Yoko Nakajima

Edaravone, a novel free radical scavenger, reduces high-mobility group box 1 and prolongs survival in a neonatal sepsis model.

Free radicals play an important role in the inflammatory process of sepsis. We hypothesized that edaravone, a novel free radical scavenger, can suppress pathophysiological events and prolong survival in a neonatal sepsis cecal ligation and perforation (CLP) model. Of 32 3-day-old anesthetized and mechanically ventilated piglets, 11 received CLP only, 10 received CLP and edaravone treatment starting 30 min after CLP, and 11 constituted a sham (control) group. Mean arterial pressure (MAP), heart rate, cardiac output, arterial blood gas, serum total hydroperoxide, nitrite and nitrate, TNF-&agr;, and high-mobility group box 1 (HMGB1) were measured before CLP and at 1, 3, and 6 h after CLP. Compared with the CLP group, the edaravone group showed higher MAP at 6 h, lower heart rate at 1 and 3 h, lower total hydroperoxide at 1 h, lower nitrite and nitrate at 3 and 6 h, and higher (although not significantly so) mean cardiac output at 1, 3, and 6 h. TNF-&agr; elevation was delayed from 1 h in the CLP group to 3 h in the edaravone group. In the edaravone group, HMGB1 did not change significantly at any time, whereas in the CLP group, it increased at 6 h. Survival times were longer in the edaravone group than in the CLP group (15.4 ± 1.4 vs. 10.2 ± 1 h; P < 0.005). In addition, each of the serial dilutions of edaravone had a higher biological antioxidant potential than tempol does. In conclusion, edaravone suppressed free radicals, delayed the TNF-&agr; surge, and prevented HMGB1 elevation, thereby maintaining MAP and prolonging survival time in a neonatal sepsis CLP model.

Evaluation of valproate effects on acylcarnitine in epileptic children by LC-MS/MS

BACKGROUND Valproate (VPA) is a simple fatty acid and a substrate for the fatty acid β-oxidation pathway. Previous data suggested that the toxicity of VPA may be provoked by carnitine deficiency and the inhibition of mitochondrial β-oxidation. OBJECTIVE The aim of the present study was to elucidate the effect of VPA treatment on carnitine and isomer-differentiated acylcarnitine disposition, and determined the relationships between acylcarnitines and blood VPA levels in long-term treated patients with VPA and/or other antiepileptic drugs. METHODS Serum samples were obtained from children aged 1-15 years old treated for at least 6 months with VPA alone (n=28) or VPA combined with other anticonvulsants (n=23) and untreated controls (n=23). Serum acylcarnitines were separated from their isomers and quantified using high-performance liquid chromatography-tandem mass spectrometry. RESULTS We found higher 3-hydroxyisovalerylcarnitine levels and trace amounts of valproylcarnitine in both VPA monotherapy and polytherapy patients. Other acylcarnitines, hexanoylcarnitine, C12, C14:1-carnitines and the ratio of long-chain acylcarnitine to free carnitine were also higher in VPA polytherapy individuals than in controls. VPA monotherapy does not result in decreases in free carnitine or in the accumulation of long-chain acylcarnitines. Blood VPA concentrations correlated positively with hexanoylcarnitine, C12, C14:1, C16:1, C18:1-carnitines in all VPA-treated children (n=51). CONCLUSION Long-term VPA treatment in pediatric patients could affect some specific acylcarnitines, which is enhanced by the concomitant use of other anticonvulsants, and the formation of valproylcarnitine alone seems insufficient to develop severe carnitine deficiency at therapeutic doses of VPA.

Children undergoing liver transplantation for treatment of inherited metabolic diseases are prone to higher oxidative stress, complement activity and transforming growth factor-β1.

BACKGROUND Main indications for liver transplantation in the pediatric population include biliary atresia and inherited metabolic diseases. The present study evaluated whether there are differences between pediatric patients undergoing living-related liver transplantation due to the two diseases in terms of their oxidative and immunological status during their regular outpatient follow-up visits. MATERIAL AND METHODS A clinical outpatient study measuring serum oxidative stress index (calculated as serum oxidant/antioxidant ratio, in the form of serum total hydroperoxide/serum biological antioxidative potential), serum terminal complement component 5a, as an indicator of complement activity and immunological status, and transforming growth factor-ß1, as a marker of liver fibrosis, in 16 patients (6 males and 10 females, 2.5-15 years old) who received living-related liver transplantation due to inherited metabolic diseases (n=6; in the form of propionic acidemia [n=1], methylmalonic acidemia [n=1], arginase deficiency [n=1], tyrosinemia [n=2], and glycogen storage disease type 1b [n=1], with an age range of 2.4-14.6 years old) and due to biliary atresia ([n=10], with an age range of 2.9-14.5 years old). RESULTS Serum oxidative stress index, complement component-5a, and transforming growth factor-ß1 were significantly higher in the inherited metabolic diseases group than in the biliary atresia group. In all patients, serum oxidative stress index correlated positively with complement component-5a and transforming growth factor-ß1. CONCLUSIONS Patients who receive living-related liver transplantation due to inherited metabolic diseases are prone to higher oxidative stress, complement activity, and serum transforming growth factor-ß1.

Establishment and directed differentiation of induced pluripotent stem cells from glycogen storage disease type Ib patient

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose‐6‐phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell‐based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2‐year‐old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC‐derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase‐3 and caspase‐9, consistent with the GSDIb patients neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.

Pitfall in the Diagnosis of Fructose-1,6-Bisphosphatase Deficiency: Difficulty in Detecting Glycerol-3-Phosphate with Solvent Extraction in Urinary GC/MS Analysis.

Fructose-1,6-bisphosphatase (FBPase), an enzyme involved in gluconeogenesis, catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. FBPase deficiency is an autosomal recessive inherited disorder, characterized by episodic attacks of hypoglycemia, ketosis, and lactic acidosis during fasting. In general, urinary organic acid analysis using gas chromatography-mass spectrometry (GC/MS) is very useful for the diagnosis of FBPase deficiency, because the appearance of glycerol or glycerol-3-phosphate in the urine is characteristic of this disease. Here, we report a case of FBPase deficiency in a girl with a history of several severe lactic acidosis events, both as a neonate and after the age of 12 months. The patient was identified as a compound heterozygote with two mutations in the FBPase 1 gene: c.841G>A (p.Glu281Lys) and c.960_961insG (p.Ser321fs). The c.841G>A is a newly identified pathogenic mutation. An abnormal level of glycerol-3-phosphate was not detected in the conventional urinary organic acid analysis using GC/MS after solvent extraction. This method, which is a widely used diagnostic standard, could not detect increased levels of glycerol or glycerol-3-phosphate in the patients urine, which was sampled during the episode. However, glycerol and glycerol-3-phosphate were detected in the same sample, when it was analyzed using GC/MS with the urease pretreatment non-extraction method. Patients with FBPase deficiency have good glycemic control after correct treatment. Therefore, accurate and early diagnosis is essential for a good prognosis. Accordingly, when a patient presents with hypoglycemia and lactic acidosis, it is important to select the appropriate method of urinalysis for organic acids by GC/MS.

Successful living donor liver transplantation for classical maple syrup urine disease

MSUD is an autosomal recessive condition characterized by a deficiency in the enzyme, BCKDH, which catalyzes the breakdown of BCAAs. If left untreated, MSUD can result in mental retardation, central nervous system disorders, and even death. Most patients with MSUD are treated with a restricted protein diet and milk from which BCAAs have been removed. LT has been shown effective in patients with MSUD. This report describes the case of a 15‐month‐old boy who received a liver graft from his mother. Transplantation was successful, and the patient was then able to ingest a normal diet. Despite episodes of acute rejection, chylous ascites, and high fever (40 °C), he has shown no evidence of MSUD recurrence. These findings indicate that patients with MSUD can be successfully treated by LDLT, even when the donor is a heterozygous carrier of a mutated BCKDH gene.

Kinetic and molecular orbital analyses of dicarboxylic acylcarnitine methylesterification show that derivatization may affect the screening of newborns by tandem mass spectrometry

Newborns are routinely screened for organic acidemias by acylcarnitine analysis. We previously reported the partial catalytic methylesterification of dicarboxylic acylcarnitines by benzenesulfonic acid moiety in the solid extraction cartridge during extraction from serum. Since the diagnosis of organic acidemias by tandem mass spectrometry is affected by the higher molecular weight of these derivatized acylcarnitines, we investigated the methylesterification conditions. The kinetic constants for the methylesterification of carboxyl groups on the acyl and carnitine sides of carnitine were 2.5 and 0.24h(-1), respectively. The physical basis underlying this difference in methylesterification rates was clarified theoretically, illustrating that methylesterification during extraction proceeds easily and must be prevented.

Identification of a novel synonymous mutation in the human β -Ureidopropionase Gene UPB1 affecting pre-mRNA splicing.

β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyzes the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, and ammonia and CO2. To date, only 16 genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report the clinical, biochemical, and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Mutation analysis of the UPB1 gene showed that the patient was compound heterozygous for a novel synonymous mutation c.93C >T (p.Gly31Gly) in exon 1 and a previously described missense mutation c.977G >A (p.Arg326Gln) in exon 9. The in silico predicted effect of the synonymous mutation p.Gly31Gly on pre-mRNA splicing was investigated using a minigene approach. Wild-type and the mutated minigene constructs, containing the entire exon 1, intron 1, and exon 2 of UPB1, yielded different splicing products after expression in HEK293 cells. The c.93C >T (p.Gly31Gly) mutation resulted in altered pre-mRNA splicing of the UPB1 minigene construct and a deletion of the last 13 nucleotides of exon 1. This deletion (r.92_104delGCAAGGAACTCAG) results in a frame shift and the generation of a premature stop codon (p.Lys32SerfsX31). Using a minigene approach, we have thus identified the first synonymous mutation in the UPB1 gene, creating a cryptic splice-donor site affecting pre-mRNA splicing.

Parieto-occipital encephalomalacia in neonatal hyperammonemia with ornithine transcarbamylase deficiency: A case report

Urea cycle disorders are congenital metabolic disorders that often cause episodic hyperammonemia. Neuroimaging in episodic hyperammonemia demonstrates several patterns of brain injuries, including focal lesions in the lentiform nucleus, insula, cingulate gyrus, and perirolandic fissure, as well as diffuse cerebral edema. In cases with neonatal onset of hyperammonemia, similar lesions have also been reported. We herein report a boy with severe neonatal hyperammonemia caused by ornithine transcarbamylase deficiency. He presented with parieto-occipital encephalomalacia, which resembles severe neonatal hypoglycemia on magnetic resonance imaging. This radiological finding may indicate parieto-occipital vulnerability not only to hypoglycemia but also to hyperammonemia.

Development of tandem mass spectrometry-based creatinine measurement using dried blood spot for newborn mass screening

BackgroundCongenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent etiologies of pediatric chronic kidney disease (CKD). However, no robust mass screening methods have been developed to detect patients with CAKUT, making early intervention to prevent progressive renal failure challenging.MethodsWe applied tandem mass spectrometry (MS/MS) to measure the creatinine (Cr) value from dried blood spot (DBS) analysis, which has been used for newborn mass screening. Subsequently, we evaluated the correlation between DBS Cr measured by MS/MS and serum Cr measured by the conventional method in pediatric patients with CKD. Finally, DBS Cr was measured in 190 full-term, healthy newborns on days 4–6 after birth.ResultsWe established a system of MS/MS-based measurement of Cr from DBS. Measured DBS Cr in the pediatric patients showed a strong association with serum Cr (r=0.86; P<0.01). The median DBS Cr value in newborns was 0.222 (interquartile range: 0.189, 0.269) mg/dl. No significant correlations were found between DBS Cr values and body weight, Apgar score, gestational age, and sex in newborns.ConclusionWe successfully established a method for MS/MS-based measurement of Cr for newborn screening and determined normal reference values for full-term newborns.