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Boronic Derivatization of Monoacylglycerol and Monitoring in Biofluids Mengle Zhu, Xiaowei Xu, Yuanlong Hou, Jialing Han, Jin Wang, Qiuling Zheng, and Haiping Hao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00805 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019
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Analytical Chemistry
Boronic Derivatization of Monoacylglycerol and Monitoring in Biofluids Mengle Zhu, ‡a,b Xiaowei Xu, ‡a Yuanlong Hou,a Jialing Han,a Jin Wang,a Qiuling Zheng*a,b and Haiping Hao*a Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key Laboratory of Natural Medicines, College of Pharmacy, China Pharmaceutical University, Tongjiaxiang #24, Nanjing, Jiangsu, 210009, China. b Department of Pharmaceutical Analysis, College of Pharmacy, China Pharmaceutical University, Tongjiaxiang #24, Nanjing, Jiangsu, 210009, China. a
ABSTRACT: Monoacylglycerols (MAGs) are active mediators involved in multiple biological processes closely related to the pathological development of diabetes, obesity and cancers. Sensitive and unambiguous detection of MAG is thus essential; however, previous methods are both indirect and labor-intensive. Herein, we developed a straightforward approach by derivatization of MAGs with 3-nitrophenylboronic acid (3-NPB) for sensitive and selective analysis in cell lysates, tissues and serums by mass spectrometry (MS). Reaction occurred between boronic acid and cis-diol moiety of MAGs blocked the formation of multiple adduct ions and tuned MAGs to negatively charged carrying species. In addition, the characteristic isotopic distribution of boron specialized the presence of modified MAGs in MS and lead to distinctive identification. To eliminate endogenous interferences, we further introduced isotopic labeled d4-NPB equivalently pre-mixed with d0-NPB to perform MAG derivatization, which resulted in rapid identification of modified MAGs among matrix by displaying doublet peak characteristics. A comparative quantification approach was thereafter evoluted to reveal the amount variation of MAGs by d0-NPB and d4-NPB separately derivatization in different pathological tissue and serum samples. The presently developed NPB-based derivatization approach is expected essential in the metabolic study of MAG-related diseases.
Introduction Lipids are essential for cellular energy supply and structure formation, and their metabolism is related with the regulation of many diseases1-4. For instance, the level of triglyceride has been widely studied and become a commonly tracked indicator in the diagnosis of cardiovascular disease, visceral obesity, metabolic syndrome and diabetes2, 5-6. Lipid metabolism initiates from the synthesis of lipids, followed by the storage within tissues and cells, and undergoes lipolysis, which are regulated by corresponding lipases7. Therefore, the monitoring of their amount variation is essential for understanding the mechanism involved in lipid-related diseases, including diabetes, obesity and cancers8-9. In lipid metabolism, monoacylglycerols (MAGs) are precursors of final lipolysis process and could be broken down to generate free fatty acids (FFAs) and glycerol by MAG lipases7. The role of MAGs in pathophysiological conditions are, however, neglected due to their trace amount in biofluids that would be easily suppressed during detection, as well as confused by the existence of stereoisomers. It is now realized to be important as a group of active signalling molecules and their content differences are discovered interrelated with numerous cancer research1, 10-11. Conventional methods involve preliminary thin-layer chromatographic (TLC) separation of MAGs, followed by degradation to glycerol and their corresponding FFAs. The obtained FFAs are then subjected to secondary gas chromatographic mass spectrometry (GC-MS) for identification and quantification, which is considered to be indirectly, tedious and inaccurate12-16. Besides, direct detection
of MAGs is usually obscured by the formation of multiple adducts in the positive ion mode of MS due to the existence of dihydroxyl structure. Accordingly, it remians a big challenge for the unambiguous identification and quantification analysis of MAGs in biological systems. Therefore, a sensitive detection and quantification method is urgently required for the analysis of MAGs in biofluids. Boronic acid has been widely used for saccharide modification to improve their MS detection selectivity and sensitivity due to its highly reactivity towards cis-diol moiety, which is also the structure feature of MAGs17-22. Herein, we reported a boronic derivatization approach for direct and sensitive identification and comparative quantification of MAGs in biofluids using 3-nitrophenylboronic acid (3-NPB). According to the designed derivatization reaction, boronic acid would selectively react with cis-diol moiety of MAGs under mild conditions and quickly generated negatively charged species during MS ionization process. In addition, the presence of boron contributed a characteristic isotopic distribution in MS spectra, which benefited the identification of modified MAGs. For MAGs in complex biofluids, we synthesized d4-NPB by one step reaction and applied an equivalently light/heavy-labeled NPB (denoted as d0/d4-NPB below, structures shown in Scheme 1A) derivatization strategy that highly improved MAG identification among matrices by presenting doublet characteristic peaks. On this basis, we further integrated a comparative quantitative approach by applying d0- and d4-NPB modification of different physiological samples separately for
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monitoring MAG amount variation. The results suggested that the designed NPB derivatization strategy was valuable for monitoring MAG variation under pathological states.
(ppm): 147.88, 140.65, 131.94, 129.10, 124.50. (Figure S1B, SI). HRMS: C6H3D4BNO5 [M-H]+, calcd 170.0568 found 170.0625. m.p.: 283-285 °C.
Reagents and materials 3-NPB was purchased from Sigma–Aldrich (St. Louis, MO, USA); d4-3-nitroaniline was purchased from Wako Pure Chemical Industries, Ltd. (Japan); sodium nitrite (NaNO2) was obtained from Sam Sa-en Chemical Technology Co., Ltd. (Shanghai, China); 4-tolylboronic acid, 4fluorobenzeneboronic acid, phenylboronic acid and tetrahydroxydiboron (B2(OH)4) was purchased from AdamasBeta (Shanghai, China); sodium acetate (CH3COONa) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); hydrochloric acid (HCl) was obtained from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China); potassium carbonate was obtained from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). 14:0 was purchased from Larodan (Stockholm, Sweden); 18:0 and 16:0 were purchased from Toronto Research Chemicals (Toronto, Canada); 18:1, 18:2, 20:4, were purchased from SHANGHAI ZZBIO CO., LTD.(Shanghai, China). Ultrapure H2O was supplied by the Milli-Q Pure Water System (Millipore, USA). Acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany).
Derivatization procedure of MAGs by NPB MAG standards were prepared at concentration of 100 μM. d0- and d4-NPB stock solutions were prepared in MeOH at 10 mM. For derivatization of MAG standards, 10 μL 10 mM of d0/d4-NPB were pre-mixed equivalently before applied to 100 μL of MAG (or MAG mixture) and reacted at 400 rpm, 37 °C for 2 hrs. Centrifugation was performed at 18,000 rpm for 10 min and 50 μL of supernatant was transferred for LC-MS analysis.
Animals and Treatments Male healthy C57BL/6 and OB mice, were obtained from Nanjing University (Jiangsu, China). They were kept in an airconditioned animal quarter at temperature of 25 ± 2 °C and relative humidity of 50 ± 10% with 12-hour light/dark cycles. Water and food were allowed ad libitum. Animals were acclimatized to the facilities for 1 week and then randomly divided into different groups for research. Animals were feeding 8 weeks and livers were taken. All animal studies were approved by the Animal Ethics Committee of China Pharmaceutical University. Synthesis of Derivatization Reagent d4-NPB d4-3-nitroaniline (50 mg, 0.36 mmol) was dissolved in H2O(1.0 mL) and added hydrochloric acid (5% in H2O, 0.6 mL, 0.90 mmol). The mixture was stirred at room temperature for 1 min. NaNO2 (30 mg, 0.43 mmol) dissolved in H2O (0.5 mL) was added by drops into the mixture and stirred for 15 min at 0°C. B2(OH)4 (64.9 mg, 0.72 mmol), CH3COONa (59.36 mg, 0.72 mmol) and H2O (6.0 mL) were added subsequently to the mixture and stirred for 20 min at room temperature. After the addition of EtOAc (25 mL), the pH of the mixture was adjusted to 8 by saturated K2CO3. After that, the mixture was separated and the organic layer was extracted with saturated K2CO3 (4×10 mL). The combined aqueous layer was acidified until pH6 with HCl (5% in H2O) and was extracted by EtOAc three times (10 mL each time). The combined organic layer was washed by H2O (2×10 mL), brine (10.0 mL) and dried over anhydrous sodium sulfate23. After suction filtration, the solvent was removed in vacuum to afford (3-nitrophenyl-2, 4, 5, 6-d4) boronic acid (d4-NPB) as off-white solid (18 mg, 29%). 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.49 (s, 2H) (Figure S1A, Supporting Information). 13C NMR (75 MHz, DMSO-d6) δ
Cell lysate/tissue/serum sample derivatization procedure For biological sample preparation, cell suspension was mixed with 400 μL of MeOH and lysed by sonication on ice; tissue sample were weighted and 400 μL of MeOH was added for homogenate for 1 min; 50 μL of serum sample were added to 550 μL of MeOH. MAG extraction was performed by vortex for 10 min and followed up with protein precipitation at -20 °C for 20 min. The application of pure MeOH benefited the elimination of endogenous saccharides by solubility difference and glycopeptides/proteins via precipitation. Centrifugation was performed at 18,000 rpm for 10 min and resulted supernatant was transferred to subsequent derivatization reaction, which was described above. Due to the complexity of serum samples, 30 mg sodium sulfate anhydrous was added to the mixture during reaction process to remove water and enhanced the reaction efficiency. After derivatization reaction, the resulted solution underwent centrifugation at 4 °C, 18,000 rpm for 10 min twice prior to LC-MS analysis. Ultra Performance Liquid Chromatography and Mass Spectrometry (UPLC-MS) A 2.1×100 mm high strength silica (HSS) T3 column was used for separation. UPLC (Waters ACQUITY UPLC I-Class) conditions were set as follows: column temperature: 40 °C; mobile phase: H2O (solvent A) and ACN (solvent B); flow rate: 0.4 mL/min; and injection volume: 1 μL. A gradient elution condition was applied as follows: 10% B for 0–0.2 min, 10– 80% B for 0.2–1min, 80–98% B for 1–3 min, maintained for 3 min, then returned to 10% B for 6–6.5 min and re-equilibrated for 2.5 min. Waters Synapt G2 Si Q-TOF was operated to acquire data in the negative ion mode and the parameters were set as follows: m/z range: 50-1200 Da; capillary voltage: -2.5 kV; sampling cone: 40 V; desolvation temperature: 500 °C; source temperature: 120 °C; cone gas flow: 50 L/h; desolvation gas flow: 800 L/h; nebulizer gas pressure at 6.5 bar. Results and discussion (A)
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which MAG identification sensitivity among complicated matrices would be improved.
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Scheme 1. (A) Chemical structures of d0- and d4-NPB; and (B) designed deravertization reaction mechanism of NPB with MAG.
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Derivatization of MAG standards Theoretically, boronic acid of NPB could react with cis-diol moiety of MAGs to form a stable five-membered ring. Due to the electron withdrawing property of boron, the derivatized products would turn to negatively charged species with distinctive MS response (Scheme 1B). MAG 16:0 was first selected as an example to verify the designed strategy. Four commercially available boronic acid derivatives were selected to test the derivatization reaction, including 4-tolylboronic acid, 4-fluorobenzeneboronic acid, phenylboronic acid and 3-NPB. Upon derivatization and MS detection, only 3-NPB yielded expected product at m/z 477.3 (monoisotopic peak) (Figure 1A) via loss of two H2O molecules. Particularly, the characteristic isotopic distribution of boron brought isotopic peak at m/z 478.3 dominant. The structure could be confirmed upon CID spectrum shown in Figure S2A by yielding glycol side chain loss, nitrobenzene loss fragment ions. Likewise, d4-NPB derivatized 16:0 was detected at m/z 482.3 (Figure 1B) carrying similar boronic distribution, but with 4 Da mass shift. Different temperatures (20 ℃, 37 ℃ and 50 ℃) and reaction time periods (1 hr, 2 hrs and 3hrs) were tested to obtain optimal derivatization using MAG 16:0 as a standard. According to the obtained MS intensity of modified 16:0, 37 ℃ resulted to higher intensity than that of 20 ℃ and 50 ℃ (Figure S2C, SI). On this basis, 2 hrs led to similar intensity with that of 1 hr and higher than that of 3 hrs while less fluctuation and more reproducible (Figure S2D, SI). Based on the principle of under physiological and mild reaction conditions to avoid unexpected side effects in biological systems, 37 ℃ and 2 hrs were selected all and after. The limit of detection (LOD) for d0-NPB derivatized 16:0 was determined as low as 20 nM with well-resolved isotopic peak distribution (data not shown). Subsequently, the calibration curve of d0-NPB derivatized 16:0 was build up (Figure S2B, SI) within the concentration ranging from 2 to 500 μM and linear trend R2=0.9639 could be achieved. On this basis, four more common MAGs, including 18:2, 18:1, 18:0, and 20:4 were selected to validate method generality. Upon derivatization, selected MAGs could be modified and detected with boronic isotopic distribution (Figure S3, SI). Thus, the designed NPB derivatization specially tuned MAGs distinctive in MS spectra by showing characteristic isotopic distribution of boron, upon
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Analytical Chemistry
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Figure 2. UPLC-MS spectra of d0/d4-labeled MAGs (A) 14:0, (B) 16:0, (C) 18:2, (D) 18:1, (E) 18:0, and (F) 20:4. In comparison with above MAG standard derivatization and detection, their screening among biological matrices is considered to be more complicated and challenged due to the existence of diverse endogenous interferences. Accordingly, we introduced equivalently pre-mixed d0- and d4-NPB to perform derivatization reaction, and followed up with UPLC separation and MS detection. We started with derivatization of a mixture containing six common MAG standards. The observed coeluent of d0/d4-labeled MAGs indicated that deuterium label would not affect the chromatographic behaviour and all MAGs appeared as doublet in MS with mass interval of 4 Da (Figure 2A-F). In particular, the relative intensity of each pair was investigated to be compatible by observing the intensity fluctuation within 9.3 % (Table S1, SI), which suggested that the utilization of d4-NPB would not reduce the derivatization efficiency nor the ionization efficiency of modified MAGs. Furthermore, the display of doublet peak of equal intensity with mass interval of 4 Da provided another dimension for MAG screening and identification among complex matrices. Derivatization of MAGs in Cell Lysates Based on the derivatization of MAG standards, we next asked the application of d0/d4-NPB derivatization in complex cell lysates. Herein, we chose HepG2 and MCF-7 cell lysates as exemplary. In the case of HepG2, d0- and d4-NPB were premixed equivalently and then doped into cell lysate to initiate the derivatization reaction. The resulted reaction solution was subjected to UPLC separation and MS detection. The acquired MS spectra in Figure 3 showed the expected results that total eleven MAGs emerged and were located rapidly due to their doublet peaks and the isotopic characteristic of boron. The acquired MS spectra showed that 16:0, in this case, mainly formed H2O adduct and was detected at m/z 496.3/500.3. This may due to the much complicate aqueous environment of cell
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Analytical Chemistry lysate. In addition, the MS intensity ratio of for each pair of modified MAG (d0/d4-labeled) was considered to be equal by detecting a fluctuation within ±13.1% (Table S2, SI), which could be reasoned by the minor signal suppression and matrix effects from complex cell lysate during chromatographic separation. Similarity, among MCF-7 cell lysate six MAGs, including 8:0, 12:1, 12:0, 14:2, 14:1 and 14:0 (Figure S4, SI), could be monitored as doublets with d0/d4-labeled peaks intensity fluctuation within 6.2% (Table S3, SI). Therefore, by utilizing equivalently d0/d4-labeled NPB derivatization, trace amount of MAGs could be precisely tracked among complex biofluids.
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Table 2. MS intensity of detected MAGs and ratios between CA and N serum samples.
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Accordingly, the intensity ratios of 14:2, 14:0, 16:0, and 18:1 were calculated as 22.7%, 37.4%, 32.4%, and 57.6% (OB/C %), which suggested that in OB liver tissues the amount of 14:2, 14:0, 16:0, and 18:1 reduced by 77.3%, 62.6%, 67.6% and 42.4%, respectively. On the contrast, the intensity ratios of 17:0 and 18:0 were detected significantly increased by 282.9% and 190.5%, which indicated that in OB sample, the formation of 17:0, 18:0 had increased by 182.9% and 90.5% compared with that in control sample. Table 1. MS intensity of detected MAGs and ratios between OB and C livers from mice.
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Figure 3. MS spectra of d0/d4-labeled NPB derivatized from HepG2 cell lysate (A) 8:0; (B) 12:1; (C) 12:0; (D) 14:3; (E) 14:2; (F) 14:1; (G) 14:0; (H) 16:1; (I) 16:0; (J) 18:1; and (K) 18:0. Comparative Quantification of MAGs between Different Pathological Tissues and Serums According to the doublet characteristic-based MAG identification capacity, a comparative quantification strategy was thereafter evoluted to relatively quantified MAGs between different pathological samples. One pair of liver tissues from control/obesity (C/OB) mice were first chosen to verify the methodology. After tissue homogenate and MAG extraction, d0- and d4-NPB were applied to C and OB samples respectively to initiate derivatization reactions and then subjected to UPLCMS analysis. Total seven obesity-related MAGs could be tracked, including 14:3, 14:2, 14:0, 16:0, 17:0, 18:1 and 18:0, and their absolute intensities are listed in Table 1. The obtained intensity difference of d0/d4-labeled of each substance represented their abundant variation and was presented as intensity ratios. Taking MAG 14:3 as an example, d0-14:3 at m/z 444.3 from control sample had absolute intensity of 8.08e5 while d4-14:3 at m/z 448.3 from obesity sample had absolute intensity of 8.07e4, which was calculated as an intensity ratio of 10.0% (OB/C%), indicating that the amount of MAG 14:3 in OB liver decreased by 90.0% compared with that in control sample (Table 1). Note that in this regard, the intensity change between different samples would be particularly useful while additional internal standard was not required during quantification process.
In order to verify the quantitative accuracy that the intensity differences were caused by the amount variation of MAGs instead of affected by the derivertization efficiency oscillation, we further investigated a reverse labeling of OB sample with d0-NPB and the control sample with d4-NPB and followed up with intensity ratio calculation. Similarly, based on the obtained intensity of d0/d4-labeled products, 14:3 was detected as reduced by 89.3%, 14:2 reduced by 78.2%, 14:0 reduced by 58.1, 16:0 reduced by 69.7, and 18:1 reduced by 43.1%, while 17:0 was detected as increased by 219.2% and 18:0 increased by 100.0% in OB sample (Table S4, SI). More importantly, comparison between the ratio changes from Table 1 and that obtained from Table S4, , the fluctuation errors were calculated around 10%, which could be explained as matrix effect or MS signal fluctuation. With reasonable ratio fluctuation, the presented comparative quantification approach could provide the abundant variation of detected MAGs between two different pathological statements.
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Analytical Chemistry Based on the above comparative quantification approach, we next explored the amount variation of MAGs in human serum, including eight samples from breast cancer patients (CA) and eight samples from normal healthy (N) people. According to the procedure described previously, CA samples were modified by d4-NPB and healthy control were labeled by d0-NPB, respectively. With the characteristic of boron isotopic distribution, total seven major MAGs could be identified and their intensity values obtained from each sample were plotted in Figure S5, including 12:1, 12:0, 14:3, 14:2, 14:1, 14:0, and 18:0, respectively. For each MAG substance, average value was calculated according to eight sample detection and listed in Table 2 for amount variation comparison. Based on the relative intensity ratio calculation (CA/N, %), besides MAG 18:0 that slightly increased by 9.0% in cancer samples, others were all detected decreasing ranging from 19.4% to 56.6%, which suggested that under pathological status of breast cancer, dominant MAGs may be consumed or less synthesized. The accuracy of measurements were further confirmed by reversed derivatization, which displayed similar ratio changes between cancer and healthy serum, and reasonable detection fluctuation ranging from 4% to 14.0% (Table S5, SI). Conclusions This study presented a novel, unambiguous and sensitive derivatization approach for MAG screening and relative quantification in biofluids. Cis-diol moiety of MAG was derivatized by NPB under mild condition, which was considered to be green and environment friendly, reduced the possibility of various metal adduct formation and improved the MS detection sensitivity. The simplicity of MAG extraction involving MeOH during cell lysis and tissue homogenate improved the selectivity of the strategy by eliminating endogenous saccharides and glycopeptides/proteins. The characteristic isotopic distribution of boron brought specificity for the identification of modified MAGs compared with other endogenous interferences. On the basis of these advantages, we newly synthesized a deuterium labelled NPB and employed equivalently mixed d0/d4-labeled NPB derivatization strategy, which brought the feature of doublet on the appearance of modified MAGs in order to emerge from complex matrices. Furthermore, a comparative quantification approach was evoluted to monitor the MAG abundant variations between different pathological samples by applying d0 and d4-labeling separately and finally reflected by their obtained intensity ratios. Therefore, the capability of selective detection and relative quantification of MAGs by the reported NPB-based derivatization approach will be essential in the metabolic study of MAG-related diseases.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author *
[email protected] (H.H), Tel: 86-25-83271179; *
[email protected] (Q.Z.), Tel: 86-25-83271203 Author Contributions ‡These authors contributed equally.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (grants 81430091, 81421005, 81720108032, 91429308, 81703471, and 21602254), the Natural Science Foundation of Jiangsu Province (grant BK20170740, BK20160767). 111 project (G20582017001), the project for Major New Drug Innovation and Development (2018ZX09711002-001004, 2018ZX09711001-002-003, 2015ZX09501010), State Key Laboratory of Natural Medicines at China Pharmaceutical University (SKLNMZZCX201817), "Double-First Rate" project (CPU2018GF09), and the Fundamental Research Funds for the Central Universities (2632019ZD20).
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