Infants and children suffering from a variety of heart, lung, and blood disorders are known to be at risk of developing systemic or pulmonary vascular disease. Despite progress made in clinical care, these patients continue to experience high morbidity and mortality. There is evidence that an impairment in the L-arginine-NO signaling pathway is involved in the pathogenesis of many of the vascular disorders afflicting children. By boosting NO production, L-citrulline, the amino acid precursor of the NO substrate L-arginine, has the potential to ameliorate vascular diseases in both the systemic and pulmonary circulations. This review will discuss the current status of the use of L-citrulline as a treatment to improve outcomes in pediatric patients suffering from disorders that place them at risk of developing systemic or pulmonary vascular disease. Future directions and the potential therapeutic use of L-citrulline in vascular diseases not yet under clinical investigation will also be discussed.

Acute chest syndrome (ACS) is a common and serious lung complication in sickle cell disease. A retrospective medical chart review was performed over a 6-year period in all pediatric ACS patients to investigate whether factors during the initial hospitalization were associated with recurrent ACS episodes. There were 386 episodes of ACS: 149 had only 1 episode of ACS, and 76 had > 1 episode of ACS; 172 (76.4%) had hemoglobin SS, and 39 (17.3%) had hemoglobin SC. The most common presenting features were fever (83%), pain (70%), and cough (61%), which changed with the number of ACS episodes. Children <4 years old were at greatest risk of recurrent ACS (P = 0.018). In addition, history of asthma (adjusted incident rate ratio [IRR] = 1.52; 95% confidence interval [CI], 1.22-1.98; P < 0.0001), shortness of breath (IRR, 1.29; 95% CI, 1.02-1.62; P = 0.033), and length of hospital stay (IRR, 1.04; 95% CI, 1.01-1.08; P = 0.017) were significantly associated with prospective ACS events. Multiple episodes of ACS are common in sickle cell disease, and certain risk factors during the initial hospitalization are associated with recurrent ACS.

Acute pain from vaso-occlusion (VOC) in sickle cell disease (SCD) is the most frequent cause of emergency room visits and hospital admissions, contributing to the high burden of health care costs (Lanzkron et al, 2010). While major advances in the care of patients with SCD have occurred over the last 30 years, very little progress has been made in the actual treatment of VOC. Nitric oxide (NO) is a powerful vasodilator that plays a fundamental role in VOC (Morris, 2008). NO is produced from L-citrulline and L-arginine, amino acids generated through the urea cycle from the NO synthase (NOS) family of enzymes (Moncada & Higgs, 1993). There are three NOS isoforms: neuronal (nNOS) found in neuronal tissue, inducible NOS (iNOS) found in cells and tissues, and endothelial NOS (eNOS) found in vascular endothelial cells. Shen et al (2005) showed that citrulline was the major supply for intracellular L-arginine and endothelial NO production in murine endothelial cells. Furthermore, Wijnands et al (2012) showed that L-citrulline supplementation restored intracellular NO production, which was related to the degree of eNOS phosphorylation. Moreover, enhanced arginase-induced arginine consumption is believed to play an integral role in the pathogenesis of sickle cell complications. In a more recent study, L-citrulline supplementation increased NO production and improved microcirculatory flow during conditions with arginase-induced arginine deficiency (Wijnands et al, 2015). However, intravenous citrulline has never been evaluated in human SCD. Hence, this study aimed to characterize the pharmacokinetic (PK) and safety profile of intravenous (IV) citrulline in this unique patient population. A single centre open label phase 1 trial of IV citrulline was performed in participants with SCD following approval from the University of Mississippi Medical Center (UMMC) institutional review board. The study was registered at ClinicalTrials.gov (NCT02314689; NCT02697240), where the inclusion and exclusion criteria are described. Th phase 1 study was performed in two steps. Step 1 included a dose escalation bolus infusion of IV citrulline in steady-state SCD to determine the PK and safety profile with a peak goal plasma citrulline concentration of 80–100 μmol/l (Barr et al, 2007). Step 2 of the study was performed to evaluate safety and PK during a vaso-occlusive crisis. The study drug, L-citrulline, was administered as open label vials of 50 mg/ml (5%) isotonic solution. Plasma sampling for PK studies were collected at specific time points. Briefly, for amino acid analysis, deproteinated plasma samples were subjected to cation exchange chromatography using a 4-component pH and ionic strength graded lithium citrate buffer system on a Beckmann 7300 amino acid analyser (Beckmann, Palo Alto, CA). Data obtained for each patient was fitted to a single-compartment PK model. The appearance of citrulline in plasma was described by a zero-order process (rate of citrulline appearance, Rapp) to account for endogenous production, whereas the removal of citrulline was determined by a first-order process (constant of citrulline removal, krem). It was assumed that the values of all parameters remained constant for each patient during the course of plasma sampling. Scientist v2.0 (Micromath Scientific Software, St. Louis, MO) was used to fit the plasma citrulline concentration to the PK model by a weighted, least squares procedure to obtain values for Rapp, krem, and the volume of distribution (Vd). Clearance was calculated as the product of krem and Vd. For safety assessments, the Investigator determined the intensity of any adverse event (AE) according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 4.03 (https://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf) and their causal relationship. A Data Safety Monitoring Board, comprising 3 physicians who were not related to the study, reviewed every third subject. A total of 8 subjects with SCD were enrolled in this phase 1 study of IV citrulline: four participants were enrolled in Step 1 and another four participants in Step 2. Patient demographics, genotype and baseline blood counts are shown in Table SI. In the first cohort of four participants, the IV bolus infusion of 20 mg/kg of L-citrulline yielded a mean peak level of 259 μmol/l and trough level in the range of 20–40 μmol/l at 4 h after infusion (Fig 1). Citrulline PK parameters for Step 1 with bolus infusion are shown in Table SII. Pharmacokinetic model simulations indicated a 20 mg/kg bolus dose of IV citrulline followed by 7 mg/kg per hour continuous infusion was needed to maintain the target citrulline plasma concentration of 100 μmol/l. Subsequently, four subjects with VOC were enrolled to receive IV citrulline bolus and continuous infusion. Individuals with VOC showed significantly lower baseline citrulline levels compared to steady-state (mean ± SD: 9·37 ± 1·43 vs. 22·28 ± 6·8, P = 0·01) (Figure S1). After the IV bolus, mean peak plasma concentrations was 257 μmol/l and citrulline plasma concentrations of approximately 100 μmol/l were achieved during a 7 mg/kg per hour continuous infusion. Furthermore, there was a robust and durable rise in both citrulline and arginine levels (Fig 2). The citrulline PK parameters for Step 2 with the bolus and IV continuous infusion are shown in Table SIII. The endogenous citrulline appearance rate (Rapp) was significantly lower in the VOC cohort compared to steady-state (5·0 μmol/h/kg vs 11·1 μmol/h/kg, P = 0·014). Overall, intravenous citrulline in SCD was well tolerated and safe. There were no AEs ≥ grade 2 level of toxicity. Drowsiness was noted in 6 participants, but not severe enough to discontinue study medication. One patient reported feeling cold and one patient had nausea (no vomiting) associated with the drowsiness. Due to the theoretical risk of vasodilation from the NO boost, vital signs were followed closely (Figure S2). In one subject, the diastolic blood pressure transiently dropped >20% from baseline during the first 30 min of drug administration but normalized within 1 h without any intervention. There were no significant changes in the complete blood count and renal function tests (Table SIV). However, one subject was readmitted about 2 weeks later with right upper quadrant painelevated alanine transaminase and aspartate transaminase, peaking at 367 and 335 u/l respectively, which resolved by day 31. The subject developed fever during hospitalization; tests revealed cytomegalovirus (CMV) IgM positivity and CMV polymerase chain reaction of 6300 copies, suggesting acute CMV infection as an aetiology. This was reported to the US Food and Drug Administration as a serious AE. This is the first report on the use of intravenous citrulline in SCD. While this intervention was well tolerated, drowsiness was a potential side effect of unclear aetiology that could be related to vasodilation of the cerebral vasculature or, perhaps, improvement in pain. Given the potential benefit of boosting endothelial NO and contributing to microcirculatory vasodilation, studies are needed to evaluate the efficacy of intravenous citrulline during a vaso-occlusive crisis. SM designed the study and wrote the manuscript. TRG performed the PK analysis, MH and NS conducted the study. JD analysed the data. MS designed the study. GC performed the amino acid testing. DD, RN and AC critically revised the manuscript. FB designed the study. This work was supported by a University of Mississippi Medical Center (UMMC) intramural grant (#68599340412) (S.M.). We are grateful to the patients and families for agreeing to participate in this phase 1 study. We thank the research nurse, Heather Atterberry, RN, and the study pharmacist Richard Ogletree Jr, Pharm D. We also thank Asklepion pharmaceuticals for supplying the study drug at no cost. U.S. Provisional Patent Application No. 62/463,931 (Intravenous citrulline for treatment of sickle cell crisis). Table SI. Showing the demographic, genotype and baseline complete blood count of participants Table SII. Citrulline pharmacokinetic model parameter estimates for I.V. bolus cohort. Table SIII. Citrulline pharmacokinetic model parameter estimates for intravenous bolus plus continuous infusion cohort. Rapp, rate of citrulline appearance; krem, constant of citrulline removal. Table SIV. Showing the laboratory profile after receiving the bolus and continuous intravenous L-citrulline at baseline and at 24 h (end of infusion). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:The objective of this study was to determine whether the implementation of an inhaled nitric oxide protocol (INO) in a pediatric ICU (PICU) would reduce cost associated with its use without negatively affecting patient outcomes.METHODS:This is a retrospective cohort study of 76 subjects who required INO therapy in the PICU during the study period. A nitric oxide setup and weaning protocol was implemented in the PICU. The medical records of subjects who had received INO 18 months after protocol implementation, as well as the medical records of subjects who had received INO in the 18 months before protocol implementation, were reviewed. Length of time on INO, cost of INO per subject, mortality, stay, and ventilator hours were recorded.RESULTS:There were 38 subjects in the pre-protocol group and 38 subjects in the post-protocol group. There was a statistically significant decrease in the median per subject cost of INO between the pre- and post-protocol groups (P < .01). There was no statistically significant difference in the median duration of INO use (P = .06), median PICU (P = .42) or hospital (P = .58) stay, median duration of mechanical ventilation (P = .79) or percent mortality (P = .28) between the 2 groups.CONCLUSIONS:Implementation of an INO setup and weaning protocol in a PICU reduces the cost associated with its use without a statistically significant difference in mortality. In an era of increased awareness regarding healthcare spending, implementation of evidence-based protocols can provide a way to ensure the judicious utilization of medical resources.

OBJECTIVESFew educational opportunities exist in paediatric cardiac critical care units (PCCUs). We introduced a new educational activity in the PCCU in the form of of patient-specific summaries (TPSS). Our objective was to study the role of TPSS in the provision of a positive learning experience to the multidisciplinary clinical team of PCCUs and in improving patient-related clinical outcomes in the PCCU.METHODSProspective educational intervention with simultaneous clinical assessment was undertaken in PCCU in an academic children's hospital. TPSS was developed utilizing the case presentation format for upcoming week's surgical cases and delivered once every week to each PCCU clinical team member. Role of TPSS to provide clinical education was assessed using five-point Likert-style scale responses in an anonymous survey 1 year after TPSS provision. Paediatric cardiac surgery patients admitted to the PCCU were evaluated for postoperative outcomes for TPSS provision period of 1 year and compared with a preintervention period of 1 year.RESULTSTPSS was delivered to 259 clinical team members including faculty, fellows, residents, nurse practitioners, nurses, respiratory therapists and others from the Divisions of Anesthesia, Cardiology, Cardio-Thoracic Surgery, Critical Care, and Pediatrics working in the PCCU. Two hundred and twenty-four (86%) members responded to the survey and assessed the role of TPSS in providing clinical education to be excellent based on mean Likert-style scores of 4.32 ± 0.71 in survey responses. Seven hundred patients were studied for the two time periods and there were no differences in patient demographics, complexity of cardiac defect and surgical details. The length of mechanical ventilation for the TPSS period (57.08 ± 141.44 h) was significantly less when compared with preintervention period (117.39 ± 433.81 h) (P < 0.001) with no differences in length of PCICU stay, hospital stay and mortality for the two time periods.CONCLUSIONSProvision of TPSS in a paediatric cardiac surgery unit is perceived to be beneficial in providing clinical education to multidisciplinary clinical teams and may be associated with improved clinical outcome.

Aim. Cerebral vasospasm is a leading cause of death and disability following aneurysmal subarachnoid hemorrhage (SAH). Nitric oxide (NO) is a potent mediator of vasodilation, and citrulline is a known contributor to NO production. The leukocytosis inflammatory response can increase vasoconstrictive compounds that may also contribute to vasospasm. Dexamethasone is a glucocorticosteroid commonly administered after SAH, which may alter the production of leukocytes and citrulline. The goal of this project was to study the effects of dexamethasone on leukocytosis, citrulline, and angiographic vasospasm.Methods. Experimental SAH was induced in 18 New Zealand white rabbits. Intravenous dexamethasone was administered to one group (N.=9) at 2 mg/kg/day. A placebo group (N.=9) was given a saline infusion with otherwise identical procedures. CSF citrulline, leukocytes, protein, and glucose, as well as plasma citrulline were measured at baseline and 3 days post-SAH in a blinded fashion. Basilar artery angiography was performed at baseline and repeated 3 days post-SAH.Results. The change in CSF citrulline from day 0 to day 3 was significantly lower in the dexamethasone group compared to controls (P=0.002). The change in CSF white blood cells was also significantly lower (P=0.005). There was no significant change in plasma citrulline levels or angiographic vasospasm.Conclusion. Dexamethasone significantly decreases CSF citrulline and CSF leukocytosis after experimental SAH. It is possible this could lead to a relative vasoconstriction and vasodilation, respectively. These processes could cancel-out opposing effects of dexamethasone on cerebral vasospasm, partially contributing to the recognized, multifactorial, inconsistent effects of glucocorticoids on vasospasm.

Background: Acute lower respiratory tract illness is a major cause of hospitalization, respiratory failure, and death in children worldwide. However, infectious agents are not identified in >50% of these clinical illnesses using traditional methods such as rapid antigen detection assays or viral culture. Objective: To conduct a pilot study using RT-PCR to determine the frequency of respiratory viruses in critically ill children with respiratory compromise. Methods: Critically ill children admitted to the Pediatric Critical Care Unit during 2 winter seasons with respiratory compromise or failure were prospectively enrolled. Respiratory tract specimens (tracheal lavage or nasal wash) were tested for 11 different viruses using realtime RT-PCR, including respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza A (IAV) and B (IBV), parainfluenza viruses (PIV) 1-3, coronaviruses OC43, 229E, and Netherlands, and human rhinovirus (HRV). Results: Thirty-two children were enrolled, 28 (88%) of which required mechanical ventilation. Clinical diagnoses included bronchiolitis, croup, asthma, apnea, and pneumonia. Of these patients, 28/32 (88%) tested positive for one of eleven viruses by RT-PCR. Compared to antigen testing for RSV, IAV, and IBV, RT-PCR detected an additional nine infections for an increased yield of 53%. RT-PCR also detected coronavirus infections in five patients, including three patients with Netherlands coronavirus. Co-infection with more than one virus was detected in 8/32 (25%). None of the 32 patients tested positive for HMPV, PIV 1, or PIV 3. Conclusions: We detected a respiratory virus in 88% of children with respiratory failure. RT-PCR significantly increased the identification of viruses compared to antigen testing. RSV, influenza A, and coronaviruses were the most common viruses detected. Our data suggest that a viral etiology can be identified in the majority of childhood episodes of severe respiratory disease and that RT-PCR is a useful addition to the diagnostic approach.

The introduction of inhaled nitric oxide (iNO) for the treatment of hypoxemic respiratory failure in neonates ushered in a new era in neonatal intensive care. This inhalational therapeutic redefined the medical management of the infant with persistent pulmonary hypertension of the newborn (PPHN). With a selective pulmonary vasodilator in hand, a kinder, gentler approach to ventilation was embraced in many neonatal intensive care units, aggressive hyperventilation and alkaline therapy were abandoned, and the number of infants referred for extracorporeal membrane oxygenation (ECMO) declined. Yet, iNO is not a panacea. A significant number of infants fail to respond or sustain a response to iNO. Furthermore, iNO is being increasingly used off-label for populations in which its safety and efficacy have not been adequately studied. In this chapter on iNO in children, we will review the spectrum of approved and off-label uses of iNO in pediatrics, and address gaps in our knowledge regarding the optimal dosing, weaning, and patient selection.

To determine the incidence and clinical and biomarker predictors of perioperative thrombosis in children with single ventricle physiology undergoing staged palliation.

Objective: Septic shock heterogeneity has important implications for clinical trial implementation and patient management. We previously addressed this heterogeneity by identifying three putative subclasses of children with septic shock based exclusively on a 100-gene expression signature. Here we attempted to prospectively validate the existence of these gene expression-based subclasses in a validation cohort. Design: Prospective observational study involving microarray-based bioinformatics. Setting: Multiple pediatric intensive care units in the United States. Patients: Separate derivation (n = 98) and validation (n = 82) cohorts of children with septic shock. Interventions: None other than standard care. Measurements and Main Results: Gene expression mosaics of the 100 class-defining genes were generated for 82 individual patients in the validation cohort. Using computer-based image analysis, patients were classified into one of three subclasses (“A,” “B,” or “C”) based on color and pattern similarity relative to reference mosaics generated from the original derivation cohort. After subclassification, the clinical database was mined for phenotyping. Subclass A patients had higher illness severity relative to subclasses B and C as measured by maximal organ failure, fewer intensive care unit-free days, and a higher Pediatric Risk of Mortality score. Patients in subclass A were characterized by repression of genes corresponding to adaptive immunity and glucocorticoid receptor signaling. Separate subclass assignments were conducted by 21 individual clinicians using visual inspection. The consensus classification of the clinicians had modest agreement with the computer algorithm. Conclusions: We have validated the existence of subclasses of children with septic shock based on a biologically relevant, 100-gene expression signature. The subclasses have relevant clinical differences.

Objective: To validate a diagnostic instrument for pediatric delirium in critically ill children, both ventilated and nonventilated, that uses standardized, developmentally appropriate measurements.Design and Setting: A prospective observational cohort study investigating the Pediatric Confusion Assessment Method for Intensive Care Unit (pCAM-ICU) patients in the pediatric medical, surgical, and cardiac intensive care unit of a university-based medical center.Patients: A total of 68 pediatric critically ill patients, at least 5 years of age, were enrolled from July 1, 2008, to March 30, 2009.Interventions: None.Measurements: Criterion validity including sensitivity and specificity and interrater reliability were determined using daily delirium assessments with the pCAM-ICU by two critical care clinicians compared with delirium diagnosis by pediatric psychiatrists using Diagnostic and Statistical Manual, 4th Edition, Text Revision criteria.Results: A total of 146 paired assessments were completed among 68 enrolled patients with a mean age of 12.2 yrs. Compared with the reference standard for diagnosing delirium, the pCAM-ICU demonstrated a sensitivity of 83% (95% confidence interval, 66-93%), a specificity of 99% (95% confidence interval, 95-100%), and a high interrater reliability (kappa = 0.96; 95% confidence interval, 0.74-1.0).Conclusions: The pCAM-ICU is a highly valid reliable instrument for the diagnosis of pediatric delirium in critically ill children chronologically and developmentally at least 5 yrs of age. Use of the pCAM-ICU may expedite diagnosis and consultation with neuropsychiatry specialists for treatment of pediatric delirium. In addition, the pCAM-ICU may provide a means for delirium monitoring in future epidemiologic and interventional studies in critically ill children. (Crit Care Med 2011; 39:150-157)

Glutathione plays a crucial role in free radical scavenging, oxidative injury, and cellular homeostasis. Previously, we identified a non-synonymous polymorphism (P462S) in the gene encoding the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in glutathione biosynthesis. This polymorphism is present only in individuals of African descent. Presently, we report that this ethnic-specific polymorphism (462S) encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. In addition, overexpression of the 462P wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the 462S isoform. We also demonstrate that apoptotically stimulated mammalian cells overexpressing the 462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type 462P enzyme. Finally, we genotyped several African and African-descent populations and demonstrate that the 462S polymorphism is in Hardy–Weinberg disequilibrium, with no individuals homozygous for the 462S polymorphism identified. These findings describe a glutathione production pathway polymorphism present in individuals of African descent with significantly decreased in vitro activity.

Acta PaediatricaVolume 99, Issue 7 p. 1109-1109 Case studies in paediatric critical care Frederick (Rick) E. Barr, Frederick (Rick) E. Barr rick.barr@vanderbilt.eduSearch for more papers by this author Frederick (Rick) E. Barr, Frederick (Rick) E. Barr rick.barr@vanderbilt.eduSearch for more papers by this author First published: 01 June 2010 https://doi.org/10.1111/j.1651-2227.2010.01844.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume99, Issue7July 2010Pages 1109-1109 RelatedInformation

Children with congenital heart defects are at risk for perioperative pulmonary hypertension if they require corrective or palliative surgery in the first week of life or if they have defects associated with significant pulmonary overcirculation. In addition, children undergoing cavopulmonary connections for single ventricle lesions require low pulmonary vascular resistance for surgical success. Treatment of perioperative pulmonary hypertension with inhaled nitric oxide has become standard therapy in many centers. Related drugs that increase nitric oxide synthesis, including arginine and citrulline, have also been studied in the perioperative period. In this article, previous clinical trials of inhaled nitric oxide, intravenous arginine, and intravenous and oral citrulline in children with perioperative pulmonary hypertension or elevated pulmonary vascular resistance after a cavopulmonary connection are reviewed. In addition, recommendations are presented for each agent on the clinical use in the perioperative setting including clinical indications, assessment of clinical effect, and length of therapy.

Much of current knowledge and momentum in the field of pulmonary hypertension (PH) has not penetrated the increasingly heterogeneous pediatric population managed by pediatric subspecialists worldwide. It is critical to prospectively test specific agents in children. Yet, the broad spectrum of pediatric pulmonary vascular diseases and the lack of clinically meaningful end points appropriate for the pediatric population pose challenges for the conduct of sufficiently powered pediatric studies. It is therefore no surprise that in the midst of significant advances in the field, there are no agents approved for use in children afflicted with PH with the exception of inhaled nitric oxide (iNO) for infants with persistent PH of the newborn (PPHN). The current and widely accepted WHO framework by which pediatric subspecialists are guided is not easily translated into the expanding range of situations encountered in pediatric practice. Like PPHN, it may be necessary to approach these pediatric pulmonary hypertensive conditions from outside the frameworks proposed for typical adult-onset diseases. Exploration of the potential intrinsic differences between children and adults may yield important discoveries regarding the genesis of disease in children and adults alike Fortunately, most children with PH are seen within specialized pediatric centers where there are untapped opportunities for multicenter collaboration. Our aim in this chapter is to approach pediatric PH over the age and development continuum from the vantage point of the pediatric subspecialists who encounter these patients in practice. We will review the abnormalities of pulmonary vascular tone and structure that impact the health and survival of children currently cared for in pediatric PH centers. Many of these disease processes occur commonly from our vantage point, yet are underrepresented in the broader field of PH research or current classification schemes. We will also address the challenges that pediatric subspecialists face in defining PH and assessing its severity in infants or children across the developmental and disease spectrums.