Excessive Tryptophan and Phenylalanine Induced Pancreatic Injury and Glycometabolism Disorder in Grower-finisher Pigs.

BACKGROUND: The increase in circulating insulin levels is associated with the onset of type 2 diabetes (T2D), and the levels of branched-chain amino acids and aromatic amino acids (AAAs) are altered in T2D, but whether AAAs play a role in insulin secretion and signaling remains unclear. OBJECTIVES: This study aimed to investigate the effects of different AAAs on pancreatic function and on the use of insulin in finishing pigs. METHODS: A total of 18 healthy finishing pigs (Large White) with average body weight of 100 ± 1.15 kg were randomly allocated to 3 dietary treatments: Con, a normal diet supplemented with 0.68% alanine; Phe, a normal diet supplemented with 1.26% phenylalanine; and Trp, a normal diet supplemented with 0.78% tryptophan. The 3 diets were isonitrogenous. There were 6 replicates in each group. RESULTS: Herein, we investigated the effects of tryptophan and phenylalanine on pancreatic function and the use of insulin in finishing pigs and found that the addition of tryptophan and phenylalanine aggravated pancreatic fat deposition, increased the relative content of saturated fatty acids, especially palmitate (C16:0) and stearate (C18:0), and the resulting lipid toxicity disrupted pancreatic secretory function. We also found that tryptophan and phenylalanine inhibited the growth and secretion of ß-cells, downregulated the gene expression of the PI3K/Akt pathway in the pancreas and liver, and reduced glucose utilization in the liver. CONCLUSIONS: Using fattening pigs as a model, multiorgan combined analysis of the insulin-secreting organ pancreas and the main insulin-acting organ liver, excessive intake of tryptophan and phenylalanine will aggravate pancreatic damage leading to glucose metabolism disorders, providing new evidence for the occurrence and development of T2D.

Traditional medicinal use is linked with apparency, not specialized metabolite profiles in the order Caryophyllales.

PREMISE: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism. METHODS: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF). RESULTS: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use. CONCLUSIONS: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.

A metabolomics approach reveals metabolic disturbance of human cholangiocarcinoma cells after parthenolide treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanacetum parthenium (L.) Schultz-Bip, commonly known as feverfew, has been traditionally used to treat fever, migraines, rheumatoid arthritis, and cancer. Parthenolide (PTL), the main bioactive ingredient isolated from the shoots of feverfew, is a sesquiterpene lactone with anti-inflammatory and antitumor properties. Previous studies showed that PTL exerts anticancer activity in various cancers, including hepatoma, cholangiocarcinoma, acute myeloid leukemia, breast, prostate, and colorectal cancer. However, the metabolic mechanism underlying the anticancer effect of PTL remains poorly understood. AIM OF THE STUDY: To explore the anticancer activity and underlying mechanism of PTL in human cholangiocarcinoma cells. MATERIAL AND METHODS: In this investigation, the effects and mechanisms of PTL on human cholangiocarcinoma cells were investigated via a liquid chromatography/mass spectrometry (LC/MS)-based metabolomics approach. First, cell proliferation and apoptosis were evaluated using cell counting kit-8 (CCK-8), flow cytometry analysis, and western blotting. Then, LC/MS-based metabolic profiling along with orthogonal partial least-squares discriminant analysis (OPLS-DA) has been constructed to distinguish the metabolic changes between the negative control group and the PTL-treated group in TFK1 cells. Next, enzyme-linked immunosorbent assay (ELISA) was applied to investigate the changes of metabolic enzymes associated with significantly alerted metabolites. Finally, the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established using MetaboAnalyst 5.0 and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database. RESULTS: PTL treatment could induce the proliferation inhibition and apoptosis of TFK1 in a concentration-dependent manner. Forty-three potential biomarkers associated with the antitumor effect of PTL were identified, which primarily related to glutamine and glutamate metabolism, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, arginine and proline metabolism, glutathione metabolism, nicotinate and nicotinamide metabolism, pyrimidine metabolism, fatty acid metabolism, phospholipid catabolism, and sphingolipid metabolism. Pathway analysis of upstream and downstream metabolites, we found three key metabolic enzymes, including glutaminase (GLS), γ-glutamyl transpeptidase (GGT), and carnitine palmitoyltransferase 1 (CPT1), which mainly involved in glutamine and glutamate metabolism, glutathione metabolism, and fatty acid metabolism. The changes of metabolic enzymes associated with significantly alerted metabolites were consistent with the levels of metabolites, and the metabolic network related to key metabolic enzymes, metabolites, and metabolic pathways was established. PTL may exert its antitumor effect against cholangiocarcinoma by disturbing metabolic pathways. Furthermore, we selected two positive control agents that are considered as first-line chemotherapy standards in cholangiocarcinoma therapy to verify the reliability and accuracy of our metabolomic study on PTL. CONCLUSION: This research enhanced our comprehension of the metabolic profiling and mechanism of PTL treatment on cholangiocarcinoma cells, which provided some references for further research into the anti-cancer mechanisms of other drugs.

Supplementation for Performance and Health in Patients with Phenylketonuria: An Exercise-Based Approach to Improving Dietary Adherence.

Supplementation is crucial for improving performance and health in phenylketonuria (PKU) patients, who face dietary challenges. Proteins are vital for athletes, supporting muscle growth, minimizing catabolism, and aiding muscle repair and glycogen replenishment post-exercise. However, PKU individuals must limit phenylalanine (Phe) intake, requiring supplementation with Phe-free amino acids or glycomacropeptides. Tailored to meet nutritional needs, these substitutes lack Phe but fulfill protein requirements. Due to limited supplement availability, athletes with PKU may need higher protein intake. Various factors affect tolerated Phe levels, including supplement quantity and age. Adhering to supplement regimens optimizes performance and addresses PKU challenges. Strategically-timed protein substitutes can safely enhance muscle synthesis and sports performance. Individualized intake is essential for optimal outcomes, recognizing proteins' multifaceted role. Here, we explore protein substitute supplementation in PKU patients within the context of physical activity, considering limited evidence.

Exploration of Raw Pigmented-Fleshed Sweet Potatoes Volatile Organic Compounds and the Precursors.

Sweet potato provides rich nutrients and bioactive substances for the human diet. In this study, the volatile organic compounds of five pigmented-fleshed sweet potato cultivars were determined, the characteristic aroma compounds were screened, and a correlation analysis was carried out with the aroma precursors. In total, 66 volatile organic compounds were identified. Terpenoids and aldehydes were the main volatile compounds, accounting for 59% and 17%, respectively. Fifteen compounds, including seven aldehydes, six terpenes, one furan, and phenol, were identified as key aromatic compounds for sweet potato using relative odor activity values (ROAVs) and contributed to flower, sweet, and fat flavors. The OR sample exhibited a significant presence of trans-ß-Ionone, while the Y sample showed high levels of benzaldehyde. Starch, soluble sugars, 20 amino acids, and 25 fatty acids were detected as volatile compounds precursors. Among them, total starch (57.2%), phenylalanine (126.82 ± 0.02 g/g), and fatty acids (6.45 µg/mg) were all most abundant in Y, and LY contained the most soluble sugar (14.65%). The results of the correlation analysis revealed the significant correlations were identified between seven carotenoids and trans-ß-Ionone, soluble sugar and nerol, two fatty acids and hexanal, phenylalanine and 10 fatty acids with benzaldehyde, respectively. In general, terpenoids and aldehydes were identified as the main key aromatic compounds in sweet potatoes, and carotenoids had more influence on the aroma of OR than other cultivars. Soluble sugars, amino acids, and fatty acids probably serve as important precursors for some key aroma compounds in sweet potatoes. These findings provide valuable insights for the formation of sweet potato aroma.

[Clinical metabolomics of Zicuiyin in treatment of diabetic kidney disease].

This study aimed to analyze the therapeutic effect of Zicuiyin on diabetic kidney disease(DKD) and explore the possible targets of this formula. Eighteen DKD patients treated in the endocrine department or nephrology department of Second Affilia-ted Hospital of Tianjin University of Traditional Chinese Medicine from January to December in 2019 were enrolled and assigned into a test group(n=10) and a control group(n=8). Both groups received routine chemical medicine treatment. In addition, the test group was treated with Zicuiyin and the control group with Huangkui Capsules for 8 weeks. The clinical trial was approved by the Ethics Committee of Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, with the ethical approval No. 2017-023-01, and all the patients signed the informed consent form. The results showed that the 8-week treatment with Zicuiyin lowered the level of glycosylated hemoglobin(HbA1c) and recovered the 24 h urinary protein(24hUP), 24 h urinary microalbumin(24hmAlb), urine albumin-to-creatinine ratio(UACR), and estimated glomerular filtration rate(eGFR) of the patients with 24hUP<3.5 g. According to the different levels in 24hUP, all the patients were divided into two subgroups(subgroup A with 24hUP<3.5 g and subgroup B with 24hUP≥3.5 g). The ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS)-based non-targeted metabolomics analysis was conducted on the baseline serum samples from diffe-rent subgroups of patients. Nineteen biomarker candidates were identified to distinguish the metabolic differences between the two subgroups, and their correlations with clinical indicators were analyzed. Zicuiyin lowered the levels of phenylalanine, pseudouridine, and adenosine [fold change(FC)<0.5, P<0.05] in subgroup A. The results indicated that Zicuiyin was more effective on the DKD patients with low urinary protein levels, and its targets were involved in phenylalanine metabolism and nucleoside metabolism.

Exploring metabolic responses and pathway changes in CHO-K1 cells under varied aeration conditions and copper supplementations using 1 H NMR-based metabolomics.

The optimization of bioprocess for CHO cell culture involves careful consideration of factors such as nutrient consumption, metabolic byproduct accumulation, cell growth, and monoclonal antibody (mAb) production. Valuable insights can be obtained by understanding cellular physiology to ensure robust and efficient bioprocess. This study aims to improve our understanding of the CHO-K1 cell metabolism using 1 H NMR-based metabolomics. Initially, the variations in culture performance and metabolic profiles under varied aeration conditions and copper supplementations were thoroughly examined. Furthermore, a comprehensive metabolic pathway analysis was performed to assess the impact of these conditions on the implicated pathways. The results revealed substantial alterations in the pyruvate metabolism, histidine metabolism, as well as phenylalanine, tyrosine and tryptophan biosynthesis, which were especially evident in cultures subjected to copper deficiency conditions. Conclusively, significant metabolites governing cell growth and mAb titer were identified through orthogonal partial least square-discriminant analysis (OPLS-DA). Metabolites, including glycerol, alanine, formate, glutamate, phenylalanine, and valine, exhibited strong associations with distinct cell growth phases. Additionally, glycerol, acetate, lactate, formate, glycine, histidine, and aspartate emerged as metabolites influencing cell productivity. This study demonstrates the potential of employing 1 H NMR-based metabolomics technology in bioprocess research. It provides valuable guidance for feed medium development, feeding strategy design, bioprocess parameter adjustments, and ultimately the enhancement of cell proliferation and mAb yield.

Unraveling the pharmacodynamic substances and possible mechanism of Trichosanthis Pericarpium in the treatment of coronary heart disease based on plasma pharmacochemistry, network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Coronary heart disease (CHD) is a chronic disease that seriously threatens people's health and even their lives. Currently, there is no ideal drug without side effects for the treatment of CHD. Trichosanthis Pericarpium (TP) has been used for several years in the treatment of diseases associated with CHD. However, there is still a need for systematic research to unravel the pharmacodynamic substances and possible mechanism of TP in the treatment of coronary heart. AIM OF THE STUDY: The purpose of current study was to explore the pharmacodynamic substances and potential mechanisms of TP in the treatment of CHD via integrating network pharmacology with plasma pharmacochemistry and experimental validation. MATERIALS AND METHODS: The effect of TP intervention in CHD was firstly assessed on high-fat diet combined with isoprenaline-induced CHD rats and H2O2-induced H9c2 cells, respectively. Then, the LC-MS was utilized to identify the absorbed components of TP in the plasma of CHD rats, and this was used to develop a network pharmacology prediction to obtain the possible active components and mechanisms of action. Molecular docking and immunohistochemistry were used to explore the interaction between TP and key targets. Subsequently, the efficacy of the active ingredients was investigated by in vitro cellular experiments, and their metabolic pathways in CHD rats were further analyzed. RESULTS: The effects of TP on amelioration of CHD were verified by in vivo and in vitro experiments. Plasma pharmacochemistry and network pharmacology screened six active components in plasma including apigenin, phenylalanine, quercetin, linoleic acid, luteolin, and tangeretin. The interaction of these compounds with potential key targets AKT1, IL-1ß, IL-6, TNF-α and VEGFA were preliminarily verified by molecular docking. And immunohistochemical results showed that TP reduced the expression of AKT1, IL-1ß, IL-6, TNF-α and VEGFA in CHD rat hearts. Then cellular experiments confirmed that apigenin, phenylalanine, quercetin, linoleic acid, luteolin, and tangeretin were able to reduce the ROS level in H2O2-induced HUVEC cells and promote the migration and tubule formation of HUVEC cells, indicating the pharmacodynamic effects of the active components. Meanwhile, the metabolites of TP in CHD rats suggested that the pharmacological effects of TP might be the result of the combined effects of the active ingredients and their metabolites. CONCLUSION: Our study found that TP intervention in CHD is characterized by multi-component and multi-target regulation. Apigenin, phenylalanine, linoleic acid, quercetin, luteolin, and tangeretin are the main active components of TP. TP could reduce inflammatory response and endothelial damage by regulating AKT1, IL-1ß, IL-6, TNF-α and VEGFA, reduce ROS level to alleviate the oxidative stress situation and improve heart disease by promoting angiogenesis to regulate endothelial function. This study also provides an experimental and scientific basis for the clinical application and rational development of TP.

Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

Outcomes in 14 live births resulting from Pegvaliase-treated pregnancies in PKU-affected females.

BACKGROUND: Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range that is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring resulting from pregnancies with poor Phe control in women with PKU. Enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet and have plasma Phe levels in a safe range for pregnancy but pegvaliase has not been approved for use in pregnant females with PKU. We report the results of chart review of 14 living offspring of females affected with PKU who were responsive to pegvaliase and chose to remain on pegvaliase throughout their pregnancy. METHODS: Fourteen pregnancies (one triplet pregnancy) and their offspring were identified at eight PKU treatment centers and medical records from pregnancy and birth were submitted for this study. Institutional Review Board approval was obtained. Responses to a dataset were provided to a single center and analyzed. RESULTS: Six females and eight males were born without congenital anomalies and all offspring had normal growth parameters. While mothers had preexisting comorbidities, no additional comorbidities were reported in the offspring. Four of eleven infants (excluding triplet pregnancies) were delivered preterm (36%), a higher rate than the general population (12%). A single first trimester (eight weeks) miscarriage in a 40y was not counted in this cohort of 14 live born infants. CONCLUSION: This retrospective study suggests that pegvaliase is effective at maintaining safe maternal blood Phe levels during pregnancy without deleterious effects on mother or child. A tendency toward premature birth (4/11; 36%) is higher than expected.

Volatile Organic Compounds (VOCs) Produced by Levilactobacillus brevis WLP672 Fermentation in Defined Media Supplemented with Different Amino Acids.

Fermentation by lactic acid bacteria (LAB) is a promising approach to meet the increasing demand for meat or dairy plant-based analogues with realistic flavours. However, a detailed understanding of the impact of the substrate, fermentation conditions, and bacterial strains on the volatile organic compounds (VOCs) produced during fermentation is lacking. As a first step, the current study used a defined medium (DM) supplemented with the amino acids L-leucine (Leu), L-isoleucine (Ile), L-phenylalanine (Phe), L-threonine (Thr), L-methionine (Met), or L-glutamic acid (Glu) separately or combined to determine their impact on the VOCs produced by Levilactobacillus brevis WLP672 (LB672). VOCs were measured using headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). VOCs associated with the specific amino acids added included: benzaldehyde, phenylethyl alcohol, and benzyl alcohol with added Phe; methanethiol, methional, and dimethyl disulphide with added Met; 3-methyl butanol with added Leu; and 2-methyl butanol with added Ile. This research demonstrated that fermentation by LB672 of a DM supplemented with different amino acids separately or combined resulted in the formation of a range of dairy- and meat-related VOCs and provides information on how plant-based fermentations could be manipulated to generate desirable flavours.

LipiFlow for the treatment of dry eye disease.

BACKGROUND: Meibomian gland dysfunction (MGD) is the most common underlying cause of dry eye disease (DED). MGD leads to pathological alteration of the composition or quantity of meibum, or both, which subsequently results in tear evaporation and the typical signs and symptoms associated with DED. The LipiFlow Thermal Pulsation System (LipiFlow) is a medical device used to treat MGD in office; however, it is unclear if LipiFlow can outperform other DED treatments. OBJECTIVES: To evaluate the effectiveness of LipiFlow for treating DED signs and symptoms and the safety of LipiFlow compared with sham or other available treatments for MGD in adults. SEARCH METHODS: The Cochrane Eyes and Vision Information Specialist searched the electronic databases for randomized controlled trials. There were no restrictions on language or date of publication. We searched the Cochrane Central Register of Controlled Trials (CENTRAL, including the Cochrane Eyes and Vision Trials Register; 2022, Issue 6), MEDLINE Ovid, Embase.com, PubMed, LILACS (Latin American and Caribbean Health Science Information database), ClinicalTrials.gov, and World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) electronic databases. We also examined the reference lists of identified trials, review articles, and guidelines for information about relevant trials that may not have been identified by our search strategy. We contacted investigators regarding ongoing trials. The last database search was performed on 24 October 2022. SELECTION CRITERIA: We included studies conducted in adults (over 18 years of age) with DED or MGD as defined by the primary trial investigators. We imposed no restrictions on race, ethnicity, or sex. We considered trials involving contact lens wearers if they were equally represented between groups. DATA COLLECTION AND ANALYSIS: We applied standard Cochrane methodology. MAIN RESULTS: We included 13 trials that randomized a total of 1155 participants (28 to 236 participants randomized per study). Six trials were conducted in the USA, three in China, two in Thailand, one in France, and one in Italy. Eight trials were of single-center design, while four trials were of multicenter design; one trial did not report the number of participating centers. Study characteristics The study population of the included trials was 66% female (range 48% to 80%), with an age range of 19 to 86 years. LipiFlow, used as a stand-alone intervention, was compared with basic warm compresses in five studies, thermostatic device in five studies, oral intervention in one trial, and topical dry eye medications in one trial. LipiFlow was also evaluated together with eyelid hygiene product versus eyelid hygiene products alone in one trial. Findings Five trials compared LipiFlow with a basic warm compress applied for varying durations and frequencies during the trial period; only one of these trials combined a warm compress with eyelid massage. Analyzing symptom scores by different questionnaires (Ocular Surface Disease Index [OSDI] and Standard Patient Evaluation of Eye Dryness [SPEED]) yielded conflicting evidence of a difference in symptoms between LipiFlow and basic warm compresses after four weeks. There was no evidence of a difference in meibomian gland expression, meibum quality, or tear breakup time when comparing LipiFlow with basic warm compresses. Another five trials compared LipiFlow with thermostatic devices. Analysis of symptom scores at four weeks showed that thermostatic devices had reduced OSDI scores by a mean difference (MD) of 4.59 (95% confidence interval [CI] 1.23 to 7.95; I2 = 0, P = 0.007; 553 participants; very low certainty evidence) as compared with LipiFlow. When we compared LipiFlow plus eyelid hygiene with eyelid hygiene alone, there was no evidence of difference in signs or symptoms at any time point evaluated. Only one trial compared LipiFlow with a topical DED medication (lifitegrast 5%). The single-trial estimate suggested that 5% lifitegrast may increase meibomian gland expression scores compared with LipiFlow at day 42 (MD -1.21, 95% CI -2.37 to -0.05; 50 participants; low certainty evidence) by using a meibomian gland expression scale of 0 to 8. One trial compared LipiFlow with an oral intervention (doxycycline), finding that LipiFlow may result in significantly better SPEED scores than doxycycline at three months (MD -4.00, 95% CI -7.33 to -0.67; 24 participants; very low certainty evidence). No other significant differences in signs or symptoms were found between LipiFlow and doxycycline at three months. We did not find any other statistically significant differences in symptoms or signs for any other analysis performed in this review at the one- to four-week time point. Adverse events No trial reported any intervention-related, vision-threatening adverse events. AUTHORS' CONCLUSIONS: LipiFlow performs similarly to other commonly used DED treatments with regard to DED signs and symptoms. The best available evidence was deemed to have a high level of bias, leading to low or very low certainty evidence. Additional research with adequate masking, a standardized testing methodology, and a sample representative of the MGD population is therefore needed before any firm conclusions can be drawn regarding comparative benefits and harms.

The impact of forearm immobilization and acipimox administration on muscle amino acid metabolism and insulin sensitivity in healthy, young volunteers.

Although the mechanisms underpinning short-term muscle disuse atrophy and associated insulin resistance remain to be elucidated, perturbed lipid metabolism might be involved. Our aim was to determine the impact of acipimox administration [i.e., pharmacologically lowering circulating nonesterified fatty acid (NEFA) availability] on muscle amino acid metabolism and insulin sensitivity during short-term disuse. Eighteen healthy individuals (age: 22 ± 1 years; body mass index: 24.0 ± 0.6 kg·m-2) underwent 2 days forearm immobilization with placebo (PLA; n = 9) or acipimox (ACI; 250 mg Olbetam; n = 9) ingestion four times daily. Before and after immobilization, whole body glucose disposal rate (GDR), forearm glucose uptake (FGU; i.e., muscle insulin sensitivity), and amino acid kinetics were measured under fasting and hyperinsulinemic-hyperaminoacidemic-euglycemic clamp conditions using forearm balance and l-[ring-2H5]-phenylalanine infusions. Immobilization did not affect GDR but decreased insulin-stimulated FGU in both groups, more so in ACI (from 53 ± 8 to 12 ± 5 µmol·min-1) than PLA (from 52 ± 8 to 38 ± 13 µmol·min-1; P < 0.05). In ACI only, and in contrast to our hypothesis, fasting arterialized NEFA concentrations were elevated to 1.3 ± 0.1 mmol·L-1 postimmobilization (P < 0.05), and fasting forearm NEFA balance increased approximately fourfold (P = 0.10). Forearm phenylalanine net balance decreased following immobilization (P < 0.10), driven by an increased rate of appearance [from 32 ± 5 (fasting) and 21 ± 4 (clamp) preimmobilization to 53 ± 8 and 31 ± 4 postimmobilization; P < 0.05] while the rate of disappearance was unaffected by disuse or acipimox. Disuse-induced insulin resistance is accompanied by early signs of negative net muscle amino acid balance, which is driven by accelerated muscle amino acid efflux. Acutely elevated NEFA availability worsened muscle insulin resistance without affecting amino acid kinetics, suggesting increased muscle NEFA uptake may contribute to inactivity-induced insulin resistance but does not cause anabolic resistance.NEW & NOTEWORTHY We demonstrate that 2 days of forearm cast immobilization in healthy young volunteers leads to the rapid development of insulin resistance, which is accompanied by accelerated muscle amino acid efflux in the absence of impaired muscle amino acid uptake. Acutely elevated fasting nonesterified fatty acid (NEFA) availability as a result of acipimox supplementation worsened muscle insulin resistance without affecting amino acid kinetics, suggesting increased muscle NEFA uptake may contribute to inactivity-induced insulin resistance but does not cause anabolic resistance.

Biochemical Pathways of Salicylic Acid Derived from l-Phenylalanine in Plants with Different Basal SA Levels.

As a plant hormone, salicylic acid (SA) has diverse regulatory roles in plant growth and stress resistance. Although SA is widely found in plants, there is substantial variation in basal SA among species. Tea plant is an economically important crop containing high contents of SA whose synthesis pathway remains unidentified. The phenylalanine ammonia-lyase (PAL) pathway is responsible for basal SA synthesis in plants. In this study, isotopic tracing and enzymatic assay experiments were used to verify the SA synthesis pathway in tea plants and evaluate the variation in phenylalanine-derived SA formation among 11 plant species with different levels of SA. The results indicated that SA could be synthesized via PAL in tea plants and conversion efficiency from benzoic acid to SA might account for variation in basal SA among plant species. This research lays the foundation for an improved understanding of the molecular regulatory mechanism for SA biosynthesis.

Phenylalanine requirements using the direct amino acid oxidation technique, and the effects of dietary phenylalanine on food intake, gastric emptying, and macronutrient metabolism in adult cats.

Despite Phe being an indispensable amino acid for cats, the minimum Phe requirement for adult cats has not been empirically defined. The objective of study 1 was to determine the minimum Phe requirement, where Tyr is in excess, in adult cats using the direct amino acid oxidation (DAAO) technique. Four adult male cats were used in an 8 × 4 Latin rectangle design. Cats were adapted to a basal diet for 7 d, top dressed with Phe to meet 140% of the adequate intake (NRC, 2006. Nutrient requirements of dogs and cats. Washington, DC: Natl. Acad. Press). Cats were randomly assigned to one of eight experimental Phe diets (0.29%, 0.34%, 0.39%, 0.44%, 0.54%, 0.64%, 0.74%, and 0.84% Phe in the diet on a dry matter [DM] basis). Following 1 d of diet adaptation, individual DAAO studies were performed. During each DAAO study, cats were placed into individual indirect calorimetry chambers, and 75% of the cat's daily meal was divided into 13 equal meals supplied with a dose of L-[1-13C]-Phe. Oxidation of L-[1-13C]-Phe (F13CO2) during isotopic steady state was determined from the enrichment of 13CO2 in breath. Competing models were applied using the NLMIXED procedure in SAS to determine the effects of dietary Phe on 13CO2. The mean population minimum requirement for Phe was estimated at 0.32% DM and the upper 95% population confidence limit at 0.59% DM on an energy density of 4,200 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors. In study 2, the effects of a bolus dose of Phe (44 mg kg-1 BW) on food intake, gastric emptying (GE), and macronutrient metabolism were assessed in a crossover design with 12 male cats. For food intake, cats were given Phe 15 min before 120% of their daily food was offered and food intake was measured. Treatment, day, and their interaction were evaluated using PROC GLIMMIX in SAS. Treatment did not affect any food intake parameters (P > 0.05). For GE and macronutrient metabolism, cats were placed into individual indirect calorimetry chambers, received the same bolus dose of Phe, and 15 min later received 13C-octanoic acid (5 mg kg-1 BW) on 50% of their daily food intake. Breath samples were collected to measure 13CO2. The effect of treatment was evaluated using PROC GLIMMIX in SAS. Treatment did not affect total GE (P > 0.05), but cats receiving Phe tended to delay time to peak enrichment (0.05 < P ≤ 0.10). Overall, Phe at a bolus dose of 44 mg kg-1 BW had no effect on food intake, GE, or macronutrient metabolism. Together, these results suggest that the bolus dose of Phe used may not be sufficient to elicit a GE response, but a study with a greater number of cats and greater food intake is warranted.

Two studies were conducted to evaluate 1) the minimum requirement for dietary Phe and 2) the effects of Phe on gastric emptying (GE) and food intake in adult cats. In study 1, the minimum Phe requirement was estimated using the direct amino acid oxidation (DAAO) technique. Four cats were used and received all diets in random order in a Latin rectangle design (0.29%, 0.34%, 0.39%, 0.44%, 0.54%, 0.64%, 0.74%, and 0.84% Phe in the diet on a dry matter [DM] basis). The minimum Phe requirement, in the presence of excess of Tyr, for adult cats was estimated to be 0.59% DM on an energy density of 4,200 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors; higher than current recommendations set in place by the National Research Council and the American Association of Feed Control Officials. In study 2, we first validated the use of the 13C-octanoic acid breath test (13C-OABT) in cats. Then, the effects of an oral bolus of Phe on food intake, GE, and macronutrient metabolism were evaluated. Phe supplementation did not influence food intake, macronutrient metabolism, or total GE, but tended to delay the time to peak GE.

Changes in the Antioxidant Potential of Camellia sinensis Cultures under the Influence of Phenolic Precursors.

The viability, productivity and survival of higher plants under the adverse factors influence are largely determined by the functional activity of the antioxidant system. The aim of our work was to investigate changes in formation of high-molecular (superoxide dismutase and peroxidase) and low-molecular (phenolics, including flavanols and proanthocyanidins) antioxidants in callus culture of Camellia sinensis under influence of phenolic precursors (L-phenylalanine-3 mM, trans-cinnamic acid-1 mM, naringenin-0.5 mM). According to the data obtained, the effect of precursors on tea callus cultures did not lead to significant increasing of superoxide dismutase and peroxidase activity in most cases. However, it led to the increased accumulation of the total phenolics content, as well as flavanols and proanthocyanidins contents. For C. sinensis callus cultures, the most promising regulator of phenolic compounds was L-phenylalanine, in the presence of which its content increased almost twice. Thus, the exogenous effect of various precursors is possible to use for the targeted regulation of certain phenolics classes accumulation in plant cells.

Minimum methionine requirement in adult cats as determined by indicator amino acid oxidation.

There is a lack of empirical data on the dietary Met requirement, in the presence of Cys or cystine, in adult cats. Thus, the aim of this study was to determine the Met requirement, in the presence of excess Cys, in adult cats at maintenance using the indicator amino acid oxidation (IAAO) technique. Six adult neutered male cats were initially selected and started the study. Cats were adapted to the basal diet sufficient in Met (0.24% dry matter, DM) for 14 d prior to being randomly allocated to one of eight dietary levels of Met (0.10%, 0.13%, 0.17%, 0.22%, 0.27%, 0.33%, 0.38%, and 0.43% DM). Different dietary Met concentrations were achieved by supplementing the basal diet with Met solutions. Alanine was additionally included in the solutions to produce isonitrogenous and isoenergetic diets. Cats underwent a 2-d adaptation period to each experimental diet prior to each IAAO study day. On IAAO study days, 13 meals were offered corresponding to 75% of each cat's daily food allowance. The remaining 25% of their daily food intake was offered after each IAAO study. A bolus dose of NaH13CO3 (0.44 mg kg-1) and l-[1-13C]-phenylalanine (13C-Phe; 4.8 mg kg-1) were provided in fifth and sixth meals, respectively, followed by a constant dose of 13C-Phe (1.04 mg kg-1) in the next meals. Breath samples were collected and total production of 13CO2 was measured every 25 min through respiration calorimetry chambers. Steady state of 13CO2 achieved over at least three breath collections was used to calculate oxidation of 13C-Phe (F13CO2). Competing models were applied using the NLMIXED procedure in SAS to determine the effects of dietary Met on 13CO2. Two cats were removed from the study as they did not eat all meals, which is required to achieve isotopic steady. A breakpoint for the mean Met requirement, with excess of Cys, was identified at 0.24% DM (22.63 mg kg-1) with an upper 95% confidence limit of 0.40% DM (37.71 mg·kg-1), on an energy density of 4,164 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors. The estimated Met requirement, in the presence of excess of Cys, is higher than the current recommendations proposed by the National Research Council's Nutrient Requirement of Dogs and Cats, the Association of American Feed Control Officials, and the European Pet Food Industry Federation.

The objective of this study was to determine the minimum Met requirement, when Cys was provided in excess, of adult cats using a highly sensitive and noninvasive technique, the indicator amino acid oxidation (IAAO). Six adult cats were fed experimental diets with varying levels of methionine (0.10%, 0.13%, 0.17%, 0.22%, 0.27%, 0.33%, 0.38%, and 0.43% on a dry matter [DM] basis) for 2 d prior to each IAAO study day. Although not all cats completed the study, a breakpoint was still defined in the statistical models applied, resulting in an estimated minimum Met requirement of 0.40% DM (37.71 mg kg−1), on an energy density of 4,164 kcal of metabolizable energy/kg DM calculated using the modified Atwater factors. The Met requirement, in the presence of excess of Cys, estimated in our study is higher than the current recommendations proposed by the National Research Council's Nutrient Requirement of Dogs and Cats, the Association of American Feed Control Officials, and the European Pet Food Industry Federation.

An Offset Patterned Cross-ß Structure in Assemblies of C3 -Symmetric Peptide Amphiphiles.

Developing peptide-based materials with controlled morphology is a critical theme of soft matter research. Herein, we report the formation of a novel, patterned cross-ß structure formed by self-assembled C3 -symmetric peptide amphiphiles based on diphenylalanine and benzene-1,3,5-tricarboxamide (BTA). The cross-ß motif is an abundant structural element in amyloid fibrils and aggregates of fibril-forming peptides, including diphenylalanine. The incorporation of topological constraints on one edge of the diphenylalanine fragment limits the number of ß-strands in ß-sheets and leads to the creation of an unconventional offset-patterned cross-ß structure consisting of short 3×2 parallel ß-sheets stabilized by phenylalanine zippers. In the reported assembly, two patterned cross-ß structures bind parallel arrays of BTA stacks in a superstructure within a single-molecule-thick nanoribbon. In addition to a threefold network of hydrogen bonds in the BTA stack, each molecule becomes simultaneously bound by hydrogen bonds from three ß-sheets and four phenylalanine zippers. The diffuse layer of alkyl chains with terminal polar groups prevents the nanoribbons from merging and stabilizes cross-ß-structure in water. Our results provide a simple approach to the incorporation of novel patterned cross-ß motifs into supramolecular superstructures and shed light on the general mechanism of ß-sheet formation in C3 -symmetric peptide amphiphiles.

Effects of gene polymorphisms on delayed MTX clearance, toxicity, and metabolomic changes after HD-MTX treatment in children with acute lymphoblastic leukemia.

This study aims to assess the role of methotrexate-related gene polymorphisms in children with acute lymphoblastic leukemia (ALL) during high-dose methotrexate (HD-MTX) therapy and to explore their effects on serum metabolites before and after HD-MTX treatment. The MTHFR 677C>T, MTHFR 1298A>C, ABCB1 3435C>T, and GSTP1 313A>G genotypes of 189 children with ALL who received chemotherapy with the CCCG-ALL-2020 regimen from January 2020 to April 2023 were analyzed, and toxic effects were reported according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0). Fasting peripheral blood serum samples were collected from 27 children before and after HD-MTX treatment, and plasma metabolites were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). The results of univariate and multivariate analyses showed that MTHFR 677C>T and ABCB1 3435 C>T gene polymorphisms were associated with the delayed MTX clearance (P < 0.05) and lower platelet count after treatment in children with MTHFR 677 mutation compared with wild-type ones (P < 0.05), and pure mutations in ABCB1 3435 were associated with higher serum creatinine levels (P < 0.05). No significant association was identified between MTHFR 677C>T, MTHFR 1298A>C, ABCB1 3435 C>T, and GSTP1 313A>G genes and hepatotoxicity or nephrotoxicity (P > 0.05). However, the serum metabolomic analysis indicated that the presence of the MTHFR 677C > T gene polymorphism could potentially contribute to delayed MTX clearance by influencing L-phenylalanine metabolism, leading to the occurrence of related toxic side effects. CONCLUSION: MTHFR 677C>T and ABCB1 3435 C>T predicted the risk of delayed MTX clearance during HD-MTX treatment in children with ALL. Serum L-phenylalanine levels were significantly elevated after HD-MTX treatment in children with the MTHFR 677C>T mutation gene. TRIAL REGISTRATION: This study was registered at the Chinese Clinical Trial Registry (registration number: ChiCTR2000035264; registration: 2020/08/05; https://www.chictr.org.cn/ ). WHAT IS KNOWN: • MTX-related genes play an important role in MTX pharmacokinetics and toxicity, but results from different studies are inconsistent and the mechanisms involved are not clear. WHAT IS NEW: • Characteristics, prognosis, polymorphisms of MTX-related genes, and metabolite changes were comprehensively evaluated in children treated with HD-MTX chemotherapy. • Analysis revealed that both heterozygous and pure mutations in MTHFR 677C>T resulted in a significantly increased risk of delayed MTX clearance, and that L-phenylalanine has the potential to serve as a predictive marker for the metabolic effects of the MTHFR 677C>T polymorphism.

Solriamfetol and m-chlorophenylpiperazine cause false positive amphetamine results on urine drug screening.

Urine drug screening by immunoassay is a common method to quickly identify drug exposures in the emergency setting and to detect unexpected drug exposures in a variety of patient care and occupational health settings. Although they provide rapid results, immunoassays are susceptible to cross-reactivity with other medications and metabolites. Herein we evaluate the performance of the Thermo Scientific DRI Amphetamines immunoassay for reactivity with trazodone, aripiprazole, atomoxetine, solriamfetol and relevant metabolites. Each of these compounds were spiked into drug-free urine across a range of concentrations and assessed for positivity on amphetamine screen. We demonstrate that the Thermo Scientific DRI assay is susceptible to interferences from m-chlorophenylpiperazine (mCPP), the main metabolite of trazodone, and solriamfetol. Characterization of assay-specific interferences in toxicology screening is instrumental for accurate interpretation of toxicology results, evaluation of patients in emergent settings and supporting patient care.