The impact of obesity-associated glycine deficiency on the elimination of endogenous and exogenous metabolites via the glycine conjugation pathway.

Background: Glycine is an integral component of the human detoxification system as it reacts with potentially toxic exogenous and endogenously produced compounds and metabolites via the glycine conjugation pathway for urinary excretion. Because individuals with obesity have reduced glycine availability, this detoxification pathway may be compromised. However, it should be restored after bariatric surgery because of increased glycine production. Objective: To examine the impact of obesity-associated glycine deficiency on the glycine conjugation pathway. We hypothesize that the synthesis rates of acylglycines from endogenous and exogenous sources are significantly reduced in individuals with obesity but increase after bariatric surgery. Methods: We recruited 21 participants with class III obesity and 21 with healthy weight as controls. At baseline, [1,2-13C2] glycine was infused to study the glycine conjugation pathway by quantifying the synthesis rates of several acylglycines. The same measurements were repeated in participants with obesity six months after bariatric surgery. Data are presented as mean ± standard deviation, and p-value< 0.05 is considered statistically significant. Results: Baseline data of 20 participants with obesity were first compared to controls. Participants with obesity were significantly heavier than controls (mean BMI 40.5 ± 7.1 vs. 20.8 ± 2.1 kg/m2). They had significantly lower plasma glycine concentration (168 ± 30 vs. 209 ± 50 µmol/L) and slower absolute synthesis rates of acetylglycine, isobutyrylglycine, tigylglycine, isovalerylglycine, and hexanoylglycine. Pre- and post-surgery data were available for 16 participants with obesity. Post-surgery BMI decreased from 40.9 ± 7.3 to 31.6 ± 6.0 kg/m2. Plasma glycine concentration increased from 164 ± 26 to 212 ± 38 µmol/L) and was associated with significantly higher rates of excretion of acetylglycine, isobutyrylglycine, tigylglycine, isovalerylglycine, and hexanoylglycine. Benzoic acid (a xenobiotic dicarboxylic acid) is excreted as benzoylglycine; its synthesis rate was significantly slower in participants with obesity but increased after bariatric surgery. Conclusion: Obesity-associated glycine deficiency impairs the human body's ability to eliminate endogenous and exogenous metabolites/compounds via the glycine conjugation pathway. This impairment is ameliorated when glycine supply is restored after bariatric surgery. These findings imply that dietary glycine supplementation could treat obesity-associated metabolic complications due to the accumulation of intramitochondrial toxic metabolites. Clinical trial registration: https://clinicaltrials.gov/study/NCT04660513, identifier NCT04660513.

Plasma amino acid signatures define types of pediatric diabetes.

BACKGROUND & AIMS: Metabolic biomarkers with pathophysiological relevance is lacking in pediatric diabetes. We aimed to identify novel metabolic biomarkers in pediatric type 1 (T1D) and type 2 diabetes (T2D). We hypothesized that (1) targeted plasma metabolomics, focused on plasma amino acid concentrations, could identify distinctively altered patterns in children with T1D or T2D, and (2) there are specific changes in concentrations of metabolites related to branch chain amino acids (BCAA) and arginine metabolism in children with T2D. METHODS: In a pilot study, we enrolled children with T1D (n = 15) and T2D (n = 13), and healthy controls (n = 15). Fasting plasma amino acid concentrations were measured by ultra-performance liquid chromatography, and compared between the groups after adjustment for confounding factors. RESULTS: The mean age (SD) of participants was 16.4 (0.9) years. There were no group differences in age, gender, race/ethnicity, or 24-h protein intake. Mean BMI percentile was higher in the T2D than the T1D group or controls (p < 0.001). The T2D group had lower arginine, citrulline, glutamine, glycine, phenylalanine, methionine, threonine, asparagine and symmetric dimethylarginine (SDMA) but higher aspartate than controls, after adjusting for BMI percentiles (all p < 0.05). Children with T2D also had lower glycine but higher ornithine, proline, leucine, isoleucine, valine, total BCAA, lysine and tyrosine than those with T1D after adjusting for confounding factors (all p < 0.05). Children with T1D had lower phenylalanine, methionine, threonine, glutamine, tyrosine, asymmetric dimethylarginine (ADMA) and SDMA than controls (all p < 0.05). CONCLUSIONS: Children with T2D and T1D have distinct fasting plasma amino acid signatures that suggest varying pathogenic mechanisms and could serve as biomarkers for these conditions.

Supplementing Glycine and N-Acetylcysteine (GlyNAC) in Older Adults Improves Glutathione Deficiency, Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Physical Function, and Aging Hallmarks: A Randomized Clinical Trial.

BACKGROUND: Elevated oxidative stress (OxS), mitochondrial dysfunction, and hallmarks of aging are identified as key contributors to aging, but improving/reversing these defects in older adults (OA) is challenging. In prior studies, we identified that deficiency of the intracellular antioxidant glutathione (GSH) could play a role and reported that supplementing GlyNAC (combination of glycine and N-acetylcysteine [NAC]) in aged mice improved GSH deficiency, OxS, mitochondrial fatty-acid oxidation (MFO), and insulin resistance (IR). To test whether GlyNAC supplementation in OA could improve GSH deficiency, OxS, mitochondrial dysfunction, IR, physical function, and aging hallmarks, we conducted a placebo-controlled randomized clinical trial. METHODS: Twenty-four OA and 12 young adults (YA) were studied. OA was randomized to receive either GlyNAC (N = 12) or isonitrogenous alanine placebo (N = 12) for 16-weeks; YA (N = 12) received GlyNAC for 2-weeks. Participants were studied before, after 2-weeks, and after 16-weeks of supplementation to assess GSH concentrations, OxS, MFO, molecular regulators of energy metabolism, inflammation, endothelial function, IR, aging hallmarks, gait speed, muscle strength, 6-minute walk test, body composition, and blood pressure. RESULTS: Compared to YA, OA had GSH deficiency, OxS, mitochondrial dysfunction (with defective molecular regulation), inflammation, endothelial dysfunction, IR, multiple aging hallmarks, impaired physical function, increased waist circumference, and systolic blood pressure. GlyNAC (and not placebo) supplementation in OA improved/corrected these defects. CONCLUSION: GlyNAC supplementation in OA for 16-weeks was safe and well-tolerated. By combining the benefits of glycine, NAC and GSH, GlyNAC is an effective nutritional supplement that improves and reverses multiple age-associated abnormalities to promote health in aging humans. Clinical Trials Registration Number: NCT01870193.

De Novo Glycine Synthesis Is Reduced in Adults With Morbid Obesity and Increases Following Bariatric Surgery.

Background: Glycine is a dietary non-essential amino acid that is low in obesity and increases following bariatric surgery. However, the exact mechanism responsible remains unclear and it is unknown whether hypoglycinemia is a cause or consequence of insulin resistance. Objective: Using multiple isotopically labeled tracers, we aimed to determine the underlying kinetic changes responsible for hypoglycinemia in obesity by: 1) Comparing glycine kinetics between participants with morbid obesity (BMI ≥ 32.5 kg/m2) to those with healthy weight (BMI < 25 kg/m2), and 2) Comparing glycine kinetic changes in participants with morbid obesity after bariatric surgery. Methods: [1,2-13C2] glycine, [2,3,3-2H3] serine, and [2H5] phenylalanine were infused to compare the glycine kinetic parameters between 21 participants with morbid obesity and 21 controls with healthy weight. Participants with morbid obesity then underwent bariatric surgery and 17 were re-studied 6 months later. Data were analyzed by non-parametric methods and presented as median (interquartile range). Results: Compared to controls, participants with morbid obesity had significantly lower plasma glycine concentrations at 163 (153-171) vs. 201 (172-227) µmol/L and significantly reduced de novo glycine synthesis rate at 86.2 (64.5-111) vs.124 (103-159) µmol·kg LBM-1·h1, p < 0.001. Following surgery, body weight and insulin resistance decreased and this was accompanied by significant increases in plasma glycine concentration to 210 (191-243) µmol/L as well as the de novo glycine synthesis rate to 127 (98.3-133) µmol·kg LBM-1·h-1, p < 0.001 vs. baseline. Conclusion: Hypoglycinemia in participants with morbid obesity was associated with impaired de novo glycine synthesis. The increase in plasma glycine concentration and de novo glycine synthesis plus the marked improvement in insulin resistance after bariatric surgery suggest that hypoglycinemia may be secondary to impaired glycine synthesis because of obesity-induced insulin resistance. Clinical Trial Registration: [https://tinyurl.com/6wfj7yss], identifier [NCT04660513].

Tryptophan oxidation in young children with environmental enteric dysfunction classified by the lactulose rhamnose ratio.

BACKGROUND: In young children, associations between linear growth faltering, environmental enteric dysfunction (EED), and the plasma kynurenine (Kyn)/tryptophan (Trp) ratio (KTR) have led to the proposal that higher Trp catabolism in response to intestinal/systemic inflammation limits Trp availability for protein synthesis, resulting in impaired growth. OBJECTIVES: We sought to estimate the Trp oxidation rate and the Trp conversion rate to Kyn in young children with and without EED. METHODS: Children aged 18-24 mo, from urban slums, were assigned to EED (n = 19) or no-EED (n = 26) groups on the basis of a urinary lactulose/rhamnose ratio (LRR) cutoff based on mean + 2 SDs of LRR (≥0.068) in normal age- and sex-matched, high-socioeconomic status children. Plasma KTR and fecal biomarkers of EED were measured. Trp oxidation in the fed state was measured using 13C1-Trp in an oral plateau feeding protocol. RESULTS: The median (quartile 1, quartile 3) fasted KTR was 0.089 (0.066, 0.110) in children with EED compared with 0.070 (0.050, 0.093) in children with no EED (P = 0.077). However, there was no difference in fed-state Trp oxidation [median (quartile 1, quartile 3) 3.1 (1.3, 5.8) compared with 3.9 (1.8, 6.0) µmol/kg FFM/h, respectively, P = 0.617] or Trp availability for protein synthesis [42.6 (36.5, 45.7) compared with 42.5 (37.9, 46.9) µmol/kg FFM/h, respectively, P = 0.868] between the groups. In contrast, the median (quartile 1, quartile 3) fractional synthesis rates of Kyn [12.5 (5.4, 20.0) compared with 21.3 (16.1, 24.7) %pool/h, P = 0.005] and the fraction of Ala derived from Trp [0.007 (0.005, 0.015) compared with 0.012 (0.008, 0.018), P = 0.037], respectively, in the plasma compartment were significantly slower in the EED group. Fecal biomarkers of EED did not differ between the groups. CONCLUSIONS: The static plasma KTR value is not a good indicator of the dynamic Trp flux down its oxidative pathway. In a poor sanitary environment, children without EED actually have a faster Kyn synthesis rate, which might be beneficial, because of the cytoprotective and anti-inflammatory functions of downstream metabolites. This study was registered in the Clinical Trials Registry of India as CTRI/2017/02/007921.

Metabolomics Profiling of Patients With A-ß+ Ketosis-Prone Diabetes During Diabetic Ketoacidosis.

When stable and near-normoglycemic, patients with "A-ß+" ketosis-prone diabetes (KPD) manifest accelerated leucine catabolism and blunted ketone oxidation, which may underlie their proclivity to develop diabetic ketoacidosis (DKA). To understand metabolic derangements in A-ß+ KPD patients during DKA, we compared serum metabolomics profiles of adults during acute hyperglycemic crises, without (n = 21) or with (n = 74) DKA, and healthy control subjects (n = 17). Based on 65 kDa GAD islet autoantibody status, C-peptide, and clinical features, 53 DKA patients were categorized as having KPD and 21 type 1 diabetes (T1D); 21 nonketotic patients were categorized as having type 2 diabetes (T2D). Patients with KPD and patients with T1D had higher counterregulatory hormones and lower insulin-to-glucagon ratio than patients with T2D and control subjects. Compared with patients withT2D and control subjects, patients with KPD and patients with T1D had lower free carnitine and higher long-chain acylcarnitines and acetylcarnitine (C2) but lower palmitoylcarnitine (C16)-to-C2 ratio; a positive relationship between C16 and C2 but negative relationship between carnitine and ß-hydroxybutyrate (BOHB); higher branched-chain amino acids (BCAAs) and their ketoacids but lower ketoisocaproate (KIC)-to-Leu, ketomethylvalerate (KMV)-to-Ile, ketoisovalerate (KIV)-to-Val, isovalerylcarnitine-to-KIC+KMV, propionylcarnitine-to-KIV+KMV, KIC+KMV-to-C2, and KIC-to-BOHB ratios; and lower glutamate and 3-methylhistidine. These data suggest that during DKA, patients with KPD resemble patients with T1D in having impaired BCAA catabolism and accelerated fatty acid flux to ketones-a reversal of their distinctive BCAA metabolic defect when stable. The natural history of A-ß+ KPD is marked by chronic but varying dysregulation of BCAA metabolism.

Glycine and N-acetylcysteine (GlyNAC) supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: Results of a pilot clinical trial.

BACKGROUND: Oxidative stress (OxS) and mitochondrial dysfunction are implicated as causative factors for aging. Older adults (OAs) have an increased prevalence of elevated OxS, impaired mitochondrial fuel-oxidation (MFO), elevated inflammation, endothelial dysfunction, insulin resistance, cognitive decline, muscle weakness, and sarcopenia, but contributing mechanisms are unknown, and interventions are limited/lacking. We previously reported that inducing deficiency of the antioxidant tripeptide glutathione (GSH) in young mice results in mitochondrial dysfunction, and that supplementing GlyNAC (combination of glycine and N-acetylcysteine [NAC]) in aged mice improves naturally-occurring GSH deficiency, mitochondrial impairment, OxS, and insulin resistance. This pilot trial in OA was conducted to test the effect of GlyNAC supplementation and withdrawal on intracellular GSH concentrations, OxS, MFO, inflammation, endothelial function, genotoxicity, muscle and glucose metabolism, body composition, strength, and cognition. METHODS: A 36-week open-label clinical trial was conducted in eight OAs and eight young adults (YAs). After all the participants underwent an initial (pre-supplementation) study, the YAs were released from the study. OAs were studied again after GlyNAC supplementation for 24 weeks, and GlyNAC withdrawal for 12 weeks. Measurements included red-blood cell (RBC) GSH, MFO; plasma biomarkers of OxS, inflammation, endothelial function, glucose, and insulin; gait-speed, grip-strength, 6-min walk test; cognitive tests; genomic-damage; glucose-production and muscle-protein breakdown rates; and body-composition. RESULTS: GlyNAC supplementation for 24 weeks in OA corrected RBC-GSH deficiency, OxS, and mitochondrial dysfunction; and improved inflammation, endothelial dysfunction, insulin-resistance, genomic-damage, cognition, strength, gait-speed, and exercise capacity; and lowered body-fat and waist-circumference. However, benefits declined after stopping GlyNAC supplementation for 12 weeks. CONCLUSIONS: GlyNAC supplementation for 24-weeks in OA was well tolerated and lowered OxS, corrected intracellular GSH deficiency and mitochondrial dysfunction, decreased inflammation, insulin-resistance and endothelial dysfunction, and genomic-damage, and improved strength, gait-speed, cognition, and body composition. Supplementing GlyNAC in aging humans could be a simple and viable method to promote health and warrants additional investigation.

Supplementing Glycine and N-acetylcysteine (GlyNAC) in Aging HIV Patients Improves Oxidative Stress, Mitochondrial Dysfunction, Inflammation, Endothelial Dysfunction, Insulin Resistance, Genotoxicity, Strength, and Cognition: Results of an Open-Label Clinical Trial.

Background: Patients with HIV (PWH) develop geriatric comorbidities, including functional and cognitive decline at a younger age. However, contributing mechanisms are unclear and interventions are lacking. We hypothesized that deficiency of the antioxidant protein glutathione (GSH) contributes to multiple defects representing premature aging in PWH, and that these defects could be improved by supplementing the GSH precursors glycine and N-acetylcysteine (GlyNAC). Methods: We conducted an open label clinical trial where eight PWH and eight matched uninfected-controls were studied at baseline. PWH were studied again 12-weeks after receiving GlyNAC, and 8-weeks after stopping GlyNAC. Controls did not receive supplementation. Outcome measures included red-blood cell and muscle GSH concentrations, mitochondrial function, mitophagy and autophagy, oxidative stress, inflammation, endothelial function, genomic damage, insulin resistance, glucose production, muscle-protein breakdown rates, body composition, physical function and cognition. Results: PWH had significant defects in measured outcomes, which improved with GlyNAC supplementation. However, benefits receded after stopping GlyNAC. Conclusions: This open label trial finds that PWH have premature aging based on multiple biological and functional defects, and identifies novel mechanistic explanations for cognitive and physical decline. Nutritional supplementation with GlyNAC improves comorbidities suggestive of premature aging in PWH including functional and cognitive decline, and warrants additional investigation.

Branched-Chain Amino Acid Oxidation Is Elevated in Adults with Morbid Obesity and Decreases Significantly after Sleeve Gastrectomy.

BACKGROUND: Plasma concentrations of branched-chain amino acids (BCAAs) are elevated in obese individuals with insulin resistance (IR) and decrease after bariatric surgery. However, the metabolic mechanisms are unclear. OBJECTIVES: Our objectives are to compare leucine kinetics between morbidly obese and healthy-weight individuals cross-sectionally, and to prospectively evaluate changes in the morbidly obese after sleeve gastrectomy. We hypothesized that leucine oxidation is slower in obese individuals and increases after surgery. METHODS: Ten morbidly obese [BMI (in kg/m2) ≥32.5, age 21-50 y] and 10 healthy-weight participants (BMI <25), matched for age (median ∼30 y) but not gender, were infused with [U-13C6] leucine and [2H5] glycerol to quantify leucine and glycerol kinetics. Morbidly obese participants were studied again 6 mo postsurgery. Primary outcomes were kinetic parameters related to BCAA metabolism. Data were analyzed by nonparametric methods and presented as median (IQR). RESULTS: Participants with obesity had IR with an HOMA-IR (4.89; 4.36-8.76) greater than that of healthy-weight participants (1.32; 0.99-1.49; P < 0.001) and had significantly faster leucine flux [218; 196-259 compared with 145; 138-149 µmol · kg fat-free mass (FFM)-1 · h-1], oxidation (24.0; 17.9-29.8 compared with 16.1; 14.3-18.5 µmol · kg FFM-1 · h-1), and nonoxidative disposal (204; 190-247 compared with 138; 129-140 µmol · kg FFM-1 · h-1) (P < 0.017 for all). After surgery, the morbidly obese had a marked improvement in IR (3.54; 3.06-6.08; P = 0.008) and significant reductions in BCAA concentrations (113; 95-157 µmol/L) and leucine oxidation (9.37; 6.85-15.2 µmol · kg FFM-1 · h-1) (P = 0.017 for both). Further, leucine flux in this group correlated significantly with IR (r = 0.78, P < 0.001). CONCLUSIONS: BCAA oxidation is not impaired but elevated in individuals with morbid obesity. Plasma BCAA concentrations are lowered after surgery owing to slower breakdown of body proteins as insulin's ability to suppress proteolysis is restored. These findings suggest that IR is the underlying cause and not the consequence of elevated BCAAs in obesity.

Preovulatory exposure to a protein-restricted diet disrupts amino acid kinetics and alters mitochondrial structure and function in the rat oocyte and is partially rescued by folic acid.

BACKGROUND: Detrimental exposures during pregnancy have been implicated in programming offspring to develop permanent changes in physiology and metabolism, increasing the risk for developing diseases in adulthood such as hypertension, diabetes, heart disease and obesity. This study investigated the effects of protein restriction on the metabolism of amino acids within the oocyte, liver, and whole organism in a rat model as well as effects on mitochondrial ultrastructure and function in the cumulus oocyte complex. METHODS: Wistar outbred female rats 8-11 weeks of age (n = 24) were assigned to three isocaloric dietary groups, including control (C), low protein (LP) and low protein supplemented with folate (LPF). Animals were superovulated and 48 h later underwent central catheterization. Isotopic tracers of 1-13C-5C2H3-methionine, 2H2-cysteine, U-13C3-cysteine and U-13C3-serine were administered by a 4 h prime-constant rate infusion. After sacrifice, oocytes were denuded of cumulus cells and liver specimens were obtained. RESULTS: Oocytes demonstrated reduced serine flux in LP vs. LPF (p < 0.05), reduced cysteine flux in LP and LPF vs. C (p < 0.05), and a trend toward reduced transsulfuration in LP vs. C and LPF. Folic acid supplementation reversed observed effects on serine flux and transsulfuration. Preovulatory protein restriction increased whole-body methionine transmethylation, methionine transsulfuration and the flux of serine in LP and LPF vs. C (p = 0.003, p = 0.002, p = 0.005). The concentration of glutathione was increased in erythrocytes and liver in LP and LPF vs. C (p = 0.003 and p = 0.0003). Oocyte mitochondrial ultrastructure in LP and LPF had increased proportions of abnormal mitochondria vs. C (p < 0.01 and p < 0.05). Cumulus cell mitochondrial ultrastructure in LP and LPF groups had increased proportions of abnormal mitochondria vs. C (p < 0.001 and p < 0.05). Preovulatory protein restriction altered oocyte expression of Drp1, Opa-1, Mfn1/2, Parl and Ndufb6 (p < 0.05) and Hk2 (p < 0.01), which are genes involved in mitochondrial fission (division) and fusion, mitochondrial apoptotic mechanisms, respiratory electron transport and glucose metabolism. CONCLUSIONS: Preovulatory protein restriction resulted in altered amino acid metabolism, abnormal cumulus oocyte complex mitochondrial ultrastructure and differential oocyte expression of genes related to mitochondrial biogenesis.

Alterations in branched-chain amino acid kinetics in nonobese but insulin-resistant Asian men.

Background: Branched-chain amino acids (BCAAs) are elevated in the insulin-resistant (IR) state. The reasons for this increase remain unclear, but it may be related to abnormalities in BCAA metabolism and free fatty acid (FFA) metabolism. Objective: In this study, we quantified BCAA and FFA kinetics of IR and insulin-sensitive (IS) nonobese Asian men with the use of stable-isotope tracers. We hypothesized that in addition to greater substrate flux, the BCAA oxidative pathway is also impaired to account for the higher plasma BCAA concentration in the IR state. Design: We recruited 12 IR and 14 IS nonobese and healthy Asian men. Oral-glucose-tolerance tests (OGTTs) were performed to quantify insulin sensitivity, and subjects underwent 2 stable-isotope infusion studies. [U-13C6]Leucine was infused to measure leucine flux and oxidation as indexes of BCAA metabolism, whereas [U-13C16]palmitate was infused to measure palmitate flux and oxidation to represent FFA metabolism, The 2H2O dilution method was used to estimate body composition. Results: IR subjects had greater adiposity and significantly higher fasting and post-OGTT glucose and insulin concentrations compared with the IS group. However, none of the subjects were diabetic. Despite similar dietary protein intake, IR subjects had a significantly higher plasma BCAA concentration and greater leucine flux. Leucine oxidation was also greater in the IR group, but the relation between leucine oxidation and flux was significantly weaker in the IR group than in the IS group (r = 0.530 compared with 0.695, P < 0.0388 for differences between slope). FFA oxidation was, however, unaffected despite higher FFA flux in the IR group. Conclusion: The higher plasma BCAA concentration in healthy nonobese individuals with IR is associated with a weaker relation between BCAA oxidation and BCAA flux and this occurs in the presence of accelerated FFA flux and oxidation.

Glutathione metabolism in type 2 diabetes and its relationship with microvascular complications and glycemia.

AIMS/HYPOTHESES: We hypothesized that there is decreased synthesis of glutathione (GSH) in type 2 diabetes (T2DM) especially in the presence of microvascular complications, and this is dependent on the degree of hyperglycemia. METHODS: In this case-control study, we recruited 16 patients with T2DM (7 without and 9 with microvascular complications), and 8 age- and sex-matched non-diabetic controls. We measured GSH synthesis rate using an infusion of [2H2]-glycine as isotopic tracer and collection of blood samples for liquid chromatography mass spectrometric analysis. RESULTS: Compared to the controls, T2DM patients had lower erythrocyte GSH concentrations (0.90 ± 0.42 vs. 0.35 ± 0.30 mmol/L; P = 0.001) and absolute synthesis rates (1.03 ± 0.55 vs. 0.50 ± 0.69 mmol/L/day; P = 0.01), but not fractional synthesis rates (114 ± 45 vs. 143 ± 82%/day; P = 0.07). The magnitudes of changes in patients with complications were greater for both GSH concentrations and absolute synthesis rates (P-values ≤ 0.01) compared to controls. There were no differences in GSH concentrations and synthesis rates between T2DM patients with and without complications (P-values > 0.1). Fasting glucose and HbA1c did not correlate with GSH concentration or synthesis rates (P-values > 0.17). CONCLUSIONS: Compared to non-diabetic controls, patients with T2DM have glutathione deficiency, especially if they have microvascular complications. This is probably due to reduced synthesis and increased irreversible utilization by non-glycemic mechanisms.

Arginine Metabolism Is Altered in Adults with A-ß + Ketosis-Prone Diabetes.

Background: A-ß + ketosis-prone diabetes (KPD) is a subset of type 2 diabetes in which patients have severe but reversible ß cell dysfunction of unknown etiology. Plasma metabolomic analysis indicates that abnormal arginine metabolism may be involved. Objective: The objective of this study was to determine the relation between gut microbiome and arginine metabolism and the relation between arginine availability and ß cell function in KPD patients compared with control participants. Methods: Kinetics of arginine and related metabolites were measured with stable isotope tracers, and insulin secretory responses to arginine and glucose were determined under euglycemic and hyperglycemic conditions in 6 KPD patients and 6 age-, gender-, and body mass index-matched control participants. Glucose potentiation of arginine-induced insulin secretion was performed in a different set of 6 KPD and 3 control participants. Results: Arginine availability was higher in KPD patients during euglycemia [53.5 ± 4.3 (mean ± SEM) compared with 40.3 ± 2.4 µmol · kg lean body mass (LBM)-1 · h-1, P = 0.03] but declined more in response to hyperglycemia (Δ 10.15 ± 2.6 compared with Δ 3.20 ± 1.3 µmol · kg LBM-1 · h-1, P = 0.041). During hyperglycemia, ornithine flux was not different between groups but after an arginine bolus, plasma ornithine AUC trended higher in KPD patients (3360 ± 294 compared with 2584 ± 259 min · µmol · L-1, P = 0.08). In both euglycemia and hyperglycemia, the first-phase insulin responses to glucose stimulation were lower in KPD patients (euglycemic insulin AUC 282 ± 108 compared with 926 ± 257 min · µU · mL-1, P = 0.02; hyperglycemic insulin AUC 358 ± 79 compared with 866 ± 292 min · µU · mL-1, P = 0.05), but exogenous arginine restored first-phase insulin secretion in KPD patients to the level of control participants. Conclusion: Compared with control participants, KPD patients have increased arginine availability in the euglycemic state, indicating a higher requirement. This is compromised during hyperglycemia, with an inadequate supply of arginine to sustain metabolic functions such as insulin secretion. Exogenous arginine administration restores a normal insulin secretory response.

Combined Vitamin B-12 and Balanced Protein-Energy Supplementation Affect Homocysteine Remethylation in the Methionine Cycle in Pregnant South Indian Women of Low Vitamin B-12 Status.

Background: Low-quality dietary protein intake and vitamin B-12 deficiency could interact to decrease methionine transmethylation and remethylation rates during pregnancy and may affect epigenetic modifications of the fetal genome.Objective: The objective of this randomized, partially open-labeled intervention trial was to examine the effect of supplemental high-quality protein and vitamin B-12 on third-trimester methionine kinetics in pregnant Indian women with a low vitamin B-12 status.Methods: Pregnant women with low serum vitamin B-12 concentrations (<200 pmol/L) were randomly assigned to 1 of 3 groups: the first group received balanced protein-energy supplementation of 500 mL milk/d plus a 10-µg vitamin B-12 tablet/d (M+B-12 group; n = 30), the second group received milk (500 mL/d) plus a placebo tablet (M+P group; n = 30), and the third group received a placebo tablet alone (P group; n = 33). Third-trimester fasting plasma amino acid kinetics were measured by infusing 1-13C,methyl-2H3-methionine, ring-2H5-phenylalanine, ring-2H4-tyrosine,1-13C-glycine, and 2,3,3-2H3,15N-serine in a subset of participants. Placental mRNA expression of genes involved in methionine pathways, placental long interspersed nuclear elements 1 (LINE-1) methylation, and promoter methylation levels of vascular endothelial growth factor (VEGF) were analyzed.Results: Remethylation rates in the M+B-12, M+P, and P groups were 5.1 ± 1.7, 4.1 ± 1.0, and, 5.0 ± 1.4 µmol ⋅ kg-1 ⋅ h-1, respectively (P = 0.057), such that the percentage of transmethylation remethylated to methionine tended to be higher in the M+B-12 group (49.5% ± 10.5%) than in the M+P group (42.3% ± 8.4%; P = 0.053) but neither differed from the P group (44.2% ± 8.1%; P > 0.1). Placental mRNA expression, LINE-1, and VEGF promoter methylation did not differ between groups.Conclusions: Combined vitamin B-12 and balanced protein-energy supplementation increased the homocysteine remethylation rate in late pregnancy. Thus, vitamin B-12 along with balanced protein-energy supplementation is critical for optimal functioning of the methionine cycle in the third trimester of pregnancy in Indian women with low serum vitamin B-12 in early pregnancy. This trial was registered at clinicaltrials.gov as CTRI/2016/01/006578.

Impaired nitric oxide production in children with MELAS syndrome and the effect of arginine and citrulline supplementation.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. The pathogenesis of this syndrome is not fully understood and believed to result from several interacting mechanisms including impaired mitochondrial energy production, microvasculature angiopathy, and nitric oxide (NO) deficiency. NO deficiency in MELAS syndrome is likely to be multifactorial in origin with the decreased availability of the NO precursors, arginine and citrulline, playing a major role. In this study we used stable isotope infusion techniques to assess NO production in children with MELAS syndrome and healthy pediatric controls. We also assessed the effect of oral arginine and citrulline supplementations on NO production in children with MELAS syndrome. When compared to control subjects, children with MELAS syndrome were found to have lower NO production, arginine flux, plasma arginine, and citrulline flux. In children with MELAS syndrome, arginine supplementation resulted in increased NO production, arginine flux, and arginine concentration. Citrulline supplementation resulted in a greater increase of these parameters. Additionally, citrulline supplementation was associated with a robust increase in citrulline concentration and flux and de novo arginine synthesis rate. The greater effect of citrulline in increasing NO production is due to its greater ability to increase arginine availability particularly in the intracellular compartment in which NO synthesis takes place. This study, which is the first one to assess NO metabolism in children with mitochondrial diseases, adds more evidence to the notion that NO deficiency occurs in MELAS syndrome, suggests a better effect for citrulline because of its greater role as NO precursor, and indicates that impaired NO production occurs in children as well as adults with MELAS syndrome. Thus, the initiation of treatment with NO precursors may be beneficial earlier in life. Controlled clinical trials to assess the therapeutic effects of arginine and citrulline on clinical complications of MELAS syndrome are needed.

Unlike pregnant adult women, pregnant adolescent girls cannot maintain glycine flux during late pregnancy because of decreased synthesis from serine.

During pregnancy, glycine and serine become more important because they are the primary suppliers of methyl groups for the synthesis of fetal DNA, and more glycine is required for fetal collagen synthesis as pregnancy progresses. In an earlier study, we reported that glycine flux decreased by 39% from the first to the third trimester in pregnant adolescent girls. As serine is a primary precursor for glycine synthesis, the objective of this study was to measure and compare glycine and serine fluxes and inter-conversions in pregnant adolescent girls and adult women in the first and third trimesters. Measurements were made after an overnight fast by continuous intravenous infusions of 2H2-glycine and 15N-serine in eleven adolescent girls (17·4 (se 0·1) years of age) and in ten adult women (25·8 (se 0·5) years of age) for 4 h. Adolescent girls had significantly slower glycine flux and they made less glycine from serine in the third (P<0·05) than in the first trimester. Baby birth length was significantly shorter of adolescent girls (P=0·04) and was significantly associated with third trimester glycine flux. These findings suggest that the pregnant adolescent cannot maintain glycine flux in late pregnancy compared with early pregnancy because of decreased synthesis from serine. It is possible that the inability to maintain glycine synthesis makes her fetus vulnerable to impaired cartilage synthesis, and thus linear growth.

Energy and Protein Supplementation Does Not Affect Protein and Amino Acid Kinetics or Pregnancy Outcomes in Underweight Indian Women.

BACKGROUND: In India, the prevalence of low birth weight is high in women with a low body mass index (BMI), suggesting that underweight women are not capable of providing adequate energy and protein for fetal growth. Furthermore, as pregnancy progresses, there is increased need to provide methyl groups for methylation reactions associated with the synthesis of new proteins and, unlike normal-BMI American women, low-BMI Indian women are unable to increase methionine transmethylation and remethylation rates as pregnancy progresses from trimester 1 to 3. This also negatively influences birth weight. OBJECTIVE: The aim was to determine the effect of dietary supplementation with energy and protein from 12 ± 1 wk of gestation to time of delivery compared with no supplement on pregnancy outcomes, protein kinetics, and the fluxes of the methyl group donors serine and glycine. METHODS: Protein kinetics and serine and glycine fluxes were measured by using standard stable isotope tracer methods in the fasting and postprandial states in 24 pregnant women aged 22.9 ± 0.7 y with low BMIs [BMI (in kg/m(2)) ≤18.5] at 12 ± 1 wk (trimester 1) and 30 ± 1 wk (trimester 3) of gestation. After the first measurement, subjects were randomly assigned to either receive the supplement (300 kcal/d, 15 g protein/d) or no supplement. RESULTS: Supplementation had no significant effect on any variable of pregnancy outcome, and except for fasting state decreases in leucine flux (125 ± 7.14 compared with 113 ± 5.06 µmol ⋅ kg(-1) ⋅ h(-1); P = 0.04) and nonoxidative disposal (110 ± 6.97 compared with 101 ± 3.69 µmol ⋅ kg(-1) ⋅ h(-1); P = 0.02) from trimesters 1 to 3, it had no effect on any other leucine kinetic variable or urea, glycine, and serine fluxes. CONCLUSION: We conclude that in Indian women with a low BMI, supplementation with energy and protein from week 12 of pregnancy to time of delivery does not improve pregnancy outcome, whole-body protein kinetics, or serine and glycine fluxes.

Arginine metabolic endotypes in pulmonary arterial hypertension.

Decreased synthesis of nitric oxide (NO) by NO synthases (NOS) is believed to play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Multiple factors may contribute to decreased NO bioavailability, including increased activity of arginase, the enzyme that converts arginine to ornithine and urea, which may compete with NOS for arginine; inadequate de novo arginine production from citrulline; and increased concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS. We hypothesized that PAH patients with the lowest arginine availability secondary to increased arginase activity and/or inadequate de novo arginine synthesis might have a slower rate of NO synthesis and greater pulmonary vascular resistance. Nine patients with group 1 PAH and 10 healthy controls were given primed, constant intravenous infusions of (15)N2-arginine, (13)C,(2)H4-citrulline, (15)N2-ornithine, and (13)C-urea in the postabsorptive state. The results showed that, compared with healthy controls, PAH patients had a tendency toward increased arginine clearance and ornithine flux but no difference in arginine and citrulline flux, de novo arginine synthesis, or NO synthesis. Arginine-to-ADMA ratio was increased in PAH patients. Two endotypes of patients with low and high arginase activity were identified; compared with the low-arginase group, the patients with high arginase had increased arginine flux, slower NO synthesis, and lower plasma concentrations of ADMA. These results demonstrate that increased breakdown of arginine by arginase occurs in PAH and affects NO synthesis. Furthermore, there is no compensatory increase in de novo arginine synthesis to overcome this increased utilization of arginine by arginase.

Indian women of childbearing age do not metabolically conserve arginine as do American and Jamaican women.

BACKGROUND: In a previous study in pregnant American women, we reported that arginine flux and nitric oxide synthesis increased in trimester 2. More recently, we reported that Indian women do not increase arginine flux during pregnancy as their American or Jamaican counterparts do. OBJECTIVE: The purpose of this study was to determine whether Indian women of childbearing age are producing less arginine and/or catabolizing more arginine and therefore have less available for anabolic pathways than do Jamaican and American women. METHODS: Thirty healthy women aged 28.3 ± 0.8 y from the United States, India, and Jamaica (n = 10/group) were given 6 h primed, constant intravenous infusions of guanidino-¹5N2-arginine, 5,5-²H2-citrulline, ¹5N2-ornithine, and ring-²H5-phenylalanine, in addition to primed, oral doses of U-¹³C6-arginine in both the fasting and postprandial states. An oral dose of deuterium oxide was also given to determine fat-free mass (FFM). RESULTS: Compared with American women, Indian and Jamaican women had greater ornithine fluxes (µmol · kg fat FFM⁻¹ · h⁻¹) in the fasting and postprandial states (27.3 ± 2.5 vs. 39.6 ± 3.7 and 37.2 ± 2.0, respectively, P = 0.01), indicating greater arginine catabolism. However, Jamaican women had a higher endogenous arginine flux than did Indian and American women in the fasting (66.1 ± 3.1 vs. 54.2 ± 3.1 and 56.1 ± 2.1, respectively, P = 0.01) and postprandial (53.8 ± 2.2 vs. 43.7 ± 4.9 and 42.8 ± 3.1, respectively, P = 0.06) states. As a consequence, Indian women had lower arginine bioavailability (µmol · kg FFM⁻¹ · h⁻¹) in the fasting state (42.0 ± 2.6) than did American (49.9 ± 1.3, P = 0.045) and Jamaican (55.5 ± 3.5, P = 0.004) women, as well as in the postprandial state (40.7 ± 3.5 vs. 51.8 ± 1.2 and 57.5 ± 3.2, respectively, P = 0.001). CONCLUSION: Compared with American and Jamaican women, Indian women of childbearing age have a decreased arginine supply because of increased arginine catabolism without an increase in arginine flux.

The Microbiome, Intestinal Function, and Arginine Metabolism of Healthy Indian Women Are Different from Those of American and Jamaican Women.

BACKGROUND: Indian women have slower arginine flux during pregnancy compared with American and Jamaican women. Arginine is a semi-essential amino acid that becomes essential during periods of rapid lean tissue deposition. It is synthesized only from citrulline, a nondietary amino acid produced mainly in the gut. The gut is therefore a key site of arginine and citrulline metabolism, and gut microbiota may affect their metabolism. OBJECTIVE: The objective of this study was to identify differences in the gut microbiota of nonpregnant American, Indian, and Jamaican women and characterize the relations between the gut microbiota, gut function, and citrulline and arginine metabolism. METHODS: Thirty healthy American, Indian, and Jamaican women (n = 10/group), aged 28.3 ± 0.8 y, were infused intravenously with [guanidino-15N2]arginine, [5,5-2H2]citrulline, and [15N2]ornithine and given oral [U-13C6]arginine in the fasting and postprandial states. Fecal bacterial communities were characterized by 16S rRNA gene sequencing. RESULTS: In the fasting state, Indian women had lower citrulline flux than did American and Jamaican women [7.0 ± 0.4 compared with 9.1 ± 0.4 and 8.9 ± 0.2 µmol ⋅ kg fat-free mass (FFM)-1 ⋅ h-1, P = 0.01] and greater enteral arginine conversion to ornithine than did American women (1.4 ± 0.11 compared with 1.0 ± 0.08 µmol ⋅ kg FFM-1 ⋅ h-1, P = 0.04). They also had lower mannitol excretion than American and Jamaican women (154 ± 37.1 compared with 372 ± 51.8 and 410 ± 39.6 mg/6 h, P < 0.01). Three dominant stool community types characterized by increased abundances of the genera Prevotella, Bacteroides, and Bacteroides with Clostridium were identified. Indian women had increased mean relative abundances of Prevotella (42%) compared to American and Jamaican women (7% and < 1%, P = 0.03) which were associated with diet, impaired intestinal absorptive capacity, and arginine flux. CONCLUSIONS: These findings suggest that dysregulated intestinal function and a unique gut microbiome may contribute to altered arginine metabolism in Indian women.