Masaki Takayanagi

Experimental evidence that phenylalanine is strongly associated to oxidative stress in adolescents and adults with phenylketonuria

Few studies have looked at optimal or acceptable serum phenylalanine levels in later life in patients with phenylketonuria (PKU). This study examined the oxidative stress status of adolescents and adults with PKU. Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, and blood anti-oxidant levels were examined. Nitric oxide (NO) production was also examined as a measure of oxidative stress. Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level (P<0.01). Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level (P<0.001). Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level (P<0.01). Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level (P<0.001). The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 μmol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q(10) were also lower (P<0.001), although the decreases did not correlate significantly with the phenylalanine level. Serum nitrite/nitrate levels, as stable NO products, were higher together with low serum asymmetric dimethylarginine, as an endogenous NO inhibitor. Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine level in should be maintained PKU below 700-800 μmol/l even in adult patients.

Congenital absence of portal vein with multiple hyperplastic nodular lesions in the liver

Congenital absence of the portal vein is an extremely rare anomaly, in which enteric blood bypasses the liver and drains into the inferior vena cava. A 16-year-old girl was referred to our hospital presenting with liver tumor. Although she had suffered from galactosemia soon after birth, the galactosemia had improved spontaneously 1 year later. Between the ages of 8 and 12 years, chronic hepatitis with a mild elevation of aspartate transaminase (AST) and alanine transaminase (ALT) was observed, but liver tumor had not been detected on computed tomography (CT) in regular medical examinations. However, at age 16, liver tumors, 10 cm in diameter, were found. Abdominal angiography indicated complete absence of the portal vein, suggesting that enteric blood was bypassing the liver and draining into the inferior vena cava. In biopsy specimens obtained under ultrasonographic guidance, liver tumors were confirmed histologically as hyperplastic nodules. In addition to this case report, the clinical features of 25 reported cases of congenital absence of the portal vein are reviewed.

Pilot study of gas chromatographic–mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease

Gas chromatographic-mass spectrometric (GC-MS) techniques for urinary organic acid profiling have been applied to high-risk screening for a wide range of diseases, mainly for inborn errors of metabolism (IEM), rather than to low-risk screening or mass screening. Using a simplified procedure with urease-pretreatment and the GC-MS technique, which allows simultaneous determination of organic acids, amino acids, sugars and sugar acids, we performed a pilot study of the application of this procedure to neonatal urine screening for 22 IEM. Out of 16,246 newborns screened, 11 cases of metabolic disorders were chemically diagnosed: two each of methylmalonic aciduria and glyceroluria, four of cystinuria, and one each of Hartnup disease, citrullinemia and alpha-aminoadipic aciduria/alpha-ketoadipic aciduria. The incidence of IEM was thus one per 1477, which was higher than the one per 3000 obtained in the USA in a study targeting amino acids and acylcarnitines in newborn blood spots by tandem mass spectrometry. Also, 227 cases were found to have transient metabolic abnormalities: 108 cases with neonatal tyrosinuria, 99 cases with neonatal galactosuria, and 20 cases with other transient metabolic disorders. Two hundred and thirty-eight cases out of 16,246 neonates (approximately 1/68) were thus diagnosed using this procedure as having either persistent or transient metabolic abnormalities.

Relationship between oxidative stress and antioxidant systems in the liver of patients with Wilson disease: Hepatic manifestation in wilson disease as a consequence of augmented oxidative stress

The role of oxidative stress in the pathogenesis of liver disease in Wilson disease (WD), a genetic disorder characterized by excess hepatic deposition of copper that generates free radicals, remains unclear. This study investigates oxidative stress on the liver and hepatic antioxidant responses in WD using liver specimens from affected patients showing mild liver damage (group I, n = 3), moderate or greater liver damage (group II, n = 5), and fulminant hepatic failure (group III, n = 5) and from asymptomatic carriers (n = 2). Decreased ratios of reduced glutathione (GSH) to oxidized glutathione (GSSG) and increased thiobarbituric acid reactive substance (TBARS), a lipid peroxidation product, were found in every affected patient, especially in group II and III patients. Activities and protein expressions of Mn-dependent superoxide dismutase (Mn-SOD), CuZn-dependent superoxide dismutase (CuZn-SOD), and catalase were decreased in all patients, especially in group III patients. Glutathione peroxidase (GPx) activity was decreased only in group III patients. Asymptomatic carriers without any clinical manifestations showed normal TBARS level and GSH/GSSG ratio with increases in both GSH and GSSG levels. Their CuZn-SOD, Mn-SOD, and catalase activities were increased. These results suggest that excessive copper-derived oxidants contribute to development and progression of liver disease in WD.

Two novel gene mutations (Glu174→Lys, Phe383→Tyr) causing the “hepatic” form of carnitine palmitoyltransferase II deficiency

Abstract Carnitine palmitoyltransferase II (CPT II) deficiency has two different clinical forms, one with “hepatic” and the other with “muscular” symptoms. We studied the molecular basis of the “hepatic” form in two Japanese siblings. Their CPT II activity in lymphoblasts was reduced to 3% of the level observed in normal controls. cDNA analysis showed that the proband was a compound heterozygote. One allele carried a new mutation, G621→A (Glu174→Lys). The other carried three single-base substitutions; a new mutation, T1249→A (Phe383→Tyr), and two previously reported polymorphisms. The brother had the same four substitutions. Neither of the two new mutations in this study was detected in the 60 alleles of 30 Japanese control subjects. Secondary structure prediction analysis of the mutated CPT II protein was different from that of the normal protein. We concluded that these mutations caused the “hepatic” form of CPT II deficiency in the probands.

Fluctuating liver functions in siblings with MPV17 mutations and possible improvement associated with dietary and pharmaceutical treatments targeting respiratory chain complex II

BACKGROUND/AIMS To describe the clinical and biological findings of two Japanese siblings with novel MPV17 gene mutations (c.451insC/c.509C > T) manifesting hepatic mitochondrial DNA depletion syndrome. METHODS We observed these brothers and sought to determine the efficacy of treatment targeting respiratory chain complex II for the younger brother. RESULTS A 3-month-old boy had presented with profound liver dysfunction, failure to thrive, and watery diarrhea. Although he was then placed on a carbohydrate-rich diet, his liver function thereafter fluctuated greatly in association with viral infections, and rapidly deteriorated to liver failure. He underwent liver transplantation at 17 months of age but died at 22 months of age. The younger brother, aged 47 months at the time of this writing, presented with liver dysfunction from 8 months of age. His transaminase levels also fluctuated considerably fluctuations in association with viral infections. At 31 months of age, treatment with succinate and ubiquinone was initiated together with a lipid-rich diet using ketone milk. Thereafter, his transaminase levels normalized and never fluctuated, and the liver histology improved. CONCLUSIONS These cases suggested that the clinical courses of patients with MPV17 mutations are greatly influenced by viral infections and that dietary and pharmaceutical treatments targeting the mitochondrial respiratory chain complex II may be beneficial in the clinical management of MPV17 mutant patients.

Histological findings in the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency

Aim:  To characterize the histological features of the livers of patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), we studied specimens from 30 patients diagnosed with NICCD by genetically analyzing the SLC25A13 gene.

A novel mutation (c.951C>T) in an exonic splicing enhancer results in exon 10 skipping in the human mitochondrial acetoacetyl-CoA thiolase gene.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inherited disorder affecting isoleucine catabolism and ketone body metabolism. A Japanese female developed a severe ketoacidotic attack at the age of 7 months. Urinary organic acid analysis showed elevated excretion of 2-methyl-3-hydroxybutyrate but not tiglylglycine. She was diagnosed as having T2 deficiency by enzyme assay using fibroblasts. Mutation analysis revealed a compound heterozygote of c.556G>T(D186Y) and c.951C>T(D317D). Since c.951C>T does not cause amino acid change, we performed cDNA analysis and found that exon 10 skipping had occurred in the c.951C>T allele. A computer search using an ESE finder showed that an exonic splicing enhancer sequence, SF2/ASF, was located in CTGA(951)CGC. We hypothesized that the exonic splicing enhancer is necessary for accurate splicing since the first nucleotide of exon 10 is C, which weakens the splice acceptor site of intron 9. We made a mini gene construct including exon 9-truncated intron 9-exon 10-truncated intron 10-exon 11 for a splicing experiment. We also made three mutant constructs which alter the SF2/ASF site (947C>T, 951C>T, 952G>A). An min-gene splicing experiment clearly showed that exon 10 skipping was induced in all three mutant constructs. Moreover, additional substitution of G for C at the first nucleotide of exon 10 resulted in normal splicing in these three mutants. These results confirmed that c.951C>T diminished the effect of the exonic splicing enhancer and caused exon 10 skipping.

Cloning and sequence analysis of a full length cDNA encoding human mitochodrial 3-oxoacyl-CoA thiolase

The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.

Molecular and clinical analyses of Japanese patients with carbamoylphosphate synthetase 1 (CPS1) deficiency.

AbstractCarbamoylphosphate synthetase I deficiency (CPS1D) is a urea-cycle disorder characterized by episodes of life-threatening hyperammonemia. Correct diagnosis is crucial for patient management, but is difficult to make from clinical presentation and conventional laboratory tests alone. Enzymatic or genetic diagnoses have also been hampered by difficult access to the appropriate organ and the large size of the gene (38 exons). In this study, in order to address this diagnostic dilemma, we performed the largest mutational and clinical analyses of this disorder to date in Japan. Mutations in CPS1 were identified in 16 of 18 patients with a clinical diagnosis of CPS1D. In total, 25 different mutations were identified, of which 19 were novel. Interestingly, in contrast to previous reports suggesting an extremely diverse mutational spectrum, 31.8% of the mutations identified in Japanese were common to more than one family. We also identified two common polymorphisms that might be useful for simple linkage analysis in prenatal diagnosis. The accumulated clinical data will also help to reveal the clinical presentation of this rare disorder in Japan.