Study Of Protein Metabolism Research Articles

Urinary excretion of gluten immunoreactive peptides as an indicator of gastrointestinal function after fasting and dietary provocation in healthy volunteers.

Understanding intestinal permeability is paramount for elucidating gastrointestinal health and pathology. The size and nature of the molecule traversing the intestinal barrier offer crucial insights into various acute and chronic diseases, as well as the evolution of some conditions. This study aims to assess the urinary excretion kinetics of gluten immunogenic peptides (u-GIP), a unique class of dietary peptides detectable in urine, in volunteers under controlled dietary conditions. This evaluation should be compared to established probes like lactulose, a non-digestible disaccharide indicative of paracellular permeability, and mannitol, reflecting transcellular permeability. Fifteen participants underwent simultaneous ingestion of standardized doses of gluten (10 g), lactulose (10 g), and mannitol (1 g) under fasting conditions for at least 8 hours pre-ingestion and during 6 hours post-ingestion period. Urine samples were collected over specified time intervals. Excretion patterns were analyzed, and correlations between the lactulose-to-mannitol ratio (LMR) and u-GIP parameters were assessed. The majority of u-GIP were detected within the first 12 hours post-ingestion. Analysis of the variability in cumulative excretion across two sample collection ranges demonstrated that lactulose and u-GIP exhibited similar onset and excretion dynamics, although GIP reached its maximum peak earlier than either lactulose or mannitol. Additionally, a moderate correlation was observed between the LMR and u-GIP parameters within the longest urine collection interval, indicating potential shared characteristics among permeability pathways. These findings suggest that extending urine collection beyond 6 hours may enhance data reliability. This study sheds light on the temporal dynamics of u-GIP in comparison to lactulose and mannitol, established probes for assessing intestinal permeability. The resemblance between u-GIP and lactulose excretion patterns aligns with the anticipated paracellular permeability pathway. The capacity to detect antigenic food protein fragments in urine opens novel avenues for studying protein metabolism and monitoring pathologies related to the digestive and intestinal systems.

Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs.

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-0,75. d-1), breakdown (3.26 ± 0.18 g.kg-0.75.d-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies.

Protein turnover in pigs: A review of interacting factors.

Protein turnover defines the balance between two continuous and complex processes of protein metabolism, synthesis and degradation, which determine their deposition in tissues. Although the liver and intestine have been studied extensively for their important roles in protein digestion, absorption and metabolism, the study of protein metabolism has focused mainly on skeletal muscle tissue to understand the basis for its growth. Due to the high adaptability of skeletal muscle, its protein turnover is greatly affected by different internal and external factors, contributing to carcass lean-yield and animal growth. Amino acid (AA) labelling and tracking using isotope tracer methodology, together with the study of myofiber type profiling, signal transduction pathways and gene expression, has allowed the analysis of these mechanisms from different perspectives. Positive stimuli such as increased nutrient availability in the diet (e.g., AA), physical activity, the presence of certain hormones (e.g., testosterone) or a more oxidative myofiber profile in certain muscles or pig genotypes promote increased upregulation of translation and transcription-related genes, activation of mTORC1 signalling mechanisms and increased abundance of satellite cells, allowing for more efficient protein synthesis. However, fasting, animal aging, inactivity and stress, inflammation or sepsis produce the opposite effect. Deepening the understanding of modifying factors and their possible interaction may contribute to the design of optimal strategies to better control tissue growth and nutrient use (i.e., protein and AA), and thus advance the precision feeding strategy.

Production of Large Amounts of [13C]Leucine-Enriched Milk Proteins by Lactating Cows

During protein metabolism kinetic studies, oral tracers are administered as labeled free amino acids and do not necessarily represent the metabolic fate of amino acids ingested as proteins. However, sufficient quantities of 13C-labeled proteins are not currently available. We here present a new methodology for producing large amounts of milk proteins intrinsically labeled with [13C]leucine. After surgical preparation, two lactating cows were infused with 80–90 g of L-[1-13C]leucine for 24–32 h, and milk was collected during and after the infusion. Casein and whey protein fractions were purified by membrane separation techniques. Arteriovenous balance across the udder indicated a very efficient extraction of leucine by the mammary gland. Five batches of pure casein and whey proteins, totaling 3854 g of protein of excellent bacteriological quality, were obtained. Two thirds of these proteins had [13C]leucine enrichments ranging from 10.5 to 19.4% ([13C]leucine atom percent excess). The overall tracer recoveries were 22 and 27% (cows 1 and 2, respectively). Thus, pure milk proteins were produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies.

Discussion on Protein Metabolism and Requirement of Aerobics Athletes during Training Based on Multisensor Data Fusion.

Competitive aerobics has emerged as a highly competitive sport beyond its own physical limit. Modern competitive aerobics competition is very fierce; athletes cannot only rely on a specific competitive skill to achieve good results. Protein is the physical basis of life activity. The life activity of human body is closely related to protein, and protein is closely related to human exercise ability. This article aims to study protein metabolism and demand of aerobics athletes during training based on multisensor data fusion. A total of 26 female aerobics athletes were randomly divided into two groups: exercise group and exercise + nutrition group. According to the characteristics of human motion, a comprehensive measurement acquisition sensor system for collecting human motion information is designed and implemented, and the system is used to monitor the subject's protein condition in real time. The subjects took protein nutrient solution before breakfast every day. The dynamic recognition algorithm designed in this paper also has shortcomings, and the monitoring protein method based on gait and other signs is not completely correct. The experiment lasted for 7 weeks. The results showed that the level of serum transferrin receptor decreased significantly in the quiet + nutrition group for 4 weeks, which was significantly different from that at 0 and 3 weeks in the same group (P < 0.01) and was significantly different from that in the same group at 7 weeks (P < 0.05). In the exercise group, the level of serum transferrin receptor increased significantly at 5 weeks, compared with the same group at 0 and 3 weeks (P < 0.05).

Principles of stable isotope research – with special reference to protein metabolism

SummaryThe 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.

Protein status of holshtinized black and white cattle

We studied protein metabolism in Holsteinized black-and-white cattle. The researches were conducted in the Kuzbass at OJC «Vaganovo». Blood sample was taken from each cow, according to generally accepted of methods and analyzed in the laboratory of biochemistry from Novosibirsk State Agrarian University by using standard kits of biochemical studies on a biochemical semiautomatic analyzer Photometer 5010 V5 +. The indicators of protein metabolism were established in the blood serum of Holsteinized black-and-white cattle in average values; creatinine was characterized by the lowest phenotypic variability. The greatest positive relationship was found between albumin/globulin coefficients and albumin (r = 0.765). The reference intervals were calculated for cows of the 3rd lactation of the Holsteinized black-and-white breed with a high level of milk productivity (9-10 thousand kg): total protein 62.02-87.58 g / l; albumin 21.05-36.09 g / l; urea 2.2-4 mmol / l; creatinine 50-71 μmol / l; albumin-globulin coefficients 0.35-1.16. The data obtained can be used for assess the interior of animals.

In vitro Reconstitution Assays of Arabidopsis 20S Proteasome

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously reported methods. The main strategy to obtain the 20S core proteasome here is to strip away the 19S regulatory subunits from the 26S proteasome. The protocol has two major parts: 1) Affinity purification of 20S proteasomes from stable transgenic lines expressing epitope-tagged PAG1, an essential component of the 20S proteasome (Procedures A-D) and 2) an in vitro 20S proteasome degradation assay (Procedure E). We anticipate that these protocols will provide simple and effective approaches to study in vitro degradation by the 20S proteasome and advance the study of protein metabolism in plants.

Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques

Nitrogen is a component of essential nutrients critical for the productivity of ruminants. If excreted in excess, N is also an important environmental pollutant contributing to acid deposition, eutrophication, human respiratory problems, and climate change. The complex microbial metabolic activity in the rumen and the effect on subsequent processes in the intestines and body tissues make the study of N metabolism in ruminants challenging compared with nonruminants. Therefore, using accurate and precise measurement techniques is imperative for obtaining reliable experimental results on N utilization by ruminants and evaluating the environmental impacts of N emission mitigation techniques. Changeover design experiments are as suitable as continuous ones for studying protein metabolism in ruminant animals, except when changes in body weight or carryover effects due to treatment are expected. Adaptation following a dietary change should be allowed for at least 2 (preferably 3) wk, and extended adaptation periods may be required if body pools can temporarily supply the nutrients studied. Dietary protein degradability in the rumen and intestines are feed characteristics determining the primary AA available to the host animal. They can be estimated using in situ, in vitro, or in vivo techniques with each having inherent advantages and disadvantages. Accurate, precise, and inexpensive laboratory assays for feed protein availability are still needed. Techniques used for direct determination of rumen microbial protein synthesis are laborious and expensive, and data variability can be unacceptably large; indirect approaches have not shown the level of accuracy required for widespread adoption. Techniques for studying postruminal digestion and absorption of nitrogenous compounds, urea recycling, and mammary AA metabolism are also laborious, expensive (especially the methods that use isotopes), and results can be variable, especially the methods based on measurements of digesta or blood flow. Volatile loss of N from feces and particularly urine can be substantial during collection, processing, and analysis of excreta, compromising the accuracy of measurements of total-tract N digestion and body N balance. In studying ruminant N metabolism, nutritionists should consider the longer term fate of manure N as well. Various techniques used to determine the effects of animal nutrition on total N, ammonia- or nitrous oxide-emitting potentials, as well as plant fertilizer value, of manure are available. Overall, methods to study ruminant N metabolism have been developed over 150 yr of animal nutrition research, but many of them are laborious and impractical for application on a large number of animals. The increasing environmental concerns associated with livestock production systems necessitate more accurate and reliable methods to determine manure N emissions in the context of feed composition and ruminant N metabolism.

Invited Article: Visualizing protein synthesis in mice with in vivo labeling of deuterated amino acids using vibrational imaging

Proteins are one of the major components of biological systems, and understanding their metabolism is critical to study various biochemical processes in living systems. Despite extensive efforts to study protein metabolism such as autoradiography, mass spectrometry, and fluorescence microscopy, visualizing the spatial distribution of overall protein metabolism in mammals at subcellular resolution is still challenging. A recent study from our group reported imaging newly synthesized proteins in cultured mammalian cells, tissues, or even in mice using stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (dAA). However, our previous method of dAA administration via drinking water, albeit convenient, is insufficient for in vivo studies. This is due to poor labeling efficiency and limited access to many important organs such as the brain, pancreas, or tumor. In this study, we have significantly improved and optimized the in vivo administration method by intra-carotid arterial injection of dAA in mice and obtained imaging contrast of protein metabolic activity in many more organs and tissues, such as cerebral and cerebellar cortex and hippocampal regions in the mouse brain. We also imaged newly formed proteins in the choroid plexus and pancreas at different time points, illustrating the metabolic dynamics of proteins in these important secretory organs. In addition, we visualized the metabolic heterogeneity of protein synthesis in colon tumor xenografts, which can be used to distinguish tumor and normal tissues. In summary, this combination of a new dAA administration technique and SRS imaging platform demonstrates an effective tool for the in vivo study of complex protein metabolism in mammals, in both physiological and pathological states.

Динаміка показників білкового обміну при комплексній антигомотоксичній терапії собак, хворих на катаральну бронхопневмонію

Бронхопневмонія у собак зустрічається часто, так як слизова оболонка респіраторного тракту легко піддається дії несприятливих умов навколишнього середовища та збудників, що присутні в повітрі. Одним з важливих діагностичних показників при катаральній бронхопневмонії є показники білкового обміну, а саме: загальний білок, альбуміни, глобуліни, альбуміно-глобуліновий коефіцієнт. Метою наших досліджень було вивчення динаміки показників білкового обміну при комплексній антигомотоксичній терапії собак, хворих на катаральну бронхопневмонію та порівняння з такими при алопатичній терапії. Матеріалом для дослідження були сорок собак, підібраних за принципом аналогів, хворих на гостру катаральну бронхопневмонію, яких було розділено на дві групи: 10 тварин в контрольній групі і тридцять – в дослідній. В контрольній групі для лікування застосовувалась алопатична терапія, в дослідній – комплексна антигомотоксична терапія. Кожна тварина перебувала під наглядом двадцять діб. За цей період тварин піддавали щоденному клінічному дослідженню. Окрім того, на перший, десятий та двадцятий день проводились дослідження показників білкового обміну. При аналізі даних, отриманих в результаті клінічних досліджень, видно, що схема лікування тварин дослідної групи виявилася більш ефективною, ніж у тварин конотрольної групи. Це проявляється у вигляді меншого терміну лікування, більшого відсотку тварин, що одужали та відсутності летальних наслідків. Результати досліджень показників білкового обміну показали, що при катаральній бронхопневмонії собак спостерігається зниження вмісту альбумінів та альбуміно-глобулінового коефіцієнту і підвищення вмісту глобулінів. Також спостерігається незначне коливання загального білку в межах норми. Протягом лікування спостерігається нормалізація даних показників, причому найбільш наочно це видно у тварин дослідної групи. Таким чином видно, що застосування комплексної антигомотоксичної терапії є ефективнішим та доцільнішим у порівнянні з традиційною терапією. Ключові слова: антигомотоксична терапія, катаральна бронхопневмонія, лімфотропне введення, траумель, фосфор-гомаккорд, клінічне дослідження, білок, альбуміни, глобуліни, альбуміно-глобуліновий коефіцієнт

24-Hour protein, arginine and citrulline metabolism in fed critically ill children – A stable isotope tracer study

The reference method to study protein and arginine metabolism in critically ill children is measuring plasma amino acid appearances with stable isotopes during a short (4-8h) time period and extrapolate results to 24-h. However, 24-hmeasurements may be variable due to critical illness related factors and a circadian rhythm could be present. Since only short duration stable isotope studies in critically ill children have been conducted before, the aim of this study was to investigate 24-happearance of specific amino acids representing protein and arginine metabolism, with stable isotope techniques in continuously fed critically ill children. In eight critically ill children, admitted to the pediatric (n=4) or cardiovascular (n=4) intensive care unit, aged 0-10 years, receiving continuous (par)enteral nutrition with protein intake 1.0-3.7g/kg/day, a 24-hstable isotope tracer protocol was carried out. L-[ring-2H5]-phenylalanine, L-[3,3-2H2]-tyrosine, L-[5,5,5-2H3]-leucine, L-[guanido-15N2]-arginine and L-[5-13C-3,3,4,4-2H4]-citrulline were infused intravenously and L-[15N]-phenylalanine and L-[1-13C]leucine enterally. Arterial blood was sampled every hour. Coefficients of variation, representing intra-individual variability, of the amino acid appearances of phenylalanine, tyrosine, leucine, arginine and citrulline were high, on average 14-19% for intravenous tracers and 23-26% for enteral tracers. No evident circadian rhythm was present. The pattern and overall 24-hlevel of whole body protein balance differed per individual. In continuously fed stable critically ill children, the amino acid appearances of phenylalanine, tyrosine, leucine, arginine and citrulline show high variability. This should be kept in mind when performing stable isotope studies in this population. There was no apparent circadian rhythm. NCT01511354 on clinicaltrials.gov.

The Streptomyces metabolite anhydroexfoliamycin ameliorates hallmarks of Alzheimer’s disease in vitro and in vivo

Anhydroexfoliamycin (1) and undecylprodigiosin (2) have been previously described as neuroprotective molecules against oxidative stress in neurons. Since oxidative stress is strongly correlated with neurodegenerative diseases, we have evaluated their effects over the principal hallmarks of Alzheimer’s disease (AD). Both compounds were tested in vitro in two different neuroblastoma cellular models, one for amyloid precursor protein metabolism studies (BE(2)-M17) and another one specific for taupathology in AD (SH-SY5Y-TMHT441). Amyloid-beta (Aβ) levels, β-secretase (BACE1) activity, tau phosphorylation, extracellular signal-regulated kinase (ERK) and glycogen synthase kinase-3beta (GSK3β) expression were analyzed and while undecylprodigiosin (2) produced poor results, anhydroexfoliamycin (1) strongly inhibited GSK3β, reducing tau phosphorylation in vitro (0.1μM). A competitive assay of anhydroexfoliamycin (1) and the specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, showed that the reduction of the phosphorylated tau levels is mediated by the JNK pathway in SH-SY5Y-TMHT441 cells. Thus, this compound was tested in vivo by intraperitoneal administration in 3xTg-AD mice, confirming the positive results registered in the in vitro assays. This work presents anhydroexfoliamycin (1) as a promising candidate for further studies in drug development against neurodegenerative diseases.

Slight chronic elevation of C-reactive protein is associated with lower aerobic fitness but does not impair meal-induced stimulation of muscle protein metabolism in healthy old men.

Ageing impairs the muscle anabolic effect of food intake, which may explain muscle loss and an increased risk of sarcopenia. Ageing is also associated with low grade inflammation (LGI), which has been negatively correlated with muscle mass and strength. In rodents, the muscle anabolic resistance observed during ageing and sarcopenia has been ascribed to the development of the LGI. We aimed to investigate this relationship in humans. We studied protein metabolism and physical fitness in healthy elderly volunteers with slight chronic C-reactive protein. Two groups of healthy elderly volunteers were selected on the presence (or not) of a chronic, slight, elevation of CRP (Control: <1; CRP+: >2 mg l(-1) and <10 mg l(-1) , for 2 months). Body composition, short performance battery test, aerobic fitness and muscle strength were assessed. Whole body and muscle protein metabolism and the splanchnic extraction of amino acids were assessed using [(13) C]leucine and [(2) H]leucine infusion. The anabolic effect of food intake was measured by studying the volunteers both at the post-absorptive and post-prandial states. Slight chronic CRP elevation resulted in neither an alteration of whole body, nor skeletal muscle protein metabolism at both the post-absorptive and the post-prandial states. However, CRP+ presented a reduction of physical fitness, increased abdominal fat mass and post-prandial insulin resistance. Plasma cytokines (interleukin-1, interleukin-6, tumour necrosis factor α) and markers of endothelial inflammation (intercellular adhesion molecule, vascular cell adhesion molecule, selectins) were similar between groups. An isolated elevated CRP in healthy older population does not indicate an impaired skeletal muscle anabolism after food intake, nor an increased risk of skeletal muscle wasting. We propose that a broader picture of LGI (notably with elevated pro-inflammatory cytokines) is required to impact muscle metabolism and mass. However, an isolated chronic CRP elevation could predict a decrease in aerobic fitness and insulin resistance installation in elderly individuals.

Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling.

Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans.

Hens Produce Artificially Enriched 13C Egg Proteins for Metabolic Tracer Studies

Clinicians and researchers studying protein metabolism in vivo, typically use isotopically-labeled free amino acids as metabolic tracers rather than isotopically-labeled proteins because such proteins are commercially unavailable. However, the use of free amino acids in lieu of protein tracers violates the critical assumption that tracer molecules undergo the identical biochemical reactions as the tracee molecules of interest. To address this problem we synthesized 13C-labeled proteins using egg laying hens and investigated the relationship between tracer dose and method of delivery on 13C-protein production. We enriched hens with one of two isotope tracers (13C-1-leucine or a uniformly labeled 13C-amino acid mixture) mixed in their food or dissolved in their drinking water at different dosing levels (86-432 mg day-1). The recovery of 13C in egg white proteins of the hen fed 13C-leucine ranged from 14% to 21%; recovery rates were highest at the lowest dosing level. At the highest dosing level egg whites were enriched more than 150‰ above background levels of 13C. The time required for half maximal 13C enrichment depended chiefly on the mode of tracer administration, and ranged from 2.5 days for 13C-leucine dissolved in water to 4.9 days for 13C-leucine mixed in food. Relative rates of 13C recovery in the egg protein were lowest for hens fed the uniformly 13C labeled amino acid mixture, presumably because of the high proportion of nonessential amino acids. The time required for the 13C-enrichment in eggs to return to background levels at the end of the enrichment period was about twice that required to initially reach isotopic equilibrium with the diet, indicating significant biochemical discrimination of endogenous 13C amino acids. We conclude that delivering small amounts of 13C amino acid tracers in the drinking water of hens is the most effective way to produce 13C-enriched proteins to for tracer studies that do not require delt 13C-enrichment above 200‰.

Amino Acid Supplementation Does Not Alter Whole-Body Phenylalanine Kinetics in Arabian Geldings 3

Stable isotope infusion methods have not been extensively used in horses to study protein metabolism. The objectives were to develop infusion and sampling methodologies for [1-13C] phenylalanine and apply these methods to determine whether the addition of supplemental amino acids to a control diet affected whole-body phenylalanine kinetics in mature horses. Arabian geldings were studied using a 6-h primed (9 μmol/kg), constant (6 μmol · kg-1 · h-1) i.v. infusion of ʟ-[1-13C] phenylalanine, with blood and breath sampled every 30 min, to measure whole-body phenylalanine kinetics in response to receiving the control diet (n = 12) or the control diet supplemented with equimolar amounts of glutamate (+Glu; 55 mg · kg-1 · d-1; n = 5), leucine (+Leu; 49 mg · kg-1 · d-1; n = 5), lysine (+Lys; 55 mg · kg-1 · d-1; n = 5), or phenylalanine (+Phe; 62 mg · kg-1 · d-1; n = 6). The plasma concentrations of the supplemented amino acid in horses receiving the +Leu, +Lys, and +Phe diets were 58, 53, and 36% greater, respectively, than for the control treatment (P< 0.05). Isotopic plateau was attained in blood [1-13C] phenylalanine and breath 13CO2 enrichments by 60 and 270 min, respectively. Phenylalanine flux (+20%) and oxidation (+110%) were greater (P < 0.05) in horses receiving the +Phe treatment than in those fed the control diet. There was no effect of treatment diet on nonoxidative phenylalanine disposal or phenylalanine release from protein breakdown. The developed methods are a valuable way to study protein metabolism and assess dietary amino acid adequacy in horses and will provide a useful tool for studying amino acid requirements in the future.

Studies in Protein Metabolism.

Studies in Protein Metabolism.

Determination of 13C isotopic enrichment of valine and threonine by GC–C–IRMS after formation of the N(O,S)-ethoxycarbonyl ethyl ester derivatives of the amino acids

We describe a new method of assessing, in a single run, (13)C isotopic enrichment of both Val and Thr by gas chromatography-combustion-isotope-ratio mass spectrometry (GC-C-IRMS). This method characterised by a rapid one-step derivatisation procedure performed at room temperature to form the N(O,S)-ethoxycarbonyl ethyl ester derivatives, and a polar column for GC. The suitability of this method for Val and Thr in in-vivo samples (mucosal hydrolysate) was demonstrated by studying protein metabolism with two tracers ((13)C-valine or (13)C-threonine). The intra-day and inter-day repeatability were both assessed either with standards or with in-vivo samples at natural abundance and at low (13)C isotopic enrichment. For inter-day repeatability CVs were between 0.8 and 1.5% at natural abundance and lower than 5.5% at 0.112 and 0.190 atom% enrichment for Val and Thr, respectively. Overall isotopic precision was studied for eleven standard amino acid derivatives (those of Val, Ala, Leu, Iso, Gly, Pro, Asp, Thr, Ser, Met, and Phe) and was assessed at 0.32 per thousand. The (13)C isotopic measurement was then extended to the other amino acids (Ala, Val, Leu, Iso, Gly, Pro, Thr, and Phe) at natural abundance for in-vivo samples. The isotopic precision was better than 0.002 atom% per amino acid (for n = 4 rats). This analytical method was finally applied to an animal study to measure Thr utilization in protein synthesis.

Protein requirements in male adolescent soccer players

Few investigations have studied protein metabolism in children and adolescent athletes which makes difficult the assessment of daily recommended dietary protein allowances in this population. The problematic in paediatric competitors is the determination of additional protein needs resulting from intensive physical training. The aim of this investigation was to determine protein requirement in 14-year-old male adolescent soccer players. Healthy male adolescent soccer players (N = 11, 13.8 +/- 0.1 year) participated in a short term repeated nitrogen balance study. Diets were designed to provide proteins at three levels: 1.4, 1.2 and 1.0 g protein per kg body weight (BW). Nutrient and energy intakes were assessed from 4 day food records corresponding to 4 day training periods during 3 weeks. Urine was collected during four consecutive days and analysed for nitrogen. The nitrogen balances were calculated from mean daily protein intake, mean urinary nitrogen excretion and estimated faecal and integumental nitrogen losses. Nitrogen balance increased with both protein intake and energy balance. At energy equilibrium, the daily protein intake needed to balance nitrogen losses was 1.04 g kg(-1) day(-1). This corresponds to an estimated average requirement (EAR) for protein of 1.20 g kg(-1) day(-1) and a recommended daily allowance (RDA) of 1.40 g kg(-1) day(-1) assuming a daily nitrogen deposition of 11 mg kg(-1). The results of the present study suggest that the protein requirements of 14-year-old male athletes are above the RDA for non-active male adolescents.