Metabolization of the Advanced Glycation End Product N-?-Carboxymethyllysine (CML) by Different Probiotic E. Coli Strains

Article abstract of DOI:10.1021/acs.jafc.8b06748

N-?-Carboxymethyllysine (CML) is formed during glycation reactions (synonym, Maillard reaction). CML is degraded by the human colonic microbiota, but nothing is known about the formation of particular metabolites. In the present study, six probiotic E. coli strains were incubated with CML in the presence or absence of oxygen in either minimal or nutrient-rich medium. CML was degraded by all strains only in the presence of oxygen. HPLC-MS/MS was applied for identification of metabolites of CML. For the first time, three bacterial metabolites of CML have been identified, namely N-carboxymethylcadaverine (CM-CAD), N-carboxymethylaminopentanoic acid (CM-APA), and the N-carboxymethyl-?¹-piperideinium ion. During 48 h of incubation of CML with five different E. coli strains in minimal medium in the presence of oxygen, 37-66% of CML was degraded, while CM-CAD (1.5-8.4% of the initial CML dose) and CM-APA (0.04-0.11% of the initial CML dose) were formed linearly. Formation of the metabolites is enhanced when dipeptide-bound CML is applied, indicating that transport phenomena may play an important role in the "handling" of the compound by microorganisms.

Full text of DOI:10.1021/acs.jafc.8b06748

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Bioactive Constituents, Metabolites, and Functions
Metabolization of the advanced glycation end product N-#-
carboxymethyllysine (CML) by different probiotic E. coli strains
Michael Hellwig, Christian Auerbach, Nicole Müller, Pauline Samuel,
Sophie Kammann, Falco Beer, Florian Gunzer, and Thomas Henle
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06748 • Publication Date (Web): 31 Jan 2019
Downloaded from http://pubs.acs.org on January 31, 2019
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Journal of Agricultural and Food Chemistry
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Metabolization of the advanced glycation end product N-ε-carboxymethyllysine (CML)
by different probiotic E. coli strains
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Michael Hellwig,1 Christian Auerbach,2 Nicole Müller,1,2 Pauline Samuel,1,2 Sophie
Kammann,1,2 Falco Beer,1 Florian Gunzer,2 Thomas Henle1
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1 Chair of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany
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Institut für Medizinische Mikrobiologie und Hygiene, Medizinische Fakultät Carl Gustav
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Carus, Technische Universität Dresden, Fetscherstr. 74, 01307 Dresden, Germany
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Abstract
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N-ε-carboxymethyllysine (CML) is formed during glycation reactions (synonym, Maillard
reaction). CML is degraded by the human colonic microbiota, but nothing is known about the
formation of particular metabolites. In the present study, six probiotic E. coli strains were
incubated with CML in the presence or absence of oxygen either in minimal or nutrient-rich
medium. CML was degraded by all strains only in the presence of oxygen. HPLC-MS/MS was
applied for identification of metabolites of CML. For the first time, three bacterial metabolites
of CML have been identified, namely N-carboxymethylcadaverine (CM-CAD), N-
carboxymethylaminopentanoic acid (CM-APA) and the N-carboxymethyl-Δ1-piperideinium
ion. During 48 h of incubation of CML with five different E. coli strains in minimal medium in
the presence of oxygen, 37–66% of CML was degraded, while CM-CAD (1.5–8.4% of the
initial CML dose) and CM-APA (0.04–0.11% of the initial CML dose) were formed linearly.
Formation of the metabolites is enhanced when dipeptide-bound CML is applied, indicating
that transport phenomena may play an important role in the “handling” of the compound by
microorganisms.
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Keywords
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Maillard reaction; N-ε-Carboxymethyllysine (CML); E. coli; metabolism; biogenic amine;
probiotic strain
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Introduction
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N-ε-carboxymethyllysine (CML) is an advanced glycation end product formed during the
Maillard reaction (synonyms—non-enzymatic browning, glycation) in food, but also under
physiological conditions. In the Maillard reaction, reducing sugars first react with amino and
imino groups of free amino acids, peptides, and proteins to form the so-called Amadori
rearrangement products (ARPs). By this reaction, N-ε-fructosyllysine is formed at the ε-amino
group of lysine. “Blockage” by this compound can afford for the modification of up to 30% of
the side-chains of the essential amino acid lysine in milk products and up to 80% in bakery
products.1,2 ARPs degrade in the second step of the reaction, what gives rise to 1,2-dicarbonyl
compounds such as 3-deoxyglucosone, methylglyoxal, and glyoxal. These compounds can also
be formed directly from reducing sugars.3,4 In the last stage of the Maillard reaction, reactive
1,2-dicarbonyl compounds react with the nucleophilic side-chains of protein-bound amino acids
to form the so-called “advanced glycation end products (AGEs)”, mainly alkylated lysine
derivatives, pyrrole and pyridinyl compounds that include the ε-amino group of lysine and
hydroimidazolones at the guanidino group of arginine.1,5 The alkylated lysine derivative N-ε-
carboxymethyllysine (CML, Figure 1) has first been described as an unusual metabolite in
children’s urine and only later as to result from the Maillard reaction.6,7 Different routes of
formation have been described for this compound: The reaction of glyoxal with lysine including
an intramolecular Cannizzaro reaction, the oxidative degradation of intermediate Schiff bases
(Namiki pathway) and ARPs, respectively, which is facilitated in alkaline environments.8,9,10,11
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The most reliable quantitative data for CML in food are based on chromatographic means such
as high-pressure liquid chromatography with tandem mass-spectrometric detection (HPLC-
MS/MS). CML is most abundant in bakery products such as bread and biscuits (1.3–46 mg/kg),
high-heat processed meat products (0.9–48 mg/kg), evaporated milk (4.7–46 mg/kg), processed
nuts (2.1–48 mg/kg), pasta products (3–16 mg/kg), and chocolate (5–37 mg/kg).12,13,14,15,16
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daily dietary intake between 2.1 and 11.3 mg CML was estimated.17,18,19 Reliability of
quantitation of CML by an immunological method, which counterintuitively detected the
highest quantities of CML in fat-rich and virtually protein-free foods and nearly none in bakery
products,20 is more and more put into question.21,22,23 However, based on this latter
methodology, many in vivo studies resulted in the detection of adverse effects of CML intake
such as inflammatory processes, endothelial dysfunction, insulin resistance, and oxidative
stress. Most of these studies are not considered conclusive due to methodological shortcomings
(for reviews, see references 24,25,26).
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The metabolic fate of CML has been a subject of several works in the recent decade.
Predominantly protein-bound in foods,27 CML is released from proteins during intestinal
luminal digestion into absorbable peptides.28 Works on Caco-2 cells suggest that CML is taken
up into enterocytes when it is bound in dipeptides and then may get into circulation to a small
extent.29 Between 15 and 25% of dietary CML is excreted via the urine in humans; the higher
the ingested amount, the lower the fraction that is excreted renally.18 Incorporation of dietary
CML into plasma and tissue proteins, especially the heart, has been suggested but is still a
matter of controversy.17,30,31 The predominant part of dietary CML is transferred into the large
intestine because it is either not absorbed or released from desquamated enterocytes. Between
20 and 30% of ingested CML is excreted via the feces in humans; the higher the ingested
amount, the higher the fraction that is excreted with the feces.18 Human and animal studies
consistently show that dietary CML is not fully recovered in urine and feces.18,32,33,34
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It was shown that CML is degraded by the human colonic microbiota with strong interindividual
differences.35 Positive effects of heat-treated foods and especially of CML on intestinal
dysbiosis have been shown in colitic mice.36,37 Biological effects of dietary constituents in an
organism may not only be evoked by the compounds themselves, but also by their metabolites,
especially when the microbiota is involved. As there are no studies available on metabolites of
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CML, we intended to elucidate possible metabolization pathways of this compound in the
present study by using well-characterized probiotic E. coli strains as model
microorganisms.38,39,40
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Materials and Methods
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Chemicals. Luria-Bertani agar (Miller) and M9 minimal salts (5×) were purchased from Becton
Dickinson (Heidelberg, Germany). Glacial acetic acid and D-glucose were from Roth
(Karlsruhe, Germany). Nonafluoropentanoic acid (NFPA), o-dianisidine, N-Boc-cadaverine,
palladium on activated charcoal (10%), 5-aminopentanoic acid and brain heart infusion (BHI)
broth were obtained from Sigma-Aldrich (Steinheim, Germany). Calcium chloride dihydrate,
L-cysteine, sodium citrate dihydrate and magnesium sulfate were obtained from Merck
Millipore (Darmstadt, Germany) and [2H2]CML from PolyPeptide (Strasbourg, France).
Acetonitrile was from Fisher Scientific (Schwerte, Germany), and methanol and hydrochloric
acid were from VWR (Darmstadt, Germany). Resazurin sodium salt, glyoxylic acid
monohydrate and 5-aminopentanol were purchased from Alfa Aesar (Karlsruhe, Germany).
The cation exchange resin DOWEX 50WX8 and formic acid were obtained from Acros
Organics (Geel, Belgium). Sodium hydroxide and potassium iodide were from Grüssing
(Filsum, Germany), and ninhydrin from Serva (Heidelberg, Germany). The syntheses of CML
and alanyl-(N-ε-carboxymethyl)-lysine (Ala-CML) were performed according to the
literature.29
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Preparation of strains for metabolic experiments. E. coli strains (Table 1) were supplied by
SymbioPharm (Herborn-Hörbach, Germany). The strains were routinely precultured using
Luria-Bertani agar (LB agar) with concentrations of 15.0 g/L agar, 5.0 g/L yeast extract, 10.0
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g/L sodium chloride, and 10.0 g/L tryptone, which were dissolved in doubly distilled water (pH
= 7.0 ± 0.2) followed by autoclaving (122 °C, 20 min). Liquid agar was poured into Petri dishes
(d = 9.4 cm) after cooling to 50 °C. BHI medium for aerobic experiments was prepared by
dissolving 7.40 g of BHI broth in 200 mL of doubly distilled water (pH = 7.4 ± 0.2) followed
by autoclaving (122 °C, 20 min). Minimal medium for aerobic experiments was prepared as
follows: An autoclaved stock solution of M9 minimal salts was diluted with autoclaved water
and added with a sterile filtered glucose-salt mix to yield final concentrations of 5 g/L
ammonium chloride, 0.01 g/L calcium chloride, 33.9 g/L disodium hydrogen phosphate, 3.91
g/L D-glucose, 0.24 g/L magnesium sulphate, 15.0 g/L potassium dihydrogen phosphate, and
2.5 g/L sodium chloride (pH = 6.8 ± 0.2). For anaerobic experiments, 0.4 g/L L-cysteine and
0.001 g/L resazurin were added to the media. Media were aliquoted into Hungate tubes (Ochs,
Bovenden-Lenglern, Germany) and autoclaved (122 °C, 20 min) after three cycles of
alternating vacuum application (0.04 bar) and nitrogen overpressure (0.4 bar).35
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Cryocultures of E. coli strains in glycerol medium were spread on LB agar plates by use of an
inoculating loop and incubated at 37 °C overnight in an incubator (TH30, Edmund Bühler,
Bodelshausen, Germany). Single colonies were transferred into 5 mL of autoclaved liquid
medium in aerobic culture tubes (12 mL) and the suspensions were incubated overnight at 37
°C in a table-top incubator under agitation (225 rpm). On the next day, the OD600 of the bacterial
suspension was measured after appropriate dilution with PBS buffer using the spectrometer
Evolution 201 UV (Thermo Scientific, Dreieich, Germany) in cuvettes (V = 1.5 mL) at
extinctions not exceeding 0.3. In order to perform the incubation of test substrates in the
exponential growth phase, the overnight cultures were diluted with the respective media to yield
OD600 values of 0.06 in BHI medium, or 0.3 in minimal medium, respectively. After 80 min of
incubation under exclusion of light (37 °C, 225 rpm), the OD600 was checked again, and
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incubations with MRPs were only started if the optical density had reached values between 0.5
and 0.7.
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For the preparation of anaerobic cultures in BHI medium, single colonies of E. coli were
inoculated in 100 µL of BHI medium and transferred into a Hungate tube containing 5.7 mL of
anaerobic BHI medium. After overnight incubation, 100 µL of the suspension was transferred
to a new Hungate tube containing 5.7 mL of anaerobic BHI medium. The suspensions were
preincubated for 80 min (37 °C, 225 rpm) before the addition of MRP solution.
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For anaerobic cultures in minimal medium, each of two cultures of single colonies of E. coli
was inoculated in 124 µL of glucose-salt mix (1 M glucose, 0.1 M MgSO4, 4.5 mM CaCl2),
added to 5.7 mL anaerobic minimal medium in a Hungate tube by means of a sterile syringe
and grown overnight. Both overnight cultures were combined and after centrifugation and
removal of the supernatant, the residue was suspended in 0.4 mL of minimal medium. Then,
0.1 mL was mixed with 124 µL of glucose-salt mix and transferred to 5.6 mL of anaerobic
minimal medium in a Hungate tube by means of a sterile syringe. The suspensions were
preincubated for 80 min (37 °C, 225 rpm) before the addition of MRP solution.
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Incubation of MRPs in the presence or absence of E. coli strains. The following substances
were subjected to incubations with E. coli: CML, Ala-CML, N-carboxymethylcadaverine (CM-
CAD),
N-carboxymethylaminopentanoic
acid
(CM-APA),
and
N-
carboxymethylaminopentanol (CM-APO). Sterile-filtered (0.2 µm) MRP solutions were added
to the preincubated bacterial suspensions in order to yield a final concentration of 250 µM as
in our previous study.35 Two additional control samples were run in parallel: In order to monitor
the stability of the MRPs in the absence of bacteria, MRPs were incubated in the medium alone
at a concentration of 250 µM. Metabolization of medium components was monitored by
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incubating bacterial suspensions without adding MRPs. The mixtures were incubated at 37 °C
for up to 72 h under constant agitation (225 rpm).
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Samples (200 µL) were taken directly after mixing (0 h), and after 4 h, 24 h, 48 h, and 72 h and
stored at -22 °C until analysis. The optical density of the suspensions was measured in parallel.
When incubations were performed in Hungate tubes, 200 µL samples were taken by means of
a sterile syringe.
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Amino acid analysis. CML, Ala-CML, and lysine were quantitated with the amino acid
analyser S4300 (Sykam, Fürstenfeldbruck, Germany) using a PEEK column filled with the
cation exchange resin LCA K07/Li (150 × 4.6 mm, 7 µm). The respective lithium buffers were
also obtained from Sykam and employed for custom gradient programs utilized previously.28,29
Post-column derivatization with ninhydrin was performed, and the absorbance of the eluate was
monitored with a two-channel photometer (λ = 440 nm, 570 nm). For amino acid analysis, 50
µL of the samples taken during the incubations was diluted with 50 µL of methanol. After
lyophilization, 100 µL of 10 mM NFPA was added, and 50 µL of the solution was mixed with
150 µL of 0.12 N lithium citrate buffer, pH 2.20. External calibration was performed with the
standards of CML and Ala-CML synthesized previously.29 The injection volume was 50 µL.
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High-Pressure Liquid Chromatography with Mass Spectrometric Detection. This was
performed using the Agilent system 1200 Series (Agilent Technologies, Böblingen, Germany),
consisting of a binary pump, an online degasser, a column oven and an autosampler. The
column used was a stainless steel column (Zorbax 300 SB-C18, 50 mm × 2.1 mm, 3.5 µm;
Agilent). Solutions of 10 mM NFPA in doubly distilled water (solvent A) and of 10 mM NFPA
in acetonitrile (solvent B) were used as the eluents. The chromatographic system was connected
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to the mass spectrometer 6410 Triple Quad (Agilent), working in the positive mode with a
capillary voltage of 4000 V and a source temperature of 350 °C. The gas flow was 11 L/min
(N2), and the nebulizer pressure was set at 35 psi. Data acquisition and evaluation were
performed with the software Mass Hunter B.02.00 (Agilent).
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System 1 was utilized for qualitative screening for potential metabolites. A linear gradient from
2% B to 47% B in 11 minutes was applied at a flow rate of 0.25 mL/min and a column
temperature of 35 °C. During 3 to 11 min, the detector was operated in the SCAN mode (m/z
80–400; dwell time, 500 ms; fragmentor voltage, 100 V). Samples taken during incubation (100
µL) were mixed with 100 µL of methanol. The mixtures were lyophilized and taken up in 200
µL of 10 mM NFPA. After centrifugation (10,621 × g, 20 °C, 10 min), the supernatants were
transferred to HPLC vials, and 5 µL was injected. The same method was used for recording
product ion scans by modifying fragmentor voltage (60–135 V) and collision energy (0–30 eV)
at preset m/z ratios.
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System 2 was utilized for the quantitation of CML by a stable-isotope dilution assay.35 A linear
gradient from 10% B to 34% B in 8 minutes was applied at a flow rate of 0.25 mL/min and a
column temperature of 35 °C. The transitions m/z 205→130 (Fragmentor voltage, 100 V;
collision energy, 9 eV) and m/z 207→130 (80 V; 5 eV) were recorded for quantitation of CML
and [2H2]CML, respectively. Simultaneously, the transitions m/z 205→142 (100 V; 9 eV) and
m/z 207→144 (80 V; 12 eV) were recorded for confirming the presence of the analytes.
Transitions were recorded between 3 and 9 min (dwell time, 250 ms). External calibration was
performed with a standard of CML between 0.9 and 26.8 pmol per injection. Prior to HPLC-
MS/MS analysis, 50 µL of the samples taken during incubation was mixed with 50 µL of
methanol. Then, 900 µL of HPLC eluent A (10 mM NFPA) was added. After mixing and
centrifugation (16.060 × g, 10 min, 4 °C), 100 µL of the supernatant was diluted with 60 µL of
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eluent A and 40 µL of a 0.25 mg/L solution of [2H2]CML in water. After mixing, the solution
was transferred to an HPLC vial, and 5 µL was injected.
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System 3 was utilized for the analysis of metabolites of CML. The same device and eluents
were used as described above, but a gradient from 2% B to 66% B was formed during 15 min.
The transitions used for the quantitation of the analytes (quantifier, Q) and for the confirmation
of the presence of the analytes (qualifier, q) are compiled in Table 2. Standard addition was
performed for quantitation as follows: For the first sample, 100 µL of the supernatant was mixed
with 20 µL of water. For the second sample, 100 µL of the supernatant was mixed with 10 µL
of water and 10 µL of a standard solution. For the third sample, 100 µL of the supernatant was
mixed with 20 µL of the standard solution. In the standard solution, the CML metabolites had
the following concentrations: CM-CAD (132.4 µg/L), CM-APA (105.1 µg/L), CM-APO (93.7
µg/L). Different addition standard solutions were utilized when the metabolites were incubated
in the presence of E. coli strain G1/2. The concentration of the incubated metabolite was
increased by a factor of 100, while the concentrations of the other compounds were left as stated
above.
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Semi-preparative ion exchange chromatography – general procedures. All separations of
synthesis mixtures were accomplished by using a column (1.5 cm × 50 cm) filled with 80 mL
of the strong cation-exchange resin DOWEX 50WX8 in the sodium form. The column was
reconditioned before each fractionation by rinsing first with 250 mL of 6 M HCl, 250 mL of
water, 250 mL of 1 M NaOH, 250 mL of water, and lastly with 250 mL of 0.1 N sodium citrate
buffer, pH 3.0 (loading buffer). Samples were prepared in loading buffer and membrane filtered
(0.45 µm). After adjustment of the pH value (≤ 3.0), samples were applied by gravity. The
column was first rinsed with 40 mL of loading buffer and then with the buffers as mentioned
below. Starting from sample application, the eluate was collected in 6-mL fractions using a
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fraction collector (Model 2110, Bio-Rad Laboratories, München, Germany). The presence of
products in the samples was monitored by a spotting test. Aliquots (1 µL) of each fraction were
transferred to a TLC plate and sprayed with ninhydrin or modified Reindel-Hoppe reagent41
after drying. For preparation of the ninhydrin reagent, 100 mg ninhydrin was dissolved in 100
mL ethanol, and 3 mL glacial acetic acid was added. Prior to detection with the second reagent,
plates were stored in a chlorine atmosphere for 30 min and then air-dried for 30 min. The
reagent was prepared by dissolving 16 mg o-dianisidine in a mixture of 50 mL water and 3 mL
glacial acetic acid, to which 100 mg potassium iodide was added. Fractions containing primary
or secondary amines showed characteristic black spots. Target fractions were then pooled, and
the pH of the solution was adjusted to 2.0 with 6 M HCl. Desalting was performed with a
column (2.5 cm × 15 cm) filled with 70 mL of the strong cation-exchange resin DOWEX
50WX8. The column was reconditioned by rinsing first with 250 mL of 6 M HCl and then 250
mL of water. After application of the sample, the column was rinsed with 250 mL of water, 250
mL of 1 M HCl, and 250 mL of 4 M HCl. The fraction eluting with 4 M HCl was evaporated
to dryness, taken up in water and lyophilized.
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Characterization of synthesized compounds. Proton NMR spectra were recorded on an
AVANCE III HD Nanobay 400 MHz UltraSield device from Bruker (Rheinstetten, Germany)
at 400.13 MHz. Chemical shifts are given in parts per million (ppm), relative to the internal
HOD signal (4.70 ppm). Coupling constants (J) are reported in Hz. Elemental analysis data
were obtained on a Vario Micro Cube CHNS elemental analyser (Elementar, Hanau, Germany).
For calculating the product content of synthesized compounds, the percentage of nitrogen
determined in the preparation was divided by the theoretical percentage of nitrogen and the
content expressed in per cent by weight. HPLC-MS/MS system 1 was employed for the
determination of molecular mass and fragmentation behaviour of the synthesized compounds.
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Synthesis of N-carboxymethylcadaverine (CM-CAD) 2. Boc-cadaverine (809 mg, 4.0 mmol)
and glyoxylic acid monohydrate (369.2 mg, 4.0 mmol) were dissolved in 30 mL of water, and
the pH was adjusted to 8.75 with conc. NaOH. After addition of 50.0 mg palladium on activated
charcoal, the mixture was stirred under a hydrogen atmosphere overnight. Then, the catalyst
was filtered off, water was added to the mixture to a final volume of 50 mL, and 50 mL 6 M
HCl was added. The solution was stirred at room temperature for 1 h to remove the Boc
protecting group. The mixture was evaporated to dryness using a rotary evaporator and taken
up in 30 mL 0.1 N sodium citrate buffer, pH 3.0 before chromatographic separation. Elution
was performed first with 300 mL of 0.3 N sodium citrate buffer, pH 5.2, and then with 600 mL
of 0.3 N sodium citrate buffer, pH 5.5. The product eluted between 230 mL and 550 mL of the
latter buffer. After desalting of the respective fractions and lyophilization, 2 was obtained as a
light yellow product and stored at -18 °C.
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Analytical data: H-NMR (400 MHz, D2O), δ [ppm]: 1.42 (m, 2H, H-3); 1.69–1.75 (m, 4H,
H-2, H-4); 2.96 (t, 2H, J = 6.9 Hz, H-1); 3.07 (t, 2H, J = 7.6 Hz, H-5); 3.83 (s, 2H, H-6). HPLC-
MS/MS (system 1): tR, 9.5 min; fragmentation (100 V, 10 eV) of [M + H]+ (m/z 161): 86 (100),
144 (44), 98 (42), 161 (6). Elemental analysis: C7H16N2O2 (MW = 160.21), calculated, C
52.48%, H 10.07%, N 17.48%; found, C 35.53%, H 7.48%, N 11.78%; content = 67.4%, based
on nitrogen. Yield: 446.9 mg (47%).
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Synthesis of N-carboxymethylaminopentanoic acid (CM-APA) 3. Aminopentanoic acid
(468.5 mg, 4.0 mmol) and glyoxylic acid monohydrate (368.0 mg, 4.0 mmol) were dissolved
in 30 mL of water, and the pH was adjusted to 8.75 with conc. NaOH. After addition of 50.0
mg palladium on activated charcoal, the mixture was stirred under a hydrogen atmosphere
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overnight. Then, the catalyst was filtered off, and water was added to the mixture to a final
volume of 75 mL. After addition of 500 mg sodium citrate dihydrate, the pH was adjusted to
3.0 with 6 M HCl before chromatographic separation. Elution was performed first with 250 mL
of 0.15 N sodium citrate buffer, pH 3.25, and then with 250 mL of 0.15 N sodium citrate buffer,
pH 3.5. The product eluted between 10 mL and 100 mL of the latter buffer. After desalting of
the respective fractions and lyophilization, 49.2 mg of a white product was obtained, which was
stored at -18 °C.
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Analytical data: 1H-NMR (400 MHz, D2O), δ [ppm]: 1.59–1.75 (m, 4H, H-3, H-4); 2.40 (t, 2H,
J = 7.0 Hz, H-2); 3.07 (t, 2H, J = 7.6 Hz, H-5); 3.81 (s, 2H, H-6). HPLC-MS/MS (system 1):
tR, 4.8 min; fragmentation (100 V, 10 eV) of [M + H]+ (m/z 176): 112 (100), 101 (58), 55 (33),
84 (33), 176 (23), 83 (20), 76 (12), 140 (10), 130 (8), 59 (8), 158 (3). Elemental analysis:
C7H13NO4 (MW = 175.18), calculated, C 47.99%, H 7.48%, N 8.00%; found, C 35.85%, H
6.09%, N 6.19%; content = 77.4%, based on nitrogen. Yield: 49.2 mg (5%).
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Synthesis of N-carboxymethylaminopentanol (CM-APO) 4. 5-Aminopentanol (416.0 mg,
4.0 mmol) and glyoxylic acid monohydrate (370.9 mg, 4.0 mmol) were dissolved in 30 mL of
water, and the pH was adjusted to 8.75 with conc. NaOH. After addition of 50.0 mg palladium
on activated charcoal, the mixture was stirred under a hydrogen atmosphere overnight. Then,
the catalyst was filtered off, and water was added to the mixture to a final volume of 50 mL.
After addition of 500 mg sodium citrate dihydrate, the pH was adjusted to 3.0 with 6 M HCl
before chromatographic separation. Elution was performed with 350 mL of 0.2 N sodium citrate
buffer, pH 3.5. The product eluted between 80 mL and 200 mL of the elution buffer. After
desalting of the respective fractions and lyophilization, 4 was obtained as a white product and
stored at -18 °C.
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Analytical data: H-NMR (400 MHz, D2O), δ [ppm]: 1.38 (m, 2H, H-3); 1.53 (m, 2H, H-2);
1.69 (m, 2H, H-4); 3.06 (m, 2H, H-5); 3.65 (t, 2H, J = 6.4 Hz, H-1); 3.83 (s, 2H, H-6). HPLC-
MS/MS (system 1): tR, 5.4 min; fragmentation (100 V, 10 eV) of [M + H]+ (m/z 162): 69 (100),
162 (76), 116 (68), 98 (68), 76 (59), 144 (18), 87 (18). Elemental analysis: C7H15NO3 (MW =
161.20), calculated, C 52.16%, H 9.38%, N 8.69%; found, C 41.32%, H 7.48%, N 7.12%;
content = 81.9%, based on nitrogen. Yield: 146.4 mg (19%).
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Results and discussion
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Identification of metabolites of CML formed by E. coli. As a model organism for our studies
on CML metabolism, E. coli was chosen due to its quantitative importance in the gut
microbiome and its ability to grow both under aerobic and anaerobic conditions. The strains
were the six probiotic strains that constitute the probiotic formulation Symbioflor 2.38,42
Properties of individual strains are compiled in Table 1. Initially, strain G1/2 was regarded as a
representative of the Symbioflor strains, and growth and CML metabolizing activity of this
strain were investigated under different conditions. The strain was able to grow both under
aerobic and anaerobic conditions in BHI and minimal media (Figures 2A and 2B). Under the
experimental conditions used, the stationary growth phase was reached at the latest after 24 h.
CML was fully stable in the utilized media when they were not inoculated with bacteria as
determined by the stable-isotope dilution assay. Degradation of CML was observed only in the
presence of bacteria under aerobic conditions and was more pronounced in minimal medium
than in BHI medium (Figures 2C and 2D). The apparent stability of CML in M9 medium under
anaerobic conditions cannot be ascribed to metabolic inactivity of the strain, since degradation
of 250 µM lysine was observed to be complete after 24 h (data not shown). In BHI medium,
CML degradation was delayed as compared to minimal medium and started only after 24 h
when the stationary growth phase had been reached. This shows that E. coli G1/2 does not revert
to “unusual” substrates unless it faces inadequate nutrient conditions after consumption of the
medium. The same effect was observed with the other strains (data not shown). Considering all
strains except G5 which did not grow in minimal medium, 96 ± 7% of CML had remained after
24 h when strains were grown in BHI medium under aerobic conditions, but significantly less
CML (89 ± 6%, P < 0.05) had remained after 24 h in the presence of strains in minimal medium.
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As BHI medium is very rich in amino acids that may interfere with the detection of specific
metabolites from CML, all following experiments were performed with the use of minimal
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medium under aerobic conditions. First, strain G1/2 was incubated with CML for 24 h, and LC-
MS/MS analyses were performed in the scan mode (m/z 100–250) in comparison to a G1/2
culture that had been incubated in parallel without CML. Mass-by-mass chromatogram
extraction revealed differences in the peak patterns of the extracted ion chromatograms for m/z
161 and m/z 176. The respective peaks were present in the cultures that had been incubated with
CML, but not in those incubated without CML. They were formed during incubation, because
they were not present in the cultures at the beginning of the experiment. Since mainly
protonated monoisotopic molecular ions are detected by ESI-MS under the experimental
conditions chosen in this study, we concluded that the substances causing these peaks should
have molecular masses of 160 Da and 175 Da, respectively, and should be equivalent to the
biogenic amine CM-CAD 2 and the acid CM-APA 3, respectively (Figure 1). The potential
metabolites were synthesized from the respective 5-aminopentane derivatives by reductive
alkylation in the presence of glyoxylic acid.29 In addition to the acid 3 and the amine 2, CM-
APO 4 as a further possible metabolite from the Ehrlich pathway of amino acids was
synthesized.43,44 This compound has been used as a linker in the course of studies concerning
RNA backbone modification.45 CM-CAD 2 was used as a linker in the synthesis of antibiotic
conjugates.46 The acid 3 had been detected as an acid hydrolysis product of an enzymatic
metabolite of the alkali-induced amino acid L-D-lysinoalanine (LAL).47 None of these
compounds has yet been discussed as a metabolite of CML. In order to analyze these
metabolites, a method was established based on HPLC-MS/MS working in the MRM mode
with quantitation by standard addition. The limits of detection and quantitation as well as the
conversion rate of CML to the respective metabolite at the concentration of the LOD are
compiled in Table 3.
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In parallel to the decrease of CML as measured by amino acid analysis (Figure 3A), the
formation of CM-CAD 2 was observed by HPLC-MS/MS (Figure 3B). The formation of 2 was
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already observable after 4 hours and linearly increased during the incubation time to reach 2.5
± 0.4% of the initial CML dose after 48 h of incubation. Regarding the decrease in the CML
concentration of 34.5 ± 6.1% in the same time, apparently not more than 7% of the decline can
be explained by the formation of this metabolite by this strain. CM-APA 3 as a further
metabolite was not observed before 24 h of incubation and accounted for only 0.1% of the initial
CML dose thus explaining ca. 0.3% of the degradation of CML. CM-APO 4 was not formed.
We conclude that further metabolites must exist which were not discovered under the
experimental conditions used in this study. The formation of CM-APA 3 requires the
intermediate formation of the aldehyde 5, which unites a secondary amine group and an
aldehyde group in the same molecule in close proximity. A chemical degradation of such an
intermediate through formation of the piperidine derivative 6 and further dehydration to the N-
carboxymethyl-Δ1-piperideinium cation 7 may not be excluded (Figure 4). This cation was
searched for by comparison of extracted ion chromatograms recorded after incubation of CML
with the strain G4/9. The respective peak of a substance showing an m/z of 142 was detected
only when CML had been incubated in the presence of E. coli. It was neither produced in the
absence of CML nor in the absence of E. coli, nor was it present at the beginning of the
experiment (Figure 5A). Moreover, it was detected when the lyophilization step which could
theoretically invoke dehydration of 6, was omitted. The product ion spectrum of m/z 142
recorded at the retention time of the peak revealed that the main fragments of the ion m/z 142
are m/z 114 and m/z 96 (Figure 5B). The transition m/z 142 → 96 can be explained by
elimination of HCOOH as proposed in Figure 5C. Loss of elements of HCOOH is common to
amino acid fragmentation reactions.48 The transition m/z 142 → 114 may be due to retro-Diels-
Alder reaction under elimination of ethylene.49 The formation of N-substituted piperideine
derivatives, which necessarily are cations, may be a unique feature of N-ε-alkylated lysine
derivatives during microbial metabolism. Piperideine as such has been detected as a metabolite
of lysine in the biosynthesis of piperidine alkaloids in Nicotiana species.50
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The detection of a metabolic pathway of an unusual amino acid in a whole organism may be
precluded by the inappropriate choice of the substrate. In S. cerevisiae, metabolization of
glycated amino acids became visible only after application of glycated dipeptides instead of
glycated amino acids.43 Therefore, alanyl-(N-ε-carboxymethyl-)-lysine (Ala-CML) was applied
to E. coli strain G1/2 as a glycated dipeptide in minimal medium under aerobic conditions. The
dipeptide was degraded very fast under formation mainly of CML (Figures 6A and 6B). A
certain chemical instability of Ala-CML was observed when it was incubated in M9 medium in
the absence of E. coli strains: After 24 h, 84 ± 8% of the compound was left, but the metabolites
2 and 3 were not detected. As the majority of E. coli peptidases are located in the cytoplasm,51
we postulate that Ala-CML is transported into the cell and cleaved to alanine and CML. Then,
CML is either metabolized or removed from the cells by (an) efflux system(s). This postulate
is substantiated by the observation that metabolite formation turns into a biphasic course as
compared to the application of free CML (Figure 6C). The initial strong increase of the
metabolite concentrations may be ascribed to fast peptide transport providing high amounts of
substrates intracellularly. From 7 h on, the rate of metabolite formation is approximating the
time course observed during the incubation of free CML, because extracellular peptide is
expended and now free CML must be internalized by (less effective) amino acid transport
systems. Transport of unusual amino acids and peptides by E. coli, but also generally by
intestinal microorganisms, needs to be explored more intensely.
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Further, we investigated whether E. coli strain G1/2 is able to metabolize the compounds 2, 3,
and 4, because literature data indicated that some E. coli strains can degrade biogenic amines.52
When strain G1/2 was incubated with 250 µM of CM-CAD 2 or CM-APA 3, no significant
change in the concentrations was detected during 48 h of incubation. However, the
concentration of CM-APO 4 decreased by 15.4 ± 0.7% during 48 h. CM-APA 3 and CM-CAD
2 as potential metabolites were not detectable. We conclude that CM-CAD 2 and CM-APA 3
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are stable end products of CML metabolism of the E. coli strains investigated in this study. A
possible pathway of metabolization of CM-APO 4 may be the oxidation to the aldehyde 5 and
the further degradation of the compound (Figure 4).
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Phenotypic comparison of six E. coli strains. The probiotic formulation Symbioflor is
constituted by a mixture of six E. coli strains (Table 1) that slightly differ in their genome.38 In
a preliminary experiment, we wanted to elucidate as to whether these differences have an
influence on the degradation of CML and on the formation of metabolites. Strain G5 was not
culturable in minimal medium and had to be excluded from these investigations. The other
strains were cultivated in the same way as described for strain G1/2.
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During 48 h of incubation with or without addition of CML to the minimal medium, all strains
reached optical densities between 1.0 and 1.3. As becomes apparent from the calculation of the
relative OD, all strains reproducibly grew better when CML had been added to minimal medium
than in the absence of the compound (Table 4). This may be indicative of a use of CML as a
nitrogen and/or carbon source by the strains. Differences were detected in the ability of the
strains to degrade CML and to produce the metabolites CM-CAD 2 and CM-APA 3. These
differences were qualitatively, but not quantitatively reproducible. Therefore, Table 4 presents
the data of one experiment with two biological replicates from the same stock culture. The
genetically closely related strains G1/2, G6/7, and G8 behaved very similarly in degrading CML
and in producing both CM-APA 3 and CM-CAD 2.38 In the presence of strain G4/9, the highest
proportion of CML was removed and the formation of CM-CAD 2 was most pronounced. Strain
G3/10 was least able to degrade CML and to produce the metabolites, although it grew as well
as strain G4/9. Higher production of CM-CAD 2 correlated with a higher production of CM-
APA 3. This may indicate that the pathways of metabolization have a similar activity relative
to another, and that metabolization of free CML in general should depend on the ability of the
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compound to get into the bacterial cell. Thus, differences in the metabolization may be due to
differences in amino acid membrane transport. Differences in intracellular metabolic activity
resulting from potential differences in gene expression, enzyme activity or substrate affinity
may also not be excluded. Further insight into the biochemical background of our findings may
be gained by metatranscriptomic analysis of the strains. This would allow the identification of
genes that are potentially involved in CML transport or metabolism. Such genes later could be
searched for in the genomes of other intestinal bacteria. Together with the incubation
experiments as described in the present study, this would help to identify further species that
are able to metabolize CML.
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Discussion. CML 1 is a polyfunctional amino acid that may undergo metabolic reactions at
different molecular sites (Figure 1). The biogenic amine 2 was identified as the main metabolite
of CML in this study. Decarboxylation of amino acids with the formation of biogenic amines
is a common metabolic pathway in many gut bacterial species, among them Clostridia,
Bifidobacteria, and Enterobacteriaceae such as E. coli and Lactobacilli.53,54 The production of
biogenic amines from amino acids was described as to provide cellular protection for
Enterobactericeae in an acidic environment, which prevails in the large intestine (pH
5.5–6.8).55 The ability to produce biogenic amines is widespread among intestinal bacteria with
pronounced inter-species variations in the individual amines that are formed (e.g., putrescine,
cadaverine, tyramine, spermine, spermidine).52 The acid 3 was identified as a further CML
metabolite. Similarly to other glycated amino acids,43 CML 1 may be subject to the Ehrlich
pathway, a metabolic route that is present not only in yeasts, but also in lactic acid bacteria.44
The Ehrlich pathway involves first the transamination of an amino acid to an α-keto acid 8,
which is then oxidatively decarboxylated to an aldehyde 5. In the case of CML, the aldehyde 5
may be reduced to the alcohol CM-APO 4 or oxidized to the fatty acid CM-APA 3.44 The α-
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keto acid 8 may also be formed through oxidation by L-amino acid oxidases, which can be
present in bacteria.56 Lastly, CM-CAD 2 may be a precursor of 3, because biogenic amines can
be oxidized to aldehydes via amine oxidases that also have been described in several
microorganisms.57
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In summary, the present study shows for the first time that E. coli strains are able to degrade
CML to defined metabolites with the biogenic amine CM-CAD 2 as the main product.
Metabolization may be oxygen-dependent at least for the six strains studied. Despite the
quantitative abundance of E. coli in feces, other (facultatively) anaerobic bacteria than E. coli
must have been responsible for the degradation of CML in the presence of the human intestinal
microbiota in our previous study.35 Provided that the metabolites are also formed by the
complex human gut microbiota, further studies need to be performed concerning the
physiological relevance of these compounds: Do the metabolites pass into the circulation and
can they be detected in urine? Does 2 address trace amine-associated receptors?58 Does 2 have
an influence on digestive processes as described for other intestinally produced amines?59 The
present study lays the foundation for a new view on structure-activity relationships of MRPs in
human physiology. Not only glycated amino acids as such need to be investigated, but also the
physiological effects of their metabolites.
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Abbreviations Used
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480
AGE, advanced glycation end product; Ala-CML, alanyl-(N-ε-carboxymethyl)-lysine; ARP,
Amadori rearrangement product; BHI, brain heart infusion; CM-APA, N-
carboxymethylaminopentanoic acid; CM-APO, N-carboxymethylaminopentanol; CM-CAD,
N-carboxymethylcadaverine; CML, N-ε-Carboxymethyllysine; LOD, limit of detection; LOQ,
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limit of quantitation; MRM, multiple reaction monitoring; MRP, Maillard reaction product;
NFPA, nonafluoropentanoic acid; OD, optical density.
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Acknowledgments
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The authors thank Dr. Kai Schwedtmann and Philipp Lange (Chair of Inorganic Molecular
Chemistry, TU Dresden) for recording the NMR spectra and for performing the elemental
analyses as well as Karla Schlosser (Chair of Food Chemistry, TU Dresden) for performing the
amino acid analyses.
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491
Notes
The authors declare no competing financial interest.
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Parts of this manuscript were presented as a lecture at the 13th International Symposium on the
Maillard Reaction, in Montreal/Canada, in September 2018.
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Figure captions
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Figure 1. Putative pathways of CML metabolization by microorganisms (Literature see text).
Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; DEC,
decarboxylase; MAO, monoamine oxidase; TAM, transaminase.
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510
Figure 2. Time course of the optical density (OD600) of inocula of E. coli strain G1/2 in (A)
nutrient-rich medium (BHI broth) and (B) minimal medium (M9) under aerobic or anaerobic
conditions. Time course of the concentration of CML in (C) nutrient-rich medium and (D)
minimal medium under aerobic or anaerobic conditions. Initial concentration, 250 µM. Open
signs, minimal medium; closed signs, BHI medium; black squares, aerobic conditions; grey
circles, anaerobic conditions. Data are given as means ± S.D. (n = 2–6).
511
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514
515
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518
Figure 3. Incubation of CML in the presence of E. coli strain G1/2 in minimal medium under
aerobic conditions. (A) Amino acid analysis chromatograms with focus on the CML peak. (B)
HPLC-MS/MS chromatograms recorded in the MRM mode at the most intense transition of N-
carboxymethylcadaverine (CM-CAD). (C) Time course of the formation of the metabolites
CM-CAD and N-carboxymethylaminopentanoic acid (CM-APA). The concentrations are given
in percent relative to the initial CML concentration (250 µM). Data are given as means ± S.D.
(n = 2).
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521
Figure 4. Proposed pathway of the chemical degradation of N-carboxymethylaminopentanal 5
as a possible intermediate in the formation of CM-APA.
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Figure 5. (A) Extracted ion chromatograms (m/z = 142) of cultures of E. coli strain G4/9 at (a)
0 h of incubation in the absence of CML, (b) 48 h of incubation in the absence of CML, (c) 0 h
of incubation in the presence of CML, (d) 48 h of incubation in the presence of CML. (B)
Product ion spectrum of the peak eluting at 3.4 min (fragmentor voltage, 100 V; collision
energy, 10 eV). (C) Proposed fragmentation reactions leading to the formation of the abundant
fragments in the product ion spectrum.
529
530
531
532
533
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535
Figure 6. Incubation of Ala-CML in the presence of E. coli strain G1/2 in minimal medium
under aerobic conditions. (A) Amino acid analysis chromatograms of Ala-CML and CML. (B)
HPLC-MS/MS chromatograms recorded in the MRM mode at the most intense transition of N-
carboxymethylaminopentanoic acid (CM-APA). (C) Time course of the formation of the
metabolites CM-CAD and CM-APA. The concentrations are given in percent relative to the
initial CML concentration (250 µM). Data are given as means ± S.D. (n = 2).
536
537
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