Non-enzymatic Reaction Research Articles

Pistachio green hull and pomegranate peel extracts as two natural antiglycation agents.

Advanced glycation end products (AGEs) are formed in the final step of the nonenzymatic Maillard reaction, which can contribute to various health problems such as diabetes mellitus, renal failure, and chronic inflammation. Bioactive compounds with antiglycation properties have the potential to inhibit AGE-related diseases. This study investigated the antiglycation potential of pistachio green hull (PGH) and pomegranate peel (PP) extracts, which are polyphenol-rich agro-residues, against fluorescent AGE formation and compared the results with pyridoxine (vitamin B6), metformin, and EDTA (as usual chemical antiglycation agents). The results showed that PGH and PP effectively inhibited the formation of AGEs in bovine serum albumin-glucose (BSA-Glu) and BSA-fructose (BSA-Fru) with antiglycation activities ranging from 92% to 97%. PP extract (with an IC50 of 94 mg ml-1) had a greater antiglycation ability than PGH extract (with an IC50 of 142 mg ml-1). Also, results indicated that the antiglycation activities of the extracts were comparable to that of pyridoxine, and higher than metformin and EDTA. These findings suggest that the two studied extracts can be used for sustainable production of high-added-value food products with a positive effect on consumers' health.

Discovery of Glycation Products: Unraveling the Unknown Glycation Space Using a Mass Spectral Library from In Vitro Model Systems.

The nonenzymatic reaction between amino acids (AAs) and reducing sugars, also known as the Maillard reaction, is the primary source of free glycation products (GPs) in vivo and in vitro. The limited number of MS/MS records for GPs in public libraries hinders the annotation and investigation of nonenzymatic glycation. To address this issue, we present a mass spectral library containing the experimental MS/MS spectra of diverse GPs from model systems. Based on the conceptional reaction processes and structural characteristics of products, we classified GPs into common GPs (CGPs) and modified AAs (MAAs). A workflow for annotating GPs was established based on the structural and fragmentation patterns of each GP type. The final spectral library contains 157 CGPs, 499 MAAs, and 2426 GP spectra with synthetic model system information, retention time, precursor m/z, MS/MS, and annotations. As a proof-of-concept, we demonstrated the use of the library for screening GPs in unidentified spectra of human plasma and urine. The AAs with the C6H10O5 modification, fructosylation from Amadori rearrangement, were the most found GPs. With the help of the model system, we confirmed the existence of C6H10O5-modified Valine in human plasma by matching both retention time, MS1, and MS/MS without reference standards. In summary, our GP library can serve as an online resource to quickly screen possible GPs in an untargeted metabolomics workflow, furthermore with the model system as a practical synthesis method to confirm their identity.

The Impact of Activating Agents on Non-Enzymatic Nucleic Acid Extension Reactions.

Non-enzymatic template-directed primer extension is increasingly being studied for the production of RNA and DNA. These reactions benefit from producing RNA or DNA in an aqueous, protecting group free system, without the need for expensive enzymes. However, these primer extension reactions suffer from a lack of fidelity, low reaction rates, low overall yields, and short primer extension lengths. This review outlines a detailed mechanistic pathway for non-enzymatic template-directed primer extension and presents a review of the thermodynamic driving forces involved in entropic templating. Through the lens of entropic templating, the rate and fidelity of a reaction are shown to be intrinsically linked to the reactivity of the activating agent used. Thus, a strategy is discussed for the optimization of non-enzymatic template-directed primer extension, providing a path towards cost-effective in vitro synthesis of RNA and DNA.

Development of simultaneous quantitation method for 20 free advanced glycation end products using UPLC–MS/MS and clinical application in kidney injury

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93–5.94 % and 2.30–8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1–3.13 ng/mL and 0.5–6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R2 >0.9974) across a wide linear range (1.56–400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N6-carboxyethyl-L-arginine (CEA), N6-carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N6-formyl-lysine, N6-carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease.

Types of Membrane Transporters and the Mechanisms of Interaction between Them and Reactive Oxygen Species in Plants.

Membrane transporters are proteins that mediate the entry and exit of substances through the plasma membrane and organellar membranes and are capable of recognizing and binding to specific substances, thereby facilitating substance transport. Membrane transporters are divided into different types, e.g., ion transporters, sugar transporters, amino acid transporters, and aquaporins, based on the substances they transport. These membrane transporters inhibit reactive oxygen species (ROS) generation through ion regulation, sugar and amino acid transport, hormone induction, and other mechanisms. They can also promote enzymatic and nonenzymatic reactions in plants, activate antioxidant enzyme activity, and promote ROS scavenging. Moreover, membrane transporters can transport plant growth regulators, solute proteins, redox potential regulators, and other substances involved in ROS metabolism through corresponding metabolic pathways, ultimately achieving ROS homeostasis in plants. In turn, ROS, as signaling molecules, can affect the activity of membrane transporters under abiotic stress through collaboration with ions and involvement in hormone metabolic pathways. The research described in this review provides a theoretical basis for improving plant stress resistance, promoting plant growth and development, and breeding high-quality plant varieties.

Non-enzymatic browning reaction of isomaltulose

Isomaltulose is a naturally occurring reducing disaccharide composed of glucose and fructose monomers. It is gaining interest as an alternative sweetener to sucrose in past years. It has been successfully used in various food products like chocolate, breakfast cereals, chewing gums, and dairy products especially, ice cream and yogurt. Nonenzymatic browning reactions in foods continue to be an active area of research because of their important roles in color, flavor and nutritional quality. The present study aimed to investigate the influence of different environmental factors on the non-enzymatic browning reaction of isomaltulose. The formation of colored products as a result of the non-enzymatic browning reaction was monitored. The most significant color change occurs in isomaltulose solution under the studied conditions (pH, temperature and amino component), which is a clear indication of a more intense course of the non-enzymatic browning reaction. The most intense staining was observed at pH 10, t = 80°C with the amino component glycine. It has been found that while glucose solutions are coloured more intensely in the presence of lysine, isomaltulose solutions are coloured more intensely in the presence of glycine. In addition to the type of amino component, the degree of staining is influenced by the pH value and the temperature. The results obtained showed that the use of isomaltulose in the composition of foods, that are subject to heat treatment, will affect the color formation of the final product.

Screening stabilisers for cyanoenone triterpenoid TX101 in rat plasma samples by simultaneous analysis of parent drug and the epoxidation product.

In the development of bioanalytical methods, stabilizing drug molecules in biological matrices is crucial for ensuring reliable exposure data in pharmacokinetic and toxicokinetic sample analyses. This study focuses on the evaluation of stabilizing effects on the synthetic triterpenoid TX101, a cyanoenone triterpenoid Nrf2 activator with known instability in plasma samples. The molecule's unsaturated double bond is susceptible to oxidation, either nonenzymatically via oxygen or enzymatically through cytochrome P450 enzyme-catalyzed epoxidation. The research explores the impact of antioxidants (L-ascorbic acid, sodium metabisulfite, sodium sulfite) and P450 enzyme inhibitors (sodium diethyldithiocarbamate, memantine hydrochloride, 1-aminobenzotriazole) on TX101 stability in rat plasma samples. Results reveal that adding 2.5 mg/mL sodium sulfite or sodium metabisulfite effectively inhibits the nonenzymatic oxidation of TX101 to TX101-epoxide, while L-ascorbic acid shows minimal stabilizing effect. Among P450 enzyme inhibitors, sodium diethyldithiocarbamate and memantine hydrochloride exhibit modest stabilizing effects, likely attributed to their antioxidant activity. The developed High-formance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method, incorporating Supported Liquid Extraction for sample cleanup, allows simultaneous monitoring of TX101 and TX101-epoxide. Application of this method in a rat dose-range finding study confirms successful inhibition of TX101-epoxide formation in samples treated with sodium sulfite or sodium metabisulfite. Overall, the study emphasizes the importance of stabilizers in preventing nonenzymatic oxidation reactions during sample storage, providing valuable insights for bioanalytical method development and validation.

Effect of the ripening stage on the pulsed vacuum drying behavior of goji berry (Lycium barbarum L.): Ultrastructure, drying characteristics, and browning mechanism

In current work, the effect of ripening stages (I, II, and III) on pulsed vacuum drying (PVD) behavior of goji berry was explored. The shortest drying time of goji berry was observed at stage I (6.99 h) which was 13.95 %, and 28.85 % shorter than those at stages II, and III, respectively. This phenomenon was closely associated with the ripening stage, as contributed by the initial physiochemical differences, ultrastructure alterations, and moisture distribution. In addition, lower maturity suffered more severe browning, primarily due to the enzymatic and non-enzymatic reactions of phenolics, followed by pigment degradation and the Maillard reaction. Additionally, the PVD process promoted the rupture and transformation of the pectin fractions, also causing browning either directly or indirectly through participation in other chemical reactions. These findings suggest that the appropriate ripening stage of goji berry should be considered as having a significant impact on drying behaviors and quality attributes.

Inhibition of Streptococcus mutans growth and biofilm formation through protein acetylation.

Numerous cellular processes are regulated in response to the metabolic state of the cell, and one such regulatory mechanism involves lysine acetylation. Lysine acetylation has been proven to play an important role in the virulence of Streptococcus mutans, a major cariogenic bacterial species. S. mutans' glucosyltransferases (Gtfs) are responsible for synthesizing extracellular polysaccharides (EPS) and contributing to biofilm formation. One of the most common nonsteroidal anti-inflammatory drugs is acetylsalicylic acid (ASA), which can acetylate proteins through a nonenzymatic transacetylation reaction. Herein, we investigated the inhibitory effects of ASA on S. mutans. ASA treatment was observed to impede the growth of S. mutans, leading to a reduction in the production of water-insoluble EPS and the formation of biofilm. Moreover, ASA decreased the enzyme activity of Gtfs while increasing the protein acetylation level. The in vivo anticaries efficacy of ASA has further been proved using the rat caries model. In conclusion, ASA as an acetylation agent attenuated the cariogenic virulence of S. mutans, suggesting the potential value of protein acetylation on antimicrobial and anti-biofilm applications to S. mutans.

Hexose phosphorylation for a non-enzymatic glycolysis and pentose phosphate pathway on early Earth

Glycolysis and pentose phosphate pathways play essential roles in cellular processes and are assumed to be among the most ancient metabolic pathways. Non-enzymatic metabolism-like reactions might have occurred on the prebiotic Earth and been inherited by the biological reactions. Previous research has identified a part of the non-enzymatic glycolysis and the non-enzymatic pentose phosphate pathway from glucose 6-phosphate and 6-phosphogluconate, which are intermediates of these reactions. However, how these phosphorylated molecules were formed on the prebiotic Earth remains unclear. Herein, we demonstrate the synthesis of glucose and gluconate from simple aldehydes in alkaline solutions and the formation of glucose 6-phosphate and 6-phosphogluconate with borate using thermal evaporation. These results imply that the initial stages of glycolysis-like and pentose phosphate pathway-like reactions were achieved in borate-rich evaporative environments on prebiotic Earth, suggesting that non-enzymatic metabolism provided biomolecules and their precursors on prebiotic Earth.

Breath of fresh air: Investigating the link between AGEs, sRAGE, and lung diseases.

Advanced glycation end products (AGEs) are compounds formed via non-enzymatic reactions between reducing sugars and amino acids or proteins. AGEs can accumulate in various tissues and organs and have been implicated in the development and progression of various diseases, including lung diseases. The receptor of advanced glycation end products (RAGE) is a receptor that can bind to advanced AGEs and induce several cellular processes such as inflammation and oxidative stress. Several studies have shown that both AGEs and RAGE play a role in the pathogenesis of lung diseases, such as chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, cystic fibrosis, and acute lung injury. Moreover, the soluble form of the receptor for advanced glycation end products (sRAGE) has demonstrated its ability to function as a decoy receptor, possessing beneficial characteristics such as anti-inflammatory, antioxidant, and anti-fibrotic properties. These qualities make it an encouraging focus for therapeutic intervention in managing pulmonary disorders. This review highlights the current understanding of the roles of AGEs and (s)RAGE in pulmonary diseases and their potential as biomarkers and therapeutic targets for preventing and treating these pathologies.

Longer fatty acid-protected GalNAz enables efficient labeling of proteins in living cells with minimized S-glyco modification.

Metabolic glycoengineering provides a powerful tool to label proteins with chemical tags for cell imaging and protein enrichment. The structures of per-O-acetylation on unnatural sugars facilitate membrane permeability and increase cellular uptake and are widely used for metabolic glycan labeling. However, unexpected S-glyco modification was discovered via a non-enzymatic reaction with protein cysteines, which was initially conducted with the hydrolysis of anomeric acetate by esterase. Herein, we synthesized a series of GalNAz derivatives that were protected with various lengths of short-chain fatty acid, including acetate, propionate, butyrate, valerate and pivalate, to detect differences in labeling efficiencies and occurrence of S-glyco modification. Our results demonstrate that all the GalNAz derivatives could effectively label proteins in HeLa cells, except the pivalate group. Of note, But4GalNAz exhibited excellent labeling abilities compared with Ac4GalNAz from the results for western blot, flow cytometry and confocal laser scanning microscopy. Moreover, the results for the S-glyco-modification assay by western blot and chemoproteomic analysis indicated that But4GalNAz generated negligible unexpected labeling signals compared to Ac4GalNAz. Our study uncovers the distinct labeling efficiency of different protected groups on unnatural sugars, which provides an alternative strategy to explore novel glycan probes.

Effect of Total Hemoglobin on Glycated Hemoglobin (HbA1c) in Type 2 Diabetes Mellitus

Background: Glycated hemoglobin (HbA1c) is a pivotal marker in the diagnosis, prognosis, and therapeutic monitoring of diabetes mellitus. Given the involvement of hemoglobin in non-enzymatic glycation reactions, it is hypothesized that total hemoglobin concentration might impact HbA1c levels alongside blood glucose concentrations. Objectives: This study aimed to estimate and compare plasma fasting glucose level and total hemoglobin level in type 2 diabetes mellitus (T2DM) patients with and without anemia. The study also aimed to estimate HbA1C and compare with calculated HbA1C using formula in T2DM patients with and without anemia and to correlate between total hemoglobin levels with directly measured HbA1c in T2DM patients with and without anemia. Methodology: This cross-sectional study, employing purposive sampling, investigated 30 anemic and 30 non-anemic Type 2 Diabetes Mellitus (T2DM) patients at the Central Diagnostic Laboratory. Total Hemoglobin, HbA1C, and Fasting Plasma Glucose were measured for the patients and the data was analyzed. Results: Independent 't' tests revealed significant differences (p < 0.0001) in estimated HbA1c, fasting blood sugar, calculated HbA1c, and Hb levels between the two groups. Subgroup analyses exhibited significant differences in estimated HbA1c for both groups individually (p = 0.003 for T2DM without anemia and p<0.0001 for T2DM with anemia). Pearson's correlation analysis indicated no significant correlation between HbA1c and hemoglobin in either subgroup. Similarly linear regression analysis treating HbA1c as the dependent variable yielded a non-significant p-value, suggesting that hemoglobin levels do not significantly influence HbA1c. Conclusion: In conclusion, regardless of anemia status, our findings suggest that hemoglobin is not a significant predictor of HbA1c levels in T2DM patients, offering valuable insights into factors influencing HbA1c variability in diabetic populations.

Manganese(II) promotes prebiotically plausible non-enzymatic RNA ligation reactions.

Using different prebiotically plausible activating reagents, the RNA ligation yield was significantly increased in the presence of Mn(II). The mechanism of the activation reaction has been investigated using 5'-AMP as an analogue.

Non-enzymatic synthesis of C-methylated fluostatins: discovery and reaction mechanism.

Two C-methylated fluostatins (FSTs) B3 (1) and B4 (2) were synthesized from flavin-mediated nonenzymatic epoxide ring-opening reactions of FST C. The structures of 1 and 2 were elucidated by HRESIMS, NMR, and ECD spectroscopic analyses. A subsequent 13C labeling study demonstrated that the C-methyl groups of 1 and 2 were derived from DMSO and enabled the mechanistic proposal of a nonenzymatic C-methylation.

Association of total and different food-derived advanced glycation end-products with risks of all-cause and cause-specific mortality.

Background: advanced glycation end-products (AGEs), formed through a series of non-enzymatic reactions, can promote inflammation and oxidative stress. Their accumulation in the body has been linked to cardiovascular disease (CVD) and cancer. However, the association of total AGEs and AGEs from different food sources with risks of all-cause, CVD, and cancer mortality is still unknown. Methods: we conducted a prospective cohort study of a nationally representative sample of 22 124 participants from the National Health and Nutrition Examination Survey (NHANES) III (1988-1994) and NHANES 2003-2006. A food frequency questionnaire (FFQ) was utilized to calculate total and different food-derived AGE intake. Associations between various dietary AGE scores and the risk of all-cause, CVD, and cancer mortality were assessed by weighted Cox proportional hazard regression models. Results: over a median follow-up period of 27.1 years, we found that in the general population, AGE scores of both baked foods and meat were risk factors for all-cause, CVD, and cancer mortality. Specially, higher AGE scores in total and those derived from 10 of the 13 food groups were statistically associated with an increased risk of CVD mortality. Egg-, fruit-, and vegetable-derived AGE scores were positively correlated with the risk of cancer mortality. Additionally, there were positive multiplicative and additive interactions between smoking and meat-derived AGE scores on all-cause mortality. Conclusions: high amounts of AGE consumption is associated with an increased risk of CVD mortality, and meat and baked food-derived AGEs were positively linked to all-cause, CVD, and cancer mortalities. Adherence to unhealthy lifestyles, such as smoking, may increase mortality from leading causes in individuals with AGE-enriched diet habits.

Inhibition of Advanced Glycation End Products (AGEs) by Fermented Foods Using Lactic Acid Bacteria.

Glycation is an important nonenzymatic reaction between reducing sugars and amines. Advanced glycation end products (AGEs) accumulation in the human body is associated with secondary complications related to diabetes in hyperglycemic environments. These observations suggest that the inhibition of AGEs formation is important for preventing diabetes mellitus (DM) progression and the development of diabetes-related complications. Lactic acid bacteria (LAB) are probiotics commonly used in fermented foods and food additives. Therefore, it is necessary to identify starter strains of LAB to produce fermented food to decrease the risk of DM and its complications. This chapter introduces the protocols that are inhibition assay of fermented food using LAB on AGEs such as Nω-(carboxymethyl) arginine (CMA), Nε-(carboxymethyl) lysine (CML), and fluorescent AGEs.

Epigenetic contributions to cancer: Exploring the role of glycation reactions.

Advanced Glycation End-products (AGEs), with their prolonged half-life in the human body, are emerging as potent diagnostic indicators. Early intervention studies, focusing on AGE cross-link breakers, have shown encouraging results in heart failure patients, paving the way for disease progression monitoring and therapy effectiveness evaluation. AGEs are the byproducts of a non-enzymatic reaction where sugars interact with proteins, lipids, and nucleic acids. These compounds possess the power to alter numerous biological processes, ranging from disrupting molecular conformation and promoting cross-linking to modifying enzyme activity, reducing clearance, and impairing receptor recognition. The damage inflicted by AGEs through the stimulation of intracellular signaling pathways is associated with the onset of chronic diseases across various organ systems. This review consolidates the characteristics of AGEs and the challenges posed by their expression in diverse physiological and pathological states. Furthermore, it highlights the clinical relevance of AGEs and the latest research breakthroughs aimed at reducing AGE accumulation.

The dynamic roles of advanced glycation end products.

Advanced glycation end products (AGEs) are a heterogeneous group of potentially harmful molecules that can form as a result of a non-enzymatic reaction between reducing sugars and proteins, lipids, or nucleic acids. The total body pool of AGEs reflects endogenously produced AGEs as well as exogeneous AGEs that come from sources such as diet and the environment. Engagement of AGEs with their cellular receptor, the receptor for advanced glycation end products (RAGE), which is expressed on the surface of various cell types, converts a brief pulse of cellular activation to sustained cellular dysfunction and tissue destruction. The AGEs/RAGE interaction triggers a cascade of intracellular signaling pathways such as mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinases, transforming growth factor beta, c-Jun N-terminal kinases (JNK), and nuclear factor kappa B, which leads to the production of pro-inflammatory cytokines, chemokines, adhesion molecules, and oxidative stress. All these events contribute to the progression of several chronic diseases. This chapter will provide a comprehensive understanding of the dynamic roles of AGEs in health and disease which is crucial to develop interventions that prevent and mitigate the deleterious effects of AGEs accumulation.

Development of Yellow Discoloration in Sawai (Pangasianodon hypophthalmus) Muscle due to Lipid Oxidation.

In this study, we investigated the impact of lipid oxidation on the discoloration of Sawai (Pangasianodon hypophthalmus) lipids and proteins. Sawai microsomes, liposomes, and salt-soluble myofibrillar proteins were prepared and subjected to lipid oxidation process. The results revealed that the levels of thiobarbituric acid-reactive substances, yellowness (as indicated by b* values), and pyrrole compounds increased when Sawai liposomes and microsomes were oxidized using iron and ascorbate. Meanwhile, the levels of free amines decreased, particularly as the iron content (25∼100 μM) and incubation time (0∼20 h) increased. The impact of oxidized liposomes at different levels (1, 2, and 5%) on the salt-soluble Sawai myofibrillar proteins was also evaluated. The findings revealed that lipid oxidation products reduced the sulfhydryl content and increased the surface hydrophobicity and carbonyl content of the salt-soluble Sawai myofibrillar proteins. These results imply that the formation of yellow discoloration in Sawai muscle could be due to nonenzymatic browning reactions occurring between lipid oxidation products and amines in the muscle protein.