Biomarkers for liver disease in urea cycle disorders.

BACKGROUND: Urea cycle disorders (UCDs) are among the most common inborn errors of liver metabolism. As therapies for hyperammonemia associated with urea cycle dysfunction have improved, chronic complications, such as liver disease, have become increasingly apparent in individuals with UCDs. Liver disease in UCDs may be associated with hepatic inflammation, hepatic fibrosis, portal hypertension, liver cancer and even liver failure. However, except for monitoring serum aminotransferases, there are no clear guidelines for screening and/or monitoring individuals with UCDs for liver disease. Thus, we systematically evaluated the potential utility of several non-invasive biomarkers for liver fibrosis in UCDs. METHODS: We evaluated grey-scale ultrasonography, liver stiffness obtained from shear wave elastography (SWE), and various serum biomarkers for hepatic fibrosis and necroinflammation, in a cohort of 28 children and adults with various UCDs. RESULTS: Overall, we demonstrate a high burden of liver disease in our participants with 46% of participants having abnormal grey-scale ultrasound pattern of the liver parenchyma, and 52% of individuals having increased liver stiffness. The analysis of serum biomarkers revealed that 32% of participants had elevated FibroTest™ score, a marker for hepatic fibrosis, and 25% of participants had increased ActiTest™ score, a marker for necroinflammation. Interestingly, liver stiffness did not correlate with ultrasound appearance or FibroTest™. CONCLUSION: Overall, our results demonstrate the high overall burden of liver disease in UCDs and highlights the need for further studies exploring new tools for identifying and monitoring individuals with UCDs who are at risk for this complication. TRIAL REGISTRATION: This study has been registered in ClinicalTrials.gov (NCT03721367).

From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity.

Argininosuccinate synthase as a novel biomarker for inflammatory conditions.

Argininosuccinate synthase (ASS) plays an important role in regulating metabolic functions in mammals. We previously reported that hepatic ASS is released into circulation at very high concentrations in response to endotoxin and acute liver injury. We propose that ASS may serve as a novel biomarker for various inflammatory conditions. Our data showed that ASS accumulated in serum and urine of septic, obese or tumor mice in a condition-dependent fashion. Moreover, ASS significantly increased in urine within the first week after tumor cell implantation in mice which subsequently develop tumors. These results suggest that ASS is a novel biomarker increased upon diverse inflammatory conditions.

Study of serum argininosuccinate lyase determination for diagnosis of liver diseases.

The objectives of this research were to establish an automatic analysis method for the determination of serum argininosuccinate lyase (ASL) and to investigate the value of serum ASL test in the diagnosis of various liver disorders. According to the chemical reaction catalyzed by ASL, an enzyme-coupled reaction system was designed, and a methodology evaluation of this method was performed. A total of 291 patients with various liver diseases, 247 patients with nonliver disease and 32 healthy controls, were recruited, their serum levels of ASL and traditional hepatopathy markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and total bilirubin (TBil), were all determined, and their diagnostic values in liver diseases were analyzed and compared. Liver biopsy and the score of histopathological inflammation grading were performed in 31 patients with hepatopathy to explore the correlation between serum ASL level and hepatic histopathological change. A continuous monitoring assay method of serum ASL activity was established, which could be performed with automatic biochemistry analyzer. Methodological evaluation exhibited that the precision of this method was good indicated by the 4.0% intraassay coefficient of variation (CV), and 5.9% interassay CV. The mean recovery was 100.5%, linear range was from 0 to 167.7 U/L, and the lowest detection limit was approximately 0 U/L. All of the tested hepatopathy markers listed above were significantly increased in the liver disease group. However, levels of traditional markers of hepatopathy were all significantly increased at different degrees (all P<0.001) in patients with nonliver diseases; in contrast, there were no significantly increased ASL levels in all non-hepatopathy groups (P=0.335). The receiver operating characteristic (ROC) curve showed that the sensitivity and specificity of ASL were 100% and 91.1% (cutoff value=8 U/L), respectively, in the assessment of liver diseases. In contrast, ALT levels were 97.6% and 24.7%, and AST levels were 83.8% and 28.3% (both cutoff values=40.0 U/L), respectively. A positive correlation (r=0.417, P=0.019) was observed between serum ASL levels (86.9+/-26.5) and scores of histopathological inflammation grading (SHIG) (9.83+/-3.36). The sensitivity and specificity of ALS is much higher than that of ALT and AST for the diagnosis of liver diseases. ASL may be a more valuable marker for estimating hepatopathy.

[Determination of serum argininosuccinate lyase in diagnosing liver diseases].

OBJECTIVE: To investigate the value of the determination of the levels of serum argininosuccinate lyase (ASL) in diagnosing various liver diseases. METHODS: Two hundred and ninety-one patients with various liver diseases, 257 patients with non-liver disease, and 32 healthy controls were recruited for this study and their serum ASL, ALT, AST, GGT, LDH, ALP, and total bilirubin (TBil) levels were determined. Liver biopsies were performed on 31 patients with hepatopathy. RESULTS: Receiver operating characteristic (ROC) curve analysis showed that the sensitivity and specificity of ASL in assessing liver diseases were 100% and 91.1% (at cut-off values of 8 U/L), those of ALT were 97.6% and 24.7% and those of AST were 83.8% and 28.3% (both at cut-off values = 40.0 U/L), respectively. The levels of ASL in various liver disease patients were: in liver cancer - acute hepatitis - liver cirrhosis - chronic hepatitis. A positive correlation (r = 0.417) was observed between serum ASL levels (86.9+/-26.5) and scores of histopathological inflammation grading (9.83+/-3.36). CONCLUSION: ASL is of higher sensitivity and specificity than those of ALT and AST for diagnosing liver diseases. ASL may be used as a useful marker in estimating hepatopathy.

Argininosuccinate lyase deficiency: mutational spectrum in Italian patients and identification of a novel ASL pseudogene.

Argininosuccinic aciduria (ASAuria) is an inborn error of metabolism caused by mutations in the argininosuccinate lyase (ASL) gene, which leads to the accumulation of argininosuccinic acid (ASA) in body fluids and severe hyperammonemia. A severe neonatal form and a milder late-onset variant are described. We report a novel ASL pseudogene located in the centromeric region of chromosome 7, 14 novel mutations in the ASL gene, and a novel intronic polymorphism found in a cohort of Italian patients. Our approach relied exclusively on genomic DNA analysis. We found seven missense mutations, two nonsense, three small insertions/deletions, and two splicing mutations. Only two patients harbored previously described mutations, and among the novel variants only two were present in more than one kindred. The pathogenicity of the splicing mutations was demonstrated by a functional splicing assay that employed a hybrid minigene. We also performed molecular modeling using the reported three-dimensional structure of ASL to predict the functional consequences of the missense mutations. There was no genotype-phenotype correlation. Patients with neonatal onset display developmental delay and seizures despite adequate metabolic control. Moreover, hepatomegaly, fibrosis, and abnormal liver function tests are common complications in these patients, but not in patients with the late infancy form. We stress the importance of mutation analysis in patients with ASAuria, to confirm the clinical diagnosis, and to perform DNA-based prenatal diagnosis in future pregnancies of these families.

Isoniazid-induced hepatic necrosis and steatosis in rabbits: absence of effect of gender.

Isoniazid, a highly effective drug for the chemoprophylaxis and treatment of tuberculosis, is associated with severe hepatotoxicity in 1-2% of individuals. In a rabbit model of isoniazid-induced hepatotoxicity, we have measured hepatic necrosis (quantitated by elevation of plasma argininosuccinic acid lyase (ASAL) activity), hepatic steatosis (quantitated by elevation of hepatic triglyceride content), and elevation in plasma triglyceride concentration in 15 rabbits. Eight of 15 rabbits were male, and 14 of 15 were rapid acetylators of sulfamethazine. Administration of isoniazid to rabbits resulted in a 27-fold increase in plasma ASAL activities, a 7.5-fold increase in hepatic triglyceride content, and a 13-fold increase in plasma triglyceride levels. This study demonstrated no effect of gender on these three pathological changes that occur in this model of isoniazid-induced hepatotoxicity in rabbits.

Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits.

Isoniazid (INH) continues to be a highly effective drug in the chemoprophylaxis and treatment of tuberculosis; however, its use is associated with hepatotoxicity (predominantly hepatic necrosis) in 1-2% of individuals. The INH metabolites, acetylhydrazine and hydrazine, have each been implicated as the causative hepatotoxin in INH-induced hepatotoxicity. Using a model of INH-induced hepatotoxicity in rabbits, in which INH-induced hepatotoxicity manifests as hepatic necrosis, hepatic steatosis (hepatic fat accumulation) and hypertriglyceridaemia (elevated plasma triglycerides), we compared the severity of these measures of toxicity with plasma levels of INH, acetylhydrazine and hydrazine. Plasma INH and acetylhydrazine were not correlated with markers of INH-induced hepatic necrosis or fatty changes. Plasma hydrazine at 32 h was correlated significantly with plasma argininosuccinic acid lyase (ASAL, a sensitive marker of hepatic necrosis) activity as area under the curve (r2 = 0.54, P < 0.002) and log plasma ASAL activity at 48 h after the first dose of INH (r2 = 0.53, p < 0.005), but not with fatty changes. These results show in this model of INH-induced hepatotoxicity in rabbits that hydrazine, and not INH or acetylhydrazine, is most likely involved in the pathogenic mechanism of hepatic necrosis.

A model of isoniazid-induced hepatotoxicity in rabbits.

Isoniazid (INH) continues to be an effective drug used for chemoprophylaxis and treatment of tuberculosis. Unfortunately, INH is associated with significant hepatotoxicity in up to 2% of individuals exposed, and if this adverse event is not recognized early it can be fatal. Research on INH-induced hepatotoxicity has been hampered by the lack of a suitable animal model that closely resembles the toxicity in humans. The mechanism of INH-induced hepatotoxicity is still unknown. The present study describes the development of a reliable model of INH-induced hepatotoxicity in rabbits. The protocol involves repeated injections of INH over a 2-day period, resulting in significant hepatic necrosis as indicated by elevations of plasma argininosuccinic acid lyase activity. Pretreatment with phenobarbital increased the occurrence of INH-induced hepatic necrosis from approximately 60% (9 out of 15 rabbits) with INH alone to more than 90% (13 out of 14 rabbits). Morphological indices were used to demonstrate the presence of INH-induced hepatotoxicity, and biochemical indices were used to demonstrate both the presence and severity of INH-induced hepatotoxicity in this model. This model may prove useful for further investigations into the mechanism of INH-induced hepatotoxicity.

Determination of argininosuccinate lyase in human serum by ion-pair reversed phase liquid chromatography.

A new assay for argininosuccinate lyase based on the separation of the enzymatic reaction mixture components by an ion-pair reversed phase mechanism is reported. The determination of enzyme activity is performed after direct injection by UV detection of the fumarate formed. The chromatographic analysis time is 8 min and a detection limit of 0.4 U/L is achieved. The HPLC method is highly accurate, sensitive and precise. The simple procedure makes this method suitable for the routine determination of ASAL activity in human serum samples.

Dietary management reverses grooving and abnormal polarization of hair shafts in argininosuccinase deficiency.

We have observed that the fragile hair of two untreated patients with argininosuccinic aciduria showed abnormal alternating zones of bright and dark banding by polarizing microscopy. Scanning electron microscopy documented discontinuous grooves with a 50 to 100 microns periodicity. Results of amino acid analysis of the hair were essentially normal. After the patients were treated with a low-protein, arginine-supplemented diet, the hair assumed a normal appearance. Five patients already treated with diet showed no hair abnormalities. The pathogenesis of the hair changes in unknown, but our findings suggest that products generated in the disease can adversely affect metabolically active tissue such as hair.

Evaluation of 31P NMR spectroscopy as an indicator of chemically induced hepatic toxicity in the rat: comparison with serum enzyme levels and pathology.

The progression of carbon tetrachloride-induced liver damage, determined by 31P NMR spectroscopy, was compared with selected serum enzyme and histological changes in rats. ATP levels declined as early as 8 h post-CCl4 administration, with partial recovery observed at 168 h. The results show that ATP reduction correlates with necrosis. In addition, early decline in ATP occurring prior to significant hepatocellular necrosis indicates abnormal energy metabolism.

Determination of argininosuccinate lyase and arginase activities with an amino acid analyzer.

The measurement of argininosuccinate lyase (ASase) and arginase, both in liver and erythrocytes, was developed by using a commercial amino acid analyzer. The method is based upon the use of two different substrates, argininosuccinate and arginine for ASase and arginase, respectively, and the measurement of only one final metabolite: ornithine. The use of ornithine as a marker of biological activity of ASase is related to the fact that in the urea cycle, the specific activity of arginase is much higher than that of ASase; thus, during in vitro determinations, arginine, which is the product of ASase, is rapidly converted to ornithine. The sensitivity of the methods is very high since we were able to detect both activities using very diluted rat liver homogenates (0.10 mg protein/ml) or few microliters of human blood. In rat liver the Vmax for ASase and arginase were respectively 0.54 and 140 mumol/h/mg protein; the apparent Km values 1.25 and 13.5 mM. In human erythrocytes the Vmax for the same enzymes were 7.2 and 170 nmol/h/mg Hb and the apparent Km values were 0.66 and 9.5 mM. In 10 healthy volunteers the specific activity of ASase and arginase determined in blood were respectively 8.60 +/- 0.46 and 124.1 +/- 14.5 nmol/h/mg Hb. The results obtained from 2 patients suffering from argininosuccinic aciduria were also reported. In these latter cases while ASase was not detectable in blood, arginase activity was at the lowest end of the confidence limits determined in healthy volunteers.

Clearance of argininosuccinate synthetase from the circulation in acute liver disease.

Argininosuccinate synthetase is an enzyme which has been found to be a specific marker for liver damage. In patients with acute hepatitis, the concentration in serum increases at the onset of the disease, but later decreases more quickly, so that the time required for normalization is shorter than that of alanine aminotransferase. This is probably caused by rapid clearance of argininosuccinate synthetase from the serum. Rapid clearance was demonstrated in experimental animals given purified enzymes intravenously. Argininosuccinate synthetase disappeared from the serum with a half life of about 15 min, while the half lives of alanine aminotransferase and aspartate aminotransferase were 4 and 5 h, respectively, under the same conditions.

An enzymatic method for the assay of serum argininosuccinate lyase.

A rapid enzymatic method was developed for the assay of serum argininosuccinate lyase (ASAL: EC 4.3.2.1) which is a useful marker enzyme for diagnosis of parenchymal liver diseases. Fumarate, liberated from argininosuccinate in the lyase-mediated reaction, was converted to pyruvate via L-malate by the actions of fumarase and malic enzyme in the presence of NADP+. The NADPH formed was then oxidized with a diaphorase-resazurin system to give a highly fluorescent resorufin. All the enzymatic reactions proceeded continuously in 0.1 M Tris-HCl buffer (pH 7.5) and allowed direct assay of ASAL in serum by monitoring the increase in the fluorescence intensity due to resorufin. The method is rapid and sensitive; only 50 microliter of serum is required. This method was used to detect increases in the activities in sera from patients with liver diseases.

Urea cycle and protein content in leucocytes from normal persons and uraemics.

It was found experimentally that the activity of the urea-cycle enzymes in the leucocytes of uraemic rats was parallel to that in their liver. Conclusions as to the activity of the urea-cycle enzymes in the liver can thus be drawn from the urea-cycle enzyme activity in the leucocytes. Investigation of the urea-cycle enzyme in the leucocytes of patients with renal insufficiency, in the pre-dialytic and dialytic states, showed that in both groups the ornithine carbamoyl transferase and the argininosuccinate lyase activity was lower in both groups of patients than in normal persons. The dialysis patients show higher argininosuccinate synthetase activity. In both groups of patients the protein content of the leucocytes is markedly lowered.