Water-Soluble Coffee Melanoidins Inhibit Digestive Proteases.

Coffee is one of the most popular beverages around the world and its consumption contributes to the daily intake of dietary melanoidins. Despite the emerging physiological role of food melanoidins, their effect on digestive processes has not been studied so far. In this study, the activity of the gastrointestinal enzymes pepsin and trypsin was investigated in the presence of water-soluble coffee melanoidins. The gastric enzyme pepsin is only slightly affected, whereas the intestinal enzyme trypsin is severely inhibited by coffee melanoidins. The intestinal digestibility of casein was significantly inhibited by coffee melanoidins at a concentration achievable by regular coffee consumption. The inhibition of proteolytic enzymes by coffee melanoidins might decrease the nutritional value of dietary proteins.

The Effect of Free and Protein-Bound Maillard Reaction Products N-ε-Carboxymethyllysine, N-ε-Fructosyllysine, and Pyrraline on Nrf2 and NFκB in HCT 116 Cells.

SCOPE: Maillard reaction products (MRPs) are believed to interact with the receptor for advanced glycation endproducts (RAGE) and lead to a pro-inflammatory cellular response. The structural basis for this interaction is scarcely understood. This study investigates the effect of individual lysine modifications in free form or bound to casein on human colon cancer cells. METHODS AND RESULTS: Selectively glycated casein containing either protein-bound N-ε-carboxymethyllysine (CML), N-ε-fructosyllysine (FL), or pyrraline is prepared and up to 94%, 97%, and 61% of lysine modification could be attributed to CML, FL, or pyrraline, respectively. HCT 116 cells are treated with free CML, pyrraline, FL, or modified casein for 24 h. Native casein is used as control. Intracellular MRP content is analyzed by UPLC-MS/MS. Microscopic analysis of the transcription factors shows no activation of NFκB by free or protein-bound FL or CML, whereas casein containing protein-bound pyrraline activates Nrf2. RAGE expression is not influenced by free or casein-bound MRPs. Activation of Nrf2 by pyrraline-modified casein is confirmed by analyzing Nrf2 target proteins NAD(P)H dehydrogenase (quinone 1) (NQO1) and heme oxygenase-1 (HO-1). CONCLUSION: Studies on the biological effects of glycated proteins require an individual consideration of defined structures. General statements on the effect of "AGEs" in biological systems are scientifically unsound.

3-Phenyllactic Acid and Polyphenols Are Substances Enhancing the Antibacterial Effect of Methylglyoxal in Manuka Honey.

Manuka honey is known for its unique antibacterial activity, which is due to methylglyoxal (MGO). After establishing a suitable assay for measuring the bacteriostatic effect in a liquid culture with a time dependent and continuous measurement of the optical density, we were able to show that honey differs in its growth retardingeffect on Bacillus subtilis despite the same content of MGO, indicating the presence of potentially synergistic compounds. In model studies using artificial honey with varying amounts of MGO and 3-phenyllactic acid (3-PLA), it was shown that 3-PLA in concentrations above 500 mg/kg enhances the bacteriostatic effect of the model honeys containing 250 mg/kg MGO or more. It has been shown that the effect correlates with the contents of 3-PLA and polyphenols in commercial manuka honey samples. Additionally, yet unknown substances further enhance the antibacterial effect of MGO in manuka honey. The results contribute to the understanding of the antibacterial effect of MGO in honey.

Assessing Bioavailability and Bioactivity of 4-Hydroxythiazolidine-2-Thiones, Newly Discovered Glucosinolate Degradation Products Formed During Domestic Boiling of Cabbage.

Glucosinolates are plant secondary metabolites found in cruciferous vegetables (Brassicaceae) that are valued for their potential health benefits. Frequently consumed representatives of these vegetables, for example, are white or red cabbage, which are typically boiled before consumption. Recently, 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were identified as a class of thermal glucosinolate degradation products that are formed during the boiling of cabbage. Since these newly discovered compounds are frequently consumed, this raises questions about their potential uptake and their possible bioactive functions. Therefore, 3-allyl-4-hydroxythiazolidine-2-thione (allyl HTT) and 4-hydroxy-3-(4-(methylsulfinyl) butyl)thiazolidine-2-thione (4-MSOB HTT) as degradation products of the respective glucosinolates sinigrin and glucoraphanin were investigated. After consumption of boiled red cabbage broth, recoveries of consumed amounts of the degradation products in urine collected for 24 h were 18 ± 5% for allyl HTT and 21 ± 4% for 4-MSOB HTT (mean ± SD, n = 3). To investigate the stability of the degradation products during uptake and to elucidate the uptake mechanism, both an in vitro stomach and an in vitro intestinal model were applied. The results indicate that the uptake of allyl HTT and 4-MSOB HTT occurs by passive diffusion. Both compounds show no acute cell toxicity, no antioxidant potential, and no change in NAD(P)H dehydrogenase quinone 1 (NQO1) activity up to 100 µM. However, inhibition of glycogen synthase kinases-3 (GSK-3) in the range of 20% for allyl HTT for the isoform GSK-3ß and 29% for 4-MSOB HTT for the isoform GSK-3α at a concentration of 100 µM was found. Neither health-promoting nor toxic effects of 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were found in the four tested assays carried out in this study, which contrasts with the properties of other glucosinolate degradation products, such as isothiocyanates.

Fasting Concentrations and Postprandial Response of 1,2-Dicarbonyl Compounds 3-Deoxyglucosone, Glyoxal, and Methylglyoxal Are Not Increased in Healthy Older Adults.

Dicarbonyl stress describes the increased formation of 1,2-dicarbonyl compounds and is associated with age-related pathologies. The role of dicarbonyl stress in healthy aging is poorly understood. In a preliminary study, we analyzed 1,2-dicarbonyl compounds, namely 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal (MGO) in plasma of older (25 months, n = 11) and younger (5 months, n = 14) male C57BL/6J (B6) mice via ultra performance liquid chromatography tandem mass spectrometry. Postprandial 3-DG was higher in younger compared to older mice, whereas no differences were found for GO and MGO. Subsequently, in the main study, we analyzed fasting serum of older women (OW, 72.4 ± 6.14 years, n = 19) and younger women (YW, 27.0 ± 4.42 years, n = 19) as well as older men (OM, 74.3 ± 5.20 years, n = 15) and younger men (YM, 27.0 ± 3.34, n = 15). Serum glucose, insulin, 1,2-dicarbonyl concentrations, and markers of oxidative stress were quantified. In a subgroup of this cohort, an oral dextrose challenge was performed, and postprandial response of 1,2-dicarbonyl compounds, glucose, and insulin were measured. In women, there were no age differences regarding fasting 1,2-dicarbonyl concentrations nor the response after the oral dextrose challenge. In men, fasting MGO was significantly higher in OM compared to YM (median: 231 vs 158 nM, p = .006), whereas no age differences in fasting 3-DG and GO concentrations were found. Glucose (310 ± 71.8 vs 70.8 ± 11.9 min·mmol/L) and insulin (7 149 ± 1 249 vs 2 827 ± 493 min·µIU/mL) response were higher in OM compared to YM, which did not translate into a higher 1,2-dicarbonyl response in older individuals. Overall, aging does not necessarily result in dicarbonyl stress, indicating that strategies to cope with 1,2-dicarbonyl formation can remain intact.

Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases.

Generation of reactive oxygen species and related oxidants is an inevitable consequence of life. Proteins are major targets for oxidation reactions, because of their rapid reaction rates with oxidants and their high abundance in cells, extracellular tissues, and body fluids. Additionally, oxidative stress is able to degrade lipids and carbohydrates to highly reactive intermediates, which eventually attack proteins at various functional sites. Consequently, a wide variety of distinct posttranslational protein modifications is formed by protein oxidation, glycoxidation, and lipoxidation. Reversible modifications are relevant in physiological processes and constitute signaling mechanisms ("redox signaling"), while non-reversible modifications may contribute to pathological situations and several diseases. A rising number of publications provide evidence for their involvement in the onset and progression of diseases as well as aging processes. Certain protein oxidation products are chemically stable and formed in large quantity, which makes them promising candidates to become biomarkers of oxidative damage. Moreover, progress in the development of detection and quantification methods facilitates analysis time and effort and contributes to their future applicability in clinical routine. The present review outlines the most important classes and selected examples of oxidative protein modifications, elucidates the chemistry beyond their formation and discusses available methods for detection and analysis. Furthermore, the relevance and potential of protein modifications as biomarkers in the context of disease and aging is summarized.

Advanced glycation end products and protein carbonyl levels in plasma reveal sex-specific differences in Parkinson's and Alzheimer's disease.

Neurodegenerative diseases (NDD) such as Alzheimer's (AD) and Parkinson's disease (PD) are distinct clinical entities, however, the aggregation of key neuronal proteins, presumably leading to neuronal demise appears to represent a common mechanism. It has become evident, that advanced glycation end products (AGEs) trigger the accumulation of such modified proteins, which eventually contributes to pathological aspect of NDDs. Increased levels of AGEs are found in amyloid plaques in AD brains and in both advanced and early PD (incidental Lewy body disease). The molecular mechanisms by which AGE dependent modifications may modulate the susceptibility towards NDDs, however, remain enigmatic and it is unclear, whether AGEs may serve as biomarker of NDD. In the present study, we examined AGEs (CML: Carboxymethyllysine and CEL: Carboxyethyllysine), markers of oxidative stress and micronutrients in the plasma of PD and AD patients and controls. As compared to healthy controls, AD females displayed lower levels of CEL while higher levels of CML were found in AD and PD patients. A somewhat similar pattern was observed for protein carbonyls (PC), revealing lower values exclusively in AD females, whereas AD males displayed significantly higher values compared to healthy controls and PD. Sex-specific differences were also observed for other relevant markers such as malondialdehyde, 3-nitrotyrosine, γ -tocopherols, retinol, plasma proteins and α-carotene, while α-tocopherols, ß-carotene, lutein/zeaxanthin, ß-cryptoxanthin and lycopene showed no relevant association. Taken together, our study suggests yet unappreciated differences of the distribution of AGEs among the sexes in NDD. We therefore suggest to make a clear distinction between sexes when analyzing oxidative (AGEs)-related stress and carbonyl-related stress and vitamins.

Proteasomal degradation of glycated proteins depends on substrate unfolding: Preferred degradation of moderately modified myoglobin.

The Maillard reaction generates protein modifications which can accumulate during hyperglycemia or aging and may have inflammatory consequences. The proteasome is one of the major intracellular systems involved in the proteolytic degradation of modified proteins but its role in the degradation of glycated proteins is scarcely studied. In this study, chemical and structural changes of glycated myoglobin were analyzed and its degradation by 20S proteasome was studied. Myoglobin was incubated with physiological (5-10 mM), moderate (50-100 mM) and severe levels (300 mM) of glucose or methylglyoxal (MGO, 50 mM). Glycation increased myoglobin's fluorescence and surface hydrophobicity. Severe glycation generated crosslinked proteins as shown by gel electrophoresis. The concentration of advanced glycation endproducts (AGEs) N-ε-carboxymethyl lysine (CML), N-ε-carboxyethyl lysine (CEL), methylglyoxal-derived hydroimidazolone-1 (MG-H1), pentosidine and pyrraline was analyzed after enzymatic hydrolysis followed by UPLC-MS/MS. Higher concentrations of glucose increased all analyzed AGEs and incubation with MGO led to a pronounced increase of CEL and MG-H1. The binding of the heme group to apo-myoglobin was decreased with increasing glycation indicating the loss of tertiary protein structure. Proteasomal degradation of modified myoglobin compared to native myoglobin depends on the degree of glycation: physiological conditions decreased proteasomal degradation whereas moderate glycation increased degradation. Severe glycation again decreased proteolytic cleavage which might be due to crosslinking of protein monomers. The activity of the proteasomal subunit ß5 is influenced by the presence of glycated myoglobin. In conclusion, the role of the proteasome in the degradation of glycated proteins is highly dependent on the level of glycation and consequent protein unfolding.