Phenylalanine Tracer Research Articles

Dose-Response of Myofibrillar Protein Synthesis To Ingested Whey Protein During Energy Restriction in Overweight Postmenopausal Women: A Randomized, Controlled Trial

BackgroundDiet-induced weight loss is associated with a decline in lean body mass, as mediated by an impaired response of muscle protein synthesis (MPS). The dose-response of MPS to ingested protein, with or without resistance exercise, is well characterized during energy balance but limited data exist under conditions of energy restriction in clinical populations. ObjectiveTo determine the dose-response of MPS to ingested whey protein following short-term diet-induced energy restriction in overweight, postmenopausal, women at rest and postexercise. DesignForty middle-aged (58.6±0.4 y), overweight (BMI: 28.6±0.4), postmenopausal women were randomly assigned to 1 of 4 groups: Three groups underwent 5 d of energy restriction (∼800 kcal/d). On day 6, participants performed a unilateral leg resistance exercise bout before ingesting either a bolus of 15g (ERW15, n = 10), 35g (ERW35, n = 10) or 60g (ERW60, n = 10) of whey protein. The fourth group (n = 10) ingested a 35g whey protein bolus after 5 d of an energy balanced diet (EBW35, n = 10). Myofibrillar fractional synthetic rate (FSR) was calculated under basal, fed (FED) and postexercise (FED-EX) conditions by combining an L-[ring-13C6] phenylalanine tracer infusion with the collection of bilateral muscle biopsies. ResultsMyofibrillar FSR was greater in ERW35 (0.043±0.003%/h, P = 0.013) and ERW60 (0.042±0.003%/h, P = 0.026) than ERW15 (0.032 ± 0.003%/h), with no differences between ERW35 and ERW60 (P = 1.000). Myofibrillar FSR was greater in FED (0.044 ± 0.003%/h, P < 0.001) and FED-EX (0.048 ± 0.003%/h, P < 0.001) than BASAL (0.027 ± 0.003%/h), but no differences were detected between FED and FED-EX (P = 0.732) conditions. No differences in myofibrillar FSR were observed between EBW35 (0.042 ± 0.003%/h) and ERW35 (0.043 ± 0.003%/h, P = 0.744). ConclusionA 35 g dose of whey protein, ingested with or without resistance exercise, is sufficient to stimulate a maximal acute response of MPS following short-term energy restriction in overweight, postmenopausal women, and thus may provide a per serving protein recommendation to mitigate muscle loss during a weight loss program. Trial registryclinicaltrials.gov (ID: NCT03326284).

Uptake of dietary amino acids into arterial blood during continuous enteral feeding in critically ill patients and healthy subjects

Amino acid availability is a regulatory factor of protein anabolism and is partly dependent on enteral amino acid uptake. During continuous enteral feeding, enteral amino acid uptake may vary considerably, but this has not been documented systematically. In this pragmatic study, we investigated patients in the intensive care unit (n=10) and healthy adults (n=10). The time course of essential amino acid concentrations in arterial plasma and the uptake of dietary phenylalanine were recorded during 12hours of continuous enteral feeding, using a 13C-labeled phenylalanine tracer. Plasma essential amino acid concentrations and 13C-phenylalanine enrichment reached a tentative steady state after no more than 4.5h from start of tracer infusion. There was a large intra- and inter-individual variability in both cohorts. No periodicity could be detected in the temporal variation. During continuous enteral feeding, uptake of amino acids shows large intra- and inter-individual variation. A tentative steady state of 13C-phenylalanine uptake is eventually reached. Registered at Australian New Zealand Clinical Trials Registry, trial ID ACTRN12616000593437.

Post Meal Energy Boluses Do Not Increase the Duration of Muscle Protein Synthesis Stimulation in Two Anabolic Resistant Situations.

Background: When given in the long term, whey proteins alone do not appear to be an optimal nutritional strategy to prevent or slow down muscle wasting during aging or catabolic states. It has been hypothesized that the digestion of whey may be too rapid during a catabolic situation to sustain the anabolic postprandial amino acid requirement necessary to elicit an optimal anabolic response. Interestingly, it has been shown recently that the duration of the postprandial stimulation of muscle protein synthesis in healthy conditions can be prolonged by the supplementary ingestion of a desynchronized carbohydrate load after food intake. We verified this hypothesis in the present study in two different cases of muscle wasting associated with anabolic resistance, i.e., glucocorticoid treatment and aging. Methods: Multi-catheterized minipigs were treated or not with glucocorticoids for 8 days. Muscle protein synthesis was measured sequentially over time after the infusion of a 13C phenylalanine tracer using the arterio-venous method before and after whey protein meal ingestion. The energy bolus was given 150 min after the meal. For the aging study, aged rats were fed the whey meal and muscle protein synthesis was measured sequentially over time with the flooding dose method using 13C Valine. The energy bolus was given 210 min after the meal. Results: Glucocorticoid treatment resulted in a decrease in the duration of the stimulation of muscle protein synthesis. The energy bolus given after food intake was unable to prolong this stimulation despite a simultaneous increase of insulin and glucose following its absorption. In old rats, a similar observation was made with no effect of the energy bolus on the duration of the muscle anabolic response following whey protein meal intake. Conclusions. Despite very promising observations in healthy situations, the strategy aimed at increasing muscle protein synthesis stimulation by giving an energy bolus during the postprandial period remained inefficient in our two anabolic resistance models.

Supplementing Breakfast with a Vitamin D and Leucine–Enriched Whey Protein Medical Nutrition Drink Enhances Postprandial Muscle Protein Synthesis and Muscle Mass in Healthy Older Men

Background: A promising strategy to help older adults preserve or build muscle mass is to optimize muscle anabolism through providing an adequate amount of high-quality protein at each meal.Objective: This "proof of principle" study investigated the acute effect of supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink on postprandial muscle protein synthesis and longer-term effect on muscle mass in healthy older adults.Methods: A randomized, placebo-controlled, double-blind study was conducted in 24 healthy older men [mean ± SD: age 71 ± 4 y; body mass index (in kg/m2) 24.7 ± 2.8] between September 2012 and October 2013 at the Unit of Human Nutrition, University of Auvergne, Clermont-Ferrand, France. Participants received a medical nutrition drink [test group; 21 g leucine-enriched whey protein, 9 g carbohydrates, 3 g fat, 800 IU cholecalciferol (vitamin D3), and 628 kJ] or a noncaloric placebo (control group) before breakfast for 6 wk. Mixed muscle protein fractional synthesis rate (FSR) was measured at week 0 in the basal and postprandial state, after study product intake with a standardized breakfast with the use of l-[2H5]-phenylalanine tracer methodology. The longer-term effect of the medical nutrition drink was evaluated by measurement of appendicular lean mass, representing skeletal muscle mass at weeks 0 and 6, by dual-energy X-ray absorptiometry.Results: Postprandial FSR (0-240 min) was higher in the test group than in the control group [estimate of difference (ED): 0.022%/h; 95% CI: 0.010%/h, 0.035%/h; ANCOVA, P = 0.001]. The test group gained more appendicular lean mass than the control group after 6 wk (ED: 0.37 kg; 95% CI: 0.03, 0.72 kg; ANCOVA, P = 0.035), predominantly as leg lean mass (ED: 0.30 kg; 95% CI: 0.03, 0.57 kg; ANCOVA, P = 0.034).Conclusions: Supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink stimulated postprandial muscle protein synthesis and increased muscle mass after 6 wk of intervention in healthy older adults and may therefore be a way to support muscle preservation in older people. This trial was registered at www.trialregister.nl as NTR3471.

Short-term muscle disuse lowers myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

Disuse leads to rapid loss of skeletal muscle mass and function. It has been hypothesized that short successive periods of muscle disuse throughout the lifespan play an important role in the development of sarcopenia. The physiological mechanisms underlying short-term muscle disuse atrophy remain to be elucidated. We assessed the impact of 5 days of muscle disuse on postabsorptive and postprandial myofibrillar protein synthesis rates in humans. Twelve healthy young (22 ± 1 yr) men underwent a 5-day period of one-legged knee immobilization (full leg cast). Quadriceps cross-sectional area (CSA) of both legs was assessed before and after immobilization. Continuous infusions of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine were combined with the ingestion of a 25-g bolus of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled dietary protein to assess myofibrillar muscle protein fractional synthetic rates in the immobilized and nonimmobilized control leg. Immobilization led to a 3.9 ± 0.6% decrease in quadriceps muscle CSA of the immobilized leg. Based on the l-[ring-(2)H5]phenylalanine tracer, immobilization reduced postabsorptive myofibrillar protein synthesis rates by 41 ± 13% (0.015 ± 0.002 vs. 0.032 ± 0.005%/h, P < 0.01) and postprandial myofibrillar protein synthesis rates by 53 ± 4% (0.020 ± 0.002 vs. 0.044 ± 0.003%/h, P < 0.01). Comparable results were found using the l-[1-(13)C]leucine tracer. Following protein ingestion, myofibrillar protein bound l-[1-(13)C]phenylalanine enrichments were 53 ± 18% lower in the immobilized compared with the control leg (0.007 ± 0.002 and 0.015 ± 0.002 mole% excess, respectively, P < 0.05). We conclude that 5 days of muscle disuse substantially lowers postabsorptive myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

The effects of resistance exercise training on macro‐ and micro‐circulatory responses to feeding and skeletal muscle protein anabolism in older men

Increases in limb blood flow in response to nutrition are reduced in older age. Muscle microvascular blood flow (MBF) in response to nutrition is also reduced with advancing age and this may contribute to age-related 'anabolic resistance'. Resistance exercise training (RET) can rejuvenate limb blood flow responses to nutrition in older individuals. We report here that 20 weeks of RET also restores muscle MBF in older individuals. Restoration of MBF does not, however, enhance muscle anabolic responses to nutrition. The anabolic effects of dietary protein on skeletal muscle depend on adequate skeletal muscle perfusion, which is impaired in older people. This study explores fed state muscle microvascular blood flow, protein metabolism and exercise training status in older men. We measured leg blood flow (LBF), muscle microvascular blood volume (MBV) and muscle protein turnover under post-absorptive and fed state (i.v. Glamin to double amino acids, dextrose to sustain glucose ∼7-7.5 mmol l(-1) ) conditions in two groups: 10 untrained men (72.3±1.4 years; body mass index (BMI) 26.5±1.15 kgm(2) ) and 10 men who had undertaken 20weeks of fully supervised, whole-body resistance exercise training (RET) (72.8±1.4 years; BMI 26.3±1.2 kgm(2) ). We measured LBF by Doppler ultrasound and muscle MBV by contrast-enhanced ultrasound. Muscle protein synthesis (MPS) was measured using [1, 2-(13) C2 ] leucine with breakdown (MPB) and net protein balance (NPB) by ring-[D5 ] phenylalanine tracers. Plasma insulin was measured via ELISA and indices of anabolic signalling (e.g. Akt/mTORC1) by immunoblotting from muscle biopsies. Whereas older untrained men did not exhibit fed-state increases in LBF or MBV, the RET group exhibited increases in both LBF and MBV. Despite our hypothesis that enhanced fed-state circulatory responses would improve anabolic responses to nutrition, fed-state increases in MPS (∼50-75%; P<0.001) were identical in both groups. Finally, whereas only the RET group exhibited fed-state suppression of MPB (∼-38%; P<0.05), positive NPB achieved was similar in both groups. We conclude that RET enhances fed-state LBF and MBV and restores nutrient-dependent attenuation of MPB without robustly enhancing MPS or NPB.

Tryptophan requirement of the enterally fed term infant in the first month of life.

Tryptophan not only is an amino acid essential to protein synthesis but also serves as a precursor in 2 important metabolic pathways: the serotonin and the kynurenine pathways. Tryptophan is related to sleeping patterns. The objective of the present study was to determine the tryptophan requirement of term infants using the indicator amino acid oxidation (IAAO) method with L-[1-C]phenylalanine as the indicator. Enterally fed infants were randomly assigned to tryptophan intakes ranging from 0.5 to 73 mg · kg · day as part of an elemental diet. After 1-day adaptation to the test diet, [C]bicarbonate and L-[1-C]phenylalanine tracers were given enterally. Breath samples were collected at baseline and during isotopic plateaus. The mean tryptophan requirement was determined by using the biphasic linear regression crossover analysis on the fraction of CO2 recovery from L-[1-C]phenylalanine oxidation (FCO2). Data are presented as mean ± standard deviation. A total of 30 term neonates (gestational age 39 ± 1 weeks) were studied at 9 ± 4 days. FCO2 decreased until a tryptophan intake of 15 mg · kg · day; additional increases in tryptophan intake did not affect FCO2. Mean requirement was determined to be 15 mg · kg · day. The mean tryptophan requirement for elemental formula-fed term infants is 15 mg · kg · day. This requirement is lower than the present recommended intake of 29 mg · kg · day, which is based on the average intake of a breastfed infant.

Pharmacological enhancement of leg and muscle microvascular blood flow does not augment anabolic responses in skeletal muscle of young men under fed conditions

Skeletal muscle anabolism associated with postprandial plasma aminoacidemia and insulinemia is contingent upon amino acids (AA) and insulin crossing the microcirculation-myocyte interface. In this study, we hypothesized that increasing muscle microvascular blood volume (flow) would enhance fed-state anabolic responses in muscle protein turnover. We studied 10 young men (23.2 ± 2.1 yr) under postabsorptive and fed [iv Glamin (∼10 g AA), glucose ∼7.5 mmol/l] conditions. Methacholine was infused into the femoral artery of one leg to determine, via bilateral comparison, the effects of feeding alone vs. feeding plus pharmacological vasodilation. We measured leg blood flow (LBF; femoral artery) by Doppler ultrasound, muscle microvascular blood volume (MBV) by contrast-enhanced ultrasound (CEUS), muscle protein synthesis (MPS) and breakdown (MPB; a-v balance modeling), and net protein balance (NPB) using [1,2-(13)C2]leucine and [(2)H5]phenylalanine tracers via gas chromatography-mass spectrometry (GC-MS). Indexes of anabolic signaling/endothelial activation (e.g., Akt/mTORC1/NOS) were assessed using immunoblotting techniques. Under fed conditions, LBF (+12 ± 5%, P < 0.05), MBV (+25 ± 10%, P < 0.05), and MPS (+129 ± 33%, P < 0.05) increased. Infusion of methacholine further enhanced LBF (+126 ± 12%, P < 0.05) and MBV (+79 ± 30%, P < 0.05). Despite these radically different blood flow conditions, neither increases in MPS in response to feeding (0.04 ± 0.004 vs. 0.08 ± 0.01%/h, P < 0.05) nor improvements in NPB (-4.4 ± 2.4 vs. 16.4 ± 5.7 nmol Phe·100 ml leg(-1)·min(-1), P < 0.05) were affected by methacholine infusion (MPS 0.07 ± 0.01%/h; NPB 24.0 ± 7.7 nmol Phe·100 ml leg(-1)·min(-1)), whereas MPB was unaltered by either feeding or infusion of methacholine. Thus, enhancing LBF/MBV above that occurring naturally with feeding alone does not improve muscle anabolism.

Effects of leucine and its metabolite β‐hydroxy‐β‐methylbutyrate on human skeletal muscle protein metabolism

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses–fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is ‘sensed’ have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-13C2]Leu and [2H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A–V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70%vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling ≤90 min vs. ≤30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; −57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Comparison of different mass spectrometry techniques in the measurement of L‐[ring‐13C6]phenylalanine incorporation into mixed muscle proteins

Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L-[ring-(13)C(6)]phenylalanine and a bolus dose of L-[ring-(13)C(6)]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC-combustion-isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring-(13)C(6)]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R(2)= 0.9962 and R(2) = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-(13)C(6)]phenylalanine tracer enrichment in low abundance and in small quantity samples.

A novel stable isotope tracer method to measure muscle protein fractional breakdown rate during a physiological non-steady-state condition

The measurement of the fractional breakdown rate (FBR) of muscle proteins during physiological non-steady state of amino acids (AAs) presents some challenges. Therefore, the goal of the present experiment was to modify the bolus stable isotope tracer injection approach to determine both fractional synthesis rate (FSR) and FBR of leg muscle protein during a physiological non-steady state of AAs. The approach uses the traditional precursor-product principle but is modified with the assumption that inward transport of AAs is proportional to their plasma concentrations. The FBR value calculated from the threonine tracer served as a reference to evaluate the validity of the FBR measurement from the phenylalanine tracer, which was under a non-steady-state condition due to the concomitant injection of unlabeled phenylalanine. Plasma phenylalanine concentration increased more than fourfold after the bolus injection, and thereafter it decreased exponentially, whereas the threonine concentration remained stable. FBR values were similar with the two tracers [0.133 ± 0.003 and 0.148 ± 0.003%/h (means ± SE) for the phenylalanine and threonine tracers, respectively, P > 0.05]. In addition, FSR values for the two tracers were similar (0.069 ± 0.002 and 0.067 ± 0.001%/h for the phenylalanine and threonine tracers, respectively, P > 0.05), indicating that the traditional FSR approach can also be used in the non-steady state. Accordingly, net balance (NB) values were similar (-0.065 ± 0.002 and -0.081 ± 0.002%/h for the phenylalanine and threonine tracers, respectively, P > 0.05). This new method of measuring muscle protein FBR during physiological non-steady state gives reliable results and allows simultaneous measurement of muscle protein FSR and thus a calculation of NB.

Whole body protein kinetics during hypocaloric and normocaloric feeding in critically ill patients

IntroductionOptimal feeding of critically ill patients in the ICU is controversial. Existing guidelines rest on rather weak evidence. Whole body protein kinetics may be an attractive technique for assessing optimal protein intake. In this study, critically ill patients were investigated during hypocaloric and normocaloric IV nutrition.MethodsNeurosurgical patients on mechanical ventilation (n = 16) were studied during a 48-hour period. In random order 50% and 100% of measured energy expenditure was given as IV nutrition during 24 hours, corresponding to hypocaloric and normocaloric nutrition, respectively. At the end of each period, whole body protein turnover was measured using d5-phenylalanine and 13C-leucine tracers.ResultsThe phenylalanine tracer indicated that whole-body protein synthesis was lower during hypocaloric feeding, while whole-body protein degradation and amino acid oxidation were unaltered, which resulted in a more negative protein balance, namely −1.9 ± 2.1 versus −0.7 ± 1.3 mg phenylalanine/kg/h (P = 0.014). The leucine tracer indicated that whole body protein synthesis and degradation and amino acid oxidation were unaltered, but the protein balance was negative during hypocaloric feeding, namely −0.3 ± 0.5 versus 0.6 ± 0.5 mg leucine/kg/h (P < 0.001).ConclusionIn the patient group studied, hypocaloric feeding was associated with a more negative protein balance, but the amino acid oxidation was not different. The protein kinetics measurements and the study’s investigational protocol were useful for assessing the efficacy of nutrition support on protein metabolism in critically ill patients.

Methionine requirement of the enterally fed term infant in the first month of life in the presence of cysteine

The essential amino acid methionine can be used for protein synthesis but also serves as a precursor for homocysteine and cysteine. The objective of this study was to determine the minimal obligatory methionine requirement of infants in the presence of excess cysteine (91 mg ⋅ kg(-1) ⋅ d(-1)) by using the indicator amino acid oxidation (IAAO) method with l-[1-(13)C]phenylalanine as the indicator. Fully enterally fed term infants <1 mo of age were randomly assigned to methionine intakes that ranged from 3 to 59 mg ⋅ kg(-1) ⋅ d(-1) as part of an elemental formula. After 1 d of adaptation to the test diet, [(13)C]bicarbonate and l-[1-(13)C]phenylalanine tracers were given enterally. Breath samples were collected at baseline and during isotopic plateaus. The mean methionine requirement was determined by using biphasic linear regression crossover analysis on the fraction of (13)CO(2) recovery from l-[1-(13)C]phenylalanine oxidation (F(13)CO(2)). Data are presented as means ± SDs. Thirty-three neonates (gestational age: 39 ± 1 wk) were studied at 13 ± 6 d. With increasing methionine intakes, F(13)CO(2) decreased until a methionine intake of 38 mg ⋅ kg(-1) ⋅ d(-1); additional increases in methionine intake did not affect F(13)CO(2). The mean methionine requirement was determined at 38 mg ⋅ kg(-1) ⋅ d(-1), and the upper and lower CIs were 48 and 27 mg ⋅ kg(-1) ⋅ d(-11), respectively (P < 0.0001, r(2) = 0.59). Although the current recommended methionine intake of 28 mg ⋅ kg(-1) ⋅ d(-1) is within the CIs of our study, the estimated mean requirement is substantially higher. However, most of the infant formulas provide a methionine intake of 49-80 mg ⋅ kg(-1) ⋅ d(-1), which is above the upper CI of our study. This trial was registered at www.trialregister.nl as NTR1610.

Effect of postdevelopmental myostatin depletion on myofibrillar protein metabolism

It is unclear whether the muscle hypertrophy induced by loss of myostatin signaling in mature muscles is maintained only by increased protein synthesis or whether reduced proteolysis contributes. To address this issue, we depleted myostatin by activating Cre recombinase for 2 wk in mature mice in which Mstn exon 3 was flanked by loxP sequences. The rate of phenylalanine tracer incorporation into myofibrillar proteins was determined 2, 5, and 24 wk after Cre activation ended. At all of these time points, myostatin-deficient mice had increased gastrocnemius and quadriceps muscle mass (≥27%) and increased myofibrillar synthesis rate per gastrocnemius muscle (≥19%) but normal myofibrillar synthesis rates per myofibrillar mass or RNA mass. Mean fractional myofibrillar degradation rates (estimated from the difference between rate of synthesis and rate of change in myofibrillar mass) and muscle concentrations of free 3-methylhistidine (from actin and myosin degradation) were unaffected by myostatin knockout. Overnight food deprivation reduced myofibrillar synthesis and ribosomal protein S6 phosphorylation and increased concentrations of 3-methylhistidine, muscle RING finger-1 mRNA, and atrogin-1 mRNA. Myostatin depletion did not affect these responses to food deprivation. These data indicate that maintenance of the muscle hypertrophy caused by loss of myostatin is mediated by increased protein synthesis per muscle fiber rather than suppression of proteolysis.

The significance of d-isomers in stable isotope studies in humans is dependent on the age of the subject and the amino acid tracer

d-Amino acids ( d-AAs) in stable isotope tracers may result in erroneous estimates of enrichment, particularly if urine is used as a surrogate for plasma enrichment. Previous studies suggest that a d-AA content of less than 0.2% will not result in significant error in studies with adult humans. To describe the effects of d-AA content of less than 0.2%, in 3 different AA tracers, on isotope enrichment in urine and plasma, arginine, proline, and phenylalanine (Phe) tracers were given enterally to human neonates. Enrichment was measured in urine and plasma using chiral chromatography and tandem mass spectrometry. The Phe tracer was also given parenterally to human neonates and enterally to children and adults to further characterize the d-AA effect. All isotopes had a confirmed d-AA content of less than 0.2%. Labeled d-arginine resulted in an overestimate for enrichment of 20% in plasma and 87% in urine. A smaller effect was seen for d-Phe, which resulted in a 5% overestimate for plasma and 40% in urine. d-Proline had no significant effect. Using the same Phe tracer, a developmental effect was found, with a reduction in the overestimate in children compared with infants and no effect on enrichment in adults. Investigators using commercially produced, stable isotope–labeled AAs need to be aware that there is no safe level of d contamination; a d-AA content less than 0.2% may result in significant overestimate for enrichment, even in plasma, for infants and children. This source of error can be avoided by the use of chiral chromatography.

Comparison of A‐V and precursor‐product methods for measuring muscle protein breakdown in burned patients

We have recently developed a new bolus precursor‐product method for measuring protein synthesis and breakdown simultaneously. The new method is an attractive alternative to our previously developed A‐V balance method because it does not require femoral lines or measurement of blood flow. Although we have previously shown that the two methods (A‐V vs precursor‐product) for measuring protein breakdown agree in rabbits, the new method has not been validated in humans. The purpose of this study was to compare muscle protein fractional breakdown rates (FBR) using the two methods in humans simultaneously. Results from the A‐V balance method were converted to units %/h with estimates of muscle protein bound pool size. Five burn patients not on anabolic treatments were studied at 1 and 3 weeks postburn using both the A‐V balance method (using 13C4 threonine tracers) and the new precursor‐product method (using 15N and 13C6 phenylalanine tracers). FBR was similar for A‐V (1 week: 0.29 ± 0.13%/h, n=3; 3 weeks: 0.29 ± 0.11%/h, n=5) and bolus (1 week: 0.24 ± 0.05 %/h, n=5; 3 weeks: 0.29 ± 0.05 %/h, n=4) methods. Comparable results are obtained with the new method using either arterial or peripheral venous enrichment. The new precursor‐product bolus method gives similar results to the A‐V balance method without the use of femoral catheters or measurement of blood flow.

Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

The goal of this study was to discover whether using different tracers affects the measured rate of muscle protein synthesis in human muscle. We therefore measured the mixed muscle protein fractional synthesis rate (FSR) in the quadriceps of older adults during basal, postabsorptive conditions and mixed meal feeding (70 mg protein x kg fat-free mass(-1) x h(-1) x 2.5 h) by simultaneous intravenous infusions of [5,5,5-(2)H(3)]leucine and either [ring-(13)C(6)]phenylalanine or [ring-(2)H(5)]phenylalanine and analysis of muscle tissue samples by gas chromatography-mass spectrometry. Both the basal FSR and the FSR during feeding were approximately 20% greater (P < 0.001) when calculated from the leucine labeling in muscle tissue fluid and proteins (fasted: 0.063 +/- 0.005%/h; fed: 0.080 +/- 0.007%/h) than when calculated from the phenylalanine enrichment data (0.051 +/- 0.004 and 0.066 +/- 0.005%/h, respectively). The feeding-induced increase in the FSR ( approximately 20%; P = 0.011) was not different with leucine and phenylalanine tracers (P = 0.69). Furthermore, the difference between the leucine- and phenylalanine-derived FSRs was independent of the phenylalanine isotopomer used (P = 0.92). We conclude that when using stable isotope-labeled tracers and the classic precursor product model to measure the rate of muscle protein synthesis, absolute rates of muscle protein FSR differ significantly depending on the tracer amino acid used; however, the anabolic response to feeding is independent of the tracer used. Thus different precursor amino acid tracers cannot be used interchangeably for the evaluation of muscle protein synthesis, and data from studies using different tracer amino acids can be compared qualitatively but not quantitatively.

The daily valine requirement of healthy adult Indians determined by the 24-h indicator amino acid balance approach

The 1985 FAO/WHO/UNU requirement for valine was set at 10 mg x kg(-1) x d(-1) on the basis of nitrogen balance studies carried out in Western subjects. It is likely that the requirement is higher, however, because the requirement of another branched-chain amino acid, leucine, was found to be about 3 times as high (40 mg x kg(-1) x d(-1)) as the 1985 FAO/WHO/UNU value (14 mg x kg(-1) x d(-1)). We assessed the valine requirement in healthy, well-nourished Indians by using 7 test valine intakes (5, 10, 15, 20, 25, 30, and 35 mg x kg(-1) x d(-1)) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) method, with phenylalanine as the indicator amino acid, while maintaining leucine intake at 40 mg x kg(-1) x d(-1). Eighteen healthy, well-nourished Indian men were studied during each of 3 randomly assigned 7-d diet periods supplying valine intakes that were equally placed on either side of a putative mean valine requirement of 20 mg x kg(-1) x d(-1). Twenty-four-hour IAAO and 24-h IAAB were measured on day 7 by use of a 24-h [13C]phenylalanine tracer infusion. The breakpoint in the relation between these values and the valine intake was determined. Two-phase linear regression of daily phenylalanine oxidation or balance against valine intake estimated a breakpoint in the response curve at a valine intake of 17 mg x kg(-1) x d(-1) (95% Fieller's CI: 11, > 35 and 11, 28 mg x kg(-1) x d(-1), respectively). From the 24-h IAAO/IAAB approach, a mean valine requirement of 17 mg x kg(-1) x d(-1) is proposed for healthy, well-nourished Indian adults.

The daily valine requirement of healthy adult Indians determined by the 24-h indicator amino acid balance approach

Background: The 1985 FAO/WHO/UNU requirement for valine was set at 10 mg · kg−1 · d−1 on the basis of nitrogen balance studies carried out in Western subjects. It is likely that the requirement is higher, however, because the requirement of another branched-chain amino acid, leucine, was found to be about 3 times as high (40 mg · kg−1 · d−1) as the 1985 FAO/WHO/UNU value (14 mg · kg−1 · d−1). Objective: We assessed the valine requirement in healthy, well-nourished Indians by using 7 test valine intakes (5, 10, 15, 20, 25, 30, and 35 mg · kg−1 · d−1) and the 24-h indicator amino acid oxidation (24-h IAAO) and balance (24-h IAAB) method, with phenylalanine as the indicator amino acid, while maintaining leucine intake at 40 mg · kg−1 · d−1. Design: Eighteen healthy, well-nourished Indian men were studied during each of 3 randomly assigned 7-d diet periods supplying valine intakes that were equally placed on either side of a putative mean valine requirement of 20 mg · kg−1 · d−1. Twenty-four–hour IAAO and 24-h IAAB were measured on day 7 by use of a 24-h [13C]phenylalanine tracer infusion. The breakpoint in the relation between these values and the valine intake was determined. Results: Two-phase linear regression of daily phenylalanine oxidation or balance against valine intake estimated a breakpoint in the response curve at a valine intake of 17 mg · kg−1 · d−1 (95% Fieller’s CI: 11, > 35 and 11, 28 mg · kg−1 · d−1, respectively). Conclusion: From the 24-h IAAO/IAAB approach, a mean valine requirement of 17 mg · kg−1 · d−1 is proposed for healthy, well-nourished Indian adults.

Phenylalanine Kinetics Differ between Formula-Fed and Human Milk–Fed Preterm Infants

Infants fed casein-dominant formulas have higher plasma phenylalanine and tyrosine concentrations than those fed mother's milk. Conversely, elevated plasma threonine concentrations are observed in infants fed whey-dominant formulas. We recently showed that formula-fed preterm infants have a lower capacity to degrade threonine than do preterm infants fed mother's milk. We hypothesized that these same infants (n = 18) would differ in their catabolism of phenylalanine in response to phenylalanine loads provided by formulas with increasing casein content of formulas (whey:casein 60:40, 40:60, and 20:80) compared with preterm infants fed mother's milk. Plasma phenylalanine concentrations significantly rose (49, 46, 79 micromol . L(-1) for whey:casein 60:40, 40:60, and 20:80, respectively, pooled SD 8, P < 0.05); and plasma phenylalanine concentrations in infants fed mother's milk were low (40 +/- 4 micromol . L(-1)). Using [1-(13)C]phenylalanine tracer and (13)CO(2) production in breath we found that although there was a significant positive relation between phenylalanine oxidation and phenylalanine intake in formula-fed infants (r(2) = 0.43, P = 0.03), these infants were not able to increase their oxidation of phenylalanine enough to prevent a significant rise in plasma phenylalanine when fed the 20:80 formula. Compared to infants fed mother's milk, formula-fed infants had significantly lower phenylalanine oxidation (39.1 vs. 30.7% of phenylalanine intake, respectively, P < 0.05). We conclude that one of the mechanisms for the differences in plasma amino acid concentration between formula-fed and mother's milk-fed preterm infants may be in vivo down-regulated catabolism of 2 important essential amino acids (phenylalanine in addition to threonine) in formula-fed preterm infants.