Amino Acids In Tissues Research Articles

A tomato a day keeps the beetle away - the impact of Solanaceae glycoalkaloids on energy management in the mealworm Tenebrio molitor.

Solanine (SOL), chaconine (CHA), and tomatine (TOM) are plant secondary metabolites produced mainly by the species of Solanaceae family, such as tomato Solanum lycopersicum L. These glycoalkaloids (GAs) have a wide range of biological activity, also in insects. However, their mechanisms of action are not precisely understood. The purpose of the study was to investigate how pure GAs and tomato leaf extract (EXT) affect glycolysis, Krebs cycle and β-oxidation of fatty acid pathways in Tenebrio molitor L. beetle. For this purpose, the larvae were injected with SOL, CHA, TOM, and EXT at two concentrations (10-8 and 10-5M). For experiments, fat body, gut, and heamolymph samples were collected 2 and 24h after injection. Then, the changes in the expression level of phosphofructokinase, citrate synthase, and β-hydroxyacyl-CoA dehydrogenase were measured using the RT-qPCR technique. The catalytic activity of these enzymes and the carbohydrate level in insects after GA treatment were determined by spectrophotometric method. Furthermore, the analysis of the amount of amino acids in tissues was performed with a GC-MS technique. The results obtained show that the GAs changed the activity and expression of the genes encoding key enzymes of crucial metabolic pathways. The effect depends on the type of GA compound, the tissue tested, and the incubation time after treatment. Furthermore, TOM and EXT affected trehalose concentration in the insect hemolymph and led to accumulation of amino acids in the fat body. The observed changes may indicate a protein degradation and/or enhanced catabolism reactions for the production of ATP used in detoxification processes. These results suggest that GAs alter energy metabolism in the mealworm T. molitor. The study contributes to our understanding of the mechanisms of action of secondary metabolites of plants in insects. This knowledge may allow the design of new natural biopesticides against insect pests because proper energy metabolism is necessary for the survival of the organism.

An Expedited Qualitative Profiling of Free Amino Acids in Plant Tissues Using Liquid Chromatography-Mass Spectrometry (LC-MS) in Conjunction With MS-DIAL.

The estimation of relative levels of amino acids is crucial for understanding various biological processes in plants, including photosynthesis, stress tolerance, and the uptake and translocation of nutrients. A wide range of liquid chromatography (LC; HPLC/UHPLC)-based methods is available for measuring the quantity of amino acids in plants. Additionally, the coupling of LC with mass spectrometry (MS) significantly enhanced the robustness of existing chromatographic methods used for amino acid quantification. However, accurate annotation and integration of mass peaks can be challenging for plant biologists with limited experience in analyzing MS data, especially in studies involving large datasets with multiple treatments and/or replicates. Further, there are instances when the experiment demands an overall view of the amino acids profile rather than focusing on absolute quantification. The present protocol provides a detailed LC-MS method for obtaining a qualitative amino acids profile using MS-DIAL, a versatile and user-friendly program for processing MS data. Free amino acids were extracted from the leaves of control and Tomato leaf curl Palampur virus (ToLCPalV)-infected Nicotiana benthamiana plants. Extracted amino acids were derivatized and separated using UHPLC-QTOF, with each amino acid subsequently identified by aligning mass data with a custom text library created in MS-DIAL. Further, MS-DIAL was employed for internal standard-based normalization to obtain a qualitative profile of 15 amino acids in control and virus-infected plants. The outlined method aims to simplify the processing of MS data to quickly assess any modulation in amino acid levels in plants with a higher degree of confidence.

Targeted metabolomics of muscle amino acid profles and hepatic transcriptomics analyses in grass carp (Ctenopharyngodon idellus) fed with broad beans

While tissue amino acid compositions reflect that of the dietary protein source, and the liver orchestrates amino acid metabolism. In this study, we investigated the muscle amino acid profiles in ordinary and crisp grass carp. The 22 amino acids were measured, and seventeen showed significant concentration differences. To understand the molecular mechanisms behind changes, we analyzed the liver transcriptome, and the 2519 differentially expressed genes (DEGs) were identified, with 1156 up-regulated and 1363 down-regulated genes. DEGs were enriched in ribosome-related biological processes. KEGG pathway analysis showed enrichment in tryptophan metabolism, lysine degradation, valine, leucine and isoleucine degradation, galactose metabolism, and glutathione metabolism with up-regulated genes, arginine and proline metabolism, arginine biosynthesis and alanine, aspartate, amino sugar and nucleotide sugar metabolism, N-Glycan biosynthesis and glutamate metabolism with down-regulated genes. A protein-protein interaction network with 260 nodes and 249 edges was constructed, and 3 modules were extracted. The top 10 hub genes with close connections to other nodes were ITM1, STT3B, SEL1L, UGGT1, MLEC, IL1B, ALG5, KRTCAP2, NFKB2, and IRAK3. In summary, this study identified candidate genes and focused on amino acid and glycan metabolism pathways, providing a reference for further investigation into liver amino acid metabolism in grass carp fed with broad beans.

Comparable glucagon-stimulated amino acid suppression in individuals with and without hepatic steatosis or steatohepatitis.

Hepatic amino acid (AA) metabolism and glucagon secretion are linked in a feedback cycle in which circulating AAs stimulate glucagon secretion, while glucagon stimulates hepatic AA catabolism. It has been proposed that metabolic dysfunction-associated steatotic liver disease (MASLD) leads to hepatic glucagon resistance, which may result in hyperaminoacidemia and hyperglucagonemia. We tested the glucagon effect on AA metabolism in subjects with obesity; 11 with steatohepatitis (MASH), 10 with steatosis (MAS), and seven subjects (CON) without steatosis. We performed a somatostatin clamp with infusions of insulin and low-dose followed by high-dose glucagon. We measured plasma levels of 17 AAs and assessed hepatic fat content (FF%) and body fat distribution (visceral and subcutaneous adipose tissue mass) by MRI. HighGlucagon suppressed plasma total AA equally in all groups; MASH 13% (SD 9%), MAS 14% (7%), CON 11% (5%), respectively. In univariate regression analyses visceral adipose tissue mass (β = 0.471, P = 0.011) and AA concentration at LowGlucagon (β = ─0.524, P = 0.004), but not FF% (β = ─0.243, P = 0.213), were significant predictors of AA reduction. Using a stepwise backward multiple regression approach revealed similar results. Total and specific AA levels (glutamic acid, tyrosine) were higher in both MASLD groups during the study and FF% was positively correlated to a number of individual AAs. Though finding elevated AA concentrations in subjects with MASLD, we conclude that in MASLD patients that do not have elevated glucagon at baseline, glucagon suppresses circulating AA levels equally in subjects with and without MASLD. NCT04042142.

167 Using ‘omics’ to optimize nutrition and health outcomes

Abstract It is clear that diet is linked with health outcomes, yet we are still far from fully understanding the impact of diet on systemic metabolic health. In the new era of ‘omics’ technology, the ability to optimize nutrition across the lifespan of companion animals is within reach. We have been using ‘omics’ technologies to understand how changes in diet composition impact metabolism using a neonatal piglet model. By measuring the composition of sow milk over the first week of lactation, we created an optimized pig milk replacer, and studied the impact of varying the whey protein composition of the milk replacer using a combination of metabolomics on blood, urine, liver, and brain tissue, as well as transcriptomics of intestinal, liver and brain tissue. Our results revealed a substantial impact of the type of whey protein on piglet metabolism that included differences in cortisol, blood and tissue amino acid profiles, and immunity. This work paves the way for additional studies to optimize animal nutrition in the neonatal period and beyond.

A secondary β-hydroxybutyrate metabolic pathway linked to energy balance.

β-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.

Modern plants and sulfur isoscapes - A review, discussion, and construction of a pilot δ34S isoscape for mobility and provenance studies.

Sulfur isotopes are increasingly used as mobility indicators in humans and animals in biology, archaeology, and forensics. However, there has been a lack of modern sulfur isotope baseline "isoscape" studies using modern plants and animals, largely due to the possibility of contamination of the S isotope values by modern pollution. We collected plants from across a 900-km east-west transect of British Columbia Canada and measured their sulfur isotope values. We then used a random forest model to determine which variables best explained the isotope data patterning and produced a sulfur isoscape for the southern region of British Columbia. We see clear patterning in the plant sulfur isotope values that relate to geographical location and rainfall. Our model also shows that for this study area, it is unlikely that there is a significant influence of anthropogenic pollution on plant δ34S values. We also discuss the use of plants as a substrate for sulfur isoscapes and possible explanations for the often-observed difference between plant and animal δ34S values from the same region, related to differing sources of sulfur in plants compared to amino acids in human and animal tissues. We found that for areas of the world where sulfur pollution is likely less widespread, it is possible to produce a modern plant S isoscape that should be an accurate baseline for mobility studies. Using random forest modelling, we have produced a baseline sulfur isoscape map of southern British Columbia that can be used for ecology, forensic and archaeological studies.

Alterations in one‑carbon metabolism and protein synthesis signals due to methionine supplementation and lipopolysaccharide challenge in Holstein fetal liver explants

One‑carbon metabolism (OCM) fueled by methionine (Met), choline, and folic acid is key for embryo development and fetal growth. We investigated effects of lipopolysaccharide (LPS) to induce inflammation in fetal liver tissue with or without Met on components of OCM and protein synthesis activity. Fetal liver harvested at slaughter from six multiparous pregnant Holstein dairy cows (37 ± 6 kg milk/d, 100 ± 3 d gestation) were incubated (0.2 ± 0.02 g) for 4 h at 37 °C with each of the following: ideal profile of amino acids (control; Lysine:Met 2.9:1), control plus LPS (1 μg/mL), increased Met supply (Met, Lys:Met 2.5:1), and Met+LPS. Data were analyzed as a 2 × 2 factorial (PROC MIXED, SAS 9.4). Ratios of mechanistic target of rapamycin (p-mTOR:mTOR) and eukaryotic elongation factor 2 (p-eEF2:eEF2) protein were lowest (P < 0.0 5) with LPS and highest with Met. Tissue amino acid concentrations were lowest (P < 0.0 5) with Met regardless of LPS suggesting enhanced use via mTOR. The marked increase (P = 0.02) in phosphorylation of S6 ribosomal protein (p-RPS6) with LPS suggested a pro-inflammatory response that was partly alleviated with Met+LPS. No effect (P = 0.4 5) on methionine adenosyl transferase 1 A (MAT1A) protein abundance was detected. Activity of betaine-homocysteine S-methyltransferase (BHMT) was greatest with Met, but Met+LPS dampened this effect (P = 0.0 5). Overall, fetal liver responds to inflammatory challenges and Met supply. The latter can stimulate protein synthesis via mTOR and alter some OCM reactions while having a modest anti-inflammatory effect.

Mass Spectrometry Imaging of Amino Acids Enabled by Quaternized Pyridinium Salt MALDI Probe.

The distribution of small biomolecules, particularly amino acids (AAs), differs between normal cells and cancer cells. Imaging this distribution is crucial for gaining a deeper understanding of their physiological and pathological significance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) provides accurate in situ visualization information. However, the analysis of AAs remains challenging due to the background interference by conventional MALDI matrices. On tissue chemical derivatization (OTCD) MSI serves as an important approach to resolve this issue. We designed, synthesized, and tested a series of pyridinium salt probes and screened out the 1-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-2,4,6-triphenylpyridin-1-ium (DCT) probe with the highest reaction efficiency and the most effective detection. Moreover, a quantum chemistry calculation was executed to address mechanistic insight into the chemical nature of the novel probes. DCT was found to map 20 common AAs in normal mouse tissues for the first time, which allowed differentiation of AA distribution in normal, normal interstitium, tumor, and tumor interstitium regions and provided potential mechanistic insights for cancer research and other biomedical studies.

Haemonchus contortus alters distribution and utilization of protein and amino acids in different tissues of host sheep

The objective was to determine host animal protein/amino acid redistribution and use among the abomasum, duodenum and muscle of sheep infected with Haemonchus contortus. Sixteen male Ujumqin sheep (32.4 ± 3.9 kg) were dewormed and randomly assigned to two groups, infected or not infected with H. contortus (GIN and CON). The GIN group had lower (P < 0.05) dry matter intake, average daily gain, and live body weight than CON, with extensive focal infiltration of lymphocytes in the lamina propria and bottom of the abomasal epithelium. In the abomasum and duodenum, there were 100 and 220 genes, respectively, that were up-regulated, whereas 56 and 149 were down-regulated. In the abomasum, the most enriched KEGG pathways were related to immunity and inflammation reaction, including: viral protein interaction with cytokine and cytokine receptor (P = 0.017), influenza A (P = 0.030), IL-17 signaling pathway (P = 0.030). In the duodenum, KEGG pathways were more enriched in nutrient metabolism, including pancreatic secretion (P < 0.001), protein digestion and absorption (P < 0.001), graft-versus-host disease (P = 0.004). Furthermore, most genes related with the above KEGG pathways were increased in the abomasum but decreased in the duodenum. Amino acid profiles in abomasum and duodenum of CON and GIN groups were clustered in a partial least-squares discriminant analysis model, with significant changes in 36 and 19 metabolites in abomasal and duodenal chyme, respectively. Further confirmed by transcriptome-targeted metabolome association analysis, GIN mainly enhanced metabolism of arginine and sulphur amino acids in abomasum and those metabolic pathways were associated. Meanwhile, GIN mainly decreased pyruvate related amino acid metabolism in duodenum. Moreover, concentrations of Arg (P = 0.036), His (P = 0.027), and Cys (P = 0.046) in longissimus thoracis et lumborum were decreased in GIN, whereas concentrations of Gly (P = 0.012) and Ala (P = 0.046) were increased. In conclusion, H. contortus enhanced metabolism of arginine and sulphur amino acids in the abomasum; decreased pyruvate metabolism in the duodenum; and drove more protein/amino acids for abomasal tissues to resist physical and immune damage, reducing protein and amino acids in duodenum and muscle for support host growth. Specific nutrients (such like arginine, histidine, and cysteine) may play important role in control gastrointestinal nematode infection for ruminant.

Nitrite and sulfide stress affects physiological and metabolic functions of gills in crab Eriocheir sinensis

Nitrite and sulfide are environmental factors that affect crabs survival and immunity. Gills are important respiratory and immune organs of Chinese mitten crabs Eriocheir sinensis, and both nitrite and sulfide can affect the physiological function stability of gill tissues. The present study investigated the acute toxicity of 10 mg/L nitrite and 5 mg/L sulfide, single and in combination, on the morphological, immune, osmoregulatory, and metabolic functions of the gill tissues of Chinese mitten crab. Both single and combined stressors were observed to disrupt the structural integrity of gill tissues. Oxidative stress-related indicators, including MDA, H2O2, and ·O2− content, exhibited an increase in all stress groups. The activities of antioxidant enzymes such as CAT and SOD and the expression levels of antioxidant genes fluctuated to varying degrees in all stress groups. The expression levels of osmotic regulating genes such as aqp, vatp, and nhe increased, as did the expression levels of lipid metabolism-related genes such as pparγ, rxr, fabp1, gyk, and dgat1. Furthermore, nitrite and sulfide stress affected the functions of metabolic pathways related to arachidonic acid, linoleic acid, D-amino acids, phenylalanine, and other substances in Chinese mitten crabs gill tissues. Additionally, the study identified 18 potential biomarkers related to stress responses. The nitrite and sulfide stressors disrupt the structure of crab gill tissues, cause osmoregulation disturbances, immune dysfunction, induce amino acid and lipid metabolic changes in gill tissue.

Metabolomic Analysis of Amino Acid and Organic Acid Profiles in Tissue From Ovarian Cancer Patients Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) and Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS)

Targeted metabolomics studies on organic acids (OAs) and amino acids (AAs) were performed to search for biomarkers for ovarian cancer progression. Simultaneous AA profiling analysis method was developed through LC-MS/MS. Under optimal conditions, method showed good linearity (r ≥ 0.998) with limits of detection (LOD) ≤ 0.22 ng/mL and limits of quantification (LOQ) of ≤0.72 ng/mL. Repeatability varied from 1.4 to 17.3 relative standard deviation (%RSD) and accuracy varied from −20.6% to 12.1% relative error (% RE). Twenty-six AAs and 13 OAs were detected in the analyses of ovarian tissue from patients with benign ovarian tumors (BOT), borderline tumors (BT), and ovarian serous carcinoma (OSC). Star pattern recognition analysis was performed by normalizing each group to the BOT group. The star symbol plots in the BT and OSC groups were distinct and easily distinguished from those in the BOT group. The BT and the OSC groups exhibited higher 3-hydroxybutyric acid and lactic acid levels, lower pyruvic acid and OA levels in the TCA cycle, and alterations in AA metabolic patterns in comparison to the BOT group. Especially, citrulline and leucine were significantly reduced, while fumaric acid and malic acid significantly increased in the OSC group compared to the BT group. The BT and the OSC groups were completely separated in orthogonal partial least squares-discriminant analysis (OPLS-DA) and heatmap analysis. These findings, elevated ketone body levels, increased anaerobic glycolysis, reduced energy production related to mitochondrial dysfunction in the TCA cycle, and AA metabolic patterns associated with the impaired antioxidant system, may explain the characteristic metabolic reprogramming and Warburg effect related to the progression of the OSC into malignancy.

Enhancement of Nutrient Composition and Non-Volatile Flavor Substances in Muscle Tissue of Red Drum (Sciaenops ocellatus) Through Inland Low Salinity Saline-Alkaline Water Culture.

The red drum (Sciaenops ocellatus), a globally significant marine aquaculture species, boasts formidable osmoregulatory capabilities and remarkable adaptability to low salinity, making it an ideal candidate for commercial cultivation in inland low salinity saline-alkaline waters. However, studies on the fundamental nutritional composition and flavor quality of S. ocellatus in these inland low salinity saline-alkaline waters remain unreported. This study delves into the impact of inland low salinity saline-alkaline environments on the basic nutritional components and nonvolatile flavor substances (including free amino acids and free nucleotides) in the muscle tissue of S. ocellatus. The findings reveal that redfish cultivated in these conditions exhibit a significant increase in the crude fat, ash, and protein content in their dorsal muscle tissue, coupled with a decrease in moisture content (p < 0.05), indicating an enhancement in the nutritional value of the dorsal muscle tissue. Furthermore, this cultivation environment significantly elevates the content of free amino acids in the muscle tissue (p < 0.05), particularly those contributing to umami and sweet tastes, while reducing the relative content of bitter amino acids. Although the total content of free nucleotides decreased, the equivalent umami concentration (EUC) in the muscle tissue markedly increased (p < 0.05) due to the synergistic effect of umami amino acids and flavor nucleotides, enhancing the umami taste characteristics. Therefore, inland low salinity saline-alkaline aquaculture not only elevates the nutritional value of S. ocellatus muscle tissue but also improves its umami flavor characteristics. This discovery opens new perspectives for further research into the impact of inland low salinity saline-alkaline environments on the flavor properties of marine animals.

Metabolomic insights into the browning inhibition of fresh-cut apple by hydrogen sulfide

Hydrogen sulfide (H2S) is known to effectively inhibit the browning of fresh-cut apples, but the mechanism at a metabolic level remains unclear. Herein, non-targeted metabolomics was used to analyze metabolic changes in surface and internal tissues of fresh-cut apple after H2S treatment. The results showed that prenol lipids were the most up-accumulated differential metabolites in both surface and inner tissue of fresh-cut apple during browning process, which significantly down-accumulated by H2S treatment. H2S treatment reduced the consumption of amino acid in surface tissue. Regarding inner tissue, H2S activated defense response through accumulation of lysophospholipid signaling and induced the biosynthesis of phenolic compounds. We therefore propose that H2S inhibited the surface browning of fresh-cut apple by reducing the accumulation of prenol lipids, directly delaying amino acid consumption in surface tissue and indirectly regulating defense response in inner tissue, which provides fundamental insights into browning inhibition mechanisms by H2S.

Effectiveness evaluation of the infl uence of protein concentrates on the chemical composition of fish meat

Due to the high demand, cost, growing shortage and low quality of fi sh-meal, there is a constant search for affordable alternative feed raw materials in feeding aquaculture fi shing grounds. The use of high-protein products of plant origin is the most effective option for replacing fi sh-meal in fi sh feed. The purpose of the work was to evaluate the eff ectiveness of the infl uence of protein concentrates on the chemical composition of rainbow trout meat. Testing of compound feed for rainbow trout, the formulation of which included high-protein feed concentrates, was carried out on the basis of IP Kalmykova I. O. and under the conditions of the Research Center “Breeding valuable sturgeon breeds” at the Volgograd State Agrarian University. It was found on base of the results obtained that more intensive growth was observed in the groups in which the mustard protein-containing feed concentrate Gorlinka was introduced into the feed formulation with partial or complete replacement of sunfl ower meal. Here, the increase in the total and average daily live weight gain was noted by 2,83–6,93 % and by 1,62–1,68 %. The use of the Agro-Matic concentrate during the period of the experiment had a positive eff ect on the growth dynamics of rainbow trout, increasing productivity indicators. Thus, live weight indicators increased by 1,29–4,15 %, daily gain indicators increased by 1,44–4,46 % relative to individuals receiving compound feed with fi sh-meal. When studying the chemical composition of trout meat with the addition of Gorlinka concentrate to the feed formulation in the experimental groups, the increase in protein content was revealed by 0,13–0,41 abs.%, inorganic matter by 0,04–0,12 abs.%, also the total amount of amino acids increased by 0,18–0,46 abs.%, while the fat content on the contrary decreased by 0,05–0,17 abs.% compared to the control. In the experimental trout groups, when introducing the Agro-Matic protein concentrate into the diet instead of fi shmeal the protein content increased by 0,03–0,40 abs.%, inorganic matter by 0,08–0,24 abs.%, total amino acids increased by 0,11–0,58 abs.%, there was a slight decrease in the amount of fat by 0,10–0,36 abs.% compared to the 1st control group. The use of protein concentrates has a positive eff ect on the qualitative composition of the muscle tissue of rainbow trout by increasing the protein content and the amount of essential amino acids in the muscle tissue of individuals surpassing the 1st control group in key indicators.

Histomorphological Comparison of Tissues Fixed in Conventional Formalin and Eco-friendly Jaggery Solution: A Cross-sectional Study

Introduction: Tissue fixation is an essential step in the microscopic preparation of tissues to preserve them by preventing autolysis, bacterial putrefaction, and increasing the tissue's refractive index. The conventional fixative used is 40% formaldehyde. Short-term exposure to formaldehyde can cause irritation to the eyes and respiratory tract, leading to breathlessness and coughing. The International Agency for Research on Cancer (IARC) has classified formaldehyde as a Class 1 human carcinogen capable of potentially causing various neoplasms, including nasopharyngeal carcinoma. Therefore, an innovative approach is being explored to replace formalin with harmless and eco-friendly jaggery. Jaggery possesses cytoprotective, antioxidant, and tissue-preservative properties. At a low pH, the fructose in jaggery breaks down into aldehydes, which cross-link with tissue amino acids, resulting in tissue fixation similar to formaldehyde. Aim: To compare the histological and gross morphological features of tissues fixed in formalin and jaggery. Materials and Methods: This cross-sectional study was conducted in Department of Pathology of a tertiary care centre. Surgical specimens obtained fresh, which were not suspicious of malignancy, were included. A sample size of 23 was selected. Surgical specimens already placed in formalin were excluded. A 30% jaggery solution was prepared by dissolving 300 grams of finely powdered jaggery in 1000 mL of distilled water, which was then filtered using filter paper. A 40% formaldehyde solution was used to prepare a 10% formalin fixative. Tissue bits from each specimen were cut into two halves and placed in formalin and jaggery separately. After 24 hours of fixation, the tissue bits were evaluated for gross morphological features, including tissue shrinkage, consistency, and colour. Tissue shrinkage was classified as mild, moderate, or marked, while consistency ranged from soft to firm to hard. The colour of the specimens varied case by case. Stained slides from jaggeryfixed and formalin-fixed tissues were assessed for histological parameters, such as nuclear details, cytoplasmic details, cellular outline, and overall staining quality. A blinded method was used to compare the stained slides using a microscope. Ratings were assigned to each case on a scale of 1-4. The data were statistically analysed using R software. Results: When comparing the formalin-fixed and jaggery-fixed specimens, no significant differences were observed in gross morphological features. All jaggery-fixed specimens appeared brown grossly. Histological features also showed no significant difference (p-value&gt;0.05) except for cytoplasmic details. Therefore, it was observed that tissue preservation using the jaggery solution was comparable to that of formalin. Conclusion: The tissue-preservative properties of jaggery are on par with formalin. Hence, jaggery solution can be used as an eco-friendly substitute for formalin. Further research with larger sample sizes can pave the way for the effective replacement of hazardous formalin with natural jaggery

Development and Validation of a Method for Hydrolysis and Analysis of Amino Acids in Ruminant Feeds, Tissue, and Milk Using Isotope Dilution Z-HILIC Coupled with Electrospray Ionization Triple Quadrupole LC-MS/MS.

Current analytical methods for amino acid (AA) analysis in ruminant nutrition are time-consuming and expensive. This study aimed to develop a method for AA analysis that is faster, more efficient, rugged, and accessible. Four representative matrixes were selected for method development and validation: milk, tissue, feed, and soy flour standard reference material from National Institute of Standards and Technology. Acid and alkaline hydrolysis were used to analyze 18 AA. Separation of AA was performed using a Z-HILIC column in an 18-min run coupled to a triple quadrupole LC/MS system in positive and negative electrospray ionization for identification and quantitation. The method was evaluated for recovery, precision, calibration curve linearity, and limits of detection (LODs) and limits of quantitation (LOQs) and applied to other feed samples. Good quantitation results were achieved for all AA, with coefficients of determination (R2) over 0.995; LODs at 0.2-28.2 and LOQs at 0.7-94.1 ng/mL; intraday and interday precision <14.9% relative standard deviation; blank recovery between 75.6 and 116.2%; and sample recovery between 75.6 and 118.0%. Overall, AA concentrations were similar to literature values, and there was a tendency for higher N recovery as AA. In conclusion, an efficient and robust method was validated to routinely analyze AA for appropriate characterization in diet formulation for dairy cattle.

Untargeted UHPLC-MS deferential screening method and targeted UHPLC-MS/MS quantitative method for the determination of amino acids in mouse brain tissue

An untargeted UHPLC-MS method was developed for the preliminary screening of differential amino acids between two mouse brain tissue samples, and a targeted UHPLC-MS/MS method was developed for the quantitative determination of amino acids in mouse brain tissue samples. The sample processing procedure, chromatographic conditions and MS parameters were optimized. The mouse brain tissue samples were homogenized with HCl solution (0.01 mol/L), protein precipitated with methanol and centrifugation. For the untargeted method, one sample was derivatized with butanol (D0)-HCl and another sample was derivatized with deuterated butanol (D9)-HCl, then combined together and analyzed with UHPLC-MS. Chromatographic peak area ratio for each amino acid was then used for differential analyses between two samples. Results showed good accuracy with standard spiked samples (83.4–113.0 %). For the targeted quantitative method, the sample supernatants were dried and residues were derivatized with butanol (D0)-HCl. Internal standards of amino acids derivatized with deuterated butanol (D9)-HCl were added before analyzed with UHPLC-MS/MS. The intra- and inter-day precisions were in the intervals of 0.5–8.1 % and 0.3–9.1 %. The LOD and the LOQ were ranged from 0.03 × 10−3 to 0.92 × 10−3 µg mL−1 and 0.08 × 10−3 to 3.08 × 10−3 µg mL−1, respectively. Spiked recoveries were from 96.8 % to 111.7 %. Both methods were applied to analyze amino acids in brain tissue of C57BL/6 mice treated and untreated with piperine and the result showed that the levels of glutamic acid, aspartic acid, histidine, asparagine, γ-aminobutyric acid, alanine, leucine and isoleucine significantly decreased in the piperine treated group comparing with the untreated group.

The Growth, Leaf Antioxidant Enzymes and Amino Acid Content of Tomato as Affected by Grafting on Wild Tomato Rootstocks 1 (S. Pimpinellifolium and S. Habrochaites) Under Salt Stress

Salinity in soil or water is a serious threat to plant growth, which reduces yields and threatens food security. It is possible to reduce the negative effects of salinity on tomato scions through grafting. To evaluate the effects of salt stress on graft combinations in hydroponic conditions, SP3, SP4, SP5 (S. pimpinellifolium), SH1, SH3, SH5 (S. habrochaites) and two hybrids L × SP5 (S. lycopersicum L. × S. pimpinellifolium) and L × SH3 (S. lycopersicum L. × S. habrochaites) were used as rootstock and two varieties Galaxy and H2274 (S. lycopersicum L.) were used as scion. Plants were evaluated in terms of biomass parameters and antioxidant enzyme activities and amino acid contents in leaf tissues under control and salt stress conditions. Salt stress decreased the fresh weight of shoots and roots by 68.71 and 23.59, respectively, and increased H2O2 content (93.90 %) and MDA content (63.71 %). In addition, salt stress in leaf tissues POD (204.49 %), CAT (83.87 %), SOD (204.49 %), GR (88.23 %), GST (100.16 %), G6PD (32.85 %), 6GPD (46.19 %), APX (157.29 %) caused an increase in enzymes and total amino acid content (160.57 %). In salt stress conditions, SH5/Galaxy and L × SP5/Galaxy are the graft combinations with the highest fresh shoot and root weight, leaf number and root volume. These graft combinations contain the most isoleucine, proline lysine, alanine arginine, sucrosine leucine, methionine, hydroxyproline glutamate, glutamine and histidine amino acids in leaf tissues and have the lowest MDA content. Ungrafted H2274 and Galaxy plants have the highest MDA and content under salt stress conditions, these ungrafted plants have the least other amino acids and enzymes other than theonine amino acid in their leaf tissues. Furthermore, ungrafted plants under saline conditions had the lowest shoot and root fresh weight, leaf number and root volume. MDA content correlated negatively with shoot and root fresh weight, leaf number and root volume. H2O2 correlated positively with CAT, SOD, GST and peroxidase enzymes and negatively with APX, GR and G6PD enzymes. It seems that the antioxidant enzyme activity and amino acid content in leaf tissues influence salinity response and the appropriate rootstock/scion combination could be a viable method for tomato cultivation in saline conditions.

262 Weaning-Induced Changes in Tissue Cysteine Utilization in Pigs

Abstract Weaning stress in pigs is associated with low and variable feed intake, poor nutrient utilization, and high feed costs. Cysteine is a sulfur amino acid with key roles in pig production, but how cysteine metabolism and requirements are affected by weaning stress should be better defined. The objective of this study was to determine weaning-induced changes in tissue cysteine metabolism. Pigs (initial BW = 7.23 ± 0.68 kg) were either weaned at 21 d age and euthanized at 23 d age (W; n = 9) or remained with the sow and euthanized at 23 d age (NW; n = 9). Weaned pigs were fasted to simulate acute nutritional stress but had free access to water. At euthanasia, blood, liver, jejunum, and ileum tissues were collected. Plasma and tissue amino acids and amino thiols were analyzed by HPLC. The abundance of proteins central to liver cysteine utilization were determined by immunoblot and enzyme activity assays. Glutathione (GSH) concentrations were less in jejunum (1,379 versus 1,720 ± 70 nmol/g; P &amp;lt; 0.01) and ileum (1,497 versus 1,740 ± 74 nmol/g; P &amp;lt; 0.05) in W than NW pigs. However, liver GSH concentration was not different between W and NW pigs (P &amp;gt; 0.10). While cysteine in jejunum and ileum were not affected by weaning (P &amp;gt; 0.10), γ-glutamylcysteine (γ-GC), the immediate precursor of GSH, declined in both jejunum (9.7 versus 14.3 ± 1.4 nmol/g; P &amp;lt; 0.01) and ileum (6.4 versus 11.2 ± 0.8 nmol/g; P &amp;lt; 0.001) in W compared with NW pigs. In liver, cysteine concentration was greater in W compared with NW pigs (417 versus 298 ± 33 nmol/g; P &amp;lt; 0.05). Although liver cysteine was greater in W pigs, liver activity of glutamate cysteine ligase (GCL; 2.49 versus 4.15 ± 0.25 nmol γ-GC/(mg protein•min); P &amp;lt; 0.001) and glutathione synthetase (GSS; 1.54 versus 1.92 nmol GSH/(mg protein•min); P &amp;lt; 0.001), enzymes needed for GSH production, were less in W than NW pigs. Immunoblot analysis of the catalytic subunit of GCL (GCLC; P &amp;lt; 0.05) and modifier subunit of GCL (GCLM; P = 0.057) were also lower in W compared with NW pigs, but GSS was not different between groups (P &amp;gt; 0.10). While plasma taurine was lower in W compared with NW pigs (64 versus 128 ± 7 µmol/L, P &amp;lt; 0.001), liver taurine was greater in W compared with NW pigs (4,726 versus 2,250 ± 471 nmol/g; P &amp;lt; 0.01). Despite an increase systemic and liver availability of cysteine, the rate-limiting amino acid for GSH synthesis, small intestine GSH concentrations decline. This suggests that systemic cysteine is not effectively utilized for gut GSH production in pigs after weaning.