The Anabolic Response to a Ground Beef Patty and Soy-Based Meat Alternative: A Randomized Controlled Trial.

BACKGROUND: Soy-based meat alternatives (SBMA) are becoming increasingly popular, but it is unclear if they have the same anabolic effect on skeletal muscle as animal meat. OBJECTIVE: We aimed to compare the stimulation of skeletal muscle protein synthesis by consumption of one or two 4 oz patties of SBMA with 4 oz (80%protein/20%fat) beef. METHODS: The study design was a randomized controlled trial. Participants were aged 18 to 40 years of age and in good general health with a BMI between 20 and 32 kg/m2. Stable isotope tracer methods were used (L-[ring-2H5] phenylalanine, [U-13C9-15N]- tyrosine and L-[ring-2H4] tyrosine) to quantify the response of muscle protein fractional synthetic rate to consumption of a single beef (4 oz), single SBMA (4 oz), or two 4 oz SBMA patties (8 oz). Whole-body rates of protein synthesis, breakdown and net balance, as well as plasma essential amino acid (EAA) concentrations, were also measured. RESULTS: The increase above basal in muscle protein FSR following consumption of the 4 oz beef patty (0.020 ± 0.016%/hour) was significantly greater than the increase following consumption of 4 oz SBMA (p = 0.021; 0.003 ± 0.010%/hour) but not 8 oz SBMA (p = 0.454; 0.013 ± 0.016%/hour). The maximal EAA concentration was significantly correlated (p = 0.046; r = 0.411) with the change in muscle FSR from the basal to postprandial period. In addition, the change in muscle FSR from the basal to postprandial period was significantly correlated (p = 0.046; r = 0.412) with the corresponding change in whole-body protein synthesis. CONCLUSION: Consumption of a 4 oz beef patty stimulates muscle and whole -body protein synthesis more than a 4 oz SBMA patty and similarly to 8 oz of SBMA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05197140.

The effects of whey, pea, and collagen protein supplementation beyond the recommended dietary allowance on integrated myofibrillar protein synthetic rates in older males: a randomized controlled trial.

BACKGROUND: Skeletal muscle mass is determined predominantly by feeding-induced and activity-induced fluctuations in muscle protein synthesis (MPS). Older individuals display a diminished MPS response to protein ingestion, referred to as age-related anabolic resistance, which contributes to the progression of age-related muscle loss known as sarcopenia. OBJECTIVES: We aimed to determine the impact of consuming higher-quality compared with lower-quality protein supplements above the recommended dietary allowance (RDA) on integrated MPS rates. We hypothesized that increasing total protein intake above the RDA, regardless of the source, would support higher integrated rates of myofibrillar protein synthesis. METHODS: Thirty-one healthy older males (72 ± 4 y) consumed a controlled diet with protein intake set at the RDA: control phase (days 1-7). In a double-blind, randomized controlled fashion, participants were assigned to consume an additional 50 g (2 × 25g) of whey (n = 10), pea (n = 11), or collagen (n = 10) protein each day (25 g at breakfast and lunch) during the supplemental phase (days 8-15). Deuterated water ingestion and muscle biopsies assessed integrated MPS and acute anabolic signaling. Postprandial blood samples were collected to determine feeding-induced aminoacidemia. RESULTS: Integrated MPS was increased during supplemental with whey (1.59 ± 0.11 %/d; P < 0.001) and pea (1.59 ± 0.14 %/d; P < 0.001) when compared with RDA (1.46 ± 0.09 %/d for the whey group; 1.46 ± 0.10 %/d for the pea group); however, it remained unchanged with collagen. Supplemental protein was sufficient to overcome anabolic signaling deficits (mTORC1 and rpS6), corroborating the greater postprandial aminoacidemia. CONCLUSIONS: Our findings demonstrate that supplemental protein provided at breakfast and lunch over the current RDA enhanced anabolic signaling and integrated MPS in older males; however, the source of additional protein may be an important consideration in overcoming age-related anabolic resistance. This trial was registered clinicaltrials.gov as NCT04026607.

Metabolomic and proteomic applications to exercise biomedicine.

Objectives: 'OMICs encapsulates study of scaled data acquisition, at the levels of DNA, RNA, protein, and metabolite species. The broad objectives of OMICs in biomedical exercise research are multifarious, but commonly relate to biomarker development and understanding features of exercise adaptation in health, ageing and metabolic diseases. Methods: This field is one of exponential technical (i.e., depth of feature coverage) and scientific (i.e., in health, metabolic conditions and ageing, multi-OMICs) progress adopting targeted and untargeted approaches. Results: Key findings in exercise biomedicine have led to the identification of OMIC features linking to heritability or adaptive responses to exercise e.g., the forging of GWAS/proteome/metabolome links to cardiovascular fitness and metabolic health adaptations. The recent addition of stable isotope tracing to proteomics ('dynamic proteomics') and metabolomics ('fluxomics') represents the next phase of state-of-the-art in 'OMICS. Conclusions: These methods overcome limitations associated with point-in-time 'OMICs and can be achieved using substrate-specific tracers or deuterium oxide (D2O), depending on the question; these methods could help identify how individual protein turnover and metabolite flux may explain exercise responses. We contend application of these methods will shed new light in translational exercise biomedicine.

Application of multiple stable isotopes to aid identification of the origin of regional and organic animal products in Hesse, Germany.

There is an increasing global demand for regional and organic produce. However, the growth of these markets depends on consumers' trust. Thus, novel methods must be developed to aid the verification of the origin of produce. We built on our previous study to identify the geographical origin and production method of animal-derived food products. Thirty-samples of eggs, 99 of milk, 34 of beef, and 62 of pork were collected from different regions in central Germany and analysed for their stable isotopic composition. The analysis followed a single-variate authentification approach using five isotope signatures, δ18O, δ2H, δ13C, δ15N, and δ34S. The best-performing indicators for verification of the geographical origin were δ15N and δ34S for beef; δ18O, δ2H, and δ13C for milk, and δ2H and δ13C for pork. These tracers indicated statistically significant differences among regions with the exception of pork; the results recorded for eggs were inconclusive. It was possible to distinguish between production methods by means of δ15N and δ34S (beef); all five tracers (eggs), and δ13C, δ15N, and δ34S (milk). This study demonstrated how the analysis of stable isotopes can be employed to determine the geographic region of origin and production method of animal-derived products in Germany.

Sex related differences in muscle health and metabolism in chronic obstructive pulmonary disease.

BACKGROUND & AIMS: Sex differences in muscle function and mass, dyspnea, and clinical outcomes have been observed in patients with Chronic Obstructive Pulmonary Disease (COPD) despite a similar level of airflow obstruction. Protein and amino acid metabolism is altered in COPD, however, it remains unclear whether a difference in metabolic signature exists between males and females with COPD that may explain the observed differences in muscle health and clinical outcomes. METHODS: In 234 moderate to severe COPD patients (males/females: 113/121) and 182 healthy controls (males/females: 77/105), we assessed, besides presence of comorbidities and clinical features, muscle function by handgrip and leg dynamometry, and body composition by dual-energy x-ray absorptiometry. In the postabsorptive state, a mixture of 18 stable isotopes of amino acids was administered by pulse and arterialized blood was sampled for 2 h. Amino acid concentrations and enrichments were analyzed by LC-MS/MS to calculate whole body (net) protein breakdown (WBnetPB) and whole body production (WBP) rates (µmol/hour) of the amino acids playing a known role in muscle health. Statistics was done by ANCOVA to examine the effects of sex, COPD, and sex-by-COPD interaction with as covariates age and lean mass. Significance was set as p < 0.05. RESULTS: Lung function was comparable between males and females with COPD. Being a female and presence of COPD were independently associated with lower appendicular lean mass, muscle strength, and WBnetPB (p < 0.05). Being a male was associated with higher visceral adipose tissue, C-reactive protein (CRP) (p < 0.05), and higher prevalence of heart failure and obstructive sleep apnea. Sex-by-COPD interactions were found indicating lower fat mass (p = 0.0005) and WBPs of phenylalanine (measure of whole body protein turnover) and essential amino acids (p < 0.05), particularly in COPD females. Higher visceral adipose tissue (p = 0.025), CRP (p < 0.0001), and WBP of tau-methylhistidine (p = 0.010) (reflecting enhanced myofibrillar protein breakdown) were observed in COPD males. CONCLUSIONS: Presence of sex specific changes in protein and amino acid metabolism and cardiometabolic health in COPD need to be considered when designing treatment regimens to restore muscle health in males and females with COPD. CLINICAL TRIAL REGISTRY: www. CLINICALTRIALS: gov, NCT01787682, NCT01624792, NCT02157844, NCT02065141, NCT02770092, NCT02780219, NCT03327181, NCT03796455, NCT01173354, NCT01154400.

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis.

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered 13C3, 15N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.

Stochastic simulation algorithm for isotope-based dynamic flux analysis.

Carbon isotope labeling method is a standard metabolic engineering tool for flux quantification in living cells. To cope with the high dimensionality of isotope labeling systems, diverse algorithms have been developed to reduce the number of variables or operations in metabolic flux analysis (MFA), but lacks generalizability to non-stationary metabolic conditions. In this study, we present a stochastic simulation algorithm (SSA) derived from the chemical master equation of the isotope labeling system. This algorithm allows to compute the time evolution of isotopomer concentrations in non-stationary conditions, with the valuable property that computational time does not scale with the number of isotopomers. The efficiency and limitations of the algorithm is benchmarked for the forward and inverse problems of 13C-DMFA in the pentose phosphate pathways, and is compared with EMU-based methods for NMFA and MFA including the central carbon metabolism. Overall, SSA constitutes an alternative class to deterministic approaches for metabolic flux analysis that is well adapted to comprehensive dataset including parallel labeling experiments, and whose limitations associated to the sampling size can be overcome by using Monte Carlo sampling approaches.

De Novo Glycine Synthesis Is Reduced in Adults With Morbid Obesity and Increases Following Bariatric Surgery.

Background: Glycine is a dietary non-essential amino acid that is low in obesity and increases following bariatric surgery. However, the exact mechanism responsible remains unclear and it is unknown whether hypoglycinemia is a cause or consequence of insulin resistance. Objective: Using multiple isotopically labeled tracers, we aimed to determine the underlying kinetic changes responsible for hypoglycinemia in obesity by: 1) Comparing glycine kinetics between participants with morbid obesity (BMI ≥ 32.5 kg/m2) to those with healthy weight (BMI < 25 kg/m2), and 2) Comparing glycine kinetic changes in participants with morbid obesity after bariatric surgery. Methods: [1,2-13C2] glycine, [2,3,3-2H3] serine, and [2H5] phenylalanine were infused to compare the glycine kinetic parameters between 21 participants with morbid obesity and 21 controls with healthy weight. Participants with morbid obesity then underwent bariatric surgery and 17 were re-studied 6 months later. Data were analyzed by non-parametric methods and presented as median (interquartile range). Results: Compared to controls, participants with morbid obesity had significantly lower plasma glycine concentrations at 163 (153-171) vs. 201 (172-227) µmol/L and significantly reduced de novo glycine synthesis rate at 86.2 (64.5-111) vs.124 (103-159) µmol·kg LBM-1·h1, p < 0.001. Following surgery, body weight and insulin resistance decreased and this was accompanied by significant increases in plasma glycine concentration to 210 (191-243) µmol/L as well as the de novo glycine synthesis rate to 127 (98.3-133) µmol·kg LBM-1·h-1, p < 0.001 vs. baseline. Conclusion: Hypoglycinemia in participants with morbid obesity was associated with impaired de novo glycine synthesis. The increase in plasma glycine concentration and de novo glycine synthesis plus the marked improvement in insulin resistance after bariatric surgery suggest that hypoglycinemia may be secondary to impaired glycine synthesis because of obesity-induced insulin resistance. Clinical Trial Registration: [https://tinyurl.com/6wfj7yss], identifier [NCT04660513].

13C15N: glucagon-based novel isotope dilution mass spectrometry method for measurement of glucagon metabolism in humans.

BACKGROUND: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. METHODS: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. RESULTS: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. CONCLUSIONS: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

δ18O as a tracer of PO43- losses from agricultural landscapes.

Accurately tracing the sources and fate of excess PO43- in waterways is necessary for sustainable catchment management. The natural abundance isotopic composition of O in PO43- (δ18OP) is a promising tracer of point source pollution, but its ability to track diffuse agricultural pollution is unclear. We tested the hypothesis that δ18OP could distinguish between agricultural PO43- sources by measuring the integrated δ18OP composition and P speciation of contrasting inorganic fertilisers (compound vs rock) and soil textures (sand, loam, clay) in southwestern Australia. δ18OP composition differed between the three soil textures sampled across six livestock farms: sandy soils had lower overall δ18OP values (21 ± 1‰) than the loams (23 ± 1‰), which corresponded with a smaller, but more readily leachable, PO43- pool. Fertilisers had greater δ18OP variability (∼8‰), with fluctuations due to type and manufacturing year. Consequently, catchment 'agricultural soil leaching' δ18OP signatures could span from 18 to 25‰ depending on both fertiliser type and timing (lag between application and leaching). These findings emphasise the potential of δ18OP to untangle soil-fertiliser P dynamics under controlled conditions, but that its use to trace catchment-scale agricultural PO43- losses is limited by uncertainties in soil biological P cycling and its associated isotopic fractionation.

Cheese Ingestion Increases Muscle Protein Synthesis Rates Both at Rest and During Recovery from Exercise in Healthy, Young Males: A Randomized Parallel-Group Trial.

BACKGROUND: Protein ingestion increases muscle protein synthesis rates. The food matrix in which protein is provided can strongly modulate the postprandial muscle protein synthetic response. So far, the muscle protein synthetic response to the ingestion of whole foods remains largely unexplored. OBJECTIVES: To compare the impact of ingesting 30 g protein provided as milk protein or cheese on postprandial plasma amino acid concentrations and muscle protein synthesis rates at rest and during recovery from exercise in vivo in young males. METHODS: In this randomized, parallel-group intervention trial, 20 healthy males aged 18-35 y ingested 30 g protein provided as cheese or milk protein concentrate following a single-legged resistance-type exercise session consisting of 12 sets of leg press and leg extension exercises. Primed, continuous intravenous L-[ring-13C6]-phenylalanine infusions were combined with the collection of blood and muscle tissue samples to assess postabsorptive and 4-h postprandial muscle protein synthesis rates at rest and during recovery from exercise. Data were analyzed using repeated measures Time × Group (× Leg) ANOVA. RESULTS: Plasma total amino acid concentrations increased after protein ingestion (Time: P < 0.001), with 38% higher peak concentrations following milk protein than cheese ingestion (Time × Group: P < 0.001). Muscle protein synthesis rates increased following both cheese and milk protein ingestion from 0.037 ± 0.014 to 0.055 ± 0.018%·h-1 and 0.034 ± 0.008 to 0.056 ± 0.010%·h-1 at rest and even more following exercise from 0.031 ± 0.010 to 0.067 ± 0.013%·h-1 and 0.030 ± 0.008 to 0.063 ± 0.010%·h-1, respectively (Time: all P < 0.05; Time × Leg: P = 0.002), with no differences between cheese and milk protein ingestion (Time × Group: both P > 0.05). CONCLUSION: Cheese ingestion increases muscle protein synthesis rates both at rest and during recovery from exercise. The postprandial muscle protein synthetic response to the ingestion of cheese or milk protein does not differ when 30 g protein is ingested at rest or during recovery from exercise in healthy, young males.

Cheese Ingestion Increases Muscle Protein Synthesis Rates Both at Rest and During Recovery from Exercise in Healthy, Young Males: A Randomized Parallel-Group Trial.

BACKGROUND: Protein ingestion increases muscle protein synthesis rates. The food matrix in which protein is provided can strongly modulate the postprandial muscle protein synthetic response. So far, the muscle protein synthetic response to the ingestion of whole foods remains largely unexplored. OBJECTIVES: To compare the impact of ingesting 30 g protein provided as milk protein or cheese on postprandial plasma amino acid concentrations and muscle protein synthesis rates at rest and during recovery from exercise in vivo in young males. METHODS: In this randomized, parallel-group intervention trial, 20 healthy males aged 18-35 y ingested 30 g protein provided as cheese or milk protein concentrate following a single-legged resistance-type exercise session consisting of 12 sets of leg press and leg extension exercises. Primed, continuous intravenous L-[ring-13C6]-phenylalanine infusions were combined with the collection of blood and muscle tissue samples to assess postabsorptive and 4-h postprandial muscle protein synthesis rates at rest and during recovery from exercise. Data were analyzed using repeated measures Time × Group (× Leg) ANOVA. RESULTS: Plasma total amino acid concentrations increased after protein ingestion (Time: P < 0.001), with 38% higher peak concentrations following milk protein than cheese ingestion (Time × Group: P < 0.001). Muscle protein synthesis rates increased following both cheese and milk protein ingestion from 0.037 ± 0.014 to 0.055 ± 0.018%·h-1 and 0.034 ± 0.008 to 0.056 ± 0.010%·h-1 at rest and even more following exercise from 0.031 ± 0.010 to 0.067 ± 0.013%·h-1 and 0.030 ± 0.008 to 0.063 ± 0.010%·h-1, respectively (Time: all P < 0.05; Time × Leg: P = 0.002), with no differences between cheese and milk protein ingestion (Time × Group: both P > 0.05). CONCLUSION: Cheese ingestion increases muscle protein synthesis rates both at rest and during recovery from exercise. The postprandial muscle protein synthetic response to the ingestion of cheese or milk protein does not differ when 30 g protein is ingested at rest or during recovery from exercise in healthy, young males.

De novo lipogenesis in non-alcoholic fatty liver disease: Quantification with stable isotope tracers.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is characterized as an abnormal accumulation of triglyceride in hepatocytes. Hepatic de novo lipogenesis may play an important role in the accumulation of lipids in the liver during NAFLD. Due to the importance of lipid biosynthetic fluxes in NAFLD and T2D, tracer methodologies have been developed for their study and quantification. Here, we address novel approaches to measure and quantify DNL using stable isotope tracers. Deuterated water is a widely used tracer for quantifying DNL rates in both animal models and humans. Enrichment of lipid hydrogens from 2 H2O can be resolved and quantified by 2 H NMR and MS spectroscopy of isolated lipids. NMR provides a much higher level of positional enrichment information compared with MS which yields a more detailed picture of lipid biosynthetic. It can also be used to quantify low levels of lipid 13 C enrichment from a second tracer such as [U-13 C]sugar with minimal interference of one tracer with the other. CONCLUSIONS: Despite the clear association between elevated DNL activity and increased hepatic triglyceride levels, implementation of non-destructive and novel methods to quantify DNL and its contribution to NAFLD are also of huge interest.

Albumin and fibrinogen kinetics in sepsis: a prospective observational study.

BACKGROUND: The measurement of circulating substrate concentrations does not provide information about substrate kinetics. It, therefore, remains unclear if a decrease in plasma concentration of albumin, as seen during critical illness, is a consequence of suppressed production in the liver or increased peripheral clearance. In this study, using stable isotope tracer infusions, we measured albumin and fibrinogen kinetics in septic patients and in a control group of non-septic subjects. METHODS: With the approval from the institutional Research Ethics Board and after obtaining written informed consent from patients or their substitute decision maker, mechanically ventilated patients with sepsis and patients scheduled for elective coronary artery bypass grafting were enrolled. Patients in the non-sepsis group were studied on the day before surgery. The stable isotope L-[ring-2H5]phenylalanine was used to measure absolute synthesis rates (ASR) of albumin and fibrinogen. A priming dose of L-[ring-2H5]phenylalanine (4 µmol/kg) was given followed by a six-hour infusion at a rate of 0.15 µmol/kg/min. At baseline and hourly thereafter, blood was drawn to measure isotope enrichments by gas chromatography/mass spectrometry. Very low density lipoprotein apolipoprotein-B 100 isotopic enrichment was used to represent the isotopic enrichment of the phenylalanine precursor pool from which the liver synthesizes proteins. Plasma albumin and fibrinogen concentrations were also measured. RESULTS: Mean plasma albumin in septic patients was decreased when compared to non-septic patients, while synthesis rates were comparable. Mean plasma fibrinogen and ASR in septic patients was increased when compared to non-septic patients. In non-septic patients, no statistically significant correlation between plasma albumin and ASR was observed but plasma fibrinogen significantly correlated with ASR. In septic patients, plasma albumin and fibrinogen significantly correlated with ASR. CONCLUSIONS: While septic patients showed lower plasma albumin levels than non-septic patients, albumin synthesis was similar in the two groups suggesting that hypoalbuminemia during sepsis was not caused by suppressed hepatic production but a result of enhanced clearance from the circulation. Hyperfibrinogenemia in septic patients was a consequence of increased fibrinogen production. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02865408 (registered on August 12, 2016) and ClinicalTrials.gov: NCT02549443 (registered on September 15, 2015).

Principles of stable isotope research - with special reference to protein metabolism.

The key to understanding the mechanisms regulating disease stems from the ability to accurately quantify the dynamic nature of the metabolism underlying the physiological and pathological changes occurring as a result of the disease. Stable isotope tracer technologies have been at the forefront of this for almost 80 years now, and through a combination of both intense theoretical and technological development over these decades, it is now possible to utilise stable isotope tracers to investigate the complexities of in vivo human metabolism from a whole body perspective, down to the regulation of sub-nanometer cellular components (i.e organelles, nucleotides and individual proteins). This review therefore aims to highlight; 1) the advances made in these stable isotope tracer approaches - with special reference given to their role in understanding the nutritional regulation of protein metabolism, 2) some considerations required for the appropriate application of these stable isotope techniques to study protein metabolism, 3) and finally how new stable isotopes approaches and instrument/technical developments will help to deliver greater clinical insight in the near future.

NMR Methods for Determining Lipid Turnover via Stable Isotope Resolved Metabolomics.

Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical diversity and dynamic range in cells makes detailed analysis very complex. Furthermore, although stable isotope tracers enable the determination of synthesis and degradation of complex lipids, the numbers of distinguishable molecules increase enormously, which exacerbates the problem. Although LC-MS-MS (Liquid Chromatography-Tandem Mass Spectrometry) is the standard for lipidomics, NMR can add value in global lipid analysis and isotopomer distributions of intact lipids. Here, we describe new developments in NMR analysis for assessing global lipid content and isotopic enrichment of mixtures of complex lipids for two cell lines (PC3 and UMUC3) using both 13C6 glucose and 13C5 glutamine tracers.

Metabolic Evaluation of the Dietary Guidelines' Ounce Equivalents of Protein Food Sources in Young Adults: A Randomized Controlled Trial.

BACKGROUND: The Dietary Guidelines for Americans (DGAs) published an "ounce equivalents" recommendation to help consumers meet protein requirements with a variety of protein food sources. However, the metabolic equivalency of these varied protein food sources has not been established. OBJECTIVE: We have investigated the hypothesis that the anabolic responses to consumption of ounce equivalents of protein food sources would be directly related to the essential amino acid (EAA) content of the protein food source. METHODS: Following 3 d of dietary control, a total of 56 healthy young adults underwent an 8.5-h metabolic study using stable isotope tracer methodology. The changes from baseline following consumption of 1 of 7 different protein food sources were compared with the baseline value for that individual (n = 8 per group). RESULTS: Consumption of ounce equivalents of animal-based protein food sources (beef sirloin, pork loin, eggs) resulted in a greater gain in whole-body net protein balance above baseline than the ounce equivalents of plant-based protein food sources (tofu, kidney beans, peanut butter, mixed nuts; P < 0.01). The improvement in whole-body net protein balance was due to an increase in protein synthesis (P < 0.05) with all the animal protein sources, whereas the egg and pork groups also suppressed protein breakdown compared with the plant protein sources (P < 0.01). The magnitude of the whole-body net balance (anabolic) response was correlated with the EAA content of the protein food source (P < 0.001). CONCLUSION: The "ounce equivalents" of protein food sources as expressed in the DGAs are not metabolically equivalent in young healthy individuals. The magnitude of anabolic response to dietary proteins should be considered as the DGAs develop approaches to establish healthy eating patterns.

High-throughput LC-MS method to investigate postprandial lipemia: considerations for future precision nutrition research.

Elevated postprandial lipemia is an independent risk factor for cardiovascular disease, yet methods to quantitate postmeal handling of dietary lipids in humans are limited. This study tested a new method to track dietary lipid appearance using a stable isotope tracer (2H11-oleate) in liquid meals containing three levels of fat [low fat (LF), 15 g; moderate fat (MF), 30 g; high fat (HF), 60 g]. Meals were fed to 12 healthy men [means ± SD, age 31.3 ± 9.2 yr, body mass index (BMI) 24.5 ± 1.9 kg/m2] during four randomized study visits; the HF meal was administered twice for reproducibility. Blood was collected over 8 h postprandially, triglyceride (TG)-rich lipoproteins (TRL), and particles with a Svedberg flotation rate >400 (Sf > 400, n = 8) were isolated by ultracentrifugation, and labeling of two TG species (54:3 and 52:2) was quantified by LC-MS. Total plasma TRL-TG concentrations were threefold greater than Sf > 400-TG. Both Sf > 400- and TRL-TG 54:3 were present at higher concentrations than 52:2, and singly labeled TG concentrations were higher than doubly labeled. Furthermore, TG 54:3 and the singly labeled molecules demonstrated higher plasma absolute entry rates differing significantly across fat levels within a single TG species (P < 0.01). Calculation of fractional entry showed no significant differences in label handling supporting the utility of either TG species for appearance rate calculations. These data demonstrate the utility of labeling research meals with stable isotopes to investigate human postprandial lipemia while simultaneously highlighting the importance of examining individual responses. Meal type and timing, control of prestudy activities, and effects of sex on outcomes should match the research goals. The method, optimized here, will be beneficial to conduct basic science research in precision nutrition and clinical drug development.NEW & NOTEWORTHY A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.

A guide to metabolic flux analysis in metabolic engineering: Methods, tools and applications.

The field of metabolic engineering is primarily concerned with improving the biological production of value-added chemicals, fuels and pharmaceuticals through the design, construction and optimization of metabolic pathways, redirection of intracellular fluxes, and refinement of cellular properties relevant for industrial bioprocess implementation. Metabolic network models and metabolic fluxes are central concepts in metabolic engineering, as was emphasized in the first paper published in this journal, "Metabolic fluxes and metabolic engineering" (Metabolic Engineering, 1: 1-11, 1999). In the past two decades, a wide range of computational, analytical and experimental approaches have been developed to interrogate the capabilities of biological systems through analysis of metabolic network models using techniques such as flux balance analysis (FBA), and quantify metabolic fluxes using constrained-based modeling approaches such as metabolic flux analysis (MFA) and more advanced experimental techniques based on the use of stable-isotope tracers, i.e. 13C-metabolic flux analysis (13C-MFA). In this review, we describe the basic principles of metabolic flux analysis, discuss current best practices in flux quantification, highlight potential pitfalls and alternative approaches in the application of these tools, and give a broad overview of pragmatic applications of flux analysis in metabolic engineering practice.

The impact of Hayward green kiwifruit on dietary protein digestion and protein metabolism.

PURPOSE: The purpose of the study was to determine if an actinidin protease aids gastric digestion and the protein anabolic response to dietary protein. METHODS: Hayward green kiwifruit (containing an actinidin protease) and Hort 16A gold kiwifruit (devoid of actinidin protease) were given in conjunction with a beef meal to healthy older subjects. Twelve healthy older males (N = 6) and females (N = 6) were studied with a randomized, double-blinded, crossover design to assess muscle and whole-body protein metabolism before and after ingestion of kiwifruit and 100 g of ground beef. Subjects consumed 2 of each variety of kiwifruit daily for 14 d prior to each metabolic study, and again during each study with beef intake. RESULTS: Hayward green kiwifruit consumption with beef resulted in a more rapid increase in peripheral plasma essential amino acid concentrations. There were significant time by kiwifruit intake interactions for plasma concentrations of EAAs, branched chain amino acids (BCAAs), and leucine (P < 0.01). However, there was no difference in the total amount of EAAs absorbed. As a result, there were no differences between kiwifruit in any of the measured parameters of protein kinetics. CONCLUSION: Consumption of Hayward green kiwifruit, with a beef meal facilitates protein digestion and absorption of the constituent amino acids as compared to Hort 16A gold kiwifruit. CLINICAL TRIAL: NCT04356573, April 21, 2020 "retrospectively registered".