Substitution of high-dose sucrose with fructose in high-fat diets resulted in higher plasma concentrations of aspartic acid, cystine, glutamic acid, ornithine and phenylalanine, and higher urine concentrations of arginine and citrulline.

High fructose intake has been shown to increase circulating alanine transaminase in humans, which could reflect damage to the liver by fructose but could also be linked to higher level of transamination of amino acids in liver. Therefore, we hypothesized that a diet with high content of fructose would affect the amino acid composition in rat plasma and urine differently from a diet with high sucrose content. Because high intake of sucrose and fructose is often accompanied with high intake of saturated fat in the Western-style diet, we wanted to compare the effects of high fructose/sucrose in diets with normal or high content of coconut oil on individual free amino acids plasma and urine. Male Wistar rats were fed diets with normal (10 wt%) or high (40 wt%) content of sucrose or fructose, with normal or high fat content (7 or 22 wt%) and 20 wt% protein (casein). Rats fed high-fructose high-fat diet had higher plasma concentrations of aspartic acid, cystine, glutamic acid, ornithine, and phenylalanine and higher urine concentrations of arginine and citrulline when compared to rats fed high-sucrose high-fat diet. Substituting normal content of sucrose with fructose in the diets had little impact on amino acids in plasma and urine. Serum concentrations of alanine transaminase, aspartate transaminase, and creatinine, and urine cystatin C and T cell immunoglobulin mucin-1 concentrations were comparable between the groups and within normal ranges. To conclude, substituting high-dose sucrose with high-dose fructose in high-fat diets affected amino acid compositions in plasma and urine.

Urinary Metabolic Profiling via LC-MS/MS Reveals Impact of Bovine Lactoferrin on Bone Formation in Growing SD Rats.

Lactoferrin (LF) exerts a promoting bone health function. The effects of LF on bone formation at the metabolic level have been less explored. Urinary metabolic profiling of growing Sprague-Dawley (SD) rats LF-supplemented (1000 mg/kg bw) for four weeks were explored by Liquid chromatography-tandem mass spectrometry (LC-MS/MS). The serum markers of bone formation and bone resorption, the bone mass, and the osteogenesis markers of femur were measured by an enzyme-linked immunosorbent assay, micro-computerized tomography, and immunohistochemistry, respectively. Compared with the control, LF supplementation improved bone formation (p < 0.05), reduced bone resorption (p < 0.05), enhanced femoral bone mineral density and microarchitecture (p < 0.05), and upregulated osteocalcin, osterix, and Runx-2 expression (p < 0.05) of femur. LF upregulated 69 urinary metabolites. KEGG and pathway enrichment analyses of those urinary metabolites, and the Person's correlation analyses among those urinary metabolites and bone status revealed that LF impacted on bone formation via regulatory comprehensive pathways including taurine and hypotaurine metabolism, arginine and proline metabolism, cyanoamino acid metabolism, nitrogen metabolism, nicotinate and nicotinamide metabolism, and fatty acid biosynthesis. The present study indicated the metabolomics is a useful and practical tool to elucidate the mechanisms by which LF augments bone mass formation in growing animals.

Whole-body synthesis of L-homoarginine in pigs and rats supplemented with L-arginine.

Recent studies suggest an important role for L-homoarginine in cardiovascular, hepatic and neurological functions, as well as the regulation of glucose metabolism. However, little is known about whole-body L-homoarginine synthesis or its response to dietary L-arginine intake in animals. Four series of experiments were conducted to determine L-homoarginine synthesis and catabolism in pigs and rats. In Experiment 1, male and female pigs were fed a corn- and soybean meal-based diet supplemented with 0.0-2.42 % L-arginine-HCl. In Experiment 2, male and female rats were fed a casein-based diet, while receiving drinking water containing supplemental L-arginine-HCl to provide 0.0-3.6 g L-arginine/kg body-weight/day. In both experiments, urine collected from the animals for 24 h was analyzed for L-homoarginine and related metabolites. In Experiment 3, pigs and rats received a single oral dose of 1 or 10 mg L-homoarginine/kg body-weight, respectively, and their urine was collected for 24 h for analyses of L-homoarginine and related substances. In Experiment 4, slices of pig and rat tissues (including liver, brain, kidney, heart, and skeletal-muscle) were incubated for 1 h in Krebs-bicarbonate buffer containing 5 or 50 µM L-homoarginine. Our results indicated that: (a) animal tissues did not degrade L-homoarginine in the presence of physiological concentrations of other amino-acids; (b) 95-96 % of orally administered L-homoarginine was recovered in urine; (c) L-homoarginine was quantitatively a minor product of L-arginineg catabolism in the body; and (d) dietary L-arginine supplementation dose-dependently increased whole-body L-homoarginine synthesis. These novel findings provide a new framework for future studies of L-homoarginine metabolism and physiology in animals and humans.

The L-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids.

The L-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N (G),N (G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC-MS or GC-MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.

Inhibition of natriuretic factors increases blood pressure in rats.

Renal dopamine and nitric oxide contribute to natriuresis during high-salt intake which maintains sodium and blood pressure homeostasis. We wanted to determine whether concurrent inhibition of these natriuretic factors increases blood pressure during high-sodium intake. Male Sprague-Dawley rats were divided into the following groups: 1) vehicle (V)-tap water, 2) NaCl-1% NaCl drinking water, 3) 30 mM l-buthionine sulfoximine (BSO), an oxidant, 4) BSO plus NaCl, and 5) BSO plus NaCl with 1 mM tempol (antioxidant). Compared with V, NaCl intake for 10 days doubled sodium intake and increased urinary dopamine level but reduced urinary nitric oxide content. NaCl intake also reduced basal renal proximal tubular Na-K-ATPase activity with no effect on blood pressure. However, NaCl intake in BSO-treated rats failed to reduce basal Na-K-ATPase activity despite higher urinary dopamine levels. Also, dopamine failed to inhibit proximal tubular Na-K-ATPase activity and these rats exhibited reduced urinary nitric oxide levels and high blood pressure. Tempol supplementation in NaCl plus BSO-treated rats reduced blood pressure. BSO treatment alone did not affect the urinary nitric oxide and dopamine levels or blood pressure. However, dopamine failed to inhibit proximal tubular Na-K-ATPase activity in BSO-treated rats. BSO treatment also increased basal protein kinase C activity, D1 receptor serine phosphorylation, and oxidative markers like malondialdehyde and 8-isoprostane. We suggest that NaCl-mediated reduction in nitric oxide does not increase blood pressure due to activation of D1 receptor signaling. Conversely, oxidative stress-provoked inhibition of D1 receptor signaling fails to elevate blood pressure due to presence of normal nitric oxide. However, simultaneously decreasing nitric oxide levels with NaCl and inhibiting D1 receptor signaling with BSO elevated blood pressure.

Role of nitric oxide deficiency in the development of hypertension in hydronephrotic animals.

Hydronephrotic animals develop renal injury and hypertension, which is associated with an abnormal tubuloglomerular feedback (TGF). The TGF sensitivity is coupled to nitric oxide (NO) in the macula densa. The involvement of reduced NO availability in the development of hypertension in hydronephrosis was investigated. Hydronephrosis was induced by ureteral obstruction in young rats. Blood pressure and renal excretion were measured in adulthood, under different sodium conditions, and before and after chronic administration of either N(G)-nitro-l-arginine methyl ester (l-NAME) or l-arginine. Blood samples for ADMA, SDMA, and l-arginine analysis were taken and the renal tissue was used for histology and determination of NO synthase (NOS) proteins. TGF characteristics were determined by stop-flow pressure technique before and after administration of 7-nitroindazole (7-NI) or l-arginine. Hydronephrotic animals developed salt-sensitive hypertension, which was associated with pressure natriuresis and diuresis. The blood pressure response to l-NAME was attenuated and l-arginine supplementation decreased blood pressure in hydronephrotic animals, but not in the controls. Under control conditions, reactivity and sensitivity of the TGF response were greater in the hydronephrotic group. 7-NI administration increased TGF reactivity and sensitivity in control animals, whereas, in hydronephrotic animals, neuronal NOS (nNOS) inhibition had no effect. l-Arginine attenuated TGF response more in hydronephrotic kidneys than in controls. The hydronephrotic animals displayed various degrees of histopathological changes. ADMA and SDMA levels were higher and the renal expressions of nNOS and endothelial NOS proteins were lower in animals with hydronephrosis. Reduced NO availability in the diseased kidney in hydronephrosis, and subsequent resetting of the TGF mechanism, plays an important role in the development of hypertension.

Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond.

There is abundant evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO), an endogenous messenger molecule formed in healthy vascular endothelium from the amino acid precursor L-arginine. Endothelial dysfunction is caused by various cardiovascular risk factors, metabolic diseases, and systemic or local inflammation. One mechanism that explains the occurrence of endothelial dysfunction is the presence of elevated blood levels of asymmetric dimethylarginine (ADMA)--an L-arginine analogue that inhibits NO formation and thereby can impair vascular function. Supplementation with L-arginine has been shown to restore vascular function and to improve the clinical symptoms of various diseases associated with vascular dysfunction.

Plasma guanidino compounds are altered by oral creatine supplementation in healthy humans.

Although creatine is one of the most widely used nutritional supplements for athletes as well as for patients with neuromuscular disorders, the effects of oral creatine supplementation on endogenous creatine synthesis in humans remains largely unexplored. The aim of the present study was to investigate the metabolic consequences of a frequently used, long-term creatine ingestion protocol on the circulating creatine synthesis precursor molecules, guanidinoacetate and arginine, and their related guanidino compounds. For this purpose, 16 healthy young volunteers were randomly divided to ingest in a double-blind fashion either creatine monohydrate or placebo (maltodextrine) at a dosage of 20 g/day for the first week (loading phase) and 5 g/day for 19 subsequent wk (maintenance phase). Fasting plasma samples were taken at baseline and at 1, 10, and 20 wk of supplementation, and guanidino compounds were determined. Plasma guanidinoacetate levels were reduced by 50% after creatine loading and remained approximately 30% reduced throughout the maintenance phase. Several circulating guanidino compound levels were significantly altered after creatine loading but not during the maintenance phase: homoarginine (+35%), alpha-keto-delta-guanidinovaleric acid (+45%), and argininic acid (+75%) were increased, whereas guanidinosuccinate was reduced (-25%). The decrease in circulating guanidinoacetate levels suggests that exogenous supply of creatine chronically inhibits endogenous synthesis at the transamidinase step in humans, supporting earlier animal studies showing a powerful repressive effect of creatine on l-arginine:glycine amidinotransferase. Furthermore, these data suggest that this leads to enhanced utilization of arginine as a substrate for secondary pathways.

Improving treatment of guanidinoacetate methyltransferase deficiency: reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation.

Guanidinoacetate methyltransferase (GAMT) deficiency (McKusick 601240), an inborn error of creatine biosynthesis, is characterized by creatine depletion and accumulation of guanidinoacetate (GAA) in the brain. Treatment by oral creatine supplementation had no effect on the intractable seizures. Based on the possible role of GAA as an epileptogenic agent, we evaluated a dietary treatment with arginine restriction and ornithine supplementation in order to achieve reduction of GAA. In an 8-year-old Kurdish girl with GAMT deficiency arginine intake was restricted to 15 mg/kg/day (0.4 g natural protein/kg/day) and ornithine was supplemented with 100 mg/kg/day over a period of 14 months. The diet was enriched with 0.4 g/kg/day of arginine-free essential amino acid mixture and creatine treatment remained unchanged (1.1 g/kg/day). Guanidino compounds in blood, urine, and CSF were measured by means of cation-exchange chromatography. The combination of arginine restriction and ornithine supplementation led to a substantial and permanent decrease of arginine without disturbance of nitrogen detoxification. Formation of GAA was effectively reduced after 4 weeks of treatment and sustained thereafter. Biochemical effects were accompanied by a marked clinical improvement. Distinctly reduced epileptogenic activities in electroencephalography accompanied by almost complete disappearance of seizures demonstrates the positive effect of GAA reduction. This indicates for the first time that GAA may exert an important epileptogenic potential in man. Arginine restriction in combination with ornithine supplementation represents a new and rationale therapeutic approach in GAMT deficiency.

Guanidino compound analysis as a complementary diagnostic parameter for hyperargininemia: follow-up of guanidino compound levels during therapy.

The aim of this collaborative study was to investigate whether guanidino compound analyses in the biologic fluids can be used as a complementary diagnostic parameter for hyperargininemia. Guanidino compounds were determined in the biologic fluids of all known living hyperargininemic patients using a cation exchange chromatographic system with a fluorescence detection method. The serum arginine, homoarginine, alpha-keto-delta-guanidino-valeric acid, argininic acid, and N-alpha-acetylarginine levels of all the hyperargininemic patients are higher than the normal range. Similar increases were seen for the urinary excretion of alpha-keto-delta-guanidinovaleric acid and argininic acid. Untreated hyperargininemic patients have the highest guanidino compound levels in cerebrospinal fluid. However, even under therapy, the arginine, homoarginine, alpha-keto-delta-guanidinovaleric acid, and argininic acid levels in cerebrospinal fluid are still increased. Protein restriction alone is not sufficient to normalize the hyperargininemia, but protein restriction together with supplementation of essential amino acids with or without sodium benzoate decreases further the arginine levels. However, whereas the argininemia can be normalized, the catabolites of arginine are still increased. We conclude that the urinary amino acid levels may remain normal in hyperargininemia, whereas consistent increases of the guanidino compounds are observed. Thus, guanidino compound analyses can be used as a complementary biochemical diagnostic parameter for hyperargininemia. Although the argininemia can be normalized by therapy, the levels of the catabolites of arginine are still elevated.

High-performance liquid chromatographic evaluation of PCF 39, a new immunomodulator agent.

An high-performance liquid chromatographic analysis of PCF 39, N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine, with ultraviolet detection, has been devised and validated. The main pharmacokinetic results encountered for rats treated intravenously with PCF 39 at a dose of 100 mg/kg are described.

Arginine-responsive asymptomatic hyperammonemia in the premature infant.

We found that more than 50% of premature infants have elevated plasma ammonium levels during the first 2 months of life. Ammonium levels were twice normal and were unaccompanied by clinical symptoms of vomiting or lethargy. Ten of these infants were given supplements of arginine (1 to 2 mmol/kg/day PO) for 1 to 2 weeks preceded and followed by control periods. In each infant, plasma ammonium levels fell significantly within 2 days of start of arginine supplementation, and increased once arginine was discontinued. We studied 59 additional premature infants, of whom 26 had normal ammonium levels and 33 were hyperammonemic. Plasma arginine and ornithine levels were significantly lower in the hyperammonemic group, but there was no difference in urinary excretion of arginine or ornithine between groups. Half of the hyperammonemic infants received arginine supplementation between 2 and 8 weeks of age. Plasma ammonium levels in the arginine group was 33 + 1 mumol/L., compared to 45 + 2 mumol/L in the untreated group. Follow-up at 18 months of age showed similar IQ scores in all groups, suggesting that significant neurologic deficits do not result from this transient metabolic defect. The mechanism of the hyperammonemia is unclear.

Arginine requirement of the young pig.

Four experiments were conducted with an arginine (Arg)-deficient, semipurified diet to determine the Arg requirement of the weanling pig. The semipurified diet contained .18% bioavailable Arg as determined by a chick bioassay procedure and, when properly supplemented with Arg, allowed rat growth equal to that obtained with a 19.2% protein corn-soybean meal diet. A preliminary experiment with pigs indicated that the semi-purified diet was markedly deficient in Arg. The Arg requirement of the pig was estimated by adding L-Arg to the semipurified diet such that final dietary Arg levels were .18, .23, .28, .38 and .48% (Exp. 2 and 3) or .48, .58 and .68% (Exp. 4). Weight gain and efficiency of feed utilization were maximized at .48% dietary Arg. Plasma urea-N was increased and plasma NH3-N decreased by .48% Arg compared with lower levels. A slight orotic aciduria was observed in pigs fed Arg levels below .48%. From these results, it is concluded that the Arg requirement of the weanling pig is .48% of the diet, a level twice the current NRC estimate.

[Lysinuric protein intolerance: a severe hyperammonemia secondary to l-arginine deficiency (author's transl)]]. / L'intolérance aux protéines avec lysinurie: une hyperammoniémie sévère par carence en L-arginine.

Lysinuric protein intolerance is an autosomal recessive disease, due to a defect in intestinal, renal and hepatic dibasic amino acid transport. Two new cases in the same family are reported. The disease appears progressively during the first months of life with failure to thrive, anorexia, vomiting, diarrhea, hepatosplenomegaly, muscular weakness, osteoporosis, anemia, leukothrombocytopenia, hyperammonemia and orotic aciduria after a high-protein intake. Hyperdibasicamino-aciduria was associated with subnormal plasma concentrations of the same aminoacids. Oral l-arginine, l-ornithine, l-lysine, and lysyl-glycine loads confirmed the diagnosis. The supplementation of the diet with l-citrulline resulted in normal levels of blood ammonia. However, hepatosplenomegaly, muscular weakness, osteoporosis remained unchanged and growth was not improved. These may be due to lysine deficiency.