Method for measuring methylglyoxal or glyoxal

Abstract

The present invention relates to a method for measuring methylglyoxal and / or glyoxal.

Description

[0001] METHOD FOR MEASURING METHYLGLYOXAL OR GLYOXAL

[0002] The present invention relates to a method for measuring methylglyoxal and / or glyoxal.

[0003] BACKGROUND

[0004] Methylglyoxal (MGO) and glyoxal are both physiological alpha dicarbonyl compounds that can be generated in vivo during the glycolysis.

[0005] MGO and glyoxal can react with nucleic acids and lipids and are particularly reactive with some residues of proteins, e.g. arginine, lysine and cysteine, to form “Advanced Glycation End products” (AGEs). It has been known that AGEs are implicated in aging process and the development of many degenerative diseases, such as diabetes, renal diseases, Parkinson disease and Alzheimer disease (Beisswenger et al., 2013; Maessen et al., 2015). The formation of AGEs leads to protein structure modifications, such as intra- and / or inter-molecular crosslinks, and results in protein function modifications. These modifications may play a causative role in the development of some diseases (Matafome et al., 2016). For example, MGO or glyoxal can react with crystallins, the proteins in lens and cornea of eyes, and forms AGEs. These MGO / glyoxal- related AGEs stimulate further glycation, oxidation, and protein aggregation of crystallins and may lead to the formation of cataract (Ahmed et al. 2003). , Interestingly, MGO can also react with insulin, inactivating the hormone. Both MGO and the AGEs are known to be involved in the development of insulin resistance in the peripheric organs (Riboulet-Chavey et al., 2006), a pathophysiological condition that precedes diabetes type 2. Besides modifying protein structures and functions, AGEs may produce other harmful effects, i.e. activating pro- inflammatory pathway by activating AGEs receptors (Matafome et al., 2016).

[0006] In addition, MGO is also known to lead to the increase of cellular oxidative stress (Seo et al., 2014), which also contributes to the development of some disease, e.g. diabetes (Brouwers et al., 2010).

[0007] All these studies confirm that MGO, glyoxal and MGO / glyoxal-related AGEs are harmful for human beings and animals.

[0008] Besides endogenous formation, MGO and glyoxal may also be generated during processing and preparation of foods and, as a result, be found in foods, especially in strongly heated foods and / or in foods high in fat and carbohydrates. For example, significant levels of MGO and glyoxal have been reported in coffee, soft drinks, fermented products such as whisky, beer and soy sauce (Maasen et al., 2021). It is thus important and necessary to be able to detect and measure methylglyoxal and / or glyoxal in biological samples or in food samples.

[0009] So far, high performance liquid chromatography (HPLC) or ultra performance liquid chromatography (UPLC) is the conventional method for measuring the level(s) of MGO and glyoxal in a sample (Maasen et al., 2021). This requires derivatization of the sample with O- phenylenediamine (OPD) in order to obtain 2-methylquinoxaline (for MGO) and quinoxaline (for glyoxal). This technology is laborious, time-consuming, and requires specialized staff and expensive equipment.

[0010] Another method reported for measuring the level of MGO is based on immunoassay, i.e. ELISA assay. Some methylglyoxal ELISA kits are commercially available in the market. However, it is worth noting that these ELISA kits do not detect MGO but rather proteins modified by MGO or a specific bond formed between MGO and a protein. Most importantly, these ELISA kits do not provide a satisfactory sensibility for quantitatively detecting MGO and glyoxal in samples.

[0011] Another approach for detecting methylglyoxal is based on biochemical reactions between MGO and other chemical compounds, e.g. a chromophore or a fluorophore. For example, a kit commercialized by Abeam uses a chromophore for detecting the presence of MGO. The reduced chromophore, final product of the assay, produces a signal which can be quantified at 450 nm. However, the underlying chemical reactions of this multi-step assay are not revealed in the kit documentation, nor the specificity of the assay, so it is unsure whether it is exclusively MGO that is measured. Several fluorophores are also described in the art as being able to detect MGO. For example, Wang et al. (Wang et al., 2013) described a fluorescent sensor using methyl-diaminobenzene-BODIPY / MBo for detecting MGO. Vidal et al. described an assay using the fluorophore NDB-H (7-hydrazino-4-nitrobenzo-2,l,3-oxadiazole) or TRI (6- hydroxy-2, 4, 5 -triaminopyrimidine) for detecting MGO. In general, this kind of technology is based on the change of the fluorescence emitted by a MGO-fluorophore complex with respect to that emitted by a fluorophore alone. However, these fluorophores often also react with other compounds commonly present in a biological sample, such as nitric oxide (NO), or other dicarbonyl compounds and, more problematic, the fluorescence emitted from these complexes is very close to the fluorescence emitted from a MGO-fluorophore complex. Therefore, the methods using these fluorophores are not enough specific for measuring the quantity of MGO and may give an overestimate of the concentration of MGO in the sample. In addition, these methods cannot detect glyoxal specifically. Therefore, there is still a need to develop a new method which can be easily implemented and can detect methylglyoxal and / or glyoxal with high specificity and sensitivity.

[0012] SUMMARY OF THE INVENTION

[0013] Investigating the role of MGO in type 2 diabetes, the Inventors have found out that a compound, dihydroethidium (DHE), can react with methylglyoxal or glyoxal and form a complex with these compounds. Most interestingly, these complexes emit a fluorescence at about 500 nm and 520 nm respectively, which is well distinguished from the fluorescence emitted by DHE alone at 420 nm or by oxidized DHE at 590 nm, respectively. In addition, DHE does not react with 3-deoxyglucosone, another physiological alpha dicarbonyl compound, nor with compounds structurally similar to MGO and glyoxal, such as butanedione, oxalic acid, 2,3-pentanedione, 2,3-hexanedione, dehydroascorbic acid, malondialdehyde, 1,4-butanedial, lactaldehyde, acetaldehyde, hydroxyacetone, dihydroxyacetone, acetone, glyceraldehyde, propionaldehyde, diacetyl, propanal, acetaldehyde, formaldehyde, glutaraldehyde, glyoxylic acid and pyruvic acid. In conclusion, DHE specifically reacts with MGO and glyoxal. Therefore, the fluorescence emitted from DHE-MGO and DHE-glyoxal complexes can be used as a reliable indication of the presence of MGO and / or glyoxal in a sample, or for measuring the quantity of MGO and / or glyoxal in a sample.

[0014] Moreover, the Inventors have shown that, by using additionally a scavenger of MGO, DHE can also specifically detect or measure MGO or glyoxal in a sample comprising both of these compounds. Thanks to the different sensitivity of MGO and glyoxal to a scavenger of MGO, in the presence of said scavenger, the reaction between DHE and MGO is partially inhibited, while the reaction between DHE and glyoxal is completely unaffected, allowing in first instance the specific detection of MGO and in second instance, the calculation of the amount of glyoxal in the sample..

[0015] Based on these unique properties of DHE, the Inventors provide herein a new method for detecting and / or measuring MGO and / or glyoxal in a sample. The method of the present invention allows to detect the presence of MGO and / or glyoxal in a sample with high sensitivity and high specificity. The method of the present invention can detect ; MGO and glyoxal levels as low as about 50 nM and 300 nM, respectively and can accurately measure the quantity of MGO or glyoxal in a sample up to several millimolar. The method of the invention can easily be implemented and is compatible with a high-throughput process using 96-well plates and compatible with fully automated high-throughput robotic platforms. The present invention relates to the use of dihydroethidium for the detection or the measure of methylglyoxal and / or of glyoxal in a sample.

[0016] In a particular embodiment, dihydroethidium is used with a scavenger of methylglyoxal.

[0017] In another aspect, the present invention provides a method for detecting or measuring methylglyoxal and / or glyoxal in a sample by using dihydroethidium and optionally a scavenger of methylglyoxal.

[0018] In a particular embodiment, said method comprises:

[0019] (a) contacting the sample with dihydroethidium;

[0020] (b) measuring the intensity of fluorescence emitted by the sample after the contacting step (a) at a wavelength between 490 and 550 nm, wherein the emission of fluorescence in step (b) is indicative of the presence of or of the level of methylglyoxal and / or of glyoxal in the sample.

[0021] In a more particular embodiment, the method comprises the steps of:

[0022] (a) providing two identical samples,

[0023] (b) contacting the first sample with dihydroethidium,

[0024] (c) measuring the intensity of fluorescence emitted by the first sample after the contacting step (b) at a wavelength between 490 and 550 nm,

[0025] (d) contacting the second sample with dihydroethidium and a dicarbonyl scavenger, in particular a scavenger of methylglyoxal and

[0026] (e) measuring the intensity of fluorescence emitted by the second sample after the contacting step (d) at a wavelength between 490 and 550 nm, wherein:

[0027] (i) the concentration of MGO in the sample is calculated from the difference of fluorescence intensity between step (c) and step (e), and

[0028] (ii) the concentration of glyoxal in the sample is obtained from calculating the difference of fluorescence intensity measured in step (c) and the fluorescence intensity of MGO in the sample that is calculated from calculation (i).

[0029] A third aspect of the invention relates to a kit for detecting or measuring methylglyoxal and / or glyoxal in a sample, the kit comprising :

[0030] - a container comprising dihydroethidium, and - a container comprising a dicarbonyl scavenger, in particular a scavenger of methylglyoxal, and

[0031] - optionally a container comprising reaction buffer.

[0032] The present invention also provides a complex formed by dihydroethidium with methylglyoxal. In particular, said complex can be used for the screening of novel scavengers of methylglyoxal. The identification of a novel potent scavenger of methylglyoxal may lead to the inclusion of the scavenger in stored food (e.g. food stored in tin or aluminum cans) or lead to a novel scavenger-containing food complement.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] Figure 1: Excitation and emission spectra of DHE (A), DHE-MGO (B), DHE-glyoxal (C) and DHE oxidized by superoxide (D). For each compound, the excitation spectrum ( on the left) under fixed emission wavelength and the spectrum emissions ( right) under fixed excitation wavelength were analyzed. Fixed wavelengths are shown on the top bars of each figure. For each compound, the excitation spectrum was analyzed at the most optimal emission wavelength ('em'), vice versa, the emission spectrum was analyzed at three excitation wavelengths ('exc'), which correspond to three peaks of the excitation spectrum. In figures IB and 1C, the native DHE fluorescence is subtracted. The dot (•) in figure 1C indicates the wavelengths of the MGO- DHE peaks.

[0035] Figure 2: Fluorescence emission by DHE alone (A, D, G), by 5 pM DHE in the presence of MGO (B, E, H) and by 5 pM DHE in the presence of xanthine (C, F, I). The fluorescence was measured in three conditions respectively: excitation at 340 nm and emission at 420 nm (A, B, C), excitation at 340 nm and emission at 520 nm (D, E, F), excitation at 544 nm and emission at 590 nm (G, H, I).

[0036] Figure 3: Increase in fluorescence emitted from MGO-DHE complex. DHE was incubated with different concentrations of MGO (0-600 pM) for 1 hour at 37°C. Fluorescence excitation: 340 nm; fluorescence emission : 520 nm.

[0037] Figure 4: Effect of scavengers and antioxidants on MGO ( measured by DHE), superoxide (SO, measured by DHE) and hydrogen peroxide H2O2 (measured by OPD). MGO (represented by diamonds), a superoxide-generating system (combination of xanthine and xanthine oxidase; represented by squares) and H2O2 (represented by triangles) were incubated with 3 concentrations of three MGO scavengers, i.e. arginine (Fig. 4A), aminoguanidine (Fig. 4B) and creatine (Fig. 4C), and four antioxidants, i.e. superoxide dismutase (SOD)(Fig. 4D), trolox (the water-soluble form of vitamin E)(Fig. 4E), vitamin C (Fig. 4F) and glutathione (Fig. 4G). Remaining signal of fluorescence (MGO and SO) or absorbance (H2O2) was measured and expressed as percentage of a control incubation without scavenger / anti-oxidant.

[0038] Figure 5: Sensitivity of DHE to three alpha-dicarbonyl compounds, i.e. methylglyoxal, glyoxal and 3-deoxyglucosone. Three alpha-dicarbonyl compounds (0 to 500 M) were incubated for 1 hour at 37° C, in the presence(Fig. 5B) or absence(Fig. 5A) of 5 pM DHE. The increase in fluorescence (340 nm excitation and 520 nm emission) during the incubation was measured.

[0039] Figure 6: Different sensitivity of MGO and glyoxal to arginine. MGO and glyoxal (500 pM) were incubated for 60 min at 37°C with 5 pM DHE and different concentrations of arginine (0.8 mM to 125 mM). The fluorescence (340 nm excitation and 520 nm emission) was measured and expressed as a percentage of the fluorescence of the control incubations without arginine. The dotted line indicates the fluorescence corresponding to 50% of control fluorescence. At the concentration of arginine indicated by the arrow (6.3 mM), methylglyoxal is partially scavenged by arginine, but glyoxal not at all.

[0040] Figure 7: Determination of MGO and glyoxal concentrations in a same sample using DHE and arginine. Sixteen mixtures of MGO and glyoxal were prepared with different concentrations: MGO (A) at 0, 75, 150, 225 pM and glyoxal (B) at 0, 400, 800, 1200 pM. Mixtures were incubated with DHE in the absence and presence of 5 mM arginine. Fluorescence was measured at 340 nm for excitation and 520 nm for emission. The concentration of MGO in each sample was determined through the difference in fluorescence of samples with and without arginine. Glyoxal concentrations in the same samples were then determined through the concentration of determined MGO and the fluorescence of the samples without arginine. The empty bars of both figures represent the real concentrations of MGO and glyoxal in the mixtures which are indicated under Fig. 7B. The full bars of both figures represent the calculated concentrations of MGO and glyoxal, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides a novel use of dihydroethidium (also known as hydroethidine) for the detection or the measure of methylglyoxal and / or of glyoxal in a sample. It is based on the unexpected finding that dihydroethidium (DHE) can form DHE-MGO and DHE-glyoxal complexes which emit a fluorescence with maximum emission wavelengths from 490 nm to 550 nm.

[0042] DHE produces inherent blue fluorescence with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 420 nm (medchemexpress.com). Dihydroethidium is known as a superoxide indicator. When oxidized by superoxide into 2- hydroxyethidium, it emits fluorescence with maximum excitation and emission wavelengths of 490 nm and 590 nm, respectively.

[0043] Therefore, the fluorescence emitted from a DHE-MGO complex and a DHE-glyoxal complex is well distinguished from that emitted from DHE alone or from oxidized DHE.

[0044] The present invention also provides a method for detecting or measuring methylglyoxal and / or glyoxal in a sample by using DHE and optionally a dicarbonyl scavenger, in particular a scavenger of MGO.

[0045] The term of “a dicarbonyl scavenger” refers to a compound which reacts with carbonyl function group of a physiological alpha dicarbonyl compound and thus abolishes the interaction of this dicarbonyl compound with any other molecule.

[0046] A dicarbonyl scavenger includes, without being limited to, a scavenger of MGO or a scavenger of glyoxal.

[0047] The term “scavenger of MGO” refers to a dicarbonyl scavenger that reacts more efficiently with MGO than with glyoxal under the same condition, i.e. a dicarbonyl scavenger whose reaction rate coefficient when reacting with MGO is higher than when reacting with glyoxal. A scavenger of MGO, through its reaction with methylglyoxal, neutralizes MGO, fully abolishing the interaction of MGO with any other molecule.

[0048] In particular, said method comprises the steps of :

[0049] (a) contacting the sample with dihydroethidium;

[0050] (b) measuring the intensity of fluorescence emitted by the sample after the contacting step (a) at a wavelength between 490 and 550 nm, wherein the emission of fluorescence in step (b) is indicative of s the presence or of the level of methylglyoxal and / or of glyoxal in the sample. Said method measures the fluorescence emitted both by DHE-MGO and DHE-glyoxal complexes. Therefore, said method allows to evaluate the total level of MGO and glyoxal in a sample, which is indicative of the total dicarbonyl reactivity in the sample.

[0051] The term “level of MGO”, as used herein, refers to the concentration of MGO in a sample.

[0052] The term “level of glyoxal”, as used herein, refers to the concentration of glyoxal in a sample.

[0053] Generally speaking, the sample that may be measured by the method of the present invention can be any biological or chemical sample. In a particular embodiment, said sample is a food sample or a biological sample. Examples of biological samples include, without being limited to, an in vitro cell culture, a tissue sample or a body fluid sample, e.g. a blood, serum, plasma sample, or a urine sample.

[0054] Preferably, the contact of DHE with a sample for the implementation of the method of the invention is performed in liquid condition. In a particular embodiment, said sample may be previously prepared in a reactional medium. In another particular embodiment, both the sample and DHE may be added simultaneously or successively to a reactional medium during the step of contacting. Said reactional medium may be any reactional medium which does not change the chemical properties of MGO or glyoxal. Examples of reactional medium include, without being limited to, buffers containing phosphate or tris and buffers according to Good et al (1966).

[0055] In some embodiments, the sample is reacted with a reactional medium comprising DHE at a concentration from 1 to 500 pM, preferably at a concentration from 5 to 200, more preferably from 100 to 200 p M.

[0056] The reaction time between a sample and DHE can be from 30 minutes to 24 hours, in particular from 1 to 3 hours. Generally speaking, the reaction time is the time required to obtain best results : lowest possible detection limit and fully linear concentration curve. This reaction time is not necessarily equivalent to the time required to complete the reaction between DHE and MGO and / or glyoxal in a sample (i.e. when there is no more increase in fluorescence intensity), which can take as long as 24 hours.

[0057] Particularly, the contact of DHE with a sample is performed at a temperature from 20 to 80 °C, particularly from 20 to 40°C, in particular at 37°C.

[0058] The fluorescence emitted by DHE-MGO and DHE-glyoxal complexes may be measured by any conventional method and by using any conventional fluorescence apparatus, e.g. fluorescent spectroscopy. The intensity of fluorescence emitted by DHE-MGO complex and DHE-glyoxal complex is measured at a wavelength between 490 and 550 nm. Particularly, the intensity of fluorescence emitted by those complexes is measured at a wavelength between 500 and 540 nm. More particularly, the intensity of fluorescence emitted by those complexes is measured at 515 nm.

[0059] The excitation spectrum for a fluorescence emission in the range between 490 and 550 nm are determined empirically using the mentioned emission wavelengths and cannot be determined by the conventional methods of absorbance measurement.

[0060] In a particular embodiment, the excitation wavelength of DHE-MGO and DHE-glyoxal complexes is comprised at a wavelength between 250 and 475 nm. More particularly, the fluorescence of DHE-MGO and DHE-glyoxal complexes is excited at a wavelength between 275 and 290 nm, between 320 and 335 nm and between 405 and 440 nm, corresponding to the three peaks of the excitation spectrum (Fig. 1). More particularly, the complexes are exited at 440 nm wavelength.

[0061] In a particular embodiment, DHE is used in combination with a dicarbonyl scavenger, in particular with a scavenger of MGO that reacts more efficiently with MGO than with glyoxal. Therefore, at submaximal concentrations, the scavenger can partially inhibit the reaction between MGO and DHE, while unaffecting the reaction between glyoxal and DHE. In some embodiment, said submaximal concentration of a scavenger is at a concentration ranged from 0.5 to 10 mM, in particular from 1 to 7 mM. Particularly, the submaximal concentration of arginine is 5 mM. The use of such a scavenger of methylglyoxal is particularly advantageous when both MGO and glyoxal may be present in a sample, since the use of this scavenger allows at first the determination of the concentration of MGO in the sample and secondly the determination of the concentration of glyoxal.

[0062] Suitable scavengers of methylglyoxal can be any known scavengers of MGO described in the art. Examples of scavengers of MGO include, without being limited to, arginine, aminoguanidine, creatine and pyridoxamine. In a preferred embodiment, the scavenger of MGO is arginine.

[0063] In a more particular embodiment, the invention provides a method for detecting or measuring separately methylglyoxal and / or glyoxal in a sample. Said method comprises the steps of:

[0064] (a) providing two identical samples, (b) contacting the first sample with dihydroethidium,

[0065] (c) measuring the intensity of fluorescence emitted by the first sample after the contacting step (b) at a wavelength between 490 and 550 nm,

[0066] (d) contacting the second sample with dihydroethidium and a scavenger of methylglyoxal, and

[0067] (e) measuring the intensity of fluorescence emitted by the second sample after the contacting step (d) at a wavelength between 490 and 550 nm, wherein:

[0068] (i) the concentration of MGO in the sample is calculated from the difference of fluorescence intensity between step (c) and step (e), and

[0069] (ii) the concentration of glyoxal in the sample is obtained from calculating the difference of fluorescence intensity measured in step (c) and the fluorescence intensity of MGO in the sample that is calculated from calculation (i).

[0070] In a particular embodiment, the concentration of glyoxal in the sample is obtained from calculating the fluorescence in (c), corresponding to the concentration of MGO determined under the calculation (i) by comparing with a MGO calibration curve. The subtraction of this fluorescence from total fluorescence measured in step (c) results in the fluorescence of DHE- glyoxal, which can thus be determined using an included glyoxal concentration curve.

[0071] Thanks to the use of a scavenger of MGO, said method allows to detect or measure separately MGO and glyoxal in a sample.

[0072] By “two identical samples”, it is meant that two samples have the same volume and the same quantities of all components and are originating from the same source or starting material. For example, the two identical samples may be derived from the same initial sample, which have been divided in two, three or more subsamples.

[0073] Said method relies on parallel measurements of two identical samples, one is reacted with DHE to obtain the fluorescence emitted both by DHE-MGO complex and DHE-glyoxal complex; another is reacted with DHE and a scavenger of MGO to obtain the same fluorescence emitted by DHE-glyoxal complex but a significantly reduced fluorescence signal emitted by the MGO-DHE complex.

[0074] In said method, the fluorescence emitted after contacting the first sample with dihydroethidium (step b) corresponds to the fluorescence emitted by both of DHE-MGO and DHE-glyoxal complexes; the fluorescence emitted after contacting the second sample with DHE and a scavenger of MGO (step d) corresponds to the same fluorescence emitted by the DHE-glyoxal complex and a reduced amount of fluorescence emitted by the MGO-DHE complex.

[0075] The difference of fluorescence intensity between step (c) and step (e) corresponds therefore to the fluorescence emitted by remaining DHE-MGO complex in the presence of a scavenger of methylglyoxal.

[0076] In some embodiments, the sample is reacted with a scavenger of MGO at a concentration ranged from 0.5 to 10 mM, in particular from 1 to 7 mM.

[0077] In a particular embodiment, the scavenger of MGO is selected from arginine, aminoguanidine, creatine, pyridoxamine, or any other dicarbonyl scavenger that reacts more efficiently with MGO than with glyoxal. More particularly, the scavenger is arginine. In a more particular embodiment, the sample is reacted with arginine at a concentration of 5 mM.

[0078] In another particular embodiment, said method may further comprise the following steps after step (e) for determining the respective levels of MGO and glyoxal,: comparing the difference of fluorescence intensity between step (c) and step (e) with fluorescence intensity obtained from MGO standards which correspond to known concentrations of MGO and also react with DHE and the scavenger in the same condition, calculating the fluorescence intensity emitted from DHE-MGO complex in the sample measured in step (c), calculating the fluorescence intensity emitted from DHE-glyoxal complex in the sample measured in step (c) to determine the concentration of glyoxal in the sample

[0079] The difference of fluorescence intensity between step (c) and step (e) is indicative of the concentration of MGO in the sample. The Inventors have shown that at every concentration of MGO, a scavenger of MGO, e.g. arginine, reduces the fluorescence of the DHE-MGO complex by the same percentage. Thus, the difference of fluorescence intensity between step (c) and step (e) is indicative of the concentration of MGO in the sample. The concentration of MGO in the sample may be determined, e.g., by comparing said difference of fluorescence intensity with those emitted from a series of MGO standards which also react with DHE and the scavenger in the same condition. The fluorescence intensity emitted from total DHE-MGO complex in the sample measured in step (c) can then be calculated.

[0080] The fluorescence intensity of DHE-glyoxal complex can be further obtained from calculating the difference of fluorescence intensity measured in step (c) and the calculated fluorescence intensity of total DHE-MGO in the sample. The concentration of glyoxal in the sample may be determined by comparison with a series of glyoxal standards which also react with DHE in the same condition.

[0081] The present invention also provides a kit for detecting or measuring methylglyoxal and / or glyoxal in a sample.

[0082] Said kit comprises or consists of a container comprising dihydroethidium and a container comprising a scavenger of methylglyoxal. Said scavenger may be selected from arginine, aminoguanidine, creatine, pyridoxamine, or any other dicarbonyl scavenger. More particularly, said scavenger is arginine.

[0083] Said kit is particularly suitable to be used for detecting or measuring MGO and / or glyoxal in a food sample or a biological sample.

[0084] The kit may further comprise any reagent and / or buffer suitable for sample preparation, such as at least one reaction buffer, a series of methylglyoxal standards, and a series of glyoxal standards. In a particular embodiment, said kit further comprises a container comprising a reaction buffer, at least one container containing a methylglyoxal standard, and at least one container containing a glyoxal standard.

[0085] In a particular embodiment, the kit consists of a container comprising dihydroethidium and a container comprising arginine.

[0086] In another aspect, the present invention relates to a complex formed by dihydroethidium with methylglyoxal. Said complex is particular useful, for example for screening more specific, non-toxic scavengers of methylglyoxal. Said scavengers may have wide application, for example as food additives for neutralizing MGO in food products or for the production of novel scavenger-containing food supplements, aimed at targeting disease- and age-related complications.

[0087] The present invention also relates to the use of said complex for screening a scavenger of methylglyoxal from a candidate molecules library.

[0088] Particularly, the present invention provides a method for screening a scavenger of methylglyoxal, comprising :

[0089] (a) providing two identical samples containing methylglyoxal,

[0090] (b) contacting the first sample with dihydroethidium,

[0091] (c) measuring the intensity of fluorescence emitted by the sample after the contacting step (b) at a wavelength between 490 and 550 nm, (d) contacting the second sample with dihydroethidium and the candidate compound, and

[0092] (e) measuring the intensity of fluorescence emitted by the sample after the contacting step (d) at a wavelength between 490 and 550 nm, wherein the difference of fluorescence intensity between step (c) and step (e) is indicative that the candidate compound is a scavenger of MGO.

[0093] The present invention is illustrated in more detail in the following examples.

[0094] Examples

[0095] Materials and methods

[0096] DHE was purchased from AAT Bioquest, MGO, glyoxal and arginine were purchased from Sigma- Aldrich. Fluorescence was detected and measured in black 96 well plates using a FluoStar Optima fluorescence microtiter plate reader from BMG Technologies and an EnSight microtiter plate reader from Perkin elmer.

[0097] Results

[0098] 1. Excitation and emission spectra of different forms of DHE

[0099] The excitation and emission spectra of four forms of DHE, i.e. native DHE, methylglyoxylated DHE, glyoxylated DHE, and superoxide-oxidized DHE (2- hydroxy ethidium), were analyzed. After incubation of DHE for 60 minutes at 37°C alone (A), or with MGO (B), glyoxal (C), or superoxide which is produced via xanthine / xanthine oxidase (D), excitation spectra of these forms of DHE were generated at fixed emission wavelengths and emission spectra were generated at fixed excitation wavelengths. For each form of DHE, the excitation spectrum was analyzed at the most optimal (peak) emission wavelength. Vice versa, the emission spectrum was established at three excitation wavelengths that corresponded to three wavelength peaks of the excitation spectrum.

[0100] Results of Fig. IB and 1C show that DHE-MGO and DHE-glyoxal complexes at three excitation wavelengths produce a fluorescence with optimal emission at 499 nm and 514 nm, respectively. Excitation and emission spectra of DHE-MGO and DHE-glyoxal are very similar. On the contrary, they are very different from the spectra of native and superoxide-oxidized DHE (Fig. 1A, Fig. ID). 2. Different forms of DHE generate distinctive fluorescence

[0101] In order to further confirm that DHE-MGO complex produces a fluorescence which can be distinguished from that produced by DHE itself or by oxidized-DHE, different concentrations of DHE were measured at different wavelengths of fluorescence excitation and emission. DHE was incubated for 60 minutes at 37°C in three different ways: alone at various concentrations (A, D, G), at 5 pM with various concentrations of MGO (B, E, H) and at 5 pM with the enzyme xanthine oxidase and various concentrations of xanthine (C, F, I). Of note, xanthine oxidase generates superoxide in the presence of xanthine. After the incubation, fluorescence was measured at three conditions: 340 nm excitation and 420 nm emission (A,B,C), 340 nm excitation and 520 nm emission (D,E,F) and 544 nm excitation and 590 nm emission (G,H,I).

[0102] The results show that DHE itself (native DHE) emits fluorescence specifically at 340 nm excitation and 420 nm emission (Fig. 2A); that DHE, upon reaction with MGO, emits fluorescence at 340 nm excitation and 520 nm emission (Fig. 2E) and that DHE, once oxidized by superoxide, emits fluorescence at 544 nm excitation and 590 nm emission (Fig. 21). In conclusion, these results confirm that it is possible to measure distinctly and simultaneously three different forms of DHE: native DHE, methylglyoxylated DHE and superoxide-oxidized DHE.

[0103] 3. DHE-MGO fluorescence intensity increases with MGO quantity

[0104] 5pM of DHE was incubated for 60 minutes at 37°C with various concentrations of MGO (0-600 pM), followed by the measurement of fluorescence at 340 nm excitation and 520 nm emission.

[0105] The results of Fig. 3 show that DHE-MGO fluorescence intensity increases with MGO quantity. The higher the concentration of MGO is, the more important the increase of fluorescence intensity is observed. This result indicates that it is possible to quantify MGO concentration up to 600 pM in a sample by measuring the intensity of the fluorescence produced by DHE-MGO complex.

[0106] 4. Effect of scavengers and antioxidants on MGO

[0107] Effect of scavengers and anti-oxidants on MGO and reactive oxygen species was analyzed. Three concentrations of three scavengers of MGO (arginine, aminoguanidine and creatine) and four anti-oxidants (superoxide dismutase (SOD), a water-soluble form of vitamin E (trolox), vitamin C and glutathione) were added to a MGO solution, a solution of superoxide- generating system which is a combination of xanthine and xanthine oxidase (SO), and a solution of hydrogen peroxide, respectively.

[0108] The quantity of MGO was evaluated by measuring the fluorescence produced by DHE- MGO complex (excitation 340 nm, emission 520 nm). The quantity of superoxide was evaluated by measuring the fluorescence produced by oxidized DHE (excitation 544 nm, emission 590 m). The quantity of hydrogen peroxide was evaluated by measuring the absorbance of OPD at 450 nm in the presence of horseradish peroxidase (HRP).

[0109] The results show that all three scavengers of MGO can specifically neutralize MGO and reduce the formation of DHE-MGO complex (Fig. 4A, B and C), while the activity of antioxidants on MGO or oxygen reactive species may not be specific (Fig. 4D, E, F and G).

[0110] These results also show that arginine is a particular efficient scavenger of MGO.

[0111] 5. Sensitivity of DHE to three alpha-dicarbonyl compounds.

[0112] In order to verify the specificity of DHE with respect to physiological dicarbonyls, three physiological dicarbonyls (MGO, glyoxal and 3-deoxyglucosone) at different concentrations (0-500 pM) were incubated for 60 minutes at 37°C in the absence or presence of 5 pM DHE. Only an increase in fluorescence intensity was observed for the incubations with MGO or glyoxal. No change of fluorescence intensity was observed for the incubation with 3- deoxyglucosone. (Fig. 5)

[0113] This result shows that DHE is only specific to MGO and glyoxal.

[0114] 6. Methylglyoxal and glyoxal have different sensitivity to arginine

[0115] MGO and glyoxal were incubated for 60 minutes at 37 °C with 5 pM DHE and various concentrations of arginine. The percentage of remaining MGO or glyoxal after the reaction was calculated. The result of Fig. 6 shows that MGO and glyoxal have very different sensitivity to arginine, a well-known scavenger of dicarbonyls . At some concentrations of arginine, for example at 5 mM (arrow), MGO is partially neutralized by arginine, while glyoxal is completely unaffected .

[0116] 7. Determination of MGO and glyoxal concentrations in a same sample

[0117] Sixteen mixtures of MGO and glyoxal were prepared with different concentrations: MGO at 0, 75, 150, 225 pM and glyoxal at 0, 400, 800, 1200 pM. These mixtures were incubated with DHE in the absence and presence of 5 mM arginine, respectively. After measuring fluorescence at 340 nm excitation and 520 nm emission, the concentration of MGO in each sample was determined through the difference in fluorescence of samples with and without arginine (i.e. 5 mM arginine does not scavenge glyoxal). More particularly, said calculated difference is compared with a series of MGO standards which also react with DHE and arginine in the same condition to determine the concentration of MGO in the samples. The fluorescence intensity emitted from DHE-MGO in the samples without arginine may be further deduced, which allows to obtain the fluorescence intensity emitted from DHE-glyoxal complex in said samples. Glyoxal concentrations in the same samples were then determined by comparison with a series of glyoxal standards.

[0118] The results of Fig. 7A and Fig. 7B show that for each of the 16 samples, the calculated concentrations of MGO and glyoxal were highly similar comparable to the concentrations of MGO and glyoxal that were added to the samples. These results confirm that both the concentrations of MGO and the concentration glyoxal in a same sample can be specifically and accurately measured by using arginine and DHE.

[0119] References

[0120] Beisswenger, P.J. et al. (2013) Early progression of diabetic nephropathy correlates with methylglyoxal -derived advanced glycation end products. Diabetes Care 36 (10), 3234-9.

[0121] Maessen, D.E. et al. (2015) The role of methylglyoxal and the glyoxalase system in diabetes and other age-related diseases. Clin Sci (Lond) 128 (12), 839-61.

[0122] Matafome, P. et al. (2016) Methylglyoxal in Metabolic Disorders: Facts, Myths, and Promises. Med Res Rev 37 (2), 368-403.

[0123] Ahmed, N. et al. (2003) Methylglyoxal-derived hydroimidazolone advanced glycation endproducts of human lens proteins. Invest Ophthalmol Vis Sci 44 (12), 5287-5292.

[0124] Riboulet-Chavey, A. et al. (2006) Methylglyoxal impairs the insulin signaling pathways independently of the formation of intracellular reactive oxygen species. Diabetes 55 (5), 1289- 99.

[0125] Seo, K. et al. (2014) Methylglyoxal induces mitochondrial dysfunction and cell death in liver. Toxicol Res 30 (3), 193-8.

[0126] Brouwers, O. et al. (2010) Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress. Diabetologia 53 (5), 989-1000.

[0127] Maasen, K. et al. (2021) Quantification of dicarbonyl compounds in commonly consumed foods and drinks; presentation of a food composition database for dicarbonyls. Food Chem 339, 128063.

[0128] Wang, T. et al. (2013) A “Turn-On” fluorescent sensor for methylglyoxal. J. Am. Chem Soc. 135, 12429-12433.

[0129] Vidal, N. et al. (2014) High throughput assay for evaluation of reactive carbonyl scavenging capacity. Redox Biol 2, 590-598.

[0130] Good, N. E., et al. (1966) Hydrogen ion buffers for biological research. Biochemistry 5, 467- 477.

Claims

Claims1. Use of dihydroethidium for the detection or the measure of methylglyoxal and / or of glyoxal in a sample.

2. The use according to claim 1, wherein dihydroethidium is used with a scavenger of methylglyoxal.

3. The use according to claim 2, wherein the scavenger is selected from arginine, aminoguanidine, creatine and pyridoxamine.

4. A method for detecting or measuring methylglyoxal and / or glyoxal in a sample, comprising the steps of :(a) contacting the sample with dihydroethidium;(b) measuring the intensity of fluorescence emitted by the sample after the contacting step (a) at a wavelength between 490 and 550 nm, wherein the emission of fluorescence in step (b) is indicative of the presence or of the level of methylglyoxal and / or of glyoxal in the sample.

5. The method according to claim 4, wherein the method comprises the steps of:(a) providing two identical samples,(b) contacting the first sample with dihydroethidium,(c) measuring the intensity of fluorescence emitted by the first sample after the contacting step (b) at a wavelength between 490 and 550 nm ,(d) contacting the second sample with dihydroethidium and a dicarbonyl scavenger, in particular a scavenger of methylglyoxal; and(e) measuring the intensity of fluorescence emitted by the second sample after the contacting step (d) at a wavelength between 490 and 550 nm, wherein:(i) the concentration of MGO in the sample is calculated from the difference of fluorescence intensity between step (c) and step (e), and(ii) the concentration of glyoxal in the sample is obtained from calculating the difference of fluorescence intensity measured in step (c) and the fluorescence intensity of MGO in the sample that is calculated from calculation (i).

6. The method according to claim 5, wherein the scavenger of methylglyoxal is selected from arginine, aminoguanidine, creatine and pyridoxamine.

7. The method according to any one of claims 4 to 6, wherein dihydroethidium is at a concentration from 1 to 500 pM, in particular from 5 to 200 p M.

8. The method according to any one of claims 5 to 7, wherein the dicarbonyl scavenger is at a concentration from 0.5 to 10 mM.

9. The method according to any one of claims 4 to 8, wherein the excitation wavelength is comprised between 250 and 475 nm.

10. A kit for detecting or measuring methylglyoxal and / or glyoxal in a sample, the kit comprising :- a container comprising dihydroethidium, and- a container comprising a dicarbonyl scavenger, in particular a scavenger of methylglyoxal,- optionally, a container comprising reaction buffer,- optionally, at least a container containing a methylglyoxal standard, and- optionally, at least a container containing a glyoxal standard.

11. The use according to any one of claims 1 to 3 or the method according to any one of claims 4 to 9 or the kit according to claim 10 wherein the sample is a food sample or a biological sample.

12. A complex formed by dihydroethidium with methylglyoxal.

13. Use of the complex according to claim 12 for the screening of novel scavengers of methylglyoxal.

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