Detection of Glycine as a Model Protein in Blood Serum Using 2D-IR Spectroscopy.

Glycine (Gly) is used as a model system to evaluate the ability of ultrafast two-dimensional infrared (2D-IR) spectroscopy to detect and quantify the low-molecular-weight proteinaceous components of blood serum. Combining data acquisition schemes to suppress absorption bands of H2O that overlap with the protein amide I band with analysis of peak patterns appearing in the off-diagonal region of the 2D-IR spectrum allows separation of the Gly spectral signature from that of the dominant protein fraction of serum in a transmission-mode 2D-IR measurement without any sample manipulation, e.g., filtration or drying. 2D-IR spectra of blood serum samples supplemented with varying concentrations of Gly were obtained, and a range of data analysis methods compared, leading to a detection limit of ∼3 mg/mL for Gly. The reported methodology provides a platform for a critical assessment of the sensitivity of 2D-IR for measuring the concentrations of amino acids, peptides, and low-molecular-weight proteins present in serum samples. We conclude that, in the case of several clinically relevant diagnostic molecules and their combinations, the potential exists for 2D-IR to complement IR absorption methods as the benefits of the second frequency dimension offered by 2D-IR spectroscopy outweigh the added technical complexity of the measurement.

Maternal arginine supplementation enhances thermogenesis in the newborn lamb.

Body temperature maintenance is one of the most important physiological processes initiated after birth. Brown adipose tissue (BAT) is an essential mediator of thermogenesis in many species and is responsible for 50% of the heat generated in the newborn lamb. To determine if maternal arginine supplementation could enhance thermogenesis in the neonate, we randomly assigned 31 multiparous Suffolk ewes, gestating singletons or twins, to receive intravenous injections of either l-arginine (27 mg/kg body weight; n = 17) or sterile saline (n = 14) three times daily from day 75 to 125 of gestation (term = 147). Following parturition, lambs were removed from their mothers and subjected to 0 °C cold challenges at 4 and 22 h of age. Rectal temperatures were higher for the duration of the cold challenges in lambs from arginine-treated ewes compared with lambs from saline-treated ewes (P < 0.05). Elevated rectal temperatures were associated with increased (P < 0.05) circulating glycine and serine concentrations in lambs. The mRNA expression of genes related to BAT function changed over time, but not between lambs from arginine-treated vs. saline-treated ewes. Results indicate that maternal arginine treatment increases neonatal thermogenesis after birth. Although the underlying mechanisms remain to be elucidated, these data are a first step in improving neonatal survival in response to cold.

Substitution of Dietary Sulfur Amino Acids by dl-2-Hydroxy-4-Methylthiobutyric Acid Reduces Fractional Glutathione Synthesis in Weaned Piglets.

BACKGROUND: Cys is limiting for reduced glutathione (GSH) synthesis and can be synthesized from Met. We hypothesized that the dietary Met hydroxyl analogue dl-2-hydroxy-4-methylthiobutyric acid (dl-HMTBA) affects Cys and GSH metabolism and oxidative stress defense differently than Met. OBJECTIVE: The objective was to elucidate whether dl-HMTBA supplementation of a Met-deficient diet affects Cys flux, GSH fractional synthetic rate (FSR), and the basal oxidative stress level relative to Met supplementation in pigs. METHODS: Twenty-nine male German Landrace piglets aged 28 d were allocated to 3 dietary groups: a basal diet limiting in Met (69% of Met plus Cys requirement) supplemented with either 0.15% l-Met (LMET; n = 9), 0.15% dl-Met (DLMET; n = 11), or 0.17% dl-HMTBA (DLHMTBA; n = 9) on an equimolar basis. At age 54 d the pigs received a continuous infusion of [1-13C]-Cys to calculate Cys flux and Cys oxidation. After 3 d, GSH FSR was determined by [2,2-2H2]-glycine infusion, and RBC GSH and oxidized GSH concentrations were measured. At age 62 d the animals were killed to determine hepatic mRNA abundances of enzymes involved in GSH metabolism, GSH concentrations, and plasma oxidative stress defense markers. RESULTS: The Cys oxidation was 21-39% and Cys flux 5-15% higher in the fed relative to the feed-deprived state (P < 0.001). On average, GSH FSR was 49% lower (P < 0.01), and RBC GSH and total GSH concentrations were 12% and 9% lower, respectively, in DLHMTBA and DLMET relative to LMET pigs (P < 0.05). In the feed-deprived state, Gly flux, the GSH:oxidized glutathione (GSSG) ratio, RBC GSSG concentrations, plasma oxidative stress markers, and the hepatic GSH content did not differ between groups. CONCLUSIONS: Although GSH FSR was higher in LMET compared with DLMET or DLHMTBA feed-deprived pigs, these differences were not reflected by lower oxidative stress markers and antioxidant defense enzymes in LMET pigs.

A Previously Unknown Drug-Drug Interaction Is Suspected in Delayed Elimination of Plasma Methotrexate in High-Dose Methotrexate Therapy.

Background: High-dose methotrexate (HD-MTX) therapy is widely implemented for leukemia, osteosarcoma, and lymphoma. Although various measures have been taken to avoid toxicity from high serum MTX concentrations, there are many cases of delayed elimination of MTX. Objective: We suspected that delayed elimination of serum MTX was caused by unknown interactions between MTX and concomitant drugs. Methods: Concerning concomitant drugs in the case of delayed elimination of MTX, we performed screening tests in 35 patients who had undergone HD-MTX therapy. We then investigated the risk factors for delayed MTX elimination in 94 patients with leukemia, lymphoma, or osteosarcoma retrospectively. Results: The percentages of concomitant use of Stronger Neo-Minophagen C (SNMC), a glycyrrhizin preparation, and vincristine were higher in the delayed group. The percentage of delayed MTX elimination in patients receiving HD-MTX therapy was 41%. Multiple logistic regression analysis revealed that the concomitant use of SNMC solely was a significant risk factor for delayed MTX (odds ratio = 12.20; 95% CI = 1.06-139.84). Conclusion and Relevance: Concomitant use of SNMC was shown to be related to delayed elimination of serum MTX, and our results suggested a previously unknown drug-drug interaction between MTX and SNMC.

The Hepatoprotective Effects of Zinc Glycine on Liver Injury in Meat Duck Through Alleviating Hepatic Lipid Deposition and Inflammation.

Dietary zinc status was recently approved to exert a powerful influence on liver health, and zinc deficiency results in hepatic injury caused by fat deposition, inflammation, and oxidant stress, but the effect of zinc on hepatic lipid metabolism and liver injury in meat duck has not been well defined. To determine the hepatoprotective effects of graded zinc glycine in meat ducks. A total of 384 1-day-old male meat ducks were subjected to 5 weeks feeding program with three experimental diets: (1) low-zinc diet, (2) adequate-zinc diet, and (3) high-zinc diet. Blood and liver samples were collected for biochemical analysis, gene expression analysis, and histopathological study. Diet with low zinc increased hepatic lipid content and triglyceride concentration. Meat ducks fed low-zinc diet exhibited considerably increased serum alanine aminotransferase (ALT) activity than birds fed other diets among all groups (P < 0.05). Low zinc administration also notably induced hepatocyte apoptosis and stimulated hepatic inflammatory gene expression. Adequate or high zinc supplementation increased hepatic zinc level, reduced hepatic lipid deposition and hepatosomatic indices through suppressing the expression of lipogenic genes including fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) (P < 0.05), and upregulated the mRNA expression of both fatty acid secretion and ß-oxidation, including carnitine palmitoyltransferase 1a (Cpt1a), peroxisome proliferator-activated receptor (PPAR)α, and apolipoprotein B (ApoB) (P < 0.05). Dietary zinc addition also declined hepatic mRNA expression of interleukin (IL)-1ß and IL-6 (P < 0.05). Furthermore, diets with adequate or high zinc significantly decreased serum ALT activity and hepatocyte apoptosis. These data revealed that supplementing adequate- or high-zinc glycine efficiently protects liver injury by attenuating lipid deposition and hepatic inflammation.

Associations between Dietary Patterns and Bile Acids-Results from a Cross-Sectional Study in Vegans and Omnivores.

Bile acids play an active role in fat metabolism and, in high-fat diets, elevated concentrations of fecal bile acids may be related to an increased risk of colorectal cancer. This study investigated concentrations of fecal and serum bile acids in 36 vegans and 36 omnivores. The reduced rank regression was used to identify dietary patterns associated with fecal bile acids. Dietary patterns were derived with secondary and conjugated fecal bile acids as response variables and 53 food groups as predictors. Vegans had higher fiber (p < 0.01) and lower fat (p = 0.0024) intake than omnivores. In serum, primary and glycine-conjugated bile acids were higher in vegans than in omnivores (p ≤ 0.01). All fecal bile acids were significantly lower in vegans compared to omnivores (p < 0.01). Processed meat, fried potatoes, fish, margarine, and coffee contributed most positively, whereas muesli most negatively to a dietary pattern that was directly associated with all fecal bile acids. According to the pattern, fat intake was positively and fiber intake was inversely correlated with bile acids. The findings contribute to the evidence that, in particular, animal products and fat may play a part in higher levels of fecal bile acids.

Serial measurement of glyphosate blood concentration in a glyphosate potassium herbicide-intoxicated patient: A case report.

INTRODUCTION: This report describes changes in blood and urine concentrations of glyphosate potassium over time and their correlations with clinical symptoms in a patient with acute glyphosate potassium poisoning. CASE REPORT: A 67-year-old man visited the emergency center after ingesting 250 mL of a glyphosate potassium-based herbicide 5 h before. He was alert but presented with nausea, vomiting, and bradyarrhythmia with atrial fibrillation (tall T waves). Laboratory findings revealed a serum potassium level of 6.52 mEq/L. After treatment with an injection of calcium gluconate, insulin with glucose, bicarbonate, and an enema with polystyrene sulfonate, the patient's serum potassium level normalized and the bradyarrhythmia converted to a normal sinus rhythm. During admission, the blood and urine concentration of glyphosate and urine aminomethylphosphonic acid (AMPA, a glyphosate metabolite) was measured at regular time intervals. The patient's glyphosate blood concentration on admission was 11.48 mg/L, and it had decreased rapidly by 16 h and maintained about 1mgl/L by 70 h after admission. Urine glyphosate and AMPA levels had also decreased rapidly by 6 h after admission. DISCUSSION: Glyphosate potassium poisoning causes hyperkalemia. Blood concentrations of glyphosate were decreased rapidly by 16 h after admission, and urine concentrations were also decreased by 6 h after admission.

Activated glycine receptors may decrease endosomal NADPH oxidase activity by opposing ClC-3-mediated efflux of chloride from endosomes.

Receptor-mediated activation of NADPH oxidase complexes commonly occurs in endosomes; the hydrogen peroxide produced by the dismutation of superoxide generated within the endosomes often functions to boost receptor function by reversibly inhibiting protein tyrosine phosphatases or by promoting formation of signaling complexes. NADPH oxidase-mediated formation of superoxide entails transfer of two electrons (provided by NADPH) from the cytosol to the endosomal lumen, where two molecules of superoxide are generated. This charge transfer must be balanced if NADPH oxidase activity is to be sustained. In many cells, this balance is achieved by ClC-3, a chloride-proton antiporter which can extrude two chlorides from the endosome to balance the importation of two electrons. The efficiency of this chloride extrusion will evidently be contingent on the cytosolic chloride level. Pro-inflammatory hormones which stimulate NADPH oxidase activity in endosomes have been shown to promote chloride extrusion from the cell, thereby expediting endosomal chloride export. Conversely, high cytosolic chloride could potentially slow endosomal NADPH oxidase activity by impeding ClC-3-mediated chloride export. Glycine-activated, strychnine-inhibitable chloride channels, which boost intracellular chloride in cells which maintain intracellular chloride levels lower than that of plasma, have shown anti-inflammatory and anti-angiogenic activity in cell culture and rodent studies. It is proposed that many of these effects may be attributable to glycine-mediated suppression of endosomal NADPH oxidase activity. This model suggests that supplemental glycine may have utility for prevention and control of atherosclerosis, heart failure, angiogenesis associated with cancer or retinal disorders, and a range of inflammation-driven syndromes - including metabolic syndrome; and it might complement the suppression of NADPH oxidase activity achievable with phycocyanobilin-enriched spirulina extracts.

Hydroxyproline Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice: Involvment of the NF-κB Signaling and Oxidative Stress.

SCOPE: Inflammatory bowel disease (IBD) is a chronic disease of gastrointestinal tract in which oxidative stress and overactivation of inflammatory response are implicated. The aim of the present study is to test the hypothesis that hydroxyproline (Hyp), an amino acid with an antioxidative property, attenuates dextran sulfate sodium (DSS)-induced colitis in mice. METHODS AND RESULTS: Male C57BL/6 mice supplemented with or without 1% Hyp are subjected to 2.5% DSS in drinking water to induce colitis. Hyp attenuates the severity of colitis as evidenced by reduced disease activity index scores, decreased myeloperoxidase activity, histological damage, and apoptosis. Furthermore, DSS-induced increases in reactive oxygen species accumulation, TNF-α and IL-6 secretion, and malonyldialdehyde activity and a decrease in reduced glutathione in the colon are ameliorated by Hyp. The enhanced phosphorylation of STAT3 and NF-κB following DSS administration is mitigated by Hyp, which is also observed in LPS-treated RAW264.7 macrophages. Moreover, the inhibitory effect of Hyp on IL-6 expression is mainly mediated by the NF-κB signaling, because the induction of STAT3 and IL-6 by LPS is markedly reversed by Bay11-7085, a specific inhibitor NF-κB. CONCLUSION: In summary, Hyp is a critical nutrient with an ability to attenuate DSS-induced colonic damage in mice. This beneficial effect of Hyp is partially mediated by inhibiting the NF-κB/IL-6 signaling and the restoration of redox homeostasis.

Effects of single and combined metformin and L-citrulline supplementation on L-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications.

The L-arginine/nitric oxide synthase (NOS) pathway is considered to be altered in muscular dystrophy such as Becker muscular dystrophy (BMD). We investigated two pharmacological options aimed to increase nitric oxide (NO) synthesis in 20 male BMD patients (age range 21-44 years): (1) supplementation with L-citrulline (3 × 5 g/d), the precursor of L-arginine which is the substrate of neuronal NO synthase (nNOS); and (2) treatment with the antidiabetic drug metformin (3 × 500 mg/d) which activates nNOS in human skeletal muscle. We also investigated the combined use of L-citrulline (3 × 5 g/d) and metformin (3 × 500 mg/d). Before and after treatment, we measured in serum and urine samples the concentration of amino acids and metabolites of L-arginine-related pathways and the oxidative stress biomarker malondialdehyde (MDA). Compared to healthy subjects, BMD patients have altered NOS, arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) pathways. Metformin treatment resulted in concentration decrease of arginine and MDA in serum, and of homoarginine (hArg) and guanidinoacetate (GAA) in serum and urine. L-Citrulline supplementation resulted in considerable increase of the concentrations of amino acids and creatinine in the serum, and in their urinary excretion rates. Combined use of metformin and L-citrulline attenuated the effects obtained from their single administrations. Metformin, L-citrulline or their combination did not alter serum nitrite and nitrate concentrations and their urinary excretion rates. In conclusion, metformin or L-citrulline supplementation to BMD patients results in remarkable antidromic changes of the AGAT and GAMT pathways. In combination, metformin and L-citrulline at the doses used in the present study seem to abolish the biochemical effects of the single drugs in slight favor of L-citrulline.

Hydrophilic interaction chromatography coupled to tandem mass spectrometry as a method for simultaneous determination of guanidinoacetate and creatine.

The biosynthesis of creatine (Cr) is closely related to the bioavailability of guanidinoacetate (GAA). The lack of one or the other may compromise their role in the energy transport and cell signaling. A reliable estimate of their levels in biological samples is imperative since they are important markers of many metabolic disorders. Therefore, a new LC-MS/MS method for simultaneous determination and quantification of GAA and Cr by multiple reaction monitoring (MRM) mode was developed based on the hydrophilic interaction chromatography (HILIC) and response surface methodology (RSM) for the optimization of chromatographic parameters. The optimized parameters ensured good separation of these similar, very polar molecules (chromatographic resolution > 1.5) without prior derivatization step in a short analysis run (6 min). The developed method was validated to ensure accurate (R, 75.1-101.6%), precise (RSD < 20%) and low quantification (LOQ of 0.025 µg mL-1 for GAA and 0.006 µg mL-1 for Cr) of the tested analytes and the use of matrix-matched calibration eliminated variable effects of complex matrices such as human plasma and urine. Therefore, this method can be implemented in medical laboratories as a tool for the diagnostics of creatine deficiencies and monitoring of guanidinoacetate and creatine supplementation regimes in biological samples.

Effects of guanidinoacetic acid on growth performance, creatine and energy metabolism, and carcass characteristics in growing-finishing pigs.

The aim of this study was to investigate the effects of dietary supplementation with guanidinoacetic acid (GAA) on the growth performance, creatine and energy metabolism, and carcass characteristics in growing-finishing pigs. In Exp. 1, Duroc × Landrace × Yorkshire pigs (n = 180, 33.61 ± 3.91 kg average BW) were blocked by weight and sex, and allotted to 5 treatments with 6 replicates (3 gilts and 3 barrows per replicate pen). Diets were corn-soybean meal-basal diets supplemented with 0, 300, 600, 900, and 1,200 mg/kg of GAA and fed to the pigs for 98 d. From days 1 to 98, G:F increased (linear, P < 0.05) with increasing addition of dietary GAA. Using a broken-line model, the optimum level of dietary GAA was 300 mg/kg during the overall experimental period (days 1 to 98) to maximize G:F. Hot carcass weight, carcass length, and lean percentage showed a tendency to increase (quadratic, 0.05 < P < 0.10) with increasing addition of dietary GAA. On day 98, serum GAA and liver creatine tended to increase (linear, P = 0.10, 0.07) as dietary GAA increased. In addition, serum ATP on day 98 increased linearly (linear, P < 0.01), and muscle ATP and adenosine monophosphate increased quadratically (quadratic, P = 0.05) with incremental GAA supplementation. In Exp. 2, Duroc × Landrace × Yorkshire pigs (n = 180, 53.19 ± 5.63 kg average BW) were blocked by weight and sex, and allotted to 5 treatments with 6 replicates (3 gilts and 3 barrows per replicate pen). Diets were corn-soybean meal-basal diets supplemented with 0, 150, 300, 600, and 1,200 mg/kg of GAA for 35 d. As dietary GAA increased, final BW, ADG, and G:F increased quadratically (quadratic, P < 0.01), and 300 mg/kg of GAA maximized ADG and final BW (P < 0.05).The results indicate that dietary GAA could increase the creatine and ATP load in the tissues of pigs and accordingly improve growth performance. Dietary supplementation with 300 mg/kg of GAA was suitable to maximize the growth performance of growing-finishing pigs.

Effect of guanidinoacetic acid supplementation on live performance, meat quality, pectoral myopathies and blood parameters of male broilers fed corn-based diets with or without poultry by-products.

Creatine is a nitrogenous compound naturally occurring in animal tissues and is obtained from dietary animal protein or de novo synthesis from guanidinoacetic acid (GAA). The dietary supply of this semi-essential nutrient could be adversely compromised when feeding purely vegetable-based diets. The objective of this experiment was to evaluate the effects of GAA supplementation in broilers fed corn-based diets with or without the inclusion of poultry by-products (PBP) on live performance, carcass and cut up yields, meat quality, pectoral muscle myopathies, differential blood count, blood clinical chemistry, serum GAA and its metabolites. The treatments consisted of PBP inclusion in the diets at 0 and 5%, with or without GAA supplementation (0 or 0.06%). A total of 1,280 one-d-old male Ross 708 broiler chicks were randomly placed in 64 floor pens with 16 replicates per treatment combination. At 0, 14, 35, 48, and 55 d, pen BW and feed intake were recorded. BW gain and FCR were calculated at the end of each phase. Individual BW was obtained at 55 d and one broiler per pen was selected for blood collection. Additionally, four broilers per pen were selected (including the chicken for blood collection) for processing. Data were analyzed as a randomized complete block design in a 2 × 2 factorial arrangement with PBP and GAA supplementation as main effects. An improvement (P < 0.05) on FCR of 0.019 (g:g) was detected at 55 d due to GAA supplementation. The probability of having breast meat with low severity of wooden breast (score 2) was increased (P < 0.05) by GAA inclusion in diets without PBP. An interaction effect (P < 0.05) was detected on GAA concentration in blood. The supplementation with GAA and PBP inclusion resulted in higher (P < 0.05) GAA serum concentration. Generally, meat quality parameters were not affected by GAA. In conclusion, GAA supplementation improved FCR regardless of dietary PBP and reduced wooden breast severity by increasing score 2 in diets without PBP.

Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic profiling.

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

Vitamin B6 is essential for serine de novo biosynthesis.

Pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.

Dietary methionine source and level affect hepatic sulfur amino acid metabolism of broiler breeder hens.

A study was carried out to investigate the effects of dietary methionine source and level on plasma free amino acids patterns and the expression of genes involved in hepatic methionine metabolism in broiler breeders. A total of 2184 broiler breeders were assigned to 13 dietary treatments, with eight replicates per treatment. The 13 treatments included one control group and 12 additional treatments employing two sources and six levels (0.05, 0.10, 0.15, 0.20, 0.25 and 1.00%). Higher plasma methionine concentration was measured for DL-methionine (DLM) treated hens. Plasma alanine concentration was linearly increased as DLM or 2-hydroxy-4-(methylthio) butanoic acid (HMTBA) supplementation level increased. There was a linear increase in concentrations of tyrosine, valine, glycine and serine as dietary DLM supplementation level increased. Hens treated with DLM had higher relative expression of ADA than those fed HMTBA. The expression of MS, ADA, SAHH and MAT2A changed quadratically as HMTBA supplementation level increased, while the expression of GNMT and SAHH changed quadratically as DLM supplementation level increased. In conclusion, the effects of HMTBA on plasma free amino acid patterns and the expression of hepatic genes involved with methionine are different from DLM.

Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy.

Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.

Impaired "Glycine"-mia in Type 2 Diabetes and Potential Mechanisms Contributing to Glucose Homeostasis.

The onset and/or progression of type 2 diabetes (T2D) can be prevented if intervention is early enough. As such, much effort has been placed on the search for indicators predictive of prediabetes and disease onset or progression. An increasing body of evidence suggests that changes in plasma glycine may be one such biomarker. Circulating glycine levels are consistently low in patients with T2D. Levels of this nonessential amino acid correlate negatively with obesity and insulin resistance. Plasma glycine correlates positively with glucose disposal, and rises with interventions such as exercise and bariatric surgery that improve glucose homeostasis. A role for glycine in the regulation of glucose, beyond being a potential biomarker, is less clear, however. Dietary glycine supplementation increases insulin, reduces systemic inflammation, and improves glucose tolerance. Emerging evidence suggests that glycine, a neurotransmitter, also acts directly on target tissues that include the endocrine pancreas and the brain via glycine receptors and as a coligand for N-methyl-d-aspartate glutamate receptors to control insulin secretion and liver glucose output, respectively. Here, we review the current evidence supporting a role for glycine in glucose homeostasis via its central and peripheral actions and changes that occur in T2D.

Personal model-assisted identification of NAD+ and glutathione metabolism as intervention target in NAFLD.

To elucidate the molecular mechanisms underlying non-alcoholic fatty liver disease (NAFLD), we recruited 86 subjects with varying degrees of hepatic steatosis (HS). We obtained experimental data on lipoprotein fluxes and used these individual measurements as personalized constraints of a hepatocyte genome-scale metabolic model to investigate metabolic differences in liver, taking into account its interactions with other tissues. Our systems level analysis predicted an altered demand for NAD+ and glutathione (GSH) in subjects with high HS Our analysis and metabolomic measurements showed that plasma levels of glycine, serine, and associated metabolites are negatively correlated with HS, suggesting that these GSH metabolism precursors might be limiting. Quantification of the hepatic expression levels of the associated enzymes further pointed to altered de novo GSH synthesis. To assess the effect of GSH and NAD+ repletion on the development of NAFLD, we added precursors for GSH and NAD+ biosynthesis to the Western diet and demonstrated that supplementation prevents HS in mice. In a proof-of-concept human study, we found improved liver function and decreased HS after supplementation with serine (a precursor to glycine) and hereby propose a strategy for NAFLD treatment.

NMR Metabolomics Investigates the Influence of Flavonoid-Enriched Rations on Chicken Plasma.

The use of flavonoids as dietary supplements is well established, mainly due to their intense antioxidant and anti-inflammatory properties. In the present study, hesperidin, naringin, and vitamin E were used as additives at different concentrations in poultry rations in order to achieve meat of improved quality. NMR metabolomics was applied to chicken blood serum samples to discern whether and how the enriched rations affected the animals' metabolic profile. Variations in the metabolic patterns according to sustenance consumption were traced by orthogonal projections to latent structures discriminant analysis (OPLS-DA) models and were attributed to specific metabolites by using S-line plots. In particular, serum samples from chickens fed with vitamin E displayed higher concentrations of glycine and succinic acid compared to control samples, which were mainly characterized by betaine, formic acid, and lipoproteins. Samples from chickens fed with hesperidin were characterized by increased levels of lactic acid, citric acid, creatine, carnosine, creatinine, phosphocreatine, anserine, glucose, and alanine compared to control samples. Lastly, naringin samples exhibited increased levels of citric and acetic acids. Results verify the scalability of NMR metabolomics to highlight metabolite variations among chicken serum samples in relation to food rations.