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.

Evidence That Differences in Fructosamine-3-Kinase Activity May Be Associated With the Glycation Gap in Human Diabetes.

The phenomenon of a discrepancy between glycated hemoglobin levels and other indicators of average glycemia may be due to many factors but can be measured as the glycation gap (GGap). This GGap is associated with differences in complications in patients with diabetes and may possibly be explained by dissimilarities in deglycation in turn leading to altered production of advanced glycation end products (AGEs). We hypothesized that variations in the level of the deglycating enzyme fructosamine-3-kinase (FN3K) might be associated with the GGap. We measured erythrocyte FN3K concentrations and enzyme activity in a population dichotomized for a large positive or negative GGap. FN3K protein was higher and we found a striking threefold greater activity (323%) at any given FN3K protein level in the erythrocytes of the negative-GGap group compared with the positive-GGap group. This was associated with lower AGE levels in the negative-GGap group (79%), lower proinflammatory adipokines (leptin-to-adiponectin ratio) (73%), and much lower prothrombotic PAI-1 levels (19%). We conclude that FN3K may play a key role in the GGap and thus diabetes complications such that FN3K may be a potential predictor of the risk of diabetes complications. Pharmacological modifications of its activity may provide a novel approach to their prevention.

Lysine-Derived Protein-Bound Heyns Compounds in Bakery Products.

Fructose and dicarbonyl compounds resulting from fructose in heated foods have been linked to pathophysiological pathways of several metabolic disorders. Up to now, very little has been known about the Maillard reaction of fructose in food. Heyns rearrangement compounds (HRCs), the first stable intermediates of the Maillard reaction between amino components and fructose, have not yet been quantitated as protein-bound products in food. Therefore, the HRCs glucosyllysine and mannosyllysine were synthesized and characterized by NMR. Protein-bound HRCs in cookies containing various sugars and in commercial bakery products were quantitated after enzymatic hydrolysis by RP-HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Protein-bound HRCs were quantitated for the first time in model cookies and in commercial bakery products containing honey, banana, and invert sugar syrup. Concentrations of HRCs from 19 to 287 mg/kg were found, which were similar to or exceeded the content of other frequently analyzed Maillard reaction products, such as N-ε-carboxymethyllysine (10-76 mg/kg), N-ε-carboxyethyllysine (2.5-53 mg/kg), and methylglyoxal-derived hydroimidazolone 1 (10-218 mg/kg) in the analyzed cookies. These results show that substantial amounts of HRCs form during food processing. Analysis of protein-bound HRCs in cookies is therefore useful to evaluate the Maillard reaction of fructose.

Association between Advanced Glycation End Products and Impaired Fasting Glucose: Results from the SALIA Study.

Advanced glycation end products (AGEs) may contribute to the development of type 2 diabetes and related complications, whereas their role in the early deterioration of glycaemia is unknown. While previous studies used antibody-based methods to quantify AGEs, data from tandem mass spectrometry coupled liquid chromatography (LC-MS/MS)-based measurements are limited to patients with known diabetes. Here, we used the LC-MS/MS method to test the hypothesis that plasma AGE levels are higher in individuals with impaired fasting glucose (IFG) than in those with normal fasting glucose (NFG). Secondary aims were to assess correlations of plasma AGEs with quantitative markers of glucose metabolism and biomarkers of subclinical inflammation. This study included on 60 women with NFG or IFG (n = 30 each, mean age 74 years) from the German SALIA cohort. Plasma levels of free metabolites (3-deoxyfructose, 3-deoxypentosone, 3-deoxypentulose), two hydroimidazolones, oxidised adducts (carboxymethyllysine, carboxyethyllysine, methionine sulfoxide) and Nε-fructosyllysine were measured using LC-MS/MS. Plasma concentrations of all tested AGEs did not differ between the NFG and IFG groups (all p>0.05). Associations between plasma levels of AGEs and fasting glucose, insulin and HOMA-IR as a measure of insulin resistance were weak (r between -0.2 and 0.2, all p>0.05). The association between 3-deoxyglucosone-derived hydroimidazolone with several proinflammatory biomarkers disappeared upon adjustment for multiple testing. In conclusion, plasma AGEs assessed by LC-MS/MS were neither increased in IFG nor associated with parameters of glucose metabolism and subclinical inflammation in our study. Thus, these data argue against strong effects of AGEs in the early stages of deterioration of glucose metabolism.

A new HPLC-based assay for the measurement of fructosamine-3-kinase (FN3K) and FN3K-related protein activity in human erythrocytes.

BACKGROUND: An impact on glycation, and possibly on diabetic complications, is attributed to fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) because they degrade Amadori compounds in vivo. Little is known about individual differences in FN3K-RP activity, which might contribute to an individual risk for diabetic complications. METHODS: An HPLC-based activity assay for FN3K-RP in erythrocytes with the substrate N-α-hippuryl-N-ε-psicosyllysine was developed. The activities of FN3K and FN3K-RP were also analysed in erythrocytes of 103 consecutive participants of a health-care survey amongst a high-risk group for diabetes. The potential associations of these activities with the subjects' health background (anthropometric data, glucose tolerance and HbA1c, blood lipids, history of metabolic diseases in the subjects and their families, and medication) were examined. RESULTS: The interindividual variability of FN3K-RP is less pronounced than that of FN3K [60-135 vs. 2.8-12.5 mU/g haemoglobin (Hb)]. No correlations with age, sex, body weight, blood cholesterol, or plasma glucose in an oral glucose tolerance test were observed. Subjects with kidney disease had higher activity of mainly FN3K-RP [111±15 vs. 98±18 mU/g Hb, mean±standard deviations (SDs), n=16 vs. 87, p=0.009], whereas subjects whose parents or siblings had a stroke showed lower FN3K activity (6.2±1.6 vs. 7.1±1.8 mU/g Hb, mean±SD, n=24 vs. 66, p=0.040). CONCLUSIONS: There is a likely impact of FN3K and FN3K-RP on the glycation cascade in vivo with potential positive and negative effects. The new screening method enables further studies to elucidate the function and importance of FN3K-RP.

Diagnostic performance and prognostic impact of FDG-PET in suspected recurrence of surgically treated non-small cell lung cancer.

PURPOSE: The differentiation of recurrent lung cancer and post-therapeutic changes remains a problem for radiological imaging, but FDG-PET allows biological characterisation of tissues by visualising glucose metabolism. We evaluated the diagnostic performance and prognostic impact of FDG-PET in cases of suspected relapse of lung cancer. METHODS: In 62 consecutive patients, 73 FDG-PET scans were performed for suspected recurrence after surgical therapy of lung cancer. FDG uptake by lesions was measured as the standardised uptake value (SUV). PET results were compared with the final diagnosis established by biopsy or imaging follow-up. SUV and clinical parameters were analysed as prognostic factors with respect to survival. RESULTS: FDG-PET correctly identified 51 of 55 relapses and gave true negative results in 16 of 18 remissions (sensitivity, specificity, accuracy: 93%, 89%, 92%). SUV in recurrent tumour was higher than in benign post-therapeutic changes (10.6+/-5.1 vs 2.1+/-0.6, p<0.001). Median survival was longer for patients with lower FDG uptake in recurrent tumour (SUV<11: 18 months, SUV > or = 11: 9 months, p<0.01). Long-term survival was observed mainly after surgical re-treatment (3-year survival rate 38%), even if no difference in median survival for surgical or non-surgical re-treatment was detected (11 vs 12 months, p=0.0627). For patients subsequently treated by surgery, lower FDG uptake predicted longer median survival (SUV<11: 46 months, SUV> or = 11: 3 months, p<0.001). SUV in recurrent tumour was identified as an independent prognostic factor (p<0.05). CONCLUSION: FDG-PET accurately detects recurrent lung cancer. SUV in recurrent tumour is an independent prognostic factor. FDG-PET helps in the selection of patients who will benefit from surgical re-treatment.