Comprehensive Identification of Amadori Compound-Modified

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Article Cite This: Chem. Res. Toxicol. 2019, 32, 1449−1457

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Comprehensive Identification of Amadori Compound-Modified Phosphatidylethanolamines in Human Plasma Xiaobo He,† Guan-Yuan Chen,† and Qibin Zhang*,†,‡ †

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Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, North Carolina 28081, United States ‡ Department of Chemistry & Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27412, United States S Supporting Information *

ABSTRACT: Amadori compound modified lipids are the result of nonenzymatic glycation and play an important role in several physiological and pathological processes. However, glycation of phosphatidylethanolamine (PE), the most abundant aminecontaining lipid in blood plasma, is underexplored and so far only a few glycated PEs have been reported. Herein, we report comprehensive profiling of Amadori-PE and -LysoPE species in human plasma. Using synthetic standards, we first optimized the enrichment procedure for extracting Amadori-PE/LysoPE from plasma. On the basis of the characteristic neutral losses of 303 Da in positive and 162 Da in negative ionization mode, we then applied neural loss scanning-liquid chromatography tandem mass spectrometry (LC-NLS-MS) to identify potentially glycated PE and LysoPE, which was followed by targeted product ion scanning (LC-PIS-MS) to confidently confirm the fatty acyl substitutions of the modified lipids. A total of 20 Amadori-LysoPE and 62 Amadori-PE species, including diacyl, plasmanyl, and plasmenyl, were identified. Among them, the concentrations of 12 Amarodi-LysoPE and 54 Amadori-PE were also quantified in native human plasma, using stable isotope labeled Amadori lipids as internal standards. reported that Amadori-PE can trigger lipid peroxidation.9 Furthermore, Amadori-PE was also found to play a role in vascular disease, diabetes, cancer, and in other diseases as well.3,10−14 Considering 1348 of glycerophosphoethanolamine structures listed on the LIPID MAPS Web site (http://www. lipidmaps.org), it would be expected to see many Amadori-PE/ LysoPE species being identified already. However, despite the importance of Amadori lipids, there is a lack of in-depth characterization of Amadori-PE/LysoPE in biological samples. Studies reported previously only focused on limited species of Amadori-PE with well-defined structures.15−17 For example, Nakagawa et al. identified 13 Amadori-PEs in human plasma and quantified 3 of them.15 The lack of identification could be due to inefficient recovery of Amadori lipids from complex biological samples, as only Folch method was reported to extract Amadori-PEs in human plasma,15,16,18 which is not suitable for extraction of Amadori-LysoPE because of its much higher hydrophilicity than Amadori-PE. To better understand the roles of Amadori-PE/LysoPE in physiological and pathological processes, it is essential to obtain a comprehensive profiling of Amadori-PE/LysoPE species in human plasma.

1. INTRODUCTION Nonenzymatic glycation, also known as Maillard reaction, plays an important role in many physiological and pathological processes, such as normal aging and complications of diabetes mellitus. The carbonyl group of reducing sugars reacts with primary amino groups in biomolecules to form an unstable Schiff base, which undergoes Amadori rearrangement to form a more stable Amadori product.1 Amadori-modified compounds can further undergo complex reactions to form advanced glycation end products (AGEs), which together with Amadori compounds are involved in age-related diseases and complications of diabetes mellitus.1−4 Phospholipids are major components of cell membranes, and they were also reported as important factors in signal transduction and membrane transport.5 Phosphatidylethanolamine (PE) is the second most abundant mammalian membrane phospholipid, composing ∼45% of total phospholipids in the brain and ∼20% of total phospholipids in the liver.6,7 In human plasma, PE (including LysoPE) composes ∼18.4% of total phospholipids on a molar basis.8 Beyond serving as a structural building block in membranes, PE has been proved to play key roles in many biochemical and physiological processes in mammalian cells.6,7 Aminophospholipids, such as PE, can be nonenzymatically glycated in vivo to form Amadori-phospholipids.3 Oak et al. © 2019 American Chemical Society

Received: April 11, 2019 Published: June 12, 2019 1449

DOI: 10.1021/acs.chemrestox.9b00158 Chem. Res. Toxicol. 2019, 32, 1449−1457

Article

Chemical Research in Toxicology

2.3.2. Phenylboronic Acid (PBA) SPE. The resultant residues from MeOH-AA extraction mentioned above were reconstituted in MeOH and then diluted 10 times in 20% MeOH containing 100 mM ammonium formate (pH 8) (loading solvent). After that, the diluted extracts were loaded onto Bond Elut PBA cartridges (100 mg) equilibrated in loading solvent. PBA cartridges were rinsed with 2 mL of MeOH containing 0.1% (v/v) ammonium hydroxide, and then eluted with 90% MeOH containing 1% formic acid. The eluant was collected from 0.5 to 3 mL and nitrogen-dried and, then, reconstituted in 50 μL of MeOH for LC-MS/MS analysis. 2.4. Glycation of Human Plasma in Vitro. Pooled human plasma (1 mL) underwent MeOH-AA extraction as described in section 2.3.1. The resultant extract was reconstituted in 0.5 mL of MeOH, mixed with 20 mg of D-glucose, and then stirred at 600 rpm in a Thermomix R Mixer at 50 °C for 48 h. The resultant reaction mixture were further processed as described in section 2.3. 2.5. LC-MS/MS Analysis. Qualitative and quantitative analysis of Amadori-PE/LysoPE were performed on a Vanquish UPLC coupled to a TSQ Quantiva triple quadrupole mass spectrometer (ThermoFisher Scientific). Separation was carried out on an Accucore C30 column (2.6 μm, 2.1 mm × 150 mm, ThermoFisher Scientific) at 40 °C. Chromatographic conditions were similar as previously reported for global lipidomic analysis,20 which contained mobile phase A (ACN:H2O, 50:50, v/v) and B (IPA:ACN, 90:10, v/v), both containing 10 mM ammonium formate and 0.1% formic acid and with the following gradient: −3−0 min, 30% B; 0−5 min, 30−43% B; 5−5.1 min, 43−50% B; 5.1−14 min, 50−70% B; 14.1−23 min, 70− 99% B; 23−26 min, 99% B; 26−26.1 min, 99−30% B; 26.1−30 min, 30% B. The TSQ Quantiva mass spectrometer was equipped with a heated electrospray ionization source, which was set at 3500 V in positive ion mode and 3000 V in negative ion mode. Sheath, auxiliary, and sweep gases were set at 20, 7, and 1 (arbitrary units), respectively. Vaporizer and ion transfer tube temperatures were both 300 °C. Argon collision gas pressure was 1.5 mTorr. For LC-NLS-MS, collision energy was 27 eV and mass scan range was m/z 550−1000; for LC-PIS-MS, the optimized collision energy was 20 eV for Amadori-PE/LysoPE species in positive ion mode, while 35 eV for Amadori-PE and 27 eV for Amadori-LysoPE in negative ion mode. 2.6. Identification and Relative Quantitation of AmadoriPE/LysoPE in Human Plasma. LC-NLS-MS and LC-PIS-MS were performed in tandem to identify Amadori-PE/LysoPE species from human plasma extract and in vitro glycated human plasma, which resulted in a list of Amadori-PE/LysoPE species identifiable in human plasma. The concentrations of these identified species were determined by LC-NLS-MS analysis using stable isotope labeled ISTDs. Human plasma samples (200 μL) spiked with 4 nM of ISTDs ([13C6]Amadori-PE(15:0/15:0) and -LysoPE(13:0/0:0)) underwent MeOH-AA extraction followed by PBA SPE, as described in sections 2.3.1. and 2.3.2. The elution effluents were nitrogen-dried and reconstituted in 50 μL of MeOH, followed by centrifugation at 10 000 × g for 10 min. Clear supernatants were subjected to LC-NLS-MS analysis with a 10 μL injection volume. Neutral losses of 303 and 309 Da in positive mode, and 162 and 168 Da in negative mode were used for natural isotopic Amadori-PE/LysoPE species and ISTDs, respectively. Quantification was performed based on the ratio between peak areas of Amadori-PE and -LysoPE and their corresponding ISTDs. All measurements were performed in triplicate.

Ideally, the analytical method for comprehensive Amadori lipids analysis should meet the following requirements: (a) optimal recovery of analytes from complex biological matrix; (b) unbiased characterization of all Amadori-lipid species; and (c) sensitive, reproducible, and accurate quantitative analysis using stable isotope labeled standard. Here, in this work, we tried to achieve these for an accurate and comprehensive analysis of Amadori-PE/-LysoPE in human plasma. We optimized extraction procedure for simultaneous enrichment of Amadori-PE and -LysoPE from human plasma and used LC coupled neutral loss scanning and product ion scanning tandem mass spectrometry (LC-NLS-MS and LC-PIS-MS) in tandem for comprehensive and confident identification of Amadori lipids. As a result, concentrations of 12 AmadoriLysoPE and 54 Amadori-PE species were determined in native human plasma using stable isotope labeled standards and LCNLS-MS.

2. MATERIALS AND METHODS 2.1. Chemicals, Reagents, and Materials. 1,2-Dipentadecanoyl-sn-glycero-3-phosphoethanolamine (PE(15:0/15:0)), 1-palmitoyl2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1(9Z)), and 1-tridecanoyl-sn-glycero-3-phosphoethanolamine (LysoPE(13:0/ 0:0)) were purchased from Avanti Polar Lipids (Alabaster, AL). [U−13C6]-D-glucose (99%) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). D-Glucose and all solvents, including methanol (MeOH), acetonitrile (ACN), isopropanol (IPA), chloroform (CHCl3), and water (H2O) of LC-MS grade and HPLC grade, were purchased from Fisher Scientific (Pittsburgh, PA). Pooled human plasma samples collected from self-reported healthy donors with K2-EDTA as anticoagulant were purchased from BioIVT (Westbury, NY, Lot no. BRH1524451, donor metadata are summarized in Table S1). 2.2. Standards Preparation. Amadori-PE/LysoPE standards (Amadori-PE(15:0/15:0), -PE(16:0/18:1(9Z)), and -LysoPE(13:0/ 0:0)) were synthesized by incubation of their respective substrates with D-glucose in anhydrous MeOH, as described previously.19 Briefly, for synthesis of Amadori-PE, 7 μmol of PE substrates were mixed with 161 μmol of D-glucose in 1 mL of MeOH and, then, stirred at 600 rpm in a Thermomix R Mixer (Eppendorf, Germany) at 60 °C for 3 days; for synthesis of Amadori-LysoPE, 12 μmol of LysoPE substrate was mixed with 215 μmol of D-glucose in 1 mL of MeOH, and stirred at 50 °C for 3 days. Amadori-PE(15:0/15:0), -PE(16:0/18:1(9Z)), and -LysoPE(13:0/ 0:0) from synthesis were purified through boronic affinity SPE and preparative HPLC separation in order, as described previously.19 Briefly, Amadori-PE/LysoPE were separated from their phospholipid substrates by boronate affinity SPE (Bond Elut PBA cartridges, 500 mg, Agilent); then, they were further purified with a Kromasil C18 column (250 × 10 mm, 10 μm, AkzoNobel, Bohus, Sweden) on a Shimadzu 20A HPLC. Isocratic mobile phases of 100% and 80% MeOH both containing 5 mM ammonium formate were used for Amadori-PE and Amadori-LysoPE, respectively, at a flow rate of 3 mL/min. Elution was monitored at 220 nm with a multiple wavelength UV detector. The synthesis and purification of stable isotope-labeled AmadoriPE/LysoPE standards were similarly performed. Highly purified (>95%) Amadori-PE/LysoPE and [13C6]AmadoriPE/LysoPE were used as standards and internal standards (ISTDs), respectively, for quantification of Amadori-PE/LysoPE in human plasma. Stock solutions of standards and ISTDs were prepared in MeOH at the concentration of 10 μM. The working solutions were diluted with MeOH from stock solutions. 2.3. Sample Preparation. 2.3.1. Extraction by Acidic MeOH (MeOH-AA). Human plasma samples (200 μL) were mixed with 10fold volume of MeOH (containing 0.5% acetic acid) and, then, vortexed for 20 s. After centrifugation at 10 000 × g for 10 min, supernatants were collected and nitrogen-dried.

3. RESULTS AND DISCUSSION 3.1. Optimization of Amadori-PE/LysoPE Extraction from Human Plasma. Among several methods of lipid/ glycolipid extraction from biological samples, Folch method is the most commonly used for extracting lipids and is the only method reported for extracting Amadori-PEs from human plasma.15,16,18 Bligh and Dyer method uses a different ratio of CHCl3/CH3OH/H2O compared to Folch method, and both methods use CHCl3 as the primary solvent for solubilization. MTBE (methyl-tert-butyl ether) and BUME (butanol-meth1450

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suppression for Amadori-PE could be effectively reduced by PBA SPE cleaning up. 3.2. Identification of Amadori-PE/LysoPE Species in Human Plasma. To identify Amadori-PE/LysoPE species in human plasma, LC-NLS-MS and LC-PIS-MS were performed in tandem, and both native and in vitro glycated human plasma were used to broaden the coverage. First, LC-NLS-MS was performed to scan precursor ions, which had neutral loss of 303 Da in positive mode and 162 Da in negative mode. These two neutral loss are characteristic of Amadori-PE/LPE as reported previously.17,19 LC-NLS-MS in positive and negative modes led to two lists of possible Amadori-PE/LysoPE species. Species shared by the two lists were defined as candidate Amadori-PE/LysoPE species. For verification and more specific fatty acyl sn-position identification, LC-PIS-MS was further performed to obtain molecular fragment information on these candidate species. Fragmentation patterns of Amadori-PE/LysoPE had been obtained in our previous study,19 which made it possible to exclude false identifications that were not conforming to the common patterns (Figure 2). 3.2.1. Diacyl Amadori-PE. Structural information on fatty acid chains could be obtained from spectra in both positive and negative ion modes. Regarding diacyl Amadori-PE, the intensity ratio between sn-1 and sn-2 carboxylate anions was used to assign sn-1/sn-2 position. Fragmentation experiments using serially elevated collision energies showed that the ratio of the sn-1/sn-2 carboxylate anions of Amadori-PE(16:0/18:1) synthetic standard was less than 1 at collision energies of 15− 50 eV. As shown in Figure 2C, the intensity of m/z 281 is significantly higher than m/z 255, and they were assigned as sn2 and sn-1 carboxylate anions, respectively. Correspondingly, the intensity of m/z 452 (loss of glucose moiety and sn-2 fatty acyl substituent as a ketene) is higher than m/z 478 (loss of glucose moiety and sn-1 fatty acyl substituent as a ketene). It is notable that m/z 452 is much more abundant than ion m/z 434 (loss of glucose moiety and sn-2 as an acid) in Figure 2C, demonstrating loss of fatty acyl substituent as a ketene is more facile than as an acid, which is consistent with results of PE fragmentation by Hsu et al.25 As shown in Figure 2D, the similar fragmentation patterns occurred for Amadori-PE(18:0/ 18:2) present in human plasma. 3.2.2. Amadori-LysoPE. With respect to Amadori-LysoPE, the intensity ratio of fragment ions m/z 304 to 224 in positive mode was used to distinguish between sn-1 and sn-2 isomers. Ions at m/z 304 and 224 reflected headgroup cations containing and lacking phosphate group, respectively. Fragmentation experiments using serially elevated collision energies showed that sn-2 isomer had much higher ratio of m/z 304/224 than sn-1 isomer at collision energies of 5−20 eV. For example, significant difference of m/z 304/224 ratio can be observed between Amadori-LysoPE(18:0/0:0) (Figure 3A and B) and Amadori-LysoPE(0:0/18:0) (Figure 3C and D). Dong et al. had similar observation in their work on lysophosphatidylcholine fragmentation.26 This is most likely attributed to electron-withdrawing effect of the sn-2 ester group, causing more facile cleavage of the phosphate-glycerol bond, which resulted in the more abundant headgroup cation (m/z 304) for Amadori sn-2 isomer. Under the same mechanism, sn-2 isomer had more facile neutral loss of 303 Da (headgroup) than sn-1 isomer. For example, much higher intensity ratio of m/z 341 ([M+H-303]+) to m/z 560 ([M+H-84]+, neutral loss of 3H2O + HCHO) could be observed for Amadori-LysoPE(0:0/18:0) (Figure 3C) than Amadori-LysoPE(18:0/0:0) (Figure 3A).

anol) methods were reported to have similar extraction properties for total lipids and significantly higher recoveries for acidic polar lipids, compared with that of the Folch method.21,22 Huang et al. used MeOH to extract gangliosides from human plasma for quantification.23 Okudaira et al. used acidic MeOH (pH 4.0) to extract lysophospholipids from mouse plasma, serum, and tissues, and their work showed that the relatively lower pH could help to inhibit the migration of acyl chain in lysophospholipids from sn-2 to sn-1 position.24 Alternatively, we used phenyl boronate affinity (PBA) resin to separate Amadori-PE from PE during synthesis of Amadori-PE standards.19 Herein, using synthetic standards of Amadori-PE and -LysoPE, we compared extraction recoveries between methods of Folch, BUME, MeOH, MeOH−0.5% acetic acid (v/v) (MeOH-AA), and MeOH-AA followed, by PBA SPE (MeOHAA + PBA). As shown in Figure 1A, the first four methods

Figure 1. Comparison between various methods for extraction of Amadori-PE and -LysoPE in human plasma. Extraction efficiency (%) and matrix effect (%) of various methods were presented in panels A and B, respectively. The values are expressed as mean ± RSD (n = 3). Extraction efficiency was measured as the ratio between the prespiked and postspiked samples, and matrix effect was measured as the ratio between the postspiked sample and standard in neat solution.

have similar extraction efficiencies (EE%) for Amadori-PE, while Folch and BUME methods have lower EE% for Amadori-LysoPE than MeOH and MeOH-AA methods. MeOH and MeOH-AA have similar extraction performance for both Amadori-PE and -LysoPE. It is notable that Folch, BUME, MeOH, and MeOH-AA methods have similar level of matrix effects (ME%), with values of 38−48% for Amadori-PE and around 100% for Amadori-LysoPE as shown in Figure 1B, although MeOH and MeOH-AA processing were used to simply precipitate protein without liquid−liquid extraction. On the other hand, by comparison between MeOH-AA and MeOH-AA + PBA methods, we can find PBA SPE resulted in a loss of recovery for Amadori-LysoPE and Amador-PE. However, in term of matrix effect, the matrix-induced ion 1451

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Figure 2. Representative MS/MS spectra of synthetic Amadori-PE(16:0/18:1) (A) in positive mode and (C) in negative mode and MS/MS spectra of Amadori-PE(18:0/18:2) present in human plasma (B) in positive mode and (D) in negative mode. Collision energy: 20 eV in positive mode and 35 eV in negative mode.

Figure 4. Representative MS/MS spectra of (A) Amadori-LysoPE(18:0/0:0) and (B) Amadori-LysoPE(0:0/18:0) in negative ion mode. Insets show the magnified m/z 190−220 regions of panels A and B. Collision energy was 35 eV.

loss of fatty acyl substituent as a ketene at sn-2 than sn-1, which is consistent with PE fragmentation results reported by Hsu et al.25 One disadvantage in using intensity ratio of m/z 214/196 to distinguish Amadori-LysoPE sn-isomers is the low intensity of these two ions in MS/MS spectra, even when different collision energies were tried. A much more easily observed difference between Amadori-LysoPE sn-1/2 isomers is the intensity of fatty acid anion. Similar to Amadori-PE, the elimination of fatty acyl chain as an acid anion for AmadoriLysoPE was more facile at sn-2 position than at sn-1 position. As shown in Figure 4, Amadori-LysoPE(0:0/18:0) has higher ratio of m/z 283 to 480 than Amadori-LysoPE(18:0/0:0),

Figure 3. Representative MS/MS spectra of (A) Amadori-LysoPE(18:0/0:0) and (C) Amadori-LysoPE(0:0/18:0) in positive ion mode. Panels B and D are magnified m/z 210−320 regions of panels A and C, respectively. Collision energy was 20 eV.

Furthermore, the intensity ratio of m/z 214 to 196 in negative mode was also used to verify the discrimination between Amadori-LysoPE regioisomers. Fragment ions of m/z 214 and 196 reflect loss of glucose moiety plus fatty acyl ketene and fatty acid, respectively. As shown in Figure 4, Amadori-LysoPE(0:0/18:0) has higher ratio of m/z 214 to 196 than Amadori-LysoPE(18:0/0:0), demonstrating more facile 1452

DOI: 10.1021/acs.chemrestox.9b00158 Chem. Res. Toxicol. 2019, 32, 1449−1457

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Chemical Research in Toxicology

Figure 5. Representative MS/MS spectra of (A) Amadori-PE(18:0/18:1), (B) Amadori-PE(P-18:0/18:1), and (C) Amadori-PE(O-18:0/18:1) in positive ion mode. Collision energy was 20 eV.

which pattern occurred for all Amadori-LysoPE sn-1/2 isomers we identified in reference human plasma. 3.2.3. Plasmenyl and Plasmanyl Amadori-PE/LysoPE. Aside from diacyl-PE, PE also contains substantial amounts of ether-linked lipidsplasmenyl PE (P-PE) and plasmanyl PE (O-PE), which usually have a 1-O-alk-1′-enyl and 1-O-alkyl group, respectively, at the sn-1 position, and an acyl group at the sn-2 position.27 In human plasma, P-PE and O-PE compose 43% of PE by mass.8 The existing of P-PE and OPE increases the analytical complexity of PE species present in biological samples. Hsu et al. reported differentiation of P-PE and O-PE by multiple-stage linear ion-trap MS (MS3/MS4) in negative ion mode,28 and another approach using [M+Li]+ ions in positive ion mode.25 Zemski Berry et al. reported CID of [M + H]+ of P-PE resulted in two prominent fragment ions, being characteristic of the sn-1 position and sn-2 position, respectively.27 In our study, plasmenyl and plasmanyl Amadori-PE (PAmadori-PE and O-Amadori-PE) present in human plasma were also detected. To identify P- and O-Amadori-PE species, first, the intensity of fragment cation arising from neutral loss of 303 Da was used to distinguish between diacyl- and P-/OAmadori-PE. For example, as shown in Figure 5, Am-PE(P18:0/18:1) and Am-PE(O-18:0/18:1) have much lower intensity ratio of [M + H − 303]+ to [M + H]+ than that of Am-PE(18:0/18:1), demonstrating neutral loss of 303 Da (headgroup) in P- and O-Amadori-PE was not as facile as that in diacyl Amadori-PE. This is in agreement with what observed by Zemski Berry et al. for plasmenyl PEs, where neutral loss of headgroup was reduced as a result of nonacyl substitution at sn-1 position.27 Second, discrimination between P- and OAmadori-PE was made possible by their different fragmentation patterns during CID in positive ion mode. For example, the spectrum of Am-PE(P-18:0/18:1) (Figure 5B) contains ion at m/z 554, arising from loss of glycerol backbone with sn-

2 fatty acyl chain (Scheme 1A). On the basis of the fragmentation pathways of PE plasmalogens proposed by Scheme 1. Proposed Fragmentation Pathways and the Fragment Ions Observed for [M + H]+ Ion of Plasmenyl Amadori-PE

The proposed fragmentation pathways are adapted from Zemski Berry & Murphy.27

Zemski Berry et al.,27 we reasoned that fragment ion at m/z 554 was generated involving the formation of a new O−P bond, and this ion is characteristic of the sn-1 position. As a verification, m/z 536 (554-H2O), 518 (554−2H2O), 500 (554−3H2O), and 392 (554-glucose moiety) could also be observed. On the other hand, cation at m/z 339 corresponding to glycerol backbone plus sn-2 fatty acyl group (Scheme 1B) was characteristic of the sn-2 position. In contrast, no fragment ions mentioned above could be observed in spectrum of AmPE(O-18:0/18:1) (Figure 5C), which indicated the double bond of the vinyl ether at sn-1 position is important for the pathway described in Scheme 1. Moreover, the cation at m/z 304 corresponding to Amadori headgroup, which always existed for diacyl- and P-Amadori-PE, could not be observed 1453

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Chemical Research in Toxicology

Figure 6. Representative MS/MS spectra of (A) Amadori-PE(18:0/18:1), (B) Amadori-PE(P-18:0/18:1), and (C) Amadori-PE(O-18:0/18:1) in negative ion mode. Collision energy was 35 eV.

3.3. Relative Quantification of Amadori-PE/LysoPE in Native Human Plasma. After identification of Amadori-PE/ LysoPE species, the concentrations of identified species in native human plasma were determined by LC-NLS-MS using stable isotope-labeled ISTDs. Limit of detection (LOD), precision, accuracy, recovery, matrix effect, and process stability were also investigated for the relative quantitative analysis. The LOD values for Amadori-LysoPE and -PE were 0.16 and 0.08 nM, respectively. The intraday (n = 3) and interday (n = 3) precision and accuracy were investigated using high (25 nM), medium (5 nM), and low (1 nM) QC levels of standards (Amadori-PE(15:0/15:0) and Amadori-LysoPE(13:0/0:0)), which were named as HQC, MQC, and LQC, respectively. As summarized in Table S3, the intraday and interday precision were 0.9−7.8% and 3.7−9.5%, respectively. In term of accuracy, best accuracies were obtained at MQC for both Amadori-PE and -LysoPE because MQC had the closest concentration to ISTDs. The accuracy got worse when the concentrations of analytes deviated from ISTDs’ concentration, while still stayed in acceptable range (80−120%). Matrix effects and recoveries (extraction efficiencies) of Amadori-PE/LysoPE at HQC, MQC, and LQC were summarized in Table S4. In consistence with comparison of extraction methods described in section 3.1, matrix effects were 86.0−91.7% and 92.8−97.9% for Amadori-PE and -LysoPE, respectively; extraction efficiencies were 47.3−57.5% and 39.1−51.4% for Amadori-PE and -LysoPE, respectively. Effects of various sampling conditions on the stability of analytes at low and high concentrations were investigated under various sampling conditions. As shown in Table S5, no obvious effects could be observed for processed sample and freeze-and-thaw handling. However, keeping at room temperature for 24 h could reduce the recovery of Amadori-LysoPE to 82.3%, which suggests temperature may be a key factor related to stability of Amadori-phospholipids, and long-time exposure to room temperature should be avoided. Among Amadori-PE/LysoPE species identified in this study, 54 Amadori-PEs and 12 Amadori-LysoPEs could be clearly detected in quantitative analysis. The concentrations of these species in native human plasma were summarized in Table 1.

for O-Amadori-PE. This indicated that lack of double bond beside the ether O atom also affect the cleavage of the phosphate-glycerol bond. MS/MS spectra in negative ion mode could also provide fragmentation information to help the discrimination between diacyl-, P-, and O-Amadori-PE species. For example, AmadoriPE(18:0/18:1), Amadori-PE(P-18:0/18:1), and AmadoriPE(O-18:0/18:1) had different substitutions at the sn-1 position, and all these 3 species had an 18:1 fatty acyl group at the sn-2 position, which was fragmented into fatty acid anion at m/z 281 (Figure 6). Amadori-PE(18:0/18:1) also had sn-1 fatty acid anion (m/z 283) in its spectrum, while AmadoriPE(P-18:0/18:1) and Amadori-PE(O-18:0/18:1) had no fragment anions observed reflecting the elimination of 1-Oalk-1′-enyl and 1-O-alkyl group at sn-1 position. Anions arising from loss of glucose moiety plus sn-2 fatty acyl group as a ketene (m/z 480, 464, 466) and loss of glucose moiety plus sn1 fatty acyl group as an acid (m/z 462, 446, 448) could help to verify the identification of sn-2 position, that is, given the m/z of [M − H]− and sn-2 fatty acid anion, the mass of sn-1 substituent could be calculated. In summary, diacyl, P-, and O-Amadori-PE could be clearly distinguished by methods mentioned above. As a result, the subclass, carbon chain length, and double bond number of Amadori-PE species detected in human plasma can be readily determined. Furthermore, under the LC condition in our study, Amadori-LysoPE and -PE were always eluted at 2−5.5 and 11−18 min, respectively. The big gap of retention time could help to differentiate between Amadori-LysoPE and -PE rapidly. It is notable that the sn-1 substituent of P-, and OAmadori-PE species identified in our study always exist as P16:0, P-18:0, P-18:1, P-20:0, P-22:0, O-16:0, O-18:0, and O20:0, which is consistent with the O- and P-PE species listed in LIPID MAPS. In spite of all efforts mentioned above, there were still isobaric species that could not be differentiated even they were eluted at different retention times, which were proposed as isomers with different double bond positions. In summary, a total of 20 Amadori-LysoPE and 62 Amadori-PE species were identified in native and in vitro glycated human plasma together (Table S2). 1454

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Chemical Research in Toxicology Table 1. Concentration of Amadori-PE and -LysoPE Quantified in Native Human Plasmaa species

class

Rt (min)

Am-LPE(0:0/22:6) Am-LPE(0:0/20:4) Am-LPE(22:6/0:0) Am-LPE(0:0/18:2) Am-LPE(20:4/0:0) Am-LPE(18:2/0:0) Am-LPE(0:0/16:0) Am-LPE(16:0/0:0) Am-LPE(0:0/18:1) Am-LPE(18:1/0:0) Am-LPE(0:0/18:0) Am-LPE(18:0/0:0) Am-PE(18:2/20:4) Am-PE(16:0/20:5) Am-PE(18:2/18:2) Am-PE(16:0/18:3) Am-PE(16:0/22:6) Am-PE(18:1/22:6) Am-PE(16:0/20:4) Am-PE(16:0/18:2) Am-PE(18:1/20:4)-1 Am-PE(18:1/18:2) Am-PE(P-16:0/22:6) Am-PE(18:1/20:4)-2 Am-PE(P-18:1/22:6)-1 Am-PE(16:0/20:3) Am-PE(O-16:0/22:6) Am-PE(18:0/20:5) Am-PE(P-18:0/22:6)-1 Am-PE(P-16:0/20:4) Am-PE(P-18:1/20:4)-1 Am-PE(P-16:0/18:2) Am-PE(P-18:1/18:2) Am-PE(16:0/22:4) Am-PE(O-16:0/22:5)

Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-LPE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE

2.44 2.54 2.55 2.60 2.71 2.79 3.23 3.51 3.52 3.81 5.00 5.45 11.76 11.83 11.91 11.96 12.51 12.60 12.78 12.93 12.93 13.09 13.16 13.18 13.30 13.37 13.42 13.43 13.50 13.52 13.63 13.72 13.78 13.83 13.86

concentration (mean ± SD, nM) 0.81 1.27 0.66 3.02 1.73 7.35 0.85 4.94 0.76 4.26 0.60 7.70 0.36 0.15 1.20 0.14 17.28 2.87 6.13 9.70 3.01 7.28 1.01 0.62 0.28 0.90 0.88 1.05 0.18 1.77 1.77 0.56 1.66 0.33 1.55

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.14 0.10 0.15 0.44 0.15 0.24 0.13 0.45 0.12 0.72 0.11 1.00 0.06 0.03 0.10 0.03 2.04 0.16 0.32 0.90 0.23 1.10 0.13 0.08 0.06 0.18 0.12 0.19 0.01 0.58 0.19 0.16 0.19 0.12 0.22

species

class

Rt (min)

Am-PE(P-18:1/20:4)-2 Am-PE(18:0/22:6) Am-PE(16:0/18:1) Am-PE(P-16:0/20:3) Am-PE(P-16:0/22:5) Am-PE(18:1/18:1) Am-PE(18:0/20:4) Am-PE(18:0/22:5)-1 Am-PE(18:0/18:2) Am-PE(P-18:0/22:6)-2 Am-PE(O-16:0/22:4) Am-PE(P-16:0/18:1) Am-PE(18:0/20:3) Am-PE(18:0/22:5)-2 Am-PE(O-16:0/18:1) Am-PE(O-18:0/22:6) Am-PE(P-18:0/20:4) Am-PE(P-18:0/22:5)-1 Am-PE(P-18:0/18:2) Am-PE(O-18:0/20:4) Am-PE(18:0/22:4) Am-PE(16:0/18:0) Am-PE(O-18:0/18:2) Am-PE(18:0/18:1) Am-PE(P-18:0/20:3) Am-PE(P-18:0/22:4) Am-PE(P-18:0/18:1) Am-PE(O-20:0/20:4) Am-PE(O-18:0/18:1) Am-PE(P-20:0/20:4) Am-PE(P-20:0/18:2)

Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE Am-PE

13.91 14.04 14.07 14.09 14.14 14.22 14.35 14.46 14.51 14.74 14.82 14.87 14.91 14.95 14.95 15.01 15.06 15.16 15.25 15.32 15.35 15.42 15.49 15.56 15.65 16.01 16.23 16.24 16.44 16.52 16.66

concentration (mean ± SD, nM) 0.14 3.35 3.48 0.14 0.33 2.63 15.92 0.75 18.66 0.60 0.28 0.28 1.49 0.32 0.35 0.53 2.98 0.27 1.18 1.10 0.38 0.18 0.20 2.78 0.17 0.13 0.17 0.13 0.18 0.26 0.09

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.38 0.10 0.02 0.04 0.19 0.84 0.10 1.15 0.13 0.03 0.02 0.30 0.07 0.04 0.06 0.25 0.07 0.40 0.10 0.03 0.02 0.10 0.31 0.04 0.04 0.05 0.05 0.06 0.05 0.04

a

All measurements were performed in triplicate. -1 and -2: Possible undefined CC positional isomer.

LC-PIS-MS in tandem, 62 of Amadori-PE and 20 of AmadoriLysoPE species were confidentially identified, which was facilitated by extensive investigation of the CID fragmentation patterns of diacyl-, lyso-, plasmenyl-, and plasmanyl-AmadoriPE under both positive and negative ion modes. Using LCNLS-MS and stable isotope labeled standards, we performed relative quantitation of these identified species, and levels of 54 Amadori-PEs and 12 Amadori-LysoPEs in human plasma from healthy subjects were determined. To our knowledge, this is the first comprehensive characterization and quantification of Amadori compound-modified phosphatidylethanolamines in native human plasma. The fragmentation rules obtained from CID-MS/MS analysis can facilitate identification of novel AmPEs, the plasma concentrations of these species also provide valuable reference values for future studies of Amadori-PEs in human health and disease. In this respect, having the capability to accurately quantify a comprehensive panel of potentially glycated PEs would enable establishment of a solid correlation between plasma levels of Am-PEs and disease status and facilitate mechanistic investigations of the roles of Am-PEs in various diseases, such as increased incidence rate of cardiovascular diseases in diabetic patients.

Amadori-PEs were found to contain 25 diacyl Amadori-PEs, 20 Amadori-P-PEs, and 9 Amadori-O-PEs, with a total concentration of 101, 14, and 5 nM, respectively. Amadori-LysoPEs were found to have a total concentration of 34 nM. It is of note that for polyunsaturated Amadori-LysoPEs, such as those containing 22:6 and 20:4 fatty acyls, we observed similar levels of sn-1 and sn-2 Amadori-LysoPEs (Table 1). The sn-isomers of lysophospholipids vary in their distribution in tissue and plasma/serum depending on the lipid class and acyl chain length.24,29 The polyunsaturated LysoPEs exist primarily in sn2 position in tissues and in sn-1 position in serum/plasma because of a quick intramolecular acyl migration from sn-2 to sn-1 in biofluid,24,29 and whether, in Amadori-LysoPEs, a similar distribution in both sn positions is due to inhibition of intramolecular acyl migration by Amadori modification remains to be determined in the future.

4. CONCLUSION In this study, we developed an analytical method for comprehensive profiling of Amadori-PE/LysoPE in human plasma. Various methods for simultaneous extraction of Amadori-PE and -LysoPE were compared, and MeOH-AA followed by PBA SPE was adopted. Using LC-NLS-MS and 1455

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Chemical Research in Toxicology

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dyslipidemia of diabetes and renal insufficiency. Proc. Natl. Acad. Sci. U. S. A. 91, 9441−9445. (12) Sookwong, P., Nakagawa, K., Fujita, I., Shoji, N., and Miyazawa, T. (2011) Amadori-glycated phosphatidylethanolamine, a potential marker for hyperglycemia, in streptozotocin-induced diabetic rats. Lipids 46, 943−952. (13) Eitsuka, T., Nakagawa, K., Ono, Y., Tatewaki, N., Nishida, H., Kurata, T., Shoji, N., and Miyazawa, T. (2012) Amadori-glycated phosphatidylethanolamine up-regulates telomerase activity in PANC1 human pancreatic carcinoma cells. FEBS Lett. 586, 2542−2547. (14) Oak, J.-H., Nakagawa, K., Oikawa, S., and Miyazawa, T. (2003) Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells. FEBS Lett. 555, 419−423. (15) Nakagawa, K., Oak, J. H., Higuchi, O., Tsuzuki, T., Oikawa, S., Otani, H., Mune, M., Cai, H., and Miyazawa, T. (2005) Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes. J. Lipid Res. 46, 2514−2524. (16) Kodate, A., Otoki, Y., Shimizu, N., Ito, J., Kato, S., Umetsu, N., Miyazawa, T., and Nakagawa, K. (2018) Development of quantitation method for glycated aminophospholipids at the molecular species level in powdered milk and powdered buttermilk. Sci. Rep 8, 8729. (17) Nakagawa, K., Ibusuki, D., Suzuki, Y., Yamashita, S., Higuchi, O., Oikawa, S., and Miyazawa, T. (2007) Ion-trap tandem mass spectrometric analysis of squalene monohydroperoxide isomers in sunlight-exposed human skin. J. Lipid Res. 48, 2779−2787. (18) Miyazawa, T., Oak, J. H., and Nakagawa, K. (2005) Tandem mass spectrometry analysis of Amadori-glycated phosphatidylethanolamine in human plasma. Ann. N. Y. Acad. Sci. 1043, 280−283. (19) He, X., and Zhang, Q. (2018) Synthesis, Purification, and Mass Spectrometric Characterization of Stable Isotope-Labeled AmadoriGlycated Phospholipids. ACS Omega 3, 15725−15733. (20) Narváez-Rivas, M., and Zhang, Q. (2016) Comprehensive untargeted lipidomic analysis using core-shell C30 particle column and high field orbitrap mass spectrometer. J. Chromatogr A 1440, 123−134. (21) Lofgren, L., Stahlman, M., Forsberg, G. B., Saarinen, S., Nilsson, R., and Hansson, G. I. (2012) The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma. J. Lipid Res. 53, 1690−1700. (22) Löfgren, L., Forsberg, G. B., and Ståhlman, M. (2016) The BUME method: a new rapid and simple chloroform-free method for total lipid extraction of animal tissue. Sci. Rep 6, 27688. (23) Huang, Q., Zhou, X., Liu, D., Xin, B., Cechner, K., Wang, H., and Zhou, A. (2014) A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma. Anal. Biochem. 455, 26−34. (24) Okudaira, M., Inoue, A., Shuto, A., Nakanaga, K., Kano, K., Makide, K., Saigusa, D., Tomioka, Y., and Aoki, J. (2014) Separation and quantification of 2-acyl-1-lysophospholipids and 1-acyl-2lysophospholipids in biological samples by LC-MS/MS. J. Lipid Res. 55, 2178−2192. (25) Hsu, F., and Turk, J. (2009) Electrospray Ionization with Lowenergy Collisionally Activated Dissociation Tandem Mass Spectrometry of Glycerophospholipids: Mechanisms of Fragmentation and Structural Characterization. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877, 2673−2695. (26) Dong, J., Cai, X., Zhao, L., Xue, X., Zou, L., Zhang, X., and Liang, X. (2010) Lysophosphatidylcholine profiling of plasma: discrimination of isomers and discovery of lung cancer biomarkers. Metabolomics 6, 478−488. (27) Zemski Berry, K. A., and Murphy, R. C. (2004) Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids. J. Am. Soc. Mass Spectrom. 15, 1499− 1508. (28) Hsu, F.-F., and Turk, J. (2007) Differentiation of 1-O-alk-1′enyl-2-acyl and 1-O-alkyl-2-acyl Glycerolphospholipids by Multiple-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00158. List of blood donor metadata, Amadori-PE/LysoPE species identified from in vitro glycated and native human plasma extract; precision, accuracy, and matrix effects of the method for quantification of Amadori-PE/ LysoPE; and stability of Amadori-PE/LysoPE under various conditions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Qibin Zhang: 0000-0002-6135-8706 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge funding from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (R01 DK116731) and the American Heart Association (17CSA33570025) for support of this research.

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REFERENCES

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