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Starch is the major source of food glucose and its digestion requires small intestinal alpha-glucosidic activities provided by the 2 soluble amylases and 4 enzymes bound to the mucosal surface of enterocytes. Two of these mucosal activities are associated with sucrase-isomaltase complex, while another 2 are named maltase-glucoamylase (Mgam) in mice. Because the role of Mgam in alpha-glucogenic digestion of starch is not well understood, the Mgam gene was ablated in mice to determine its role in the digestion of diets with a high content of normal corn starch (CS) and resulting glucose homeostasis. Four days of unrestricted ingestion of CS increased intestinal alpha-glucosidic activities in wild-type (WT) mice but did not affect the activities of Mgam-null mice. The blood glucose responses to CS ingestion did not differ between null and WT mice; however, insulinemic responses elicited in WT mice by CS consumption were undetectable in null mice. Studies of the metabolic route followed by glucose derived from intestinal digestion of (13)C-labeled and amylase-predigested algal starch performed by gastric infusion showed that, in null mice, the capacity for starch digestion and its contribution to blood glucose was reduced by 40% compared with WT mice. The reduced alpha-glucogenesis of null mice was most probably compensated for by increased hepatic gluconeogenesis, maintaining prandial glucose concentration and total flux at levels comparable to those of WT mice. In conclusion, mucosal alpha-glucogenic activity of Mgam plays a crucial role in the regulation of prandial glucose homeostasis. [ABSTRACT FROM AUTHOR]

In disadvantaged populations, recurrent infections lead to a loss of body nitrogen and worsen nutritional status. The resulting malnutrition, in its turn, produces a greater susceptibility to infection. This study aimed to examine the ability of a new minimally invasive tracer protocol to measure leucine oxidation, and then to use it to quantify the effect of vaccination on leucine kinetics and oxidation. Undernourished men (n = 5; body mass index 16.3 +/- 0.9 kg/m(2)) and children (n = 9; age 4.1 +/- 0.6 y; weight-for-age Z-score -2.3 +/- 0.7) underwent metabolic studies 6 d before and 1 d after vaccination with diphtheria, pertussis and tetanus (DPT). The tracer protocol was performed in the fed state and involved two 3-h sequential periods of frequent (20 min) oral doses of NaH(13)CO(3) or [1-(13)C] leucine. Frequent breath samples and urine collections were made. Blood samples were obtained from the men and used for the determination of the isotopic enrichment of alpha-ketoisocaproic acid. The prevaccination oxidation of leucine (percentage of dose +/- SD) was 18.1 +/- 2.3 (men) and 16.7 +/- 3.8 (children). One day after vaccination, these values had risen to 19. 9 +/- 1.9 (P < 0.05) in the men and to 19.5 +/- 4.6 (P < 0.01) in the children. In the adults, vaccination was associated with a rise in whole-body protein breakdown [mg protein/(kg.h)] from 200 +/- 40 to 240 +/- 10 (P < 0.05). A minor simulated infection increases leucine catabolism in undernourished humans and this new, minimally invasive protocol is sufficiently sensitive to measure these changes. [ABSTRACT FROM AUTHOR]

The changes in protein metabolism of severe childhood malnutrition are generally perceived as a metabolic adaptation to chronic protein undernutrition. However, severe malnutrition is invariably accompanied by infections which also have profound effects on protein metabolism. This study aimed to distinguish the effect of protein undernutrition from that of an inflammatory stimulus on muscle and plasma protein synthesis rates. Two groups of five piglets consumed diets containing either 23% or 3% protein for 4 wk. They then were infused intravenously with 2H3-leucine before and 48 h after subcutaneous injections of turpentine to measure the fractional synthesis rates (FSR) of muscle protein and both the FSR and the absolute synthesis rates (ASR) of albumin and fibrinogen. Prior to turpentine injection, compared to control piglets, protein-deficient piglets had significantly lower muscle FSR and plasma concentrations of both albumin and fibrinogen, although only albumin had lower FSR and ASR. Turpentine injection decreased muscle FSR but increased the FSR, ASR and plasma concentrations of both albumin and fibrinogen in control piglets. In protein-deficient piglets, the inflammatory stress caused a further decrease in muscle protein FSR and in plasma albumin concentration despite marked increases in albumin FSR and ASR. Fibrinogen FSR, ASR and plasma concentration were increased. We conclude that protein undernutrition and inflammation elicit the same kinetic response in muscle protein but different kinetic responses in plasma proteins. Furthermore, whereas protein deficiency reduces the plasma albumin pool via a reduction in albumin synthesis, inflammation reduces it through a stimulation of catabolism and/or loss from the intravascular space. [ABSTRACT FROM AUTHOR]

Presents a study which estimated the contributions of dietary nucleotides and nucleotides synthesized de novo to ribonucleic acid synthesis in pregnant mice. Means of estimation; Isolation and analysis of ribonucleic acid, protein, nucleosides and amino acids; Interpretation of isotopomer distribution; Utilization of dietary nucleic acids; Nutritional relevance.

The use of plasma protein concentrations to assess protein-nutritional status has been questioned because concentrations and kinetics are affected by factors other than protein intake. To determine the effect of protein deficiency on plasma protein concentration and synthesis, two groups of four piglets consumed diets containing either 20 or 3% protein. After 8 wk, 2H3-leucine was infused intravenously to measure the fractional and absolute synthesis rates (FSR and ASR) of albumin, transferrin, retinol binding protein (RBP), transthyretin (TTR), a new peptide called TTR2, the high density apolipoprotein (HDL-apoA-1), fibrinogen, and haptoglobin. Compared with controls, protein-deficient pigs had significantly lower (P < 0.05) plasma albumin, RBP and TTR2 concentrations, significantly slower (P < 0.05) FSR of fibrinogen, HDL-apoA-1, transferring and TTR2, significantly lower (P < 0.05) ASR of albumin, fibrinogen, transferrin, and TTR2, and a significantly higher (P < 0.05) ASR of TTR. Fibrinogen and transferrin concentrations did not differ between groups, but transthyretin concentration was higher in protein-deficient pigs. These results suggest that protein-nutritional status cannot be predicted from the concentrations of all plasma proteins, that chronic protein deficiency affects the rate of synthesis of only some plasma proteins, and that the kinetic response of plasma proteins to protein restriction cannot be predicted from measurements of plasma concentrations. [ABSTRACT FROM AUTHOR]

To investigate the utilization of dietary amino acids for hepatic protein synthesis, seven female pigs ( 28 d old, 7.5 kg) were implanted with catheters in a carotid artery, the jugular and portal veins, and the stomach. A portal flow probe was also implanted. The pigs were fed a high protein diet once hourly and infused intragastrically with [U-13C]algal protein for 6 h. Amino acid labeling was measured in arterial and portal blood, in the hepatic free and protein-bound pools and in apolipoprotein B-100 (apoB-100), albumin and fibrinogen. The isotopic enrichments of apoB-100-bound [U-13C]threonine, leucine, lysine and phenylalanine were 33, 100, 194 and 230% higher than those of their respective hepatic free amino acid pools (P < 0.01). Using the labeling of apoB-100 to estimate that of the protein synthetic precursor, the fractional rate of hepatic protein synthesis was 42 +/- 2%/d. Between 5 and 8% of the dietary tracer amino acids was used for hepatic protein synthesis. In contrast to the small intestinal mucosa, in which the majority of the metabolized amino acids were apparently catabolized, protein synthesis utilized from 48% (threonine) to 90% (lysine) of the hepatic uptake of tracer amino acids. It appears that hepatic protein synthesis consumes nutritionally significant quantities of dietary essential amino acids in first pass and that extracellular, especially portal, essential amino acids are channeled to hepatic protein synthesis in the fed state. [ABSTRACT FROM AUTHOR]

To investigate the contribution of dietary carbohydrate to glutamate and acetyl CoA synthesis, two groups of adult mice were fed a high- (HCD) or a low-carbohydrate diet (LCD) in which 5% of the carbohydrate was [U-13C]-glucose. Four animals from each dietary group were killed after 1, 2 and 5 d. The tracer:tracee ratios of [13C3] and [13C6]blood glucose and of the [13C2] and [13C3] isotopomers of blood, mucosal, hepatic and muscle alanine and glutamate were used to calculate the fractional contribution of glucose to the 3-carbon, acetyl CoA and oxaloacetate pools of each tissue. In the HCD mice, glucose contributed 66, 33 and 31% of the acetyl CoA pool of muscle, liver and mucosa, respectively. The contribution of glucose to acetyl CoA was lowered by 33% (P < 0.05) and 55% (P < 0.01) in the liver and muscle of the LCD group, respectively, but was unaltered in the mucosa. Glucose made a minor contribution to glutamate synthesis via oxaloacetate in the liver (23%) and muscle (10%) of the HCD group. The fraction of hepatic and muscle glutamate synthesis derived from glucose was not affected by the diet. We conclude that glucose oxidation in liver and muscle parallels the contribution of carbohydrate to dietary energy and that glucose is not a major carbon precursor for muscle glutamate synthesis. Net glutamate synthesis in extraintestinal tissues is preserved when dietary carbohydrate is restricted. [ABSTRACT FROM AUTHOR]

To investigate the extent of first-pass intestinal metabolism of dietary amino acids, seven female pigs (28 d old, 8.0 kg) were implanted with arterial, venous, portal and gastric catheters and with an ultrasonic portal blood flow probe. The pigs were fed a milk-based diet once hourly and infused intragastrically with [U-13C]algal protein. On average, 56% of the essential amino acid (EAA) intake appeared in the portal blood. However, the net portal balance of methionine (48% of intake) and threonine (38% of intake) tended (P = 0.08) to be lower than the mean of all EAA. The net portal balance (expressed as a percentage of intake) of alanine (205%), tyrosine (167%) and arginine (137%) exceeded their intake. Net portal outflow of ammonia accounted for 18% of total amino acid nitrogen intake. As a percentage of the enteral tracer input, there was substantial first-pass metabolism of lysine (35%), leucine (32%), phenylalanine (35%) and threonine (61%). However, only 18, 21, 18 and 12% of the total first-pass metabolism of lysine, leucine, phenylalanine and threonine, respectively, were recovered in mucosal protein. We conclude that roughly one third of dietary intake of EAA is consumed in first-pass metabolism by the intestine and that amino acid catabolism by the mucosal cells is quantitatively greater than amino acid incorporation into mucosal protein. [ABSTRACT FROM AUTHOR]

Neonates deposit protein at a very high rate and efficiently utilize dietary amino acids for protein deposition. This high efficiency is associated with an elevated stimulation of tissue protein synthesis by feeding. Our recent studies have focused on identification of the factors that mediate this response in the neonate. A positive curvilinear relationship between skeletal muscle protein synthesis and plasma insulin concentration was identified in fasted and fed suckling pigs; the relationship changes with development. To test the specific effects of insulin on protein metabolism in the neonate, a procedure to clamp amino acids, under hyperinsulinemic conditions, was developed. By using this technique, we showed that insulin-stimulated whole-body amino acid disposal is elevated in the neonate, and this response may account for the efficient use of dietary amino acids for protein accretion. More recent studies suggest that the enhanced stimulation of skeletal muscle protein synthesis by feeding in the neonate is primarily insulin mediated; however, the stimulation of liver protein synthesis by feeding seems to be largely a function of amino acid concentration. [ABSTRACT FROM AUTHOR]

Increased morbidity and mortality are associated with lower plasma protein concentrations in children with severe protein-energy malnutrition. However, the kinetic changes responsible for repletion of the plasma pools of nutrient transport proteins and the rapidity of their replenishment in these children have not been determined. This study was undertaken to determine whether an increased rate of synthesis is the mechanism responsible for repletion of the plasma retinol-binding protein, transthyretin and high density lipoprotein-apolipoprotein A1 concentrations of children with severe malnutrition during nutritional rehabilitation. The plasma concentrations and synthesis rates of retinol-binding protein, transthyretin and high density lipoprotein-apolipoprotein A1 were measured using a constant intragastric infusion of 2H3-leucine in 22 children with severe protein-energy malnutrition, at approximately 2 d postadmission (study 1), approximately 8 d post-admission when infections were under control (study 2) and approximately 59 d postadmission at recovery (study 3). In study 1 the plasma concentrations and rates of synthesis of all the proteins were lower compared with values at recovery. In study 2, retinol-binding protein and transthyretin concentrations and absolute synthesis rates increased to the recovered values seen in study 3, but the high density lipoprotein-apolipoprotein A1 concentration and synthesis rate remained significantly lower. These results suggest that repletion of the plasma pool of these three nutrient transport proteins occurs at different rates, through an increase in the rate of synthesis. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Background: A-β + ketosis-prone diabetes (KPD) is a subset of type 2 diabetes in which patients have severe but reversible β 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 β 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.

Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient energy to meet the needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications in this disease. NO is synthesized from arginine by NO synthase, which catalyzes the conversion of arginine to NO and citrulline. Citrulline can be recycled into arginine, and therefore, both arginine and citrulline support NO synthesis. The use of 15 N 2 -arginine and 13 C-, 2 H 4 -citrulline stable isotope infusion allows measuring arginine flux; citrulline flux; citrulline-to-arginine flux, which represents the de novo arginine synthesis rate; and arginine-to-citrulline flux, which represents the NO production rate. The objective of this review is to highlight the utility of this method in providing additional evidence for NO deficiency in MELAS syndrome, adding more insight into the potential mechanisms of NO deficiency in this syndrome, and allowing for the assessment of the effects of supplementation with the NO donors, arginine and citrulline, on improving NO production in MELAS syndrome., Competing Interests: Author disclosures: AWE-H and FJ, no conflicts of interest., (© 2017 American Society for Nutrition.)

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- 13 C,methyl- 2 H 3 -methionine, ring- 2 H 5 -phenylalanine, ring- 2 H 4 -tyrosine,1- 13 C-glycine, and 2,3,3- 2 H 3 , 15 N-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., Competing Interests: 3: Author disclosures: S Devi, A Mukhopadhyay, P Dwarkanath, T Thomas, J Crasta, A Thomas, CN Sheela, JW Hsu, GJ Tang, F Jahoor, and AV Kurpad, no conflicts of interest., (© 2017 American Society for Nutrition.)

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., (© 2016 American Society for Nutrition.)

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., (© 2015 American Society for Nutrition.)

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., (© 2016 American Society for Nutrition.)

Although 2 earlier studies reported that aromatic amino acid (AAA) supplementation of children with severe acute malnutrition (SAM) improved whole-body protein anabolism during the early postadmission (maintenance) phase of rehabilitation, it is not known whether this positive effect was maintained during the catch-up growth and recovery phases of treatment. This study aimed to determine whether supplementation with an AAA cocktail (330 mg · kg(-1) · d(-1)) vs. isonitrogenous Ala would improve measures of protein kinetics in 22 children, aged 4-31 mo, during the catch-up growth and recovery phases of treatment for SAM. Protein kinetics were assessed by measuring leucine, phenylalanine, and urea kinetics with the use of standard stable isotope tracer methods in the fed state. Supplementation started at the end of the maintenance period when the children were clinically/metabolically stable and continued up to full nutritional recovery. Three experiments were performed: at the end of maintenance (at ∼13 d postadmission), at mid-catch-up growth (at ∼23 d post- admission when the children had replenished 50% of their weight deficit), and at recovery (at ∼48 d postadmission when they had achieved at least 90% weight for length). Children in the AAA group had significantly faster protein synthesis compared with those in the Ala group at mid-catch-up growth (101 ± 10 vs. 72 ± 7 μmol phenylalanine · kg(-1) · h(-1); P < 0.05) and better protein balance at mid-catch-up growth (49 ± 5 vs. 30 ± 2 μmol phenylalanine · kg(-1) · h(-1); P < 0.05) and at recovery (37 ± 8 vs. 11 ± 3 μmol phenylalanine · kg(-1) · h(-1); P < 0.05). We conclude that dietary supplementation with AAA accelerates net protein synthesis in children during nutritional rehabilitation for SAM.

VLDL apo B-100 is essential for the secretion of liver fat. It is thought that synthesis of this lipoprotein is impaired in childhood severe acute malnutrition (SAM), especially in the edematous syndromes, and that this contributes to the common occurrence of hepatic steatosis in this condition. However, to our knowledge, it has not been confirmed that VLDL apo B-100 synthesis is slower in edematous compared with nonedematous SAM and that it is impaired in the malnourished compared with the well-nourished state. Therefore, VLDL apo B-100 kinetics were measured in 2 groups of children with SAM (15 edematous and 7 nonedematous), aged 4-20 mo, at 3 stages during treatment. Measurements were done at 4 ± 1 d postadmission, mid- catch-up growth in weight, and at recovery (normal weight-for-length). VLDL apo B-100 synthesis was determined using stable isotope methodology to measure the rate of incorporation of (2)H(3)-leucine into its apoprotein moiety. The fractional and absolute synthesis of VLDL apo B-100 did not differ between the groups or from the initial malnourished stage to the recovery stage. Concentrations of VLDL apo B-100 were greater in the edematous than in the nonedematous group (P < 0.04) and did not differ from the initial stage to recovery. The data indicate that VLDL apo B-100 synthesis is not reduced when children develop either edematous or nonedematous SAM.

The high rate of muscle protein accretion in neonates is sustained by the marked increase in muscle protein synthesis in response to feeding. Little is known about the role of proteolysis in the regulation of protein accretion in response to feeding during the neonatal period. To determine the feeding-induced response of protein breakdown at the whole-body level and in the hindlimb of neonates, 10- and 28-d-old piglets that had been food deprived overnight were infused (7 h) with [1-13C]phenylalanine and [ring-2H4]tyrosine during an initial food deprivation period (3 h), followed by a feeding period (4 h). During feeding, endogenous flux of phenylalanine decreased (P < 0.01) in both the whole body and the hindlimb. Feeding reduced (P < 0.01) whole-body proteolysis but increased hindlimb proteolysis (P = 0.04), suggesting that tissues other than the hindlimb are involved in the reduction in whole-body proteolysis during feeding. Overnight food deprivation resulted in a net mobilization of phenylalanine from whole-body proteins (P < 0.01) but not hindlimb proteins. These responses were unaffected by age. The results suggest that the hindlimb requires a continuous supply of free amino acids to sustain the high rate of muscle protein turnover in neonates and that adaptive mechanisms provide free amino acids to sustain skeletal muscle protein accretion in early postnatal life when the amino acid supply is limited.

We investigated whether supplementation with an aromatic amino acid (AAA) cocktail consisting of 0.5 mmol each of phenylalanine, tryptophan, and tyrosine compared with isonitrogenous amounts of alanine (Ala) would improve measures of protein kinetics in 14 (8 with AAA, 6 Ala) children with edematous malnutrition (aged 6-24 mo) during the infected acute malnourished state. Supplementation started immediately after the baseline experiment, 2 d postadmission and continued to the end of the acute phase of treatment. The second (postsupplementation) experiment was done approximately 12 d postadmission. We measured leucine kinetics, phenylalanine and tyrosine fluxes, using an i.g. 8-h prime continuous infusion of (2)H(3)-leucine, and an i.v. 6-h prime continuous infusion of (13)C-leucine, (2)H(2)-tyrosine, and (2)H(5)-phenylalanine in the fed state. Leucine flux tended to be faster (P = 0.06) in the AAA group compared with Ala group after supplementation (mean difference +/- SEM): 22.6 +/- 10.9 micromol/(kg . h). The rate of leucine appearance from protein breakdown [28.1 +/- 9.4 micromol/(kg . h)] and the nonoxidative disposal of leucine [i.e., leucine to protein synthesis; 35.4 +/- 12.9 micromol/(kg . h)] were faster (P < 0.02) in the AAA group than in the Ala group. There was no significant effect of supplementation on leucine splanchnic metabolism, phenylalanine, and tyrosine fluxes. These findings are consistent with the hypothesis that the blunting of the protein catabolic response to infection in children with edematous malnutrition syndrome is due to limited availability of aromatic amino acids.

Urea cycle disorders (UCD) are human conditions caused by the dysregulation of nitrogen transfer from ammonia nitrogen into urea. The biochemistry and the genetics of these disorders were well elucidated. Earlier diagnosis and improved treatments led to an emerging, longer-lived cohort of patients. The natural history of some of these disorders began to point to pathophysiological processes that may be unrelated to the primary cause of acute morbidity and mortality, i.e., hyperammonemia. Carbamyl phosphate synthetase I single nucleotide polymorphisms may be associated with altered vascular resistance that becomes clinically relevant when specific environmental stressors are present. Patients with argininosuccinic aciduria due to a deficiency of argininosuccinic acid lyase are uniquely prone to chronic hepatitis, potentially leading to cirrhosis. Moreover, our recent observations suggest that there may be an increased prevalence of essential hypertension. In contrast, hyperargininemia found in patients with arginase 1 deficiency is associated with pyramidal tract findings and spasticity, without significant hyperammonemia. An intriguing potential pathophysiological link is the dysregulation of intracellular arginine availability and its potential effect on nitric oxide (NO) metabolism. By combining detailed natural history studies with the development of tissue-specific null mouse models for urea cycle enzymes and measurement of nitrogen flux through the cycle to urea and NO in UCD patients, we may begin to dissect the contribution of different sources of arginine to NO production and the consequences on both rare genetic and common multifactorial diseases.

Although it is well known that the intestinal tract has a high metabolic rate, the substrates that are used to generate the necessary energy remain poorly established, especially in fed animals. Under fed conditions, the quantification of substrate used by the gut is complicated by the fact that potential oxidative precursors are supplied from both the diet and the arterial circulation. To circumvent this problem, and to approach the question of the compounds used to generate ATP in the gut, we combined measurements of portal nutrient balance with enteral and intravenous infusions of [U-(13)C]substrates. We studied rapidly growing piglets that were consuming diets based on whole-milk proteins. The results revealed that 95% of the dietary glutamate presented to the mucosa was metabolized in first pass and that of this, 50% was metabolized to CO(2). Dietary glucose was oxidized to a very limited extent, and arterial glutamine supplied no >15% of the CO(2) production by the portal-drained viscera. Glutamate was the single largest contributor to intestinal energy generation. The results also suggested that dietary glutamate appeared to be a specific precursor for the biosynthesis of glutathione, arginine and proline by the small intestinal mucosa. These studies imply that dietary glutamate has an important functional role in the gut. Furthermore, these functions are apparently different from those of arterial glutamine, the substrate that has received the most attention.

Quantification of the metabolism of dietary glucose by the splanchnic tissues is incomplete. Whether habitual carbohydrate intake affects splanchnic glucose metabolism is not known. Female mice were offered isoenergetic and isonitrogenous quantities of diets containing high (HCD) or low (LCD) amounts of carbohydrate, 5% of which was [U-13C]-glucose. Four mice from each dietary group were killed after 24, 48 and 120 h. The 13C-isotopomer distribution in blood glucose, lactate and alanine and in hepatic alanine and glycogen was measured by selected ion monitoring mass spectrometry. [U-13C]-Glucose and its products, [U-13C]-lactate and alanine, were in complete isotopic equilibrium in the blood. The tracer:tracee ratio of hepatic [U-13C]-alanine was significantly higher (P < 0.01) than that of circulating alanine. In both groups, the tracer:tracee ratio of circulating [U-13C]-glucose was significantly (P < 0.001) lower than that of the dietary carbohydrate, and the ratio of [13C3]-glucose:[U-13C]-glucose [0.57 (HCD) and 0.78 (LCD); diet effect P < 0.05], a measure of glucose metabolic cycling, was between two- and fivefold higher than published values obtained with intravenous tracer glucose. The tracer:tracee ratio of [U-13C]-glycogen glucose was significantly (P < 0.05) higher than that of arterial glucose. We conclude the following: 1) dietary glucose is extensively recycled, via pyruvate, within the liver; 2) this metabolic cycle is maintained in mice consuming low carbohydrate diets; and 3) dietary carbohydrate is channelled to hepatic glycogen. We speculate that the metabolic cycling of enteral glucose is related to the hepatic catabolism of dietary protein.

This study was undertaken to determine the following: 1) the kinetic changes responsible for the depletion and repletion of plasma transferrin (Tr) concentration in children with protein-energy malnutrition (PEM); 2) the role of infection in mediating these changes; and 3) whether plasma Tr concentration is related to body protein status. We measured plasma Tr concentration, and fractional (FSR) and absolute (ASR) Tr synthesis rates with the use of a constant intragastric infusion of 2H3-leucine in 14 children with PEM, at 2 d postadmission (study 1), 8 d postadmission when infections were under control (study 2), and at recovery (study 3). In studies 1 and 2, the children synthesized less Tr and had lower Tr concentrations compared with values at recovery. When infections were controlled, plasma Tr concentration rose, but Tr synthesis was unchanged. There were only fair correlations (P < 0. 05) between plasma Tr concentrations and indices of wasting. Concerning malnourished children, we reached the following conclusions: 1) changes in the Tr pool size are achieved mainly through changes in synthesis rate; 2) infections play a minor role in reducing the Tr pool through either changes in the rate of catabolism or loss from the intravascular space; and 3) Tr concentration is not a very good indicator of protein nutritional status.

We have examined the effect of protein malnutrition on brush border (BB) lactase phlorizin hydrolase (LPH) synthesis in young pigs. Two groups of four 3-wk-old pigs were fed diets containing either 19 g soy protein, 63 g carbohydrate and 5 g fat per 100 g diet (a protein-sufficient diet) or 3 g soy protein, 85 g carbohydrate and 5 g fat per 100 g diet (a protein-deficient diet). After 8 wk of consuming the diets, pigs were infused intravenously with 2H3-leucine for 8 h, then killed. The jejunum was collected for measurement of lactase activity, LPH mRNA abundance and the rate of LPH post-translational synthesis. Lactase activities did not differ between groups (mean 8.1 +/- 1.2 micromol x min(-1) x g mucosa(-1)). LPH mRNA abundance relative to elongation factor-1alpha mRNA (the constitutive/reference mRNA) was significantly (P < 0.05) higher in well-nourished pigs (0.36 +/- 0.03%) than in protein-malnourished pigs (0.21 +/- 0.02%). The rate constants of BB LPH post-translational synthesis were also significantly higher in the well-nourished (103 +/- 9% x d(-1)) than in the protein-malnourished pigs (66 +/- 8% x d(-1)). Further, the absolute synthesis rate of BB LPH, a measure of the amount of enzyme synthesized per gram of tissue, was significantly higher in well-nourished than in protein-malnourished pigs (in arbitrary units, 892 +/- 90 vs. 450 +/- 34, respectively). Thus, protein malnutrition affects both LPH mRNA abundance and post-translational processing in young pigs.

To determine the effect of severe chronic protein deficiency on protein synthesis in different tissues and total protein in plasma, and on plasma biochemical constituents involved in amino acid metabolism, we fed diets containing either 20 or 3% protein to two groups of four age-matched piglets. After consuming the diets for 8 wk, the pigs received a primed-constant infusion of 2 H3-leucine for 8 h to measure the fractional synthesis rates (FSR) of tissue protein and total protein in plasma. Plasma urea and amino acid concentrations, particularly indispensable amino acids, were significantly lower in protein-deficient pigs. Fractional protein synthesis rates were lower in skin by 66% (P < 0.01), in jejunal mucosa by 50% (P < 0.05), in masseter muscle by 40% (P < 0.05), and in liver by 25% (P < 0.02); the fractional synthesis rate of the longissimus muscle was not different than controls. Although plasma protein concentration was significantly (P < 0.01) lower in protein-deficient pigs, the fractional synthesis rate of the total intravascular plasma protein pool was not different. We conclude that adaptation to a low protein diet involves a reduction in the rate of protein synthesis in most body tissues, with the most marked changes occurring in skin and intestine, two tissues which frequently exhibit severe functional impairment in protein malnutrition.

We measured how feeding status affects the kinetics of multiple indispensable amino acids in four adult female subjects studied both in fed and fasted state. The subjects ingested one dose of uniformly 13C-labeled algae (Spirulina platensis). The isotopic enrichments (measured with negative chemical ionization gas chromatography-mass spectrometry) of the branched chain amino acids, phenylalanine, lysine and threonine were followed for 24 h in both the plasma and in VLDL-apolipoprotein B-100 (apoB-100). Fasting lowered body protein degradation when measured with the branched chain amino acids, increased it when measured with phenylalanine and had no statistically significant effect when determined from the kinetics of lysine and threonine. These apparent differences challenge the adequacy of current models of whole-body protein turnover. The ratio of the peak labeling of amino acids in plasma and apoB-100 was used as an estimate of the isotopic dilution in the hepatic pool. In contrast to our earlier observations during intravenous tracer amino acid administration, in the present study fasting lowered the ratio of the peak isotopic enrichments of apoB-100 and plasma amino acids. This supports our contention that feeding increases the use of hepatic portal amino acids for hepatic secretory protein synthesis.

The mechanisms responsible for depletion of systemic glutathione levels in nutritional deprivation and/or in infective and inflammatory conditions have not been fully established. We quantified the effects of protein undernutrition and experimental inflammation on the concentration and synthesis of reduced glutathione in the erythrocytes, liver and jejunal mucosa of young pigs. Two groups of five piglets consumed diets containing either 23 or 3% protein and, after 4 wk, were infused intravenously with [13C2]glycine before and 48 h after subcutaneous injections of turpentine. Erythrocyte, hepatic and intestinal mucosal reduced glutathione was quantified as the monobromobimane derivative by HPLC. Reduced glutathione synthesis was determined by measurements of the tracer/tracee ratio of reduced glutathione-bound glycine. In well-nourished piglets, turpentine injection had no effect on erythrocyte reduced glutathione concentrations or rate of synthesis. Protein undernutrition was associated with lower erythrocyte reduced glutathione concentrations (1.05 +/- 0.04 vs. 1.32 +/- 0.06 mmol/L, P < 0.01) and synthesis (42 +/- 5 vs. 60 +/- 5%/d), and turpentine inflammation caused a further fall in erythrocyte reduced glutathione concentration to 0.96 +/- 0.05 mmol/L, despite a significant (P < 0.05) increase in reduced glutathione synthesis. The combination of protein undernutrition and inflammation had a marked effect on mucosal reduced glutathione concentration (37 +/- 3% of control) and synthesis (65 +/- 5% of control). Hepatic reduced glutathione concentration and synthesis did not differ in the two groups. We conclude that the biosynthetic supply of reduced glutathione is sufficient to withstand an inflammatory challenge in well-nourished piglets but not in protein-deficient animals.

The exact relationship between the constant infusion and flooding dose techniques for measuring fractional muscle protein synthesis (FMPS) rates was determined in anesthetized dogs during the postabsorptive period, using different isotopes of leucine as tracers. First, [1,2-13C]- and [U-14C]leucine were given simultaneously by prime-constant infusion for 5 h to establish that both isotopes gave comparable FMPS rate values. The tracers gave almost identical FMPS rates and the values obtained after 3 and 5 h were not different. Next, the constant infusion and flooding dose methods were compared in the same animal by first measuring FMPS rate with a prime-constant infusion of [U-14C]leucine, then with a flooding dose of 376 mumol/kg [1,2-13C]leucine. The flooding dose method gave FMPS rates that were markedly greater than the constant infusion values. Finally, the effect of a 376 mumol/kg leucine flooding dose on FMPS rate was assessed using the prime-constant infusion of [U-14C]lysine. The large dose of leucine decreased FMPS rate by approximately 50% because acid-soluble muscle lysine (precursor) specific activity increased while the rate of incorporation of labeled lysine into protein decreased. These results indicate that the flooding dose method gives FMPS rates that are markedly greater than those obtained with the constant infusion method and that the latter method is not valid when a large dose of leucine is given.