Urea Cycle Defects Research Articles

Strategies for optimizing the serum persistence of engineered human arginase I for cancer therapy

Systemic l-arginine depletion following intravenous administration of l-arginine hydrolyzing enzymes has been shown to selectively impact tumors displaying urea cycle defects including a large fraction of hepatocellular carcinomas, metastatic melanomas and small cell lung carcinomas. However, the human arginases display poor serum stability (t1/2=4.8h) whereas a bacterial arginine deiminase evaluated in phase II clinical trials was reported to be immunogenic, eliciting strong neutralizing antibody responses. Recently, we showed that substitution of the Mn2+ metal center in human Arginase I with Co2+ (Co-hArgI) results in an enzyme that displays 10-fold higher catalytic efficiency for L-Arg hydrolysis, 12–15 fold reduction in the IC50 towards a variety of malignant cell lines and, importantly a t1/2=22h in serum. To investigate the utility of Co-hArgI for L-Arg depletion therapy in cancer we systematically investigated three strategies for enhancing the persistence of the enzyme in circulation: (i) site specific conjugation of Co-hArgI engineered with an accessible N-terminal Cys residue to 20kDa PEG-maleimide (Co-hArgI-CPEG-20K); (ii) engineering of the homotrimeric Co-hArgI into a linked, monomeric 110kDa polypeptide (Co-hArgI x3) and (iii) lysyl conjugation of 5kDa PEG-N-hydroxysuccinimide (NHS) ester (Co-hArgI-KPEG-5K). Surprisingly, even though all three formulations resulted in proteins with a predicted hydrodynamic radius larger than the cut-off for renal filtration, only Co-hArgI amine conjugated to 5kDa PEG remained in circulation for sufficiently long durations. Using Co-hArgI-KPEG-5K labeled with an end-terminal fluorescein for easy detection, we demonstrated that following intraperitoneal administration at 6mg/kg weight, a well tolerated dose, the circulation t1/2 of the protein in Balb/c mice is 63±10h. Very low levels of serum L-Arg (<5μM) could be sustained for over 75h after injection, representing a 9-fold increase in pharmacodynamic efficacy relative to similarly prepared Mn2+-containing hArgI conjugated to 5kDa PEG-NHS ester (Mn-hArgI-KPEG-5K). The favorable pharmacokinetic and pharmacodynamic properties of Co-hArgI-KPEG-5K reported here, coupled with its human origin which should reduce the likelihood of adverse immune responses, make it a promising candidate for cancer therapy.

High urgency liver transplantation in ornithine transcarbamylase deficiency presenting with acute liver failure

OTCD can present with ALF at any age. Under adequate therapy symptoms resolve quickly. We report a three-yr-old girl with the manifestation of an OTCD as ALF. Despite adequate pharmacotherapy and protein restriction, the patient deteriorated and developed hepatic encephalopathy. A high urgency liver transplantation was performed and the patient recovered completely. We conclude that in patients with ALF urea cycle defects in general and OTCD in particular should be considered as differential diagnosis. Patients should be managed in a center that has the capacity for an emergency liver transplantation.

Gene Transfer for Inborn Errors of Metabolism of the Liver: The Clinical Perspective

There are numerous inborn errors of metabolism of the liver, and they are all rare to very rare. To get a clear picture of the indications for gene transfer in these conditions, it is essential to get a clear view on the current (lack of) insight in the pathophysiology of these disorders, the current treatment options and hence on the window of opportunity for new treatments as gene transfer. The aim of this review, is to illustrate the problems related to treatment of inborn errors of metabolism of the liver. General aspects defining the quest for treatments for very rare diseases are touched upon, but for the sake of clarity, this review is restricted to five illustrative examples: Crigler-Najjar type I, the urea cycle defects, phenylketonuria, classic galactosemia and propionic acidemia. These examples reflect the problems that are currently experienced and can be expected, when gene transfer trials for these disorders are undertaken.

Das klinische Spektrum von Harnstoffzyklusdefekten im Erwachsenenalter

Urea cycle defects belong to the most common metabolic disorders with a cumulative incidence of 1:8000. A common trait of urea cycle defects is a disturbed detoxification of ammonia leading to hyperammonemia in the event of a high nitrogen load. Most patients develop symptoms in the neonatal period or in infancy, e. g. vomiting, seizures and disturbed consciousness. Depending on the affected enzyme and its residual activity, patients differ in the age at first presentation, the character and severity of symptoms and in the susceptibility to metabolic derangement. The presence of hyperammonemia and an altered plasma amino acid profile give the essential diagnostic clues. Since modern therapeutic measures have prolonged the life expectancy of these patients and provided the possibility of a first presentation in adulthood, patients with urea cycle defects have become an increasing challenge in internal medicine. The reported case series illustrates the heterogeneous clinical course of these disorders from childhood to adulthood.

Acute Management of Sick Infants with Suspected Inborn Errors of Metabolism

Diagnosis of inborn errors of metabolism (IEM) such as an organic acidemia or urea cycle defects requires high index of suspicion in a critically ill infant as these conditions mimic common pediatric illnesses. Prompt initiation of the treatment is mandatory even if a definitive diagnosis is not established immediately. Initial screening investigations may give clues and help to classify these disorders in broad categories. It is of utmost importance to preserve samples for testing.

Bacterial expression of mutant argininosuccinate lyase reveals imperfect correlation of in‐vitro enzyme activity with clinical phenotype in argininosuccinic aciduria

The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme. Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299T > C), p.V178M (c.532G > A), p.E189G (c.566A > G), p.Q286R (c.857A > G), p.K315E (c.943A > G), p.R379C (c.1135C > T) and p.R385C (c.1153C > T) in comparison to the wildtype protein. In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K(m) values for argininosuccinic acid differed significantly from the wild-type ASL protein. The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.

Bioengineered Human Arginase I with Enhanced Activity and Stability Controls Hepatocellular and Pancreatic Carcinoma Xenografts

Hepatocellular carcinoma (HCC) and pancreatic carcinoma (PC) cells often have inherent urea cycle defects rendering them auxotrophic for the amino acid L-arginine (L-arg). Most HCC and PC require extracellular sources of L-arg and undergo cell cycle arrest and apoptosis when L-arg is restricted. Systemic, enzyme-mediated depletion of L-arg has been investigated in mouse models and human trials. Non-human enzymes elicit neutralizing antibodies, whereas human arginases display poor pharmacological properties in serum. Co2+ substitution of the Mn2+ metal cofactor in human arginase I (Co-hArgI) was shown to confer more than 10-fold higher catalytic activity (kcat/Km) and 5-fold greater stability. We hypothesized that the Co-hArgI enzyme would decrease tumor burden by systemic elimination of L-arg in a murine model. Co-hArgI was conjugated to 5-kDa PEG (Co-hArgI-PEG) to enhance circulation persistence. It was used as monotherapy for HCC and PC in vitro and in vivo murine xenografts. The mechanism of cell death was also investigated. Weekly treatment of 8 mg/kg Co-hArgI-PEG effectively controlled human HepG2 (HCC) and Panc-1 (PC) tumor xenografts (P = .001 and P = .03, respectively). Both cell lines underwent apoptosis in vitro with significant increased expression of activated caspase-3 (P < .001). Furthermore, there was evidence of autophagy in vitro and in vivo. We have demonstrated that Co-hArgI-PEG is effective at controlling two types of L-arg-dependent carcinomas. Being a nonessential amino acid, arginine deprivation therapy through Co-hArgI-PEG holds promise as a new therapy in the treatment of HCC and PC.

Threshold for toxicity from hyperammonemia in critically ill children

Hyperammonemia results from reduction of hepatocyte function or enzyme of urea cycle deficiency. Hyperammonemia contributes to cerebral edema that may lead to cerebral herniation. The threshold of toxicity of ammonemia is unknown. We conducted a retrospective observational study in our pediatric intensive care unit. All children who developed hyperammonemia from January 2000 to April 2009 were included. Clinical and laboratory data at admission, specific treatments implemented, and ammonemias the first 7 days after inclusion were collected. The outcome assessed was 28 day mortality. Risk of mortality was estimated by a logistic regression model. Ninety patients with liver failure (63.3%) and primary or secondary urea cycle defect (23.3%) were included. Patients with urea cycle defects were more likely to receive ammonia scavengers than patients with liver failure (47.6% versus 3.5%). The 28 day mortality rate was 31.1%. Risk of mortality increased according to the ammonemia within 48 h: odds ratio 1.5, 1.9, 3.3, 2.4 for ammonemia above 100, 150, 200, and 300 μmol/L, respectively. Peak ammonemia ≥200 μmol/L within the first 48 h was an independent risk factor for mortality, with greater risk found in liver failure than in urea cycle defect. Our study identifies a threshold of exposure to ammonia (≥200 μmol/L) above which mortality increases significantly, especially in liver failure. Specific treatments of hyperammonemia are rarely used in liver failure when compared with urea cycle defect even though use of ammonia scavengers may help to decrease ammonemia.

Induction and Prevention of Severe Hyperammonemia in the spfash Mouse Model of Ornithine Transcarbamylase Deficiency Using shRNA and rAAV-mediated Gene Delivery

Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.

Arginase Deficiency Presenting as Intractable Hiccups With Severe Hyperammonemia at One Month of Age

Arginase deficiency is a rare urea cycle disorder and generally presents in children of preschool age with developmental delay and spasticity. Unlikely other urea cycle defects, patients rarely present hyperammonemic symptoms, especially in the early infantile period. A one-month-old male infant was admitted to our hospital because of abnormal respiratory condition with persistent hiccups and lethargy. The initial laboratory studies showed respiratory alkalosis and remarkable hyperammonemia [ammonia, 1970 ? mol/L (25 times normal)]. Amino acid chromatography showed a markedly elevated plasma arginine level (1131 µ mol/L; 10 times normal), and erythrocyte arginase activity was undetectable. Despite prompt diagnosis and normalization of ammonia and arginine levels by peritoneal dialysis, the patient developed neurodevelopmental impairment during the one-year follow-up period. In summary, this is a rare case of arginase deficiency in terms of early infantile onset and presentation of the severe form of hyperammone mia. Pathologic hiccups could be one of the clinical clues to the presence of hyperammonemia even in young infants with arginase deficiency. doi:10.4021/jmc141w

Liver Transplantation in an Adult with Citrullinaemia Type 2

Citrullinaemia is a urea cycle defect that results from a deficiency of the enzyme arginosuccinate synthetase. Type 1 disease is diagnosed in childhood, whereas Type 2 disease is adult onset. We report the outcome of a patient with citrullinemia Type 2 who received a liver transplant at our center and the implications of this diagnosis in liver transplantation.

Arginase Deficiency Presenting as Intractable Hiccups With Severe Hyperammonemia at One Month of Age

Arginase deficiency is a rare urea cycle disorder and generally presents in children of preschool age with developmental delay and spasticity. Unlikely other urea cycle defects, patients rarely present hyperammonemic symptoms, especially in the early infantile period. A one-month-old male infant was admitted to our hospital because of abnormal respiratory condition with persistent hiccups and lethargy. The initial laboratory studies showed respiratory alkalosis and remarkable hyperammonemia [ammonia, 1970 µmol/L (25 times normal)]. Amino acid chromatography showed a markedly elevated plasma arginine level (1131 µmol/L; 10 times normal), and erythrocyte arginase activity was undetectable. Despite prompt diagnosis and normalization of ammonia and arginine levels by peritoneal dialysis, the patient developed neurodevelopmental impairment during the one-year follow-up period. In summary, this is a rare case of arginase deficiency in terms of early infantile onset and presentation of the severe form of hyperammonemia. Pathologic hiccups could be one of the clinical clues to the presence of hyperammonemia even in young infants with arginase deficiency. J Med Cases. 2011;2(2):67-70 doi: https://doi.org/10.4021/jmc141w

A urinary cause of coma

Hyperammonemic coma is rare except in association with hepatic encephalopathy. When liver disease is ruled out, hyperammonemia should suggest the existence of a portosystemic shunt or an inherited urea cycle defect [1]. Urinary ureterocolic diversion (UD) that has been widely used for years to treat bladder exstrophy can be responsible for hyperammonemic coma, metabolic hyperchloremic acidosis, and subsequent colonic neoplasia [2–5]. Here, we present a 68-year-old man admitted to the ICU for a hyperammonemic coma secondary to UD by ureterosigmoidostomy. He had a history of congenital bladder exstrophy and epispadias treated in the first year of life by ureterosigmoidostomy and had a recent history of spontaneous reversible coma. Upon admission, the body temperature was 36.2 C, heart rate was 97 beats/min, and blood pressure was 185/84 mmHg. Hypotonic coma without any focal deficit was noted (Glasgow coma scale = 3). Abdominal inspection showed skin atrophy in the hypogastric area, an atrophic penis, and a large laparotomy scar. He subsequently required respiratory assistance. Blood sample results are shown in Table 1. The cerebral CT scan and lumbar puncture were normal. The ammonia level was elevated (346 lmol/L; normal level \50 lmol/L). The initial treatment is given Table 1. Urea cycle disorders were initially ruled out by serum amino acid chromatography and a portosystemic shunt by Doppler ultrasound and CT scan. Bacteriological culture of mixed urine and feces identified wild-type Escherichia coli and Enterococcus faecalis, two urea-splitting bacteria. Treatment by intravenous cefotaxime and oral vancomycin was started. Normalization of ammonemia rapidly ensued. However, a cerebral CT, performed on day 5, showed major cerebral edema in both temporal lobes (Fig. 1). The neurological status of the patient improved and could be weaned from mechanical ventilation on day 10. The cerebral CT scan performed on day 24 was normal; however, the cerebral MRI scan performed on the same day showed diffuse cortical hypersignals on diffusion-weighted images and diffuse hypersignals in both temporal lobes in the FLAIR-weighted images (Fig. 1). During a 2-month period, the patient experienced three relapses of hyperammonemic coma (Table 1). The decision to perform surgical C. Levi and C. Hussenet contributed equally to this work.

Female heterozygotes for the hypomorphic R40H mutation can have ornithine transcarbamylase deficiency and present in early adolescence: a case report and review of the literature

IntroductionOrnithine transcarbamylase deficiency is the most common hereditary urea cycle defect. It is inherited in an X-linked manner and classically presents in neonates with encephalopathy and hyperammonemia in males. Females and males with hypomorphic mutations present later, sometimes in adulthood, with episodes that are frequently fatal.Case presentationA 13-year-old Caucasian girl presented with progressive encephalopathy, hyperammonemic coma and lactic acidosis. She had a history of intermittent regular episodes of nausea and vomiting from seven years of age, previously diagnosed as abdominal migraines. At presentation she was hyperammonemic (ammonia 477 μmol/L) with no other biochemical indicators of hepatic dysfunction or damage and had grossly elevated urinary orotate (orotate/creatinine ratio 1.866 μmol/mmol creatinine, reference range <500 μmol/mmol creatinine) highly suggestive of ornithine transcarbamylase deficiency. She was treated with intravenous sodium benzoate and arginine and made a rapid full recovery. She was discharged on a protein-restricted diet. She has not required ongoing treatment with arginine, and baseline ammonia and serum amino acid concentrations are within normal ranges. She has had one further episode of hyperammonemia associated with intercurrent infection after one year of follow up. An R40H (c.119G>A) mutation was identified in the ornithine transcarbamylase gene (OTC) in our patient confirming the first symptomatic female shown heterozygous for the R40H mutation. A review of the literature and correspondence with authors of patients with the R40H mutation identified one other symptomatic female patient who died of hyperammonemic coma in her late teens.ConclusionsThis report expands the clinical spectrum of presentation of ornithine transcarbamylase deficiency to female heterozygotes for the hypomorphic R40H OTC mutation. Although this mutation is usually associated with a mild phenotype, females with this mutation can present with acute decompensation, which can be fatal. Ornithine transcarbamylase deficiency should be considered in the differential diagnosis of unexplained acute confusion, even without a suggestive family history.

439 Use of Amplitude Integrated Electroencephalography (AEEG) in Patients with Inborn Errors of Metabolism - a Review of 29 Cases

Background: Amplitude integrated EEG (aEEG) has been established in neonatal care as tool to monitor encephalopathy after asphyxia and seizures due to various aetiologies. Patterns representing seizures and different degrees of encephalopathy have been defined. Patients with inborn errors of metabolism often present with encephalopathy/ seizures but published knowledge regarding aEEG use in metabolic patients remains limited as disease frequencies are low. Aims: Review aEEG tracings of patients with inborn errors of metabolism to evaluate the potential role of this technique in evaluation/treatment of this patient group. Methods: Through an international collaboration, 29 aEEG tracings of patients with inborn errors of metabolism were collected and reviewed. Results: Diagnoses were: disorders of energy metabolism (n=10), disorders of amino or organic acid metabolism and urea cycle defects (n=14), peroxisomal disorders (n=5). Encephalopathic changes were seen in 18/29 patients (62%); ictal discharges were noted in 19/29 patients (66%). In all diagnostic groups, with the exception of peroxisomal disorders, both encephalopathic changes and seizures usually coincided; in peroxisomal disorders, seizures were seen without encephalopathic background patterns. 4/29 patients (14%) showed unusual upward shifts of the lower aEEG margins Five patients, some clinically symptomatic, had normal aEEGs. Background patterns changed in 8/29 (28%) tracings during the recording. Details of individual recordings will be presented. Conclusions: The majority of patients with metabolic disorders had abnormal aEEG tracings, some with marked and/or unusual changes. As aEEG monitoring allows for ongoing assessment of patients, it may aid in evaluation, monitoring of treatment responses and counseling in this patient group.

Recurrent liver failure in a 25-year-old female

Acute liver failure (ALF) is defined as severe and sudden liver dysfunction leading to coagulopathy and encephalopathy in a previously healthy person without preexisting liver disease. Almost half of adult cases of ALF are due to acetaminophen toxicity, with 21% labeled indeterminate or other. We present a patient with a second episode of ALF, both episodes being initiated by catabolic stress. Elevated aminotransferases, jaundice, an elevated international normalized ratio, and confusion were typical of idiopathic ALF, and a low serum ceruloplasmin level initially led to a misdiagnosis of acute Wilson disease. Citrullinemia type I, a urea cycle defect caused by a deficiency of argininosuccinate synthetase, was diagnosed on the basis of plasma amino acids and was confirmed by molecular testing. Urea cycle defects are not generally considered causes of ALF in adults and are described rarely in children beyond the neonatal period. Our case illustrates the importance of screening patients with idiopathic ALF for a metabolic disorder. A prompt diagnosis and timely treatment enabled her to recover fully without the need for liver transplantation.

Liver transplantation for inherited metabolic disorders of the liver

Liver transplantation is curative, life saving or both for a range of inherited diseases affecting the liver. Indications, timing and outcome of transplantation for these diseases are the focus of this review. Liver transplant represents a mode of gene replacement therapy for several disorders, including Wilson disease, hemochromatosis, tyrosinemia, urea cycle defects and hypercholesterolemia in which the primary defect residing in the liver results in hepatic complications or severe extrahepatic disease. Liver transplant is also an important therapeutic modality in multisystemic genetic disorders with major hepatic disease such as glycogen storage disease types I, III and IV and porphyria. For familial amyloidosis and primary hyperoxaluria, liver replacement eliminates the source of the injurious products that results in extrahepatic disease. Innovations in medical and surgical management of these patients have led to improved outcomes providing an important benchmark for future gene therapy of these disorders. Recent developments have refined the indications for liver transplant in the treatment of inherited metabolic diseases. The full potential of liver transplant in these disorders can be harnessed by careful patient selection, optimizing timing and perioperative metabolic management of these patients.

Continuous hemofiltration in the control of neonatal hyperammonemia: a 10-year experience

In a 10-year review of the utilization of continuous veno-venous hemofiltration (CVVH) for the treatment of neonatal hyperammonemia, 14 patients were identified with hyperammonemia due to either a urea cycle defect or an organic acidemia. Intensive care survival was 64%. The pretreatment level of serum ammonia and the rapidity of ammonia clearance did not differ between survivors and non-survivors (p = 0.16 and p = 0.93, respectively). Likewise, the duration of CVVH therapy did not differ between survivors and non-survivors (p = 0.1). Indicators of pretreatment physiological stress showed either a correlation with non-survival [Pediatric Risk of Mortality (PRISM) score, p = 0.006, cardioactive drug requirement, p = 0.003], or demonstrated a trend to such a correlation (serum lactate, p = 0.06). Complications associated with the CVVH technique were infrequent. Hypotension was seen in seven patients, but in only one patient did it arise de novo following the initiation of CVVH. In conclusion, neither the severity of the hyperammonemic state nor the efficacy of ammonia removal correlated with patient outcome. The pre-CVVH PRISM score and requirement for cardio-active medication were significantly greater in those patients who did not survive their acute illness. The pre-CVVH physiological condition of the neonates in this cohort was the main determinant of outcome.

Fatal ammonia toxicity in an adult due to an undiagnosed urea cycle defect: under-recognition of ornithine transcarbamylase deficiency

There is a lack of awareness of acutely presenting inborn errors of metabolism in adults, of which the X-linked urea cycle defect ornithine transcarbamylase (OTC) deficiency is an example, many comparatively mild mutations having been identified. In male hemizygotes clinical manifestations and age at presentation vary and depend on the mutation. In female heterozygotes the clinical spectrum depends on the extent to which the abnormal gene is expressed. Milder versions of the defect may not cause clear clinical symptoms and may remain unrecognized until the person is subjected to an unusually high nitrogen load when they develop severe hyperammonaemia. During acute episodes liver enzymes may be normal or only slightly elevated and occasionally accompanied by coagulopathy, but the key finding is hyperammonaemia. Boys with these milder forms may exhibit abnormal behaviour and be diagnosed with attention deficit hyperactivity disorder. This case illustrates how late presentation of OTC deficiency in a non-specialist centre can be difficult to differentiate from drug abuse, psychiatric illness or encephalopathy. Failure to measure blood ammonia in adults with unexplained key symptoms - particularly prolonged vomiting without diarrhoea and altered mental state/hallucinations, or to recognize the significance of elevated blood ammonia without evidence of liver decompensation can lead to delayed or missed diagnosis.

Favorable effect of 4-phenylacetate on liver functions attributable to enhanced bile salt export pump expression in ornithine transcarbamylase-deficient children

4-Phenylbutyrate (4-PB) acting against hyperammonemia has been administered to patients with urea cycle defects. Results of our recent experiments using animals and cultured cells strongly suggest that this agent enhances the function of bile salt export pump/ATP binding cassette B11 (BSEP/ABCB11) promoting bile acid excretion from hepatocytes to bile canaliculi, although it has not been confirmed in humans. Considering that 4-PB is converted easily into 4-phenylacetate (4-PA) in the liver, such an effect of 4-PB might occur through 4-PA. We performed retrospective analyzes of the effects of 4-PA on the liver functions of three ornithine transcarbamylase (OTC)-deficient female children receiving 4-PA. Two of the three received intravenous administration of 4-PA only at episodic periods of hyperammonemia; the remaining one received it orally at intercurrent periods. Soon after 4-PA administration, the serum total bile acid level was decreased to one-half or one-third of pre-treatment levels, but it returned to the basal levels within one month after 4-PA discontinuation. Other serum parameters for cholestasis such as gamma-glutamyl transferase also decreased markedly. Concomitantly, alanine aminotransferase and aspartate amino transferase levels decreased significantly. Western blot analyzes of the liver samples revealed that the 4-PA administration enhanced BSEP/ABCB11 protein expressions in the membranous fraction of liver cells, although the liver BSEP/ABCB11 messenger RNA level remained unchanged. These results suggest that 4-PA enhanced liver BSEP/ABCB11 function and thereby improved liver functions in OTC-deficient children. For treatment of liver disorders requiring enhancement of BSEP function, 4-PA might be a candidate.