Plasma Amino Acid Research Articles

High-Moisture Extrusion of a Dietary Protein Blend Impairs In Vitro Digestion and Delays In Vivo Postprandial Plasma Amino Acid Availability in Humans

BackgroundIndustrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. MethodsIn Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a “dry” blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. ResultsProtein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0–150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. ConclusionsThese data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein.This trial was registered at clinicaltrials.gov as NCT05584358.

Longitudinal associations of plasma amino acid levels with recovery from malarial coma.

Disordered amino acid metabolism is observed in cerebral malaria (CM). We sought to determine whether abnormal amino acid concentrations were associated with level of consciousness in children recovering from coma. We quantified 21 amino acids and coma scores longitudinally and analyzed data for associations. In a prospective observational study, we enrolled 42 children with CM. We measured amino acid levels at entry and at frequent intervals thereafter and assessed consciousness by Blantyre Coma Scores (BCS). Thirty-six healthy children served as controls for in-country normal amino acid ranges. We employed logistic regression using a generalized linear mixed-effects model to assess associations between out-of-range amino acid levels and BCS. At entry 16/21 amino acid levels were out-of-range. Longitudinal analysis revealed 10/21 out-of-range amino acids were significantly associated with BCS. Elevated phenylalanine levels showed the highest association with low BCS. This finding held when out-of-normal-range data were analyzed at each sampling time. We provide longitudinal data for associations between abnormal amino acid levels and recovery from CM. Of 10 amino acids significantly associated with BCS, we propose that elevated phenylalanine may be a surrogate for impaired clearance of ether lipid mediators of inflammation contributing to CM pathogenesis.

#655 Warm meal after a haemodialysis session saves the day (and lives?)

Abstract Background and Aims The haemodialysis (HD) process leads to chronic nutrient deficiency, particularly of proteins and amino acids. About 6-12 grams of amino acids loss and 7-8 grams of protein loss occurring during each dialysis session may contribute to hypoalbuminemia, a strong predictor of malnutrition and mortality [1]. Optimal dietary protein intake (DPI) may replenish low plasma amino acids, but DPI inadequacy is a common issue in HD patients, as well as malnutrition due to comorbidities. COVID-19 has made dramatic changes in dialysis centers’ procedures, including the transition from warm meals to cold sandwiches for patients to eat after the session. After the end of COVID-19 pandemic warm meals (meat dish included) were reintroduced. Our aim was to quantify the effect of one to three warm meals per week after the HD session (depending on the regime) on the patients’ protein status. Method Data of 68 patients (24 females and 44 males) on chronic HD were collected after signing the ICF consisting of total protein and albumin values measured two months apart. The first measurement was done while patients were on the long-term sandwich diet after every HD session. The second measurement was done 45 days after the warm meal diet was initiated after every session. Serum total protein and serum albumin were measured, with normal intervals for total serum protein and albumin being 66–81 g/L and 45–51 g/L respectively. The values were compared using Wilcoxon signed-rank test. Results The average albumin value with dry food was 35.9 g/L and for total protein 63.6 g/L, with just 6 patients having normal albumin levels (8.8%) and 21 patients having normal total protein levels (30.9%). The average albumin value after 45 days of HD with warm meals was 39.8 g/L and 66.6 g/L for total protein, with 28 patients having normal albumin levels (41.2%) and 36 patients having normal total protein levels (52.9%). This marks an increase of 4.7% in average value of total protein (3 g/L) and statistically significant increase in albumin value of 10.8% (3.9 g/L, p &amp;lt; 0.001). Greater number of patients had normal total protein and albumin values (4.7 times more patients had a normal albumin level after the initiation of warm meals, and 1.7 times more patients had normal total protein levels). There is a difference regarding male and female patients with a greater impact on males. Male patients had an average total proteins value increase of 5.3% (3.37 g/L) while female patients had 3.5% (2.29 g/L). There was a statistically significant increase in albumin value both for male (p &amp;lt; 0.001) and female patients (p = 0.002). In male patients the average increase was 12% (4.33 g/L), while in female patients it was 8.5% (3.04 g/L). After the introduction of warm meals, 50% of male patients had normal albumin values (vs. 9% before), while 25% of female patients reached normal albumin values (vs. 8.3% before). Conclusion Patients on chronic HD suffer from malnutrition which could lead to significantly higher mortality. Adequate nutrition is crucial for their wellbeing. This study has shown that having a warm meal (protein based) can lead to an increase of total plasma protein and albumin with a higher impact on male patients. In this way we could increase DPI of HD patients, reduce the chance of malnutrition and reduce the cardiovascular risk and mortality.

A panel of four plasma amino acids is a promising biomarker for newly diagnosed bladder cancer

BackgroundMetastasis and recurrence are the main causes of death in post-operative bladder cancer (BC), emphasizing the importance of exploring early-stage diagnostic markers. Serum biomarkers constitute a promising diagnostic approach for asymptomatic stage cancer as they are non-invasive, have high accuracy and low cost. AimsTo correlate concentrations of plasma amino acids with BC progression to assess their utility as an early-stage diagnostic. MethodsNewly diagnosed BC patients (n=95) and normal controls (n=96) were recruited during the period from 1 December 2018 to 30 December 2020. General and food frequency questionnaires established their basic information and dietary intake data. Venous blood samples were collected from fasting subjects and used to detect levels of plasma amino acids by liquid chromatography-mass spectrometry. Verification was performed on the GSE13507 transcriptome gene expression matrix of BC from Gene Expression Omnibus (GEO) database. ResultsEleven amino acids have been identified as altered in the plasma of newly diagnosed BC patients compared to controls (P < 0.05). Adjusted by gender, education, smoking and other factors, plasma ornithine level (OR = 0.256, 95% CI: 0.104 ∼ 0.630) is a protective factor for BC, plasma levels of methionine (OR = 3.460, 95% CI: 1.384 ∼ 8.651), arginine (OR = 3.851, 95% CI: 1.542 ∼ 9.616), and glutamate (OR = 3.813, 95% CI: 1.543 ∼ 9.419) are all risk factors for BC. ROC analysis demonstrated that the combination of plasma ornithine, methionine, arginine and glutamate could accurately diagnose BC (AUC = 0.84, 95% CI: 0.747 ∼ 0.833). In addition, the mRNA level of arginase 1 was decreased (P < 0.05), while the inducible nitric oxide synthase was increased significantly, which may be linked with the disturbance of arginine metabolism in BC patients. Further analysis of GEO database confirmed the role of arginine metabolism. ConclusionA biomarker panel containing four amino acids may provide a feasible strategy for the early diagnosis of BC. However, further validation is required through prospective studies.

Quantification and interpretation of postprandial whole-body protein metabolism using stable isotope methodology: a narrative review.

Stable isotopes are routinely applied to determine the impact of factors such as aging, disease, exercise, and feeding on whole-body protein metabolism. The most common approaches to quantify whole-body protein synthesis, breakdown, and oxidation rates and net protein balance are based on the quantification of plasma amino acid kinetics. In the postabsorptive state, plasma amino acid kinetics can easily be assessed using a constant infusion of one or more stable isotope labeled amino acid tracers. In the postprandial state, there is an exogenous, dietary protein-derived amino acid flux that needs to be accounted for. To accurately quantify both endogenous as well as exogenous (protein-derived) amino acid release in the circulation, the continuous tracer infusion method should be accompanied by the ingestion of intrinsically labeled protein. However, the production of labeled protein is too expensive and labor intensive for use in more routine research studies. Alternative approaches have either assumed that 100% of exogenous amino acids are released in the circulation or applied an estimated percentage based on protein digestibility. However, such estimations can introduce large artifacts in the assessment of whole-body protein metabolism. The preferred estimation approach is based on the extrapolation of intrinsically labeled protein-derived plasma bioavailability data obtained in a similar experimental design setting. Here, we provide reference data on exogenous plasma amino acid release that can be applied to allow a more accurate routine assessment of postprandial protein metabolism. More work in this area is needed to provide a more extensive reference data set.

Chemotherapy-Induced Changes in Plasma Amino Acids and Lipid Oxidation of Resected Patients with Colorectal Cancer: A Background for Future Studies.

Previous studies have documented that FOLFOX and XELOX therapies negatively impact the metabolism of skeletal muscle and extra-muscle districts. This pilot study tested whether three-month FOLFOX or XELOX therapy produced changes in plasma amino acid levels (PAAL) (an estimation of whole-body amino acid metabolism) and in plasma levels of malondialdehyde (MDA), a marker of lipid hyper oxidation. Fourteen ambulatory, resected patients with colorectal cancer scheduled to receive FOLFOX (n = 9) or XELOX (n = 5) therapy, after overnight fasting, underwent peripheral venous blood sampling, to determine PAAL and MDA before, during, and at the end of three-month therapy. Fifteen healthy matched subjects (controls) only underwent measures of PAAL at baseline. The results showed changes in 87.5% of plasma essential amino acids (EAAs) and 38.4% of non-EAAs in patients treated with FOLFOX or XELOX. These changes in EAAs occurred in two opposite directions: EAAs decreased with FOLFOX and increased or did not decrease with XELOX (interactions: from p = 0.034 to p = 0.003). Baseline plasma MDA levels in both FOLFOX and XELOX patients were above the normal range of values, and increased, albeit not significantly, during therapy. In conclusion, three-month FOLFOX or XELOX therapy affected plasma EAAs differently but not the baseline MDA levels, which were already high.

Dietary CP and digestion kinetics influence BW loss, litter weight gain, and reproduction by affecting postprandial amino acid metabolism in lactating sows

To avoid a high body protein mobilization in modern lean sows during lactation, an adequate dietary amino acid (AA) supply and an efficient AA utilization are crucial. This study evaluated the effects of dietary CP and in vitro protein digestion kinetics on changes in sow body condition, litter weight gain, milk composition, blood metabolites, protein utilization efficiency and subsequent reproductive performance. We hypothesized that a slower digestion of dietary protein would improve AA availability and utilization. In total, 110 multiparous sows were fed one of four lactation diets in a 2 × 2 factorial design, with two CP concentrations: 140 g/kg vs 180 g/kg, and two protein digestion kinetics, expressed as a percentage of slow protein (in vitro degradation between 30 and 240 min): 8 vs 16% of total protein. Feeding sows the high CP diets reduced sow weight loss (Δ = 7.6 kg, P < 0.01), estimated body fat loss (Δ = 2.6 kg, P = 0.02), and estimated body protein loss (Δ = 1.0 kg, P = 0.08), but only at a high percentage of slow protein. A higher percentage of slow protein increased litter weight gain throughout lactation (Δ = 2.6 kg, P = 0.04) regardless of CP concentrations, whereas a higher CP only increased litter weight gain during week 3 of lactation (Δ = 1.2 kg, P = 0.01). On Day 15 postfarrowing, serial blood samples were taken from a subsample of sows fed with the high CP diets. In these sows, a high percentage of slow protein resulted in higher plasma AA concentrations at 150 and 180 min after feeding (Δ = 0.89, P = 0.02, Δ = 0.78, P = 0.03, mmol/L, respectively) and lower increases in urea at 90 and 120 min after feeding (Δ = 0.67, P = 0.04, Δ = 0.70, P = 0.03, mmol/L, respectively). The higher dietary CP concentration increased total nitrogen loss to the environment (Δ = 604 g, P < 0.01) with a reduction of protein efficiency (Δ = 14.8%, P < 0.01). In the next farrowing, a higher percentage of slow protein increased subsequent liveborn litter size (Δ = 0.7, P < 0.05). In conclusion, feeding sows with a high dietary CP concentration alleviated maternal weight loss during lactation when the dietary protein digestion rate was slower, but lowered protein efficiency. A slower protein digestion improved litter weight gain, possibly by reducing AA oxidation and improving plasma AA availability, thus, improving protein efficiency.

Preoperative profiles of plasma amino acids and derivatives distinguish periampullary cancer and benign disease

Periampullary cancers, including pancreatic ductal adenocarcinoma, ampullary-, cholangio-, and duodenal carcinoma, are frequently diagnosed in an advanced stage and are associated with poor overall survival. They are difficult to differentiate from each other and challenging to distinguish from benign periampullary disease preoperatively. To improve the preoperative diagnostics of periampullary neoplasms, clinical or biological markers are warranted.In this study, 28 blood plasma amino acids and derivatives from preoperative patients with benign (N = 45) and malignant (N = 72) periampullary disease were analyzed by LC-MS/MS.Principal component analysis and consensus clustering both separated the patients with cancer and the patients with benign disease. Glutamic acid had significantly higher plasma expression and 15 other metabolites significantly lower plasma expression in patients with malignant disease compared with patients having benign disease. Phenylalanine was the only metabolite associated with improved overall survival (HR = 0.50, CI 0.30–0.83, P < 0.01).Taken together, plasma metabolite profiles from patients with malignant and benign periampullary disease were significantly different and have the potential to distinguish malignant from benign disease preoperatively.

Empagliflozin increases plasma levels of citrulline, histidine, and α-aminobutyric acid in patients with type 2 diabetes: effects of a sodium-glucose co-transporter 2 inhibitor on the plasma amino acid profile

ABSTRACT Background To investigate effects of empagliflozin on plasma amino acids in people with type 2 diabetes. Research design and methods In a randomized, active-controlled, open-label trial, 58 patients with type 2 diabetes were randomized to 10 mg/day empagliflozin (n = 29) or standard treatment without empagliflozin (control group, n = 29) and treated for 12 weeks. We obtained blood samples at baseline and 12 weeks and assessed the plasma amino acid profile by liquid chromatography-mass spectrometry liquid chromatography. We also calculated the Fischer ratio (the ratio of branched-chain to aromatic amino acids). Results In the empagliflozin group but not in the control group, plasma levels of citrulline, histidine, and α-aminobutyric acid (AABA), the Fischer ratio, and serum high-molecular weight (HMW) adiponectin increased significantly (p = 0.0099, 0.0277, 0.0318, 0.0135, and 0.0304, respectively) and plasma plasminogen activator inhibitor-1 (PAI-1) decreased significantly (p = 0.0014). In the empagliflozin group, the change in plasma citrulline was positively correlated with the changes in HMW adiponectin (r = 0.488, p = 0.0084) and the Fischer ratio (r = 0.393, p = 0.0353) but negatively correlated with the change in ferritin (r= −0.533,p = 0.0051); the change in plasma histidine was negatively correlated with the change in PAI-1 (r= –0.398, p = 0.0397) and urinary albumin creatinine ratio (r= −0.478, p = 0.0088). Conclusion Empagliflozin significantly increases plasma citrulline, histidine, and AABA in people with type 2 diabetes. Clinical trial registration www.umin.ac.jp identifier is UMIN000025418.

Sex differences in amino acid composition in a rodent model of ARPKD, the PCK rat

Polycystic Kidney Disease (PKD) is a severe, genetically inherited disease that leads to renal failure. Like several other chronic kidney diseases, male sex is a risk factor for accelerated PKD. Rat models of both Autosomal Recessive and Dominant forms of PKD (PCK and Han:SPRD rats, respectively) exhibit sex differences in disease progression. Previous studies have found that amino acids (AAs) and peptides are predominant constituents of cystic fluid. We hypothesized that AA homeostasis in male and female cystic fluid would demonstrate sexual dimorphism, and these differences may provide essential information about the progression of cystogenesis. We quantified the AAs in cystic fluid, plasma, and urine in PCK and control Sprague Dawley (SD) rats (only plasma and urine for SD rats). Of the 42 AAs that were measured, only asparagine, serine, aspartic acid, glutamic acid, beta-alanine, cystathionine, and leucine were NOT significantly different between SD and PCK plasma values. Interestingly, only 18 AAs were significantly different in urine of PCK rats compared to SD rats. In SD rats, between sexes, 26 AAs were significantly different in plasma and 5 were significantly different in urine. Among PCK rats, 13 AAs in plasma, 9 AAs in urine, and 4 AAs in cystic fluid were significantly different between sexes. The 4 AAs that differed between male and female cystic fluid were 1-methylhistidine, 3-methylhistidine, ethanolamine, and taurine. Moreover, taurine was the most abundant AA in cystic fluid. Taurine concentrations in both plasma (165 ± 9 vs. 128 ± 4 μM, N=6, p=0.003) and cystic fluid (7508 ± 469 vs. 4557 ± 788 μM, N=6, p=0.01) were significantly different between sexes in the PCK rats (male vs. female, respectively). Urine values for the PCK rats (9705 ± 1863 vs. 7570 ± 1631 μM, N=6) were not significantly different in males and females; however, the average urinary taurine excretion in PCK rats was nearly 4-fold greater than SD rats (8637 ± 1223 vs. 2219 ± 465 μM, N=12, p&lt;0.0001). There were no significant differences in taurine concentrations between SD male and female rat plasma (202 ± 17 vs. 194 ± 7.0 μM, N=6) or urine (2044 ± 614 vs. 2395 ± 752 μM, N=6). Western blot of the taurine transporter, TauT, detected bands at 50 and 75 kDa, and both bands were significantly reduced by ~50% in PCK kidneys compared to SD kidneys. Lastly, immunolabeling in PCK kidney sections demonstrated that TauT is present at the apical membrane of cyst epithelia. These results point to a potential role for AAs, and specifically for taurine in cystogenesis, which likely varies by sex in diseased, but not healthy kidneys. R00 HL153686, BX004024, R01 DK126720, R01 DK129227. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1033: Loss Of Serine Hydroxymethyltransferase 2 In Macrophages Blunts Glutathione And Enhances Ceramide Synthesis To Accelerate Atherosclerosis

Increasing evidence suggests an important role for amino acid metabolism in atherosclerotic cardiovascular disease (ASCVD), highlighting potential biomarkers, causative factors, and therapeutic targets. Among all amino acids, lower circulating glycine is consistently reported to be associated with enhanced ASCVD severity. The amino acid serine is the main precursor for glycine, and a lower glycine:serine ratio is associated with more advanced and unstable atherosclerotic plaques. The mitochondrial serine hydroxymethyltransferase 2 (SHMT2) catalyzes the bulk of glycine formation from serine, and is critical in development since global knockout mice are nonviable. We found that SHMT2 is highly expressed in macrophages in both human and mouse atherosclerotic plaques. LPS-treated macrophages displayed reduced SHMT2 expression concomitant with a lower intracellular glycine:serine ratio. To study the role of SHMT2 in macrophages, we developed novel myeloid-specific Shmt2 knockout mice ( Shmt2 MKO ). Metabolomics and stable isotope tracing studies revealed that macrophages lacking SHMT2 have a lower glycine:serine ratio leading to attenuated de novo synthesis of glutathione and enhanced formation of the ceramide precursor, sphinganine. Accordingly, the loss of SHMT2 in macrophages enhanced superoxide production and ceramide accumulation. To determine the role of macrophage SHMT2 in atherosclerosis, we crossed the Shmt2 MKO mice with apolipoprotein E-deficient ( Apoe -/- ) mice. After 12 weeks on Western diet, Shmt2 MKO / Apoe -/- mice showed no differences in plasma lipid or amino acid profiles as compared to Shmt2 flox / Apoe -/- mice. Nevertheless, plaque area and necrotic core size were significantly enhanced in Shmt2 MKO / Apoe -/- mice. Taken together, macrophage SHMT2 plays a critical role in atherosclerosis by regulating redox and ceramide metabolism.

Adding value to laboratory results: machine learning for plasma amino acid profile interpretation

Adding value to laboratory results: machine learning for plasma amino acid profile interpretation

Amino Acids Profile in Children with Acute Brucellosis.

Many new cases of brucella infections are seen in Turkey every year, especially in March, April, and May, due to the consumption of local unpasteurized cheese. Amino acids profiles have not been studied in brucellosis so far. The amino acid profiles may be affected by infectious diseases. Our study aims to evaluate the plasma amino acid profile in the progression of acute brucellosis. Plasma amino acid profile was performed by an 8045 LC-MS / MS device (Shimadzu 8045, Japan) using JASEM amino acid kit. Analysis of 45 amino acid profiles was made and results profiles showed significant differences in concentrations and types of amino acids in brucella patients. We observed a significant difference in terms of alanine, arginine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, ornithine, phenylalanine, proline, tyrosine, valine, alpha-aminoadipic acid, alpha-amino-pimelic acid, argininosuccinic acid, gamma-aminobutyric acid, thiaproline, 1-methylhistidine, 3-methylhistidine, hydroxylysine, hydroxyproline, cystine, serotonin, ethanolamine, and taurine (p-value < 0.05 for each). No significant differences were determined regarding asparagine, citrulline, histidine, leucine, alloisoleucine, lysine, methionine, serine, threonine, tryptophan, anserine, alpha aminobutyric acid, beta aminoisobutyric acid, beta-alanine, cystathionine, histamine, and 5-oh-trp (p-value > 0.05 for all). Patients with brucellosis have a specific profile of amino acids which may reflect sequelae of pathological and metabolic biochemical changes in the disease process due to the growth of Brucella spp. in the human body leading to an imbalance of amino acid levels.

Supplementation of Methionine Dipeptide Enhances the Milking Performance of Lactating Dairy Cows.

Methionine dipeptide (Met-Met) could improve milk protein synthesis in bovine epithelia mammary cells and lactating mice, while the effects of Met-Met on lactation performance, rumen fermentation and microbiota profile in lactating dairy cows have not been explored. For this reason, 60 Chinese lactating Holstein cows were allocated into three treatment groups: control group (CON), 6 g/d methionine dipeptide group (MM), and 6.12 g/d rumen-protected methionine dipeptide group (RPMM). The experiment lasted for 10 weeks to monitor lactation performance, plasma amino acid profile and rumen fermentation parameters and microbiota profile. Results showed that MM increased the energy-corrected milk (ECM), and RPMM increased both milk yield and ECM (p < 0.05). The milk protein concentration and yield were increased by MM and RPMM (p < 0.05). The rumen fermentation showed that RPMM increased total volatile fatty acids, acetate and valerate concentrations (p < 0.05). The relative abundance of Firmicutes, including Succiniclasticum, Selenomonas and Clostridium_XlVa, were enriched and the Prevotella was decreased by RPMM (p < 0.05). In summary, daily supplementing with 6 g of MM or RPMM in lactating dairy cows could improve milk yield and both percentage and yield of milk protein, and RPMM benefited the rumen fermentation and altered the bacterial composition. These results provided the first evidence that Met-Met supplementation can improve lactation performance of dairy cows.

Development of reliable in vitro long-term culture systems for hematopoietic cells of crayfish Cherax quadricarinatus and virus susceptibility assay

Long-term in vitro cultured crayfish cells can serve as a valuable tool for investigating various aspects of crayfish biology, encompassing cell physiology and viral mechanisms. Regrettably, they are difficult to maintain a highly active state and survive during long-term and continuous culture in vitro. In order to generate highly viable crayfish cell cultures, it is crucial to optimize dependable culture conditions and utilize a suitable cell medium. In this study, we have successfully developed reliable in vitro long-term culture systems for hematopoietic (HPT) cells of crayfish Cherax quadricarinatus by formulating an optimal crayfish cell growth medium (OCCM), including under two-dimensional (2D) and three-dimensional (3D) conditions. The novel medium was optimized by a mix of exogenous nutrients, crayfish plasma (CP) and shrimp serum-derived amino acids and saccharides within the Leibovitz's L-15 (L-15) medium. Our newly developed crayfish cell growth medium (CCM-28) enabled the culturing of HPT cells for over 142 days under 2D and over 60 days under 3D conditions, with consistent active cell proliferation observed over a 30-day incubation period. This proliferation was comparable to in vivo conditions, with a cell division rate exceeding 20%, suggesting that a high mitotic rate has been successfully achieved during long-term in vitro culture. Flow cytometry analysis further elucidated the mitosis-arrest of HPT cells cultured in vitro was predominantly observed in the G0/G1 phase. Furthermore, the cell subculture techniques have been established using mechanical dissociation method (in 2D) and substrate exchange method (in 3D). Additionally, the susceptibility of these cell cultures to covert mortality nodavirus (CMNV) and white spot syndrome virus (WSSV) was confirmed by capturing cytopathic effects (CPE) and analyzing viral copy numbers. Species identification using the cytochrome oxidase subunit I (COI) gene established the origin of the cell cultures from C. quadricarinatus. In conclusion, the successful development of highly viable crayfish cells in long-term in vitro culture systems, as demonstrated in this study, will significantly advance the research on immortalization in crayfish cells and the study of mechanisms related to viruses.

Domestic dogs maintain clinical, nutritional, and hematological health outcomes when fed a commercial plant-based diet for a year.

Domestic dogs can maintain health on complete and well-balanced canine plant-based nutrition (K9PBN). Novel insight on health outcomes in dogs consuming K9PBN is of relevance to veterinary professionals and consumers given a growing interest in non-traditional dog foods with perceived health benefits, while considering potential safety concerns. We aimed to investigate nutritional equivalence by measuring clinical health outcomes in adult dogs fed K9PBN over twelve months compared to a meat-based diet at baseline. We enrolled fifteen clinically healthy adult dogs living in households in Los Angeles County, California in a prospective cohort study and evaluated clinical, hematological, and nutritional parameters in dogs at 0, 6, and 12 months, including complete blood count (CBC), blood chemistry, cardiac biomarkers, plasma amino acids, and serum vitamin concentrations. The study found that clinically healthy, client-owned, adult dogs maintain health, based on physical exams, complete blood count, serum chemistry, plasma amino acids, serum vitamins, and cardiac biomarkers combined with client-reported observations, when fed commercial K9PBN over a twelve-month period. This study is the most comprehensive and longest known K9PBN investigation to date and provides clinically relevant evidence-based nutrition data and new knowledge on outcomes in clinically healthy dogs who thrive without consumption of animal-derived ingredients. These results also provide a valuable foundation for the future study of K9PBN as a potential nutritional intervention for clinically relevant pathologies in canine medicine. Lastly, it is of major relevance to One Health paradigms since ingredients produced independent of industrial food animal production are both more sustainable and help to circumvent ethical dilemmas for maintenance of health in domestic dogs.

Impact of Distinct Antiandrogen Exposures on the Plasma Metabolome in Feminizing Gender-affirming Hormone Therapy.

The plasma metabolome is a functional readout of metabolic activity and is associated with phenotypes exhibiting sexual dimorphism, such as cardiovascular disease. Sex hormones are thought to play a key role in driving sexual dimorphism. Gender-affirming hormone therapy (GAHT) is a cornerstone of transgender care, but longitudinal changes in the plasma metabolome with feminizing GAHT have not been described. Blood samples were collected at baseline and after 3 and 6 months of GAHT from transgender women (n = 53). Participants were randomized to different anti-androgens, cyproterone acetate or spironolactone. Nuclear magnetic resonance-based metabolomics was used to measure 249 metabolic biomarkers in plasma. Additionally, we used metabolic biomarker data from an unrelated cohort of children and their parents (n = 3748) to identify sex- and age-related metabolite patterns. We identified 43 metabolic biomarkers altered after 6 months in both anti-androgen groups, most belonging to the very low- or low-density lipoprotein subclasses, with all but 1 showing a decrease. We observed a cyproterone acetate-specific decrease in glutamine, glycine, and alanine levels. Notably, of the metabolic biomarkers exhibiting the most abundant "sex- and age-related" pattern (higher in assigned female children and lower in assigned female adults, relative to assigned males), 80% were significantly lowered after GAHT, reflecting a shift toward the adult female profile. Our results suggest an anti-atherogenic signature in the plasma metabolome after the first 6 months of feminizing GAHT, with cyproterone acetate also reducing specific plasma amino acids. This study provides novel insight into the metabolic changes occurring across feminizing GAHT.

Reduced plasma glycine concentration in healthy and chronically diseased older adults: a marker of visceral adiposity?

BackgroundPrevious studies have shown that a reduced plasma concentration of the amino acid glycine (Gly) is associated with intra-abdominal obesity, but the mechanism remains unclear. ObjectivesThis study aimed to investigate whether lower plasma Gly concentrations in older adults are independently associated with (visceral) adiposity, age, sex, presence of chronic disease, and glucose intolerance, and whether they are caused by a reduced Gly whole-body production (WBP) and/or increased Gly disposal capacity. MethodsWe studied 102 older adults (47 males/55 females, 68.5 ± standard deviation 6.4 y) without comorbidities and 125 older adults with chronic obstructive pulmonary disease (COPD) (58 males/67 females, 69.7 ± 8.6 y). We assessed body composition and visceral adipose tissue (VAT) by dual-energy x-ray absorptiometry and muscle function by dynamometry. We measured postabsorptive plasma amino acid profile and glucose, followed by pulse administration of stable isotope-labeled Gly ([2,2-2H2]), and blood sampling was performed to measure the WBP of Gly. Results are expressed as means and 95% confidence intervals (CIs). ResultsWe found a lower plasma Gly concentration in healthy males and males with COPD than in females (Healthy: 211; 95% CI: 193,230 compared with 248; 95% CI: 225,271; COPD: 200; 95% CI: 186,215 compared with 262: 95% CI: 241, 283; P < 0.0001, respectively), with no difference between healthy and COPD groups. A negative relationship was found between unadjusted plasma Gly and VAT mass (R2: 0.16; slope: −1.7; 95% CI: −2.4, −1.2; P < 0.0021), but not with total body fat or fasting glucose. The strong association between lower plasma Gly and increased VAT mass in older adults was independent of age, sex, body weight, lean mass or body mass index, and the presence of COPD. Inclusion of these covariates increased the R2 to 0.783. We found no relation between the VAT and WBP of Gly (P = 0.35) or Gly clearance (P = 0.187) when lean mass was considered. ConclusionsReduced plasma Gly in older adults can be considered a marker of visceral adiposity, independent of sex, age, body composition, presence of chronic disease, and whole-body Gly production or clearance.This study was registered on clinicaltrials.gov as NCT01787682, NCT02082418, NCT02157844, NCT02770092, NCT02780219, NCT03796455, and NCT04461236.

Dietary crude protein and protein solubility manipulation enhances intestinal nitrogen absorption and mitigates reactive nitrogen emissions through gut microbiota and metabolome reprogramming in sheep

Dietary nutrient manipulation (e.g. protein fractions) could lower the environmental footprints of ruminants, especially reactive nitrogen (N). This study investigated the impacts of dietary soluble protein (SP) levels with decreased crude protein (CP) on intestinal N absorption, hindgut N metabolism, fecal microbiota and metabolites, and their linkage with N metabolism phenotype. Thirty-two male Hu sheep, with an age of six months and an initial BW of 40.37 ± 1.18 kg, were randomly assigned to four dietary groups. The control diet (CON), aligning with NRC standards, maintained a CP content of 16.7% on a dry matter basis. Conversely, the experimental diets (LPA, LPB, and LPC) featured a 10% reduction in CP compared with CON, accompanied by SP adjustments to 21.2%, 25.9%, and 29.4% of CP, respectively. Our results showed that low-protein diets led to significant reductions in the concentrations of plasma creatinine, ammonia, urea N, and fecal total short-chain fatty acids (SCFA) (P < 0.05). Notably, LPB and LPC exhibited increased total SCFA and propionate concentrations compared with LPA (P < 0.05). The enrichment of the Prevotella genus in fecal microbiota associated with energy metabolism and amino acid (AA) biosynthesis pathways was evident with SP levels in low-protein diets of approximately 25% to 30%. Moreover, LPB and LPC diets demonstrated a decrease in fecal NH4+–N and NO2−–N contents as well as urease activity, compared with CON (P < 0.05). Concomitantly, reductions in fecal glutamic acid dehydrogenase gene (gdh), nitrite reductase gene (nirS), and nitric oxide reductase gene (norB) abundances were observed (P < 0.05), pointing towards a potential reduction in reactive N production at the source. Of significance, the up-regulation of mRNA abundance of AA and peptide transporters in the small intestine (duodenum, jejunum, and ileum) and the elevated concentration of plasma AA (e.g. arginine, methionine, aspartate, glutamate, etc.) underscored the enhancement of N absorption and N efficiency. In summary, a 10% reduction in CP, coupled with an SP level of approximately 25% to 30%, demonstrated the potential to curtail reactive N emissions through fecal Prevotella enrichment and improve intestinal energy and N utilization efficiency.

A Phase I Trial of a Methionine Restricted Diet with Concurrent Radiation Therapy

Methionine is an essential amino acid critical for cell growth and survival. Preclinical evidence suggests a methionine restricted diet (MRD) sensitizes cancer to radiation therapy (RT), without significant adverse effects. However, this has never been evaluated in humans. The purpose of this pilot study was to evaluate the safety and feasibility of concurrent MRD with standard-of-care definitive RT in adults with any non-skin cancer malignancy. The MRD extended from 2 wk before RT initiation, through 2 wk beyond RT completion. The primary endpoint of safety was assessed as rate of grade 3 or higher acute and late toxicities. Feasibility was assessed with quantitative plasma amino acid panel every 2 wk during the MRD (target plasma methionine 13 μM). Nine patients were accrued over a two-year period, with five able to complete the treatment course. The trial was closed due to slow accrual and subjects’ difficulty maintaining the diet. No grade 3 or higher adverse events were observed. Subjects’ average methionine level was 18.8 μM during treatment, with average nadir 16.8 μM. These findings suggest the safety of concurrent MRD with RT, with toxicities comparable to those expected with RT alone. However, the diet was challenging, and unacceptable to most patients.