MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

Introduction

MG (methylglyoxal) is a reactive dicarbonyl metabolite and potent glycating agent of protein and DNA. It is produced in all cells by spontaneous degradation of triosephosphates, e.g. glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, and other sources [1]. It is an arginine-directed glycating agent of proteins and precursor of the quantitatively important protein AGE (advanced glycation end-product) MG-H1 [MG-derived hydroimidazolone or Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine] [2]. MG also modifies DNA to form deoxyguanosine-derived imidazopurinone MGdG {3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one} isomers. MGdG was the major quantitative adduct of DNA damage detected in vivo [3]. MG is found in cells, tissues and body fluids, increasing in diabetes and associated vascular complications, renal failure and in vivo models of Parkinson's disease [49]. Increased concentrations of MG are linked to mitochondrial dysfunction in diabetes and aging [10,11], oxidative stress [12], dyslipidaemia [13], cell detachment and anoikis [4], apoptosis [14] and increased frequency of DNA strand breaks [3]. Glo1 (glyoxalase I) metabolizes >99% MG and thereby protects the proteome and genome. Gene deletion of GLO1 is embryonically lethal and GLO1 silencing increases MG, MG-H1 and MGdG concentrations, causing premature aging and disease [15]. Studies of MG have importance for human health, longevity and treatment of disease.

There is an increasing demand for facile measurement of MG in cell, tissues and body fluids. The concentration of MG is typically 1–4 μM in cells and 0.1–0.5 μM in physiological fluids. Other dicarbonyls such as glyoxal and 3-DG (3-deoxyglucosone) are also present. MG and other dicarbonyls are also present in medical dialysis fluids [7] and in sweetened beverages such as cola [16]. MG is typically assayed by derivatization with DB (1,2-diaminobenzene) and related compounds in cell-free extracts [4,8,17]. The assay is preferably performed under acidic conditions which stabilize triosephosphates from degradation and catalyse the dehydration rate-limited derivatization [17]. Such assays are not amenable to high sample throughput, subcellular, live-cell and in vivo estimations. A fluorogenic derivatization assay would be better suitable for these applications, similar to the fluorogenic probes approach taken for the measurement of reactive oxygen species and NO (nitric oxide), for example [18,19].

Fluorogenic derivatizing agents such as DDB (1,2-diamino-4,5-dimethoxybenzene) have been used for assay of MG, but they are unstable under physiological conditions [20,21]. Fluorescein, rhodamine and BODIPY fluorogenic probes have been developed with bright fluorescence emission in the visible wavelength range. Moreover, 1,2-diaminophenyl derivatives were synthesized in initial attempts to develop fluorogenic probes for NO detection, forming the fluorescent triazolo derivatives DAF-2 (4,5-diaminofluorescein) [22], DAR-1 (4,5-diaminorhodamine) [23] and DAMBO-P(H) [8-(3,4-diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene] [24]. Interferences with NO detection were soon found, such as high concentrations of the 1,2-dioxo metabolite dehydroascorbate [25] and cell-permeant nitric oxide synthase inhibitors such as L-NAME (NG-nitro-L-arginine methyl ester) only partly inhibited the development of fluorescence [26], but the reaction with endogenous α-oxoaldehydes such as MG was overlooked. In the present article, we describe the reaction of MG with DAF-2 and DAR-1 and the strengths and limitations of their use to detect MG and related dicarbonyl metabolites, including in live cells. A similar approach was taken independently in preparing a 1,2-diaminophenyl derivative of BODIPY [27].

Reaction of DAF-2 and DAR-1 with MG

When DAF-2 and DAR-1 (2–20 μM) was incubated with 10–100 μM MG in 100 mM sodium phosphate buffer (pH 7.4) at 37°C, there was characteristic development of fluorescence. LC–MS/MS analysis with concurrent fluorescence detection showed that the fluorescence was due to formation of 1:1 adducts of MG with DAF-2 and DAR-1, DAF-2–MG and DAR-1–MG, with characteristic fluorescent excitation and emission (Table 1 and Figures 1A and 1B). The formation of two structural isomers is assumed and indeed were resolved chromatographically for DAR-1–MG. The fluorescence excitation and emission bands overlap with those of NO adducts, DAF-2T and DAR-1T, and so MG adducts are likely to interfere in detection of NO in physiological systems in previous studies. In subsequent experiments, fluorescence measurements were made in a fluorescence microplate reader with 10 nm bandwidth filters with excitation and emission wavelengths of 435 nm and 510 nm (DAF-2 studies) and 545 nm and 570 nm (DAR-1 studies).

Table 1
Molecular and fluorescence characteristics of MG adducts of DAF-2 and DAR-1

Molecular mass, mass and fragmentation and fluorescence characteristics were determined by LC–MS/MS analysis with a scanning fluorescence detector inline immediately upstream of the mass spectrometer. Instrumentation: Acquity™ UPLC (ultrahigh-performance liquid chromatography) system with Acquity™ fluorescence detector and Quattro Premier tandem mass spectrometry (Waters). Samples: DAF-2 or DAR-1 (20 μM) incubated for 1 h at 37°C in 10 mM ammonium acetate buffer (pH 4.8) or 10 mM sodium phosphate (pH 7.4), as appropriate. UPLC conditions: BEH column, 1.2 μm, 2.1 mm×100 mm ODS column, eluted with 0.1% trifluoroacetic acid in water with a linear gradient of 0–50% acetonitrile over 10 min. Injection volume was 50 μl and the flow rate was 0.2 ml/min. Fluorescence emission and excitation wavelengths were scanned for the fluorescent adducts. MS detection conditions: positive ESI with capillary voltage 0.60 kV, ion source temperature 120°C, desolvation gas temperature 350°C and cone and desolvation gas flows 99 and 900 l/h respectively. DHA, dehydroascorbic acid.

Fluorescence characteristics
ProbeReactantAdductChromatographic retention time Rt (min)Molecular ion→fragment ion ([M+H]+/m/z; Da)Excitation λmax (nm)Emission λmax (nm)Reference
DAF-2 None DAF-2 7.9 363.1→316.9; 363.1→191.4 – – Our study 
 NO DAF-2T   491 513 [19
 DHA DAF-2–DHA   495 515 [25
 MG DAF-2–MG 8.8 399.1→353.1; 399.1→200.2 435 509 Our study 
DAR-1 None DAR-1 13.8 473.3→411.2; 473.3→385.1 – – Our study 
 NO DAR-1T   556 575 [23
 MG DAR-1–MG 11.3 509.3→465.2; 509.3→421.1 545 566 Our study 
Fluorescence characteristics
ProbeReactantAdductChromatographic retention time Rt (min)Molecular ion→fragment ion ([M+H]+/m/z; Da)Excitation λmax (nm)Emission λmax (nm)Reference
DAF-2 None DAF-2 7.9 363.1→316.9; 363.1→191.4 – – Our study 
 NO DAF-2T   491 513 [19
 DHA DAF-2–DHA   495 515 [25
 MG DAF-2–MG 8.8 399.1→353.1; 399.1→200.2 435 509 Our study 
DAR-1 None DAR-1 13.8 473.3→411.2; 473.3→385.1 – – Our study 
 NO DAR-1T   556 575 [23
 MG DAR-1–MG 11.3 509.3→465.2; 509.3→421.1 545 566 Our study 

Reaction of MG with DAF-2 and DAR-1A

Figure 1
Reaction of MG with DAF-2 and DAR-1A

Formation of structural isomeric MG adducts of DAF-2, DAF-2–MG. The molecular structure of the NO adduct DAF-2T is shown in the inset for comparison. (B) Formation of structural isomeric MG adducts of DAR-1, DAR-1–MG. The molecular structure of the NO adduct DAR-1T is shown in the inset for comparison. (CE) Development of fluorescence from the reaction of MG with DAF-2. (C) Time course, 10 μM DAF-2 and 50 μM MG were incubated in 100 mM sodium phosphate buffer (pH 7.4) at 37°C. (D and E) Dependence of initial rate of development of fluorescence (dF/dt)0 on DAF-2 concentration (D) and MG concentration (E). (FH) Development of fluorescence from the reaction of MG with DAR-1. (F) Time course, 10 μM DAR-1 and 50 μM MG were incubated in 100 mM sodium phosphate buffer (pH 7.4) at 37°C. (G and H) Dependence of initial rate of development of fluorescence (dF/dt)0 on DAR-1 concentration (G) and MG concentration (H). (I) Use of fluorogenic probe DAF-2 in an assay of Glo1 and inhibitor screening. GSH (2 mM) and MG (2 mM) were incubated in 100 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min to pre-form the MG–GSH hemithioacetal substrate. Glo1 (≥400 units/mg of protein, from yeast; Sigma) was added and then incubation continued for 30 min. The incubation was performed in triplicate without Glo1 (Control), with Glo1 (+Glo1) and with test Glo1 inhibitor for screening (+Glo1+4 μM BrBzGSH). An aliquot of reaction mixture (5 μl) was then removed and diluted 40-fold into 100 mM sodium phosphate buffer (pH 6.6) at 37°C, with 10 μM DAF-2 and the initial rate of fluorescence was recorded. The remaining MG concentration in the Glo1 activity assay is then deduced from a calibration curve run concurrently (as in E).

Figure 1
Reaction of MG with DAF-2 and DAR-1A

Formation of structural isomeric MG adducts of DAF-2, DAF-2–MG. The molecular structure of the NO adduct DAF-2T is shown in the inset for comparison. (B) Formation of structural isomeric MG adducts of DAR-1, DAR-1–MG. The molecular structure of the NO adduct DAR-1T is shown in the inset for comparison. (CE) Development of fluorescence from the reaction of MG with DAF-2. (C) Time course, 10 μM DAF-2 and 50 μM MG were incubated in 100 mM sodium phosphate buffer (pH 7.4) at 37°C. (D and E) Dependence of initial rate of development of fluorescence (dF/dt)0 on DAF-2 concentration (D) and MG concentration (E). (FH) Development of fluorescence from the reaction of MG with DAR-1. (F) Time course, 10 μM DAR-1 and 50 μM MG were incubated in 100 mM sodium phosphate buffer (pH 7.4) at 37°C. (G and H) Dependence of initial rate of development of fluorescence (dF/dt)0 on DAR-1 concentration (G) and MG concentration (H). (I) Use of fluorogenic probe DAF-2 in an assay of Glo1 and inhibitor screening. GSH (2 mM) and MG (2 mM) were incubated in 100 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min to pre-form the MG–GSH hemithioacetal substrate. Glo1 (≥400 units/mg of protein, from yeast; Sigma) was added and then incubation continued for 30 min. The incubation was performed in triplicate without Glo1 (Control), with Glo1 (+Glo1) and with test Glo1 inhibitor for screening (+Glo1+4 μM BrBzGSH). An aliquot of reaction mixture (5 μl) was then removed and diluted 40-fold into 100 mM sodium phosphate buffer (pH 6.6) at 37°C, with 10 μM DAF-2 and the initial rate of fluorescence was recorded. The remaining MG concentration in the Glo1 activity assay is then deduced from a calibration curve run concurrently (as in E).

The development of fluorescence from DAF-2 and DAR-1 with MG was initially studied in 100 mM sodium phosphate buffer (pH 7.4) at 37°C. There was a linear increase in fluorescence over the initial 30 min of reaction, although the reaction reached end point only after 6–8 h (Figures 1C–1H). The initial rate of increase in development of fluorescence, (dF/dt)0, with DAF-2 and DAR-1 was first-order with respect to the concentration of fluorogenic probe and MG. Similar fluorescence development occurred in 100 mM sodium phosphate buffer (pH 6.6) at 37°C (appropriate for the assay of Glo1 activity) and 100 mM ammonium acetate buffer (pH 4.8) (appropriate for assay of dicarbonyls in dialysis fluids; the lower the pH, the higher the rate of development of fluorescence and formation of the adduct). This development of fluorescence with formation of DAF-2–MG and DAR-1–MG could therefore be used in a kinetic assay for MG concentration using a fixed concentration of DAF-2 or DAR-1. We investigated the application of this in a fixed time point assay of Glo1 activity, assaying the remaining MG concentration and amenable to high sample throughput applications [28], detection of dicarbonyls in dialysis fluids and detection and imaging of MG in live cells.

High-throughput assay of Glo1 activity

The application of DAF-2 quantification of MG was examined in the conventional spectrophotometric assay of Glo1 activity. GSH (2 mM) and MG (2 mM) is incubated in 100 mM sodium phosphate buffer (pH 6.6) at 37°C, for 10 min to pre-form the MG–GSH hemithioacetal substrate. Glo1 (≥400 units/mg of protein, from yeast; Sigma) is then added and incubation is continued for 30 min. The incubation is performed in triplicate without Glo1 (blanks), with Glo1 (positive control) and with test Glo1 inhibitor for screening; in this case, the authentic Glo1 inhibitor BrBzGSH (S-p-bromobenzylglutathione) (4 μM). An aliquot of reaction mixture (5 μl) is then removed and diluted 40-fold into 100 mM sodium phosphate buffer (pH 6.6) at 37°C, with 10 μM DAF-2 and the initial rate of fluorescence is recorded. The remaining MG concentration in the Glo1 activity assay is then deduced from a calibration curve run concurrently (as in Figure 1E). The outcome shows that residual MG concentration in the Glo1 activity assay may be quantified, remaining above levels in the positive control when a positive hit for Glo1 inhibitor test compound is found (Figure 1I). Thus this assay could replace the Glo1 spectrophotometric assay in high-throughput screening of Glo1 inhibitors for anti-cancer drug development [29] and Glo1 inducers for dietary bioactives and pharmaceuticals to improve metabolic and vascular health [30,31].

Monitoring of 1,2-dicarbonyl compound glucose degradation products in PD (peritoneal dialysis) fluids

Dicarbonyl compounds, i.e. glyoxal, MG, 3-DG and others, are formed during heat sterilization of PD fluids with glucose osmolyte [32]. These compounds, also known as GDPs (glucose degradation products), may produce adverse effects when the PD fluids are used clinically [33], and concentrations may vary during dispatch and storage at ambient conditions at the PD centre or home of the patient [34]. A facile method for measuring GDPs could provide simple screening of GDPs and confidence in use of low GDP PD fluids at the PD clinic. Both DAF-2 and DAR-1 produced increased fluorescence, characteristic of the MG adducts when incubated with PD fluid (Figures 2A and 2E). The increased fluorescence was blocked by pre-incubation with 500 μM AG (aminoguanidine) (Figures 2B and 2F), indicating that the increased fluorescence was due to dicarbonyl derivatization. This fluorescence is likely to be produced from multiple dicarbonyl adducts as C2–C6 dicarbonyl compounds are formed in heat-sterilized PD fluids [7]. Fluorogenic monitoring of dicarbonyl GDPs with DAF-2 and DAR-1 could provide a convenient basis for batch monitoring of quality of PD fluids at the sites of storage and use.

Fluorogenic detection of dicarbonyls in dialysis fluids and human plasma

Figure 2
Fluorogenic detection of dicarbonyls in dialysis fluids and human plasma

Dialysis fluids. DAF-2 (10 μM) (A and B) and DAR-1 (E and F) incubated with PD fluid diluted 2-fold in 100 mM sodium phosphate buffer (pH 7.4) at 37°C with 0.3% sodium azide (to block peroxidase-catalysed dicarbonyl formation). Key: control, dialysis fluid alone (-·-) and probe alone (=); Dianeal PD4 (Baxter U.K.)/1.36% glucose (···); Dianeal PD4/2.27% glucose (− − −); Dianeal PD4/3.38% glucose (–). (B and F) Dianeal PD4/1.36%, 2.27% and 3.86% glucose pre-incubated with 500 μM AG for 1 h at 18°C to quench dicarbonyl GDPs and then with10 μM DAF-2 (B) or DAR-1 (F). Human plasma. Incubation of 10 μM DAF-2 (C) or DAR-1 (G) with plasma from human healthy control subjects diluted 2-fold in 100 mM sodium phosphate buffer (pH 7.4) at 37°C, with 0.3% sodium azide. Key: without further addition (–); pre-incubated with 500 μM AG for 1 h (···). Controls: (D) DAF-2; (H) DAR-1; plasma alone (-·-) and probe alone (=). Instrumentation: fluorescent measurements were made on a FLUOstar OPTIMA microplate reader (BMG Labtech) with excitation and emission wavelengths of 435 nm and 510 nm (DAF-2 studies) and 545 nm and 570 nm (DAR-1 studies). a.u., arbitrary units.

Figure 2
Fluorogenic detection of dicarbonyls in dialysis fluids and human plasma

Dialysis fluids. DAF-2 (10 μM) (A and B) and DAR-1 (E and F) incubated with PD fluid diluted 2-fold in 100 mM sodium phosphate buffer (pH 7.4) at 37°C with 0.3% sodium azide (to block peroxidase-catalysed dicarbonyl formation). Key: control, dialysis fluid alone (-·-) and probe alone (=); Dianeal PD4 (Baxter U.K.)/1.36% glucose (···); Dianeal PD4/2.27% glucose (− − −); Dianeal PD4/3.38% glucose (–). (B and F) Dianeal PD4/1.36%, 2.27% and 3.86% glucose pre-incubated with 500 μM AG for 1 h at 18°C to quench dicarbonyl GDPs and then with10 μM DAF-2 (B) or DAR-1 (F). Human plasma. Incubation of 10 μM DAF-2 (C) or DAR-1 (G) with plasma from human healthy control subjects diluted 2-fold in 100 mM sodium phosphate buffer (pH 7.4) at 37°C, with 0.3% sodium azide. Key: without further addition (–); pre-incubated with 500 μM AG for 1 h (···). Controls: (D) DAF-2; (H) DAR-1; plasma alone (-·-) and probe alone (=). Instrumentation: fluorescent measurements were made on a FLUOstar OPTIMA microplate reader (BMG Labtech) with excitation and emission wavelengths of 435 nm and 510 nm (DAF-2 studies) and 545 nm and 570 nm (DAR-1 studies). a.u., arbitrary units.

Detection of dicarbonyls in human plasma

We attempted to detect MG in human plasma. When plasma was incubated with 10 μM probe, DAF-2 showed a small increase in fluorescence compared with control pre-incubated with 500 μM AG (Figure 2C). There was, however, high background fluorescence from plasma alone (Figure 2D). The sensitivity of this method was inadequate to reliably quantify MG (and related dicarbonyls). Fluorescence development from DAR-1 added to plasma decreased with time, suggesting instability of the probe and adducts formed (Figures 2G and 2H).

Detection of dicarbonyls in live cells in vitro

The ability of the cell-permeant version of the DAF-2 probe DAF-2DA (DAF-2 diacetate) to deliver DAF-2 into cells and image cellular MG in cultured cells in vitro was evaluated. Human lung carcinoma A549 cells were loaded with the DAF-2 probe by incubation with DAF-2DA for 30 min in the presence of cell-permeant NO inhibitor, L-NAME, and imaged using confocal microscopy. With DAF-2DA, there was development of very weak green fluorescence in A549 cells (Figure 3B). When exogenous MG (100 μM) was added to cells, this fluorescence was increased markedly (Figure 3C). This suggests that loading of cells with DAF-2 by DAF-2DA produces weak fluorescence that is NO-independent which may reflect reaction with endogenous cellular MG in cells (~1–4 μM) [4]. The response is potentiated markedly by addition of exogenous MG. Similar findings were reported with the BODIPY-related probe by Wang et al. [27]. DAF-2DA was also used to assess whether increased fluorescence could be detected in cells using a microplate based-cell assay with A549 cells cultured in 96-well plates. An increase in fluorescence was not discernible after L-NAME and DAF-2DA treatments. An increase in fluorescence was seen, however, in the presence of exogenous MG and the rate of fluorescence increase was potentiated in the presence of the cell-permeant BrBzGSHCp2 (S-p-bromobenzylglutathione cyclopentyl diester) (results not shown). This suggests that loading of cells with DAF-2 by DAF-2DA could be used in screening of cell-permeant Glo1 inhibitors, particularly in the presence of added exogenous MG.

Detection of dicarbonyls in live cells in vitro

Figure 3
Detection of dicarbonyls in live cells in vitro

Confocal microscopy for MG detection in A549 cells. Cells were seeded in glass-bottomed dishes in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS and 2 mM L-glutamine. Cells were placed in a humidified (37°C, 5% CO2) incubator for 24 h or until 70% confluent. The imaging was performed in the presence of 1 mM probenecid, an ion transporter inhibitor to prevent cell export of DAF-2–and DAF-2–dicarbonyl adducts. (A) Images were captured of cells in serum-free medium to decrease background fluorescence. (B) Subsequently, cells were then pre-treated with 1 mM L-NAME for 30 min and loaded with 10 μM DAF-2DA for 30 min and images were captured with the probe alone to determine whether endogenous levels of dicarbonyls could be imaged. (C) Medium was then changed and cells were incubated with 100 μM MG for 1 h. Imaging was performed on a Zeiss confocal microscope using a ×40 oil objective (upper panels). Corresponding phase-contrast images were also taken (lower panels).

Figure 3
Detection of dicarbonyls in live cells in vitro

Confocal microscopy for MG detection in A549 cells. Cells were seeded in glass-bottomed dishes in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS and 2 mM L-glutamine. Cells were placed in a humidified (37°C, 5% CO2) incubator for 24 h or until 70% confluent. The imaging was performed in the presence of 1 mM probenecid, an ion transporter inhibitor to prevent cell export of DAF-2–and DAF-2–dicarbonyl adducts. (A) Images were captured of cells in serum-free medium to decrease background fluorescence. (B) Subsequently, cells were then pre-treated with 1 mM L-NAME for 30 min and loaded with 10 μM DAF-2DA for 30 min and images were captured with the probe alone to determine whether endogenous levels of dicarbonyls could be imaged. (C) Medium was then changed and cells were incubated with 100 μM MG for 1 h. Imaging was performed on a Zeiss confocal microscope using a ×40 oil objective (upper panels). Corresponding phase-contrast images were also taken (lower panels).

Comparison of DAF-2, DAR-1 and BODIPY-derived fluorogenic probes for MG detection

Fluorogenic derivatizing agents based on the DB scaffold have been used for the detection of NO [2224]. It can now been appreciated that such compounds may also give a fluorescence response by reaction with physiological dicarbonyl compounds, i.e. MG and others. This may account for the previously reported fluorescence response with endothelial and other cells in vitro that was not blocked by NO synthase inhibitors [26]. We have shown in our studies that MG reacts with DAF-2 and DAR-1 to produce a characteristic fluorophore and, moreover, the development of related increased fluorescence can be used to report on MG and other dicarbonyl concentrations. Wang et al. [27] recently synthesized MBo (methyl diaminobenzene-BODIPY), a sensor for MG that has been adapted using a BODIPY scaffold with a 6-methyl substituent derivative, a sensor adopted from a benzene-BODIPY ethyl ester again used for sensing NO [24]. The limit of detection for MG in PBS was 50–100 nM MG. In our studies with DAF-2, the kinetic assay for MG, the limit of detection for MG was approximately 700 nM (analyte concentration equivalent to 3S.D. of the zero analyte control in calibration curves). MBo was also used to estimate MG in mouse plasma, finding 0.51–0.99 μM MG [27]. Interference-free estimates of MG in human plasma are lower than this at 100–300 nM [35,36]. Sensitivity for DAF-2 to MG may be increased with improved fluorimetric detection and by subtraction of background fluorescence, as described for use in NO detection [37].

It has recently been reported that peroxidase activity in physiological samples can lead to overestimation of MG by catalysing the degradation of DB to MG [17]. The DB scaffold fluorogenic probes may also be susceptible to peroxidase-catalysed degradation to MG and glyoxal, producing interference in assay and imaging. This interference may be prevented by the addition of 0.3% sodium azide during derivatization, where appropriate. Addition of AG to scavenge MG provides a good control for MG-unrelated fluorescence in fluorogenic assay of MG. In cell samples, controls with a cell-permeant NO synthase inhibitor, such as L-NAME, are also required as AG is both a MG scavenger and a NO synthase inhibitor [38].

Conclusion

The use of fluorogenic probes originally designed for detection of NO in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. To date, the studies performed on such probes, i.e. studies described in the present article and the recent report by Wang et al. [27], show that these probes have the potential to facilitate protocols for measurement of MG. Importantly, a high-throughput assay for MG would probably aid Glo1-based therapeutic development, i.e. Glo1 inhibitors and Glo1 inducers [30,39]. Both DAF-2 and DAR-1 can be used to qualitatively measure MG and other dicarbonyls in a cell-free based assay; specifically, they could replace the spectrophotometric assay currently used to measure Glo1 activity. This would greatly facilitate high-throughput screening of Glo1 inhibitors and inducers. Currently, DAF-2 and related BODIPY probes have limited utility to detect and image MG in live cells in vitro, except in the presence of exogenous MG and cell-permeant Glo1 inhibitors. This may nevertheless provide a route to cell-based screening of Glo1 inhibitors where cellular MG concentration is expected to increase 6-fold or more [15]. Further development of the probes with greater sensitivity for MG and related dicarbonyl detection would provide a route to facile detection of MG in live cells in vitro and possibly lead to small animal imaging of MG in vivo. A DB scaffold probe for near-IR fluorescence has been reported [40]. We anticipate that the use of fluorogenic probes could be important in clarifying the biological role of MG and other dicarbonyls in human health and disease.

Glyoxalase Centennial: 100 Years of Glyoxalase Research and Emergence of Dicarbonyl Stress: A Biochemical Society Focused Meeting held at the University of Warwick, U.K., 27–29 November 2013. Organized and Edited by Naila Rabbani and Paul Thornalley (University of Warwick, U.K.).

Abbreviations

     
  • AG

    aminoguanidine

  •  
  • AGE

    advanced glycation end-product

  •  
  • BrBzGSH

    S-p-bromobenzylglutathione

  •  
  • DAF-2

    4,5-diaminofluorescein

  •  
  • DAF-2DA

    DAF-2 diacetate

  •  
  • DAR-1

    4,5-diaminorhodamine

  •  
  • DB

    1,2-diaminobenzene

  •  
  • 3-DG

    3-deoxyglucosone

  •  
  • GDP

    glucose degradation product

  •  
  • Glo1

    glyoxalase I

  •  
  • MBo

    methyl diaminobenzene-BODIPY

  •  
  • MG

    methylglyoxal

  •  
  • MGdG

    3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one

  •  
  • MG-H1

    MG-derived hydroimidazolone or Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine

  •  
  • L-NAME

    NG-nitro-L-arginine methyl ester

  •  
  • PD

    peritoneal dialysis

We thank the sponsors of the Glyoxalase Centennial conference and past and present members of our research team for their contributions to glyoxalase research.

Funding

We thank the Warwick Impact Fund for support for our research.

References

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