, 1H), 6.8 (dd, J ϭ 8.0, 1.4
oxythreosone (1-DT), 3-DT, threosone, MGO, ethanedial (glyoxal, Hz, 1H), 7.0 (m, 1H), 7.36 (dd, J ϭ 8.0, 1.5 Hz, 1H), 7.96 (m, 2H),
GO), 2-oxopropanoic acid (pyruvic acid), oxoethanoic acid (glyoxylic 8.17 (m, 1H), 8.23 (m, 1H). 13C NMR (100 MHz, DMSO-d6): ␦
acid), and ethanedioic acid (oxalic acid) and was used to screen an (ppm) ϭ 63.3, 72.0, 74.5, 116.4, 116.6, 122.9, 126.4, 127.2, 128.9,
initial set of plasma samples from 15 healthy human subjects and 24 129.5, 131.0, 132.0, 139.6, 141.2, 143.2, 147.4, 156.0, 165.1.
uremic patients undergoing hemodialysis (HD patients). The results HR-MS: m/z 393.09645 (found); m/z 393.09596 (calculated for
ϩ
are discussed with respect to the sources and mechanistic relation- C18H18O4N4K [M ϩ K] ).
ships of the ␣-DCs detected.
3-(D-erythro-Glycerol-1-yl)-quinoxaline-2-carboxylic ␥-Lac-
tone (DHA-Q)—A suspension of precursor DHA-Q (500 mg) in
water (10 ml) was treated with 0.1 M hydrochloric acid (25 ml)
EXPERIMENTAL PROCEDURES
Materials and Plasma Samples—Chemicals of the highest and stirred at room temperature. The reaction process was fol-
grade available were obtained from Sigma-Aldrich and Fisher lowed by thin layer chromatography (TLC; EtOAc, UV detec-
unless otherwise indicated. NMR solvents were purchased from tion). The reaction mixture was extracted with EtOAc (3 ϫ
OCTOBER 10, 2014•VOLUME 289•NUMBER 41
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␣-Dicarbonyl Compounds in Vivo
35 ml), the organic layers were combined, and the solvent was against the authentic reference ␣-DC-Q standards. Due to
evaporated. The residue was purified by column chromatogra- the lack of authentic reference for the quinoxaline of 3,4-
phy (silica gel 60, 63–200 ␮m (Merck), EtOAc). Fractions dideoxyglucosone-3-ene (3,4-DGE-Q), quantitation was
including the compound with Rf 0.24 (TLC: EtOAc, UV detec- done with 3-DGal-Q under the assumption of an equal
tion) were collectively concentrated in vacuo to afford 180 mg extinction coefficient.
(66%) of light yellow needles. NMR results were in line with
De Novo Formation of MGO-Q from G3P and DHAP with
those of Nemet and Monnier (25). HR-MS: m/z 269.05301 OPD under Various Assay Conditions—G3P and DHAP (50 and
(found); m/z 269.05328 (calculated for C12H10O4N2Na [M ϩ 100 ␮M, which corresponds to a plasma concentration of 100
ϩ
Na] ).
and 200 ␮M, respectively) were incubated under assay condi-
2,3-DKG—2,3-DKG as sodium salt was synthesized accord- tions in amber glass vials under argon atmosphere at 22 °C in
ing to the method of Otsuka et al. (42). The product was various buffer solutions containing 0.55 mM OPD and 3.4 mM
obtained as a white hygroscopic precipitate. It was character- EDTA. The buffers used were potassium phosphate buffer (0.4
ized by the LC-MS/MS method mentioned below after M, pH 7.0), sodium formate buffer (0.4 M, pH 3.0), and perchlo-
derivatization with OPD and directly utilized for quinoxaline ric acid (0.4 M, pH Ͻ0). Vials were tightly sealed by a screw cap
synthesis.
with integrated polytetrafluoroethylene/silicone septum. Reac-
3-((1S,2R)-1,2,3-Trihydroxypropyl)-quinoxaline-2-carboxylic tion mixtures were subjected to LC-UV analysis as described
Acid (2,3-DKG-Q)—The corresponding quinoxaline of 2,3-DKG below not only after 24 h but immediately after the addition of
was obtained according to Nemet and Monnier (25). Purification OPD and after a 1-h incubation time to account for possible
of the quinoxaline solution was done by preparative HPLC-UV MGO impurities of the starting materials. The yield of MGO
(tR ϭ 62 min). Fractions including the pure compound were was determined against the authentic reference ␣-DC-Q
collectively evaporated under reduced pressure, dissolved in standards.
water, and lyophilized to afford 2,3-DKG-Q as a light yellow
Assay of ␣-DCs in Plasma—Sodium formate buffer (2 M, pH
powder in quantitative yield. NMR data were in line with the liter- 3.0, 200 ␮l), the internal standard pyruvic acid-13C2-Q (1.25
ature (25). HR-MS: m/z 263.06749 (found); m/z 263.06735 (calcu- ␮M, 100 ␮l) and the derivatizing agent OPD (2.75 mM, 200 ␮l)
ϩ
lated for C12H11O5N2 [M Ϫ H] ).
were added to 500 ␮l of blood plasma. The sample was incu-
Isotopically Labeled 2-Hydroxy-3-methyl-2,3-13C2-quinoxa- bated for 24 h in the dark at room temperature under argon
line (Pyruvic Acid-13C2-Q)—Pyruvic acid-13C2-Q was synthe- atmosphere. TFA (2 M, 250 ␮l) was added, and the incubation
sized according to the method of Arun et al. (43). Briefly, a continued for 1 h under the same conditions. The pH of the
solution of OPD in distilled water (306.9 mg in 8 ml of ultrapure sample was then adjusted to pH 3.0 with ammonium hydroxide
water, 2.84 mM) was added to 1,2-13C2-pyruvic acid (250.0 mg (4 M, 415 ␮l). Water was added (85 ␮l) to give a total sample
in 8 ml ultrapure water, 2.84 mM) dropwise with constant stir- volume of 1750 ␮l. The protein precipitate was separated by
ring. The precipitated pale yellow colored compound was fil- centrifugation (16,000 ϫ g and 20 °C). The supernatant (storage
tered, washed with water, and lyophilized. The crude sample at Ϫ80 °C) was administered to LC-MS/MS analysis.
was recrystallized from 50% ethanol absolute (404.7 mg, 88%).
HPLC-UV—A Besta HD 2-200 pump (Wilhelmsfeld, Ger-
NMR data were in line with the literature. HR-MS: m/z many) was used at a flow rate of 15 ml/min. Elution of material
163.0779 (found); m/z 163.0776 (calculated for C713C2H8ON2 was monitored by a UV detector (Jasco UV-2075 with a prepar-
ϩ
[M ϩ H] ).
ative flow cell (Gross-Umstadt, Germany)). The detection
Time Course of the OPD Reaction of 3-DG, MGO, GO, Glyox- wavelength was 320 nm. Chromatographic separations were
ylic Acid, Pyruvic Acid, Oxalic Acid, DHA Precursor-Q, and performed on a stainless steel column (KNAUER, 250 ϫ 20
2,3-DKG-Q under Assay Conditions—All compounds (20 ␮M) mm, Eurospher-100 C18, 10 ␮m (Berlin, Germany)). The
were incubated under assay conditions in amber glass vials mobile phase used consisted of water (solvent A) and metha-
under argon atmosphere at 22 °C in 0.4 M formate buffer (pH nol/water (7:3 (v/v), solvent B). To both solvents (A and B) 0.8
3.0) containing 0.55 mM OPD and 3.4 mM EDTA. Vials were ml/liter formic acid was added. Samples were injected at 5% B
tightly sealed by a screw cap with integrated polytetrafluoroeth- for 2,3-DKG-Q and at 35% B for 3-DGal-Q and purified under
ylene/silicone septum. At various time points, aliquots of the isocratic conditions.
reaction mixtures were subjected to LC-UV analysis as
NMR Spectroscopy—NMR spectra were recorded on a Varian
described below. After 24 h, trifluoroacetic acid (TFA; 0.4 M) VXR 400 spectrometer operating at 400 MHz for 1H and 100
was added, and the incubation was continued for 1 h. Then pH MHz for 13C or on a Varian Unity Inova 500 instrument oper-
3.0 was adjusted again with 4 M ammonium hydroxide. The ating at 500 MHz for H and 125 MHz for 13C, respectively.
1
injection volume was adjusted to account for the dilution of the Chemical shifts are given relative to external SiMe4.
reaction mixture. The yields were determined against the
Accurate Mass Determination (High Resolution MS)—The
authentic reference ␣-DC-Q standards.
high resolution positive and negative ion electrospray ioniza-
Time Course of the OPD Reaction of ASA, DHA, 2,3-DKG, tion mass spectra (electrospray ionization-high resolution MS)
and 3-DG-Q under Assay Conditions—All compounds (20 ␮M) were taken on a Bruker Apex III Fourier transform ion cyclo-
were incubated as described above over a prolonged time tron resonance mass spectrometer (Bruker Daltonics, Billerica,
period. No TFA was added after 24 h. At various time points, MA) equipped with an Infinity cell, a 7.0-tesla superconducting
aliquots of the reaction mixtures were subjected to LC-UV magnet, a radio frequency-only hexapole ion guide, and an
analysis as described below. The yields were determined external off-axis electrospray source (Apollo; Agilent, Santa
28678 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 41•OCTOBER 10, 2014

␣-Dicarbonyl Compounds in Vivo
TABLE 1
LC-MS/MS parameters for quinoxaline quantitation
Precursor ion
Product ion 1a
Product ion 2b
Product ion 3b
Quinoxaline and letter
assignment (see Fig. 2)
Retention
time
m/z
DPc
m/z
CEd
CXPe
m/z
CE
CXP
m/z
CE
CXP
min
amuf
265.1
251.1
162.9
221.1
235.2
247.1
235.2
191.2
205.1
147.1
205.1
363.3
175.1
355.3
160.9
163.1
175.1
189.1
131.1
145.0
V
amu
185.1
173.1
90.1
eV
V
amu
221.1
161.1
145.1
161.0
187.1
185.3
145.0
144.1
145.1
119.1
158.1
159.1
129.2
247.2
91.9
eV
V
amu
161.0
233.2
117.1
132.1
217.2
171.1
157.1
161.2
169.2
102.1
174.1
219.1
145.0
337.2
64.9
eV
V
2,3-DKG (a)
Glucosone (b)
Oxalic acid (c)
Pentosone (d)
1-DG (e)
9.5
40
55
70
50
55
55
55
45
50
65
50
65
55
52
60
60
45
55
32
50
23.0
24.0
42.0
22.0
28.0
18.0
24.0
17.5
21.0
26.5
19.5
27.0
24.0
19.0
28.0
28.0
22.0
25.0
40.0
41.0
11.0
16.0
15.0
14.0
12.0
20.0
17.0
14.0
14.0
9.0
17.0
30.0
25.0
27.0
27.0
20.0
31.0
37.0
27.0
30.0
34.0
29.0
35.0
15.0
40.0
40.0
35.0
25.0
30.0
30.5
17.0
13.0
10.0
13.0
11.0
15.0
9.0
27.0
19.0
31.0
45.0
18.0
33.0
12.0
24.0
30.0
39.0
32.5
24.0
25.5
15.0
54.0
54.0
55.0
52.0
11.0
16.0
7.0
9.9
10.5
11.2
11.9
12.0
13.9
14.0
16.8
17.1
17.1
18.4
19.2
19.5
19.5
19.5
19.6
20.5
20.9
22.3
173.1
175.1
229.0
199.1
173.1
187.1
129.0
187.2
187.1
157.2
109.2
133.1
134.0
157.1
171.2
77.0
10.0
16.0
10.0
30.0
12.0
11.0
8.5
DHA (f)
3-DG (g)
Threosone (h)
1-DP (i)
11.0
12.0
10.0
14.0
10.0
9.0
Glyoxylic acid (j)
3-DP (k)
10.0
16.0
10.0
8.0
13.0
12.0
10.0
12.0
11.0
11.0
5.0
Lederer’s glucosone
3-DT (l)
DHA precursor (m)
Pyruvic acid (n)
Pyruvic acid-13C2
1-DT (o)
16.0
7.0
10.0
10.0
7.0
92.1
7.0
64.9
156.1
145.1
104.0
118.1
15.0
11.0
8.0
89.1
3,4-DDP (p)
GO (q)
15.0
6.0
102.1
16.0
MGO (r)
77.0
5.0
7.0
65.0
45.0
5.0
a MRM transition used for quantitation (quantifier).
b MRM transition used for confirmation (qualifier).
c
DP, declustering potential.
d CE, collision energy.
e CXP, collision cell exit potential.
f amu, atomic mass units.
Clara, CA). Nitrogen was used as a drying gas at 150 °C. The to separate aliquots of the sample after the workup procedure. The
samples were dissolved in methanol, and the solutions were aliquots were analyzed, and a regression of response versus con-
introduced continuously via a syringe pump at a flow rate of 120 centration is used to determine the concentration of the analyte in
␮l/h. The data were acquired with 256,000 data points and the sample. Spikes were run for one of approximately every 30
zero-filled to 1,024,000 by averaging 32 scans.
samples. Calibration with this method resolves potential matrix
High Performance Liquid Chromatography with Coupled interferences. Potential losses during the workup procedure and
Ultraviolet-Mass Spectrometry Detection (LC-UV-MS/MS)— intrabatch changes of instrument sensitivity were corrected with
The HPLC apparatus (Jasco, Gross-Umstadt, Germany) con- pyruvic acid-13C2 as an internal standard.
sisted of a pump (PU-2080 Plus) with degasser (LG-2080-02)
To obtain fragmentation spectra of ␣-DCs-Q in plasma
and quaternary gradient mixer (LG-2080-04), a column oven workup solutions, target material was first enriched by repeated
(Jasco Jetstream II), an autosampler (AS-2057 Plus), and a UV collection from the above HPLC system. After solvent evapora-
detector (UV-2075). Mass spectrometric detection was con- tion in a vacuum concentrator (Savant SpeedVac Plus SC 110 A
ducted on an API 4000 QTrap LC-MS/MS system (Applied combined with a Vapor Trap RVT 400, Thermo Fisher Scien-
Biosystems/MDS Sciex, Concord, Canada) equipped with a tific GmbH (Dreieich, Germany)), the residue was dissolved in
turbo ion spray source using electrospray ionization in positive water and reinjected, using a CID experiment. The fragmenta-
mode: sprayer capillary voltage, 4.5 kV; nebulizing gas flow, 50 tion spectra of the authentic references were obtained with the
ml/min; heating gas, 60 ml/min at 550 °C; and curtain gas, 40 same parameters. For 1-DG-Q, the following parameters were
ml/min.
used: declustering potential, 50 V; collision energy, 30 eV; col-
Chromatographic separation was performed on a stainless lision cell exit potential, 10 V; scan range, m/z 130–250 (4 s). All
steel column packed with RP-18 material (KNAUER, 250 ϫ 4.6 plasma workup samples and incubations were analyzed in sin-
mm, Eurospher-100 C18, 5 ␮m (Berlin, Germany)) using a flow gle batches to exclude interassay variations.
rate of 1.0 ml/min. The mobile phase used was water (solvent A)
Validation of the Quantitation Method—Intraassay coeffi-
and methanol/water (7:3 (v/v), solvent B). To both solvents (A and cients of variation were determined by repeated workup and
B), 0.6 ml/liter heptafluorobutyric acid was added. Analysis was analysis of a plasma sample (n ϭ 6). In addition, the limit of
performed at 25 °C column temperature using gradient elution: detection and limit of quantitation with all steps of the analysis
5% B (0–0.1 min) to 35% B (0.5 min) to 47% B (12 min) to 100% B included were estimated according to DIN 32645 (n ϭ 6, con-
(17–25 min). For mass spectrometric detection, the scheduled fidence level p ϭ 0.95, k ϭ 3) (44). To determine recovery rates,
multiple-reaction monitoring mode was used, utilizing collision- the respective alpha-DCs-Q were added at four different con-
induceddissociation(CID)oftheprotonatedmoleculeswithcom- centrations to three parallel sets of blood samples of one sub-
pound-specific orifice potentials and fragment-specific collision ject. The native and the spiked plasma sample were subjected to
energies(Table1). Quantitationwasperformedusingthestandard the assay of ␣-DCs in plasma as described above. The recovery
addition method. More precisely, increasing concentrations of rate was estimated as the quotient of (spiked ␣-DC-Q
authentic ␣-DC-Q reference compounds at factors of 0.5, 1, 2, and amount Ϫ amount ␣-DC-Q in native plasma sample)/amount
3 times the concentration of the analyte in the sample were added of added ␣-DC-Q ϫ 100%.
OCTOBER 10, 2014•VOLUME 289•NUMBER 41
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␣-Dicarbonyl Compounds in Vivo
RESULTS
Development of an LC-MS/MS Method for 17 Relevant Dicar-
bonyl Compounds as Their Corresponding Quinoxalines—The
complex matrix and the low concentration of most target ana-
lytes hamper the use of UV detectors for qualitative and quan-
titative analysis. Tandem mass spectrometric detection in the
multiple-reaction monitoring mode was prerequisite for the
detection of the 17 relevant ␣-DCs in blood plasma. To achieve
highly sensitive detection utilizing collision-induced dissocia-
tion of the protonated molecules, the compound-specific ori-
fice potentials and fragment-specific collision energies had to
be determined by authentic reference material (Table 1).
HPLC separation was based on a RP-18 phase with methanol
␣-Dicarbonyl Compounds in Blood Plasma of Healthy
Human Subjects and Hemodialysis Patients—Plasma was
obtained from 15 healthy subjects with normal renal function
and no proteinuria and 24 uremic patients undergoing hemo-
dialysis (HD patients). Details are given in Table 2. Normal
renal function was defined as a serum creatinine level below 102
␮mol/liter. To ensure the absence of inflammatory reactions,
C-reactive protein had to be 5 ␮mol/liter or below for all
healthy subjects.
After derivatization of target ␣-DCs to the respective qui-
noxalines and separation from the protein residue, the plasma
samples were subjected to the described LC-MS/MS method. as the organic eluent. Optimization of gradient, column tem-
All plasma workup samples of controls and HD patients were
each analyzed in single batches to exclude interassay variations.
Intraassay coefficients of variation were determined by inde-
pendent analyses of three different blood samples for each sub-
ject and were Ͻ10% in all cases. The results are presented in
Table 3.
perature, and ion pair reagent led to almost baseline separation
of all critical quinoxalines possessing the same molecular
weight, which therefore could not be distinguished via MS
detection (1-DG-Q versus 3-DG-Q versus 4-DG-Q versus
3-DGal-Q, 1-DP-Q versus 3-DP-Q, 1-DT-Q versus 3-DT-Q).
Fig. 2 shows the typical chromatogram of a plasma sample after
derivatization with OPD.
In selected cases, the identity of the detected compounds in
plasma was confirmed by comparison of the native fragmenta-
tion spectrum with authentic reference standards. The results
were in full compliance and are exemplarily shown for 1-DG-Q
Verification of the Derivatization Procedure—The reactivity
of ␣-DCs required conversion into stable quinoxaline deriva-
tives. After the blood sample is drawn from the living subject,
the complex system blood is prone to significant alterations
regarding its biological activity and chemical composition,
especially under exposure to oxygen. Immediate plasma gener-
ation and instant start of the derivatization procedure under
well defined conditions is therefore prerequisite for reproduc-
ible quantitative results.
ϩ
in Fig. 1. The protonated molecular ion [M ϩ H] at m/z 235.2
is expected to undergo dehydration to the ion at m/z 217.2 and
199.2. Subsequent decay leads to signals at m/z 191.0, 187.1,
175.1, 171.2, and 158.1.
TABLE 2
Profile of subjects examined in this study
To prevent underestimation of rather slow reacting com-
pounds, OPD was chosen as the derivatization agent. It has a
comparatively high conversion rate of ␣-DCs to the respective
quinoxaline derivatives. To ensure complete derivatization,
selected native ␣-DCs (GO, MGO, glyoxylic acid, pyruvic acid,
oxalic acid, and 3-DG) were incubated with OPD under stan-
Healthy subjects
HD patients
No. of participants
15
24
Sex, female/male
6/9
6/18
Age (years)
24–34
37 Ϯ 2
82 Ϯ 13
Ͻ5
30–85
40 Ϯ 17
861 Ϯ 322
11 Ϯ 21
HbA1c (mmol/mol)
Serum creatinine (␮mol/liter)
C-reactive protein (mg/liter)
TABLE 3
Levels of all relevant ␣-dicarbonyl compounds in human blood plasma
Values shown are the mean Ϯ S.D. of 15 healthy subjects and 24 patients undergoing hemodialysis (HD patients), replicate analyses, n ϭ 3. Student’s t test was used for
statistical evaluation of significant differences between both groups: *, p Ͻ 0.01 versus healthy subjects; **, p Ͻ 0.001 versus healthy subjects.
Healthy subjects
Mean ؎ S.D.
HD patients
Quinoxaline
Range
Mean ؎ S.D.
Range
pmol/ml
pmol/ml
Glucosone
46 Ϯ 11
22 Ϯ 3
28–67
96 Ϯ 49**
57–276
1-DG
16–28
35–56
30 Ϯ 16
12–82
3-DG
43 Ϯ 5
65 Ϯ 20**
7.0 Ϯ 2.5a
11 Ϯ 5a
36–125
Lederer’s glucosone
Pentosone
1-DP
ϽLOD
ϽLOD–13
ϽLOD–23
3.2–15
15 Ϯ 10a
3.6 Ϯ 1.2a
11 Ϯ 2
ϽLOD–32
2.6–7.8
6.2 Ϯ 2.6a**
33 Ϯ 9**
3-DP
9–17
20–53
Threosone
1-DT
5.4 Ϯ 0.7a
3.1 Ϯ 0.3
10 Ϯ 2
4.2–6.6
9.0 Ϯ 3.5a**
3.8 Ϯ 1.0*
45 Ϯ 11**
219 Ϯ 129**
1273 Ϯ 980**
4.2–19
2.6–3.8
2.0–6.6
3-DT
8–14
28–75
MGO
61 Ϯ 7
51–76
42–617
GO
491 Ϯ 47
7250 Ϯ 2549
1264 Ϯ 353
40 Ϯ 21
6.0 Ϯ 1.0
687 Ϯ 351b
405–564
2818–12,038
783–1942
18–82
400–4914
8399–97,935
1026–3514
16–70
Pyruvic acid
Glyoxylic acid
Oxalic acid
3,4-DDP
DHA precursor
DHA
35,874 Ϯ 19,080**
2031 Ϯ 608**
36 Ϯ 12
4.4–7.7
19 Ϯ 8**
6–42
337–1303
6809–23,967
567–2669
1925 Ϯ 1106b**
12,538 Ϯ 7183b
411 Ϯ 238c**
308–4829
2737–26,680
145–937
15,400 Ϯ 4019b
1741 Ϯ 674c
2,3-DKG
a
LOD Ͻ ϫ Ͻ LOQ.
b The sum of DHA-Q and DHA precursor-Q account for approximately 38% of the true DHA content.
c Values adjusted as described under “Disussion.”
28680 JOURNAL OF BIOLOGICAL CHEMISTRY
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␣-Dicarbonyl Compounds in Vivo
FIGURE 1. Verification of 1-DG by CID of m/z 235.2 [M ؉ H]؉. A, authentic reference; B, plasma workup.
FIGURE 2. LC-MS/MS chromatogram of a plasma sample after standard
workup procedure. For clarity, only the quantifier mass transition for each
analyte of the scheduled multiple-reaction monitoring is shown. For letter
assignments (a–r), see Table 1.
FIGURE 3. Reactions of selected dicarbonyl compounds with OPD in for-
mate buffer (0.4 M, pH 3.0) at room temperature: 3-DG (ࡗ), MGO (●), GO
(f), pyruvic acid (ꢀ), glyoxylic acid (Œ), and oxalic acid (hexagons).
estimation of ␣-DCs in trace amounts, avoiding overestimation
dard assay conditions, and the formation of respective quinoxa- of certain dicarbonyl quinoxalines is an even bigger challenge.
lines was monitored between 0.5 and 48 h. The results pre- For example, de novo formation of glucosone-Q and GO-Q
sented in Fig. 3 revealed complete reaction (Ϯ5%) within 11 h, during derivatization with OPD are described in the literature
with the short-chained substances reacting much more rapidly, (22, 36, 45). The main reasons are the strong oxidative condi-
which is in accordance with our previous findings (36). The tions imposed on the system by the addition of OPD in combi-
exception was oxalic acid, which showed no reaction with OPD. nation with oxygen and trace amounts of metal ions. However,
Furthermore, the stability of all quinoxaline compounds with our derivatization procedure, a stable plateau for all ana-
included in our method was monitored with authentic refer- lytes was reached within the relevant incubation time, indicat-
ences under the derivatization conditions for 24 h. No degra- ing no or negligible de novo formation in the plasma matrix. In
dation was observed.
this regard, it is important to note that the incubation took
In addition, the derivatization of a plasma sample was mon- place under argon atmosphere to minimize the impact of oxy-
itored by repeated injection of aliquots and LC-MS/MS ana- gen. Furthermore, EDTA as a metal ion-chelating agent was
lyses of the complete ␣-DC spectrum. Besides the risk of under- present in the assay.
OCTOBER 10, 2014•VOLUME 289•NUMBER 41
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␣-Dicarbonyl Compounds in Vivo
TABLE 4
Conversion rate in mol % of G3P and DHAP, respectively, to MGO over
an incubation period of 24 h in the indicated medium in presence of
0.55 mM OPD and 3.4 mM EDTA
G3P
DHAP
100 ␮M 200 ␮M 100 ␮M 200 ␮M
mol %
mol %
pH Ͻ0; 0.4 M perchloric acid
21.3
21.2
8.5
8.3
pH 3.0; 0.4 M sodium formate buffer 1.4
pH 7.0; 0.4 M potassium phosphate 18.3
buffer
1.5
7.7
7.6
19.8
96.6
98.6
FIGURE 4. Degradation of 3-DG-Q at pH 0.6 (open symbols) and pH 3.0
(closed symbols): 3-DG (ࡗ), 3-DGal (●), and 3,4-DGE (Œ).
The plasma samples were diluted in formate buffer (pH 3.0).
McLellan and Thornalley (31) applied different derivatization
procedures to biological systems and discovered significant
interferences from G3P and DHAP by spontaneous elimination
of phosphate to form MGO during sample processing, depend-
ing on the chosen pH value. The rate of reaction increased with
increasing pH. To avoid the degradation of G3P and DHAP, we
conducted the derivatization step under strong acidic condi-
tions with perchloric acid. However, under these harsh condi-
tions, we discovered significant degradation of 3-DG-Q,
FIGURE 5. Reaction of DHA with OPD in formate buffer (0.4 M, pH 3.0) at
room temperature yields DHA precursor-Q (E), DHA-Q (●), and 2,3-
DKG-Q (Œ).
1-DP-Q, 3-DT-Q, and threosone-Q (data not shown). Espe- ences found were inevitable, and the results were comparable
cially 3-DG-Q with a half-life of 80 h was rapidly degraded to with the well established method of McLellan. To ensure the
form the diastereomer 3-DGal-Q (Fig. 4). Mittelmaier et al. (46) absence of contaminants in water or chemicals used in the assay
already investigated the formation of 3-DGal in peritoneal dial- possibly leading to overestimation, a reactant blank was incu-
ysis fluids under sterilization conditions. They postulated a bated along with the plasma samples.
reaction mechanism that includes a reversible dehydration of
Mechanistic Relationship of DHA, 2,3-DKG, and Their Cor-
free 3-DG leading to 3,4-dideoxyglucosone-3-ene (3,4-DGE). responding Quinoxalines under Assay Conditions—Investiga-
Subsequently, 3,4-DGE undergoes hydration to form 3-DGal. tion of the derivatization reaction of DHA in sodium formate
Obviously, this type of reaction also takes place with the corre- buffer (0.4 M, pH 3.0) with OPD (0.55 mM) gave rather surpris-
sponding quinoxalines under harsh acidic assay conditions. ing results shown in Fig. 5 and in the scheme in Fig. 6. Initially,
Whereas with the native ␣-DC, the carbonyl moiety is the driv- a precursor compound (DHA precursor-Q) with two molecules
ing force, with the quinoxaline, it is the extension of conjugated of OPD was formed. The precursor then converted to DHA-Q.
unsaturation.
However, after 24 h, no plateau was reached, and the precursor
We monitored the degradation of 3-DG-Q in sodium for- compound was still detectable in significant amounts. A quan-
mate solution at different pH values (data not shown). Based on titative reaction of DHA with OPD was only achieved after
our findings, we chose more gentle conditions and employed a more than 6 days, which is not feasible for the present analytical
formate buffer (0.4 M, pH 3.0). Here, no degradation of 3-DG-Q assessment. In addition, oxidative conditions imposed by OPD
at 21 °C for 48 h was observed. In addition, formation of MGO lead to formation of DHA from ASA. When 20 ␮M ASA was
caused by oxidative degradation of nucleic acids and related incubated under assay conditions, 50 mol % of the ASA was
compounds during the derivatization process is accelerated by converted to DHA-Q after 6 days, whereas after 24 h, de novo
strong acids like perchloric acid but negligible with milder formation of DHA-Q from ASA was negligible (Ͻ2%).
acidic treatment (47, 48).
In contrast, conversion of 2,3-DKG to its corresponding qui-
To ensure reliable MGO quantitation with the method noxaline is completed after 8 h (data not shown). Obviously,
described herein, we conducted control experiments at differ- DHA is the least reactive of all ␣-DCs assessed (with the excep-
ent pH conditions with 100 and 200 ␮M concentrations of G3P tion of oxalic acid). Consequently, it is important to note that
and DHAP, respectively (Table 4). The chosen concentrations the plasma levels of DHA-Q and DHA precursor-Q given in
were based on the experiments conducted by McLellan and Table 3 have to be added and account for ϳ38% of the true
Thornalley (31). The conversion rate at pH 3.0 after 24 h in mol DHA plasma content.
% was below 2% for G3P and below 8% for DHAP, independent
To monitor the conversion of DHA precursor-Q to DHA-Q,
from the starting concentration of both. The minor interfer- the precursor was synthesized independently. The authentic
28682 JOURNAL OF BIOLOGICAL CHEMISTRY
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␣-Dicarbonyl Compounds in Vivo
FIGURE 8. Degradation of 2,3-DKG-Q (Œ) under standard workup condi-
tions yields DHA-Q (●).
DKG-Q were detected (7%). A reverse reaction of the quinoxa-
lines to form free DHA with subsequent hydrolytic opening of
the lactone ring system to yield 2,3-DKG and its corresponding
quinoxaline 2,3-DKG-Q can be excluded. Thus, DHA-Q has to
be hydrolyzed slowly to form 2,3-DKG-Q directly. To further
investigate the relationship of the two quinoxalines, 2,3-
DKG-Q was incubated under assay conditions, including the
protein precipitation step with TFA after 24 h. The results in
Fig. 8 show conversion of 2,3-DKG-Q to DHA-Q, which was
significantly triggered at the lower pH during the 1-h precipi-
tation step. 2,3-DKG-Q yields ϳ25% DHA-Q after the com-
plete workup procedure. Obviously, there is a steady state
between DHA-Q and 2,3-DKG-Q with preferred DHA-Q for-
mation as the more stable product under acidic conditions.
Validation of the Proposed Method—The limit of detection
and limit of quantitation with all steps of the analytical method
included were calculated as described under “Experimental
Procedures.” The limit of detection differs from 0.5 to 42.2
pmol/liter. The accuracy of the method was determined as the
recovery rate at three different concentration levels and ranged
between 82 and 120%. Repeatability was expressed as the coef-
ficient of variation and was 10% or better. The complete valida-
tion data are shown in Table 5.
FIGURE 6. Mechanistic relationship of DHA, 2,3-DKG, and their corre-
sponding derivatization products.
DISCUSSION
Up to now, only GO, MGO, 3-DG, and DHA were quanti-
tated in human plasma using established methods, although the
spectrum of ␣-DCs in vivo is supposed to be far more complex.
Major endogenous sources are the degradation of blood glu-
cose and ascorbic acid. As a result, the corresponding degrada-
tion products already identified in model experiments should
also be present in vivo. Beyond that, lipid peroxidation as well as
enzymatic and non-enzymatic pathways irrespective of Mail-
FIGURE 7. Formation of DHA-Q (●) and 2,3-DKG-Q (Œ) starting from DHA
precursor-Q (E) under standard workup conditions.
reference DHA precursor-Q was then incubated under assay lard reactions contribute to the content of ␣-DCs. It is specu-
conditions (Fig. 7). A half-life of 5 h was observed. After 24 h, lated that even exogenous sources like foods add to the plasma
81% of DHA precursor-Q was converted to DHA-Q. Residual level of ␣-DCs.
amounts of DHA precursor-Q (10% based on initial concentra-
Because derivatization with OPD is prone to both underesti-
tion) remained and had to be considered for the calculation of mation of ␣-DC levels and de novo formation during the
DHA concentration. Interestingly, also small amounts of 2,3- derivatization procedure, recovery levels for each compound
OCTOBER 10, 2014•VOLUME 289•NUMBER 41
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␣-Dicarbonyl Compounds in Vivo
TABLE 5
of the triose phosphates G3P and DHAP are known sources for
MGO. Oxidation of acetone in the catabolism of ketone bodies,
oxidation of aminoacetone in the catabolism of threonine, deg-
radation of proteins glycated by glucose, and degradation of
ASA also contribute to the total plasma content of MGO. GO is
formed by DNA oxidation, lipid peroxidation (52), sugar autox-
idation (53), and oxidative degradation of glycated proteins
(11). GO and MGO are both detoxified mainly by the glyoxalase
system with glutathione as a cofactor to give glycolate and lac-
tate, respectively (55). Because C2- and C3-fragments originate
by various non-enzymatic and enzymatic pathways besides the
breakdown of glucose and ASA, they must be evaluated as very
vague parameters from the mechanistic point of view. The
same applies for pyruvic acid as a central intermediate in several
metabolic processes, which explains its high concentration in
plasma.
Coefficient of variation (CV), recovery rates, limit of detection (LOD),
and limit of quantitation (LOQ) (all steps of the analysis included) of
plasma samples
Quinoxaline
CVa
Recoveryb
LODb
LOQb
%
%
109
112
97
pmol/ml
pmol/ml
18.3
6.6
Glucosone
6
6.1
1-DG
4
2.2
3-DG
5
5.4
16.2
10.5
24.9
7.5
c
Lederer’s glucosone
Pentosone
1-DP
—
114
98
3.5
7
9
8
3
3
3
8
9
2
4
7
3
8.3
82
2.5
3-DP
93
3.5
10.5
12.6
1.5
Threosone
1-DT
90
4.2
91
0.5
3-DT
98
2.0
6.0
MGO
106
98
3.0
9.0
GO
6.9
20.7
42.3
126.6
20.4
2.4
Pyruvic acid
Glyoxylic acid
Oxalic acid
3,4-DDP
DHA precursor
DHA
99
14.1
42.2
6.8
92
89
110
0.8
d
d
—
—
2.9
8.7
The C6-dicarbonyl compound glucosone and its analogues
1-DG and 3-DG do not arise from ascorbate degradation and
therefore are markers for glucose-derived ␣-DCs in the context
of Maillard chemistry (see scheme in Fig. 9). 3-DG is formed
non-oxidatively from the Amadori product of glucose via 1,2-
enolization and dehydration, whereas 2,3-enolization yields
1-DG (56). Oxidation of the Amadori compound leads to glu-
10
6
91
22.9
6.1
68.7
18.3
2,3-DKG
119
a Repeatability conditions, n ϭ 6.
b Replicate analyses, n ϭ 3.
c Estimation not possible because analyte was below the limit of detection.
d Estimation not possible because compound is the intermediate in DHA-Q
formation.
were thoroughly evaluated, and conversion conditions were cosone (37). Besides from the Maillard reaction, an important
kept strictly constant. A blank was prepared for each derivatiza- endogenous route leading to 3-DG formation from glucose is
tion set to monitor the quality of all incorporated reagents. The the enzymatic polyol pathway (57). Although the bioconversion
derivatization period was adjusted to provide quantitative con- of glucose into glucosone by pyranose oxidase for synthetic
version of all ␣-DCs except DHA and oxalic acid, which will be purposes is described in the literature (58), no such enzymatic
discussed below. The sample deproteinization step by the addi- pathway has been identified in vivo so far.
tion of TFA was kept as short as possible. Immediately after
3-DG is reviewed in the literature as the most abundant
protein separation, the pH was adjusted to pH 3.0 again. Under C6-dicarbonyl in vivo (59) but possesses only a very limited
these conditions, even critical quinoxaline structures like glycating reactivity (37). Thus, the chemistry of 3-DG has to
3-DG-Q proved to be stable, and oxidative degradation of be considered as of minor relevance regarding Maillard pro-
nucleic acids and related compounds as well as degradation of cesses under physiological conditions. This is supported by
␦
G3P and DHAP, which will lead to overestimation of MGO, the fact that 3-DG-derived AGEs (e.g. N -[5-hydro-5-(2,3,4-
were avoided.
trihydroxybutyl)-4-imidazolon-2-yl]ornithine (3-DG-H1), 6-(2-
␣-DCs are known to exhibit a high reactivity toward N-ter- formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine (pyrraline),
⑀
minal amino acids and amino acid residues of proteins, espe- and N -{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-
cially to the sulfhydryl group of cysteine, the guanidino group of [(2S,3R)-2,3,4-trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-
arginine, and the ⑀-amino group of lysine. Because these amino ylidene}-l-lysinate (DODIC or DOGDIC)) are only of minor
functionalities are readily available in excess in human blood quantitative importance in blood plasma. 1-DG and glucosone
plasma, we reason that nearly all ␣-DCs are bound reversibly or were identified as the central intermediates leading to C4- and
irreversibly to these residues. In the case of MGO, in vitro C5-fragments, respectively (60). Their reductone structure with
experiments of Lo et al. (49) showed that only 1% exists in a free an ␣-oxo-enediol moiety boasts significantly higher reactivity.
form. The reversibly bound MGO is in a dynamic equilibrium More generally, this applies to all analog C4- and C5-dicar-
with its free form and can be measured by trapping with OPD bonyls. As reported before, the half-life of 1-DG is about 0.5 h
(50, 51). Therefore, our approach included the separation of the under physiological conditions (61) versus 8 h for glucosone
high molecular plasma fraction only after the derivatization (37) and 40 h for 3-DG (36). Hence, 1-DG is by far the most
procedure to cover the total amount of free and reversibly reactive and thus important ␣-DC intermediate regarding glu-
bound ␣-DCs in a reliable way. Although, the definition of cose-derived AGEs. In particular, amine-induced ␤-cleavage in
“reversibly bound ␣-DCs” remains the object of critical discus- the presence of lysine leads directly to carboxylic acid amides, a
sion (48) because some reversible reactions under assay condi- novel class of amide-AGEs (14, 62) that are of quantitative
tions might be instead irreversible under physiological condi- importance in vivo (9).
tions, the good validation results proved the reliability of the
newly developed workup and derivatization procedure.
Lederer’s glucosone was not detected in the plasma of
healthy subjects, although it is a relative stable non-reductone
The formation of MGO in physiological systems was studied structure like 3-DG. However, unlike 3-DG, the required eno-
extensively in the literature, reviewed recently by Rabbani and lization along the entire carbon backbone makes this ␣-DC sus-
Thornalley (16). The enzymatic and spontaneous degradation ceptible to multiple degradation processes. The existence of
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␣-Dicarbonyl Compounds in Vivo
FIGURE 9. Formation pathways of ␣-DCs from glucose and ascorbic acid.
Lederer’s glucosone in vivo is evident from the detection of its of DHA. In addition, we confirmed that ASA and DHA did not
AGE follow-up structure glucosepane at low levels in human yield any C4- or C5-dicarbonyl degradation compounds during
blood and extracellular matrix (63, 64).
the incubation period. Indeed, the plasma level of 2,3-DKG can
DHA is a C6-dicarbonyl structure exclusively assigned to the be estimated fairly accurately, taking into account the compre-
degradation of ASA, formed by oxidation. As mentioned above, hensively investigated relationship of DHA-Q and 2,3-DKG-Q
the DHA level in plasma can only be roughly estimated by the under assay conditions. Based on our results, 6.5% of DHA-Q
analytical assay described herein, mainly because of the incom- was converted to 2,3-DKG-Q during the derivatization time of
plete conversion to its corresponding quinoxaline in the given 24 h (Figs. 7 and 8). On the other hand, 25% of the formed
time period. However, DHA in aqueous solution hydrolyzes 2,3-DKG-Q was converted to DHA-Q. The values given in
irreversibly to 2,3-DKG, which is the central intermediate of Table 3 for 2,3-DKG are already adjusted accordingly. The
ASA degradation (65). Therefore, it is important to differenti- adjustments described above are only valid if DHA-Q is in sig-
ate between DHA as part of the redox equilibrium ASA-DHA, nificant excess, which is given by the situation in blood plasma.
mediated in vivo by enzymatic pathways, and 2,3-DKG as the Only in this case, the change in DHA-Q level due to the con-
direct precursor of fragmentation products with a carbon back- version is negligible compared with that of 2,3-DKG-Q.
bone smaller than C6.
␣-DCs with a carbon skeleton smaller than C6 arise from the
The yields of 2,3-DKG-Q from DHA in Fig. 5 and from DHA degradation of glucose as well as of ASA. As established in pre-
precursor-Q in Fig. 7 after 24 h were almost equal, which cer- vious papers of our group (39, 66), the C4-dicarbonyls
tainly excludes de novo formation of 2,3-DKG via hydrolyzation threosone, 1-DT, and 3-DT are formed from both 1-DG and
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␣-Dicarbonyl Compounds in Vivo
2,3-DKG via ␤-dicarbonyl cleavage with an C4-enediol as the
The presented LC-MS/MS method provides for the first time
reactive intermediate. Oxidation of the latter leads to threosone, the opportunity to identify and quantitate the complete spec-
whereas dehydration at C3 results in 3-DT. The enediol may also trum of relevant ␣-DCs in plasma of healthy human subjects in
undergo isomerization to give 1-DT in an equivalent reaction. As a a single chromatographic run. For 14 compounds, the plasma
consequence under deaeration, which represents the situation in levels were determined. Three compounds were below the limit
vivo, 3-DT was the prominent structure because the reductone of quantitation but were unequivocally identified. 10 sub-
1-DT has to be considered a much more reactive and, thus, short stances have not been reported for human plasma samples
lived intermediate.
before. To evaluate the clinical relevance of the assay described
It has to be noted that differentiation between l-threo-pen- herein, an initial set of 24 uremic patients undergoing hemodi-
tos-2-ulose (arising from ASA and often referred to as alysis was analyzed. Uremia is related to an increase in oxidative
xylosone) and the C4-stereoisomer D-erythro-pentos-2-ulose and carbonyl stress and thus should lead to a clear shift in the
(arising from glucose) was not possible. For the applied LC- dicarbonyl spectrum. Indeed, most ␣-DCs were considerably
MS/MS method, MRM parameters were determined with higher in HD patients. Glucose-derived glucosone exhibited a
L-threo-pentos-2-ulose. In model incubations of glucose and 2-fold increase, which is expected under conditions of elevated
lysine, a signal with retention time and mass transitions identi- oxidative stress. In contrast, 1-DG does not require an oxida-
cal to those of L-threo-pentos-2-ulose was detected (data not tion step for its formation and remained nearly at the level of
shown). By definition, with glucose as precursor, the detected healthy subjects. Interestingly, plasma levels of 2,3-DKG were
compound has to be D-erythro-pentos-2-ulose. Thus, both considerably decreased in HD patients. This must be explained
structures coelute and are therefore summarized under the by a significantly accelerated degradation via oxidative path-
term pentosone regardless of its origin. Yet, the plasma levels of ways. GO and MGO are further compounds of published inter-
pentosone were comparatively high and did not fit into the est in regard to certain chronic diseases like uremia. A 3-fold
picture. Decarboxylation is a well established mechanism of elevation of GO and a 4-fold increase of MGO were observed in
ASA degradation and leads to C5-compounds, including pen- uremic plasma. This is in line with the literature (27, 29, 32),
tosone (65, 67), but under physiological conditions, the forma- although the absolute values differ significantly, depending on
tion of C4-dicarbonyls from 2,3-DKG is favored (25, 39). Glu- the respective study. The assessment of ␣-DC plasma levels
cose-derived pentosone stems from glucosone by the same depends strongly on the analytical approach, specifically on
mechanism of hydrolytic ␤-cleavage as threosone from 1-DG as workup conditions, derivatization procedure, and chromato-
the precursor. After isomerization and hydration, formic acid is graphic method. Most importantly, the derivatization was con-
cleaved off and gives an C5-1,2-enediol as the reactive interme- ducted in the presence of protein to assess both free and revers-
diate. Oxidation leads to pentosone, whereas water elimination ibly bound ␣-DCs. Therefore, a direct comparison of the
yields 3-DP or, after 2,3-enolization, 1-DP (60). Considering the plasma levels of the present study and those reported previously
need for an oxidation step in order to obtain pentosone from is not possible.
both 2,3-DKG or glucosone, formation of 1-DP and 3-DP
should be favored. The results therefore strongly suggest an tion of ␣-DC compounds in plasma has been carried out exten-
additional alternative source for pentosone formation in vivo. sively regarding the formation of artifacts and mechanistic rela-
Nevertheless, validation of the present method for the detec-
In Maillard chemistry, glyoxylic acid is assigned to disaccha- tionships to exclude false quantitative data. In general, the
ride chemistry (38) but can also arise from oxidation of glyoxal elevated levels of ␣-DCs found explain the elevated levels of
(68) and degradation of DHA (65, 69). However, this cannot AGEs in uremia. The newly developed method has now to be
account solely for the plasma levels, which were 2.5-fold higher extended to follow-up studies with patients with various com-
than GO and in the same range as 2,3-DKG. An alternative plications. The results for a wide range of highly reactive car-
source is the degradation of hydroxyproline with the subse- bonyl intermediates will help us to understand the complex
quent glyoxylate metabolism in the human organism, which is mechanisms and factors that influence ␣-DC formation and
rather complex, involving several enzymatic and non-enzy- consequently open new perspectives regarding the formation
matic reactions, and has been subject to recent investigation and relevance of AGE chemistry in vivo.
(54).
Oxalic acid can originate via ␤-dicarbonyl fragmentation as
well as via oxidative ␣-DC cleavage from 2,3-DKG and is the
main degradation product of the latter (39). In addition, oxalate
is also part of the glyoxylate metabolism mentioned above.
However, under assay conditions, the dicarboxylic acid oxalic
acid is not converted to its corresponding quinoxaline. This is
expected, because at the chosen workup pH, the carboxylic acid
groups do not show sufficient carbonyl activity to react with
OPD. Consequently, there must be alternative precursors for
oxalic acid-Q formation to oxalic acid itself. 3,4-DDP is a
known intermediate of maltose degradation but was found in
neither the glucose nor ASA reaction systems (38). Hence, the
origin of the detected 3,4-DDP-Q remains unknown.
Acknowledgment—We thank Dr. J. Schmidt (Leibnitz Institute of
Plant Biochemistry, Halle, Germany) for performing accurate mass
determination.
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