Simultaneous determination of carbonyl compounds related to thermal treatment and oxidative stability of infant formulas by gas-diffusion microextraction and high-performance liquid chromatography with ultraviolet detection.

Infant formulae are the only possible alternative to breastfeeding during the first year of life, so it is crucial to assure their innocuousness. Infant formula undergoes heat treatments to ensure safety and shelf life. However, such processes impact health as they lead to the formation of malondialdehyde, acrolein, and α-dicarbonyl compounds, related to Maillard reaction. Thus, there is a need for improved analytical methods to ensure the safety, quality, and nutritional value of infant formulae, and also exploring the potential of specific compounds as indicators for quality control and monitoring purposes. We developed and validated a novel, efficient, and cost-effective method using gas-diffusion microextraction for the simultaneous quantification of carbonyl compounds in infant formula. Malondialdehyde, acrolein, glyoxal, methylglyoxal, and diacetyl were detected as o-phenylenediamine derivatives using HPLC with UV detection. Parameters influencing extraction efficiency were studied using an asymmetric screening design. The validated method has shown excellent linearity, sensitivity, accuracy, and precision. It was applied to analyze 26 infant formula samples, including starter, follow-up, and special formulated powdered infant formula. Methylglyoxal was found in all samples (0.201-3.153 µg mL-1), while malondialdehyde was present only in certain starter formulas (1.033-1.802 µg mL-1). Acrolein (0.510-3.246 µg mL-1), glyoxal (0.109-1.253 µg mL-1), and diacetyl (0.119-2.001 µg mL-1) were detected in various sample types. Principal components and hierarchical cluster analyses have showcased distinct sample clustering based on analyte contents. This study presents a novel methodology for the analysis of markers of thermal treatment and oxidative stability in infant formula. It contributes to the characterization of the products' composition and quality control of infant formulae, thereby enhancing their safety and nutritional adequacy. This study also presents the first reported quantification of acrolein in infant formula and introduces the application of the acrolein-o-phenylenediamine derivative for food analysis.

Application of tea polyphenols as additives in brown fermented milk: Potential analysis of mitigating Maillard reaction products.

Brown fermented milk (BFM) is favored by consumers in the dairy market for its unique burnt flavor and brown color. However, Maillard reaction products (MRP) from high-temperature baking are also noteworthy. In this study, tea polyphenols (TP) were initially developed as potential inhibitors of MRP formation in BFM. The results showed that the flavor profile of BFM did not change after adding 0.08% (wt/wt) of TP, and its inhibition rates on 5-hydroxymethyl-2-furaldehyde (5-HMF), glyoxal (GO), methylglyoxal (MGO), Nε-carboxymethyl lysine (CML), and Nε-carboxyethyl lysine (CEL) were 60.8%, 27.12%, 23.44%, 57.7%, and 31.28%, respectively. After 21 d of storage, the levels of 5-HMF, GO, MGO, CML, and CEL in BFM with TP were 46.3%, 9.7%, 20.6%, 5.2%, and 24.7% lower than the control group, respectively. Moreover, a smaller change occurred in their color and the browning index was lower than that of the control group. The significance of this study was to develop TP as additives to inhibit the production of MRP in brown fermented yogurt without changing color and flavors, thereby making dairy products safer for consumers.

Antiglycation Effects of Adlay Seed and Its Active Polyphenol Compounds: An In Vitro Study.

This study aimed to evaluate the antiglycation effects of adlay on protein glycation using in vitro glycation assays. Adlay seed was divided into the following four parts: the hull (AH), testa (AT), bran (AB), and polished adlay (PA). A solvent extraction technique and column chromatography were utilized to investigate the active fractions and components of adlay. Based on a BSA-glucose assay, the ethanolic extracts of AT (ATE) and AB (ABE) revealed a greater capacity to inhibit protein glycation. ATE was further consecutively partitioned into four solvent fractions with n-hexane, ethyl acetate (ATE-Ea), 1-butanol (ATE-BuOH), and water. ATE-BuOH and -Ea show marked inhibition of glucose-mediated glycation. Medium-high polarity subfractions eluted from ATE-BuOH below 50% methanol with Diaion HP-20, ATE-BuOH-c to -f, exhibited superior antiglycation activity, with a maximum inhibitory percentage of 88%. Two phenolic compounds, chlorogenic acid and ferulic acid, identified in ATE-BuOH with HPLC, exhibited potent inhibition of the individual stage of protein glycation and its subsequent crosslinking, as evaluated by the BSA-glucose assay, BS-methylglyoxal (MGO) assay, and G.K. peptide-ribose assay. In conclusion, this study demonstrated the antiglycation properties of ATE in vitro that suggest a beneficial effect in targeting hyperglycemia-mediated protein modification.

An "AND"-logic-gate-based fluorescent probe with dual reactive sites for monitoring extracellular methylglyoxal level changes of activated macrophages.

An "AND"-logic-gate-based fluorescent probe NAP-DCP-4 with dual reactive sites is reported, which has improved selectivity for methylglyoxal over glyoxal, featuring formaldehyde-enhanced methylglyoxal detection and irreversible and reversible turn-on fluorescence responses at different excitation wavelengths. Its cell-impermeability enables facile monitoring of extracellular methylglyoxal level changes in the supernatant of activated macrophages.

ICT based water-soluble fluorescent probe for discriminating mono and dicarbonyl species and analysis in foods.

A unique and highly water-soluble ICT-based fluorescent probe is developed for efficient detection and discrimination of reactive monocarbonyl formaldehyde (FA) from dicarbonyl methylglyoxal (MGO)/glyoxal (GO) by modulating the ICT process, which was confirmed by photophysical and TD-DFT analysis. The probe is applied in cellular imaging and quantifying FA in preserved food and MGO in manuka honey.

Extracellular glucose is crucially involved in the fate decision of LPS-stimulated RAW264.7 murine macrophage cells.

Pyroptosis, a type of inflammatory cell death, is dependent on the inflammatory caspase-mediated cleavage of gasdermin D (GSDMD), and the subsequent pore formation on plasma membranes through which interleukin (IL)-1ß and IL-18 are released from cells. During proinflammatory activation, macrophages shift their metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis. Hypoxia-inducible factor (HIF)1α is involved in the induction of IL-1ß gene expression as well as the metabolic shift towards glycolysis. However, the relationships between pyroptosis and glycolysis, as well as between pyroptosis and HIF1α are poorly investigated. Here we show that lipopolysaccharide (LPS) stimulation of RAW264.7 murine macrophage cells results in pyroptosis when cells are cultured in high glucose medium. During pyroptosis, HIF1α activation occurs transiently followed by downregulation to sub-basal levels. HIF1α downregulation and pyroptosis are observed when cells are stimulated with LPS under high glucose conditions. We also found that intracellular levels of methylglyoxal (MGO), a side product of glycolysis, increase when cells are stimulated with LPS under high glucose conditions. The addition of glycolysis inhibitor and rapamycin suppresses HIF1α downregulation and pyroptosis. These results show that glycolysis plays a crucial role not only in pro-inflammatory activation, but also in pyroptosis in LPS-stimulated RAW264.7 macrophages.

Methylglyoxal and D-lactate in cisplatin-induced acute kidney injury: Investigation of the potential mechanism via fluorogenic derivatization liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) proteomic analysis.

Nephrotoxicity severely limits the chemotherapeutic efficacy of cisplatin (CDDP). Oxidative stress is associated with CDDP-induced acute kidney injury (AKI). Methylglyoxal (MG) forms advanced glycation end products that elevate oxidative stress. We aimed to explore the role of MG and its metabolite D-lactate and identify the proteins involved in CDDP-induced AKI. Six-week-old female BALB/c mice were intraperitoneally administered CDDP (5 mg/kg/day) for 3 or 5 days. Blood urea nitrogen (42.6 ± 7.4 vs. 18.3 ± 2.5; p < 0.05) and urinary N-acetyl-ß-D-glucosaminide (NAG; 4.89 ± 0.61 vs. 2.43 ± 0.31 U/L; p < 0.05) were significantly elevated in the CDDP 5-day group compared to control mice. Histological analysis confirmed AKI was successfully induced. Confocal microscopy revealed TNF-α was significantly increased in the CDDP 5-day group. Fluorogenic derivatized liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) showed the kidney MG (36.25 ± 1.68 vs. 18.95 ± 2.24 mg/g protein, p < 0.05) and D-lactate (1.78 ± 0.29 vs. 1.12 ± 0.06 mol/g protein, p < 0.05) contents were significantly higher in the CDDP 5-day group than control group. FD-LC-MS/MS proteomics identified 33 and nine altered peaks in the CDDP 3-day group and CDDP 5-day group (vs. control group); of the 35 proteins identified using the MOSCOT database, 11 were antioxidant-related. Western blotting confirmed that superoxide dismutase 1 (SOD-1) and parkinson disease protein 7 (DJ-1) are upregulated and may participate with MG in CDDP-induced AKI. This study demonstrates TNF-α, MG, SOD-1 and DJ-1 play crucial roles in CDDP-induced AKI.

An activatable near-infrared fluorescent probe for methylglyoxal imaging in Alzheimer's disease mice.

Visual detection of the methylglyoxal (MGO) level in the brain is critical for understanding its role in the onset and progression of AD. Herein, we disclosed a NIR fluorescent probe, DBTPP, for detecting MGO by utilizing a thiadiazole-fused o-phenylenediamine moiety as a MGO-specific sensing unit. DBTPP exhibits a series of distinct advantages, such as NIR emission, high selectivity and sensitivity, excellent acid-stability, and a huge off-on ratio. The probe could accurately monitor both exogenous and endogenous MGO variations in SH-SY5Y cells. Besides, it was able to image the endogenous MGO in a transgenic AD mouse model successfully, suggesting the great potential of MGO as a biomarker for early AD diagnosis.

Quantitation of α-Dicarbonyls and Advanced Glycation Endproducts in Conventional and Lactose-Hydrolyzed Ultrahigh Temperature Milk during 1 Year of Storage.

A comprehensive quantitative characterization of Maillard reaction products was carried out for conventional (CON) and lactose-hydrolyzed (LH) ultrahigh temperature (UHT) milk during storage at 20, 30, and 40 °C for 1 year. The accumulation of 3-deoxyglucosone (3-DG) and 3-deoxygalactosone (3-DGal) in LH-UHT milk ranged from 20-fold (at 20 °C) to 44-fold (at 40 °C) higher than that in CON-UHT milk. High temperature storage (40 °C) significantly accelerated the accumulation of 3-DG, 3-DGal, and 5-hydroxymethyl furfural but not the majority of the analyzed advanced glycation endproducts (AGEs). The concentrations of major AGEs including N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), methylglyoxal-hydroimidazolone isomers (MG-H1/H3), glyoxal-hydroimidazolone isomers (G-H1/H3), and G-H2 detected in CON milk during storage were in the range 12-700, 1-14, 8-45, 4-13, and 1-30 µM, respectively, while they were 30-570, 2-88, 17-150, 9-20, and 5-34 µM, respectively, in LH milk. Pyrraline, S-(carboxymethyl)cysteine (CMC), and glyoxal-lysine dimer were detected in lower levels, while MG-H2, methylglyoxal-lysine dimer, argpyrimidine, glyoxal-lysine-amide, glycolic acid-lysine-amide, and pentosidine were not detected in any of the milk samples. This work demonstrates for the first time that five of the analyzed AGEs (CML, CEL, MG-H1/H3, G-H1/H3, and G-H2) could be selected as markers for evaluation of the extent of the Maillard reaction in UHT milk. These results contribute to a better understanding of how Maillard reactions progress during storage of UHT milk and can be used to develop strategies to inhibit Maillard reactions in LH milk.

Formation of Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine during black tea processing.

This study selected common processing methods for orthodox black tea and investigated changes in the levels of Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL), lysine, and polyphenols during each processing stage and using different parameters of each processing step. The effects of epicatechin gallate, epigallocatechin, epigallocatechin gallate, and gallic acid on the levels of CML, CEL, fructoselysine, glyoxal, and methylglyoxal were investigated by chemical model systems study under black tea processing conditions. In tea samples, CML and CEL levels significantly increased during drying (could reach 51.8 and 8.7 µg/g tea, respectively), while natural withering and extensive rolling and fermentation times facilitated the formation of CML and CEL by altering the substrate concentrations and the cellular structure of tea leaves to be dried. The results of model systems (containing lysine, glucose, and fructose) indicated that polyphenols were able to enhance the production of CML and CEL, and the levels of CML and CEL increased 1.2-3.2- and 1.6-3.5-fold, respectively. Furthermore, the main pathways responsible for CML and CEL formation during black tea processing likely involve fructoselysine and others but not glyoxal or methylglyoxal.

Fabrication of electrochemical biosensor with vanadium pentoxide nano-interface for the detection of methylglyoxal in rice.

Increased consumption of raw and par-boiled rice results in the formation of methylglyoxal (MG) at higher concentration and leads to complications in diabetic patients. Highly sensitive electrochemical biosensor was developed using glutathione (GSH) as a co-factor with vanadium pentoxide (V2O5) as a nano-interface for MG detection in rice samples. The Pt/V2O5/GSH/Chitosan bioelectrode displayed two well-defined redox peaks in its cyclic voltammograms for MG reduction. This occurred as two electron transfer process where MG gained two electrons from oxidized glutathione disulfide and formed hemithioacetal. The current density response of the fabricated bioelectrode was linear towards MG in the concentration range of 0.1-100 µM with the correlation coefficient of 0.99, sensitivity of 1130.86 µA cm-2 µM-1, limit of detection of 2 nM and response time of less than 18 s. The developed bioelectrode was used for the detection of MG in raw and par-boiled rice samples.

Lipidomic analysis for carbonyl species derived from fish oil using liquid chromatography-tandem mass spectrometry.

Lipid peroxidation gives rise to carbonyl species, some of which are reactive and play a role in the pathogenesis of numerous human diseases. Oils are ubiquitous sources that can be easily oxidized to generate these compounds under oxidative stress. In this present work, we developed a targeted lipidomic method for the simultaneous determination of thirty-five aldehydes and ketones derived from fish oil, the omega-3 fatty acid-rich source, by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytes include highly toxic reactive carbonyl species (RCS) such as acrolein, crotonaldehyde, trans-4-hydroxy-2-hexenal (HHE), trans-4-hydroxy-2-nonenal (HNE), trans-4-oxo-2-nonenal (ONE), glyoxal and methylglyoxal, all of which are promising biomarkers of lipid peroxidation. They were formed using in vitro Fe(II)-mediated oxidation, and derivatized using 2,4-dinitrophenylhydrazine (DNPH) for the feasibility of quantitative assay. Before analysis, solid phase extraction (SPE) was used to clean samples further. Uniquely different patterns of carbonyl compound generation between omega-3 and 6 fatty acids were observed using this lipidomic approach. The method developed was both validated, and successfully applied to monitor formation of carbonyl species by lipid peroxidation using ten different fish oil products. Hypotheses of correlations between the monitored dataset of analytes and their parent fatty acids were also tested using the Pearson's correlation test. Results indicate our method is a useful analytical tool for lipid peroxidation studies.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

High glucose, glucose fluctuation and carbonyl stress enhance brain microvascular endothelial barrier dysfunction: Implications for diabetic cerebral microvasculature.

We previously demonstrated that in normal glucose (5mM), methylglyoxal (MG, a model of carbonyl stress) induced brain microvascular endothelial cell (IHEC) dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC). Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced IHEC dysfunction. MG-induced loss of transendothelial electrical resistance (TEER) was potentiated in IHECs cultured for 7 or 12 days in 25 mM glucose (hyperglycemia); moreover, barrier function remained disrupted 6h after cell transfer to normal glucose media (acute glycemic fluctuation). Notably, basal occludin glycation was elevated under these glycemic states. TEER loss was exaggerated by inhibition of glutathione (GSH) synthesis and abrogated by NAC, which corresponded to GSH decreases and increases, respectively. Significantly, glyoxalase II activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and GSH inhibition increased MG accumulation, consistent with a compromised capacity for MG elimination. α-Oxoaldehydes (MG plus glyoxal) levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG-occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG-occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes.

Formation of 4(5)-methylimidazole and its precursors, α-dicarbonyl compounds, in Maillard model systems.

Glyoxal, methylglyoxal, and diacetyl formed from sucrose alone and from a D-glucose/ammonia Maillard model system were analyzed by gas chromatography. They are known as precursors of 4(5)-methylimidazole (MI). Glyoxal and methylglyoxal formed more in acidic conditions than in basic conditions, whereas diacetyl formed the most at the highest basic condition of pH 12. Glyoxal formation from sucrose ranged from 0.33 to 32.90 µg/g under four different time and temperature conditions. Amounts of glyoxal, methylglyoxal, and diacetyl formed in Maillard model systems ranged from 2.98 to 46.12 µg/mL, from 8.27 to 156.61 µg/mL, and from 14.94 to 1588.45 µg/mL, respectively. 4(5)-MI formation in the same model systems ranged from 28.56 to 1269.71 µg/mL. Addition of sodium sulfite reduced formation of these chemicals significantly. Total α-dicarbonyl compounds in 12 commercial soft drinks ranged from 5.75 to 50.72 µg/mL. 4(5)-MI was found in levels ranging from 1.76 to 28.11 ng/mL in 10 commercial soft drinks.

A quick, convenient and economical method for the reliable determination of methylglyoxal in millimolar concentrations: the N-acetyl-L-cysteine assay.

The determination of methylglyoxal (MG) concentrations in vivo is gaining increasing importance as high levels of MG are linked to various health impairments including complications of diabetes. In order to standardize the measurements of MG in body fluids, it is necessary to precisely determine the concentration of MG stock solutions used as analytical standards. The "gold standard" method for the determination of MG concentration in the millimolar range is an enzyme-catalyzed endpoint assay based on the glyoxalase I catalyzed formation of S-lactoylglutathione. However, as this assay used purified glyoxalase I enzyme, it is quite expensive. Another method uses a derivation reaction with 2,4-dinitrophenylhydrazine, but this substance is explosive and needs special handling and storage. In addition, precipitation of the product methylglyoxal-bis-2,4-dinitrophenylhydrozone during the reaction limits the reliability of this method. In this study, we have evaluated a new method of MG determination based on the previously published fast reaction between MG and N-acetyl-L-cysteine at room temperature which yields an easily detectable condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine. When comparing these three different assays for the measurement of MG concentrations, we find that the N-acetyl-L-cysteine assay is the most favorable, providing an economical and robust assay without the need for the use of hazardous or expensive reagents.

Acetilação radicalar de amino ácidos, peptídeos e nucleobases pelos sistemas biacetilo/peroxinitrito e metilglioxal/peroxinitrito / Radical acetylation of aminoacids, peptides, and nucleobases by the biacetyl or methylglyoxal/peroxynitrite systems

Biacetilo (2,3-butanediona) é um contaminante de comida e cigarro, também implicado na hepatoxicidade do álcool e em doenças pulmonares. O metilglioxal (MG), um α-oxoaldeído reativo frequentemente associado ao diabetes e envelhecimento, é produto da fragmentação oxidativa de trioses fosfato, acetona e aminoacetona. Por sua vez, peroxinitrito - um potente oxidante, agente nitrante e nucleófilo formado in vivo pela reação controlada por difusão do ânion radical superóxido com o radical óxido nítrico (k ~1010 M-1s-1) é capaz de se adicionar a CO2 e compostos carbonílicos, gerando produtos potencialmente tóxicos ou sinalizadores celulares. Aminoácidos, peptídeos e nucleobases podem ser acetilados nos grupos amina e na porção desoxiribose. Relativamente ao tratamento com peroxinitrito isolado, níveis superiores de 3-nitrotirosina foram detectados quando tirosina foi tratada com peroxinitrito/biacetilo ou metilglioxal. Ambos os grupos amina de lisina (Lys) ou um deles de derivados de lisina bloqueados e um deles (Ac-Lys-OMe, Z-Lys-OMe) foram acetilados pelo sistema biacetilo ou metilglioxal/peroxinitrito. Em tetrapeptídeos sintéticos contendo lisina como aminoácido amino-terminal (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), a lisina foi acetilada pelo sistemas dicarbonilico/peroxinitrito no grupo α-amina (em maior extensão) e/ou no ε-amina (em menor extensão). No conjunto, estes resultados podem ser interpretados à luz do mecanismo proposto para a reação de compostos α-dicarbonílicos com peroxinitrito, o qual envolve sequencialmente: (i) adição nucleofílica de peroxinitrito à carbonila; (ii) homólise do aduto peroxinitroso formado, liberando •NO2 e um radical oxila do reagente carbonílico; (iii) β-clivagem do radical oxila a um ácido carboxílico (ácido acético no caso de biacetilo e ácido fórmico, a partir de metilglioxal) e radical acetila; (iv) captação do radical acetila pelo oxigênio molecular dissolvido dando acetato, ou por aminoácido ou nucleobase...

Diacetyl (2,3-butanedione) is a food and cigarette contaminant recently implicated in alcohol hepatotoxicity and lung disease. In turn, methylglyoxal (MG) is an α-oxoaldehyde frequently associated with diabetes and aging that is putatively formed by the oxidative fragmentation of trioses phosphate, acetone and aminoacetone. Peroxynitrite - a potent oxidant, nitrating agent and nucleophile formed in vivo by the diffusion-controlled reaction of superoxide radical with nitric oxide (k ~1010 M-1s-1) - is able to form adducts with carbon dioxide and carbonyl compounds. When initially present in the reaction mixtures before addition of ONOO-, amino acids, peptides and nucleobases undergo acetylation at the amino group and purine moieties in the presence of biacetyl or methylglyoxal. Higher levels of 3-nitrotyrosine nitration were measured when peroxynitrite/biacetyl or metilglioxal was added to tyrosine, in comparison with peroxynitrite alone. Both amino groups of L-lysine or one of the amino groups of L-lysine derivatives (Z-Lys-OH and Ac-Lys-OH) were acetylated by biacetyl and methylglyoxal/peroxynitrite system. Using tetrapeptides containing lysine at the terminal amino acid (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), the lysine residue was acetylated at both or either α-amino (major adduct) and ε-amino group (minor adduct). Altogether these data can be interpreted by the mechanism proposed to describe the reaction of α-dicarbonyls with peroxynitrite as follows: (i) nucleophilic addition of peroxynitrite to the carbonyl group of the reagent; (ii) homolysis of the formed peroxynitroso carbonyl adduct to •NO2 and a carbonyloxyl radical; (iii) β-cleavage of the oxyl radical to acetyl radical plus acetic acid (from diacetyl) or formic acid (from methylglyoxal); (iv) competitive scavenging of the acetyl radical by dissolved molecular oxygen and by added amino acid, peptide or nucleobase, ultimately yielding acetate or acetylated biomolecule. If occurring in vivo...

[Methylglyoxal--a test for impaired biological functions of exotrophy and endoecology, low glucose level in the cytosol and gluconeogenesis from fatty acids (a lecture)].

In philogenesis, due to the failure to store a great deal of carbohydrates in vivo as glycogen, all animal species began synthesizing from glucose palminitic fatty acid and depositing it as triglycerides. During biological dysfunction of exotrophy (long starvation, early postnatality, hibernation), cells also accomplish a reverse synthesis of glucose from fatty acids under aerobic conditions. Under physiological conditions, acetyl-CoA that is converted to malate and pyruvate in the glyoxalate cycle is a substrate of glyconeogenesis. Under pathological conditions of hypoxia and deficiency of macroerges, gluconeogenesis occurs without ATP consumption through the methylglyoxal pathway when used as a substrate of ketone bodies via the pathway: butyric acid (butyrate) --> beta-hydroxybutyrate --> acetoacetate --> acetone --> acetol --> methylglyoxal --> S-D-lactol-glutathione --> D-lactate --> pyruvate --> D-lactate. Under physiological conditions, this gluconeogenesis pathway does not function. We believe that with low glucose levels in the cell cytosole (glycopenia), under pathological conditions of hypoxia and due to failure to mitochondria to oxidize fatty acids, gene expression and gluconeogenesis occur through the methylglyoxal pathway. At the same time, the cytosol, intercellular environment, and plasma shows the elevated levels of methylglyoxal and D-lactate that it is converted to by the action of glyoxalases I and II. Under pathological conditions, glycopenia develops in starvation, diabetes, and metabolic acidosis, neoplasms, renal failure, and possibly, metabolic syndrome. The chemical interaction of methylglyoxal with the amino acid residues of lysine and arginine results in the denaturation of circulating and structurized proteins via carbonylation--glycosylation.

Use of capillary electrophoresis to evaluate protective effects of methylglyoxal scavengers on the activity of creatine kinase.

Methylglyoxal (MGO) is a highly reactive alpha-oxoaldehyde formed endogenously in numerous enzymatic and nonenzymatic reactions. The reactions between MGO and various amino residues in proteins not only result in inactivation of enzymes, but also lead to the formation of different detrimental advanced glycation endproducts (AGEs). Recently, it was reported that creatine kinase (CK, EC 2.7.3.2) activity could be reduced or even lost under incubation with MGO in vitro. In this study, an efficient CE analytical method was developed for the evaluation of CK activity. Based on this CE method, the inhibitory effect of MGO on CK activity was confirmed. Several MGO scavengers such as aminoguanidine (AG) and some thiols showed obvious protective effects on CK activity against MGO. Furthermore, tiopronin (TP), a hepatoprotective drug, was found for the first time to counteract MGO-induced inhibition of CK activity in CK reaction. Meanwhile, TP also retained adenosine diphosphate (ADP) generation level in plasma treated with MGO, which implies that this drug may have potential protective effect on other enzymes which are associated with adenine nucleotide metabolism. Besides, the established CE approach can be utilized as a model for screening effective MGO scavengers by monitoring CK-catalyzed conversion between adenosine triphosphate and ADP.

Methylglyoxal: its presence in beverages and potential scavengers.

Nonenzymic glycation, also known as the Maillard reaction, is a complex series of reactions between reducing sugars and amino compounds. Previous studies have demonstrated that reactive dicarbonyl compounds (e.g., methylglyoxal [MG] and glyoxal [GO]), formed as intermediate products of the Maillard reaction, irreversibly and progressively modify proteins over time and yield advanced glycation end products (AGEs), which are thought to contribute to the development of diabetes mellitus and its complications. Several studies have shown that higher levels of MG are present in diabetic patients' plasma than in healthy people's plasma. Thus, decreasing the levels of MG and GO will be an effective approach to reduce the formation of AGEs and the development of diabetic complications. Here, we briefly describe our effort in searching for non- or less-toxic trapping agents of reactive dicarbonyl species from dietary sources. In addition, we have discovered that commercial beverages contain extremely high levels of MG. The potential hazardous effects of dietary MG on humans remain to be explored.