Protein Kinetics Research Articles

Measuring Protein Turnover in the Field: Implications for Military Research

Protein turnover reflects the continual synthesis and breakdown of body proteins, and can be measured at a whole-body (i.e. aggregated across all body proteins) or tissue (e.g. skeletal muscle only) level using stable isotope methods. Evaluating protein turnover in free-living environments, such as military training, can help inform protein requirements. We undertook a narrative review of published literature with the aim of reviewing the suitability of, and advancements in, stable isotope methods for measuring protein turnover in field research. The 2 primary approaches for measuring protein turnover are based on precursor- and end-product methods. The precursor method is the gold-standard for measuring acute (over several hours) skeletal muscle protein turnover, whereas the end-product method measures chronic (over several weeks) skeletal muscle protein turnover and provides the opportunity to monitor free-living activities. Both methods require invasive procedures such as the infusion of amino acid tracers and muscle biopsies to assess the uptake of the tracer into tissue. However, the end-product method can also be used to measure acute (over 9-24 h) whole-body protein turnover noninvasively by ingesting 15N-glycine, or equivalent isotope tracers, and collecting urine samples. The end-product method using 15N-glycine is a practical method for measuring whole-body protein turnover in the field over short (24 h) time frames and has been used effectively in recent military field research. Application of this method may improve our understanding of protein kinetics during conditions of high physiological stress in free-living environments such as military training.

The Photosensitizer Octakis(cholinyl)zinc Phthalocyanine with Ability to Bind to a Model Spike Protein Leads to a Loss of SARS-CoV-2 Infectivity In Vitro When Exposed to Far-Red LED.

Photodynamic inactivation of pathogenic microorganisms can be successfully used to eradicate pathogens in localized lesions, infected liquid media, and on various surfaces. This technique utilizes the photosensitizer (PS), light, and molecular oxygen to produce reactive oxygen species that kill pathogens. Here, we used the PS, water soluble octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+), to inactivate an initial 4.75–5.00 IgTCID50/mL titer of SARS-CoV-2, thereby preventing viral infection when tested in Vero E6 cell cultures. Zn-PcChol8+ in a minimally studied concentration, 1 µM and LED 3.75 J/cm2, completely destroyed the infectivity of SARS-CoV-2. To detect possible PS binding sites on the envelope of SARS-CoV-2, we analyzed electrostatic potential and simulated binding of Zn-PcChol8+ to the spike protein of this coronavirus by means of Brownian dynamics software, ProKSim (Protein Kinetics Simulator). Most of the Zn-PcChol8+ molecules formed clusters at the upper half of the stalk within a vast area of negative electrostatic potential. Positioning of the PS on the surface of the spike protein at a distance of no more than 10 nm from the viral membrane may be favorable for the oxidative damage. The high sensitivity of SARS-CoV-2 to photodynamic inactivation by Zn-PcChol8+ is discussed with respect to the application of this PS to control the spread of COVID-19.

Long-term repeatability of cartilage oligomeric matrix protein kinetics in response to walking stress

Purpose: The concentration of articular cartilage blood markers and their load induced changes can serve as surrogate parameter for investigating articular cartilage metabolism and may be useful for detecting early changes in cartilage homeostasis and ultimately predicting future structural change. One of these markers is serum cartilage oligomeric matrix protein (sCOMP), a cartilage constituent that is involved in collagen fibril organization and may represent cartilage turnover or degeneration.

RNA-binding protein kinetics

KIN-CLIP allows determination of binding and dissociation kinetics of RNA-binding proteins in cells.

β-Lactoglobulin Is Insulinotropic Compared with Casein and Whey Protein Ingestion during Catabolic Conditions in Men in a Double-Blinded Randomized Crossover Trial.

Muscle loss during acute infectious disease is mainly triggered by inflammation, immobilization, and malnutrition. The objective was to compare muscle protein kinetics and metabolism following ingestion of the dairy protein supplements β-lactoglobulin (BLG), casein (CAS), and whey (WHE) during controlled catabolic conditions. We used a randomized crossover design (registered at clinicaltrials.gov as NCT03319550) to investigate 9 healthy male participants [age: 20-40 y; BMI (in kg/m2) 20-30] who were randomly assigned servings of BLG, CAS, or WHE (0.6g protein/kg, one-third as bolus and two-thirds as sip every 20min) on 3 separate occasions separated by ∼6-8 wk. The participants received an infusion of lipopolysaccharide (1ng/kg) combined with 36h of fasting and bed rest before each study day, mimicking a clinical catabolic condition. The forearm model and isotopic tracer techniques were used to quantify muscle protein kinetics. Muscle biopsy specimens were obtained and intramyocellular signaling investigated using Western blot. BLG, CAS, and WHE improved the net balance of phenylalanine (NBphe) from baseline with ∼75% (P<0.001) with no difference between interventions (primary outcome, P<0.05). No difference in rates of appearance and disappearance of phenylalanine or in intramyocellular signaling activation was found between interventions (secondary outcomes). The incremental AUC for serum insulin was 62% higher following BLG compared with CAS (P <0.001) and 30% higher compared with WHE (P=0.002), as well as 25% higher in WHE compared with CAS (P=0.006). Following BLG consumption, plasma concentrations of glucose-dependent insulinotropic peptide (GIP) increased 70% compared with CAS (P=0.001) and increased 34% compared with WHE (P=0.06). No significant difference was found between WHE and CAS (P=0.12). BLG, WHE, and CAS have similar effects on muscle in young male participants during catabolic conditions. BLG showed specific, possibly GIP-dependent, insulinotropic properties, which may have future clinical implications.

C-reactive protein in spinal surgery: more predictive than prehistoric

PurposeIn spinal surgery, surgical site infections (SSI) after dorsal spondylodesis lead to severe short- and long-term complications. Despite various clinical and serological evidence, the detection of a postoperative SSI remains crucial. In this retrospective cohort study, we determined the prognostic value of C-reactive protein (CRP) kinetics after open reduction and dorsal spondylodesis in the development of a SSI.MethodsWe retrospectively analyzed 192 patients from 2016 to 2018 undergoing open reduction and dorsal spondylodesis with and without SSI for 20 days at a level-I trauma center and assessed their serological and clinical characteristics.ResultsOn day 7 and 8 after surgery, patients who developed a SSI displayed significantly higher CRP levels. A second peak after the initial maximum of CRP and a restricted failure to decline as well as a maximum CRP of more than 225 mg/l predict an infectious complication with a sensitivity of 92.9%, and a specificity of 78.2%. A binary logistic regression leads to 85.7% and 69.7%, respectively. A one-phase decay exponential regression can predict 75.6% of the variance after the initial peak of CRP.ConclusionOur study demonstrates a high value of postoperative CRP kinetics in SSI detection after dorsal spondylodesis. Moreover, we observed typical CRP levels with a specific course as indicative predictors that may facilitate an early SSI detection in clinical practice.

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Whole-body protein kinetic models to quantify the anabolic response to dietary protein consumption

<h2>Summary</h2> Determination of whole body rates of protein synthesis, breakdown and net balance in human subjects still has an important role in nutrition research. Quantifying the anabolic response to dietary protein intake is a particularly important application. There are different models with which to accomplish this goal, each with advantages and limitations. The nitrogen (N)-flux method in which tracer is given orally has distinct advantages in terms of lack of invasiveness. In addition, the calculated results include all aspects of whole-body protein synthesis and breakdown. However, the prolonged timeframe of the method eliminates the possibility of the "pre-post" experimental design whereby each subject serves as their own control in the evaluation of the response to a meal. Models based on the primed-constant infusion of an essential amino acid (EAA) tracer enable the determination of baseline whole-body protein kinetics within 2 h, and can quantify a dynamic change from the basal state. The greatest challenge when using an EAA model is distinguishing exogenous and endogenous sources of the tracee in the blood. One approach is to use an intrinsically-labeled protein. This method has the advantage that the exogenous tracee is clearly distinguished from endogenous tracee. On the other hand, the intrinsically-labeled protein method suffers from unmeasured dilution that is likely to cause the systematic underestimation of the rate of appearance of exogenous tracee and thus overestimate the rate of whole-body protein breakdown. Alternatively, the "bioavailability" approach estimates the contribution of exogenous tracee to the peripheral circulation from the amount of tracee ingested, the true ileal digestibility of the tracee, and the irreversible loss of tracee prior to entry into the peripheral circulation. Errors in assumed values with the bioavailability method can potentially be significant, but are not likely to result in the systematic over- or under-estimations of rates of whole-body protein synthesis and breakdown. The optimal method depends on the degree of uncertainty regarding required assumptions in a particular circumstance. With all methods, it is advisable to calculate upper and lower bounds of whole body protein kinetics, in accord with reasonable maximal and minimal assumed values. Simultaneous use of two methods requiring different assumptions can also serve to confirm the validity of single approach.

Mimicking a Cellular Environment for RNA Folding Studies

The complexity of the cellular environment is difficult to replicate in vitro. Inert macromolecules such as dextran, Ficoll, and polyethylene glycol (PEG) are common in vitro crowders, but by themselves do not reproduce in-cell biomolecular interactions. Dilute cell lysates or lysis buffer replicate sticking trends observed in cells but do not account for crowding. Recently, we demonstrated that a combination of 150 mg/ml Ficoll and 60% lysis buffer could be used as an in vitro mimic that accounts for both steric crowding and non-steric sticking effects on cytoplasmic protein stability and kinetics.

Extracting Protein Recruitment Kinetics to DNA Damage using qFADD.py

Organisms are constantly bombarded with DNA damaging factors from sources both within and outside their cells. In response, complex repair pathways have evolved to handle all types of DNA damage, such as double and single strand breaks and base oxidation. Different pathways invoke a variety of signaling and repair enzymes, and accurate measurement of the recruitment timescales and interplay between repair protein kinetics is paramount to mapping these damage responses. One popular method to this end is laser microirradiation.

Essential amino acid-enriched whey enhances post-exercise whole-body protein balance during energy deficit more than iso-nitrogenous whey or a mixed-macronutrient meal: a randomized, crossover study

BackgroundThe effects of ingesting varying essential amino acid (EAA)/protein-containing food formats on protein kinetics during energy deficit are undetermined. Therefore, recommendations for EAA/protein food formats necessary to optimize both whole-body protein balance and muscle protein synthesis (MPS) during energy deficit are unknown. We measured protein kinetics after consuming iso-nitrogenous amounts of free-form essential amino acid-enriched whey (EAA + W; 34.7 g protein, 24 g EAA sourced from whey and free-form EAA), whey (WHEY; 34.7 g protein, 18.7 g EAA), or a mixed-macronutrient meal (MEAL; 34.7 g protein, 11.4 g EAA) after exercise during short-term energy deficit.MethodsTen adults (mean ± SD; 21 ± 4 y; 25.7 ± 1.7 kg/m2) completed a randomized, double-blind crossover study consisting of three, 5 d energy-deficit periods (− 30 ± 3% of total energy requirements), separated by 14 d. Whole-body protein synthesis (PS), breakdown (PB), and net balance (NET) were determined at rest and in response to combination exercise consisting of load carriage treadmill walking, deadlifts, and box step-ups at the end of each energy deficit using L-[2H5]-phenylalanine and L-[2H2]-tyrosine infusions. Treatments were ingested immediately post-exercise. Mixed-muscle protein synthesis (mixed-MPS) was measured during exercise through recovery.ResultsChange (Δ postabsorptive + exercise to postprandial + recovery [mean treatment difference (95%CI)]) in whole-body (g/180 min) PS was 15.8 (9.8, 21.9; P = 0.001) and 19.4 (14.8, 24.0; P = 0.001) greater for EAA + W than WHEY and MEAL, respectively, with no difference between WHEY and MEAL. ΔPB was − 6.3 (− 11.5, − 1.18; P = 0.02) greater for EAA + W than WHEY and − 7.7 (− 11.9, − 3.6; P = 0.002) greater for MEAL than WHEY, with no difference between EAA + W and MEAL. ΔNET was 22.1 (20.5, 23.8; P = 0.001) and 18.0 (16.5, 19.5; P = 0.00) greater for EAA + W than WHEY and MEAL, respectively, while ΔNET was 4.2 (2.7, 5.6; P = 0.001) greater for MEAL than WHEY. Mixed-MPS did not differ between treatments.ConclusionsWhile mixed-MPS was similar across treatments, combining free-form EAA with whey promotes greater whole-body net protein balance during energy deficit compared to iso-nitrogenous amounts of whey or a mixed-macronutrient meal.Trial registrationClinicalTrials.gov, Identifier no. NCT04004715. Retrospectively registered 28 June 2019, first enrollment 6 June 2019

Membrane composition and lipid to protein ratio modulate amyloid kinetics of yeast prion protein.

Understanding of prion aggregation in a membrane environment may help to ameliorate neurodegenerative complications caused by the amyloid forms of prions. Here, we investigated the membrane binding-induced aggregation of yeast prion protein Sup35. Using the combination of fluorescence correlation spectroscopy (FCS) at single molecule resolution and other biophysical studies, we establish that lipid composition and lipid/protein ratio are key modulators of the aggregation kinetics of Sup35. In the presence of a zwitterionic membrane (DMPC), Sup35 exhibited novel biphasic aggregation kinetics at lipid/protein ratios ranging between 20 : 1 and 70 : 1 (termed here as the optimum lipid concentration, OLC). In ratios below (low lipid concentration, LLC) and above (ELC, excess lipid concentration) that range, the aggregation was found to be monophasic. In contrast, in the presence of negatively charged membranes, we did not observe any bi-phasic aggregation kinetics in the entire range of protein to lipid ratios. Our results provide a mechanistic description of the role that membrane concentration/composition-modulated aggregation may play in neurodegenerative diseases.

Equivalent servings of free-range reindeer promote greater net protein balance compared to commercial beef

ABSTRACT Wild game consumption has been associated with health benefits but the acute influence on human protein metabolism remains unknown. We compared feeding-induced responses of equivalent amounts of free-range reindeer (FR) and commercial beef (CB) on protein kinetics using stable isotope methodology. Seven participants (age: 40 ± 14 years; body mass index: 24 ± 3 kg/m2) completed two randomised studies, ingesting 2 oz of FR or CB. L-[ring 2H5]phenylalanine & L-[ring 2H2]tyrosine were delivered via primed, continuous intravenous infusion. Blood samples were collected during the basal period and following consumption of FR or CB. Feeding-induced changes in whole-body protein synthesis (PS), protein breakdown (PB), and net protein balance (NB) were determined via plasma sample isotope enrichment analysis by gas chromatography-mass spectrometry; plasma essential amino acid (EAA) concentrations were determined by liquid chromatography-electrospray ionisation-mass spectrometry. Plasma post-prandial EAA concentrations were higher with FR compared to CB (P < 0.05). The acute feeding-induced PS response was not different, but PB was reduced and contributed to a superior level of NB (P < 0.00001) in FR compared to CB. Our results demonstrate that FR may influence more favourable protein metabolism than CB. These data support potential health benefits of wild game onf whole-body protein. Abbreviations: BMI: body mass index; DIAAS: digestible indispensable amino acid score; CB: commercial beef; EAA: essential amino acids; FR: free-range reindeer; Ra: rate of appearance; UAF: University of Alaska Fairbanks; USDA: USA Department of Agriculture

The driving force for co-translational protein folding is weaker in the ribosome vestibule due to greater water ordering.

Interactions between the ribosome and nascent chain can destabilize folded domains in the ribosome exit tunnel's vestibule, the last 3 nm of the exit tunnel where tertiary folding can occur. Here, we test if a contribution to this destabilization is a weakening of hydrophobic association, the driving force for protein folding. Using all-atom molecular dynamics simulations, we calculate the potential-of-mean force between two methane molecules along the center line of the ribosome exit tunnel and in bulk solution. Associated methanes, we find, are half as stable in the ribosome's vestibule as compared to bulk solution, demonstrating that the hydrophobic effect is weakened by the presence of the ribosome. This decreased stability arises from a decrease in the amount of water entropy gained upon the association of the methanes. And this decreased entropy gain originates from water molecules being more ordered in the vestibule as compared to bulk solution. Therefore, the hydrophobic effect is weaker in the vestibule because waters released from the first solvation shell of methanes upon association do not gain as much entropy in the vestibule as they do upon release in bulk solution. These findings mean that nascent proteins pass through a ribosome vestibule environment that can destabilize folded structures, which has the potential to influence co-translational protein folding pathways, energetics, and kinetics.

In vitro protein and starch digestion kinetics of individual chickpea cells: from static to more complex in vitro digestion approaches

Attention has been given to more (semi-)dynamic in vitro digestion approaches ascertaining the consequences of dynamic in vivo aspects on in vitro digestion kinetics. As these often come with time and economical constraints, evaluating the consequence of stepwise increasing the complexity of static in vitro approaches using easy-to-handle digestion set-ups has been the center of our interest. Starting from the INFOGEST static in vitro protocol, we studied the influence of static gastric pH versus gradual gastric pH change (pH 6.3 to pH 2.5 in 2 h) on macronutrient digestion in individual cotyledon cells derived from chickpeas. Little effect on small intestinal proteolysis was observed comparing the applied digestion conditions. Contrary, the implementation of a gradual gastric pH change, with and without the addition of salivary α-amylase, altered starch digestion kinetics rates, and extents by 25%. The evaluation of starch and protein digestion, being co-embedded in cotyledon cells, did not only confirm but account for the interdependent digestion behavior. The insights generated in this study demonstrate the possibility of using a hypothesis-based approach to introduce dynamic factors to in vitro models while sticking to simple and cost-efficient set-ups.

C-reactive protein flare-response predicts long-term efficacy to first-line anti-PD-1-based combination therapy in metastatic renal cell carcinoma.

ObjectivesImmune checkpoint blockade (IO) has revolutionised the treatment of metastatic renal cell carcinoma (mRCC). Early C‐reactive protein (CRP) kinetics, especially the recently introduced CRP flare‐response phenomenon, has shown promising results to predict IO efficacy in mRCC, but has only been studied in second line or later. Here, we aimed to validate the predictive value of early CRP kinetics for 1st‐line treatment of mRCC with αPD‐1 plus either αCTLA‐4 (IO+IO) or tyrosine kinase inhibitor (IO+TKI).MethodsIn this multicentre retrospective study, we investigated the predictive potential of early CRP kinetics during 1st‐line IO therapy. Ninety‐five patients with mRCC from six tertiary referral centres with either IO+IO (N = 59) or IO+TKI (N = 36) were included. Patients were classified as CRP flare‐responders, CRP responders or non‐CRP responders as previously described, and their oncological outcome was compared.ResultsOur data validate the predictive potential of early CRP kinetics in 1st‐line immunotherapy in mRCC. CRP responders, especially CRP flare‐responders, had significantly prolonged progression‐free survival (PFS) compared with non‐CRP responders (median PFS: CRP flare‐responder: 19.2 months vs. responders: 16.2 vs. non‐CRP responders: 5.6, P < 0.001). In both the IO+IO and IO+TKI subgroups, early CRP kinetics remained significantly associated with improved PFS. CRP flare‐response was also associated with long‐term response ≥ 12 months.ConclusionsEarly CRP kinetics appears to be a low‐cost and easy‐to‐implement on‐treatment biomarker to predict response to 1st‐line IO combination therapy. It has potential to optimise therapy monitoring and might represent a new standard of care biomarker for immunotherapy in mRCC.

The value of sepsis biomarkers and their kinetics in the prognosis of septic shock due to bacterial infections.

In this study, we aim to explore the value of procalcitonin (PCT), C-reactive protein (CRP), and serum cholinesterase (SChE) activity kinetics as useful predictors of mortality in patients with septic shock admitted to the intensive care unit (ICU). We conducted aprospective single-blinded study in the ICU of auniversity hospital during aperiod of 1 year. Were included all patients 18 years of age or older, with confirmed septic shock. For all included patients, blood samples of septic biomarkers (PCT, SChE activity, and CRP) were obtained. Serum was collected at the day of ICU admission (day 0), the day of septic shock (day 1), then 3 and 5 days after the septic shock development. During the study period, 60 patients were included. The mean age (± SD) was 47.7 ± 19 years. There were 46 male (74%) and 14 female (26%) patients. Mean SAPSII on ICU admission was 40.7 ± 16 (median: 37), and mean SOFA score on ICU admission was 16 ± 4 (median: 7). During their ICU stay, out of the 60 included patients, 37 patients died (61%). The comparison between the 2 groups (deaths and survivors) showed that the factors associated with poor outcome were age, SOFA score on ICU admission, and the need for invasive mechanical ventilation. The day of septic shock, there was no difference in the mean concentrations in those of plasma SChE activity or in the PCT and CRP plasma between survivors and non-survivors. However, the comparison of mean plasma SChE activity, and PCT and CRP plasma concentrations (on day 3 and day 5) between survivors and non-survivors, showed asignificant difference between the 2 groups. Our study suggests that, in agroup of critically ill patients with severe septic shock, arise or no change in procalcitonin and/or CRP level, and/or adecrease or no change in SChE activity should warn the clinician about the insufficiency and/or inadequacy of the therapy. However, afall in procalcitonin and/or CRP levels, and/or arise in SChE activity were associated with afavourable prognosis. Based on our study and some other data detailed above, we recommend that an estimation of SChE acti-vity, procalcitonin, and CRP on the day of septic shock, followed by estimation within the next 72-120 h, could help the prognostic assessment of critically ill patients with septic shock. Further studies are needed to define the critical values related to mortality.

Protein absorption and kinetics in critical illness.

Timing, dose, and route of protein feeding in critically ill patients treated in an ICU is controversial. This is because of conflicting outcomes observed in randomized controlled trials (RCTs). This inconsistency between RCTs may occur as the physiology of protein metabolism and protein handling in the critically ill is substantially different from the healthy with limited mechanistic data to inform design of RCTs. This review will outline the current knowledge and gaps in the understanding of protein absorption and kinetics during critical illness. Critically ill patients, both children and adults, lose muscle protein because of substantial increases in protein degradation with initially normal, and over time increasing, protein synthesis rates. Critically ill patients appear to retain the capacity to absorb dietary protein and to use it for building body protein; however, the extent and possible benefit of this needs to be elucidated. More sophisticated methods to study protein absorption and digestion have recently been described but these have yet to be used in the critically ill. Adequate understanding of protein absorption and kinetics during critical illness will help the design of better interventional studies in the future. Because of the complexity of measuring protein absorption and kinetics in the critically ill, very few investigations are executed. Recent data using isotope-labelled amino acids suggests that critically ill patients are able to absorb enteral protein and to synthesize new body protein. However, the magnitude of absorption and anabolism that occurs, and possible benefits for the patients need to be elucidated.

Comparison of basal whole-body protein kinetics and muscle protein synthesis between young and older adults.

Significant research has been dedicated to counteracting age‐related muscle loss, but underlying mechanisms have not been clearly established. Previous research examining differences in basal protein kinetics between young and older individuals has been limited by a lack of evaluation of protein breakdown and net balance. The aim of this study was to more comprehensively examine differences in basal protein kinetics between younger and older males and females. Basal whole‐body protein kinetics and muscle fractional synthetic rate (FSR) from 91 younger (18–38 years; 52% female) and 66 older (51–81 years; 53% female) healthy adults were determined using stable isotope tracer techniques (L‐[ring‐2H5]phenylalanine and L‐[ring‐2H2]tyrosine). There were no group × sex interaction effects (p > .05). Older individuals had greater whole‐body protein synthesis (mean difference old‐young (Δ) ± SE: 28.54 ± 8.15 mg/kg LBM/hr; p = .001) and breakdown (Δ: 15.44 ± 7.33 mg/kgLBM/hr; p = .038), but a less negative net balance (Mean ± SD: Young: −31.22 ± 7.42 mg/kg LBM/hr; Old: −18.11 ± 21.60 mg/kg LBM/hr; p < .001) compared to young individuals. Basal FSR was not significantly different between young and older (Δ: 0.007 ± 0.003%/hr; p = .052). Across the age range, females had greater whole‐body protein turnover (PSΔ: 19.10 ± 7.00 mg/kgLBM/hr; PBΔ: 19.22 ± 6.31 mg/kgLBM/hr; p < .01) compared to males. Results demonstrate a difference in basal whole‐body protein kinetics between young and older adults, with older adults having a higher protein turnover rate and a less negative net balance. Across the age range, females were also found to have a higher turnover rate compared to males. Differences may represent a shift in older physiology toward mechanisms that increase the efficiency of amino acid reutilization, especially in women.

Impact of habituated dietary protein intake on fasting and postprandial whole-body protein turnover and splanchnic amino acid metabolism in elderly men: a randomized, controlled, crossover trial

Efficacy of protein absorption and subsequent amino acid utilization may be reduced in the elderly. Higher protein intakes have been suggested to counteract this. We aimed to elucidate how habituated amounts of protein intake affect the fasted state of, and the stimulatory effect of a protein-rich meal on, protein absorption, whole-body protein turnover, and splanchnic amino acid metabolism. Twelve men (65-70 y) were included in a double-blinded crossover intervention study, consisting of a 20-d habituation period to a protein intake at the RDA or a high amount [1.1 g · kg lean body mass (LBM)-1 · d-1 or >2.1 g · kg LBM-1 · d-1, respectively], each followed by an experimental trial with a primed, constant infusion of D8-phenylalanine and D2-tyrosine. Arterial and hepatic venous blood samples were obtained after an overnight fast and repeatedly 4 h after a standardized meal including intrinsically labeled whey protein concentrate and calcium-caseinate proteins. Blood was analyzed for amino acid concentrations and phenylalanine and tyrosine tracer enrichments from which whole-body and splanchnic amino acid and protein kinetics were calculated. High (compared with the recommended amount of) protein intake resulted in a higher fasting whole-body protein turnover with a resultant mean ± SEM 0.03 ± 0.01 μmol · kg LBM-1 · min-1 lower net balance (P<0.05), which was not rescued by the intake of a protein-dense meal. The mean ± SEM plasma protein fractional synthesis rate was 0.13 ± 0.06%/h lower (P<0.05) after habituation to high protein. Furthermore, higher fasting and postprandial amino acid removal were observed after habituation to high protein, yielding higher urea excretion and increased phenylalanine oxidation rates (P<0.01). Three weeks of habituation to high protein intake (>2.1 g protein · kg LBM-1 · d-1) led to a significantly higher net protein loss in the fasted state. This was not compensated for in the 4-h postprandial period after intake of a meal high in protein.This trial was registered at clinicaltrials.gov as NCT02587156.