LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma.

An amino group of phosphatidylethanolamine (PE) is considered as a target for nonenzymatic glycation, and the potential involvement of lipid glycation in the pathogenesis of diabetic complications has generated interest. However, unlike an early glycation product of PE (Amadori-PE), the occurrence and roles of advanced glycation end products of PE (AGE-PE) in vivo have been unclear. Here, we developed an LC-MS/MS method for the analysis of AGE-PE [carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE)]. Collision-induced dissociation of CM-PE and CE-PE produced characteristic ions, permitting neutral loss scanning (NLS) and multiple reaction monitoring (MRM) of AGE-PE. By NLS analysis, a series of AGE-PE molecular species was detected in human erythrocytes and blood plasma. In LC-MS/MS analysis, MRM enabled the separation and determination of the predominant AGE-PE species. Between healthy subjects and diabetic patients, no significant differences were observed in AGE-PE concentrations in erythrocytes and plasma, whereas Amadori-PE concentrations were higher in diabetic patients. These results provide direct evidence for the presence of AGE-PE in human blood, and indicated that, compared with Amadori-PE, AGE-PE is less likely to be accumulated in diabetic blood. The presently developed LC-MS/MS method appears to be a powerful tool for understanding in vivo lipid glycation and its pathophysiological consequence.


Blood sample preparation
Human blood samples were collected from eight healthy volunteers (four females and four males, age 22 ± 1 years) and 10 newly detected type 2 diabetic patients (seven females and three males, age 55 ± 12 years, hemoglobin A1c 12 ± 2%). None of the diabetic patients received any medication for the control of blood glucose at the time of blood collection. This study was approved by the institutional review board of the Nippon Medical School, Tokyo, Japan, and informed consent was obtained from all participants.
Blood (10 ml) was collected into a tube containing EDTA-2Na as an anticoagulant and centrifuged at 1,000 g for 10 min at 4°C. After plasma and buffy coat were removed, erythrocytes were washed three times with phosphate-buffered saline (pH 7.4) to prepare packed cells. Total lipids were extracted from 1 ml of the packed cells using 2-propanol and chloroform ( 17 ). Plasma (1 ml) was subjected to total lipid extraction using Folch's partition in human erythrocytes and mammalian mitochondrial membranes, respectively. In contrast, Breitling-Utzmann et al. ( 14 ) reported that neither CM-PE nor CE-PE was detected in blood samples. Because of the potential consequence of AGE-PE in diabetic complications, these confl icting results imply the need for a new analytical mean to accurately measure CM-PE and CE-PE. To address this need, we aimed to develop a quantitative method to analyze CM-PE and CE-PE by using LC-MS/MS. With the developed method, we analyzed CM-PE and CE-PE in erythrocyte and plasma of healthy subjects and diabetic patients to assess the effects of hyperglycemia on the accumulation of early and advanced glycation end products of PE (Amadori-PE and AGE-PE, respectively).

Materials
All PE molecular species were purchased from Avanti Polar Lipids (Alabaster, AL). The molecular species were indicated by the carbon chain length and the unsaturation degree of the sn -1,2 acyl chains; e.g., "16:0-18:1 PE" for 1-hexadecanoyl-2-octadecenoyl-sn -glycero-3-phosphoethanolamine. CM-PE and CE-PE standards were synthesized by using the PE species as starting materials, as described by Utzmann and Lederer ( 15 ), with modifications. In brief, for the synthesis of CM-PE, PE (10 µmol), glyoxylate (100 µmol), and cyanoborohydride (10 µmol) were dissolved in 100 ml of methanol and incubated at 60°C for 4 h. For the synthesis of CE-PE, pyruvate (100 µmol) was used instead of glyoxylate. The reaction mixture was evaporated to dryness, and the residue was dissolved in 10 ml of chloroform-methanol (99:1, v/v; containing 5 mM ammonium acetate). The fl ow rate was set at 0.2 ml/min, and the column temperature was maintained at 40°C. CM-PE and CE-PE were detected using multiple reaction monitoring (MRM) for the transition of parent ions to product ions. For the quantitation of CM-PE and CE-PE in blood samples, we focused on eight molecular species ( Using synthesized AGE-PE species, we prepared standard solutions at concentrations of 5-1,000 pmol/ml (a range expected to encompass concentrations encountered in vivo). The erythrocyte extract, plasma extract, or the standard solution (2 µl each) was then subjected to LC-MS/MS, and the AGE-PE molecular species were individually detected using MRM. The concentrations of CM-PE and CE-PE in erythrocytes and plasma were calculated using the calibration curves of the synthesized AGE-PE molecules.
The MRM detection was also applied for Amadori-PE and PE. Amadori-PE and PE were analyzed using a silica column

Statistics
The data are expressed as means ± SD and analyzed using Student's t -test. Differences were considered signifi cant at P < 0.01. ( 16 ). Each extract was evaporated to dryness, and the residue was dissolved in 1 ml of chloroform-methanol (2:1, v/v).

MS/MS analysis
A 4000 QTRAP quadrupole/linear ion-trap tandem mass spectrometer (Applied Biosystems) was used for MS/MS analysis. Initially, we analyzed synthesized 18:1-18:1 CM-PE and 18:1-18:1 CE-PE as the reference compounds of AGE-PE. To evaluate the MS/MS fragmentation, product ion scanning was performed by directly injecting 18:1-18:1 CM-PE or 18:1-18:1 CE-PE solution (0.2 nmol/ml methanol) into MS/MS (5 µl/injection) using methanol as carrier solvent (0.2 ml/min). Electrospray ionization was used as an ion source with collision energy of 33 eV, transition dwell time of 100 ms, turbo gas temperature at 500°C, and spray voltage of 5,000 V. Nitrogen pressure values for turbo, nebulizer, and curtain gases were set at 30, 60, and 10 pounds per square inch, respectively. Positive ion spectra were collected in the m/z range of 100-900.
Neutral loss scanning (NLS) was performed for profi ling CM-PE and CE-PE molecular species in blood samples. The MS/MS instrument was programmed to scan parent ions that yielded a neutral loss of 199 Da for CM-PE or of 213 Da for CE-PE after fragmentation in the collision cell. Sample (erythrocyte extract or plasma extract, 5 µl each) was injected directly into MS/MS as described above. Amadori-PE and PE molecular species were also evaluated using NLS of 303 Da and 141 Da, respectively.

LC-MS/MS analysis with multiple reaction monitoring
For LC-MS/MS analysis, a Shimadzu liquid chromatography system, including a vacuum degasser, a quaternary pump, and an autosampler (Shimadzu, Kyoto, Japan), was equipped with the 4000 QTRAP mass spectrometer. CM-PE and CE-PE were analyzed using an ODS column (Xbridge, 2.1 × 100 mm; Waters, Tokyo, Japan) with an isocratic mobile phase of methanol-water   ( Fig. 4A, B ). Amadori-PE and nonglycated native PE species were also detected by NLS of 303 Da (H 2 PO 4 CH 2 CH-2 NHC 6 H 11 O 5 ) and 141 Da (H 2 PO 4 CH 2 CH 2 NH 2 ), respectively ( Fig. 4C, D ). The neutral loss spectra of CM-PE, CE-PE, Amadori-PE, and native PE indicated that PE glycation proceeded toward the formation of AGE-PE in erythrocytes. The glycated molecular species of PE (CM-PE, CE-PE, and Amadri-PE molecular species) were also observed in NLS spectra of plasma samples; however, some AGE-PE species could not be clearly detected in plasma possibly due to their low amounts (supplementary Fig. I ).  ( Fig. 3A ). The data indicated that CM-PE and CE-PE were actually produced as AGE products of PE ( Fig. 3B, C ).  method to analyze AGE-PE with high selectivity and sensitivity at the molecular species level.

Profi ling of AGE-PE molecular species in blood samples
By using the QTRAP MS/MS, we found that protonated CM-PE and CE-PE tended to generate product ions of [M+H-199] + and [M+H-213] + , respectively ( Fig. 2 ). The neutral loss of the polar head group indicates that NLS ( Fig. 2 ) and MRM ( Fig. 3 ) are adaptable for the (LC-)MS/MS analysis of AGE-PE. The NLS technique enabled us to profi le molecular species of CM-PE and CE-PE in human erythrocytes and plasma even without LC separation ( Fig. 4 , supplementary Fig. I ). These results provide direct information on the molecular species of AGE-PE in erythrocytes and plasma and indicate that PE glycation proceeds toward the formation of AGE-PE in vivo. However, compared with native PE molecular species composition, 18:0-20:4 and 18:0-22:6 species were higher in erythrocyte CM-PE composition ( Fig. 4 , Table  1 ). It suggests that 18:0-20:4 and 18:0-22:6 PE are susceptible to carboxymethylation or these CM-PE molecular species are more stable than the others in erythrocytes, but the possibilities require investigation. In this study, we analyzed glycation products of diacyl PE species; however, alkenyl-acyl PE (plasmalogen) species are also in biological specimens. It appears likely that glycated alkenyl-acyl PE species are also generated in vivo. However, due to the limited source of pure alkenyl-acyl PE species, it is diffi cult at present to prepare plasmenyl AGE-PE molecular species as authentic standards for LC-MS/MS analysis. Thus, we could not evaluate the plasmenyl AGE-PE species in this study.
MRM experiments can provide accurate quantitation of lipid molecules, as reviewed by Sullards ( 21 ). In the present study, LC-MS/MS with MRM was highly useful for the measurement of AGE-PE in blood samples. Based on the results of NLS, we focused on eight molecular species with MRM. Parameters were optimized to permit MRM detection and LC separation by using synthetic reference compounds ( Fig. 5A ). Under the optimized conditions, all calibration curves showed good linearity (0.998-0.999) ( Fig. 5B ), with detection limits of 5 fmol/injection at a signal-to-noise ratio of 3. The eight molecular species of both CM-PE and CE-PE were clearly detected in MRM chromatograms of erythrocytes ( Fig. 6 ). Most of the AGE-PE molecular species were also shown in plasma MRM chromatograms (supplementary Fig. II ). As shown in Tables 1 and 2 , no signifi cant differences were observed in CM-PE and CE-PE concentrations in erythrocytes and plasma between healthy subjects and diabetic patients. In contrast, Amadori-PE concentrations were signifi cantly higher in diabetic erythrocytes and plasma ( Tables 1 and 2 ).

DISCUSSION
For the occurrence of AGE of PE in vivo, there have been at least three confl icting reports (12)(13)(14). A recently developed QTRAP MS/MS offers specifi c benefi ts ( 18 ) for biomolecular analysis including lipid molecules ( 7,19,20 ). The QTRAP MS/MS allows product ion scanning, NLS, and MRM, providing useful structural information of the analytes even in the presence of background contaminants in complex biological materials. In the present study, using synthesized CM-PE and CE-PE as reference compounds, we developed an LC-MS/MS  Values are means ± SD (n = 8 for healthy subjects and n = 10 for diabetic patients). c Number in parenthesis represents as mmol/mol of total CM-PE, CE-PE, or Amadori-PE species against total native PE species.
According to the protein glycation mechanism, the generation of CM derivatives requires an oxidation step. The reaction proceeds via the oxidative degradation of Amadori products ( 25 ) and/or the reaction of amines with glyoxal, a product of glucose autoxidation as well as lipid peroxidation ( 26,27 ). On the other hand, CE derivatives ( 28 ) are reaction products of the C-3 unit methylglyoxal, which is a product formed by reverse aldol reaction of 3-deoxyosones, enzymatic synthesis from dihydroxyacetone phosphate, and nonenzymatic dephosphorylation of glyceraldehyde phosphate or dihydroxyacetone phosphate. Considering this literature (25)(26)(27)(28), the plausible formation mechanism of CM-PE and CE-PE is depicted in Fig. 1B . The different formation mechanisms between CM-PE and CE-PE may explain why the CM-PE level is higher than that of CE-PE in vivo ( Tables 1 and 2 ). In addition, CM-PE and CE-PE might be used as biomarkers for oxidative stress and carbonyl stress, respectively.
In the present study, among the glycation products of PE, only Amadori-PE was signifi cantly elevated in erythrocytes concentrations using MRM. For LC separation, we investigated LC conditions and adopted an ODS column for separation of AGE-PE species. A silica column under hydrophilic interaction chromatography mode was used for separation of both Amadori-PE and nonglycated PE. These LC conditions could reduce background noise and improve resolutions of the analytes. Under the present conditions, the detection limits of AGE-PE by MRM were around 5 fmol/injection, which were relatively sensitive compared with that of LC analysis of phospholipid derivatives, such as phospholipid hydroperoxides ( 22,23 ) and platelet-activating factor-like phospholipids ( 24 ) Table 2 ). and plasma of diabetic subjects ( Tables 1 and 2 ). The data suggest that hyperglycemia in diabetic patients does not affect AGE-PE concentrations in erythrocytes and plasma, whereas the Amadori-PE concentration was markedly increased under hyperglycemic conditions. Similar to the present results, Requena et al. ( 12 ) reported that no differences were observed in erythrocyte CM-PE levels between healthy and diabetic subjects. The reason given was that the diabetic patients participating in that study were free of complications ( 12 ). Thus, AGE-PE might have accumulated more in diabetic patients with severe complications, because oxidative stress and carbonyl stress under hyperglycemic conditions are considered to be involved in the pathogenesis of diabetic complications. In this study, like AGE-PE, no signifi cant differences were observed in plasma concentrations of carboxymethyllysine (one of the well-known protein AGEs) between healthy subjects and diabetic patients (data not shown). Therefore, in a future study, it should be necessary to further elucidate the involvement of AGE lipids and AGE proteins in the pathogenesis of diabetic complications by analyzing their levels between diabetic patients with and without complications. On the other hand, as mentioned above, we found that Amadori-PE, but not carboxymethyllysine, was higher in diabetic blood. The result suggests that Amadori-PE is more prone to be accumulated compared with carboxymethyllysine, even in early stages of diabetes. This may be related to the fact that Amadori-PE and carboxymethyllysine are early and advanced glycation products, respectively ( 29,30 ). To put it another way, Amadori-PE may be used as a potentially sensitive marker for refl ecting hyperglycemic conditions in the early stage of diabetes (31)(32)(33).
In summary, we developed the LC-MS/MS assay for CM-PE and CE-PE and provided direct information on the molecular species of AGE-PE in human erythrocytes and plasma. The LC-MS/MS technique with MRM will be a powerful tool for understanding the pathophysiological consequence of in vivo lipid glycation.