Amino Acid Oxidation Research Articles

Cu(I)-Catalyzed C(sp3)-H Functionalization of Amino Acids with Benzimidate and Reactive Oxygen Species (ROS) To Synthesize Triazines and 2-Pyrrolidinones.

An easily accessible Cu(I)-catalyzed regioselective oxidative C-N/C-O cross-coupling organic transformation has been disclosed for the syntheses of variably functionalized triazines and N-benzoylpyrrolidin-2-ones through the involvement of C(sp3)-H bond functionalization, which is unknown in the literature. This general synthetic method is extended for decarboxylative oxidation of amino acids to install carbonyl functionality. It facilitates the formation of 2-3 new bonds through the cross-coupling strategy involving benzimidates, amino acids, and in situ-generated reactive oxygen species (ROS) from the aerial O2 as the sole oxidant. The key utilities of the new reactions are demonstrated by its operational simplicity, regioselectivity, robustness, and broad substrate scope with high yields.

Kinetic and Mechanistic Investigation of L‐Phenylalanine Oxidation by Alkaline Cu(III) Periodate in CPC Micellar Medium

ABSTRACTAmino acid oxidation is fascinating because different oxidants produce diverse compounds. No research has examined how metal catalysts affect amino acid oxidation by diperiodatocuprate (III) (DPC) in micellar environments. This research is crucial to understanding amino acids in redox processes and identifying active species of Ru(III) and DPC. The study will evaluate how cationic surfactant affects Ru(III)‐facilitated L‐phenylalanine (L‐Pheala) oxidation utilizing DPC in an alkaline medium. The reaction's advancement has been assessed employing the pseudo‐first‐order condition as a gauge for [OH−], [DPC], ionic strength, [L‐Pheala], [Ru(III)], [IO4−], [Surfactant], and temperature. L‐Pheala and DPC interact stoichiometrically in a ratio of 1:4. Across the spectrum of concentrations examined, the reported reaction reflects less than unit order kinematics in relation to both [L‐Pheala] (0.61 in the aqueous medium and 0.58 in the CPC micellar medium) and [OH−] (0.47 in the aqueous medium and 0.51 in the CPC micellar medium), first‐order reliance on the [DPC] and [Ru(III)], and negative fractional‐order for [IO4−] (−0.54 in the aqueous medium and −0.56 in the CPC micellar medium). A zero salt effect is suggested by the observed constancy in oxidation rate with the inclusion of electrolytes. The oxidation rate is significantly enhanced by Ru(III) solution (as a catalyst) at ppm concentration. Cetylpyridinium chloride (CPC) micellar media facilitate an additional enhancement (four times) in the rate of the reaction. CPC thus exhibits an excellent compatibility with Ru(III) for the L‐Pheala oxidation using (DPC).

A new Rose Bengal glycopolymer: Photosensitization in two stages

Antimicrobial photodynamic therapy is a promising alternative to deal with antimicrobial resistance. However, both the low specificity and low local oxygen molecular concentrations decrease the antimicrobial efficiency limiting its use. An interesting approach to the problem is the use of molecules that can react reversibly with singlet oxygen by the formation of reversible endoperoxides, such as naphthalene, anthracene and pyridone derivatives. Particularly, the use of these molecules with mannosyl derivatives allow the interaction with adhesins presented on pili and fimbriae improving the localization near to bacteria. In this work, we synthesized polymeric nanoparticles able to generate singlet oxygen (under both irradiation and dark conditions) in the vicinity of a center capable of recognizing mannose and oxidize nearby biomolecules. Rose Bengal was used as photosensitizer due to its attractive photophysical properties (vis absorption, high singlet oxygen generation) and biocompatibility. The polymeric nanoparticles were obtained by radical polymerization using polyvinyl alcohol as a template, showing sizes around 300 nm with negative zeta potential by dynamic light scattering. The singlet oxygen generation was monitored following DPBF consumption and showed to be dependent on the amount of pyridone in the feed of polymers. In addition, the release of singlet oxygen was also dependent on pyridone concentration showing a slower rate constant at 40 % pyridone, while for contents of 10 % and 60 % higher rate constants were observed. The specific interaction of glycopolymers with Concanavalin A was demonstrated by successful agglutination assays, but also a low participation of unspecific interactions for polymers without mannosyl derivatives was observed. On the other hand, the oxidation of amino acids of Concanavalin A was monitored by acrylamide gel electrophoresis. Type I and Type II photosensitization were observed with the formation of dimers and fragments with lower molecular weight, while in dark conditions only products with lower molecular weight were observed, result consistent with singlet oxygen released by pyridone endoperoxides.

Vampire bats rapidly fuel running with essential or non-essential amino acids from a blood meal.

In most mammals, running is fuelled by oxidization of endogenous carbohydrates and lipids while amino acids contribute little (< 5-10%). Common vampire bats (Desmodus rotundus), however, specialize on a unique, protein-rich blood diet. Therefore, we hypothesized that (i) vampire bats would rapidly begin utilizing dietary amino acids to support running metabolism, and (ii) that relative reliance on essential and non-essential amino acids would be similar. We fed bats cow's blood enriched either with isotopically labelled glycine (non-essential amino acid) or leucine (essential amino acid). Bats were exercised at speeds of 10, 20 and 30 m min-1 on a respirometry treadmill, allowing us to assess metabolic rate (i.e. O2 consumption and CO2 production) and track the oxidation of labelled amino acids in exhaled CO2. Vampire bats oxidized amino acids as their primary fuel as indicated by a respiratory exchange ratio (RER = ratio of CO2 production to O2 consumption rates) of approximately 0.8-0.9 at all speeds, with the labelled meal accounting for as much as 60% of oxidized fuels at peak usage. Similar oxidation rates indicated bats did not discriminate between essential and non-essential amino acid use. These findings reiterate how strongly metabolism can be shaped by a specialized diet.

Towards a dynamic model of indispensable amino acid requirements of the adult human: A factorial estimate of oxidative amino acid losses

BackgroundConsensus regarding the required intake of indispensable amino acids (IDAAs) and protein (representing total AAs) in the adult is lacking. Oxidation is a major, though not exclusive, source of IDAA loss in the human body and a primary factor determining requirements; a quantitative understanding of oxidative IDAA losses is required. ObjectiveTo develop a factorial diurnal model of total oxidative IDAA and protein losses in the adult human. MethodsA factorial diurnal model of oxidative losses of protein and each IDAA at maintenance was developed by estimating the magnitude and variability of sources of oxidative loss from existing literature: inevitable catabolism (constitutive oxidation of each absorbed dietary AA), and protein turnover in the postprandial and postabsorptive states. Total oxidative losses were calculated by summing individual losses, validated against published independent nitrogen balance data and compared to current IDAA requirements. ResultsThe factorial model predicted minimum oxidative total AA losses of 390±60 mg.kgBW-1.day-1, 59% of the EAR for protein. Inevitable AA oxidation and oxidation associated with postabsorptive protein turnover were the major sources of the oxidative loss for protein, at 40% and 44%, respectively. Summed oxidative IDAA losses ranged from 64% (isoleucine) to 91% (tryptophan) of current requirements. Total oxidative losses predicted by the model were significant predictors of actual experimental oxidative losses obtained by nitrogen balance (R2=0.66; p=0.049). ConclusionsThe use of a factorial model for estimation of minimum IDAA and protein oxidative losses in the adult human provides an essential starting point for an updated understanding of protein and IDAA requirements. Further iterations of the model will estimate total protein and IDAA requirements, and account for variations in dietary protein quantity and quality, as well as different populations and physiological states. Additional data, especially for inevitable oxidation in humans, and particularly with respect to individual IDAAs, are needed.

Stability of the Glycated Amino Acid 6-(2-Formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine (Pyrraline) During Oxidation.

Food proteins may be modified during processing and storage through reactions with reducing sugars (Maillard reaction, glycation) or by reactive oxygen species (protein oxidation). Little is known about particular reactions at the interface of glycation and oxidation. In the present study, the glycated amino acid pyrraline (6-(2-formyl-5-hydroxymethyl-1-pyrrolyl)-l-norleucine) and the proteinogenic amino acids tyrosine and tryptophan were subjected to different types of oxidation. The stability of the amino acids was assessed by HPLC with UV detection, whereas oxidation products were assigned by HPLC with triple quadrupole or time-of-flight mass spectrometric detection. Conditions that lead to oxidation of aromatic proteinogenic amino acids can also lead to oxidation of pyrraline. Pyrraline was particularly unstable in the presence of permanganate, hypochlorite, and under hydroxyl radical-generating conditions (iron, ethylenediaminetetraacetic acid, ascorbic acid). Evidence obtained by high-resolution mass spectrometry revealed the oxidation of pyrraline to 6-(2,5-diformyl-1-pyrrolyl)-l-norleucine, 6-(2-carboxy-5-hydroxymethyl-1-pyrrolyl)-l-norleucine, 6-(2-formyl-5-carboxy-1-pyrrolyl)-l-norleucine, and 6-(2,5-dicarboxy-1-pyrrolyl)-l-norleucine in the presence of potassium permanganate. The latter product was isolated by semipreparative HPLC and characterized by NMR. Under hydroxyl radical-generating conditions, pyrraline is hydroxylated at the ring under formation of 6-(2-formyl-4-hydroxy-5-hydroxymethyl-1-pyrrolyl)-l-norleucine or 6-(2-formyl-3-hydroxy-5-hydroxymethyl-1-pyrrolyl)-l-norleucine. This study shows that the so-called "advanced glycation end products" are no end products of the Maillard reaction, but may undergo further chemical degradation reactions.

Protein requirements in adults with phenylketonuria and bioavailability of glycomacropeptide compared to an l-amino acid-based product.

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase deficiency. Treatment is primarily a low-Phe diet combined with l-amino acid-based products (l-AA). Protein requirements in adults with PKU have not been directly determined. A formula with glycomacropeptide (GMP) and low phenylalanine is available, yet untested for optimal protein synthesis. To determine the protein requirements in adults with PKU and the bioavailability of GMP-AA in the same patients using the indicator amino acid oxidation (IAAO) technique. Each participant was allocated to 7 separate l-AA intakes (range: 0.1-1.8 g/kg/day) in Experiment 1. In Experiment 2, the same patients participated in 4 GMP-AA intakes (range: 0.1-0.9 g/kg/day). The IAAO method with l-[1-13C]-lysine as the indicator amino acid and its oxidation to 13CO2 was used as the primary indicator of protein synthesis. Protein requirements were identified with a breakpoint, and bioavailability was determined by comparing 13CO2 slope from GMP-AA versus l-AA. Six adults with PKU (4 M: 2F) completed a total of 54 study days over the 2 experiments. The estimated average requirement (EAR) for protein was determined to be 1.11 g/kg/day (R2 = 0.20). The bioavailability of protein from GMP-AA was determined to be 99.98%, which was high and near to 100% comparable to l-AA; although, the results apply only to the tested GMP-AA blend. To our knowledge, this is the first study to directly define a quantitative protein requirement and indicates that current PKU protein recommendations for adults with PKU may be underestimated. The bioavailability of protein in the GMP-AA blend was high and optimal for protein synthesis in adults with PKU.

Metabolic Shifts Induced by Helminth Infections and Their Contribution to Stunting in Vulnerable Populations

Aims: to revisit the condition of chronic metabolic shifting caused by immunomodulatory sequence facilitated by helminth infection together with other deteriorating condition such as persistent exposure to infection which contributes to stunting formation. Discussion: During dynamic interaction between active helminth infection and the host, the helminth and its excretory/secretory products induce and arouse the type 2 immune response which drives host tolerance and plays an important role in promoting tissue repair. Helminths also activates M2 Macrophages and induce a metabolic shift, even metabolic reprogramming towards reliance on oxidative phosphorylation, lipid oxidation and amino acid metabolism. Helminth-induced activation and metabolic reprogramming of macrophages precede the improvement in overall whole-body metabolism, denoted by improved insulin sensitivity, body mass in response to high-fat diet and atherogenic index in mammals. Contributions of altered nutrient uptake, adipose tissue function and/or the intestinal microbiota with the ability of helminths to alter metabolic status play a pivotal role in increased metabolism rate and may lead to wasting and even stunting formation. Conclusion: Helminth drives the type 2 immunity and activates its cell line which is responsible for metabolic shifting and reprogramming.

Insight into the oxidation mechanism of coconut globulin by atmospheric cold plasma focusing on side chain amino acids

Atmospheric cold plasma (ACP), a novel non-thermal processing technology, generates active substances that stimulate protein oxidation in protein-based foods. Nevertheless, the precise mechanism through which ACP initiates amino acid oxidation on protein side chains remains ambiguous. This study primarily aimed to elucidate the mechanism of ACP-induced oxidation of coconut globulin, focusing on the process of amino acid oxidation. Analysis of protein oxidation products indicated a positive correlation between the extent of protein oxidation and the voltage and duration of ACP treatment. By analyzing the composition of amino acids and active ingredients, the study identified that the most significant changes amino acids were methionine, cysteine, and arginine, and •OH was the primary free radicals. The findings from oxidation kinetics and dynamic simulation indicated that •OH predominantly oxidized methionine, followed by L-cysteine and L-arginine. These results offer theoretical framework for understanding protein oxidation by ACP and suggest potential applications in protein-based food.

Flowering in aged white tea: Recovering umami taste and amplifying of stale aroma

Throughout the natural aging process from new to aged white tea, the flavor evolves into a ‘stale flavor’, despite the initial umami diminishes. The flowering process, inoculation of Eurotium cristatum to white tea, improves the flavor. The impact on sensory qualities and underlying chemical basis of flowering in aged white tea warrant investigation. Sensory analysis, non-targeted metabolomics and volatilomics together deciphered flavor modifications of flowering in aged white tea from different aging years (FAWTs). Findings indicate the flowering process can recover the umami of aged white tea, enhancing the ‘stale flavor’. These changes primarily stem from oxidations of catechins and free amino acids, enrichments of flavonols and soluble sugars, and 16 pivotal aroma compounds from degradations of lipids and glycosides. Additionally, 15 volatile and 39 non-volatile compounds were identified as potential biomarkers for FAWTs. These findings offer a viable strategy to improving the quality of aged white tea.

Novel Pink Pigments Produced by Thermal Interaction of Theaflavins, Theanine, and Glucose: Color Formation, Isolation, and Structural Characterization.

A color-deepening effect of theaflavins on the theanine-glucose thermal reaction model was revealed. Generated chromogenic intermediates in the initial stage and an accelerated browning rate through the promoted degradation of theanine-glucose Amadori rearrangement product in the intermediate and final stages are responsible for the color-deepening effect. Four pink-to-red theaflavin-theanine intermediates were verified as theaflavinies referencing the nuclear magnetic resonance and liquid chromatography-mass spectrometry information on theaflavins and l-theanine, including one accurately identified as theaflavinie 4. Theaflavinie 4 showed two maximum absorption peaks at 401 and 506 nm with parallel intensities, which resulted in a significant dichromic color change from pale pink to orange and red. Theaflavinies also could undergo further thermal reactions to yield brown polymers under higher temperatures (130 and 140 °C). This research provided new insight into realizing thermally formed polymers during black tea processing, which may be formed by oxidation products and amino acids or proteins through non-enzymatic thermal reactions.

PSIX-13 Methionine requirements across the life cycle of working Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Abstract Methionine is a limiting amino acid in canine diets, but few requirement studies have been conducted across life stages in large-breed dogs. The objective of this study was to determine the minimum requirement (MR) for methionine (Met) in Labrador retrievers at 4 life stages using the indicator amino acid (AA) oxidation (IAAO) technique. In total, 24 dogs were used: 6 puppies aged 10 to 14 wk, 6 puppies aged 14 wk to 9 mo, 6 adults aged 2 to 5 yr, and 6 seniors aged over 8 yr in feeding studies to evaluate the changing requirements of methionine (Met) as canines age. After 2-d adaptation to Met-adequate basal diet (Met = 0.56% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of Met ranging from deficient to sufficient (final Met content in experimental diets was 0.1982, 0.2386, 0.279, 0.49, 0.525, 0.56% dry matter, with indispensable amino acids formulated at 1.6x NRC values. Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C]phenylalanine based on body weight was first given, followed by [1-13C]Phe doses every 30 min spanning a 4-h period), and breath samples were collected via respiration mask every 30 min. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total CO2 production measured via indirect calorimetry and isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for Met were estimated to be 0.84 ± 0.05 g/1000 kcal (mean ± 2SD) for puppies 10 to 14 wk, 0.78 ± 0.16 g/1,000kcal (mean ± 2SD) for puppies 14 wk to -9 mo, 0.68 ± 0.19 g/1,000kcal (mean ± 2SD) for adults 2 to 5 yr, and 0.62 ± 0.17 g/1,000kcal (mean ± 2SD) for seniors. This research provides important information to assist in accurate formulation of diets that contain appropriate methionine for large breed dogs at all life stages.

PSLBI-17 Total sulfur amino acid requirements across the canine life cycle in Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Abstract Methionine and cysteine are the principal sulfur-containing amino acids that are incorporated into proteins, although homocysteine and taurine also have important physiological roles in canines. As limited studies have been conducted for total sulfur-containing amino acids (TSAA) requirements across the canine life cycle, our objective was to determine the minimum requirements (MR) for TSAA at 4 life stages using the indicator amino acid oxidation (IAAO) technique. Labrador Retrievers (n = 24) were used in feeding studies to evaluate the changing requirements of TSAA as dogs mature: 6 puppies aged 10 to 14 wk, 6 puppies &amp;gt;14 wk to 9 mos, 6 adults 2 to 5 yr (yr), and 6 seniors &amp;gt; 8yr. Following a 2-d adaptation to a TSAA-adequate basal diet (TSAA = 1.12% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of TSAA ranging from deficient to sufficient (final TSAA content in experimental diets were 0.41, 0.46, 0.51, 0.98, 1.05, and 1.12% dry matter, with indispensable amino acids formulated at 1.6x NRC values). The test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was included (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 spanning 4 h). Breath samples were collected via a respiration mask every 30 min. Total CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for TSAA were estimated to be 1.55 ± 0.069 g/1,000 kcal (mean ± 2 SD) for puppies 10 to 14 wk, 1.53 ± 0.21 g/1,000 kcal (mean ± 2 SD) for puppies 14 wk to 9 mo, 1.4 ± 0.30 g/1000 kcal (mean ± 2 SD) for adults 2 to 5 yr, and 1.27 ± 0.23 g/1,000 kcal (mean ± 2 SD) for seniors. These results may contribute to the formulation of diets that meet the age-appropriate requirements of TSAA for large-breed dogs at all life stages.

PSLBI-18 Isoleucine requirements of young adult and senior Labrador retrievers

Abstract Age-appropriate diets that meet the unique nutritional needs of aging are in greater demand as senior dogs represent an increasing percentage of the pet population. Studies targeting the specific amino acid requirements of the aging canine are important because they may inform dietary formulations for this specific life stage. In this study, the indicator amino acid oxidation (IAAO) technique was used to determine the minimum requirements for isoleucine (Iso) in young adult and senior Labrador retrievers. After 2-d adaptation to Iso-adequate basal diet (Iso = 0.608% dry matter), dogs underwent individual IAOO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of isoleucine ranging from deficient to sufficient (final Iso content in experimental diets were 0.244, 0.285, 0.326, 0.532, 0.57 and 0.608% dry matter). Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 min spanning a 4 h period), and breath samples were collected via respiration mask every 30 min. CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for isoleucine were 898 ± 71 mg/1,000 kcal ME (mean ± 2 SD) for young adults and 885 ± 91 mg/1,000 kcal ME (mean ± 2 SD) in senior dogs, which is slightly greater than the National Research Council’s minimum requirement. As the pet food industry offers more specialized diets for aging canines, updating the amino acid requirements for such animals is increasingly important.

Dietary starch structure modulates nitrogen metabolism in laying hens via modifying glucose release rate

The objective of this study was to investigate the effects of starch structure (Amylopectin/Amylose, AP/AM) in a low-protein diet on production performance, nitrogen utilization efficiency, and cecal flora in laying hens. Four hundred eighty 45-wk-age Hy-Line Gray laying hens were randomly allocated to five dietary groups and subjected to a 12-wk feeding trial. The AP/AM ratios of the five experiment diets were 1.0, 1.5, 2.0, 3.0, and 4.0. The results indicated that compared to other groups, laying hens fed with AP/AM 4.0 diets showed significantly improved average egg weight and feed conversion ratio (P < 0.05). Furthermore, as the AP/AM ratio increased, there was a significant linear enhancement in intestinal amino acids apparent digestibility, apparent metabolizable energy, and villus area (P < 0.05). Compared to the high AP groups, high-AM diets significantly increased eggshell thickness, crude protein digestibility, and reduced energy supply from amino acid oxidation in ileum (P < 0.05). Additionally, moderate-AM diets enriched with short-chain fatty acid-producing bacteria in the cecum, such as Lactobacillus, Rikenellaceae_RC9_gut_group, and Christensenellaceae_R-7_group, which are associated with the promoting nitrogen utilization. These findings may offer useful information on optimizing starch structure for the design of food products and relevant therapies due to the potential effects on nutrient metabolism and gut homeostasis.

Heterotrophic nitrification by Alcaligenes faecalis links organic and inorganic nitrogen metabolism.

Heterotrophic nitrification remains a mystery for decades. It has been commonly hypothesized that heterotrophic nitrifiers oxidize ammonia to hydroxylamine and then to nitrite in a way similar to autotrophic AOA and AOB. Recently, heterotrophic nitrifiers from Alcaligenes were found to oxidize ammonia to hydroxylamine and then to N2 ("dirammox", direct ammonia oxidation) by the gene cluster dnfABC with a yet-to-be-reported mechanism. The role of a potential glutamine amidotransferase DnfC clues the heterotrophic ammonia oxidation might involving in glutamine. Here, we found Alcaligenes faecalis JQ135 could oxidize amino acids besides ammonia. We discovered that glutamine is an intermediate of the dirammox pathway and the glutamine synthetase gene glnA is essential for both A. faecalis JQ135 and the Escherichia coli cells harboring dnfABC gene cluster to oxidize amino acids and ammonia. Our study expands understanding of heterotrophic nitrifiers and challenges the classical paradigm of heterotrophic nitrification.

Chicken meal is not an appropriate reference protein for estimating protein quality of ingredients used in extruded diets intended for dogs.

The indicator amino acid oxidation (IAAO) method has been used to determine metabolic availability (MA) of amino acids in feedstuffs for pigs, humans, and preliminarily for cats. Peas are a commonly used protein source in grain-free extruded dog diets. However, peas have a poor sulfur amino acid (AA) ratio (methionine [Met]:cysteine) with Met being the first limiting AA. Furthermore, little is known about the MA of Met in peas fed to dogs. Therefore, our objective was to compare the MA of Met in peas to chicken meal (CM), as a gold-standard reference protein. The study was done as a replicated 5 × 5 complete Latin square design. Ten neutered male mixed-breed dogs (1.5 years old; 26.0 kg ± 2.4kg body weight; BW) fed to maintain ideal BW received all dietary treatments: BAS: lamb-based diet (deboned lamb and lamb meal) providing Met at 50% of its requirement (0.27g/100g dry matter [DM]), CHK: CM and lamb-based diet, and PEA: ground dried pea and lamb-based diet both providing Met at 68% of its requirement (0.35 and 0.37g/100g DM, respectively). Two other treatments were created by blending BAS with PEA (BAP) and the BAS with CHK (BAC) to create diets with Met at 59% of requirement (0.32 and 0.31g/100g DM, respectively). This resulted in three graded levels of Met for both CM and peas to allow for a slope-ratio assay approach to quantify MA with the BAS diet as the common first point. All other AAs were provided to meet at least 120% of the AAFCO recommendations for adult dogs. The BAS diet, with supplemental DL-Met, was fed for a 2-wk wash-in period. After 2 d of diet adaptation IAAO was performed. Dogs were fed 13 small meals where meal 6 contained a priming dose (9.4mg/kg BW) of L-[1-13C]-phenylalanine (Phe; 99%) as well as a constant dose (2.4mg/kg BW) in meals 6-13. Breath samples were collected and enrichment of 13CO2 was measured using isotope-ratio mass spectrometry to calculate the rate of Phe oxidation (F13CO2 umol/kg BW/h). Oxidation was analyzed via SAS using PROC GLIMMIX with dog and period as random effects, and diet, %Met, and their interaction as fixed effects. Unexpectedly, the slope of Phe oxidation, in response to increasing Met intake, from CM was 31% of that of peas, indicating a lower MA for Met in CM as compared to peas. This finding may be due to damage of AAs during rendering. At this time, CM in extruded diets is not an acceptable reference protein to determine MA of AAs in dogs, and the MA of Met from peas cannot be confidently assessed.

Facile synthesis of magnetic ionic covalent organic framework and dispersive magnetic solid phase extraction of aromatic amino acid oxidation products in thermally processed foods

Aromatic amino acid oxidation products (AAAOPs) are newly discovered risk substances of thermal processes. Due to its significant polarity and trace level in food matrices, there are no efficient pre-treatment methods available to enrich AAAOPs. Herein, we proposed a magnetic cationic covalent organic framework (Fe3O4@EB-iCOF) as an adsorbent for dispersive magnetic solid-phase extraction (DMSPE). Benefiting from the unique charged characteristics of Fe3O4@EB-iCOF, AAAOPs can be enriched through electrostatic interaction and π-π interactions. Under the optimal DMSPE conditions, the combined HPLC-MS/MS method demonstrated good linearity (R2 ≥ 0.990) and a low detection limit (0.11–7.5 μg·kg−1) for AAAOPs. In addition, the method was applied to real sample and obtained satisfactory recoveries (86.8 % ∼ 109.9 %). Especially, we applied this method to the detection of AAAOPs in meat samples and conducted a preliminarily study on its formation rules, which provides a reliable basis for assessing potential dietary risks.

Differential utilization of vitamin B12-dependent and independent pathways for propionate metabolism across human cells

Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the β-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the β-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation.

Total sulfur amino acid requirements are higher during late gestation compared with early gestation in healthy Canadian pregnancies in a repeated-measures trial

BackgroundDietary Reference Intake (DRI) Recommendations for total sulfur amino acids (TSAAs; methionine + cysteine) during pregnancy are based on factorial calculations using data from adult males. To date, no data exist on TSAA requirements obtained directly during pregnancy. ObjectivesThe objective of this study was to examine whether TSAA requirements during early (11–20 wk) and late (31–40 wk) gestation in healthy females with singleton pregnancies are different than current recommendations, and different between early and late gestation using the indicator amino acid oxidation (IAAO) technique. MethodsTwenty-five females 20–40 y with a healthy singleton pregnancy were studied using the IAAO technique in a repeated measures design for a total of 70, 8-h d. On each study day a methionine test intake (range: 0–40 mg⋅kg−1⋅d−1) was provided in 8 hourly, isonitrogenous and isocaloric meals with cysteine excluded from the diet. Breath samples were collected at baseline and isotopic steady state of orally provided L-1-13C-Phenylalanine for measurement of phenylalanine oxidation. The requirement was determined using biphasic linear regression crossover analysis to identify a breakpoint in 13CO2 production, representing the estimated average requirement (EAR). ResultsThe TSAA requirement in healthy pregnant participants in early gestation was 11.1 mg⋅kg−1⋅d−1 {R2m = 0.79, R2c = 0.79; 95% confidence interval [CI] (8.9, 13.3 mg⋅kg−1⋅d−1)} and 15.0 mg⋅kg−1⋅d−1 (R2m = 0.72, R2c = 0.79; 95% CI [13.0, 17.0 mg⋅kg−1⋅d−1]) in late gestation. The difference between confidence intervals of the 2 breakpoints was = −3.9 ± 3.0, and statistically different. ConclusionsWe directly measured TSAA requirements in healthy pregnant mothers, and our findings suggest that requirements are lower than current DRI recommendations of 20 and 25 mg⋅kg−1⋅d−1, as the EAR, and Recommended Dietary Allowance, respectively. Late gestation TSAA needs are significantly different and increased 35% compared with early gestation. Recommendations for TSAA intake need to be tailored for gestational stage.This clinical trial was registered at clinicaltrials.gov as NCT04326322.