Nobuo Sakura

Long-Term Treatment and Diagnosis of Tetrahydrobiopterin-Responsive Hyperphenylalaninemia with a Mutant Phenylalanine Hydroxylase Gene

A novel therapeutic strategy for phenylketonuria (PKU) has been initiated in Japan. A total of 12 patients who met the criteria for tetrahydrobiopterin (BH4)-responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) (EC 1.14.16.1) gene were recruited at 12 medical centers in Japan between June 1995 and July 2001. Therapeutic efficacy of BH4 was evaluated in single-dose, four-dose, and 1-wk BH4 loading tests followed by long-term BH4 treatment, and also examined in relation to the PAH gene mutations. The endpoints were determined as the percentage decline in serum phenylalanine from initial values after single-dose (>20%), four-dose (>30%), and 1-wk BH4 (>50%) loading tests. Patients with mild PKU exhibiting decreases in blood phenylalanine concentrations of >20% in the single-dose test also demonstrated decreases of >30% in the four-dose test. The 1-wk test elicited BH4 responsiveness even in patients with poor responses in the shorter tests. Patients with mild HPA, many of whom carry the R241C allele, responded to BH4 administration. No clear correlation was noted between the degree of decrease in serum phenylalanine concentrations in the single- or four-dose tests and specific PAH mutations. The 1-wk test (20 mg/kg of BH4 per day) is the most sensitive test for the diagnosis of BH4-responsive PAH deficiency. Responsiveness apparently depends on mutations in the PAH gene causing mild PKU, such as R241C. BH4 proved to be an effective therapy that may be able to replace or liberalize the phenylalanine-restricted diets for a considerable number of patients with mild PKU.

Newborn mass screening and selective screening using electrospray tandem mass spectrometry in Japan.

Electrospray tandem mass spectrometry was applied to detect a series of inherited metabolic disorders during a newborn-screening pilot study and a selective screening in Japan. In our mass screening of 102,200 newborns, five patients with propionic acidemia, two with methylmalonic acidemia, two with medium-chain acyl-CoA dehydrogenase deficiency, three with citrullinemia type II, and one with phenylketonuria were identified. In a selective screening of 164 patients with symptoms mainly related to hypoglycemia and/or hyperammonemia, 12 with fatty acid oxidation disorders and six with other disorders were found. The results indicated the importance of newborn screening using this technology in Japan.

Plasma total homocysteine concentrations in epileptic patients taking anticonvulsants

Plasma total homocysteine (tHcy) and serum folate (FA) concentrations were measured in 130 epileptic patients taking anticonvulsant drugs. A significant inverse correlation was found between FA and tHcy. This was greater in the older group (> or = 15 years) than in the younger group (1 to 14 years). There were four FA-deficient patients (FA concentration < 3 ng/mL regardless of symptoms), including three patients in the older group and one in the younger group. All FA-deficient patients had received long-term treatment (> 7 years) with multiple anticonvulsants. Their tHcy levels were higher than the 90th percentile of those in control subjects. Two patients showed extremely high levels of tHcy (57.9 and 29.1 mumol/L) and subnormal plasma methionine levels. After FA therapy, their tHcy decreased to levels the same as or less than those of control subjects and FA increased to above the normal range. Based on these findings, we conclude that measuring FA and tHcy concentrations may be useful for preventing thrombosis due to hyperhomocysteinemia in epileptic patients taking anticonvulsants, particularly those who receive long-term treatment with multiple agents.

Dermatan sulfate and heparan sulfate as a biomarker for mucopolysaccharidosis I

Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by deficiency of α-L-iduronidase leading to accumulation of its catabolic substrates, dermatan sulfate (DS) and heparan sulfate (HS), in lysosomes. This results in progressive multiorgan dysfunction and death in early childhood. The recent success of enzyme replacement therapy (ERT) for MPS I highlights the need for biomarkers that reflect response to such therapy. To determine which biochemical markers are better, we determined serum and urine DS and HS levels by liquid chromatography tandem mass spectrometry in ERT-treated MPS I patients. The group included one Hurler, 11 Hurler/Scheie, and two Scheie patients. Seven patients were treated from week 1, whereas the other seven were treated from week 26. Serum and urine DS (ΔDi-4S/6S) and HS (ΔDiHS-0S, ΔDiHS-NS) were measured at baseline, week 26, and week 72. Serum ΔDi-4S/6S, ΔDiHS-0S, and ΔDiHS-NS levels decreased by 72%, 56%, and 56%, respectively, from baseline at week 72. Urinary glycosaminoglycan level decreased by 61.2%, whereas urine ΔDi-4S/6S, ΔDiHS-0S, and ΔDiHS-NS decreased by 66.8%, 71.8%, and 71%, respectively. Regardless of age and clinical severity, all patients showed marked decrease of DS and HS in blood and urine samples. We also evaluated serum DS and HS from dried blood-spot samples of three MPS I newborn patients, showing marked elevation of DS and HS levels compared with those in control newborns. In conclusion, blood and urine levels of DS and HS provide an intrinsic monitoring and screening tool for MPS I patients.

Plasma total glutathione concentrations in epileptic patients taking anticonvulsants.

Glutathione plays an important role in protecting cells against oxidative damage as a free radical scavenger. Since several anticonvulsants have been associated with decreased intrahepatic glutathione levels, we investigated plasma concentrations of total glutathione (including reduced and oxidized forms, tGSH=GSH+GSSG) in 45 epileptic patients taking anticonvulsant drugs. Plasma tGSH concentrations were significantly lower than in controls in patients treated with carbamazepine or phenytoin monotherapy, or with multiple drugs. Plasma tGSH concentrations in patients treated with valproic acid and in patients treated with phenobarbital did not differ significantly from those in controls. In no patient group was a significant correlation evident between duration of treatment or drug concentration and plasma tGSH concentration. No significant differences in plasma total cysteine concentrations were found between any patient group and controls. We conclude that some anticonvulsant drugs can lower plasma tGSH levels, reflecting treatment-related oxidative stress.

Betaine and homocysteine concentrations in foods

Background : Betaine (Bet) supplementation is an effective strategy for dietary treatment of homocystinuria. However,previous reports on diet therapy have only examined methionine (Met) and cystine concentrations, but not those of Bet and homocysteine(Hcy) in food items. We set up a hypothesis that there are some food items, which contain a small amount of Met, but a great amountof Hcy and Bet.

Plasma total glutathione concentrations in healthy pediatric and adult subjects

Glutathione, as a free radical scavenger, plays an important role in protecting cells against oxidative damage. Since aging is associated with decreased plasma glutathione levels, we investigated plasma concentrations of total glutathione (including the reduced and oxidized forms, tGSH=GSH+GSSG) and total cysteine in 129 healthy pediatric and adult subjects. Plasma tGSH concentrations were significantly lower in infants than in adults. There were no significant differences in plasma tGSH concentrations between the children and adult group. No significant differences in plasma total cysteine concentrations were found between any pediatric and adult group. We conclude that lower plasma tGSH levels in pediatric subjects may reflect greater oxidative stress during the neonatal and infant period.

Unexpectedly high prevalence of the mild form of propionic acidemia in Japan: presence of a common mutation and possible clinical implications

Abstract. Propionic acidemia [MIM 606054] is a form of organic acidemia caused by genetic deficiency of propionyl-CoA carboxylase (PCC) and characterized by attacks of severe metabolic acidemia and hyperammonemia beginning in the neonatal period or in early infancy. There are, however, patients who have higher PCC activities and present later with unusual symptoms, such as mild mental retardation or extrapyramidal symptoms, sometimes even without metabolic acidosis. Through the neonatal screening of more than 130,000 Japanese newborns we detected a frequency of patients with propionic acidemia more than ten times higher than previously reported, most of them with milder phenotypes. The mutational spectrum was quite different from that of patients with the severe form and there was a common mutation (Y435C) in the β subunit of the PCC gene (PCCB). Since patients with the mild form could present with unusual symptoms and therefore could easily remain unrecognized, it is important to identify those patients and clarify their natural history. Molecularly, one of the mutations (A1288C) caused an unusual pattern of multiple exon skipping and another unidentified mutation lead to the absence of mRNA. Taking into consideration previous findings regarding PCCB mutations, it appears that this gene is particularly prone to posttranscriptional modifications such as missense mediated exon skipping, mRNA decay, or rapid product degradation.

Circadian rhythms in plasma cortisone and cortisol and the cortisone/cortisol ratio.

We evaluated the circadian rhythms in the plasma concentrations of cortisol, cortisone and their free forms, and in the cortisone/cortisol ratios by means of reversed-phase high performance liquid chromatography in normal adult subjects. Plasma concentrations of cortisone, as well as cortisol, exhibited a circadian rhythm. The ratios of cortisone/cortisol remained almost constant during the waking hours of normal subjects. Changes in the cortisone/cortisol ratios previously reported in patients with various diseases exceeded the diurnal changes detected in the present study. Thus, the determination of the cortisone/cortisol ratio provides information that is useful in assessing the adrenal function of patients with various diseases.

Differential diagnosis of neonatal mild hypergalactosaemia detected by mass screening: clinical significance of portal vein imaging.

Summary: The aetiology of hypergalactosaemia in 100 neonates detected by screening using the Paigen method is discussed. Hypergalactosaemia was transient in 94 cases and persistent in 6. The aetiology among transient cases was unknown in 55, delayed closure of the ductus venosus in 19, heterozygous UDP-galactose 4-epimerase (GALE) deficiency in 16, and heterozygous galactose-1-phosphate uridyltransferase (GALT) deficiency in 6. The aetiology among persistent cases was hepatic haemangioendothelioma with portovenous shunting in 2, and patent ductus venosus with hypoplasia of the intrahepatic portal vein, citrin deficiency, homozygous GALE deficiency, and heterozygous GALE deficiency in one patient each. The abnormalities of the portal system were identified ultrasonographically at the initial consultation and measurements of the total bile acid and α-fetoprotein concentrations were helpful in resolving the differential diagnosis. The causes of hypergalactosaemia varied, but a major cause was portosystemic shunt. Evaluation of patients with hypergalactosaemia should not be limited to enzymatic analysis, but should also include hepatic imaging, especially ultrasonography. Additionally, determination of total bile acids and α-fetoprotein is helpful in identifying the aetiology of hypergalactosaemia in infants.