Introduction: The kinetics of protein oxidation, monitored in breath, and its contribution to the whole body protein status is not well established. Objectives: To analyze protein oxidation in various metabolic conditions we developed/validated a 13C-protein oxidation breath test using low enriched milk proteins. Method/Design: 30 g of naturally labeled 13C-milk proteins were consumed by young healthy volunteers. Breath samples were taken every 10 min and 13CO2 was measured by Isotope Ratio Mass Spectrometry. To calculate the amount of oxidized substrate we used: substrate dose, molecular weight and 13C enrichment of the substrate, number of carbon atoms in a substrate molecule, and estimated CO2-production of the subject based on body surface area. Results: We demonstrated that in 255 min 20% ± 3% (mean ± SD) of the milk protein was oxidized compared to 18% ± 1% of 30 g glucose. Postprandial kinetics of oxidation of whey (rapidly digestible protein) and casein (slowly digestible protein) derived from our breath test were comparable to literature data regarding the kinetics of appearance of amino acids in blood. Oxidation of milk proteins was faster than that of milk lipids (peak oxidation 120 and 290 minutes, respectively). After a 3-day protein restricted diet (~10 g of protein/day) a decrease of 31% ± 18% in milk protein oxidation was observed compared to a normal diet. Conclusions: Protein oxidation, which can be easily monitored in breath, is a significant factor in protein metabolism. With our technique we are able to characterize changes in overall protein oxidation under various meta-bolic conditions such as a protein restricted diet, which could be relevant for defining optimal protein intake under various conditions. Measuring protein oxidation in new-born might be relevant to establish its contribution to the protein status and its age-dependent development.
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The capacity to utilize ingested protein for optimal support of protein synthesis and lean body mass is described within the paradigm of anabolic competence. Protein synthesis can be stimulated by physical exercise, however, it is not known if physical exercise affects post-exercise protein oxidation. Characterization of the driving forces behind protein oxidation, such as exercise, can contribute to improved understanding of whole body protein metabolism. The purpose of this study is to determine the effect of two levels of aerobic exercise intensity on immediate post-exercise exogenous protein oxidation. Sixteen healthy males with a mean (SD) age of 24 (4) years participated. The subjects' VO2-max was estimated with the Åstrand cycling test. Habitual dietary intake was assessed with a three-day food diary. Exogenous protein oxidation was measured by isotope ratio mass spectrometry. These measurements were initiated after the ingestion of a 30 g 13C-milk protein test drink that was followed by 330 minutes breath sample collection. On three different days with at least one week in between, exogenous protein oxidation was measured: 1) during rest, 2) after 15 minutes of aerobic exercise at 30% of VO2-max (moderate intensity), and 3) after 15 minutes of aerobic exercise at 60% of VO2-max (vigorous intensity). After vigorous intensity aerobic exercise, 31.8%±8.0 of the 30 g 13C-milk protein was oxidized compared to 26.2%±7.1 during resting condition (p = 0.012), and 25.4%±7.6 after moderate intensity aerobic exercise compared to resting (p = 0.711). In conclusion, exogenous protein oxidation is increased after vigorous intensity aerobic exercise which could be the result of an increased protein turnover rate.
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Muscle fiber-type specific expression of UCP3-protein is reported here for the firts time, using immunofluorescence microscopy
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This paper descibes a study that shows that glycogen-lowering exercise, performed the evening before an exercise bout in combination with glycogen restriction leads to a reduction of the oxidation rate of ingested glucose during moderate-intensity exercise
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RATIONALE: Disturbed protein metabolism may result in malnutrition. A non-invasive low cost clinical tool to measure protein metabolism is lacking. Explorative research (n=1) with a newly developed non-invasive 13C-protein breath test suggested a decrease in protein oxidation after a protein restricted diet. Now, we aimed to test the effect of protein restriction in more subjects, to assess sensitivity of the test.METHODS: In this exploratory study, 14 healthy male subjects (23±3 y) participated. Habitual intake was assessed by a 4-day food diary. Next, subjects were instructed to use a 4-day isocaloric protein restricted diet (0.25 g protein/kg bw/day). After an overnight fast, a 30 g naturally enriched 13C-milk protein test drink was consumed, followed by collection of breath samples up to 330 min. Protein oxidation was analyzed by Isotope Ratio Mass Spectrometry. 24-h urine was collected on day 4 of the habitual diet, and on every day of the 4-day protein restricted diet, to assess actual change in protein intake.RESULTS: After the protein restricted diet, 30.2%±7.7 of the 30 g 13C-milk protein was oxidized over 330 min, compared to 30.6%±6.2 (NS) after the subject’s habitual diet (1.4±0.3 g protein/kg bw/day). Within subjects, both increase and decrease in oxidation was found. During the 4-day protein restricted diet, urinary urea:creatinine ratio decreased by 56%±10, consistent with a reduction in protein intake of 44%±15 (g/day) and 53%±12 (g/kg bw/day), based on urea and food diary, respectively.CONCLUSIONS: The breath test shows variation within subjects and between diets, which could be related to the sensitivity of the test. We cannot explain the variation by the measured variables. Alternatively, our results may implicate that in some of our subjects, protein intake did not sufficiently decrease to levels that could alter protein metabolism.
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RATIONALE: Disturbed protein metabolism may result in malnutrition. A non-invasive low cost clinical tool to measure protein metabolism is lacking. Explorative research (n=1) with a newly developed non-invasive 13C-protein breath test suggested a decrease in protein oxidation after a protein restricted diet. Now, we aimed to test the effect of protein restriction in more subjects, to assess sensitivity of the test.METHODS: In this exploratory study, 14 healthy male subjects (23±3 y) participated. Habitual intake was assessed by a 4-day food diary. Next, subjects were instructed to use a 4-day isocaloric protein restricted diet (0.25 g protein/kg bw/day). After an overnight fast, a 30 g naturally enriched 13C-milk protein test drink was consumed, followed by collection of breath samples up to 330 min. Protein oxidation was analyzed by Isotope Ratio Mass Spectrometry. 24-h urine was collected on day 4 of the habitual diet, and on every day of the 4-day protein restricted diet, to assess actual change in protein intake.RESULTS: After the protein restricted diet, 30.2%±7.7 of the 30 g 13C-milk protein was oxidized over 330 min, compared to 30.6%±6.2 (NS) after the subject’s habitual diet (1.4±0.3 g protein/kg bw/day). Within subjects, both increase and decrease in oxidation was found. During the 4-day protein restricted diet, urinary urea:creatinine ratio decreased by 56%±10, consistent with a reduction in protein intake of 44%±15 (g/day) and 53%±12 (g/kg bw/day), based on urea and food diary, respectively.CONCLUSIONS: The breath test shows variation within subjects and between diets, which could be related to the sensitivity of the test. We cannot explain the variation by the measured variables. Alternatively, our results may implicate that in some of our subjects, protein intake did not sufficiently decrease to levels that could alter protein metabolism.
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Knowledge of the time of deposition is pivotal in forensic investigations. Recent studies show that changes in intrinsic fluorescence over time can be used to estimate the age of body fluids. These changes have been attributed to oxidative modifications caused by protein–lipid interactions. This pilot study aims to explore the impact of these modifications on body fluid fluorescence, enhancing the protein–lipid model system for age estimation. Lipid and protein oxidation markers, including protein carbonyls, dityrosine, advanced glycation end-products (AGEs), malondialdehyde (MDA), and 4-hydroxynonenal (HNE), were studied in aging semen, urine, and saliva over 21 days. Surface plasmon resonance imaging (SPRi), enzyme-linked immunosorbent assay (ELISA), and fluorescence spectroscopy were applied. Successful detection of AGE, dityrosine, MDA, and HNE occurred in semen and saliva via SPRi, while only dityrosine was detected in urine. Protein carbonyls were measured in all body fluids, but only in saliva was a significant increase observed over time. Additionally, protein fluorescence loss and fluorescent oxidation product formation were assessed, showing significant decreases in semen and saliva, but not in urine. Although optimization is needed for accurate quantification, this study reveals detectable markers for protein and lipid oxidation in aging body fluids, warranting further investigation.
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Using stable isotope techniques, this study shows that plasma free fatty acid oxidation is not impaired during exercise in non-obese type II diabetic patients.
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Exercise is one of the external factors associated with impairment of intestinal integrity, possibly leading to increased permeability and altered absorption. Here, we aimed to examine to what extent endurance exercise in the glycogen‐depleted state can affect intestinal permeability toward small molecules and protein‐derived peptides in relation to markers of intestinal function. Eleven well‐trained male volunteers (27 ± 4 years) ingested 40 g of casein protein and a lactulose/rhamnose (L/R) solution after an overnight fast in resting conditions (control) and after completing a dual – glycogen depletion and endurance – exercise protocol (first protocol execution). The entire procedure was repeated 1 week later (second protocol execution). Intestinal permeability was measured as L/R ratio in 5 h urine and 1 h plasma. Five‐hour urine excretion of betacasomorphin‐7 (BCM7), postprandial plasma amino acid levels, plasma fatty acid binding protein 2 (FABP‐2), serum pre‐haptoglobin 2 (preHP2), plasma glucagon‐like peptide 2 (GLP2), serum calprotectin, and dipeptidylpeptidase‐4 (DPP4) activity were studied as markers for excretion, intestinal functioning and recovery, inflammation, and BCM7 breakdown activity, respectively. BCM7 levels in urine were increased following the dual exercise protocol, in the first as well as the second protocol execution, whereas 1 h‐plasma L/R ratio was increased only following the first exercise protocol execution. FABP2, preHP2, and GLP2 were not changed after exercise, whereas calprotectin increased. Plasma citrulline levels following casein ingestion (iAUC) did not increase after exercise, as opposed to resting conditions. Endurance exercise in the glycogen depleted state resulted in a clear increase of BCM7 accumulation in urine, independent of DPP4 activity and intestinal permeability. Therefore, strenuous exercise could have an effect on the amount of food‐derived bioactive peptides crossing the epithelial barrier. The health consequence of increased passage needs more in depth studies.
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