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.

Analysis of daily energy, protein, fat, and carbohydrate intake in citrin-deficient patients: Towards prevention of adult-onset type II citrullinemia.

Patients with citrin deficiency during the adaptation/compensation period exhibit diverse clinical features and have characteristic diet of high protein, high fat, and low carbohydrate. Japanese cuisine typically contains high carbohydrate but evaluation of diet of citrin-deficient patients in 2008 showed a low energy intake and a protein:fat:carbohydrate (PFC) ratio of 19:44:37, which indicates low carbohydrate consumption rate. These findings prompted the need for diet intervention to prevent the adult onset of type II citrullinemia (CTLN2). Since the publication of the report about 10 years ago, patients are generally advised to eat what they wish under active dietary consultation and intervention. In this study, citrin-deficient patients and control subjects living in the same household provided answers to a questionnaire, filled-up a maximum 6-day food diary, and supplied physical data and information on medications if any. To study the effects of the current diet, the survey collected data from 62 patients and 45 controls comparing daily intakes of energy, protein, fat, and carbohydrate. Food analysis showed that patient's energy intake was 115% compared to the Japanese standard. The confidence interval of the PFC ratio of patients was 20-22:47-51:28-32, indicating higher protein, higher fat and lower carbohydrate relative to previous reports. The mean PFC ratio of female patients (22:53:25) was significantly different from that of male patients (20:46:34), which may explain the lower frequency of CTLN2 in females. Comparison of the present data to those published 10 years ago, energy, protein, and fat intakes were significantly higher but the amount of carbohydrate consumption remained the same. Regardless of age, most patients (except for adolescents) consumed 100-200 g/day of carbohydrates, which met the estimated average requirement of 100 g/day for healthy individuals. Finally, patients were generally not overweight and some CTLN2 patients were underweight although their energy intake was higher compared with the control subjects. We speculate that high-energy of a low carbohydrate diet under dietary intervention may help citrin-deficient patients attain normal growth and prevent the onset of CTLN2.

Metabolic basis and treatment of citrin deficiency.

Citrin deficiency is a hereditary disorder caused by SLC25A13 mutations and manifests as neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD), and adult-onset type II citrullinemia (CTLN2). Citrin is a component of the malate-aspartate nicotinamide adenine dinucleotide hydrogen (NADH) shuttle, an essential shuttle for hepatic glycolysis. Hepatic glycolysis and the coupled lipogenesis are impaired in citrin deficiency. Hepatic lipogenesis plays a significant role in fat supply during growth spurt periods: the fetal period, infancy, and puberty. Growth impairment in these periods is characteristic of citrin deficiency. Hepatocytes with citrin deficiency cannot use glucose and fatty acids as energy sources due to defects in the NADH shuttle and downregulation of peroxisome proliferator-activated receptor α (PPARα), respectively. An energy deficit in hepatocytes is considered a fundamental pathogenesis of citrin deficiency. Medium-chain triglyceride (MCT) supplementation with a lactose-restricted formula and MCT supplementation under a low-carbohydrate diet are recommended for NICCD and CTLN2, respectively. MCT supplementation therapy can provide energy to hepatocytes, promote lipogenesis, correct the cytosolic NAD+ /NADH ratio via the malate-citrate shuttle and improve ammonia detoxification, and it is a reasonable therapy for citrin deficiency. It is very important to administer MCT at a dose equivalent to the liver's energy requirements in divided doses with meals. MCT supplementation therapy is certainly promising for promoting growth spurts during infancy and adolescence and for preventing CTLN2 onset. Intravenous administration of solutions containing fructose is contraindicated, and persistent hyperglycemia should be avoided due to glucose intoxication for patients receiving hyperalimentation or with complicating diabetes.

Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis.

Cancer cells hijack and remodel existing metabolic pathways for their benefit. Argininosuccinate synthase (ASS1) is a urea cycle enzyme that is essential in the conversion of nitrogen from ammonia and aspartate to urea. A decrease in nitrogen flux through ASS1 in the liver causes the urea cycle disorder citrullinaemia. In contrast to the well-studied consequences of loss of ASS1 activity on ureagenesis, the purpose of its somatic silencing in multiple cancers is largely unknown. Here we show that decreased activity of ASS1 in cancers supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Our studies were initiated by delineating the consequences of loss of ASS1 activity in humans with two types of citrullinaemia. We find that in citrullinaemia type I (CTLN I), which is caused by deficiency of ASS1, there is increased pyrimidine synthesis and proliferation compared with citrullinaemia type II (CTLN II), in which there is decreased substrate availability for ASS1 caused by deficiency of the aspartate transporter citrin. Building on these results, we demonstrate that ASS1 deficiency in cancer increases cytosolic aspartate levels, which increases CAD activation by upregulating its substrate availability and by increasing its phosphorylation by S6K1 through the mammalian target of rapamycin (mTOR) pathway. Decreasing CAD activity by blocking citrin, the mTOR signalling, or pyrimidine synthesis decreases proliferation and thus may serve as a therapeutic strategy in multiple cancers where ASS1 is downregulated. Our results demonstrate that ASS1 downregulation is a novel mechanism supporting cancerous proliferation, and they provide a metabolic link between the urea cycle enzymes and pyrimidine synthesis.

Protective effect of resveratrol on citrullinemia type I-induced brain oxidative damage in male rats.

Citrullinemia Type I is an inborn error, which leads to accumulation of citrulline and ammonia in blood and body tissues. We evaluated the in vitro effects of citrulline, ammonia and the influence of resveratrol on oxidative stress parameters in the cerebrum of 30- and 60-day-old male Wistar rats. Citrulline (0.1, 2.5, 5.0 mM), ammonia (0.01, 0.1, 1.0 mM) and resveratrol (0.01, 0.1, 0.5 mM) were added to the assays to measure thiobarbituric acid reactive substances (TBA-RS), total sulfhydryl content and the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Citrulline (2.5 and 5.0 mM) increased TBA-RS in the cerebellum of 30-day-old and in the cerebral cortex and cerebellum of 60-day-old. Citrulline (5.0 mM) increased SOD and reduced GSH-Px in the hippocampus of 30-day-old, whereas in the cerebellum it increased GSH-Px. In the cerebral cortex, 2.5 and 5.0 mM citrulline reduced GSH-Px. In 60-day-old, 2.5 and 5.0 mM citrulline increased SOD in the cerebellum, increased GSH-Px in the cerebral cortex and 5.0 mM citrulline reduced CAT and increased SOD in the cerebral cortex. Ammonia (0.1 and 1.0 mM) reduced the sulfhydryl content in the cerebral cortex of 30- and 60-day-old, 1.0 mM ammonia increased SOD and reduced GSH-Px in the cerebellum of 30-day-old and increased SOD in the hippocampus and cerebellum of 60-day-old. Resveratrol was able to prevent the majority of these alterations. Thus, citrulline and ammonia induce oxidative stress in the cerebrum of rats; however, resveratrol was able to exert antioxidant effects against these substances.

Severity-adjusted evaluation of newborn screening on the metabolic disease course in individuals with cytosolic urea cycle disorders.

OBJECTIVE: The implementation of newborn screening (NBS) programs for citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) is subject to controversial debate. The aim of this study was to assess the impact of NBS on the metabolic disease course and clinical outcome of affected individuals. METHODS: In 115 individuals with CTLN1 and ASA, we compared the severity of the initial hyperammonemic episode (HAE) and the frequency of (subsequent) HAEs with the mode of diagnosis. Based on a recently established functional disease prediction model, individuals were stratified according to their predicted severe or attenuated phenotype. RESULTS: Individuals with predicted attenuated forms of CTLN1 and ASA were overrepresented in the NBS group, while those with a predicted severe phenotype were underrepresented compared to individuals identified after the manifestation of symptoms (SX). Identification by NBS was associated with reduced severity of the initial HAE both in individuals with predicted severe and attenuated phenotypes, while it was not associated with lower frequency of (subsequent) HAEs. Similar results were obtained when including some patients diagnosed presymptomatically (i.e. prenatal testing, and high-risk family screening) in this analysis. CONCLUSION: Since one of the major challenges of NBS outcome studies is the potential overrepresentation of individuals with predicted attenuated phenotypes in NBS cohorts, severity-adjusted evaluation of screened and unscreened individuals is important to avoid overestimation of the NBS effect. NBS enables the attenuation of the initial HAE but does not affect the frequency of subsequent metabolic decompensations in individuals with CTLN1 and ASA. Future long-term studies will need to evaluate the clinical impact of this finding, especially with regard to mortality, as well as cognitive outcome and quality of life of survivors.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Circulating tricarboxylic acid cycle metabolite levels in citrin-deficient children with metabolic adaptation, with and without sodium pyruvate treatment.

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid ß-oxidation cycle between 22 citrin-deficient children (age, 3-13years) with normal liver functions and 37 healthy controls (age, 5-13years). TCA cycle analysis showed that basal plasma citrate and α-ketoglutarate levels were significantly higher in the affected than the control group (p<0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p<0.001). The plasma level of 3-OH-butyrate derived from fatty acid ß-oxidation was significantly higher in the affected group (p<0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p<0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development.

Biochemical and molecular characteristics of citrin deficiency in Korean children.

Mutations in SLC25A13 cause citrin deficiency, which has three phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) and adult-onset type 2 citrullinemia (CTLN2). The purpose of this study was to determine the mutation spectrum and the clinical and biochemical characteristics of citrin deficiency in Korean patients. Thirty-four patients were diagnosed with citrin deficiency based on mutations in SLC25A13, as verified by direct sequencing and long PCR screening of a large transposon insertion. A total of 66 alleles from 33 unrelated families of 34 patients with citrin deficiency (27 NICCD, 2 FTTDCD and 5 CTLN2) were retrospectively identified. The common pathogenic alleles were IVS16ins3kb (33%), c.851_854del (30%) and c.1177+1G>A (12%), and three novel variants were identified. Levels of citrulline, threonine, methionine, tyrosine and arginine and the threonine-to-serine ratio were higher in children with neonatal intrahepatic cholestasis caused by NICCD compared with that in patients with idiopathic neonatal hepatitis (INH). We concluded that Korean patients with citrin deficiency showed the highest frequency of the IVS16ins3kb mutation and that plasma amino-acid profiles can be used to differentiate between NICCD and INH.

Steatogenesis in adult-onset type II citrullinemia is associated with down-regulation of PPARα.

SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial ß-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα.

Age at disease onset and peak ammonium level rather than interventional variables predict the neurological outcome in urea cycle disorders.

BACKGROUND: Patients with urea cycle disorders (UCDs) have an increased risk of neurological disease manifestation. AIMS: Determining the effect of diagnostic and therapeutic interventions on the neurological outcome. METHODS: Evaluation of baseline, regular follow-up and emergency visits of 456 UCD patients prospectively followed between 2011 and 2015 by the E-IMD patient registry. RESULTS: About two-thirds of UCD patients remained asymptomatic until age 12 days [i.e. the median age at diagnosis of patients identified by newborn screening (NBS)] suggesting a potential benefit of NBS. In fact, NBS lowered the age at diagnosis in patients with late onset of symptoms (>28 days), and a trend towards improved long-term neurological outcome was found for patients with argininosuccinate synthetase and lyase deficiency as well as argininemia identified by NBS. Three to 17 different drug combinations were used for maintenance therapy, but superiority of any single drug or specific drug combination above other combinations was not demonstrated. Importantly, non-interventional variables of disease severity, such as age at disease onset and peak ammonium level of the initial hyperammonemic crisis (cut-off level: 500 µmol/L) best predicted the neurological outcome. CONCLUSIONS: Promising results of NBS for late onset UCD patients are reported and should be re-evaluated in a larger and more advanced age group. However, non-interventional variables affect the neurological outcome of UCD patients. Available evidence-based guideline recommendations are currently heterogeneously implemented into practice, leading to a high variability of drug combinations that hamper our understanding of optimised long-term and emergency treatment.

Fatigue and quality of life in citrin deficiency during adaptation and compensation stage.

Citrin-deficient children and adolescents between adult-onset type II citrullinemia and neonatal intrahepatic cholestasis by citrin deficiency do not have clear clinical features except for unusual diet of high-fat, high-protein, and low-carbohydrate food. The aims of the present study are to characterize fatigue and quality of life (QOL) in citrin-deficient patients during adaptation and compensation stage, and to define the relationship between fatigue and QOL. The study subjects were 55 citrin-deficient patients aged 1-22years (29 males) and 54 guardians. Fatigue was evaluated by self-reports and proxy-reports of the PedsQL Multidimensional Fatigue Scale. QOL was evaluated by the PedsQL Generic Core Scales. Both scale scores were significantly lower in child self-reports (p<0.01 and p<0.05, respectively) and parent proxy-reports (p<0.01 and p<0.01, respectively) than those of healthy children. Citrin-deficient patients with scores of 50 percentile or less of healthy children constituted 67.5% of the sample for the Fatigue Scale and 68.4% for the Generic Core Scales. The PedsQL Fatigue Scale correlated with the Generic Core Scales for both the patients (r=0.56) and parents reports (r=0.71). Assessments by the patients and their parents showed moderate agreement. Parents assessed the condition of children more favorably than their children. The study identified severe fatigue and impaired QOL in citrin-deficient patients during the silent period, and that such children perceive worse fatigue and poorer QOL than those estimated by their parents. The results stress the need for active involvement of parents and medical staff in the management of citrin-deficient patients during the silent period.

Effects of supplementation on food intake, body weight and hepatic metabolites in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse model of human citrin deficiency.

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight.

[Utilization of high-resolution melting analysis to screen patients with neonatal intrahepatic cholestasis caused by citrin deficiency].

OBJECTIVE: To assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS: Based on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing. RESULTS: HRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing. CONCLUSION: HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.

Pathogenic variants of the mitochondrial aspartate/glutamate carrier causing citrin deficiency.

Citrin deficiency is a pan-ethnic and highly prevalent mitochondrial disease with three different stages: neonatal intrahepatic cholestasis (NICCD), a relatively mild adaptation stage, and type II citrullinemia in adulthood (CTLN2). The cause is the absence or dysfunction of the calcium-regulated mitochondrial aspartate/glutamate carrier 2 (AGC2/SLC25A13), also called citrin, which imports glutamate into the mitochondrial matrix and exports aspartate to the cytosol. In citrin deficiency, these missing transport steps lead to impairment of the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, and the urea cycle. In this review, we describe the geological spread and occurrence of citrin deficiency, the metabolic consequences and use our current knowledge of the structure to predict the impact of the known pathogenic mutations on the calcium-regulatory and transport mechanism of citrin.

Effects of resveratrol on alterations in cerebrum energy metabolism caused by metabolites accumulated in type I citrullinemia in rats.

We investigated the in vitro effects of citrulline (0.1, 2.5 and 5.0 mM) and ammonia (0.01, 0.1 and 1.0 mM), and the influence of resveratrol (0.01 mM, 0.1 mM and 0.5 mM) on pyruvate kinase, citrate synthase, succinate dehydrogenase (SDH), complex II, and cytochrome c oxidase activities in cerebral cortex, cerebellum and hippocampus homogenates of 60-day-old male Wistar rats. Results showed that 2.5 and 5.0 mM citrulline decreased pyruvate kinase activity in cerebral cortex and, at a concentration of 5.0 mM, increased its activity in hippocampus. Additionally, 5.0 mM citrulline increased citrate synthase activity in the cerebellum of rats. Citrulline (5.0 mM) reduced complex II and cytochrome c oxidase activities in cerebral cortex and hippocampus. With regard to ammonia, at 0.1 and 1.0 mM, decreased complex II activity in cerebral cortex and at 1.0 mM decreased its activity in cerebellum and hippocampus. Ammonia (1.0 mM) also decreased cytochrome c oxidase activity in cerebral cortex and cerebellum of rats. Resveratrol was able to prevent most of the alterations caused by these metabolites in the biomarkers of energy metabolism measured in the cerebrum of rats. Data suggest that these alterations in energy metabolism, caused by citrulline and ammonia, are probably mediated by the generation of free radicals, which can in turn be scavenged by resveratrol.

A case of adult-onset type II citrullinemia with comorbid epilepsy even after liver transplantation.

The current study using single case voxel-based morphometry (VBM) of magnetic resonance imaging (MRI) and ¹H-MR-spectroscopy (¹H-MRS) explores the neural background of unexplained seizure attacks and electroencephalography (EEG) abnormalities persisting even after liver transplantation in a patient with adult-onset type II citrullinemia (CTLN2). Although the MRI had shown no gross abnormality, the VBM revealed significantly smaller-than-normal regional volume in the left hippocampus of the patient as compared with 111 age-matched controls. ¹H-MRS further indicated reduction of all metabolite concentrations in the left hippocampus compared with those in the right homolog region, with the single exception of elevated glutamate concentration. These results are similar to those of patients with mesial temporal lobe epilepsy (TLE), although CTLN2-complicated mesial TLE has rarely been reported. In contrast to TLE, periictal asterixis and interictal slow activities on EEG support another possibility that the patient might have slight metabolic encephalopathy even after the liver transplantation.

Neonatal intrahepatic cholestasis associated with citrin deficiency (NICCD): a case series of 11 Malaysian patients.

Citrin deficiency, aetiologically linked to mutations of SLC25A13 gene, has two clinical phenotypes, namely adult-onset type II citrullinaemia (CTLN2) and neonatal/infantile intrahepatic cholestasis, caused by citrin deficiency (NICCD). Malaysian patients with NICCD, especially of Malay and East Malaysian indigenous descent, have never been reported in the literature. We present the clinical features, biochemical findings and results of molecular analysis in 11 Malaysian children with NICCD. In this case series, all patients manifested prolonged cholestatic jaundice and elevated citrulline levels. The other more variable features included failure to thrive, bleeding diathesis, hypoproteinaemia, abnormal liver enzymes, prolonged coagulation profile, hyperammonaemia, hypergalactosaemia, multiple aminoacidaemia, elevated α-feto protein and urinary orotic acid as well as liver biopsies showing hepatitis and steatosis. DNA analysis of SLC25A13 revealed combinations of 851del4(Ex9), IVS16ins3kb and 1638ins23. Most of our patients recovered completely by the age of 22 months. However, one patient had ongoing symptoms at the time of reporting and one had died of liver failure. Since a small percentage of children with NICCD will develop CTLN2 and the mechanisms leading to this is yet to be defined, ongoing health surveillance into adulthood is essential.

AGC2 (Citrin) Deficiency-From Recognition of the Disease till Construction of Therapeutic Procedures.

Can you imagine a disease in which intake of an excess amount of sugars or carbohydrates causes hyperammonemia? It is hard to imagine the intake causing hyperammonemia. AGC2 or citrin deficiency shows their symptoms following sugar/carbohydrates intake excess and this disease is now known as a pan-ethnic disease. AGC2 (aspartate glutamate carrier 2) or citrin is a mitochondrial transporter which transports aspartate (Asp) from mitochondria to cytosol in exchange with glutamate (Glu) and H+. Asp is originally supplied from mitochondria to cytosol where it is necessary for synthesis of proteins, nucleotides, and urea. In cytosol, Asp can be synthesized from oxaloacetate and Glu by cytosolic Asp aminotransferase, but oxaloacetate formation is limited by the amount of NAD+. This means an increase in NADH causes suppression of Asp formation in the cytosol. Metabolism of carbohydrates and other substances which produce cytosolic NADH such as alcohol and glycerol suppress oxaloacetate formation. It is forced under citrin deficiency since citrin is a member of malate/Asp shuttle. In this review, we will describe history of identification of the SLC25A13 gene as the causative gene for adult-onset type II citrullinemia (CTLN2), a type of citrin deficiency, pathophysiology of citrin deficiency together with animal models and possible treatments for citrin deficiency newly developing.