Addition of the anti-CML antibody did, however, not completely diminish the TNF- secretion

Addition of the anti-CML antibody did, however, not completely diminish the TNF- secretion. protein matrix and individual AGEs. It was ensured that all samples did not contain endotoxin concentrations 0.06 EU/mL. The dietary AGEs induced TNF-alpha secretion of human macrophage-like cells. This effect was decreased by the Crassicauline A addition of N()-carboxymethyllysine (CML)-antibodies or a receptor for advanced glycation endproducts (RAGE) antagonist. None of the individual AGEs induce any TNF-alpha, indicating that AGEs should be bound to proteins to exert an inflammatory reaction. These findings show that dietary AGEs directly stimulate the inflammatory response of human innate immune cells and help us define the risk of regular consumption of AGE-rich food products on human health. = 0 min) to 50% A (= 4 min.) followed by a 1 min. equilibration at the initial conditions. The column was kept at a constant temperature of 45 C. A Quattro Premier triple quadrupole mass spectrometer (Waters) was connected to the UPLC was operated in positive electrospray ionization (ESI+). The capillary voltage was set to Crassicauline A 3.0 kV, and the source and desolvation temperatures were 120 C and 450 C, respectively. The cone and desolvation gas flow were set at 100 L/h and 600 L/h, respectively. Compound-specific cone voltages and collision energies can be found in Table S1. Quantification was performed using an internal standard approach and nine-point calibration curves. MG-H1-d3 was used as Crassicauline A internal standard for the quantification of pentosidine. Quantification was performed using the precursor?product ion multiple reaction monitoring (MRM) transitions reported in Table S1. Instrumental detection and quantitation limits (IDL and IQL, respectively) were determined as the concentration from a peak with a signal-to-noise ratio of 3 and 10, respectively, in a standard chromatogram. Method detection and quantitation limits (MDL and MQL, respectively) were determined for AGEs detected in sample extracts as the concentration from a peak with a signal-to-noise ratio of 3 and 10, respectively, in a sample chromatogram. For pentosidine (not detected in the samples) the MDL and MQL were determined using a value of 3 and 10 times the noise at the retention time of pentosidine as peak area. Compound-specific detection and quantification limits are listed in Table S2. The arithmetic mean recovery ( SE) was 113 2% for CML-d2, 104 2% for CEL-d4, and 33 1% for MG-H1-d3. The relative differences observed between duplicate analyses were SCNN1A 12%, 7%, and 15% for CML, CEL, and MG-H1, respectively. 2.6. Cell Culture and Exposure THP-1 monocytes (ATCC, TIB- 202), cultured in RPMI 1640 with L-glutamine, Hepes and phenol red (Gibco, Thermo Scientific, Waltham, MA, USA) supplemented with 10% ( 0.05. Significance is indicated as: * = 0.05; ** = 0.01; *** = 0.0013. 3. Results 3.1. Analytical Characterization of Glycated Casein To assess the effects of AGEs on human macrophage-like cells, AGEs were made in Crassicauline A the form of glycated casein, combining casein, lactose, and glucose. In order to monitor the formation of AGEs in the glycated casein, AGE-specific fluorescence was recorded during the heating process. The fluorescence signal increased until 90 min of Crassicauline A heating. AGE-specific fluorescence did not further increase between 90 min to 120 min of heating (Figure 1). Open in a separate window Figure 1 AGE-specific fluorescence of glycated casein measured at ex = 370 nm/em = 440 nm in relation to the heating time of the three components casein from bovine milk (10 g/L), lactose (0.2 M) and glucose (11 mM). Values were corrected for baseline fluorescence and data are shown as mean SD, = 3. CML, CEL, MG-H1 and pentosidine were quantified in glycated casein using UPLC-MS/MS. Pentosidine was undetectable in the samples. The formation of CML, CEL and MG-H1 in glycated casein during the 120-min heating period is shown in Figure 2..