Mahroukh Rafii

Asymmetric Dimethylarginine Is Increased in Asthma

RATIONALE Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that competes with L-arginine for binding to NOS. It has been suggested that ADMA contributes to inflammation, collagen deposition, nitrosative stress, and lung function in murine models. OBJECTIVES To test the hypothesis that ADMA is increased in asthma and that NOS inhibition by ADMA contributes to airways obstruction. METHODS We assessed alterations of L-arginine, ADMA, and symmetric dimethylarginine (SDMA) levels in a murine model of allergic airways inflammation using LC-tandem mass spectrometry. Based on the levels of ADMA observed in the murine model, we further tested the direct effects of nebulized inhaled ADMA on airways responsiveness in naive control mice. We also assessed alterations of L-arginine, ADMA, and SDMA in humans in adult lung specimens and sputum samples from pediatric patients with asthma. MEASUREMENTS AND MAIN RESULTS ADMA was increased in lungs from the murine model of allergic airways inflammation. Exogenous administration of ADMA to naive mice, at doses consistent with the levels observed in the allergically inflamed lungs, resulted in augmentation of the airways responsiveness to methacholine. ADMA levels were also increased in human asthma lungs and sputum samples. CONCLUSIONS ADMA levels are increased in asthma and contribute to NOS-related pathophysiology.

Dietary Protein Requirement of Female Adults >65 Years Determined by the Indicator Amino Acid Oxidation Technique Is Higher Than Current Recommendations

BACKGROUND Studies on protein requirements in vulnerable groups such as older adults are few, and results are conflicting. OBJECTIVE The main objective of this study was to determine the protein requirements of free-living women >65 y by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO2 in response to graded intakes of protein. METHODS Twelve subjects participated in the study, with protein intakes ranging from 0.2 to 2.0 g · kg(-1) · d(-1) for a total of 82 studies. The diets provided energy at 1.5 times each subjects resting energy expenditure and were isocaloric. Protein was given as an amino acid mixture on the basis of the egg protein pattern, except for phenylalanine and tyrosine, which were maintained constant across the protein intake amounts. All subjects were adapted for 2 d before the study day to a protein intake of 1.0 g · kg(-1) · d(-1). The mean protein requirement was determined by applying a mixed-effects change-point regression analysis to F(13)CO2 (label tracer oxidation in (13)CO2 breath), which identified a breakpoint in the F(13)CO2 in response to graded amounts of protein. RESULTS The mean estimated average requirement (EAR) and upper 95% CI (approximating the RDA) protein requirement of women >65 y were 0.96 and 1.29 g · kg(-1) · d(-1), respectively. CONCLUSION These estimates of protein requirements for older women are higher than the current EAR and RDA based on nitrogen balance data, which are 0.66 and 0.80 g · kg(-1) · d(-1), respectively. This trial was registered at clinicaltrials.gov as NCT01604980.

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimers disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC-MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).

Asymmetric Dimethylarginine Contributes to Airway Nitric Oxide Deficiency in Patients with Cystic Fibrosis

RATIONALE Airway nitric oxide is reduced in cystic fibrosis airways. Asymmetric dimethylarginine is an endogenous nitric oxide synthase inhibitor that may contribute to nitric oxide deficiency in cystic fibrosis. OBJECTIVES To test the hypothesis that asymmetric dimethylarginine is increased in cystic fibrosis and contributes to nitric oxide deficiency and airway obstruction. METHODS The concentrations of asymmetric dimethylarginine, symmetric dimethylarginine, and l-arginine were measured in sputum of clinically stable patients with cystic fibrosis, in patients with cystic fibrosis before and after treatment for a pulmonary exacerbation, and in healthy control subjects, using liquid chromatography-tandem mass spectrometry. MEASUREMENTS AND MAIN RESULTS Asymmetric dimethylarginine was increased in cystic fibrosis compared with control sputum, and the l-arginine/asymmetric dimethylarginine ratio was decreased. Symmetric dimethylarginine exceeded asymmetric dimethylarginine concentrations in control sputum, but this ratio was reversed in cystic fibrosis. Treatment for pulmonary exacerbation resulted in a decrease in sputum asymmetric dimethylarginine and an improved l-arginine/asymmetric dimethylarginine ratio. The treatment-related decrease in asymmetric dimethylarginine correlated significantly with an increase in sputum nitric oxide metabolites and improvement in pulmonary function. The activity of the asymmetric dimethylarginine-metabolizing enzyme, dimethylarginine dimethylaminohydrolase, was higher in cystic fibrosis sputum before rather than after treatment, suggesting that the accumulation of asymmetric dimethylarginine is caused by increased production, not decreased degradation, of asymmetric dimethylarginine. CONCLUSIONS Asymmetric dimethylarginine is increased in cystic fibrosis airways and may contribute to airway obstruction in patients with cystic fibrosis by reducing nitric oxide formation.

Arginine Is Synthesized From Proline, Not Glutamate, in Enterally Fed Human Preterm Neonates

In neonatal mammals, arginine is synthesized in the enterocyte, with either proline or glutamate as the dietary precursor. We have shown several times in piglets that proline is the only precursor to arginine, although in vitro evidence supports glutamate in this role. Because of this uncertainty, we performed a multitracer stable isotope study to determine whether proline, glutamate, or both are dietary precursors for arginine in enterally fed human neonates. Labeled arginine (M + 2), proline (M + 1), and glutamate (M + 3) were given enterally to 15 stable, growing preterm infants (GA at birth 30–35 wk) at 1–3 wk postnatal age. Enrichment in urine of the tracer amino acids and the M + 1 and M + 3 isotopomers of arginine were measured by LC-tandem mass spectrometry to determine the contribution of proline and glutamate to arginine synthesis. Plateau enrichments of arginine and proline tracers were measurable in urine. Urinary glutamate enrichment was not detected. Conversion of proline to arginine was detected. However, the M + 3 isotopomer of arginine, which would have been synthesized from glutamate, was not detected. We conclude that, in contrast to the current consensus in the literature based on in vitro studies, proline is the major contributor to arginine synthesis in human preterm infants.

Arginine synthesis from enteral glutamine in healthy adults in the fed state

Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.

Arginine Can Be Synthesized from Enteral Proline in Healthy Adult Humans

There is considerable controversy recently in identifying dietary precursors for arginine synthesis. We have previously shown in human neonates and piglets that proline is the sole dietary precursor for arginine synthesis. It is unclear in adult humans whether proline is a dietary precursor for arginine. We performed a multi-tracer stable isotope study in adults using (15)N(2)-ureido arginine and (15)N proline to elucidate synthesis of citrulline and arginine and determine whether proline is a precursor for arginine. Primed, intermittent infusions of the labeled amino acids were given enterally to 5 healthy men consuming a standardized milkshake diet. Blood was sampled during plateau enrichment between 1.5 and 3 h. Plasma enrichment occurred for both tracers, giving enteral turnover estimates of 93 μmol · kg(-1) · h(-1) for arginine and 154 μmol · kg(-1) · h(-1) for proline. Appearance of the label from proline in arginine and the intermediaries, ornithine and citrulline, was measured in all participants. The rate of synthesis of arginine from proline was 3.7 μmol · kg(-1) · h(-1), which is estimated to be ~40% of newly synthesized arginine. In this first study in adult humans using an enteral proline tracer, we have demonstrated synthesis of arginine from this dietary amino acid. Therefore, as in newborns, proline must now be considered a dietary precursor for arginine in healthy adults.

The significance of d-isomers in stable isotope studies in humans is dependent on the age of the subject and the amino acid tracer.

d-Amino acids (d-AAs) in stable isotope tracers may result in erroneous estimates of enrichment, particularly if urine is used as a surrogate for plasma enrichment. Previous studies suggest that a d-AA content of less than 0.2% will not result in significant error in studies with adult humans. To describe the effects of d-AA content of less than 0.2%, in 3 different AA tracers, on isotope enrichment in urine and plasma, arginine, proline, and phenylalanine (Phe) tracers were given enterally to human neonates. Enrichment was measured in urine and plasma using chiral chromatography and tandem mass spectrometry. The Phe tracer was also given parenterally to human neonates and enterally to children and adults to further characterize the d-AA effect. All isotopes had a confirmed d-AA content of less than 0.2%. Labeled d-arginine resulted in an overestimate for enrichment of 20% in plasma and 87% in urine. A smaller effect was seen for d-Phe, which resulted in a 5% overestimate for plasma and 40% in urine. d-Proline had no significant effect. Using the same Phe tracer, a developmental effect was found, with a reduction in the overestimate in children compared with infants and no effect on enrichment in adults. Investigators using commercially produced, stable isotope-labeled AAs need to be aware that there is no safe level of d contamination; a d-AA content less than 0.2% may result in significant overestimate for enrichment, even in plasma, for infants and children. This source of error can be avoided by the use of chiral chromatography.

Tetrahydrobiopterin Is Present in High Quantity in Human Milk and Has a Vasorelaxing Effect on Newborn Rat Mesenteric Arteries

Breast milk reduces the incidence of necrotizing enterocolitis (NEC). BH4 is a cofactor for endothelial NOS (eNOS). Reduced BH4 levels, or its oxidation to dihydrobiopterin (BH2), uncouple eNOS resulting in formation of reactive oxygen species (ROS) that have been implicated in the pathogenesis of NEC. We evaluated colostrum and mature breast milk, as well as infant formula, BH4 and BH2 content. In addition, we tested the BH4 effect on the newborn rat mesenteric arterial vascular tone. BH4 and BH2 content increased 3-fold in mature breast milk, when compared with colostrum (p < 0.01), without a change in their ratio. Infant formula had a negligible BH4 content and lower biopterins ratio, when compared with breast milk. eNOS is the predominant synthase isoform in newborn rat mesenteric arteries. In the presence of BH4, mesenteric arteries contracted less to thromboxane A2 analog U46619 (p < 0.01) and this effect was abolished following eNOS inhibition. BH4 (10−6 M) vasorelaxed the newborn rat mesenteric arteries. We conclude that when compared with infant formula, breast milk has a high BH4 content that increases as breastfeeding continues. Given its mesenteric arterial vasorelaxing effect, BH4 may play an important role in the reduced NEC incidence among breast-fed infants.

Methionine-Adequate Cysteine-Free Diet Does Not Limit Erythrocyte Glutathione Synthesis in Young Healthy Adult Men

Most methods of determining amino acid (AA) requirements are based on endpoints that determine adequacy for protein synthesis. However, the sulfur AA (SAA) cysteine is believed to be the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant, glutathione (GSH). Our objectives were to determine whether supplementation of cysteine in a diet containing adequate SAA for protein synthesis, as methionine, increased GSH synthesis by measuring the fractional and absolute synthesis rates, and if concentration of GSH changed in response to feeding 5 graded intakes of cysteine (0, 10, 20, 30, and 40 mg x kg(-1) x d(-1)) in a random order with a fixed methionine intake of 14 mg x kg(-1) x d(-1) and a protein intake of 1 g x kg(-1) x d(-1). Each subject received a multivitamin and choline supplement during the study. Four healthy adult men each underwent 5 isotope infusion studies of 7-h duration after a 2-d adaptation to the level of cysteine intake being studied on the isotope infusion day. The isotope used was [U-(13)C(2)-(15)N]glycine. Analyses included erythrocyte GSH synthesis rates and concentration and urinary sulfate excretion. The GSH synthesis rates and concentration, measured at a methionine intake of 14 mg x kg(-1) x d(-1), did not change with increasing intakes of cysteine. Urinary sulfate excretion showed a significant positive relationship with cysteine intake (r = 0.92; P < 0.01). In conclusion, this study provides preliminary evidence that consumption of SAA adequate to meet the requirement for protein synthesis does not limit GSH synthesis in healthy adult men receiving an otherwise adequate diet.