The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

Background  

Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens.     

Enteral feeding induces diet-dependent mucosal dysfunction, bacterial proliferation, and necrotizing enterocolitis in preterm pigs on parenteral nutrition

Preterm neonates have an immature gut and metabolism and may benefit from total parenteral nutrition (TPN) before enteral food is introduced. Conversely, delayed enteral feeding may inhibit gut maturation and sensitize to necrotizing enterocolitis (NEC). Intestinal mass and NEC lesions were first recorded in preterm pigs fed enterally (porcine colostrum, bovine colostrum, or formula for 20-40 h), with or without a preceding 2- to 3-day TPN period (n = 435). Mucosal mass increased during TPN and further after enteral feeding to reach an intestinal mass similar to that in enterally fed pigs without TPN (+60-80% relative to birth). NEC developed only after enteral feeding but more often after a preceding TPN period for both sow's colostrum (26 vs. 5%) and formula (62 vs. 39%, both P < 0.001, n = 43-170). Further studies in 3-day-old TPN pigs fed enterally showed that formula feeding decreased villus height and nutrient digestive capacity and increased luminal lactic acid and NEC lesions, compared with colostrum (bovine or porcine, P < 0.05). Mucosal microbial diversity increased with enteral feeding, and Clostridium perfringens density was related to NEC severity. Formula feeding decreased plasma arginine, citrulline, ornithine, and tissue antioxidants, whereas tissue nitric oxide synthetase and gut permeability increased, relative to colostrum (all P < 0.05). In conclusion, enteral feeding is associated with gut dysfunction, microbial imbalance, and NEC in preterm pigs, especially in pigs fed formula after TPN. Conversely, colostrum milk diets improve gut maturation and NEC resistance in preterm pigs subjected to a few days of TPN after birth.     

Expression and function of arginine-producing and consuming-enzymes in the kidney

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.     

Enteral arginase II provides ornithine for citrulline synthesis

The synthesis of citrulline from arginine in the small intestine depends on the provision of ornithine. To test the hypothesis that arginase II plays a central role in the supply of ornithine for citrulline synthesis, the contribution of dietary arginine, glutamine, and proline was determined by utilizing multitracer stable isotope protocols in arginase II knockout (AII(-/-)) and wild-type (WT) mice. The lack of arginase II resulted in a lower citrulline rate of appearance (121 vs. 137 μmol·kg(-1)·h(-1)) due to a reduced availability of ornithine; ornithine supplementation was able to restore the rate of citrulline production in AII(-/-) to levels comparable with WT mice. There were significant differences in the utilization of dietary citrulline precursors. The contribution of dietary arginine to the synthesis of citrulline was reduced from 45 to 10 μmol·kg(-1)·h(-1) due to the lack of arginase II. No enteral utilization of arginine was observed in AII(-/-) mice (WT = 25 μmol·kg(-1)·h(-1)), and the contribution of dietary arginine through plasma ornithine was reduced in the transgenic mice (20 vs. 13 μmol·kg(-1)·h(-1)). Dietary glutamine and proline utilization were greater in AII(-/-) than in WT mice (20 vs. 13 and 1.4 vs. 3.7 μmol·kg(-1)·h(-1), respectively). Most of the contribution of glutamine and proline was enteral rather than through plasma ornithine. The arginase isoform present in the small intestinal mucosa has the role of providing ornithine for citrulline synthesis. The lack of arginase II results in a greater contribution of plasma ornithine and dietary glutamine and proline to the synthesis of citrulline.     

Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet

We have shown that first-pass intestinal metabolism is necessary for approximately 50% of whole body arginine synthesis from its major precursor proline in neonatal piglets. Furthermore, the intestine is not the site of increased arginine synthesis observed during dietary arginine deficiency. Primed constant intravenous (iv) and intraportal (ip) infusions of L-[U-14C]proline, and iv infusion of either L-[guanido-14C]arginine or L-[4,5-3H]arginine were used to measure first-pass hepatic arginine synthesis in piglets enterally fed either deficient (0.20 g.kg(-1).day(-1)) or generous (1.80 g.kg(-1).day(-1)) quantities of arginine for 5 days. Conversion of arginine to other urea cycle intermediates and arginine recycling were also calculated for both dietary treatments. Arginine synthesis (g.kg(-1).day(-1)) from proline was greater in piglets (P < 0.05) fed the deficient arginine diet in both the presence (generous: 0.07; deficient: 0.17; pooled SE = 0.01) and absence (generous: 0.06; deficient: 0.20; pooled SE = 0.01) of first-pass hepatic metabolism. There was no net arginine synthesis from proline during first-pass hepatic metabolism regardless of arginine intake. Arginine conversion to urea, citrulline, and ornithine was significantly greater (P < 0.05) in piglets fed the generous arginine diet. Calculated arginine fluxes were significantly lower (P = 0.01) for [4,5-3H]arginine than for [guanido-14C]arginine, and the discrepancy between the values was greater in piglets fed the deficient arginine diet (35% vs. 20%). Collectively, these findings show that first-pass hepatic metabolism is not a site of net arginine synthesis and that piglets conserve dietary arginine in times of deficiency by decreasing hydrolysis and increasing recycling.     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Antenatal and early postnatal dexamethasone treatment decreases cortisol secretion in preterm infants

Glucocorticoids are used antenatally to accelerate the maturation of fetal respiratory and cardiovascular systems when a threat of preterm delivery exists. Postnatally, they are used to prevent and treat respiratory distress syndrome. This study investigates the effects of antenatal (ACT) and early postnatal corticosteroid treatment (PCT) on serum cortisol and plasma catecholamine and adenosine 3',5'-cyclic monophosphate (cAMP) concentrations in preterm neonates. The infants in the ACT group had a significantly lower cortisol concentration than the infants in the non-ACT group on the first day of life. After birth, the infants were further divided into non-PCT and PCT groups. PCT suppressed cortisol levels significantly after 2 days, and the cortisol levels were still lower 2 days after discontinuation of PCT. No effect of PCT on plasma cAMP or catecholamine concentrations was observed. The results indicate that both ACT and a short PCT can significantly suppress basal cortisol levels in preterm infants.     

Arginine deficiency in preconfluent intestinal Caco-2 cells modulates expression of proteins involved in proliferation, apoptosis, and heat shock response

Arginine is classified as a conditionally essential amino acid required exogenously during catabolic disease states and periods of rapid growth, both characterized by increased arginine utilization. Arginine plays an important role in the intestine, where it is extensively metabolized, and enhances its immune-supportive function and mucosal repair. Cell proliferation is important for the latter process. This study aimed for a better molecular insight in the response to arginine deprivation/supplementation of preconfluent and 5-day-confluent, differentiated Caco-2 intestinal cells. The potential of citrulline to counteract the effects of arginine deprivation was investigated in preconfluent cells. 2-DE combined with MALDI-TOF-MS and the antibody microarray technology were applied. Evidence is provided that arginine deficiency modulates the protein expression profiles of preconfluent Caco-2 cells differently than that of postconfluent differentiated cells. In preconfluent cells, certain proteins changed in direct response to arginine deficiency, whereas other proteins did not, but instead responded during the recovery phase after an arginine/citrulline resupplementation. The protein changes suggest that arginine deprivation decreases cell proliferation and heat shock protein expression, and enhances the cells susceptibility to apoptosis. These processes are critical for proper cell function, and hence a state of arginine deficiency can be detrimental for intestinal cells which proliferate actively in vivo.     

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.     

Interleukin 1beta increases arginine accumulation and activates the citrulline-NO cycle in rat pancreatic beta cells.

Nitric oxide (NO) may contribute to pancreatic beta cell damage during the development of type 1 diabetes. Its formation can be triggered by cytokines which induce the expression of the inducible form of nitric oxide synthase (iNOS) in pancreatic islets. In the iNOS-catalyzed reaction, arginine is converted into citrulline and NO. Cellular NO formation may be regulated by the availability of arginine. Arginine can be provided extracellularly, entering the cell mainly through the cationic amino acid transporter system y+CAT, and intracellularly, by protein degradation or synthesis from citrulline (the citrulline-NO cycle). This study demonstrates for the first time that the citrulline-NO cycle is induced in FACS-purified rat beta cells exposed to interleukin-1beta(IL-1beta) and that extracellular arginine or citrulline is required for NO production by beta cells. Moreover, the accumulation of arginine was higher in IL-1beta-treated beta cells than in control cells.beta cells expressed mRNAs for the two y+CAT transporters CAT-2A and CAT-2B with no change in transporter expression after exposure to IL-1beta. It is concluded that the activation of the citrulline-NO cycle and an increase in arginine accumulation may be adaptive responses in cytokine-exposed beta-cells to assure an adequate arginine supply for continuous NO production in the presence of low extracellular arginine levels which may prevail during insulitis.     

Enhanced utilization and altered metabolism of arginine in inflammatory macrophages caused by raised nitric oxide synthesis

Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells).We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.     

Arginine deficiency causes runting in the suckling period by selectively activating the stress kinase GCN2

Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-kinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to ～25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2α phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2.     

危重症新生儿肾上腺皮质功能状态的研究

目的:研究危重症新生儿肾上腺皮质功能状态、肾上腺皮质功能不全(AI)的发生率及其与患儿预后的关系。方法:选择我科收治的日龄2天的危重症新生儿52例(其中早产儿23例,足月儿29例),根据新生儿危重病例评分(NCIS)分为轻度危重组和重度危重组,分别检测其血清基础皮质醇和小剂量促肾上腺皮质激素(ACTH)刺激实验30分钟后的清皮质醇峰值。血清基础皮质醇15μg/dl为合并AI。结果:危重症早产儿基础皮质醇浓度及小剂量ACTH刺激实验前后皮质醇差值均较足月儿组低[(15.08±4.98)μg/dl vs(19.65±9.18)μg/dl,P=0.027;(12.06±3.71)μg/dl vs(19.09±4.75)μg/dl,P=0.000]。危重症新生儿合并AI的发生率为38.5%,其中早产儿组为47.8%,足月儿组为34.5%。AI组新生儿死亡2例,未发现AI与患儿的死亡率有关。结论:危重症早产儿肾上腺皮质功能较足月儿差。危重症新生儿合并AI的发生率较高,但与患儿的死亡率无关。     

Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum

Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high‐affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

Arginine Depletion by Arginine Deiminase Does Not Affect Whole Protein Metabolism or Muscle Fractional Protein Synthesis Rate in Mice

Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [¹⁴C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.     

深度水解蛋白配方奶在不同体重早产儿喂养中的临床效果观察

目的:评估深度水解配方奶(eHPF)在不同体重早产儿早期喂养中临床应用效果。方法:选取2017年9月至2018年12月出生的早产儿,分为极低出生体重儿组(体重1000-1500g之间)62例和低出生体重儿(体重1500-2000g之间)100例,每组再随机分为两组,分别予以深度水解蛋白奶(eHPF)和早产儿配方奶(SPF)喂养。极低出生体重儿组于12小时后开始微量喂养,低出生体重儿12小时内适量喂养;极低出生体重儿组深度水解蛋白奶喂养2周后改早产儿奶喂养,低出生体重儿组深度水解蛋白奶1周后改早产儿奶喂养。比较深度水解蛋白奶在不同体重早产儿早期喂养中的临床应用效果,不同体重早产儿恢复出生体重时间、每日体重增长速度、胃管留置时间、完全肠内喂养天数、住院天数、喂养不耐受发生率、宫外发育迟缓发生率及尿素氮、碱性磷酸酶指标。结果:深度水解蛋白喂养组极低出生体重儿/低出生体重儿恢复出生体重天数、完全肠道喂养天数、胃管留置时间、住院天数较早产儿奶喂养组明显缩短(P0.05),每天体重增长优于早产儿组,喂养不耐受、宫外发育迟缓发生率明显低于早产儿组(P0.05),尿素氮、碱性磷酸酶无统计学差异(P0.05)。结论:深度水解蛋白奶用于不同体重早产儿早期喂养效果明显优于早产儿配方奶,其更有助于早产儿的生长发育。     

Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity in preterm infants. We hypothesize that the intestinal injury in this disease is a consequence of synergy among three of the major risk factors for NEC: prematurity, enteral feeding, and bacterial colonization. Together these factors result in an exaggerated inflammatory response, leading to ischemic bowel necrosis. Human milk may decrease the incidence of NEC by decreasing pathogenic bacterial colonization, promoting growth of nonpathogenic flora, promoting maturation of the intestinal barrier, and ameliorating the proinflammatory response.     

A multitracer stable isotope quantification of the effects of arginine intake on whole body arginine metabolism in neonatal piglets

We have previously shown that deficient arginine intake increased the rate of endogenous arginine synthesis from proline. In this paper, we report in vivo quantification of the effects of arginine intake on total endogenous arginine synthesis, on the rates of conversion between arginine, citrulline, ornithine, and proline, and on nitric oxide synthesis. Male piglets, with gastric catheters for diet and isotope infusion and femoral vein catheters for blood sampling, received a complete diet for 2 days and then either a generous (+Arg; 1.80 g x kg(-1) x day(-1); n = 5) or deficient (-Arg; 0.20 g.kg(-1).day(-1); n = 5) arginine diet for 5 days. On day 7, piglets received a primed, constant infusion of [guanido-(15)N(2)]arginine, [ureido-(13)C;5,5-(2)H(2)]citrulline, [U-(13)C(5)]ornithine, and [(15)N;U-(13)C(5)]proline in an integrated study of the metabolism of arginine and its precursors. Arginine synthesis (micromol x kg(-1) x h(-1)) from both proline (+Arg: 42, -Arg: 74, pooled SE: 5) and citrulline (+Arg: 67, -Arg: 120; pooled SE: 15) were higher in piglets receiving the -Arg diet (P < 0.05); and for both diets proline accounted for approximately 60% of total endogenous arginine synthesis. The conversion of proline to citrulline (+Arg: 39, -Arg: 67, pooled SE: 6) was similar to the proline-to-arginine conversion, confirming that citrulline formation limits arginine synthesis from proline in piglets. Nitric oxide synthesis (micromol x kg(-1) x h(-1)), measured by the rate conversion of [guanido-(15)N(2)]arginine to [ureido-(15)N]citrulline, was greater in piglets receiving the +Arg diet (105) than in those receiving the -Arg diet (46, pooled SE: 10; P < 0.05). This multi-isotope method successfully allowed many aspects of arginine metabolism to be quantified simultaneously in vivo.