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A Ratiometric Fluorescent Probe for Monitoring Endogenous Methylglyoxal in Living Cells and Diabetic Blood Samples Huiling Wang, Yulin Xu, Li Rao, Chun-tao Yang, Hong Yuan, Tingjuan Gao, Xin Chen, Hongyan Sun, Ming Xian, Chunrong Liu, and Changlin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05426 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019
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Analytical Chemistry
A Ratiometric Fluorescent Probe for Monitoring Endogenous Methylglyoxal in Living Cells and Diabetic Blood Samples Huiling Wang,†,# Yulin Xu,†,# Li Rao,† Chuntao Yang,§ Hong Yuan,† Tingjuan Gao,† Xin Chen, † Hongyan Sun,‡ Ming Xian║ Chunrong Liu,*† and Changlin Liu† †Key
Laboratory of Pesticide and Chemical Biology, Ministry of Education, Chemical Biology Center,
College of Chemistry, and International Joint Research Center for Intelligent Biosensing Technology and Health, Central China Normal University, Wuhan 430079, Hubei, China. §Key
Laboratory of Protein Modification and Degradation, School of Basic Medical Sciences,
Guangzhou Medical University, Guangzhou, China. ‡Department
of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong,
China. ║Department #These
of Chemistry, Washington State University, Pullman, Washington 99164, United States.
authors contributed equally.
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Abstract Optical imaging provides noninvasive powerful tools not only for better understanding the physiological and pathological roles of Methylglyoxal (MGO) in living systems but also for potential clinical diagnosis of MGO-related diseases such as diabetic complications. However, so far only very few “turn-on” MGO fluorescent sensors have been developed, and they are all based on the reaction between MGO and benzenediamines. Due to the possible reactions of benzenediamines with other cellular molecules such as NO and FA, these sensors suffer from limited selectivity and potential deactivation in cells. Herein, we report a novel MGO recognition reaction using 2-aminoacetamide. The reaction between MGO and 2-aminoacetamide was found to be highly efficient and specific, with no interference from NO and FA in particularly. This reaction was used to develop the first ratiometric fluorescent probe (CMFP) for MGO. We have proved that CMFP could detect MGO at physiological concentrations in both aqueous buffer and living cells with excellent selectivity and sensitivity. Furthermore, we successfully utilized CMFP to study intracellular MGO generation routes and evaluated MGO levels of clinic blood samples from healthy and diabetic patients. These results highlight the potential utility of this probe in both basic science research and clinical diagnosis.
Introduction Reactive carbonyl species (RCS) are a family of molecules with highly reactive carbonyl groups that are continuously produced via oxidation of carbohydrates, lipids, and amino acids in various living organism, from bacterial to human beings.1-5 Representative examples are 4-hydroxy-trans-2-nonenal, acrolein, methylglyoxal, glyoxal, malondialdehyde and formaldehyde. So far many of these species have been 2
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demonstrated to play important roles in physiological and pathological process.6,7 Among them, methylglyoxal (MGO), a kind of RCS with two reactive carbonyl groups endogenously produced from glycolysis, lipid peroxidation, and protein amino acids metabolism within cells,8,9 has received increased attention. It has been suggested to be a major precursor of advanced glycation end products (AGEs) by reacting with DNA, lipids and proteins (mainly through lysine, arginine and cysteine residues). The MGO-mediated AGEs could induce protein dysfunction,10 activation of membrane receptors and proinflammatory signaling,11 exerting multiple and diverse effects in cellular oxidative stress,12,13 inflammation,12,14,15 and endothelial cells dysfunction.16-18 Besides, literatures demonstrate that elevated levels of MGO are associated with pathologies of various human diseases such as obesity,19,20 cardiovascular disease,21 hyperalgesia,22,23 kidney disease,24,25 metabolic syndrome,26 colorectal cancer,27 and especially diabetes.28 The elevation of MGO levels in diabetic human bloods and urines is widely observed and thought to be involved in the mechanisms contributing to the development and progression of diabetes. More importantly, the clinical data demonstrates that hyperglycemia can’t fully explain the development of diabetes complications. And the long-used diagnostic markers blood glucose and glycated hemoglobin (HbA1c) are reported to be not insufficient to predict the progressions of diabetic complications. Owing to the correlation between MGO-induced AGEs levels and clinical features of various diabetic complications such as diabetic nephropathy, retinopathy and cardiovascular complications,28 MGO is regarded as an alternative/better diagnostic marker for diabetic complications.29,30 Despite the rising interest in MGO research, fundamental questions regarding its regulation of production, mechanism of action, and metabolism still remain unaddressed.31 One of critical debates in this field is the biologically relevant levels of MGO as current reports spanning over 103-fold concentration range.32,33 Accurate and reliable measurement of MGO concentrations in biological samples is still required for better understanding the roles of MGO in cellular processes and pathologies of related diseases. Currently, the major methods for MGO detection are electrochemical 3
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and absorbance-based assays, HPLC and LC-MS approaches.34-36 These methods often require post-mortem processing and destruction of tissues or cell lysates. Fluorescence imaging with chemical probes has emerged as a powerful tool for in situ and real-time visualization of biomolecules in biological systems owing to its high sensitivity, non-invasiveness and convenience. However, until now very few “turn-on” fluorescent probes for MGO detection have been developed, although much progress has been made in developing fluorescent probes for another RCS, such as formaldehyde. 37-40 And these MGO probes often suffer from possible poor selectivity, potential deactivation and limited quantitative capability. In one hand, the recognition moiety of these probes,41-43 o-phenylenediamine (OPD) could react with other important biologic small molecules, such as NO and formaldehyde (FA) to induce possible selectivity and potential deactivation problem in cells.41 On the other hand, the detection signal of these “turn-on” probes relies on single emission intensity change, which can be distinctly affected by instrument efficiency, light scattering, local probe concentration and microenvironment.44 In contrast, the ratiometric fluorescent probes of MGO, employing the ratio of the emission intensity at two different wavelengths, could provide a self-calibration effect to reduce most of the aforementioned interference, and enable more accurately quantitative tracking of MGO in biologic samples. Until now, there is no ratiometric fluoresce probes of MGO reported. Herein, we present the first ratiometric fluorescent probe CMFP with excellent sensitivity and selectivity based on a novel MGO recognition reaction for the detection of exogenous and endogenous MGO in living cells. Moreover, we further demonstrate the applications of CMFP in investigating possible biogenesis approaches of endogenous MGO within living cells and monitoring the MGO levels of real clinical blood samples from healthy people, diabetics and diabetic complications patients. Our results indicate that CMFP could be potentially used not only for basic science research but also for clinical sample analysis.
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Experimental Section Analog computation methods Density functional theory (DFT) calculations were performed for the theoretical investigation of ESIPT mechanism. The DFT method employed in this work is ωB97xD with excellent performance on both short and long ranges interaction.45 Both CMFP and CMFP-CP were considered. Local minima and transition state structures involved in ESIPT were obtained by geometry optimization. Excited states were simulated employing TDDFT technique.46 Frequency analysis was used to verify the nature of stationary points. Geometries were determined with 6-31+G (d) basis set while energies were determined by single point calculations with 6-311+G (d, p) basis set upon optimized structures. All calculations were performed using the Gaussian09 package.47 Some input files for DFT and TDDFT calculations employed in this work (Supporting Information). Cell culture HeLa cells were grown on T25 flask in DMEM supplemented with 10 % (v/v) FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37ºC under a humidified atmosphere containing 5 % CO2. General fluorescence imaging methods For fluorescence imaging, HeLa cells were sub-cultured and seeded in glass-bottomed dishes (NEST) at a density of 5x104 and incubated in DMEM with 10 % FBS and 1 % antibiotics for 24 hours. Cells in each dish would reach to 70 % confluence before imaging experiments. Then, the adherent cells were washed with PBS and changed into FBS-free DMEM. Next, cells were pretreated with varying concentrations of MGO or other analytes at 37ºC for appropriate time. Then, the cells were washed with PBS and incubated with the mixture of fresh DMEM and administered concentrations of CMFP at 37ºC for 40 min. Fluorescence imaging were performed after removal of the culture solution. General flow cytometry methods 5
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Cells were incubated with given concentrations of MGO, MGO scavengers or MGO initiator for certain time. Then, the cells were washed with PBS and incubated with the mixture of fresh DMEM and administered concentrations of CMFP at 37ºC for 40 min. Next, the cells were analyzed by flow cytometry (BD, AccuriTM C6) equipped with 375 nm UV laser light source, a 520 nm bandpass filter. General fluorescence imaging methods for blood samples Whole blood samples collected from healthy people and diabetics were diluted to 30 folds of PBS. Then, these samples were treated with 30 μM of CMFP and different concentrations of MGO (0, 200 μM), followed by an incubation for 2 hours at 37ºC. Cell imaging was carried out after removal of the culture solution. Data were collected at 420-480 nm and 500-650 nm upon excitation at 405 nm. Fluorescence imaging methods for leukocyte cells from blood samples Whole blood samples were collected from healthy people and diabetics were diluted to 30 folds of PBS, and treated with SDS (erythrocyte scavenger), CMFP (30 μM) and MGO (0, 200 μM), followed by an incubation for 2 hours at 37ºC. Then, these samples were co-cultured with acridine orange (leukocyte dye) for another 10 min. Cell imaging was carried out after removal of the culture solution. Data were collected at 420-480 nm and 500-650 nm upon excitation at 405 nm and 488 nm.
Results and Discussion As described above, most of the existing methods for analyzing MGO are developed by utilizing the o-phenylenediamine (OPD) moiety, which is also known as an efficient NO recognition group for NO fluorescent probe development, as recognition moiety to capture MGO through twice amino-carbonyl condensation. However, as reported in literatures,48 besides MGO, the OPD can also react with formaldehyde or NO to form benzimidazoline/benzimidazole or benzotriazole. Therefore, establishing a novel highly selective MGO recognition reaction is critical for MGO fluorescent probe development. We hypothesize that the reactions of OPD with MGO, HCHO 6
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and NO might occur via the routes described in Scheme 1A. The first step of these reactions, condensation of -NH2 with -CH=O or N=O, should be rapid equilibrium, and no stable product was formed in this step. And so, due to the higher reactivity of amino group towards C=O than C=N/N=N, we reckon that it might be possible to differentiate the MGO from HCHO and NO by attaching an adjacent carbonyl group to decrease the nucleophilicity of second amino group. With this idea in mind, three 2-aminoacetamide derivatives (1a-c) were studied in the model reactions of MGO (Scheme 1B). The reactions were carried out in a mixed solution of CH3CN/PBS (pH 7.4, 1:1 v/v). The products were analyzed after 3 hours at room temperature. When the parent compound 2-amino-acetamide with no substitution 1a or methyl substitution at 2-position 1b were treated with MGO, no desired cyclization product was obtained. However, the substrates with phenyl group at 2-position showed good reactivity and the corresponding cyclization product 2c was obtained in 53 % yields. Moreover, when compound 1c was treated with HCHO or NOC-18 (NO donor), as expected, no cyclization product was isolated. These results confirmed our hypothesis that certain 2-aminoacetamide derivatives could be used to trap MGO selectively.
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N
N A
O
O or
O
NH2
O
C1
O
N
NH2
Me
O
N
O D1
N NH2
X NH2 O C2 N N X NH2 O
HCHO
+ NH2
O
2-aminoacetamide NO
C3 NH2
O B
O
R
NH2
O
HCHO
NH2 NH2
N
HO
N
Me
2a, R=H, N.R. 2b, R=CH3, N.R. 2c, R=phenyl, 53%
1a, R=H 1b, R=CH3 1c, R=phenyl O
R PBS:CH3CN=1:1
+
+ H N 2
NH2
N N N OH O (NOC-18, NO donor)
1c
PBS:CH3CN=1:1 X N.R.
NH2
C
NH2 O
O
O
N MGO
O
O
OH O
Me
N O OH
ESIPT
O
CMFP
X
ESIPT
CMFP-CP
Scheme 1. The design principle and the reaction mechanism of MGO probe. A): Proposed recognition moiety for MGO over NO and HCHO. B): Model reactions of MGO. C): The proposed reaction mechanism of CMFP and MGO.
3-Hydroxychromone (3-HC) dyes are a type of ESIPT molecules which exhibit an excited-state intramolecular proton transfer (ESIPT) reaction to give a dual fluorescence with two emission bands, corresponding to the initially excited normal (N*) and the tautomer (T*) forms.49 We envisioned that if 2-aminoacetamide moiety was installed the into 3-HC scaffold (CMFP, Figure S1A), its cyclization mediated with MGO might affect ESIPT process to exhibit a ratiometric fluorescent response. To test the feasibility of our hypothesis, the theoretical calculation to forecast ESIPT mechanism for both CMFP and proposed MGO-mediated cyclization products (two possible isomers with methyl group at different position) CMFP-CP were carried out by using DFT. The obtained results were summarized in Figure S1B. Almost negligible difference was observed between the two isomers of CMFP-CP. In general, ESIPT process often starts from N state ground state, then changed to first excited 8
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state N* after HOMO → LUMO transition and followed by a transition stage in which the proton is transferred to the other oxygen atom to form T* state. And the final stage T* → T state transition is the source of fluorescent. ESIPT effect mainly depends on the N* → T* step, which is 0.51 eV exothermic for CMFP and 0.15/0.13 eV for CMFP-CP. Accordingly, thermodynamically ESIPT is allowed for both CMFP and CMFP-CP. In virtue of the fact that N* state could only survive for a very short time before emission, the N* → T* should be a very fast step to kinetically enable ESIPT process. Therefore, ESIPT is possible for CMFP with a low energy barrier of 0.11 eV and probably partly forbidden for CMFP-CP with a much higher energy barrier of 0.34/0.37 eV. Moreover, the difference between CMFP and CMFP-CP frontier orbitals was also illustrated in Figure S1. In the case of CMFP, the electron gains more possibility to appear on the benzene group near the attacked carboxyl after HOMO → LUMO transition, thus attracts the proton and promotes ESIPT. On the contrary, the frontier electrons mainly reside on the heterocyclic group before and after HOMO → LUMO transition in the case of two possible isomers of CMFP-CP, which significantly disfavors ESIPT. With these calculation results in hands, we therefore decided to evaluate CMFP as a ratiometric fluorescent probe for MGO detection. To this end, CMFP was synthesized from p-phthalaldehyde in seven steps (Scheme S1). With this probe in hand, we tested its fluorescence properties and responses to MGO in PBS buffer (100 mM, pH 7.4). CMFP showed strong fluorescence emission at 525 nm (λex, 350 nm) which could be assigned as the emission band corresponding to its tautomer (T*) form after ESIPT process. As expected, CMFP reacted rapidly to generate cyclization product CMFP-CP (64 % yield, Supporting Information) which exhibited a blue shift of fluorescence emission at 440 nm (λex, 350 nm) assigned as the emission band of excited normal (N*) forms, probably due to the disruption of ESIPT process by the MGO-mediated cyclization reaction. The distinct gap between these two bands is over 85 nm, which makes this probe favorable for the dual emission ratiometric imaging owing to the minimum 9
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overlap between these two bands. The effects of pH were also investigated and CMFP was found to work effectively in neutral to weak basic pH range of 7.0-8.0 (Figure S3). To demonstrate the efficiency of this probe in the measurement of MGO, varying concentrations of MGO (0-600 M) were added to the solutions of CMFP (10 M). As shown in Figure 1A-a, the emission band at 525 nm decreased slightly, while the band at 440 nm underwent a distinct increment simultaneously. The intensity ratio of the two emission bands, I440
nm/I525 nm,
was linearly related to the concentrations of
MGO in the range of 0-600 M, increased from 0.09 to 3.02 (Figure 1A-b). The detection limit50 was calculated to be around 0.24 μM, and the LOD is 0.5 μM in real experiments, indicating a high sensitivity. To test the selectivity of the probe for MGO, CMFP was treated with a series of biological relevant species including other reactive carbonyl species (HCHO, CH3CHO, CHOCH2CHO and CHOCHO), reactive oxygen and nitrogen species (H2O2 and NO), and amino acids (GSH, Cys, Glu, Ser, Lys, Asp and Ala). As expected, only MGO (500 μM) induces a dramatic increment of intensity ratio I440 nm/I525 nm
while other tested biologic relevant species trigger no change or minor
changes (Figure 1B). Deserved to be mentioned, in consistence with our model reactions, CMFP indeed displayed no response to HCHO, CH3CHO and NO. Furthermore, glyoxal (GO), another dicarbonyl species sharing a similar structure with MGO, only induced a minor change. We speculated that it might be ascribed to that glyoxal tent to form a stable homotrimer in aqueous solution which decreased its reactivity towards our probe.51,52 These results demonstrate good selectivity of CMFP for MGO, suggesting that CMFP may be useful for monitoring of MGO in biological systems.
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Figure 1. Fluorescence emission spectra and selectivity of CMFP. A: a) Fluorescence emission spectra of CMFP (10 M) with various concentrations of MGO (0, 0.5, 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600 μM) at 37ºC for 90 min. b) Linear correlation of fluorescence emission intensity ratios (I440 nm/I525 nm) towards MGO concentrations. B: Fluorescence responses of 10 µM CMFP to 500 µM of biologically relevant species in PBS (100 mM, pH 7.4, 10 % DMSO, v/v) at 37ºC for 90 min.
Then, the capacity of CMFP in monitoring MGO in cultured cells was investigated with confocal microscope. HeLa cells were pretreated with MGO at three separate concentrations (0, 100, 200 μM) for 1 hour before being incubated with CMFP (10 µM) for addition 40 min. As shown in Figure 2a-d, HeLa cells stained with CMFP alone showed strong fluorescence in the green channel and relatively weak fluorescence in blue channel. While MGO pre-treated HeLa cells further stained with CMFP exhibited ratiometric fluorescence responses in a dose-dependent manner, leading to an obvious increment of fluorescence ratio (Iblue/Igreen). To corroborate confocal imaging data, the flow cytometric assay, a high-throughput technique for fast quantification of large cell population, was carried out. HeLa cells were pre-treated with 0, 0.2, 0.5 and 1.0 mM of MGO, and then stained with CMFP for 40 min before being analyzed with flow cytometry equipped with 520 nm bandpass filter. As shown in Figure 2e-f, obvious MGO concentration-dependent enhancements of fluorescence were observed. It was in step with our results from previous confocal imagine experiments, fluorescence enhanced obviously in blue channel and decreased slightly in green channel in MGO concentration-dependent manner. In addition, the cell viability assay illuminated that CMFP has very low cytotoxicity (Figure S6). These 11
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results suggest that CMFP is cell permeable and can be used in detecting MGO within cells.
Figure 2. Confocal microscopy images and flow cytometry analysis of MGO in living HeLa cells using CMFP. Cells were pretreated with varying concentrations of MGO (a, vehicle; b, 100 μM; c, 200 μM) for 1 hour at 37ºC and then stained with 10 μM of CMFP for 40 min. Images were collected at 420-480 nm and 500-650 nm upon excitation at 405 nm. Scale bar: 50 μm. Representative images from replicate experiments (n = 5) are shown. d) Integrated emission intensity ratios of images (a-c) (Iblue/Igreen). e) Cells were pretreated with MGO (0, 0.2, 0.5, 1.0 mM) at 37ºC for 1 hour and then stained with 20 μM CMFP for 40 min before flow cytometry analysis. Excitation was provided by the 375 nm UV laser, and a 520 nm bandpass filter set was applied. f) Normalized fluorescence intensity of flow cytometry data.
As described above, the CMFP-loaded cells without external MGO still exhibited obvious fluorescence in blue channel, and we speculated it might be ascribed to the intracellular intrinsic MGO. To figure it out, we planned to pre-treat cells with N-acetylcysteine (NAC), a reported MGO scavenger,53 to set another control group. Prior to that, experiments were performed to test the applicability of NAC as MGO scavenger in our research. CMFP was incubated with NAC in aqueous 12
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buffer, and displayed no fluorescence response which excluded the possibility of NAC’s interference to the emission profile of our probe. Then, a sample of MGO pre-treated with NAC in aqueous buffer was subsequently incubated with CMFP. As shown in Figure S8, we observed a significant decrement of fluorescence intensity at 440 nm, and a slight increment at 525 nm in comparison with the NAC-free control group. These results indicated that NAC could act as an efficient MGO scavenger in fluoresce assays containing CMFP. Encouraged by these results, we then applied CMFP in tracking endogenous MGO in living cells. Results revealed that pre-treating of NAC almost diminished the fluorescence of the HeLa cells loaded with CMFP in blue channel (Figure 3c), leading to an obvious decrement of fluorescence ratio (Iblue/Igreen) from 1.2 (control group, Figure 3b) to 0.5 (Figure 3d), while exogenous MGO increased the fluorescence ratio to 2.6 (Figure 3a, 3d). This result is in coincidence with the data from flow cytometry experiments (Figure S9). To confirm the generality of its capability in detecting endogenous produced MGO, the cell imaging experiments in three more cell lines (HT-29 cells, HepG2 cells and MCF-7 cells) were carried out. And similar results with HeLa cells were achieved (Figure S10). These observations demonstrated that CMFP was able to monitor the endogenous MGO in living cells.
Figure 3. Confocal microscopy images of endogenous MGO in living HeLa cells using CMFP. After pretreating with 100 μM MGO (a), vehicle (b), or 5.0 mM NAC (c) for appropriate time, 13
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HeLa cells were incubated with 10 μM of CMFP at 37ºC for 40 min. Images were collected at 420-480 nm and 500-650 nm upon excitation at 405 nm. Scale bar: 50 μm. d) Integrated fluorescence emission intensity ratio (Iblue/Igreen) of images (a-c).
After confirming that CMFP could detect endogenous MGO in living cells, we then tested the application of CMFP in investigation of the endogenously generation routes of MGO in living cells. According to literatures reported,28-30 intracellular MGO was endogenously produced mainly during glycose metabolism, lipid peroxidation and amino acids metabolism. In that light, we utilized H2O2 (external ROS resource) and SOD1 inhibitors (ATN-224, LD34, intracellular ROS regulators) to trigger lipid peroxidation, glucose and fructose to enhance glycolysis metabolism, and aminoacetone (key transient intermediate of amino acids metabolism) to simulate amino acids metabolism pathway to study the intracellular MGO production.
54-56
HeLa cells were pre-treated with these reagents separately, incubated with CMFP and then imagined with confocal microscope or measured with flow cytometry. Data revealed that H2O2 and glucose could induce concentration-dependent increments of both fluorescence ratios (Iblue/Igreen) in cells imaging experiments and fluorescence intensity in flow cytometry experiments (Figure 4, S11). Besides, similar results were also achieved with fructose and intracellular ROS regulators (ANT-224, LD34) in flow cytometry experiments (Figure S11). However, HeLa cells pre-loaded with aminoacetone, and followed by incubation with CMFP exhibited minor change of fluorescence intensity compared with control group in flow cytometry experiments (Figure S11). Taken together, these results revealed that external ROS (H2O2), endogenous ROS regulators (ATN-224, LD-34), and reducing sugar (glucose, fructose) could increase the fluorescent ratios (Iblue/Igreen) in cell imaging experiment or fluorescent intensities in flow cytometry analysis experiment. While even high concentration of aminoacetone (5 mM), failed to increase the fluorescent intensity of CMFP stained HeLa cells in flow cytometry analysis experiment. It indicates that lipid peroxidation and glycolysis might predominantly constitute the biogenesis way of endogenous generation of MGO within HeLa cells. Moreover, our results showed 14
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that 2.0-5.0 mM of glucose and 50-100 μM of H2O2 induced increments of fluorescent ratios (Iblue/Igreen) at comparable levels (around 1.5-1.9 folds for glucose, 1.7-2.1 folds for H2O2) in cell imaging experiments. And similar results were also obtained from flow cytometry experiments. With further consideration of the physiological concentration ranges of H2O2 (1-100 nM) and glucose (mM scare), 20,57 we speculated that glycolysis might be the primary resource of endogenous MGO within HeLa cells.
Figure 4. Investigation of biogenesis approaches of endogenous MGO in HeLa cells using CMFP. a-c) HeLa cells were treated with H2O2 (a: vehicle, b: 50 μM, c: 100 μM) at 37ºC for 1 hour, and then incubated with 10 μM CMFP at 37ºC for 40 min. d) Integrated emission intensity ratios (Iblue/Igreen) of (a-c). e-g) HeLa cells were treated with glucose (e: vehicle, f: 2 mM, g: 5 mM) at 37ºC for 24 hours, and then treated with 10 μM of CMFP at 37ºC for 40 min. h) Integrated emission intensity ratios (Iblue/Igreen) of images (e-g). All images were collected at 420-480 nm and 500-650 nm upon excitation at 405 nm. Scale bar: 50 μm. Representative images from replicate experiments (n = 5) are shown.
Finally, we performed the application of CMFP in evaluating MGO in real clinical samples, the whole blood samples from diabetic and healthy people. A series of whole blood samples 1-6 from type II diabetic without complications (person 1), type II diabetic without complications (person 2), healthy people, people with type II diabetic foot, type I diabetic, people with diabetic retinopathy were collected 15
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separately. These blood samples were loaded with/without MGO, incubated with CMFP, and then imagined with confocal microscope (Figure 5, S12, S13). Acridine orange, a nucleic acid-selective fluorescent cationic dye, was employed to stain and localize with the leukocytes (Figure S12). All these samples displayed fluorescence both in blue channel and green channel, and the external MGO increased the fluorescence ratio (Iblue/Igreen) (Figure 5, S12, S13). Moreover, much brighter fluorescence and higher fluorescence ratios (Iblue/Igreen) were observed in leukocytes than blood red cells from same blood samples, which indicated that the blood leukocyte cells could uptake more CMFP and higher levels of MGO existed in blood leukocytes. Furthermore, samples from people with diabetes exhibited higher fluorescence ratios (Iblue/Igreen) within both blood red cells and leukocytes than healthy people (Figure 5, S12, S13) which was in line with the reported observations29,30 that whole bloods of people with diabetes always harbors elevated levels of MGO. Lastly, our data also showed that sample 4, 5, 6 displayed higher Iblue/Igreen than sample 1-2. These were in correspondence with literature that higher level of MGO was detected in type I diabetes (sample 5) than type II diabetes (sample 1-2),29,30 diabetic complications (samples 4,6) than diabetes without complications (sample 1-2).29,30 And more parallel samples were still required to be tested to illuminate the links between the MGO levels and different types of diabetes. Moreover, the literature reported that the main resource of MGO in blood cells was not glycolysis which was different from somatic cells such as HeLa cells, and there was no correlation between MGO and glucose concentrations.19,20 We therefore compared the fluorescent ratios of CMFP-stained blood samples (sample 2, 4, 5, 6: 0.76, 0.51, 0.80, 0.84 in blood red cells; 1.26, 0.65, 1.05, 0.88 in leukocytes) and blood glucose concentrations (sample 2, 4, 5, 6: 8.95, 5.25, 4.04, 6.72 mM, data from the first affiliated hospital of guangzhou medical university), but failed to find a clear correlation. It indicated that we could not evaluate the MGO levels in blood samples through measuring the blood glucose concentration. Therefore, it is of great significant to monitor the MGO levels in human blood samples not only for further investigations of the pathological 16
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involvement of MGO in diabetic complications but also for potential clinic diagnosis of diabetic complications. Taken together, the data shows that CMFP could detect endogenously produced MGO in human blood samples, and being potentially applicated in pathological investigations and diagnosis of diabetes.
Figure 5. Application of CMFP in leukocytes. Six fresh whole blood samples collected from diabetics and diabetics with chronic complications were diluted into 30 folds of PBS buffer (v/v) to prepare Samples 1-6. Sample 1: type II diabetic without complications (person 1), Sample 2: type II diabetic without complications (person 2), Sample 3: healthy people (person 3), Sample 4: type II diabetic foot (person 4), Sample 5: type I diabetics (person 5), Sample 6: type II diabetic retinopathy (person 6). These samples were treated with CMFP and MGO (a-f: 30 μM CMFP, g-l: 30 μM CMFP, 200 μM MGO), Then, all samples were incubated for 2 hours at 37ºC. Fluorescent images were collected at 420-480 nm and 500-650 nm upon excitation at 405 nm. Scale bar: 20 μm. Representative images from replicate experiments (n = 5) are shown. m): Integrated emission intensity ratios (Iblue/Igreen) of images.
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Conclusions In summary, we report in this study, a MGO-mediated cyclization of 2-aminoacetamide derivatives under mild conditions. This reaction proves to be specific for MGO over other biological relevant reactive species. Based on this reaction, a ratiometric fluorescent probe, CMFP, was developed by combining 2-aminoacetamide and 3-hydroxychromone for probing MGO sensitively and selectively in aqueous buffers, as well as within cells. To the best of our knowledge, CMFP is the first reported ratiometric fluorescent probe for MGO detection, and can detect the endogenous MGO in living cells. With probe CMFP, we further studied the biogenesis of intracellular MGO. Our results revealed that lipid peroxidation and glycolysis might predominantly constitute the biogenesis way of endogenous generation of MGO within cells. We also proved the applicability of CMFP in evaluating MGO levels of real clinic samples, the whole blood samples from healthy and diabetic people. These applications underscore the potential utility of this probe in both basic science research and clinical diagnosis. We are now utilizing these probes to study the contributions of MGO to physiological and pathological processes, and try to find a clear correlation between blood MGO levels and diabetes.
Author Information Corresponding Author *E-mail:
[email protected] Conflict of Interest Disclosure The authors declare no competing financial interests.
Acknowledgments This work is supported by National Natural Science Foundation of China (201502064). 18
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Supporting Information Supplemental Experimental Procedures, Schemes, Figures, Cell Images and NMR Spectrums can be found in Supporting Information. The Supporting Information is available free of charge via the internet.
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