Ammonia transporter RhBG initiates downstream signaling and functional responses by activating NFκB.

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG-MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG-MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors.

The functional impact of 1,570 individual amino acid substitutions in human OTC.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Individuals with neonatal onset appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma, and death, outcomes ameliorated by rapid diagnosis and treatment. Here, we develop a high-throughput functional assay for human OTC and individually measure the impact of 1,570 variants, 84% of all SNV-accessible missense mutations. Comparison to existing clinical significance calls, demonstrated that our assay distinguishes known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. This functional stratification allowed us to identify score ranges corresponding to clinically relevant levels of impairment of OTC activity. Examining the results of our assay in the context of protein structure further allowed us to identify a 13 amino acid domain, the SMG loop, whose function appears to be required in human cells but not in yeast. Finally, inclusion of our data as PS3 evidence under the current ACMG guidelines, in a pilot reclassification of 34 variants with complete loss of activity, would change the classification of 22 from variants of unknown significance to clinically actionable likely pathogenic variants. These results illustrate how large-scale functional assays are especially powerful when applied to rare genetic diseases.

Nicotinamide riboside rescues dysregulated glycolysis and fatty acid ß-oxidation in a human hepatic cell model of citrin deficiency.

Citrin deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate-aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic ratio of reduced versus oxidized form of nicotinamide adenine dinucleotide (NAD) and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.

NHH promotes Sepsis-associated Encephalopathy with the expression of AQP4 in astrocytes through the gut-brain Axis.

Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE.

ASS1 deficiency is associated with impaired neuronal differentiation in zebrafish larvae.

Citrullinemia type 1 (CTLN1) is a rare autosomal recessive urea cycle disorder caused by deficiency of the cytosolic enzyme argininosuccinate synthetase 1 (ASS1) due to pathogenic variants in the ASS1 gene located on chromosome 9q34.11. Even though hyperammenomia is considered the major pathomechanistic factor for neurological impairment and cognitive dysfunction, a relevant subset of individuals presents with a neurodegenerative course in the absence of hyperammonemic decompensations. Here we show, that ASS1 deficiency induced by antisense-mediated knockdown of the zebrafish ASS1 homologue is associated with defective neuronal differentiation ultimately causing neuronal cell loss and consecutively decreased brain size in zebrafish larvae in vivo. Whereas ASS1-deficient zebrafish larvae are characterized by markedly elevated concentrations of citrulline - the biochemical hallmark of CTLN1, accumulation of L-citrulline, hyperammonemia or therewith associated secondary metabolic alterations did not account for the observed phenotype. Intriguingly, coinjection of the human ASS1 mRNA not only normalized citrulline concentration but also reversed the morphological cerebral phenotype and restored brain size, confirming conserved functional properties of ASS1 across species. The results of the present study imply a novel, potentially non-enzymatic (moonlighting) function of the ASS1 protein in neurodevelopment.

Urea cycle disorders in critically Ill adults.

PURPOSE OF REVIEW: Urea cycle disorders (UCDs) cause elevations in ammonia which, when severe, cause irreversible neurologic injury. Most patients with UCDs are diagnosed as neonates, though mild UCDs can present later - even into adulthood - during windows of high physiologic stress, like critical illness. It is crucial for clinicians to understand when to screen for UCDs and appreciate how to manage these disorders in order to prevent devastating neurologic injury or death. RECENT FINDINGS: Hyperammonemia, particularly if severe, causes time- and concentration-dependent neurologic injury. Mild UCDs presenting in adulthood are increasingly recognized, so broader screening in adults is recommended. For patients with UCDs, a comprehensive, multitiered approach to management is needed to prevent progression and irreversible injury. Earlier exogenous clearance is increasingly recognized as an important complement to other therapies. SUMMARY: UCDs alter the core pathway for ammonia metabolism. Screening for mild UCDs in adults with unexplained neurologic symptoms can direct care and prevent deterioration. Management of UCDs emphasizes decreasing ongoing ammonia production, avoiding catabolism, and supporting endogenous and exogenous ammonia clearance. Core neuroprotective and supportive critical care supplements this focused therapy.

Valproate-induced hyperammonemia, neuroleptic sensitivity, and cerebellar atrophy-A clinical conundrum in the management of bipolar disorder.

OBJECTIVE: Treatment of bipolar disorder (BD) involves complexities especially when patients come with significant sensitivity to various psychotropic medications and comorbidities. The following cases aim to recapitulate and discuss some of such situations. CASES: Case 1: A 36-year-old man with intellectual development disorder and BD experienced catatonia, seizures, and hyperammonemia following valproate administration. Treatment involved electroconvulsive therapy (ECT) and a tailored medication regimen, ultimately leading to stability. Case 2: A 63-year-old man with long-standing BD exhibited resistance to lithium and valproate of late, having co-existing essential tremors and cerebellar atrophy. Multiple medication trials led to side effects, requiring ECT for symptom improvement, followed by a carefully adjusted maintenance regimen. CONCLUSION: Medication side effects can pose major challenges in treatment of BD. Comprehensive evaluation and monitoring are essential. ECT can prove valuable in such cases. There is pressing need to develop more safer treatment alternatives, especially considering the progressively ageing society.

Impact of citrulline substitution on clinical outcome after liver transplantation in carbamoyl phosphate synthetase 1 and ornithine transcarbamylase deficiency.

Carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC) deficiencies are rare urea cycle disorders, which can lead to life-threatening hyperammonemia. Liver transplantation (LT) provides a cure and offers an alternative to medical treatment and life-long dietary restrictions with permanent impending risk of hyperammonemia. Nevertheless, in most patients, metabolic aberrations persist after LT, especially low plasma citrulline levels, with questionable clinical impact. So far, little is known about these alterations and there is no consensus, whether l-citrulline substitution after LT improves patients' symptoms and outcomes. In this multicentre, retrospective, observational study of 24 patients who underwent LT for CPS1 (n = 11) or OTC (n = 13) deficiency, 25% did not receive l-citrulline or arginine substitution. Correlation analysis revealed no correlation between substitution dosage and citrulline levels (CPS1, p = 0.8 and OTC, p = 1). Arginine levels after liver transplantation were normal after LT independent of citrulline substitution. Native liver survival had no impact on mental impairment (p = 0.67). Regression analysis showed no correlation between l-citrulline substitution and failure to thrive (p = 0.611) or neurological outcome (p = 0.701). Peak ammonia had a significant effect on mental impairment (p = 0.017). Peak plasma ammonia levels correlate with mental impairment after LT in CPS1 and OTC deficiency. Growth and intellectual impairment after LT are not significantly associated with l-citrulline substitution.

A novel pathogenic variant in the carnitine transporter gene, SLC22A5, in association with metabolic carnitine deficiency and cardiomyopathy features.

BACKGROUND: Primary carnitine deficiency (PCD) denotes low carnitine levels with an autosomal recessive pattern of inheritance. Cardiomyopathy is the most common cardiac symptom in patients with PCD, and early diagnosis can prevent complications. Next-generation sequencing can identify genetic variants attributable to PCD efficiently. OBJECTIVE: We aimed to detect the genetic cause of the early manifestations of hypertrophic cardiomyopathy and metabolic abnormalities in an Iranian family. METHODS: We herein describe an 8-year-old boy with symptoms of weakness and lethargy diagnosed with PCD through clinical evaluations, lab tests, echocardiography, and cardiac magnetic resonance imaging. The candidate variant was confirmed through whole-exome sequencing, polymerase chain reaction, and direct Sanger sequencing. The binding efficacy of normal and mutant protein-ligand complexes were evaluated via structural modeling and docking studies. RESULTS: Clinical evaluations, echocardiography, and cardiac magnetic resonance imaging findings revealed hypertrophic cardiomyopathy as a clinical presentation of PCD. Whole-exome sequencing identified a new homozygous variant, SLC22A5 (NM_003060.4), c.821G > A: p.Trp274Ter, associated with carnitine transport. Docking analysis highlighted the impact of the variant on carnitine transport, further indicating its potential role in PCD development. CONCLUSIONS: The c.821G > A: p.Trp274Ter variant in SLC22A5 potentially acted as a pathogenic factor by reducing the binding affinity of organic carnitine transporter type 2 proteins for carnitine. So, the c.821G > A variant may be associated with carnitine deficiency, metabolic abnormalities, and cardiomyopathic characteristics.

Extracellular vesicles from hyperammonemic rats induce neuroinflammation in hippocampus and impair cognition in control rats.

Patients with liver cirrhosis show hyperammonemia and peripheral inflammation and may show hepatic encephalopathy with cognitive impairment, reproduced by rats with chronic hyperammonemia. Peripheral inflammation induces neuroinflammation in hippocampus of hyperammonemic rats, altering neurotransmission and leading to cognitive impairment. Extracellular vesicles (EVs) may transmit pathological effects from the periphery to the brain. We hypothesized that EVs from peripheral blood would contribute to cognitive alterations in hyperammonemic rats. The aims were to assess whether EVs from plasma of hyperammonemic rats (HA-EVs) induce cognitive impairment and to identify the underlying mechanisms. Injection of HA-EVs impaired learning and memory, induced microglia and astrocytes activation and increased TNFα and IL-1ß. Ex vivo incubation of hippocampal slices from control rats with HA-EVs reproduced these alterations. HA-EVs increased membrane expression of TNFR1, reduced membrane expression of TGFßR2 and Smad7 and IκBα levels and increased IκBα phosphorylation. This led to increased activation of NF-κB and IL-1ß production, altering membrane expression of NR2B, GluA1 and GluA2 subunits, which would be responsible for cognitive impairment. All these effects of HA-EVs were prevented by blocking TNFα, indicating that they were mediated by enhanced activation of TNFR1 by TNFα. We show that these mechanisms are very different from those leading to motor incoordination, which is due to altered GABAergic neurotransmission in cerebellum. This demonstrates that peripheral EVs play a key role in the transmission of peripheral alterations to the brain in hyperammonemia and hepatic encephalopathy, inducing neuroinflammation and altering neurotransmission in hippocampus, which in turn is responsible for the cognitive deficits.

Increased levels and activation of the IL-17 receptor in microglia contribute to enhanced neuroinflammation in cerebellum of hyperammonemic rats.

BACKGROUND: Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with mild cognitive impairment and motor incoordination. Rats with chronic hyperammonemia reproduce these alterations. Motor incoordination in hyperammonemic rats is due to increased GABAergic neurotransmission in cerebellum, induced by neuroinflammation, which enhances TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway activation. The initial events by which hyperammonemia triggers activation of this pathway remain unclear. MHE in cirrhotic patients is triggered by a shift in inflammation with increased IL-17. The aims of this work were: (1) assess if hyperammonemia increases IL-17 content and membrane expression of its receptor in cerebellum of hyperammonemic rats; (2) identify the cell types in which IL-17 receptor is expressed and IL-17 increases in hyperammonemia; (3) assess if blocking IL-17 signaling with anti-IL-17 ex-vivo reverses activation of glia and of the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway. RESULTS: IL-17 levels and membrane expression of the IL-17 receptor are increased in cerebellum of rats with hyperammonemia and MHE, leading to increased activation of IL-17 receptor in microglia, which triggers activation of STAT3 and NF-kB, increasing IL-17 and TNFα levels, respectively. TNFα released from microglia activates TNFR1 in Purkinje neurons, leading to activation of NF-kB and increased IL-17 and TNFα also in these cells. Enhanced TNFR1 activation also enhances activation of the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway which mediates microglia and astrocytes activation. CONCLUSIONS: All these steps are triggered by enhanced activation of IL-17 receptor in microglia and are prevented by ex-vivo treatment with anti-IL-17. IL-17 and IL-17 receptor in microglia would be therapeutic targets to treat neurological impairment in patients with MHE.

Hyperornithinemia-hyperammonemia-homocitrullinuria: a rare neurometabolic disorder in two siblings.

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome is an extremely rare disorder of urea cycle, with few patients reported worldwide. Despite hyperammonemia control, the long-term outcome remains poor with progressive neurological deterioration. We report the clinical, biochemical, and molecular features of two Lebanese siblings diagnosed with this disorder and followed for 8 and 15 years, respectively. Variable clinical manifestations and neurological outcome were observed. The patient with earlier onset of symptoms had a severe neurological deterioration while the other developed a milder form of the disease at an older age. Diagnosis was challenging in the absence of the complete biochemical triad and the non-specific clinical presentations. Whole exome sequencing revealed a homozygous variant, p.Phe188del, in the SLC25A15 gene, a French- Canadian founder mutation previously unreported in Arab patients. Hyperammonemia was controlled in both patients but hyperonithinemia persisted. Frequent hyperalaninemia spikes and lactic acidosis occured concomitantly with the onset of seizures in one of the siblings. Variable neurological deterioration and outcome were observed within the same family. This is the first report from the Arab population of the long-term outcome of this devastating neurometabolic disorder.

Hyperammonemic encephalopathy after tyrosine kinase inhibitors: A literature review and a case example.

OBJECTIVE: To review the evidence of uncommon but fatal adverse event of hyperammonemic encephalopathy by tyrosine kinase inhibitors (TKI) and the possible mechanisms underlying this condition and to describe the case of a patient that developed drug-induced hyperammonemic encephalopathy related to TKI. DATA SOURCES: Literature search of different databases was performed for studies published from 1 January 1992 to 7 May 2023. The search terms utilized were hyperammonemic encephalopathy, TKI, apatinib, pazopanib, sunitinib, imatinib, sorafenib, regorafenib, trametinib, urea cycle regulation, sorafenib, carbamoyl-phosphate synthetase 1, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase 1, Mitogen activated protein kinases (MAPK) pathway and mTOR pathway, were used individually search or combined. DATA SUMMARY: Thirty-seven articles were included. The articles primarily focused in hyperammonemic encephalopathy case reports, management of hyperammonemic encephalopathy, urea cycle regulation, autophagy, mTOR and MAPK pathways, and TKI. CONCLUSION: Eighteen cases of hyperammonemic encephalopathy were reported in the literature from various multitargeted TKI. The mechanism of this event is not well-understood but some authors have hypothesized vascular causes since some of TKI are antiangiogenic, however our literature review shows a possible relationship between the urea cycle and the molecular inhibition exerted by TKI. More preclinical evidence is required to unveil the biochemical mechanisms responsible involved in this process and clinical studies are necessary to shed light on the prevalence, risk factors, management and prevention of this adverse event. It is important to monitor neurological symptoms and to measure ammonia levels when manifestations are detected.

Hyperammonemia in the Pediatric Emergency Department.

ABSTRACT: Hyperammonemia is a serious clinical condition associated with significant morbidity and mortality. In the pediatric population, this is often caused by urea cycle disorders, acute liver failure, or other less common underlying etiologies. Children and teens with hyperammonemia can have a broad range of clinical findings, including vomiting, respiratory distress, and changes in mental status. As ammonia levels worsen, this presentation can progress to respiratory failure, encephalopathy, cerebral edema, seizures, and death. Given the risk of neurologic damage, timely identification and management of hyperammonemia is critical and includes initial resuscitation, early consultation with subspecialists, and initiation of appropriate therapies. It is important for pediatric emergency medicine providers to understand the clinical findings, causes, diagnosis, and management of hyperammonemia because they play a key role in the provision of effective, multidisciplinary care of these patients.

[A Case of Consciousness Disorder Due to Hyperammonemia Induced by Modified FOLFOX6 for Multiple Liver Metastasis from Rectal Cancer].

A 74-year-old man underwent laparoscopic-assisted high anterior resection with D3 lymph node dissection for rectal cancer, which was simultaneously accompanied by multiple liver metastases. The patient received mFOLFOX6 therapy for liver metastases 1 month after the surgery. Anorexia, nausea, and vomiting appeared on the second day of treatment. On the third day of treatment, impaired consciousness(JCS Ⅱ-20)and flapping tremors appeared. Blood tests revealed hyperammonemia, and the patient was diagnosed with impaired consciousness due to hyperammonemia, which was inferred to be caused by 5-fluorouracil(5-FU). Intravenous infusion and branched-chain amino acids were administered, and the patient recovered. The underlying disease of renal dysfunction, constipation, and dehydration due to chemotherapy might have induced the hyperammonemia. It is important to note that hyperammonemia can lead to a disturbance of consciousness during chemotherapy including 5-FU.

Mitochondrial carbonic anhydrase VA and VB: properties and roles in health and disease.

Carbonic anhydrase V (CA V), a mitochondrial enzyme, was first isolated from guinea-pig liver and subsequently identified in mice and humans. Later, studies revealed that the mouse genome contains two mitochondrial CA sequences, named Car5A and Car5B. The CA VA enzyme is most highly expressed in the liver, whereas CA VB shows a broad tissue distribution. Car5A knockout mice demonstrated a predominant role for CA VA in ammonia detoxification, whereas the roles of CA VB in ureagenesis and gluconeogenesis were evident only in the absence of CA VA. Previous studies have suggested that CA VA is mainly involved in the provision of HCO3 - for biosynthetic processes. In children, mutations in the CA5A gene led to reduced CA activity, and the enzyme was sensitive to increased temperature. The metabolic profiles of these children showed a reduced supply of HCO3 - to the enzymes that take part in intermediary metabolism: carbamoylphosphate synthetase, pyruvate carboxylase, propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. Although the role of CA VB is still poorly understood, a recent study reported that it plays an essential role in human Sertoli cells, which sustain spermatogenesis. Metabolic disease associated with CA VA appears to be more common than other inborn errors of metabolism and responds well to treatment with N-carbamyl-l-glutamate. Therefore, early identification of hyperammonaemia will allow specific treatment with N-carbamyl-l-glutamate and prevent neurological sequelae. Carbonic anhydrase VA deficiency should therefore be considered a treatable condition in the differential diagnosis of hyperammonaemia in neonates and young children.

Ammonia induces amyloidogenesis in astrocytes by promoting amyloid precursor protein translocation into the endoplasmic reticulum.

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer's disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aß) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes ß-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aß. Finally, we show the ammonia-induced production of Aß in astrocytic endoplasmic reticulum was specific to Aß42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aß42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.

Clearance and production of ammonia quantified in humans by constant ammonia infusion - the effects of cirrhosis and ammonia-targeting treatments.

BACKGROUND & AIMS: Hyperammonaemia is a key pathological feature of liver disease and the primary driver of hepatic encephalopathy (HE). However, the relative roles of increased ammonia production and reduced clearance are poorly understood as is the action of ammonia-targeting drugs for HE. We aimed to quantify whole-body ammonia metabolism in healthy persons and patients with cirrhosis and to validate our method by examining the effects of glycerol phenylbutyrate and lactulose + rifaximin treatment. METHODS: Ten healthy men and ten male patients with cirrhosis were investigated by 90-minute constant ammonia infusion to achieve steady-state plasma ammonia. Whole-body ammonia clearance was calculated as infusion rate divided by steady-state concentration increase and ammonia production was calculated as clearance multiplied by baseline ammonia concentration. Participants were re-investigated after the ammonia-targeting interventions. RESULTS: In healthy persons, ammonia clearance was 3.5 (3.1-3.9) L/min and ammonia production was 49 (35-63) µmol/min. Phenylbutyrate increased clearance by 11% (4-19%, p = 0.009). In patients with cirrhosis, ammonia clearance was 20% lower at 2.7 (2.1-3.3) L/min (p = 0.02) and production was nearly threefold higher at 131 (102-159) µmol/min (p <0.0001). Lactulose + rifaximin reduced production by 20% (2-37%, p = 0.03). The infusion was generally well-tolerated apart from in one hyperammonaemic patient, with cirrhosis and possible bleeding unrelated to the infusion, who developed clinical HE that reverted when infusion was discontinued. CONCLUSIONS: Whole-body ammonia clearance and production may be measured separately using the described technique. This technique identified a lower clearance and a higher production of ammonia in patients with cirrhosis, and showed that phenylbutyrate increases clearance, whereas lactulose + rifaximin reduces production. IMPACT AND IMPLICATIONS: High blood ammonia plays a key role in cirrhosis-related brain dysfunction. However, the relative roles of reduced ammonia clearance and increased ammonia production are poorly understood as is the action of ammonia-targeting treatments. This study presents a relatively simple test to measure ammonia metabolism. By using this test, it was possible to show that patients with cirrhosis exhibit decreased ammonia clearance and increased ammonia production compared to healthy persons, and to quantify the unique effects of different ammonia-targeting treatments. The test described herein may be used to examine a range of questions related to normal physiology, pathophysiology and the mechanisms of action of ammonia-targeting treatments. CLINICAL TRIAL NUMBER: ClinicalTrials.gov (1-16-02-297-20).

Extracellular vesicles from mesenchymal stem cells reduce neuroinflammation in hippocampus and restore cognitive function in hyperammonemic rats.

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFß levels and membrane expression of TGFß receptors in hippocampus. This leads to microglia activation and reduced Smad7-IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1ß which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFß in the EVs from MSCs, which activates TGFß receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7-IkB pathway. This normalizes IL-1ß, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE.

Taste-masked formulation of sodium phenylbutyrate (ACER-001) for the treatment of urea cycle disorders.

Urea cycle disorders (UCDs) are a group of rare inherited metabolic diseases caused by a deficiency of one of the enzymes or transporters that constitute the urea cycle. Defects in these enzymes lead to acute accumulation (hyperammonemic crises, HAC) or chronically elevated levels (hyperammonemia) of ammonia in the blood and/or various tissues including the brain, which can cause persistent neurological deficits, irreversible brain damage, coma, and death. Ongoing treatment of UCDs include the use of nitrogen-scavenging agents, such as sodium phenylbutyrate (salt of 4-phenylbutyric acid; NaPBA) or glycerol phenylbutyrate (GPB). These treatments provide an alternative pathway for nitrogen disposal through the urinary excretion of phenylacetylglutamine. ACER-001 is a novel formulation of NaPBA with polymer coated pellets in suspension, which is designed to briefly mask the unpleasant bitter taste of NaPBA and is being developed as a treatment option for patients with UCDs. Four Phase 1 studies were conducted to characterize the bioavailability (BA) and/or bioequivalence (BE) of ACER-001 (in healthy volunteers) and taste assessment relative to NaPBA powder (in taste panelists). ACER-001 was shown to be bioequivalent to NaPBA powder under both fed and fasting conditions. Lower systemic exposure of phenylacetate (PAA) and phenylbutyrate (PBA) was observed when ACER-001 was administered with a high-fat meal relative to a fasting state suggesting that the lower doses of PBA administered under fasting conditions may yield similar efficacy with potentially fewer dose dependent adverse effects relative to higher doses with a meal. ACER-001 appeared to be adequately taste-masked, staying below the aversive taste threshold for the first 3 min after the formulation was prepared and remaining palatable when taken within 5 min.