We report the use of ultra pressure liquid chromatography (UPLC), coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode to determine the advanced glycation endproduct, Nepsilon-(carboxymethyl)lysine (CML). The procedure was applied to acid hydrolyzates of protein isolated from a range of foods (milks processed at different temperatures, butter, cheese, infant formulae, bread, raw and cooked minced beef and olive oil). Highest levels of CML were determined in white bread crust (15.2 +/- 0.63 mmol/mol Lys), wholemeal bread crust (13.1 +/- 0.61 mmol/mol Lys) and evaporated full-fat milk (4.86 +/- 0.77 mmol/mol Lys). Lowest levels of CML were measured in raw minced beef beef (0.03 +/- 002 mmol/mol Lys), raw full-fat cow's milk (0.08 +/- 0.03 mmol/mol Lys) and pasteurized skimmed cow's milk (0.09 +/- 0.002 mmol/mol Lys). CML could not be detected in olive oil.

Apples are a rich source of polyphenols and fiber. A major proportion of apple polyphenols escape absorption in the small intestine and together with non-digestible polysaccharides reach the colon, where they can serve as substrates for bacterial fermentation. Animal studies suggest a synergistic interaction between apple polyphenols and the soluble fiber pectin; however, the effects of whole apples on human gut microbiota are less extensively studied. Three commercial apple varieties—Renetta Canada, Golden Delicious and Pink Lady—were digested and fermented in vitro using a batch culture colonic model (pH 5.5–6.0, 37 °C) inoculated with feces from three healthy donors. Inulin and cellulose were used as a readily and a poorly fermentable plant fiber, respectively. Fecal microbiota composition was measured by 16S rRNA gene Illumina MiSeq sequencing (V3-V4 region) and Fluorescence in Situ Hybridization. Short chain fatty acids (SCFAs) and polyphenol microbial metabolites were determined. The three apple varieties significantly changed bacterial diversity, increased Actinobacteria relative abundance, acetate, propionate and total SCFAs (p < 0.05). Renetta Canada and Golden Delicious significantly decreased Bacteroidetes abundance and increased Proteobacteria proportion and bifidobacteria population (p < 0.05). Renetta Canada also increased Faecalibacterium prausnitzii, butyrate levels and polyphenol microbial metabolites (p < 0.05). Together, these data suggest that apples, particularly Renetta Canada, can induce substantial changes in microbiota composition and metabolic activity in vitro, which could be associated with potential benefits to human health. Human intervention studies are necessary to confirm these data and potential beneficial effects.

Cancer cachexia is a muscle wasting condition that occurs in response to a malignant growth in the body. The mechanisms regulating cardiac muscle mass with cachexia are not well understood. Using the ApcMin/+ mouse model of colorectal cancer, we investigated how cachexia affects the regulation of 5′-adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling in the heart. Compared to age-matched C57BL/6 (BL6) mice, ApcMin/+ body mass and heart mass were lower at 12 (11±5 and 8±3%, respectively) and 20 weeks (26±3 and 6±4%, respectively) of age (P<0.05). Diminished heart mass in the 20-week-old ApcMin/+ mice coincided with a decreased rate of myofibrillar protein synthesis and increased AMPKα phosphorylation. Cachexia decreased mTOR phosphorylation and the phosphorylation of the mTOR substrates, S6 ribosomal protein and 4EBP1 independent of Akt activation. These changes in mTOR-related protein signaling were accompanied by modest increases in the amount of Beclin1 but not protein ubiquitination or cardiomyocyte apoptosis. Taken together, these data suggest that loss of cardiac mass during cachexia progression in the ApcMin/+ mouse is associated with an Akt-independent suppression of anabolic signaling and evidence of increased autophagy.

Cysteine is arguably the most reactive amino acid in protein. A wide range of cysteine derivatives is formed in vivo, resulting from oxidation, nitrosation, alkylation and acylation reactions. This review describes succination of proteins, an irreversible chemical modification of cysteine by the Krebs cycle intermediate, fumarate, yielding S-(2-succinyl)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane and develop in concert with mitochondrial and oxidative stress in diabetes. Increased succination of glyceraldehyde-3-phosphate dehydrogenase explains the loss in specific activity of this enzyme in muscle of streptozotocin-diabetic rats and increased succination of adiponectin may explain the decreased secretion of adiponectin from adipose tissue in type 2 diabetes. In addition to GAPDH and adiponectin, other succinated proteins identified in adipocytes include cytoskeletal proteins (tubulin, actin) and chaperone proteins in the endoplasmic reticulum. Succination of adipocyte protein in vitro is inhibited by uncouplers of oxidative phosphorylation and by inhibitors of ER stress. 2SC serves as a biomarker of mitochondrial stress and recent studies suggest that succination is the mechanistic link between mitochondrial and ER stress in diabetes.

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs) form when proteins are heated with reducing sugar or lipid. N(epsilon)-(Carboxymethyl)lysine (CML) is the most commonly studied AGE/ALE in foods, but the relative importance of dietary sugar and lipid as its precursors is uncertain. The aim of this study was to determine the relative amounts of CML formed from fatty acid and glucose in a model food system. Model systems were prepared by heating casein (3.2%) with glucose or fatty acid (oleic, linoleic, linolenic, or arachidonic acid) (200 mM) or a mixture of glucose and linolenic acid (200 mM of each precursor) at 95 degrees C for up to 8 h. CML was determined by ultrapressure liquid chromatography-tandem mass spectrometry. The amount of CML formed from casein and glucose incubated at 95 degrees C for 8 h was 15-fold higher than that obtained when casein was heated with arachidonic acid under the same conditions. However, the loss of lysine in the casein-arachidonic acid incubations was 83% compared to 54% loss in the casein-glucose incubations. The loss of lysine in casein-fatty acid model systems increased with degree of unsaturation of the fatty acid. The formation of lipid peroxidation products during oxidation of fatty acids might be a potent factor for loss of lysine in the casein-fatty acid systems.