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L-citrulline (L-CIT), a precursor to L-arginine (L-ARG), is a key contributor to the nitric oxide (NO) signaling pathway. Endothelial dysfunction, characterized by deficient nitric oxide synthesis, is implicated in the pathogenesis of various neonatal conditions such as necrotizing enterocolitis (NEC) and bronchopulmonary dysplasia (BPD) associated pulmonary hypertension (PH). This review summarizes the current evidence around the possible role of L-CIT supplementation in the treatment of these conditions. Detoxification of endogenously produced superoxide radicals is inadequate in preterm infants due to immature antioxidants that leads to the production of peroxynitrite, a reactive oxygen-free radical that is cytotoxic and causes damage to organelles and cellular membranes, further disrupting the coupling of endothelial NO synthase enzyme and the generation of high levels of reactive nitrogen and oxygen species. Animal studies in lipopolysaccharide-induced models of chorioamnionitis and hyperoxia- and inflammation-induced BPD-PH in rodent lung models revealed that L-CIT supplementation significantly mitigated structural changes in the pulmonary vasculature, preserved alveolar growth, and increased vascular endothelial growth factor gene expression, highlighting the anti-inflammatory and antioxidant effects of L-CIT supplementation. Similar benefits were noted in newborn piglet models of chronic hypoxia-induced PH and NEC. Pharmacokinetic studies in neonates have shown doses of 100–300 mg/kg/day to be safe and well tolerated. A few studies have shown the beneficial effects of L-CIT supplementation in pulmonary hypertension secondary to congenital heart disease, but evidence of efficacy in the neonatal population is lacking. While L-CIT shows promise in the treatment of various neonatal conditions, adequately powered studies to evaluate the safety and efficacy of L-CIT supplementation post-surgical NEC and BPD ± PH in the extremely preterm population are needed to translate this novel therapy to clinical practice. [ABSTRACT FROM AUTHOR]

Background Pelvic trauma can have long-lasting debilitating effects, including severe erectile dysfunction (ED) in men. While there are effective treatments for ED, these treat the symptoms not the cause. Those who suffer from an acute traumatic injury to the neurovascular supply of penis, may benefit from regenerative therapy. COMP4 is an all-natural herbal compound (Muira puama, Paullinia cupana , Ginger, and L-Citrulline) has potential to enhance penile vascular/smooth muscle/neuronal regeneration via Nitric Oxide synthesis. Low energy shockwave (LESW) therapy promotes nerve regeneration/angiogenesis by activating stem/progenitor cells. Aim To investigate the effects of the COMP4 and LESW for the treatment of ED in a rat pelvic trauma model. Methods The experimental design included five groups (Lewis rats aged 10-12 weeks; n = 8/group): (1) Sham, (2) Injury control, (3) Peanut Butter (PNB; Vehicle) Control, (4) COMP4, and (5) COMP4 + LESW. Pelvic neurovascular injury (PNVI) was induced by performing bilateral cavernous nerve crush injury and internal pudendal bundle ligation under general anesthesia. One week after PNVI rats received COMP4 (45 mg/kg b.wt./day) orally via PNB and/or LESW (1000 pulses at 0.06 mJ/mm2, 3 Hz, three times per week to the pelvis around penis) for 6 weeks. After 1 week of washout period, erectile function (EF) was assessed via intracavernous pressure (ICP; mmHg) measurements; rats euthanized, penile tissues collected for analyses. Outcomes COMP4 and LESW treatments improved EF recovery. Results There was a significant (P  < .01) improvement in EF (ICP) with COMP4 (153.6 ± 26.6) and COMP4 + LESW (174.1 ± 38.2) treatments compared to vehicle (PNB) control (109.4 ± 32.6). COMP4 + LESW treatment enhanced EF compared to COMP4 alone. The base level ICP before treatments (Group 2) was 117.3 ± 6 and was lower (P  < .01) than sham (165 ± 43.8) and COMP4 & COMP4 + LESW treated rats. Masson's trichrome staining of corpus cavernosum penis demonstrated reduced (P  < .001) smooth muscle-to-collagen (SM:C) ratio in injury and PNB controls compared to sham. COMP4 treatment following PNVI showed an increase (P  < .05) in SM:C ratio. Adding LESW and COMP4 enhanced (P  < .01) the SM:C ratio, suggesting a reduction in fibrosis. Western blot analysis revealed a significant increase in Endothelial Nitric Oxide Synthase and α-Smooth Muscle Actin (α-SMA) in the corpus cavernosum with COMP4 and COMP4 + LESW treatments compared to PNB control. Clinical Implications COMP4 and/or LESW can serve as an adjunct therapy to mitigate ED. Strengths and Limitations Identified novel treatment option to improve EF recovery. Unable to address in-depth cellular/molecular mechanisms. Conclusions COMP4 and LESW treatments appear to be promising to improve clinical EF recovery in pelvic trauma. [ABSTRACT FROM AUTHOR]

Watermelon is a rich natural source of Citrulline, an amino acid with potential antioxidant and vasodilation effects in the human body. An efficient Microwave-assisted aqueous two-phase extraction (MA-ATPE) technique was developed to extract and enrich L-Citrulline from watermelon rind in a one-step procedure. The aqueous two-phase system (ATPS) consisted of the ionic liquid 1-Butyl-3-methylimidazolium bromide [C4mim] [Br], and the salt dipotassium hydrogen phosphate (K2HPO4) was used as the extracting solvent. MA-ATPE conditions, such as the compositions of ATPS, particle size, liquid-solid ratio, irradiation power, and extraction time were investigated. The results indicated that the optimal conditions were as follows: salt concentration 35%, ionic liquid concentration 1.6 M, particle sizes 60 mesh, irradiation power 300 W, and extraction time 10 min. Under the optimized conditions, the extraction yields of L-Citrulline reached 87.74%. According to the DPPH results, at a concentration of 2 mg/mL, the DPPH radical-scavenging activity of the MA-ATPE extract from watermelon rind (0.388 mg/ml) was greater than that of the MAE (Microwave-assisted extraction) extract (0.456 mg/ml) and the conventional extract (0.468 mg/ml). DLS and TEM were used together to examine the microstructure of the top phase and explore the potential extraction mechanism. The findings revealed that the aggregation and embrace phenomenon were the primary driving forces for the separation. Compared with the conventional method and regular MAE, MA-ATPE exhibited a higher extraction yield and a shorter extraction time. In conclusion, MA-ATPE is an efficient, rapid, eco-friendly, and economical technique, representing an excellent alternative for the extraction of bioactive compounds. [ABSTRACT FROM AUTHOR]

Objective Palmitic acid (PA), the most abundant saturated free fatty acids, induces apoptosis in bovine mammary epithelial cells (MECs). It is suggested that oxidative stress and endoplasmic reticulum (ER) stress are key mechanisms underlying PA-induced cell death. This study aimed to investigate the interaction between ER stress and oxidative stress during PA-induced cell death in mammary alveolar cell-T (MAC-T) cells. Additionally, we examined whether L-citrulline can protect against PA-induced damage of MAC-T cells. Methods MAC-T cells were treated with 4-phenyl butyric acid (4-PBA) or N-acetyl-L-cysteine (NAC) to inhibit PA-induced ER stress and oxidative stress, respectively. MAC-T cells were pretreated with or without L-citrulline for 48 h followed by PA treatment. Cell viability was measured with MTT assays. Intracellular reactive oxygen species (ROS) levels in MAC-T cells were assessed using 5-(and-6)-chloromethyl- 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester dye. Real-time quantitative polymerase chain reaction was used to explore the regulation of genes associated with oxidative stress, and ER stress genes. Western blotting analysis was also carried out. Results 4-PBA significantly reduced PA-induced mRNA expressions of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and intracellular ROS levels. Furthermore, NAC dramatically reduced PA-induced ROS levels and the mRNA expressions of NRF2, ATF4, and CHOP. L-citrulline pretreatment effectively rescued cell viability decreased by PA. Moreover, L-citrulline pretreatment significantly downregulated the PA-induced upregulation of GRP78, ATF4, and CHOP mRNA expression, and protein expression of p-PERK and cleaved caspase-3. PA increased intracellular ROS levels and NRF2 mRNA expression, whereas L-citrulline pretreatment remarkably reduced these levels. Conclusion Both ER and oxidative stresses interact during PA-induced cell death in MAC-T cells, and L-citrulline could attenuate this cell death by inhibiting ER and oxidative stresses. Therefore, L-citrulline may be a promising supplement for protecting against PA-induced cell death in bovine MECs during the lactation period of dairy cows.

Highlights: 1. This study elucidates the effect of L-citrulline supplementation on creatine kinase MM (CK-MM) expression post-eccentric exercise, offering novel insights into its potential role in mitigating muscle damage. 2. The findings demonstrate that L-citrulline administration significantly reduces CK-MM expression levels in skeletal muscle tissue, suggesting its therapeutic potential in enhancing muscle recovery and performance following exercise-induced damage. 3. This study contributes valuable insights into the multifaceted benefits of L-citrulline supplementation for supporting overall muscle health and performance by identifying its protective effects under different mechanisms, including improved blood flow, antioxidant activity, enhanced mitochondrial function, and promotion of muscle protein synthesis. Abstract Eccentric exercise often induces muscle injuries in athletes, resulting in impaired performance and prolonged recovery time. Creatine kinase MM (CK-MM) is a biomarker for assessing muscle damage, with elevated levels indicating injury. L-citrulline, an amino acid, has shown promise in enhancing performance and reducing recovery time. However, its specific effect on CK-MM remains unclear. This study utilized immunohistochemistry analysis to investigate the effect of L-citrulline supplementation on CK-MM expression post-eccentric exercise in male BALB/c mice. This in vivo study was conducted with a post-test-only design. A total of 25 mice were divided into two control groups (normal/C1 and negative/C2) and three treatment groups (T1, T2, and T3), each containing five mice. The T1, T2, and T3 groups were daily administered 250, 500, and 1,000 mg/kg bw of L-citrulline for seven days, respectively. All mice, except the C1 group, performed a downhill running procedure. The CK-MM expression in skeletal muscle tissue post-eccentric exercise was assessed using immunohistochemistry analysis. The statistical analysis included the Shapiro-Wilk test for data distribution and the Kruskal-Wallis and Mann-Whitney post-hoc tests for significant differences (p

Eccentric exercise often induces muscle injuries in athletes, resulting in impaired performance and prolonged recovery time. Creatine kinase MM (CK-MM) is a biomarker for assessing muscle damage, with elevated levels indicating injury. Lcitrulline, an amino acid, has shown promise in enhancing performance and reducing recovery time. However, its specific effect on CK-MM remains unclear. This study utilized immunohistochemistry analysis to investigate the effect of Lcitrulline supplementation on CK-MM expression post-eccentric exercise in male BALB/c mice. This in vivo study was conducted with a post-test-only design. A total of 25 mice were divided into two control groups (normal/C1 and negative/C2) and three treatment groups (T1, T2, and T3), each containing five mice. The T1, T2, and T3 groups were daily administered 250, 500, and 1,000 mg/kg bw of L-citrulline for seven days, respectively. All mice, except the C1 group, performed a downhill running procedure. The CK-MM expression in skeletal muscle tissue post-eccentric exercise was assessed using immunohistochemistry analysis. The statistical analysis included the Shapiro-Wilk test for data distribution and the Kruskal-Wallis and Mann-Whitney post-hoc tests for significant differences (p<0.05). The results showed that CKMM expression in the C2 group (91.00±2.24%) was significantly higher (p=0.008) than that of the C1 group (70.00±10.0%). Subsequently, the T1 (68.00±9.08%, p=0.008), T2 (72.00±7.58%, p=0.008), and T3 (67.00±9.75%, p=0.008) groups exhibited significantly lower expressions than the C2 group. These results were consistent with the role of CK-MM as a marker for muscle damage, and they indicated that L-citrulline might have a protective effect against muscle damage posteccentric exercise. However, no significant differences were observed among the C1, T1, T2, and T3 groups. In conclusion, L-citrulline supplementation demonstrates promise in attenuating muscle damage following eccentric exercise, as evidenced by reduced CK-MM expression levels. These findings highlight the potential therapeutic role of L-citrulline in enhancing muscle recovery and performance. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction decreases exercise limb blood flow (BF) and muscle oxygenation. Acute L-Citrulline supplementation (CIT) improves muscle tissue oxygen saturation index (TSI) and deoxygenated hemoglobin (HHb) during exercise. Although CIT improves endothelial function (flow-mediated dilation [FMD]) in hypertensive women, the impact of CIT on exercise BF and muscle oxygenation (TSI) and extraction (HHb) are unknown. We examined the effects of CIT (10 g/day) and a placebo for 4 weeks on blood pressure (BP), arterial vasodilation (FMD, BF, and vascular conductance [VC]), and forearm muscle oxygenation (TSI and HHb) at rest and during exercise in 22 hypertensive postmenopausal women. Compared to the placebo, CIT significantly (p < 0.05) increased FMD (Δ−0.7 ± 0.6% vs. Δ1.6 ± 0.7%) and reduced aortic systolic BP (Δ3 ± 5 vs. Δ−4 ± 6 mmHg) at rest and improved exercise BF (Δ17 ± 12 vs. Δ48 ± 16 mL/min), VC (Δ−21 ± 9 vs. Δ41 ± 14 mL/mmHg/min), TSI (Δ−0.84 ± 0.58% vs. Δ1.61 ± 0.46%), and HHb (Δ1.03 ± 0.69 vs. Δ−2.76 ± 0.77 μM). Exercise BF and VC were positively correlated with improved FMD and TSI during exercise (all p < 0.05). CIT improved exercise artery vasodilation and muscle oxygenation via increased endothelial function in hypertensive postmenopausal women. [ABSTRACT FROM AUTHOR]

Obesity in adolescents is associated with non-communicable risk factors and diseases like metabolic-associated fatty liver disease (MAFLD), which is the liver manifestation of metabolic syndrome. L-citrulline is a non-protein amino acid that has shown positive effects on the degree of steatosis in animals with non-alcoholic fatty liver disease (NAFLD). The aim of the study was to evaluate the effect of oral L-citrulline supplementation on liver function and cardiovascular risk factors in adolescents with abdominal obesity and MAFLD. A prospective, double-blind clinical trial in adolescents with abdominal obesity was randomized into two groups: forty-two adolescents were supplemented with L-citrulline (6 g of L-citrulline/day) (n = 22) and placebo (n = 20) for eight weeks. The variables evaluated were anthropometry, blood pressure, glucose, insulin, HOMA-IR, L-citrulline, L-arginine, malondialdehyde, lipid profile, liver profile, urea, uric acid, and hepatic steatosis by ultrasound. After supplementation, the L-citrulline group had a decrease in liver fat accumulation (p = 0.0007); increases in body weight (p = 0.02), glucose (p = 0.03), and HOMA-IR (p = 0.03); and decreases in BMI (p = 0.002), total cholesterol (p = 0.001), HDL-C (p = 0.01), LDL-C (p = 0.002), and alkaline phosphatase (p = 0.05). L-citrulline for eight weeks decreases hepatic fat accumulation and LDL-C levels in adolescents with abdominal obesity and MAFLD. [ABSTRACT FROM AUTHOR]

L-citrulline (L-CIT), a precursor to L-arginine (L-ARG), is a key contributor to the nitric oxide (NO) signaling pathway. Endothelial dysfunction, characterized by deficient nitric oxide synthesis, is implicated in the pathogenesis of various neonatal conditions such as necrotizing enterocolitis (NEC) and bronchopulmonary dysplasia (BPD) associated pulmonary hypertension (PH). This review summarizes the current evidence around the possible role of L-CIT supplementation in the treatment of these conditions. Detoxification of endogenously produced superoxide radicals is inadequate in preterm infants due to immature antioxidants that leads to the production of peroxynitrite, a reactive oxygen-free radical that is cytotoxic and causes damage to organelles and cellular membranes, further disrupting the coupling of endothelial NO synthase enzyme and the generation of high levels of reactive nitrogen and oxygen species. Animal studies in lipopolysaccharide-induced models of chorioamnionitis and hyperoxia- and inflammation-induced BPD-PH in rodent lung models revealed that L-CIT supplementation significantly mitigated structural changes in the pulmonary vasculature, preserved alveolar growth, and increased vascular endothelial growth factor gene expression, highlighting the anti-inflammatory and antioxidant effects of L-CIT supplementation. Similar benefits were noted in newborn piglet models of chronic hypoxia-induced PH and NEC. Pharmacokinetic studies in neonates have shown doses of 100–300 mg/kg/day to be safe and well tolerated. A few studies have shown the beneficial effects of L-CIT supplementation in pulmonary hypertension secondary to congenital heart disease, but evidence of efficacy in the neonatal population is lacking. While L-CIT shows promise in the treatment of various neonatal conditions, adequately powered studies to evaluate the safety and efficacy of L-CIT supplementation post-surgical NEC and BPD ± PH in the extremely preterm population are needed to translate this novel therapy to clinical practice.

Background: The management of erectile dysfunction (ED) shows several grey zones and new treatments are required to reduce the percentage of patients discontinuing treatment. Here, we aim to evaluate the role of a natural mixture named Icarifil® (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) in the management of patients with ED. Methods: From September 2022 to March 2023, all patients attending 3 urological institutions due to ED were randomized to receive the following for 3 months: Icarifil® 1 sachet every 24 h (Group 1) or Icarifil® 1 sachet + tadalafil 5 mg 1 tablet every 24 h (Group 2) or tadalafil 5 mg 1 tablet daily (Group 3). All patients underwent urologic visits and dedicated questionnaires (IIEF-5, SEP-2, SEP-3) at enrollment and at the follow-up evaluation (3 months). Patient-Reported Outcomes (PROs) at the follow-up evaluation were used. The primary endpoint was the difference in the questionnaires at the follow-up visit compared to the one at enrollment among the study groups. Results: In the per-protocol analysis, 52 patients in Group 1, 55 in Group 2 and 57 in Group 3 were analyzed. At the follow-up evaluation, IIEF-5 scores improved in all the 3 groups between enrollment and the follow-up evaluation, but a statistically significant difference was reported between Group 2 (+7.4) and Group 1 (+4.1) or Group 3 (+5.1), (p < 0.001; p < 0.001). Moreover, 47 patients (94.0%) in Group 2 showed an improvement in the SEP questionnaires, when compared with the baseline, while 29 in Group 1 (56.9%) and 42 in Group 3 (82.3%) showed a statistically significant difference (p = 0.004; p = 0.003) among the groups. The PRO analysis reported better efficacy and patient satisfaction in Group 2 when compared with Group 1 or Group 3. Conclusions: In conclusion, Icarifil® is able to improve penile erectile function in mild–moderate ED and significantly improve the clinical efficacy of daily used tadalafil 5 mg. Icarifil® could represent an interesting alternative treatment in patients experiencing adverse effects or with contraindications for chronic treatment with PDE5-is. [ABSTRACT FROM AUTHOR]

Abstract Enzymatic detection of citrulline, a potential biomarker for various diseases, is beneficial. However, determining citrulline levels requires expensive instrumental analyses and complicated colorimetric assays. Although l-amino acid oxidase/dehydrogenase is widely used to detect l-amino acids, an l-citrulline-specific oxidase/dehydrogenase has not been reported. Therefore, in this study, we aimed to develop an l-citrulline-specific enzyme by introducing a mutation into l-arginine oxidase (ArgOX) derived from Pseudomonas sp. TPU 7192 to provide a simple enzymatic l-citrulline detection system. The ratio of the oxidase activity against l-arginine to that against l-citrulline (Cit/Arg) was 1.2%, indicating that ArgOX could recognize l-citrulline as a substrate. In the dehydrogenase assay, the specific dehydrogenase activity towards l-arginine was considerably lower than the specific oxidase activity. However, the specific dehydrogenase activity towards l-citrulline was only slightly lower than the oxidase activity, resulting in improved substrate specificity with a Cit/Arg ratio of 49.5%. To enhance the substrate specificity of ArgOX, we performed site-directed mutagenesis using structure-based engineering. The 3D model structure indicated that E486 interacted with the l-arginine side chain. By introducing the E486 mutation, the specific dehydrogenase activity of ArgOX/E486Q for l-citrulline was 3.25 ± 0.50 U/mg, which was 3.8-fold higher than that of ArgOX. The Cit/Arg ratio of ArgOX/E486Q was 150%, which was higher than that of ArgOX. Using ArgOX/E486Q, linear relationships were observed within the range of 10–500 μM l-citrulline, demonstrating its suitability for detecting citrulline in human blood. Consequently, ArgOX/E486Q can be adapted as an enzymatic sensor in the dehydrogenase system. Graphical Abstract

Postmenopausal women have augmented pressure wave responses to low-intensity isometric handgrip exercise (IHG) due to an overactive metaboreflex (postexercise muscle ischaemia, PEMI), contributing to increased aortic systolic blood pressure (SBP). Menopause-associated endothelial dysfunction via arginine (ARG) and nitric oxide deficiency may contribute to exaggerated exercise SBP responses. L-Citrulline supplementation (CIT) is an ARG precursor that decreases SBP, pulse pressure (PP) and pressure wave responses to cold exposure in older adults. We investigated the effects of CIT on aortic SBP, PP, and pressure of forward (Pf) and backward (Pb) waves during IHG and PEMI in twenty-two postmenopausal women. Participants were randomised to CIT (10 g/d) or placebo (PL) for 4 weeks. Aortic haemodynamics were assessed via applanation tonometry at rest, 2 min of IHG at 30 % of maximal strength, and 3 min of PEMI. Responses were analysed as change (Δ) from rest to IHG and PEMI at 0 and 4 weeks. CIT attenuated ΔSBP (−9 ± 2 v. −1 ± 1 mmHg, P = 0·006), ΔPP (−5 ± 2 v. 0 ± 1 mmHg, P = 0·03), ΔPf (−6 ± 2 v. −1 ± 1 mmHg, P = 0·01) and ΔPb (−3 ± 1 v. 0 ± 1 mmHg, P = 0·02) responses to PEMI v. PL. The ΔPP during PEMI was correlated with ΔPf (r = 0·743, P < 0·001) and ΔPb (r = 0·724, P < 0·001). Citrulline supplementation attenuates the increase in aortic pulsatile load induced by muscle metaboreflex activation via reductions in forward and backward pressure wave amplitudes in postmenopausal women. [ABSTRACT FROM AUTHOR]

Arginine deiminase (ADI) has been widely employed for the biological production of nutraceutical citrulline. However, sub-optimal enzyme properties, operational instability, and difficulty in reuse impede its industrial usage as well as increase the production cost. The present study describes a simple, cost-effective carrier-free immobilization technology that can circumvent these limitations. ADI was precipitated and cross-linked with glutaraldehyde to obtain novel cross-linked arginine deiminase aggregates (ADI-CLEA). A neural network-based multiobjective genetic algorithm (MOGA-NN) was employed to optimize the immobilization of ADI. MOGA-NN optimization yielded ADI-CLEA with 0.30 IU/mL activity and 61 % recovery. Structural characterization using TEM, SEM, FTIR, DLS/ZETA revealed that the developed ADI-CLEA were monodisperse, ultra-porous, and highly stable. ADI-CLEA showed an improved pH /temperature profile and resistivity against denaturants than free enzyme. The efficiency of ADI-CLEA in the industrial production of nutraceutical citrulline was also studied. ADI-CLEA produced 215.4 g/L citrulline in first cycle of usage. Reusability and storage stability analysis further showed that ADI-CLEA could be reused for more than seven consecutive cycles and stable for up to 50 days. In conclusion, this novel formulation increases the stability/robustness of ADI, eases the product separation process and could be reused for economically viable bioproduction of nutraceutical citrulline in industries. [Display omitted] • Novel cross-linked enzyme aggregates of arginine deiminase (ADI-CLEA) were reported. • Immobilization conditions were optimized by network-based multiobjective genetic algorithm. • ADI-CLEA exhibited enhanced catalytic efficiency, stability and robustness. • Improved citrulline yield (215 g/L) was obtained by ADI-CLEA. • ADI-CLEA could be recycled for seven cycles without losing its activity. [ABSTRACT FROM AUTHOR]

Purpose: L-citrulline is a non-essential and non-protein amino acid with potent antioxidant properties. In this study, the new aqueous two-phase system (ATPS) was used with and without ultrasound for the first time to isolate and purify L-citrulline from watermelon rind simultaneously. Methods: This system consists of an ionic liquid 1-Butyl-3-methylimidazolium bromide [C4mim] [Br] and dipotassium hydrogen phosphate (K2HPO4) salt. Accordingly, the impact of increasing the salt concentration, the ionic liquid, the solid–liquid ratio, and the ultrasound power and time on the extraction efficiency of L-citrulline and sugar compounds were examined. The HPLC technique Was employed to determine the concentration of L-citrulline in the sample with the highest efficiency rate. Moreover, the DLS tests were applied to determine the particle size. Results: As the concentrations of the ionic liquid, salt, and solid–liquid ratio were increased in the aqueous two-phase extraction system (ATPS) without applying the ultrasonic assistant, the L-citrulline extraction efficiency was increased initially and then decreased. Based on the results, applying the ultrasound to the sample with the highest amino acid efficiency rate, improved the extraction process and enhanced the amino acid extraction efficiency by 90.45%. The best extraction conditions obtained are related to the set, including the 1.5 M ionic liquid, 35% salt, the 1:40 solid–liquid ratio, the power of 100 W, and the ultrasonic time of 15 min. Conclusion: In general, the ionic liquid is a perfect extracting substance, and the ILUAE approach is a simple, efficient, fast, green, and environmentally friendly method for extracting ingredients. [ABSTRACT FROM AUTHOR]

Enzymatic detection of citrulline, a potential biomarker for various diseases, is beneficial. However, determining citrulline levels requires expensive instrumental analyses and complicated colorimetric assays. Although l-amino acid oxidase/dehydrogenase is widely used to detect l-amino acids, an l-citrulline-specific oxidase/dehydrogenase has not been reported. Therefore, in this study, we aimed to develop an l-citrulline-specific enzyme by introducing a mutation into l-arginine oxidase (ArgOX) derived from Pseudomonas sp. TPU 7192 to provide a simple enzymatic l-citrulline detection system. The ratio of the oxidase activity against l-arginine to that against l-citrulline (Cit/Arg) was 1.2%, indicating that ArgOX could recognize l-citrulline as a substrate. In the dehydrogenase assay, the specific dehydrogenase activity towards l-arginine was considerably lower than the specific oxidase activity. However, the specific dehydrogenase activity towards l-citrulline was only slightly lower than the oxidase activity, resulting in improved substrate specificity with a Cit/Arg ratio of 49.5%. To enhance the substrate specificity of ArgOX, we performed site-directed mutagenesis using structure-based engineering. The 3D model structure indicated that E486 interacted with the l-arginine side chain. By introducing the E486 mutation, the specific dehydrogenase activity of ArgOX/E486Q for l-citrulline was 3.25 ± 0.50 U/mg, which was 3.8-fold higher than that of ArgOX. The Cit/Arg ratio of ArgOX/E486Q was 150%, which was higher than that of ArgOX. Using ArgOX/E486Q, linear relationships were observed within the range of 10–500 μM l-citrulline, demonstrating its suitability for detecting citrulline in human blood. Consequently, ArgOX/E486Q can be adapted as an enzymatic sensor in the dehydrogenase system. Key points: ArgOX has higher substrate specificity for citrulline in the dehydrogenase assay. Through 3D modeling, ArgOX interacts with l-arginine via E486. ArgOX/E486Q can measure l-citrulline within the range of 10–500 μM. [ABSTRACT FROM AUTHOR]

Background. Eccentric exercise may trigger mechanical stress, resulting in muscle damage that may decrease athletic performance. L-citrulline potentially prevents skeletal muscle damage after acute eccentric exercise. This study aimed to assess the dose-response effect of L-citrulline as a preventive therapy for skeletal muscle damage in mice after acute eccentric exercise. Methods. This is a controlled laboratory in vivo study with a post-test-only design. Male mice (BALB/c, n=25) were randomized into the following groups: a normal control (C1) (n=5); a negative control (C2) with downhill running and placebo intervention (n=5); treatment groups: T1 (n=5), T2 (n=5), and T3 (n=5), were subjected to downhill running and 250, 500, and 1,000 mg/kg of L-citrulline, respectively, for seven days. Blood plasma was used to determine the levels of TNNI2 and gastrocnemius muscle tissue NOX2, IL-6, and caspase 3 using ELISA. NF-χB and HSP-70 expressions were determined by immunohistochemistry. Results. Skeletal muscle damage (plasma TNNI2 levels) in mice after eccentric exercise was lower after 250 and 500 mg/kg of L-citrulline. Further, changes in oxidative stress markers, NOX2, were reduced after a 1,000 mg/kg dose. However, a lower level of change has been observed in levels of cellular response markers (NF-κB, HSP-70, IL-6, and caspase 3) after administration of L-citrulline doses of 250, 500, and 1,000 mg/kg. Conclusion. L-citrulline may prevent skeletal muscle damage in mice after acute eccentric exercise through antioxidant effects as well as inflammatory and apoptotic pathways. In relation to dose-related effects, it was found that L-citrulline doses of 250, 500, and 1,000 mg/kg significantly influenced the expression of NF-κB and HSP-70, as well as the levels of IL-6 and caspase 3. Meanwhile, only doses of 250 and 500 mg/kg had an impact on TNNI2 levels, and the 1,000 mg/kg dose affected NOX2 levels. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effects of pre-exercise L-citrulline supplementation on the athletic performance of Yili speed-racing horses during a high-intensity exercise. On the 20th day of the experiment, blood samples were collected at 3 h and 6 h post-supplementation to measure the amino acid and polyamine concentrations. On the 38th day of the experiment, the horses participated in a 2000 m speed race, and three distinct blood samples were gathered for assessing blood gases, hematological parameters, the plasma biochemistry, antioxidant parameters, and NO concentrations. The results indicate that the L-citrulline group showed a significant increase in the plasma citrulline and arginine concentrations. Conversely, the concentrations of alanine, serine, and threonine were significantly decreased. The glycine concentration decreased significantly, while there was a trend towards an increase in the glutamine concentration. Additionally, the levels of putrescine and spermidine in the plasma of the L-citrulline group were significantly increased. In terms of exercise performance, L-citrulline can improve the exercise performance of sport horses, significantly reduce the immediate post-race lactate levels in Yili horses, and accelerate the recovery of blood gas levels after an exercise. Furthermore, in the L-citrulline group of Yili horses, The levels of the total protein of plasma, superoxide dismutase, catalase, and lactate dehydrogenase were significantly increased both 2 h before and 2 h after the race. The total antioxidant capacity showed a highly significant increase, while the malondialdehyde content significantly decreased. In the immediate post-race period, the creatinine content in the L-citrulline group significantly increased. In conclusion, this study demonstrates that L-citrulline supplementation can influence the circulating concentrations of L-citrulline and arginine in Yili horses, enhance the antioxidant capacity, reduce lactate levels, and improve physiological and biochemical blood parameters, thereby having a beneficial effect on the exercise performance of athletic horses.

ABSTRACT: High ambient temperature is a major environmental stressor affecting poultry production, especially in the tropical and subtropical regions of the world. Nutritional interventions have been adopted to combat thermal stress in poultry, including the use of amino acids. L-citrulline is a nonessential amino acid that is involved in nitric oxide generation and thermoregulation, however, the molecular mechanisms behind L-citrulline's regulation of body temperature are still unascertained. This study investigated the global gene expression in the hypothalamus of chickens fed either basal diet or L-citrulline-supplemented diets under different housing temperatures. Ross 308 broilers were fed with basal diet (CON) or 1% L-citrulline diet (LCT) from day-old, and later subjected to 2 environmental temperatures in a 2 by 2 factorial arrangement as follows; basal diet-fed chickens housed at 24°C (CON-TN); L-citrulline diet-fed chickens housed at 24°C (LCT-TN); basal diet-fed chickens housed at 35°C (CON-HS), and L-citrulline diet-fed chickens housed at 35°C (LCT-HS) from 22 to 42 d of age. At 42-days old, hypothalamic tissues were collected for mRNA analyses and RNA sequencing. A total of 1,019 million raw reads were generated and about 82.59 to 82.96% were uniquely mapped to genes. The gene ontology (GO) term between the CON-TN and LCT-TN groups revealed significant enrichments of pathways such as central nervous system development, and Wnt signaling pathway. On the other hand, GO terms between the CON-HS and LCT-HS groups revealed enrichments in the regulation of corticosteroid release, regulation of feeding behavior, and regulation of inflammatory response. Several potential candidate genes were identified to be responsible for central nervous system development (EMX2, WFIKKN2, SLC6A4 Wnt10a, and PHOX2B), and regulation of feed intake (NPY, AgRP, GAL, POMC, and NMU) in chickens. Therefore, this study unveils that L-citrulline can influence transcripts associated with brain development, feeding behavior, energy metabolism, and thermoregulation in chickens raised under different ambient temperatures.

Background Eccentric exercise may trigger mechanical stress, resulting in muscle damage that may decrease athletic performance. L-citrulline potentially prevents skeletal muscle damage after acute eccentric exercise. This study aimed to assess the dose-response effect of L-citrulline as a preventive therapy for skeletal muscle damage in mice after acute eccentric exercise. Methods This is a controlled laboratory in vivo study with a post-test-only design. Male mice (BALB/c, n = 25) were randomized into the following groups: a normal control (C1) (n = 5); a negative control (C2) with downhill running and placebo intervention (n = 5); treatment groups: T1 (n = 5), T2 (n = 5), and T3 (n = 5), were subjected to downhill running and 250, 500, and 1,000 mg/kg of L-citrulline, respectively, for seven days. Blood plasma was used to determine the levels of TNNI2 and gastrocnemius muscle tissue NOX2, IL-6, and caspase 3 using ELISA. NF-κB and HSP-70 expressions were determined by immunohistochemistry. Results Skeletal muscle damage (plasma TNNI2 levels) in mice after eccentric exercise was lower after 250 and 500 mg/kg of L-citrulline. Further, changes in oxidative stress markers, NOX2, were reduced after a 1,000 mg/kg dose. However, a lower level of change has been observed in levels of cellular response markers (NF-κB, HSP-70, IL-6, and caspase 3) after administration of L-citrulline doses of 250, 500, and 1,000 mg/kg. Conclusion L-citrulline may prevent skeletal muscle damage in mice after acute eccentric exercise through antioxidant effects as well as inflammatory and apoptotic pathways. In relation to dose-related effects, it was found that L-citrulline doses of 250, 500, and 1,000 mg/kg significantly influenced the expression of NF-κB and HSP-70, as well as the levels of IL-6 and caspase 3. Meanwhile, only doses of 250 and 500 mg/kg had an impact on TNNI2 levels, and the 1,000 mg/kg dose affected NOX2 levels.

Erectile dysfunction (ED) is a pathology that affects both an individual's life and their partner's, representing an increasing health concern. Currently, phosphodiesterase type 5 inhibitors (PDE5i) remain the first-line therapy for erectile dysfunction, and no other drugs are approved. However, the high cost of these drugs and related adverse events limit their use. Since the concept of nutraceuticals has been extended from health promotion to the treatment of diseases, in this work, a novel mixture of different natural substances named Icarifil (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) was studied to verify its ability to increase the proliferation of muscle epithelium cells and cell turgor, as well as, due to containing antioxidants, its ability to reduce the amount of ROS that are mediators in the pathophysiological process of ED. Moreover, Icarifil showed efficacy in inhibiting PDE5 levels higher than 65% compared to the control and 35% compared to the mixture of L-Citrulline and L-Carnitine, the major components of the formulation. In association with the low dosage of a phosphodiesterase (PDE) inhibitor (tadalafil), the inhibitory capacity of tadalafil was significantly increased and comparable to high-dosage drug response, promoting the possibility of reducing the daily dosage of the drug and consequently its adverse effects. [ABSTRACT FROM AUTHOR]

Background: Peripheral pain regulation is a very complex phenomenon due to the numerous neural pathways responsible for it. Objectives: The current study aimed to determine the anti-nociceptive activity of L-citrulline and the possible role of opioidergic, nitric oxide (NO), and serotoninergic systems in mice using the formalin and hot plate tests. Methods: In this study, 300 male NMRI mice were divided into 2 groups: 150 mice were used for the formalin test and 150 for the hot plate test (tests 1-6) with 4 sub-groups in each (n=50). The formalin test determined pain caused by the injection of formalin in the hind paw, and the hot plate test recorded pain reactions caused by heat stimulation as response latency time. Further, time mice capable of staying on the rotarod bar were determined. Results: Morphine reduced licking and biting time and latency time in the hot plate (P0.05. Conclusion: It seems that L-citrulline has anti-nociceptive effects, and its role is mediated by opioidergic, NO systems, as well as 5-HT2 and 5-HT3 receptors. Further, L-citrulline did not disturb motor coordination.

Gut microbiota play an important role in the gut ecology and development of pigs, which is always regulated by nutrients. This study investigated the effect of L-Citrulline on growth performance, carcass characteristics, and its potential regulatory mechanism. The results showed that 1% dietary L-Citrulline supplementation for 52 days significantly increased final weight, liveweight gain, carcass weight, and average backfat and markedly decreased drip loss (p < 0.05) of finishing pigs compared with the control group. Microbial analysis of fecal samples revealed a marked increase in a-diversity and significantly altered composition of gut microbiota in finishing pigs in response to L-Citrulline. In particular, these altered gut microbiota at the phylum and genus level may be mainly involved in the metabolic process of carbohydrate, energy, and amino acid, and exhibited a significant association with final weight, carcass weight, and backfat thickness. Taken together, our data revealed the potential role of L-Citrulline in the modulation of growth performance, carcass characteristics, and the meat quality of finishing pigs, which is most likely associated with gut microbiota. [ABSTRACT FROM AUTHOR]

The outbreak of coronavirus disease 2019 (COVID‐19) has caused a worldwide pandemic since 2019. A metabolic disorder is a contributing factor to deaths from COVID‐19. However, the underlying mechanism of metabolic dysfunction in COVID‐19 patients and the potential interventions are not elucidated. Here targeted plasma metabolomic is performed, and the metabolite profiles among healthy controls, and asymptomatic, moderate, and severe COVID‐19 patients are compared. Among the altered metabolites, arachidonic acid and linolenic acid pathway metabolites are profoundly up‐regulated in COVID‐19 patients. Arginine biosynthesis, alanine, aspartate, and glutamate metabolism pathways are significantly disturbed in asymptomatic patients. In the comparison of metabolite variances among the groups, higher levels of l‐citrulline and l‐glutamine are found in asymptomatic carriers and moderate or severe patients at the remission stage. Furthermore, l‐citrulline and l‐glutamine combination therapy is demonstrated to effectively protect mice from coronavirus infection and endotoxin‐induced sepsis, and is observed to efficiently prevent the occurrence of pulmonary fibrosis and central nervous system damage. Collectively, the data reveal the metabolite profile of asymptomatic COVID‐19 patients and propose a potential strategy for COVID‐19 treatment. [ABSTRACT FROM AUTHOR]

Grape seed extract (GSE) or L-citrulline supplement has been known to increase nitric oxide (NO) bioavailability and enhance endothelial-mediated vasodilation. Accordingly, to examine the additive benefits of combination of the two supplementations on hemodynamic responses to dynamic exercise, young, healthy males were recruited for this study. Effects of 7 days of 1) GSE + L-citrulline, 2) GSE, 3) L-citrulline, and 4) placebo supplementation on systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MAP), cardiac output, total vascular conductance (TVC), and oxygen (O2) consumption were examined at rest and during cycling exercise. Compared with placebo, GSE, L-citrulline, and combined supplementations did not reduce SBP, DBP, and MAP, while cardiac output (placebo; 23.6 ± 1.3 L/min, GSE; 25.7 ± 1.1 L/min; L-citrulline, 25.2 ± 1.2 L/min; GSE + L-citrulline; 25.3 ± 0.9 L/min) and TVC (placebo; 234.7 ± 11.3 ml/min/mmHg, GSE; 258.3 ± 10.6 ml/min/mmHg; L-citrulline, 255.2 ± 10.6 ml/min/mmHg; GSE + L-citrulline; 260.4 ± 8.9 ml/min/mmHg) were increased at only the 80% workload (p < 0.05). Compared with placebo and L-citrulline, GSE and combined supplementations had a reduction in VO2 across workloads (p < 0.05). However, there was no additive benefits on these variables. We conclude that supplementation with GSE, L-citrulline, and combined supplementations increased cardiac output due partially to decreased vascular resistance. Our findings suggest that GSE may act as an ergogenic aid that can improve O2 delivery to exercising muscles. [ABSTRACT FROM AUTHOR]

Mitochondrial fission is a crucial process in maintaining metabolic homeostasis in normal physiology and under conditions of stress. Its dysregulation has been associated with several metabolic diseases, including, but not limited to, obesity, type 2 diabetes (T2DM), and cardiovascular diseases. Reactive oxygen species (ROS) serve a vital role in the genesis of these conditions, and mitochondria are both the main sites of ROS production and the primary targets of ROS. In this review, we explore the physiological and pathological roles of mitochondrial fission, its regulation by dynamin-related protein 1 (Drp1), and the interplay between ROS and mitochondria in health and metabolic diseases. We also discuss the potential therapeutic strategies of targeting mitochondrial fission through antioxidant treatments for ROS-induced conditions, including the effects of lifestyle interventions, dietary supplements, and chemicals, such as mitochondrial division inhibitor-1 (Mdivi-1) and other mitochondrial fission inhibitors, as well as certain commonly used drugs for metabolic diseases. This review highlights the importance of understanding the role of mitochondrial fission in health and metabolic diseases, and the potential of targeting mitochondrial fission as a therapeutic approach to protecting against these conditions. [ABSTRACT FROM AUTHOR]

Pathophysiological conditions such as endothelial dysfunction and arterial stiffness, characterized by low nitric oxide bioavailability, deficient endothelium-dependent vasodilation and heart effort, predispose individuals to atherosclerotic lesions and cardiac events. Nitrate (NO3−), L-arginine, L-citrulline and potassium (K+) can mitigate arterial dysfunction and stiffness by intensifying NO bioavailability. Dietary compounds such as L-arginine, L-citrulline, NO3− and K+ exert vasoactive effects as demonstrated in clinical interventions by noninvasive flow-mediated vasodilation (FMD) and pulse-wave velocity (PWV) prognostic techniques. Daily L-arginine intakes ranging from 4.5 to 21 g lead to increased FMD and reduced PWV responses. Isolated L-citrulline intake of at least 5.6 g has a better effect compared to watermelon extract, which is only effective on endothelial function when supplemented for longer than 6 weeks and contains at least 6 g of L-citrulline. NO3− supplementation employing beetroot at doses greater than 370 mg promotes hemodynamic effects through the NO3−-NO2-/NO pathway, a well-documented effect. A potassium intake of 1.5 g/day can restore endothelial function and arterial mobility, where decreased vascular tone takes place via ATPase pump/hyperpolarization and natriuresis, leading to muscle relaxation and NO release. These dietary interventions, alone or synergically, can ameliorate endothelial dysfunction and should be considered as adjuvant therapies in cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Background and aimNitric oxide (NO) is the intracellular chemical responsible for initiating a penile erection. Despite conflicting clinical data, it continues to be publicized and promoted that orally administered l-arginine, the putative substrate for NO, enhances the erectile response presumably by stimulating NO production by the corporal tissues resulting in an increase in cGMP production. To shed light on this issue, an in vitro study was conducted to explore the effect of direct exogenous administration of l-arginine as well as its precursor and metabolite, l-citrulline, on the NO-cGMP pathway within the cavernosal smooth muscle (CSM) cell.Materials and methodsCSM cells obtained from 8 to 10 week old Sprague-Dawley rats were grown in Dulbecco media with 20% fetal calf serum and then incubated with or without l-arginine (L-ARG) or l-citrulline (L-CIT) in a time course and dose-response manner. Sildenafil (0.4 mM), IBMX (1 mM), l-NAME (3 μM), ODQ (5 μM) and Deta Nonoate (10 μM) were used as either inhibitors or stimulators of the NO-cGMP pathway. mRNA and protein were extracted and used for the determination of the phosphodiesterase 5 (PDE5). PDE5 activity was determined by luminometry. cGMP content was determined by ELISA. Nitrite formation, an indicator of NO production, was measured in the cell culture media by a colorimetric assay. The cationic (CAT-1) and neutral (SNAT-1) amino acid transporters for L-ARG and L-CIT, respectively, were determined by Western blot.ResultsWhen compared to untreated CSM cells, incubation with 0.25-4.0 mM of L-ARG or 0.3-4.8 mM of L-CIT anywhere between 3 and 24 h did not result in any additional nitrite or cGMP production. The addition of l-NAME, IBMX or ODQ to these L-ARG and L-CIT treated cells did not alter these results. L-CIT but not L-ARG increased PDE5 mRNA and protein content as well as the activity of the PDE5 enzyme. Both CAT-1 and SNAT-1 were expressed in the CSM cells.ConclusionsThis in vitro study demonstrates that exogenous administration of L-ARG or L-CIT failed to stimulate production of either NO or cGMP by the corporal CSM cells. A re-evaluation of the presumptive role of the exogenous administration of L-ARG in improving the synthesis of NO at least at the level of the CSM cells appears warranted.

A pre-workout supplement's (PWS; 200 mg caffeine, 3.3 g creatine monohydrate, 3.2 g β-alanine, 6 g citrulline malate and 5 g branched chained amino acid (BCAA) per dose) acute effects on the alactic (jumping, sprinting, agility), lactic (Running-Based Anaerobic Sprint Test, RAST) and aerobic performance (Yo-Yo Intermittent Recovery Test Level 1, Yo-Yo IRL1 VO2max) of well-trained basketball players was investigated in this double-blind placebo-controlled study. Thirty players (age 18–31 years, height 166–195 cm, weight 70.2–116.7 kg, body fat 10.6–26.4%) were allocated to pre-workout (PWS, n = 15) or placebo (PL, n = 15) groups. Half of the participants in each group performed the evaluations without PWS or PL, while the rest consumed PWS or PL 30 min before the assessments (1st trial) and vice versa (2nd trial). Significant improvements in counter-movement jump (CMJ) (PWS: 4.3 ± 2.1%; PL: 1.2 ± 1.0%), agility (PWS: −2.9 ± 1.8%; PL: 1.8 ± 1.7%), RAST average (PWS: 18.3 ± 9.1%; PL: −2.2 ± 2.0%), minimum power (PWS: 13.7 ± 8.9%; PL: −7.5 ± 5.9%), and fatigue index (PWS: −25.0 ± 0.9%; PL: −4.6 ± 0.6%) were observed in the PWS group vs. the PL group (p < 0.05). No differences were found regarding sprinting, aerobic performance, and blood lactate concentrations. Thus, although players' alactic and lactic anaerobic performance could be improved, peak power, sprinting and aerobic performance are not. [ABSTRACT FROM AUTHOR]

Background: The repercussions on oxidative and inflammatory stress markers under the effects of arginine and citrulline in response to exercise are not fully reached. We completed a systematic review to investigate the effects of L-Citrulline or L-Arginine on oxidative stress and inflammatory biomarkers following exercise. EMBASE, MEDLINE (PubMed), Cochrane Library, CINAHL, LILACS, and Web of Science databases were used to record the trials. This study includes randomized controlled trials (RCTs) and non-RCTs with subjects over 18 years old. Those under the intervention protocol consumed L-Citrulline or L-Arginine, and the controls ingested placebo. We recognized 1080 studies, but only 7 were included (7 studies in meta-analysis). We observed no difference between pre- vs. post-exercise for oxidative stress (subtotal = −0.21 [CI: −0.56, 0.14], p = 0.24, and heterogeneity = 0%. In the sub-group "L-Arginine" we found a subtotal = −0.29 [−0.71, 0.12], p = 0.16, and heterogeneity = 0%. For the "L-Citrulline" subgroup we observed a subtotal = 0.00 [−0.67, 0.67], p = 1.00, and heterogeneity was not applicable. No differences were observed between groups (p = 0.47), and I² = 0%) or in antioxidant activity (subtotal = −0.28 [−1.65, 1.08], p = 0.68, and heterogeneity = 0%). In the "L-Arginine" sub-group, we found a subtotal = −3.90 [−14.18, 6.38], p = 0.46, and heterogeneity was not applicable. For the "L-Citrulline" subgroup, we reported a subtotal = −0.22 [−1.60, 1.16], p = 0.75, and heterogeneity was not applicable. No differences were observed between groups (p = 0.49), and I² = 0%), inflammatory markers (subtotal = 8.38 [−0.02, 16.78], p = 0.05, and heterogeneity = 93%. Tests for subgroup differences were not applicable, and anti-inflammatory markers (subtotal = −0.38 [−1.15, 0.39], p = 0.34 and heterogeneity = 15%; testing for subgroup differences was not applicable). In conclusion, our systematic review and meta-analysis found that L-Citrulline and L-Arginine did not influence inflammatory biomarkers and oxidative stress after exercise. [ABSTRACT FROM AUTHOR]

In recent years, arginine deiminase (ADI, EC 3.5.3.6) has attracted much attention as a biocatalyst that produces the functional amino acid l‐citrulline from l‐arginine and also as an anticancer enzyme. Here, we identified and characterized a putative ADI from the thermophilic bacterium Halothermothrix orenii. The H. orenii ADI (H‐ADI) protein was expressed in Escherichia coli BL21(DE3) with a specific activity of 91.8 U/mg protein at 55°C and pH 6.5. The enzyme remained at 74% relative activity after incubation at 45°C for 180 min, only 25% at 50°C. The melting temperature was 56°C. H‐ADI is not a metal‐requiring enzyme; Ni2+ slightly improved the catalytic activity. The Km and Vmax for l‐arginine were 55.5 mM and 156.8 μmol/min/mg protein, respectively. Moreover, three residues (Arg183, Arg237, and His273) were key to the formation of l‐citrulline, as analyzed by alanine‐scanning mutagenesis. Finally, the enzymatic synthesis of l‐citrulline was carried out at 50°C with a conversion ratio reaching 99.03%. Together, these findings show that H‐ADI is a promising biocatalyst for the production of l‐citrulline. [ABSTRACT FROM AUTHOR]

Approximately 8–42% of premature infants with chronic lung disease of prematurity, bronchopulmonary dysplasia (BPD), develop pulmonary hypertension (PH). Infants with BPD-PH carry alarmingly high mortality rates of up to 47%. Effective PH-targeted pharmacotherapies are desperately needed for these infants. Although many PH-targeted pharmacotherapies are commonly used to treat BPD-PH, all current use is off-label. Moreover, all current recommendations for the use of any PH-targeted therapy in infants with BPD-PH are based on expert opinion and consensus statements. Randomized Control Trials (RCTs) are needed to determine the efficacy of PH-targeted treatments in premature infants with or at risk of BPD-PH. Prior to performing efficacy RCTs, studies need to be conducted to obtain pharmacokinetic, pharmacodynamic, and safety data for any pharmacotherapy used in this understudied and fragile patient population. This review will discuss current and needed treatment strategies, identify knowledge deficits, and delineate both challenges to be overcome and approaches to be taken to develop effective PH-targeted pharmacotherapies that will improve outcomes for premature infants with or at risk of developing BPD-PH. [ABSTRACT FROM AUTHOR]

The prevalence of type 2 diabetes (T2D) is increasing worldwide. Decreased nitric oxide (NO) bioavailability is involved in the pathophysiology of T2D and its complications. L‐citrulline (Cit), a precursor of NO production, has been suggested as a novel therapeutic agent for T2D. Available data from human and animal studies indicate that Cit supplementation in T2D increases circulating levels of Cit and L‐arginine while decreasing circulating glucose and free fatty acids and improving dyslipidemia. The underlying mechanisms for these beneficial effects of Cit include increased insulin secretion from the pancreatic β cells, increased glucose uptake by the skeletal muscle, as well as increased lipolysis and β‐oxidation, and decreased glyceroneogenesis in the adipose tissue. Thus, Cit has antihyperglycemic, antidyslipidemic, and antioxidant effects and has the potential to be used as a new therapeutic agent in the management of T2D. This review summarizes available literature from human and animal studies to explore the effects of Cit on metabolic parameters in T2D. It also discusses the possible mechanisms underlying Cit‐induced improved metabolic parameters in T2D. [ABSTRACT FROM AUTHOR]

The global market for nutritional supplements (NS) is growing rapidly, and the use of L-arginine (Arg), L-citrulline (Cit), and citrulline malate (CitMal) supplements has been shown to enhance cardiovascular health and athletic performance. Over the past decade, Arg, Cit, and CitMal supplements have received considerable attention from researchers in the field of exercise nutrition, who have investigated their potential effects on hemodynamic function, endothelial function, aerobic and anaerobic capacity, strength, power, and endurance. Previous studies were reviewed to determine the potential impact of Arg, Cit, and CitMal supplements on cardiovascular health and exercise performance. By synthesizing the existing literature, the study aimed to provide insight into the possible uses and limitations of these supplements for these purposes. The results showed that both recreational and trained athletes did not see improved physical performance or increased nitric oxide (NO) synthesis with 0.075 g or 6 g doses of Arg supplement per body weight. However, 2.4 to 6 g of Cit per day for 7 to 16 days of various NSs had a positive impact, increasing NO synthesis, enhancing athletic performance indicators, and reducing feelings of exertion. The effects of an 8 g acute dose of CitMal supplement were inconsistent, and more research is needed to determine its impact on muscle endurance performance. Based on the positive effects reported in previous studies, further testing is warranted in various populations that may benefit from nutritional supplements, including aerobic and anaerobic athletes, resistance-trained individuals, elderly people, and clinical populations, to determine the impact of different doses, timing of ingestion, and long-term and acute effects of Arg, Cit, and CitMal supplements on cardiovascular health and athletic performance. [ABSTRACT FROM AUTHOR]

Heart rate variability (HRV) provides a simple method to evaluate autonomic function in health and disease. A reduction in HRV may indicate autonomic dysfunction and is strongly associated with aspects of cardiometabolic disease, including hyperglycemia. Reduced nitric oxide (NO) bioavailability is also implicated in the development of cardiometabolic disease and autonomic dysfunction. Watermelons are natural sources of L-arginine and L-citrulline, substrates used for NO synthesis. Watermelon consumption can improve NO bioavailability. We conducted a randomized, double-blind, placebo-controlled crossover trial to test the effects of 2 weeks of daily watermelon juice (WMJ) supplementation on HRV in response to an oral glucose challenge (OGC) in healthy young adults. We also performed indirect calorimetry to assess if our intervention altered the metabolic response to the OGC. WMJ supplementation preserved high-frequency power (HF) (treatment effect, p = 0.03) and the percentage of successive differences that differ by more than 50 ms (pNN50) (treatment effect, p = 0.009) when compared to the placebo treatment. There was no difference in resting energy expenditure or substate oxidation according to treatment. We report that WMJ supplementation attenuates OGC-induced reductions in HRV. Future work should emphasize the importance of NO bioavailability in autonomic dysfunction in cardiometabolic disease. [ABSTRACT FROM AUTHOR]

Objective: To investigate the therapeutic effect of L-Citrulline (L-Cit) on preeclampsia (PE) rat model and its effect on oxidative stress and trophoblast cell invasion. Methods: After establishing the PE rat model induced by N-nitro-L-arginine methyl ester (L-NAME), the rats were divided into Control group, PE group, PE+ low, middle and high dose L-Cit groups (L-L-Cit, M-L-Cit and H-L-Cit groups in sequence, with doses of 100, 200 and 500 mg/kg in sequence), n=6, continuous administration for 7 days. On the 21st day of pregnancy, systolic blood pressure (SBP), 24-hour urinary protein, serum superoxide dismutase (SOD) and malondialdehyde (MDA) were measured, and placental tissue was stained with hematoxylin-eosin (HE). The HTR-8/Svneo cells were divided into Control group, hypoxia/reoxygenation (H/R) group, H/R+L-Cit group and H/R+L-Cit+sc-221593 group. The cells were treated with H/R, respectively, and cultured with L-Cit (200 μg/mL) and the specific ERK1/2 inhibitor sc-221593 (10 μmol/L) for 48 h. Cell invasion was determined by Transwell assay. The protein expressions of total-ERK1/2, p-ERK1/2, total-p38, p-p38, matrix metalloproteinase (MMP)-2 and MMP-9 in placental tissues and cells were detected by Western blotting. Results: Compared with PE group, the SBP and 24 h urinary protein levels of the rats in L-L-Cit group, M-L-Cit group and H-L-Cit group were decreased (P<0.05); the placental tissue morphology was significantly improved; serum SOD was increased, while MDA was decreased (P<0.05); the phosphorylation levels of ERK1/2 and p38 and the protein expression levels of MMP-2 and MMP-9 in placental tissue were increased (P<0.05). Compared with H/R group, the number of invasive cells in H/R+L-Cit group increased (P<0.05); the phosphorylation levels of ERK1/2 and p38 and the protein expression levels of MMP-2 and MMP-9 increased (P<0.05). Compared with the H/R+L-Cit group, the above changes in the H/R+L-Cit+sc-221593 group were reversed (P<0.05). Conclusion: L-Cit may alleviate the symptoms of PE by reducing oxidative stress in PE and promoting trophoblast invasion by activating the ERK/JNK pathway. [ABSTRACT FROM AUTHOR]

Nitric-oxide-stimulating dietary supplements are widely available and marketed to strength athletes and weightlifters seeking to increase muscle performance and augment training adaptations. These supplements contain ingredients classified as nitric oxide (NO) precursors (i.e., "NO boosters"). Endogenous NO is generated via a nitric oxide synthase (NOS)-dependent pathway and a NOS-independent pathway that rely on precursors including L-arginine and nitrates, with L-citrulline serving as an effective precursor of L-arginine. Nitric oxide plays a critical role in endothelial function, promoting relaxation of vascular smooth muscle and subsequent dilation which may favorably impact blood flow and augment mechanisms contributing to skeletal muscle performance, hypertrophy, and strength adaptations. The aim of this review is to describe the NO production pathways and summarize the current literature on the effects of supplementation with NO precursors for strength and power performance. The information will allow for an informed decision when considering the use of L-arginine, L-citrulline, and nitrates to improve muscular function by increasing NO bioavailability. [ABSTRACT FROM AUTHOR]

With global warming becoming of increasing concern, poultry farms are experiencing a concomitant increase in heat stress. Chickens are very sensitive to high ambient temperature (HT), so the development of novel nutrients that will help deal with the challenge posed by heat stress is vital. We revealed that L‐citrulline (L‐Cit) can reduce body temperature in chickens. Orally administered L‐Cit solution has been found to provide heat tolerance in chickens and to result in reduced food intake. Heat exposure and oral administration of L‐Cit led to increased levels of plasma insulin, whereas heat stress led to a decline in plasma thyroxine. Dietary administration of L‐Cit was also shown to be effective to reduce heat stress in broiler chickens. Moreover, L‐Cit was found to be metabolized in the liver within 1 h of its administration, and in L‐Cit‐treated broiler chicks, the Cit‐Arginine cycle and the Krebs cycle were found to be active. L‐Cit has not yet been approved for inclusion in the poultry diet, so it is important to find alternative sources of L‐Cit. Taken together, these findings suggest that L‐Cit may serve as an important novel nutrient with the ability to produce heat tolerance in chickens under HT. [ABSTRACT FROM AUTHOR]

In this work, a robust and mechanically composite hydrogel with an efficient electromagnetic interference shielding performance was successfully fabricated via the incorporation of l-citrulline-modified Ti3C2Tx MXene nanosheets into the polyacrylamide/sodium alginate hydrogels by using ferrous chloride as the adhesive. A l-citrulline-modified Ti3C2Tx MXene nanosheet was the main shielding medium; ferrous chloride could not only enhance the mechanical property of the resultant hydrogels but also slightly improve the EMI shielding efficiency. The optimal tensile strength (3.42 MPa) and the EMI shielding effectiveness (26.8 dB) were achieved for the composite hydrogels with 6.5 wt % l-citrulline-modified Ti3C2Tx MXene nanosheets and 0.6 mol/L ferrous chloride, and the high ductility (780% elongation at break) of the composite hydrogel was reached with 0.5 wt % l-citrulline-modified Ti3C2Tx MXene and 0.8 mol/L ferrous chlorides. With outstanding mechanical and EMI shielding performances, the prepared composite hydrogels could apply in the electronic skin field. [ABSTRACT FROM AUTHOR]

L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.

Background: Pelvic trauma can have long-lasting debilitating effects, including severe erectile dysfunction (ED) in men. While there are effective treatments for ED, these treat the symptoms not the cause. Those who suffer from an acute traumatic injury to the neurovascular supply of penis, may benefit from regenerative therapy. COMP4 is an all-natural herbal compound (Muira puama, Paullinia cupana , Ginger, and L-Citrulline) has potential to enhance penile vascular/smooth muscle/neuronal regeneration via Nitric Oxide synthesis. Low energy shockwave (LESW) therapy promotes nerve regeneration/angiogenesis by activating stem/progenitor cells., Aim: To investigate the effects of the COMP4 and LESW for the treatment of ED in a rat pelvic trauma model., Methods: The experimental design included five groups (Lewis rats aged 10-12 weeks; n = 8/group): (1) Sham, (2) Injury control, (3) Peanut Butter (PNB; Vehicle) Control, (4) COMP4, and (5) COMP4 + LESW. Pelvic neurovascular injury (PNVI) was induced by performing bilateral cavernous nerve crush injury and internal pudendal bundle ligation under general anesthesia. One week after PNVI rats received COMP4 (45 mg/kg b.wt./day) orally via PNB and/or LESW (1000 pulses at 0.06 mJ/mm2, 3 Hz, three times per week to the pelvis around penis) for 6 weeks. After 1 week of washout period, erectile function (EF) was assessed via intracavernous pressure (ICP; mmHg) measurements; rats euthanized, penile tissues collected for analyses., Outcomes: COMP4 and LESW treatments improved EF recovery., Results: There was a significant ( P  < .01) improvement in EF (ICP) with COMP4 (153.6 ± 26.6) and COMP4 + LESW (174.1 ± 38.2) treatments compared to vehicle (PNB) control (109.4 ± 32.6). COMP4 + LESW treatment enhanced EF compared to COMP4 alone. The base level ICP before treatments (Group 2) was 117.3 ± 6 and was lower ( P  < .01) than sham (165 ± 43.8) and COMP4 & COMP4 + LESW treated rats. Masson's trichrome staining of corpus cavernosum penis demonstrated reduced ( P  < .001) smooth muscle-to-collagen (SM:C) ratio in injury and PNB controls compared to sham. COMP4 treatment following PNVI showed an increase ( P  < .05) in SM:C ratio. Adding LESW and COMP4 enhanced ( P  < .01) the SM:C ratio, suggesting a reduction in fibrosis. Western blot analysis revealed a significant increase in Endothelial Nitric Oxide Synthase and α-Smooth Muscle Actin (α-SMA) in the corpus cavernosum with COMP4 and COMP4 + LESW treatments compared to PNB control., Clinical Implications: COMP4 and/or LESW can serve as an adjunct therapy to mitigate ED., Strengths and Limitations: Identified novel treatment option to improve EF recovery. Unable to address in-depth cellular/molecular mechanisms., Conclusions: COMP4 and LESW treatments appear to be promising to improve clinical EF recovery in pelvic trauma., Competing Interests: The authors declare no conflict of interest, financial or otherwise., (Published by Oxford University Press on behalf of The International Society for Sexual Medicine 2025.)

Background: Evidence suggests L-arginine may be effective at reducing pre-eclampsia and related outcomes. However, whether L-arginine can prevent or only treat pre-eclampsia, and thus the target population and timing of initiation, remains unknown., Objectives: To evaluate the effects of L-arginine and L-citrulline (precursor of L-arginine) on the prevention and treatment of pre-eclampsia., Search Strategy: MEDLINE, Embase, CINAHL, Global Index Medicus and the Cochrane Library were searched through 7 February 2024., Selection Criteria: Trials administering L-arginine or L-citrulline to pregnant women, with the comparison group receiving placebo or standard care, were included., Data Collection and Analysis: Meta-analyses were conducted separately for prevention or treatment trials, using random-effects models., Main Results: Twenty randomised controlled trials (RCTs) (2028 women) and three non-randomised trials (189 women) were included. The risk of bias was 'high' in eight RCTs and showed 'some concerns' in 12. In prevention trials, L-arginine was associated with a reduced risk of pre-eclampsia (relative risk [RR] 0.52; 95% confidence interval [CI], 0.35, 0.78; low-certainty evidence, four trials) and severe pre-eclampsia (RR 0.23; 95% CI, 0.09, 0.55; low-certainty evidence, three trials). In treatment trials, L-arginine may reduce mean systolic blood pressure (MD -5.64 mmHg; 95% CI, -10.66, -0.62; very low-certainty evidence, three trials) and fetal growth restriction (RR 0.46; 95% CI, 0.26, 0.81; low-certainty evidence, two trials). Only one study (36 women) examined L-citrulline and reported no effect on pre-eclampsia or blood pressure., Conclusions: L-arginine may be promising for pre-eclampsia prevention and treatment, but findings should be interpreted cautiously. More trials are needed to determine the optimal dose and time to commence supplementation and support clinical decision-making., (© 2025 The Author(s). BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Background & objectives: Malaria affects around 228 million people all over the globe. Malaria causing parasite Plasmodium infection leads to activation of immune responses. The growth of parasite and immune activation requires semi essential amino acids like L-arginine. Malaria infection leads to condition of hyperargininemia and low availability of nitric oxide. However, the effect of L-arginine supplementation in malaria infected mice has not been explored in in-vivo studies. In this study we have compared the effect of oral supplementation of nitric oxide donor, L-arginine and L-citrulline, in malaria infected mice Methods: To examine the effect of oral supplementation of L-arginine and L-citrulline, Plasmodium berghei infected mice were divided in different groups and respective groups were fed with L- arginine and L-citrulline, parasitemia was measured on different days. Mice was sacrificed and immunophenotyping was done on 10 days post infection. Results: our results show that supplementation of L-arginine induces conducive environment for Plasmodium growth due to which the infected mice dies earlier than control wild type infected mice whereas L-citrulline supplementation inhibits parasite growth and mice survives for longer period of time. Flow cytometric analysis shows that supplementation of L-arginine increases cTLA-4 on T cell population, increases Treg cells leading to immunosuppression while supplementation of L-citrulline does not have effect on T cells population and number of Treg cell decrease compared to P. berghei infected mice. Interpretation & conclusion: our results show that L-citrulline can be a better alternative than L-arginine because of lower expression of inhibitory molecules and lower parasitemia as well as increased survival of infected mice.

Introduction: Effective parenteral and enteral amino acid replacement is crucial for critically ill patients with altered amino acid metabolism. This study aimed to assess the effects of l-citrulline supplementation on the clinical and laboratory outcomes in critically patients. Methods: This was a double-blind placebo-controlled randomized clinical trial. 82 critically ill patients who were expected to receive mechanical ventilation for more than 72 hours were selected. The patients were assigned to either a placebo or an intervention group. The patients in the placebo group received 10 gr of microcrystalline cellulose and the ones in the intervention group were given l-citrulline daily for 7 days. Serum levels of fasting blood sugar (FBS), lipid profile, hepatic enzymes, serum electrolytes, urea nitrogen, creatinine, and C-reactive protein (CRP) were evaluated before and after the intervention. Duration of invasive ventilation, intensive care unit (ICU) length of stay, ventilator-free days, and 28-day mortality rate were recorded and compared between groups. Results: Eighty-two patients completed the trial. No statistically significant differences were observed between the two groups in terms of age (p = 0.46), sex (p = 0.49), body mass index (BMI) (p = 0.41), Sequential Organ Failure Assessment (SOFA) Score (p = 0.08), Clinical Pulmonary Infection Score (CPIS) score (p = 0.76), Acute Physiology and Chronic Health Evaluation (APACHE II) score (p = 0.58), risk factors (p = 0.13), ICU stay before randomization (p = 0.32), and reason of admission (p = 0.50) before the intervention. Citrulline group had a notable reduction in FBS (p = 0.04), total cholesterol (TC) (p = 0.02), low density lipoprotein (LDL-C) (p

Erectile dysfunction (ED) is a pathology that affects both an individual’s life and their partner’s, representing an increasing health concern. Currently, phosphodiesterase type 5 inhibitors (PDE5i) remain the first-line therapy for erectile dysfunction, and no other drugs are approved. However, the high cost of these drugs and related adverse events limit their use. Since the concept of nutraceuticals has been extended from health promotion to the treatment of diseases, in this work, a novel mixture of different natural substances named Icarifil (L-Citrulline, L-Carnitine, Eruca vesicaria, Panax ginseng, Tribulus terrestris, Turnera diffusa, Taurine, Vitamin E, Zinc) was studied to verify its ability to increase the proliferation of muscle epithelium cells and cell turgor, as well as, due to containing antioxidants, its ability to reduce the amount of ROS that are mediators in the pathophysiological process of ED. Moreover, Icarifil showed efficacy in inhibiting PDE5 levels higher than 65% compared to the control and 35% compared to the mixture of L-Citrulline and L-Carnitine, the major components of the formulation. In association with the low dosage of a phosphodiesterase (PDE) inhibitor (tadalafil), the inhibitory capacity of tadalafil was significantly increased and comparable to high-dosage drug response, promoting the possibility of reducing the daily dosage of the drug and consequently its adverse effects.

l-Citrulline may improve non-invasive vascular function and cardiometabolic risk markers through increases in l-arginine bioavailability and nitric oxide synthesis. A meta-analysis of randomised controlled trials (RCT) was performed to examine longer-term and postprandial effects of l-citrulline supplementation and watermelon consumption on these markers for CVD in adults. Summary estimates of weighted mean differences in vascular function and cardiometabolic risk markers with accompanying 95 % CI were calculated using random or fixed-effect meta-analyses. Seventeen RCT were included involving an l-citrulline intervention, of which six studied postprandial and twelve longer-term effects. Five studies investigated longer-term effects of watermelon consumption and five assessed effects during the postprandial phase. Longer-term l-citrulline supplementation improved brachial artery flow-mediated vasodilation (FMD) by 0·9 %-point (95 % CI 0·7, 1·1, P < 0·001). Longer-term watermelon consumption improved pulse wave velocity by 0·9 m/s (95 % CI 0·1, 1·5, P < 0·001), while effects on FMD were not studied. No postprandial effects on vascular function markers were found. Postprandial glucose concentrations decreased by 0·6 mmol/l (95 % CI 0·4, 0·7, P < 0·001) following watermelon consumption, but no other longer-term or postprandial effects were observed on cardiometabolic risk markers. To conclude, longer-term l-citrulline supplementation and watermelon consumption may improve vascular function, suggesting a potential mechanism by which increased l-citrulline intake beneficially affects cardiovascular health outcomes in adults. No effects on postprandial vascular function markers were found, while more research is needed to investigate the effects of l-citrulline and watermelon on risk markers related to cardiometabolic health. [ABSTRACT FROM AUTHOR]