Arginine Deficiency Research Articles

Adaptation of Conductometric Monoenzyme Biosensor for Rapid Quantitative Analysis of L-arginine in Dietary Supplements.

The present study reports on the development, adaptation, and optimization of a novel monoenzyme conductometric biosensor based on a recombinant arginine deiminase (ADI) for the determination of arginine in dietary supplements with a high accuracy of results. Aiming for the highly sensitive determination of arginine in real samples, we studied the effect of parameters of the working buffer solution (its pH, buffer capacity, ionic strength, temperature, and protein concentration) on the sensitivity of the biosensor to arginine. Thus, it was determined that the optimal buffer is a 5 mM phosphate buffer solution with pH 6.2, and the optimal temperature is 39.5 °C. The linear functioning range is 2.5-750 µM of L-arginine with a minimal limit of detection of 2 µM. The concentration of arginine in food additive samples was determined using the developed ADI-based biosensor. Based on the obtained results, the most effective method of biosensor analysis using the method of standard additions was chosen. It was also checked how the reproducibility of the biosensor changes during the analysis of pharmaceutical samples. The results of the determination of arginine in real samples using a conductometric biosensor based on ADI clearly correlated with the data obtained using the method of ion-exchange chromatography and enzymatic spectrophotometric analysis. We concluded that the developed biosensor would be effective for the accurate and selective determination of arginine in dietary supplements intended for the prevention and/or elimination of arginine deficiency.

L-Citrulline Supplementation Improves Leg Endothelial Function and Blood Flow Responses to Plantarflexion Exercise in Hypertensive Postmenopausal Women

Menopause and hypertension are associated with macro- and microvascular endothelial dysfunction at rest. Arginine deficiency and low nitric oxide (NO) synthesis may contribute to reduced muscle blood flow (BF) and oxygen delivery to exercising muscles. Improving BF to calf muscles may enhance physical activity. L-Citrulline supplementation (CIT) has improved resting macrovascular endothelial function via increased arginine availability for NO synthesis. Thus, the purpose of this study was to examine the effects of CIT on resting microvascular endothelial function and BF responses to plantarflexion exercise (PFE) in hypertensive postmenopausal women. We hypothesized that CIT would improve microvascular function and BF responses to PFE compared to placebo (PL). Twenty-one hypertensive postmenopausal women were randomized to 4 weeks of CIT (10g/day, n=10) or PL ( n=11). Superficial femoral macro- (flow-mediated dilation; FMD) and microvascular function (5s average peak blood velocity and flow during reperfusion) were assessed by ultrasound at 0 and 4 weeks. Femoral artery BF and vascular conductance (VC) were measured via ultrasound at rest and during 3-minute stages of rhythmic PFE at 5%, 15%, and 30% of 1-repetition maximum (1RM) at 0 and 4 weeks. BF and VC responses to PFE were analyzed as the change from rest to the last minute of each stage. There were no differences between groups in FMD, 5s peak velocity and flow during reperfusion, and vascular responses to PFE at 0 weeks. CIT significantly increased FMD (CIT:Δ1.28 ± 0.42 vs PL:Δ0.01 ± 0.40%, p = 0.04) and 5s peak velocity (CIT:Δ17.44 ± 4.21 vs PL:Δ0.09 ± 4.01 cm/s, p = 0.01) and flow (CIT:Δ220 ± 54 vs PL:Δ35 ± 51 mL/min, p = 0.02) during reperfusion compared to PL. CIT had no effect on BF and VC responses to 5% PFE ( p > 0.05). CIT significantly augmented BF responses to 15% PFE (CIT:Δ82 ± 16 vs PL:Δ22 ± 15 mL/min, p < 0.01) and 30% PFE (CIT:Δ156 ± 33 vs PL:Δ38 ± 32 mL/min, p < 0.01) and VC responses to 15% PFE (CIT:Δ0.80 ± 0.16 vs PL:Δ0.11 ± 0.15 mL*100mmHg/min, p < 0.01) and 30% PFE (CIT:Δ1.49 ± 0.29 vs PL:Δ0.24 ± 0.27 mL*100mmHg/min, p < 0.01) compared to PL. Improvements in FMD were not correlated with BF or VC responses to PFE ( p > 0.05). Improvements in 5s peak velocity were positively correlated with BF responses to 15% PFE ( r = 0.498, p = 0.02) and 30% PFE ( r = 0.532, p = 0.01), and with VC responses to 15% PFE ( r = 0.542, p = 0.01) and 30% PFE ( r = 0.633, p < 0.01). Similarly, augmented 5s flow was positively correlated with BF ( r = 0.461, p = 0.04) and VC ( r = 0.558, p = 0.01) responses to 30% PFE. These findings indicate that CIT improved superficial femoral artery macro- and microvascular endothelial function at rest. However, only improvements in microvascular endothelial function influenced vascular function during leg exercise in hypertensive postmenopausal women. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hyperammonaemia in cats

Ammonia is an important nitrogen source required for amino acid, protein and nucleic acid synthesis. In addition, it plays an essential role in the kidney's maintenance of acid–base balance. However, high concentrations of ammonia are cytotoxic and clinical signs primarily reflect neurotoxicity. The body detoxifies ammonia through the urea cycle in the liver or by consuming ammonia in the conversion of glutamate to glutamine in the liver, brain and muscle tissue. The most common cause of hyperammonaemia in cats is congenital portosystemic shunting. Additional causes include cobalamin or arginine deficiency, disruption of the urea cycle by congenital enzyme deficiencies or acute liver failure, excessive muscle activity, infections with urease-producing bacteria, kidney disease and multiple acquired portosystemic shunts.

Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation

Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine–histidine–purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

Low Arginine Bioavailability and Clinical Outcomes in Children with Sickle Cell Disease Hospitalized with Vaso-Occlusive Pain Episode

Introduction: Global arginine bioavailability ratio (GABR) (Arginine/[Citrulline+Ornithine]) is associated with clinical outcomes including pain, pulmonary hypertension, and mortality in patients with sickle cell disease (SCD). Arginine replacement therapy has demonstrated opioid-sparing effects, improved blood pressure and cardiovascular function, and shorten length of hospital stay. Arginine is a conditionally essential amino acid synthesized from citrulline in the kidneys. During a vaso-occlusive pain episode (VOE), hemolysis leads to the release of erythrocyte-arginase (arginine-metabolizing enzyme) that metabolizes arginine into ornithine. Arginine-to-ornithine ratio (arg/orn) is a biomarker of arginase activity and GABR incorporates the impact of kidney function on global arginine bioavailability. The association between arginine bioavailability and clinical outcomes has not been sufficiently explored in children with SCD-VOE. Objective: To evaluate associations between arginine bioavailability and clinical outcomes in children with SCD-VOE who received intravenous (IV) arginine replacement therapy. Methods: We conducted a secondary analysis of a prospective, single-center, double-blinded randomized controlled trial of IV arginine therapy (TID, up to 7 days) in children with SCD age 3-21 years hospitalized for VOE requiring IV opioids. Patients with significant liver/renal dysfunction or those previously enrolled were excluded. Subjects were randomized into 1 of 3 arms: 1) 100 mg/kg/dose arginine (standard dose), 2) loading dose: 200 mg/kg followed by standard dose or 3) placebo. Demographics, total parenteral opioid (TPO) use (morphine equivalents, mg/kg) time to crisis resolution (time of study drug delivery to last IV opioid in hours), pain scores, and targeted amino acids were obtained before treatment and at discharge. Mean±SD, paired t-tests, and Pearson correlation analyses between groups were performed. This trial was registered at www.clinicaltrials.goc as #NCT02536170 Results: 1,548 patients were screened, 266 were eligible, 114 consented, and 108 were randomized (54.4% were female, mean age 12.44 ± 3.90). All participants identified as African American and 75% had genotype Hb-SS. 69.1% were on hydroxyurea. Safety results of this RCT have been previously reported ( Reyes et al, Am J Hematol 2022). Plasma arginine levels were low at VOE presentation (mean 50±28 μM), with low arginine levels (<60 μM) found in 74% of patients. Arginine replacement therapy increased arginine concentration by 182% (p<0.0001) compared to 26% (p=0.24) in placebo. Arg/orn correlated to arginase concentration ( r= - 0.38, p<0.0001). Time-to-crisis-resolution strongly correlated with TPO ( r=0.72, p<0.0001). Arginine bioavailability at presentation was inversely correlated to time-to-crisis-resolution for both arg/orn and GABR ( r= -0.37 p=0.02) in the placebo arm only but not in those participants who received arginine therapy (GABR: r= - 0.05, p=0.6; arg/orn: r= - 0.07, p=0.55) (Fig1A,1B). Similar trends were observed for TPO. Pain scores at presentation positively correlated with TPO ( r= 0.33, p=0.04) in the placebo arm only, but not in the arm that received arginine therapy ( r= 0.03, p=0.76). Time-to-crisis-resolution positively correlated with age ( r= 0.2, p=0.03). Conclusion: In the absence of arginine replacement therapy, low arginine bioavailability predicted longer time-to-crisis resolution and a higher use of IV opioids in patients with SCD-VOE. Arginine bioavailability may represent a novel biomarker of SCD-pain severity. This data further confirms arg/orn as a surrogate for arginase activity reflective of hemolytic rate as previously described. Arginine replacement therapy ameliorates the influence of arginine deficiency on clinical outcomes related to pain in SCD. An NHLBI/ Pediatric Emergency Care Applied Research Network (PECARN) supported Phase-3 multi-center trial of arginine replacement therapy enrolling 360 children with SCD (SCD Treatment with Arginine Therapy - STArT) is ongoing.

Enteral L-citrulline supplementation in preterm infants is safe and effective in increasing plasma arginine and citrulline levels-a pilot randomized trial.

Deficiencies of citrulline and arginine have been associated with adverse outcomes in preterm-infants and data regarding enteral supplementation in preterm infants is limited. This randomized -trial [NCT03649932] included 42 preterm infants (gestational age ≤33 weeks) randomized to receive enteral L-citrulline in low (100 mg/kg/day), medium (200 mg/kg/day) and high-dose (300 mg/kg/day) groups for 7 days. Plasma citrulline and arginine levels were obtained pre-and-post supplementation and efficacy was determined by a significant increase in levels after supplementation. A p < 0.05 was considered significant. Safety monitoring included blood-pressure-monitoring as well as complications and death during hospitalization. A total of 40/42 (95%) of the recruits completed the 7-day supplementation with no adverse events. Plasma-citrulline levels increased significantly in all three groups while plasma-arginine levels increased significantly in the high-dose group. Enteral L-citrulline supplementation in preterm infants is safe and effective in increasing plasma citrulline and arginine levels. NCT03649932 https://clinicaltrials.gov/ct2/show/NCT03649932 .

Plasma arginine as a candidate predictive biomarker for response to immune checkpoint inhibition (ICI) in metastatic colorectal cancer (mCRC): Analysis of the CCTG CO.26 trial.

3545 Background: Nutritional stress is one of the mechanisms used by tumour cells to evade the immune system. Arginine (ARG), an amino acid involved in several cellular functions including immunomodulation, is important in regulating T-lymphocyte cell activity and the anti-tumour response. ARG deficiency in the tumour microenvironment has been shown to impair T-cell response while ARG supplementation may promote anti-tumour immune activity. In this exploratory post-hoc analysis of the Phase II CO.26 trial (NCT02870920), we investigated the role of plasma ARG in predicting response to ICI in patients (pts) with refractory mCRC. Methods: CO.26 was a phase II trial which randomized pts with refractory mCRC to durvalumab plus tremelimumab (D+T) versus best supportive care (BSC). Plasma ARG concentrations were determined from blood samples pre-treatment using HPLC-tandem mass spectrometry. The median plasma ARG value was used as a cut-off stratifying pts into ARG-high (≥10650 ng/ml) versus ARG-low ( &lt; 10650 ng/ml) groups. Progression-free (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method and compared between groups using the log-rank test. Cox proportional hazard models were used to analyze prognostic and predictive impacts of ARG on PFS and OS. Results: Of 180 pts enrolled in CO.26, 162 pts (N = 115 treated with D+T and 47 BSC) had pre-treatment blood samples for baseline ARG analysis. There were no significant differences in baseline characteristics between pts included in this analysis and the total study pts, or between ARG-high and ARG-low pts. In pts treated with D+T, ARG-high was associated with more favourable prognosis (ARG-high median OS 7.62 months vs. ARG-low 5.49 months, multivariable hazard ratio [HR] 0.60, 95% confidence interval [CI] 0.40-0.91, p = 0.016). In ARG-high pts, D+T significantly improved OS (median OS 7.62 months with D+T vs 3.61 months BSC; HR 0.61, 95% CI 0.37-0.99, p = 0.04). In ARG-low pts there was no OS benefit with D+T (median OS 5.49 months D+T vs 4.27 months BSC; HR 0.84, 95% CI 0.50-1.41, p = 0.51. Interaction p = 0.037). Baseline ARG values had no association with PFS or disease control rate. Conclusions: Baseline plasma ARG was prognostic in pts with mCRC treated with D+T, and high ARG was predictive of improved OS with ICI. Prospective studies should be done to validate ARG as a biomarker identifying mCRC pts likely to derive benefit from ICI. Therapeutic approaches targeting the ARG pathway should be investigated in future studies.

THE IMPACT OF CHOLECALCIFEROL ON THE SEX HORMONES LEVEL AND SERUM BIOCHEMICAL INDICES IN RAT MALES WITH EXPERIMENTAL GONADOPATHY

THE IMPACT OF CHOLECALCIFEROL ON THE SEX HORMONES LEVEL AND SERUM BIOCHEMICAL INDICES IN RAT MALES WITH EXPERIMENTAL GONADOPATHY

Dietary Arginine Deficiency and Gut Ammonium Infusion Alter Flux of Urea Cycle Intermediates across the Portal-Drained Viscera of Pigs

The objective of these experiments in pigs were to test the hypotheses that 1) gut synthetic processes could adapt to additional dietary glutamate or ornithine to meet tissue needs for arginine with feeding arginine-deficient diets and 2) acute elevation of ammonium in the hepatic-portal blood leads to increased glutamine production. Arterial [117 ± 5.3 (arginine-deficient) vs. 78 ± 5 (arginine-adequate) µmol/L] and portal ammonium concentrations were elevated in pigs fed arginine-deficient diets. Dietary ornithine, which elevated portal-drained visceral flux of ornithine, corrected the urinary orotic aciduria, but not the hyperammonemia seen with feeding arginine-deficient diets. Concentrations or portal drained viscera fluxes of arginine, ornithine, glutamate and glutamine were not altered even though portal and arterial ammonium concentrations were increased 8- and 3.5-fold with mesenteric infusion of ammonium. It was concluded that 1) substitution of glutamate for glycine or alanine does not alter gut production of ornithine, citrulline or arginine; 2) gut citrulline production is not altered by levels of dietary arginine, ornithine or glutamate; 3) increased ammonium challenge does not lead to increased glutamine production even though peripheral ammonium levels increased over threefold; and 4) provision of arginine for tissue needs will have to be met from dietary sources, as adaptations in gut synthetic processes seem to be refractory to dietary arginine status.

Effects of arginine replacement with L-citrulline on the arginine/nitric oxide metabolism in chickens: An animal model without urea cycle

BackgroundThis study examined the efficacy of L-citrulline supplementation on the arginine/nitric oxide metabolism, and intestinal functions of broilers during arginine deficiency. A total of 288 day-old Arbor Acre broilers were randomly assigned to either an arginine deficient basal diet (NC diet), NC diet + 0.50% L-arginine (PC diet), or NC diet + 0.50% L-citrulline (NCL diet). Production performance was recorded, and at 21 days old, chickens were euthanized for tissue collection.ResultsThe dietary treatments did not affect the growth performance of broilers (P > 0.05), although NC diet increased the plasma alanine aminotransferase, urate, and several amino acids, except arginine (P < 0.05). In contrast, NCL diet elevated the arginine and ornithine concentration higher than NC diet, and it increased the plasma citrulline greater than the PC diet (P < 0.05). The nitric oxide concentration in the kidney and liver tissues, along with the plasma and liver eNOS activities were promoted by NCL diet higher than PC diet (P < 0.05). In the liver, the activities of arginase 1, ASS, and ASL, as well as, the gene expression of iNOS and OTC were induced by PC diet greater than NC diet (P < 0.05). In the kidney, the arginase 1, ASS and ASL enzymes were also increased by PC diet significantly higher than the NC and NCL diets. Comparatively, the kidney had higher abundance of nNOS, ASS, ARG2, and OTC genes than the liver tissue (P < 0.05). In addition, NCL diet upregulated (P < 0.05) the mRNA expression of intestinal nutrient transporters (EAAT3 and PEPT1), tight junction proteins (Claudin 1 and Occludin), and intestinal mucosal defense (MUC2 and pIgR). The intestinal morphology revealed that both PC and NCL diets improved (P < 0.05) the ileal VH/CD ratio and the jejunal VH and VH/CD ratio compared to the NC fed broilers.ConclusionThis study revealed that NCL diet supported arginine metabolism, nitric oxide synthesis, and promoted the intestinal function of broilers. Thus, L-citrulline may serve as a partial arginine replacement in broiler's diet without detrimental impacts on the performance, arginine metabolism and gut health of chickens.

Streptococcal arginine deiminase regulates endothelial inflammation, mTOR pathway and autophagy

Endothelial cells (EC) are active participants in the inflammation process. During the infection, the change in endothelium properties provides the leukocyte infiltrate formation and restrains pathogen dissemination due to coagulation control. Pathogenic microbes are able to change the endothelium properties and functions in order to invade the bloodstream and disseminate in the host organism. Arginine deiminase (ADI), a bacterial arginine-hydrolyzing enzyme, which causes the amino acid deficiency, important for endothelium biology. Previous research implicates altered metabolism of arginine in the development of endothelial dysfunction and inflammation. It was shown that arginine deficiency, as well as overabundance affects the balance of mechanical target of rapamycin (mTOR)/S6 kinase (S6K) pathway, arginase and endothelial nitric oxide synthase (eNOS) resulted in reactive oxygen species (ROS) production and EC activation. ADI creating a deficiency of arginine can interfere cellular arginine-dependent processes. Thus, this study was aimed at investigation of the influence of streptococcal ADI on the metabolism and inflammations of human umbilical vein endothelial cells (HUVEC). The action of ADI was studied by comparing the effect Streptococcus pyogenes M49-16 paternal strain expressing ADI and its isogenic mutant M49-16delArcA with the inactivated gene ArcA. Based on comparison of the parental and mutant strain effects, it can be concluded, that ADI suppressed mTOR signaling pathway and enhanced autophagy. The processes failed to return to the basic level with arginine supplement. Our study also demonstrates that ADI suppressed endothelial proliferation, disrupted actin cytoskeleton structure, increased phospho-NF-κB p65, CD62P, CD106, CD54, CD142 inflammatory molecules expression, IL-6 production and lymphocytes-endothelial adhesion. In spite of the ADI-mediated decrease in arginine concentration in the cell-conditioned medium, the enzyme enhanced the production of nitric oxide in endothelial cells. Arginine supplementation rescued proliferation, actin cytoskeleton structure, brought NO production to baseline and prevented EC activation. Additional evidence for the important role of arginine bioavailability in the EC biology was obtained. The results allow us to consider bacterial ADI as a pathogenicity factor that can potentially affect the functions of endothelium.

Citrulline enteral administration markedly reduces immunosuppressive extrafollicular plasma cell differentiation in a preclinical model of sepsis.

The sustained immunosuppression associated with severe sepsis favors an increased susceptibility to secondary infections and remains incompletely understood. Plasmablast and plasma cell subsets, whose primary function is to secrete antibodies, have emerged as important suppressive populations that expand during sepsis. In particular, sepsis supports CD39hi plasmablast metabolic reprogramming associated with adenosine-mediated suppressive activity. Arginine deficiency has been linked to an increased risk of secondary infections in sepsis. Overcoming arginine shortage by citrulline administration efficiently improves sepsis-induced immunosuppression and secondary infections in the cecal ligation and puncture murine model. Here, we aimed to determine the impact of citrulline administration on B cell suppressive responses in sepsis. We demonstrate that restoring arginine bioavailability through citrulline administration markedly reduces the dominant extrafollicular B cell response, decreasing the immunosuppressive LAG3+ and CD39+ plasma cell populations, and restoring splenic follicles. At the molecular level, the IRF4/MYC-mediated B cell reprogramming required for extrafollicular plasma cell differentiation is shunted in the splenic B cells of mice fed with citrulline. Our study reveals a prominent impact of nutrition on B cell responses and plasma cell differentiation and further supports the development of citrulline-based clinical studies to prevent sepsis-associated immune dysfunction.

Computational investigation on the effect of the lysine 2-hydroxyisobutyrylation on argininosuccinate synthetase 1 conformational dynamics in Botrytis cinerea.

Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered post-translational modification in recent years, which has been identified in several species and is associated with diverse cellular functions. Botrytis cinerea, as a broad host pathogen, is very destructive and causes serious losses to agricultural economy. Argininosuccinate synthetase (ASS, citrulline-aspartate ligase) is the rate-limiting enzyme in the catalytic arginine synthesis pathway. Arginine deficiency can affect the growth of Botrytis cinerea. The Khib site Lys120 was found in functional domain of argininosuccinate synthetase 1 from Botrytis cinerea (Bcass1), which is located in conserved loop. It is worth exploring how K120hib affects the conformation of Bcass1. In this study, molecular dynamics (MD) simulations, binding free energy calculation, principal component analysis (PCA), and dynamic cross-correlation analysis were used to explore the influence of K120hib on the conformation of Bcass1. The increase of root-mean-square fluctuation (RMSF) value of related residues and PCA results suggests that K120hib increases the flexibility of some regions of Bcass1. Moreover, K120hib weakens the binding free energy between Bcass1 and the two substrates. These results will help to understand the effects of K120hib on Bcass1 and provide new ideas for regulating the pathogenicity of Botrytis cinerea.

Impact of Arginine Therapy on Clinical Outcomes, Mitochondrial Function and Oxidative Stress in Children with Sickle Cell Disease Hospitalized with Vasoocclusive Pain Episodes: A Randomized Controlled Trial

Introduction: Vaso-occlusive pain episodes (VOE) are the leading cause of emergency department (ED) visits & hospitalization in patients with sickle cell disease (SCD). During SCD-VOE, patients develop an acute arginine (Arg) deficiency. Supplementation with Arg has shown opioid-sparing effects, improves blood pressure & cardiopulmonary function & decreases length of hospital stay. Though we have previously demonstrated that intravenous (IV) Arg improves mitochondrial (mito) function, the precise mechanisms by which Arg mediates these positive effects remain unclear. Objectives: To determine the role of IV Arg as adjuvant in the management of SCD-VOE Methods: Prospective single-center double-blind randomized controlled trial (RCT) of IV Arg (TID, up to 7 days) in children with SCD age 3-21 years hospitalized for VOE requiring IV opioids. Patients with significant liver/renal dysfunction or those previously enrolled were excluded. Subjects were randomized into 1 of 3 arms: 1) 100 mg/kg/dose Arg, standard dose (SD), 2) loading dose: 200 mg/kg followed by SD or 3) placebo. Demographics, total parenteral opioid (TPO) use (morphine equivalents, mg/kg) time to crisis resolution (time of study drug delivery to last IV opioid in hours), pain scores, patient reported outcomes (PROMIS) & mito function were obtained before treatment & at discharge. The primary outcome measure was TPO use between study arms. Hypothesis tests & unadjusted & covariate-adjusted fixed & mixed effects generalized linear regression models were used, as appropriate, to compare cross-sectional & longitudinal outcomes within & between randomization arms. This protocol utilized IND#66943 (Sponsor-Morris), registered with ClinicalTrials.gov (NCT02536170). Results: 1,548 patients were screened, 266 were eligible, 114 consented, & 108 were randomized. Safety results of this RCT have been previously reported (Reyes et al, Am J Hematol 2022). Demographics & clinical outcomes of subjects randomized by treatment arm are provided in Table 1. While statistically insignificant, there was a clinically relevant decrease in TPO & time to crisis resolution in both Arg arms compared to placebo. The placebo group required 45% higher TPO & experienced >15 hours longer mean time to crisis resolution compared to combined Arg-treated groups when adjusted for HU use, continuous age and sex. Among children <17 years, the placebo group (n=33) required 80% more TPO compared to combined Arg groups (n=57; p=0.075). No differences in ED vs discharge pain scores or patient/parent PROMIS reports across arms were found. Mito Complex V activity was higher (p=0.02) & protein carbonyl levels were lower (p=0.003) at ED visit in patients on Hydroxyurea (HU). Notably mito Complex IV & V activity increased significantly in both Arg arms while there was no change in the placebo group (Fig 1, p<0.001); protein carbonyl levels in platelet rich plasma decreased in both Arg groups (Fig 1, p<0.001), suggesting a decrease in oxidative stress that increased in the placebo arm (p=0.02). Greatest mito improvement occurred with Arg loading dose. Conclusion: Arg supplementation increases mito activity & decreases oxidative stress in children with SCD-VOE. This is the first report to suggest a HU-related impact on mito activity in SCD. Prior reports show that Complex V inhibition leads to increased mito oxidant production in platelets from SCD patients. Acute improvements in Complex IV & V function & decreased oxidative markers with Arg therapy are consistent with Arg-induced reductions in mito oxidant generation. Arg therapy improves symptoms associated with MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) suggesting an impact of Arg on mito activity that may be beneficial in SCD. Given emerging data supporting a link between mito function and pain, improved mito activity may mechanistically contribute to decreased pain & ultimately less opioid requirement with IV Arg treatment, as well as have further implications for improved metabolism and oxidative signaling in SCD. In this RCT, Arg therapy also demonstrated clinically relevant opioid-sparing & a decreased time to crisis resolution that was statistically insignificant. A larger sample size may elucidate potential differences; we are currently enrolling 360 children in an ongoing PECARN-endorsed multicenter Phase 3 RCT (SCD Treatment with Arg Therapy - STArT). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A prospective real-world study evaluating serum levels of arginine and cysteine in women with chronic telogen effluvium: do we really need blanket prescription of amino acid supplementation?

Amino acids form a major component of hair fibres and are prescribed routinely in the form of nutritional supplements in patients with chronic telogen effluvium (CTE). Such a practice is based on assumption of a nutritional deficiency state in such patients. In this prospective study, we evaluated the serum levels of cysteine and arginine in 30 women with CTE and in healthy controls. We found no significant difference between the two groups in terms of serum arginine levels. Cysteine levels were higher in patients with CTE (P < 0.001). No correlation was found between levels of serum amino acids (cysteine and arginine) and either diet type (vegetarian or not) or body mass index, and no significant correlation between levels of the two amino acids and severity of disease. Our work suggests that arginine and cysteine deficiency is not present in women with CTE. Supplementation is unlikely to be of any benefit in nutrient-replete populations and only adds to the cost of therapy.

Spermidine activates authophagy but does not rescue human neuroblastoma SH-SY5Y cells from effects of arginine starvation

Background. Neuroblastoma is a malignant tumor of the sympathetic nervous system common in early childhood. Autophagy is manifested in neuroblastoma cells at basal levels, but is often upregulated in cells of the aggressive neuroblastoma forms. The aim of the study was to evaluate effects of polyamine spermidine and deficiency of arginine on cell viability and autophagy regulation in cells of human neuroblastoma. Materials and Methods. The human neuroblastoma SH-SY5Y cell line was an experimental model for the MTT assay of metabolic activity and cell viability upon exposure to different concentrations of spermidine in complete and arginine-free media. Assessing autophagy induction under arginine deficiency and spermidine treatment was carried out using fluorescence microscopy of neuroblastoma cells labeled with autophagic lysosomes-staining dye monodancylcadaverine. Results and Discussion. It was found that arginine withdrawal abrogates proliferation of SH-SY5Y cells. In the presence of arginine precursor, citrulline, in arginine-free medium, SH-SY5Y cells could not proliferate and, therefore, are auxotrophic for arginine. SH-SY5Y cells were more sensitive to arginine starvation than to starvation for indispensible amino acids lysine or leucine. It was also revealed that spermidine at low concentrations of 5-10 μM did not affect SH-SY5Y cells viability irrespective of arginine availability. However, at 50 and higher concentrations this polyamine was highly cytotoxic in arginine-sufficient or deficient media. Analysis of autophagy induction by spermidine and under arginine starvation revealed an increase in the number of autophagic lysosomes in SH-SY5Y cells and additive effect of the two stimuli. Conclusion. The described experiments revealed that arginine deprivation abrogated proliferation, led to a decrease in cell viability and induced autophagy in human neuroblastoma SH-SY5Y cells. Spermidine at the concentrations of 5-10 μM, while upinducing autophagy, did not improve survival of SH-SY5Y cells under arginine deprivation, whereas at a concentration above 50 μM spermidine had a strong cytotoxic effect. The main observation of this study is that autophagy can be readily manipulated in neuroblastoma cells by simultaneous deprivation for arginine and spermidine treatment.

Amino acid signature during sickle cell pain crisis shows significant alterations related to nitric oxide and energy metabolism.

Nitric oxide depletion secondary to arginase induced arginine deficiency has been shown to be important in the pathophysiology of vaso-occlusion in sickle cell pain crisis. Our objective of this study was to perform a comprehensive amino acid evaluation during sickle cell pain crisis. In a total of 58 subjects (29 in steady-state sickle cell disease and 29 with sickle cell pain crisis), the amino acids related to nitric oxide pathway was significantly decreased during sickle cell pain crisis compared to steady-state sickle cell disease: arginine (p = 0.001), citrulline (p = 0.012), and ornithine (p = 0.03). In addition, the amino acids related to energy metabolism was significantly decreased during a pain crisis: asparagine (p < 0.001), serine (p = 0.002), histidine (p = 0.017), alanine (p = 0.004), tyrosine (p = 0.012), methionine (p = 0.007), cystine (p = 0.016), isoleucine (p = 0.016) and lysine (p = 0.006). The amino acid related to oxidative stress were significantly higher during a sickle cell pain crisis (glutamic acid (p < 0.001). Furthermore, multivariate analysis with partial least squares-discriminant analysis (PLS-DA) showed that deficiencies of the amino acids arginine, asparagine, citrulline, methionine and alanine were the most important related to sickle cell pain crisis.

Creatine metabolism in patients with urea cycle disorders

The urea cycle generates arginine that is one of the major precursors for creatine biosynthesis. Here we evaluate levels of creatine and guanidinoacetate (the precursor in the synthesis of creatine) in plasma samples (ns = 207) of patients (np = 73) with different types of urea cycle disorders (ornithine transcarbamylase deficiency (ns = 22; np = 7), citrullinemia type 1 (ns = 60; np = 22), argininosuccinic aciduria (ns = 81; np = 31), arginase deficiency (ns = 44; np = 13)). The concentration of plasma guanidinoacetate positively correlated (p < 0.001, R2 = 0.64) with levels of arginine, but not with glycine in all patients with urea cycle defects, rising to levels above normal in most samples (34 out of 44) of patients with arginase deficiency. In contrast to patients with guanidinoacetate methyltransferase deficiency (a disorder of creatine synthesis characterized by elevated guanidinoacetate concentrations), creatine levels were normal (32 out of 44) or above normal (12 out of 44) in samples from patients with arginase deficiency. Creatine levels correlated significantly, but poorly (p < 0.01, R2 = 0.1) with guanidinoacetate levels and, despite being overall in the normal range in patients with all other urea cycle disorders, were occasionally below normal in some patients with argininosuccinic acid synthase and lyase deficiency. Creatine levels positively correlated with levels of methionine (p < 0.001, R2 = 0.16), the donor of the methyl group for creatine synthesis. The direct correlation of arginine levels with guanidinoacetate in patients with urea cycle disorders explains the increased concentration of guanidino compounds in arginase deficiency. Low creatine levels in some patients with other urea cycle defects might be explained by low protein intake (creatine is naturally present in meat) and relative or absolute intracellular arginine deficiency.

Does a bacterial infection lead to a nutritional deficiency in foraging Allenby's gerbils?

Abstract Bacterial infections can have both direct (physiological) and indirect (ecological) costs for their hosts. The direct costs of infection may include nutritional deficiencies, which reduce body condition over time, and result in the hosts modifying their foraging behaviour to compensate for any losses. Allenby’s gerbils are commonly infected with the Mycoplasma haemomuris-like bacteria, which may induce choline (essential vitamin) and arginine (amino acid) deficiencies. Gerbils should therefore alter their behaviour to make up for any shortfalls in nutrient production and retention. We tested two predictions. 1) infected gerbils would compensate for nutrient losses by spending more time foraging in patches, including, during riskier periods of owl presence, and in riskier open microhabitats. Furthermore, infected gerbils would preferentially forage from patches supplemented with choline and arginine enriched food over control food. 2) Alternatively, if infection did not create a nutrient deficiency, then infected individuals would display a similar pattern of patch use to uninfected individuals, and harvest similar amounts of food from supplemented and control patches. Our findings supported the second prediction. There was no observed difference in foraging behaviour between infected and uninfected individuals. Moreover, infected gerbils did not selectively forage more intensively from food patches enriched with either choline or arginine seed over the control patches. These results suggest that this bacteria-gerbil interaction does not result in a nutrient deficiency as observed for other Mycoplasma-rodent systems.

Nitric Oxide (NO) and Arginine as Factors for Increasing Poultry Meat Productivity

Nitric oxide (NO) is intensively synthesized in the embryo of birds. There is evidence that NO mediates myogenesis at the embryonic stage. In this regard, it might be possible to control muscle development and meat productivity by modulating NO synthesis. Using high-precision and a highly sensitive enzymatic sensor, the authors found that it is not the rate of synthesis that correlates with meat productivity, but the rate of NO oxidation to nitrate, which occurs in the tissues of the embryo. In broilers, it is several orders of magnitude higher than in layers. This indicator is solely due to the characteristics of embryonic tissues and is allelically determined. In-ovo arginine supplementation, a source of NO, did not lead to a significant increase in its synthesis and oxidation, but occasionally increased live weight gain, which was likely associated with a deficiency of free arginine and was not directly related to the effect of nitric oxide. Exogenous NO donors were oxidized with the same intensity as endogenous donors. These compounds did not have a significant effect on growth rate. Also, a reduction of 50% in the rate of NO synthesis under the action of a NO synthase inhibitor did not affect this parameter. Thus, regulation of poultry meat productivity is possible by modulating gene expression related to embryonic NO oxidation and ensuring an optimal amino acid and energy equilibrium, rather than by promoting embryonic NO synthesis.&#x0D; Keywords: Nitric oxide (NO), NO donor compounds, nitrate, arginine, live weight