Peptide Stapling by Crosslinking Two Amines with α-Ketoaldehydes through Diverse Modified Glyoxal-Lysine Dimer Linkers.

α-Ketoaldehydes play versatile roles in the ubiquitous natural processes of protein glycation. However, leveraging the reactivity of α-ketoaldehydes for biomedical applications has been challenging. Previously, the reactivity of α-ketoaldehydes with guanidine has been harnessed to design probes for labeling Arg residues on proteins in an aqueous medium. Herein, a highly effective, broadly applicable, and operationally simple protocol for stapling native peptides by crosslinking two amino groups through diverse imidazolium linkers with various α-ketoaldehyde reagents is described. The use of hexafluoroisopropanol as a solvent facilitates rapid and clean reactions under mild conditions and enables unique selectivity for Lys over Arg. The naturally occurring GOLD/MOLD linkers have been expanded to encompass a wide range of modified glyoxal-lysine dimer (OLD) linkers. In a proof-of-concept trial, these modular stapling reactions enabled a convenient two-round strategy to streamline the structure-activity relationship (SAR) study of the wasp venom peptide anoplin, leading to enhanced biological activities.

Parkinsonism-Associated Protein DJ-1 Is an Antagonist, Not an Eraser, for Protein Glycation.

Advanced glycation end-products (AGEs) are irreversible protein modifications that are strongly associated with aging and disease. Recently, the Parkinsonism-associated protein DJ-1 has been reported to exhibit deglycase activity that erases early glycation intermediates and stable AGEs from proteins. In this work, we use mass spectrometry and western blot to demonstrate that DJ-1 is not a deglycase and cannot remove AGEs from protein or peptide substrates. Instead, our studies revealed that DJ-1 antagonizes glycation through glyoxalase activity that detoxifies the potent glycating agent methylglyoxal (MGO) to lactate. We further show that attenuated glycation in the presence of DJ-1 can be attributed solely to its ability to decrease the available concentration of MGO. Our studies also provide evidence that DJ-1 is allosterically activated by glutathione. Together, this work reveals that although DJ-1 is not a genuine deglycase, it still harbors the ability to prevent AGE formation and can be used as a valuable tool to investigate metabolic stress.

Biochemical and Biophysical in Vitro Studies and Systematic Literature Review on the Antioxidant and Antiglycation Activities of Trazodone.

BACKGROUND/AIMS: Trazodone is a selective serotonin reuptake inhibitor; however, other mechanisms of the drug's anti-depressive properties have also been postulated. Hence, the aim of the study was to perform a systematic review and assess antiglycoxidative properties of trazodone in in vitro models. METHODS: Trazodone's scavenging and chelating properties were measured with spectrophotometric method. The impact of the drug on carbonyl/oxidative stress was marked in the bovine serum albumin (BSA) model where sugars (glucose, fructose, galactose, ribose) and aldehydes (glyoxal and methylglyoxal) were used as glycation agents. Aminoguanidine and N-acetylcysteine (NAC) were applied as reference glycation/free radical inhibitors. Glycation biomarkers (kynurenine, N-formylkynurenine, dityrosine as well as advanced glycation end products contents) were assessed spectrofluorometrically. Concentrations of oxidation parameters (total thiols (TTs), protein carbonyls (PCs) and also advanced oxidation protein products (AOPPs) levels) were determined spectrophotometrically. RESULTS: We demonstrated that trazodone poorly scavenged radicals (hydroxyl radical, nitric oxide, hydrogen peroxide and 2,2-diphenyl-1-picrylhydrazyl radical) and showed low ferrous ion chelating, unlike aminoguanidine and NAC. Sugars/aldehydes caused enhancement of glycation parameters, as well as a decrease of TTs and an increase of PCs and AOPPs levels compared to BSA incubated alone. Trazodone did not reduce oxidation parameters to the baseline (BSA) and significantly exacerbated glycation markers in comparison with both BSA and BSA+glycators. The content of glycation products was markedly lower in aminoguanidine and NAC than in trazodone. The molecular docking of trazodone to BSA revealed its very low affinity, which may indicate non-specific binding of trazodone, facilitating the attachment of glycation factors. CONCLUSION: According to our findings, it may be concluded that trazodone poorly counteracts oxidation and intensifies glycation in vitro. A possible mechanism for antiglycoxidative effect of trazodone in vivo may be the enhancement of the body's adaptive response, as indicated by the results of our systematic review.

An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies.

The mammalian cell nucleus contains different types of membrane-less nuclear bodies (NBs) consisting of proteins and RNAs. Microscopic imaging has been widely applied to study the organization and structure of NBs. However, current fixation methods are not optimized for such imaging: When a fixation method is chosen to maximize the quality of the RNA fluorescence in situ hybridization (FISH), it often limits the labeling efficiency of proteins or affects the ultrastructure of NBs. Here, we report that addition of glyoxal (GO) into the classical paraformaldehyde (PFA) fixation step not only improves FISH signals for RNAs in NBs via augmented permeability of the fixed nucleus and enhanced accessibility of probes, but also largely preserves protein fluorescent signals during fixation and immunostaining. We also show that GO/PFA fixation enables the covisualization of different types of nuclear bodies with minimal impact on their ultrastructures under super-resolution microscopy.

LC-MS/MS Analysis of Reaction Products of Arginine/Methylarginines with Methylglyoxal/Glyoxal.

Methylglyoxal (MGO) and glyoxal (GO) are toxic α-dicarbonyl compounds that undergo reactions with amine containing molecules such as proteins and amino acids and result in the formation of advanced glycation end products (AGEs). This study aimed at investigating the reactivity of arginine (Arg) or dimethylarginine (SDMA or ADMA) with MGO or GO. The solutions of arginine and MGO or GO were prepared in PBS buffer (pH 7.4) and incubated at 37 °C. Direct electrospray ionization-high-resolution mass spectrometry (ESI-HRMS) analysis of the reaction mixture of Arg and MGO revealed the formation of Arg-MGO (1:1) and Arg-2MGO (1:2) products and their corresponding dehydrated products. Further liquid chromatography (LC)-MS analyses revealed the presence of isomeric products in each 1:1 and 1:2 product. The [M + H]+ of each isomeric product was subjected to MS/MS experiments for structural elucidation. The MS/MS spectra of some of the products showed a distinct structure indicative fragment ions, while others showed similar data. The types of products formed by the arginines with GO were also found to be similar to that of MGO. The importance of the guanidine group in the formation of the AGEs was reflected in similar incubation experiments with ADMA and SDMA. The structures of the products were proposed based on the comparison of the retention times and HRMS and MS/MS data interpretation, and some of them were confirmed by drawing analogy to the data reported in the literature.

Glyoxal-derived advanced glycation end-products, Nε-carboxymethyl-lysine, and glyoxal-derived lysine dimer induce apoptosis-related gene expression in hepatocytes.

BACKGROUND: Advanced glycation end-products (AGEs) are proteins or lipids that have been glycated nonenzymatically by reducing sugars and their derivatives such as methylglyoxal. AGEs are known to cause inflammation, oxidative stress, and diseases in the human body. The toxic effects of AGEs and their structures on the origin of the protein being modified have not been well studied. METHODS AND RESULTS: Five different types of AGEs: AGE1 (glucose-derived), AGE2 (glyceraldehyde-derived), AGE3 (glycolaldehyde-derived), AGE4 (methylglyoxal-derived), and AGE5 (glyoxal-derived); were used to examine the effect of AGEs on HepG2 cells. AGE2 through 5 increase the production of reactive oxygen species (ROS) in liver cells, an initiating factor for apoptosis. At the mRNA and protein levels, AGE5 treatment showed the greatest increase in expression of apoptosis-related factors such as Bax, p53, and Caspase 3. Quantitative analysis revealed that Nε-carboxymethyl-lysine (CML) and glyoxal-lysine dimer (GOLD) were the important types of AGE5. The ROS generation and the expression of apoptotic factors both increased when cells were treated with CML and GOLD. CONCLUSION: These findings suggest that AGE5 treatment activates the apoptosis-related gene expression in hapatocytes, with CML and GOLD as potential major AGE compounds.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

The reaction of acetaldehyde, glyoxal, and ammonia to yield 2-methylimidazole: thermodynamic and kinetic analyses of the mechanism.

The most thermodynamically and kinetically favorable pathways for the formation of 2-methylimidazole (2MI) in the reaction of glyoxal and acetaldehyde with ammonia in aqueous solution have been determined. The formation of 2MI proceeds through a number of successive intermediates of acyclic and cyclic structures, and the most favorable route (thermodynamically and kinetically) for the formation of the imidazole ring is the condensation of amine intermediates, in contrast to the existing concepts of imine structures. The limiting stage is the stage of cyclization involving the intramolecular attack by the amino group of the precyclic intermediate on the carbon atom bound to the hydroxyl group with the simultaneous release of a water molecule according to the SN2 mechanism. Further stages of stepwise dehydration lead to the formation of a cyclic diazine, the intramolecular migration of the proton of the tertiary carbon atom to the nitrogen atom of which completes the formation of 2MI. Experimental studies on the effect of the order of mixing of initial reagents on the 2MI yield confirmed the quantum-chemically substantiated favorable pathway for the formation of 2MI during the interaction of amine intermediates, and also revealed that the selectivity of the 2MI formation is achieved by successive mixing of acetaldehyde with ammonia until the formation of hydroxyamine products and their further interaction with glyoxal.

Kinetics, Products, and Brown Carbon Formation by Aqueous-Phase Reactions of Glycolaldehyde with Atmospheric Amines and Ammonium Sulfate.

Glycolaldehyde (GAld) is a C2 water-soluble aldehyde produced during the atmospheric oxidation of isoprene and many other species and is commonly found in cloudwater. Previous work has established that glycolaldehyde evaporates more readily from drying aerosol droplets containing ammonium sulfate (AS) than does glyoxal, methylglyoxal, or hydroxyacetone, which implies that it does not oligomerize as quickly as these other species. Here, we report NMR measurements of glycolaldehyde's aqueous-phase reactions with AS, methylamine, and glycine. Reaction rate constants are smaller than those of respective glyoxal and methylglyoxal reactions in the pH range of 3-6. In follow-up cloud chamber experiments, deliquesced glycine and AS seed particles were found to take up glycolaldehyde and methylamine and form brown carbon. At very high relative humidity, these changes were more than 2 orders of magnitude faster than predicted by our bulk liquid NMR kinetics measurements, suggesting that reactions involving surface-active species at crowded air-water interfaces may play an important role. The high-resolution liquid chromatography-electrospray ionization-mass spectrometric analysis of filter extracts of unprocessed AS + GAld seed particles identified sugar-like C6 and C12 GAld oligomers, including proposed product 3-deoxyglucosone, with and without modification by reactions with ammonia to diimine and imidazole forms. Chamber exposure to methylamine gas, cloud processing, and simulated sunlight increased the incorporation of both ammonia and methylamine into oligomers. Many C4-C16 imidazole derivatives were detected in an extract of chamber-exposed aerosol along with a predominance of N-derivatized C6 and C12 glycolaldehyde oligomers, suggesting that GAld is capable of forming brown carbon SOA.

The possible role of methylglyoxal metabolism in cancer.

Tumours reprogram their metabolism to acquire an evolutionary advantage over normal cells. However, not all such metabolic pathways support energy production. An example of these metabolic pathways is the Methylglyoxal (MG) one. This pathway helps maintain the redox state, and it might act as a phosphate sensor that monitors the intracellular phosphate levels. In this work, we discuss the biochemical step of the MG pathway and interrelate it with cancer.

Developing a Glyoxal-Crosslinked Chitosan/Gelatin Hydrogel for Sustained Release of Human Platelet Lysate to Promote Tissue Regeneration.

The clinical application of human platelet lysate (HPL) holds promise for tissue regeneration, and the development of an efficient vehicle for its delivery is desired. Chitosan-based hydrogels are potential candidates, but they often exhibit weak mechanical properties. In this study, a chitosan/gelatin (CS-GE) hydrogel crosslinked by glyoxal was fabricated for sustained release of HPL. The influence of HPL on Hs68 fibroblast and human umbilical vein endothelial cell (HUVEC) culture was evaluated, and we found that supplementing 5% HPL in the medium could significantly improve cell proliferation relative to supplementing 10% fetal bovine serum (FBS). Moreover, HPL accelerated the in vitro wound closure of Hs68 cells and facilitated the tube formation of HUVECs. Subsequently, we fabricated CS-GE hydrogels crosslinked with different concentrations of glyoxal, and the release pattern of FITC-dextrans (4, 40 and 500 kDa) from the hydrogels was assessed. After an ideal glyoxal concentration was determined, we further characterized the crosslinked CS-GE hydrogels encapsulated with different amounts of HPL. The HPL-incorporated hydrogel was shown to significantly promote the proliferation of Hs68 cells and the migration of HUVECs. Moreover, the release pattern of transforming growth factor-ß1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB) from hydrogel was examined in vitro, demonstrating a sustained release profile of the growth factors. Finally, the chick chorioallantoic membrane assay revealed that HPL encapsulation in the hydrogel significantly stimulated angiogenesis in ovo. These results demonstrate the great potential of the crosslinked CS-GE hydrogel to serve as an effective delivery system for HPL to promote tissue regeneration.

Complexes of Formaldehyde and α-Dicarbonyls with Hydroxylamine: FTIR Matrix Isolation and Theoretical Study.

The interactions of formaldehyde (FA), glyoxal (Gly) and methylglyoxal (MGly) with hydroxylamine (HA) isolated in solid argon and nitrogen were studied using FTIR spectroscopy and ab initio methods. The spectra analysis indicates the formation of two types of hydrogen-bonded complexes between carbonyl and hydroxylamine in the studied matrices. The cyclic planar complexes are stabilized by O-H⋯O(C), and C-H⋯N interactions and the nonplanar complexes are stabilized by O-H⋯O(C) bond. Formaldehyde was found to form with hydroxylamine, the cyclic planar complex and methylglyoxal, the nonplanar one in both argon and nitrogen matrices. In turn, glyoxal forms with hydroxylamine the most stable nonplanar complex in solid argon, whereas in solid nitrogen, both types of the complex are formed.

Keto-Enol Tautomerism in Passerini and Ugi Adducts.

The use of arylglyoxal as starting material in Passerini and Ugi reactions affords ß-ketoamides. This has allowed to study keto-enol tautomerism in these systems and assess the way in which the presence of acyloxy or aminoacyl groups bound to the C2 position affects such tautomerism, and to investigate the reactivity of both the enol and carbonyl forms. In this work we also prove the versatility of the Passerini reaction, since depending on the conditions to which the corresponding adducts are subjected different products of synthetic interest can be obtained.

Spruce Bark-Extracted Lignin and Tannin-Based Bioresin-Adhesives: Effect of Curing Temperatures on the Thermal Properties of the Resins.

In this study, formaldehyde-free bioresin adhesives were synthesised from lignin and tannin, which were obtained from softwood bark. The extraction was done via organosolv treatment and hot water extraction, respectively. A non-volatile, non-toxic aldehyde, glyoxal, was used as a substitute for formaldehyde in order to modify the chemical structure of both the lignin and tannin. The glyoxal modification reaction was confirmed by ATR-FTIR spectroscopy. Three different resin formulations were prepared using modified lignin along with the modified tannin. The thermal properties of the modified lignin, tannin, and the bioresins were assessed by DSC and TGA. When the bioresins were cured at a high temperature (200 °C) by compression moulding, they exhibited higher thermal stability as well as an enhanced degree of cross-linking compared to the low temperature-cured bioresins. The thermal properties of the resins were strongly affected by the compositions of the resins as well as the curing temperatures.

Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging.

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.

Fluorophore-Promoted Facile Deprotonation and Exocyclic Five-Membered Ring Cyclization for Selective and Dynamic Tracking of Labile Glyoxals.

The lack of effective chemical tools capable of dynamic tracking of labile glyoxal species (GOS) [e.g., methylglyoxal (MGO) and glyoxal (GO)] levels with high selectivity over other relevant electrophilic species, particularly, formaldehyde (FA) and nitric oxide (NO), has significantly hampered the understanding of their roles in a complex metabolic network and disease progressions. Herein, we report the rational design of the bioinspired 4-(2-guanidino)-1,8-naphthalimide fluorescent probes NAP-DCP-1 and NAP-DCP-3 from arginine-specific protein modifications. These probes undergo facile reversible fluorophore-promoted deprotonation-cyclization of a guanidium ion with labile GOS to form exocyclic five-membered dihydroxyimidazolidines. The probe NAP-DCP-1 can differentiate GOS levels in the serum of diabetic mice and patients from nondiabetic ones, which correlate very well with glucose levels, providing the GOS level as a potential new biomarker for diabetes diagnosis. Notably, the endoplasmic reticulum (ER)-targeting probe NAP-DCP-3 enabled the study of GOS perturbation in ER under various stress conditions and led to the discovery that formaldehyde (FA), either exogenously added or endogenously generated, could induce GOS level increases in ER. This finding reveals the previous unknown connection of FA with upregulated GOS levels and suggests that GOS is a key metabolite in bridging one-carbon metabolism with glycolysis and the downstream cell redox status. Moreover, the probes also showed potentials in separate quantification of MGO and GO via ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and unexpected selectivity modulation for GO over MGO via two-photon excitation. It is expected that probes reported herein provide powerful tools to study GOS level modulations in complex biological networks and would facilitate GOS-associated basic research and discovery.

Glyoxals as in vivo RNA structural probes of guanine base-pairing.

Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram-positive and Gram-negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.

T-cell activation by transitory neo-antigens derived from distinct microbial pathways.

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

Food-derived 1,2-dicarbonyl compounds and their role in diseases.

Reactive 1,2-dicarbonyl compounds (DCs) are generated from carbohydrates during food processing and storage and under physiological conditions. In the recent decades, much knowledge has been gained concerning the chemical formation pathways and the role of DCs in food and physiological systems. DCs are formed mainly by dehydration and redox reactions and have a strong impact on the palatability of food, because they participate in aroma and color formation. However, they are precursors of advanced glycation end products (AGEs), and cytotoxic effects of several DCs have been reported. The most abundant DCs in food are 3-deoxyglucosone, 3-deoxygalactosone, and glucosone, predominating over methylglyoxal, glyoxal, and 3,4-dideoxyglucosone-3-ene. The availability for absorption of individual DCs is influenced by the release from the food matrix during digestion and by their reactivity towards constituents of intestinal fluids. Some recent works suggest formation of DCs from dietary sugars after their absorption, and others indicate that certain food constituents may scavenge endogenously formed DCs. First works on the interplay between dietary DCs and diseases reveal an ambiguous role of the compounds. Cancer-promoting but also anticancer effects were ascribed to methylglyoxal. Further work is still needed to elucidate the reactions of DCs during intestinal digestion and pathophysiological effects of dietary DCs at doses taken up with food and in "real" food matrices in disease states such as diabetes, uremia, and cancer.

Determination of glyoxal and methylglyoxal in serum by UHPLC coupled with fluorescence detection.

Glyoxal (GO) and methylglyoxal (MGO) are two important biomarkers in diabetes. Analytical methods for determination of GO and MGO in serum samples are either HPLC with UV-Vis (low sensitivity) or MS/MS (expensive) detection. These disadvantages have hampered the introduction of these biomarkers as a routine analyte for diabetes diagnostics into the clinical laboratory. In this study, we introduce a UHPLC method with fluorescence detection for the measurement of GO and MGO in serum samples by pre-column derivatization at neutral pH with 5, 6-diamino-2,4-dihydroxypyrimidine sulfate (DDP) to form lumazines. The method was validated as per FDA guidelines. Using this method, we have determined GO and MGO in a variety of animal serum samples, and for example, determined the GO and MGO concentration in adult bovine serum to be 852 ±â€¯27 and 192 ±â€¯10 nmol/L, respectively. In human serum, GO and MGO levels in non-diabetic subjects (n = 14) were determined to be 154 ±â€¯88 and 98 ±â€¯27 nmol/L, and in serum samples from subjects with diabetes (n = 14) 244 ±â€¯137 and 190 ±â€¯68 nmol/L, respectively. In addition, interference studies showed that physiological serum components did not lead to an artificial increase in the levels of GO and MGO.