||Yuichi Mushimoto, Yuki Hasegawa, Hironori Kobayashi, Hong Li, Jamiyan Purevsuren, Isamu Nakamura, Takeshi Taketani, Seiji Fukuda, Seiji Yamaguchi, Enzymatic evaluation of glutaric acidemia type 1 by an in vitro probe assay of acylcarnitine profiling using fibroblasts and electrospray ionization/tandem mass spectrometry (MS/MS), JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 10.1016/j.jchromb.2009.04.043, Vol.877, No.25, pp.2648-2651, 2009.09, Glutaric acidemia type 1 (GA1) is usually diagnosed with an accumulation of glutaric acid (GA) or 3-hydroxyglutaric acid by GC/MS. In some cases, however, excretion of GA is low. We investigated enzymatic evaluation of GA1 using fibroblasts and MS/MS. After loading substrates, lysine, 2-aminoadipate (2AA), or GA, in fibroblasts, and incubating for 96 h, glutarylcarnitine (C5DC) levels in the media were measured. A significant increase of C5DC was observed in GA1 patients, irrespective of substrates added. 2AA showed the largest difference between patients and controls (p = 0.0004). Results suggested enzymatic evaluation of GA1 is useful under appropriate culture conditions. (C) 2009 Elsevier B.V. All rights reserved..
||J. Purevsuren, T. Fukao, Y. Hasegawa, H. Kobayashi, S. Fukuda, S. Yamaguchi, CLINICAL AND MOLECULAR ASPECTS OF JAPANESE PATIENTS WITH MITOCHONDRIAL TRIFUNCTIONAL PROTEIN DEFICIENCY, MOLECULAR GENETICS AND METABOLISM, 10.1016/j.ymgme.2009.07.011, Vol.98, No.1-2, p.48, 2009.09.
||Mitsuru Endo, Yuki Hasegawa, Seiji Fukuda, Hironori Kobayashi, Yuka Yotsumoto, Yuichi Mushimoto, Hong Li, Jamiyan Purevsuren, Seiji Yamaguchi, In vitro probe acylcarnitine profiling assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry predicts severity of patients with glutaric aciduria type 2, JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, 10.1016/j.jchromb.2010.03.029, Vol.878, No.20, pp.1673-1676, 2010.06, Glutaric aciduria type 2 (multiple acyl-CoA dehydrogenase deficiency. MAD) is a multiple defect of mitochondrial acyl-CoA dehydrogenases due to a deficiency of election transfer flavoprotein (ETF) cm ETF dehydrogenase The clinical spectrum are relatively wide from the neonatal onset, severe form (MAD-S) to the late-onset, milder form (MAD-M) In the present study, we determined whether the in vitro probe acylcarnitine assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry (MS/MS) can evaluate then clinical severity or not Incubation of cells from MAD-S patients with palmitic acid showed large increase in palmitoylcarnitine (C16), whereas the downstream acylcarnitines, C14. C12, C10 or C8 as well as C2. were extremely low In contrast, accumulation of C16 was smaller while the amount of downstream metabolites was higher in fibroblasts from MAD-M compared to MAD-S The ratio of C16/C14. C16/C12, or C16/C10. in the culture medium was significantly higher in MAD-S compared with that in MAD-M Loading octanoic acid or myristic acid led to a significant elevation in C8 or C12, respectively in MAD-S. while their effects were less pronounced in MAD-M In conclusion, it is possible to distinguish MAD-S and MAD-M by in vitro probe acylcarnitine profiling assay with various fatty acids as substrates This strategy may be applicable for other metabolic disorders (C) 2010 Elsevier B V All rights reserved.
||Li H, Fukuda S, Hasegawa Y, Kobayashi H, Purevsuren J, Mushimoto Y, Yamaguchi S, Heat Stress Deteriorates Mitochondrial β-Oxidation of Long-chain Fatty Acids in Cultured Fibroblasts with Fatty Acid β-Oxidation Disorders, Journal of Chromatography B, 10.1016/j.jchromb.2010.01.046, Vol.878, No.20, pp.1669-1672, 2010.06.
||Hong Li, Seiji Fukuda, Yuki Hasegawa, Hironori Kobayashi, Jamiyan Purevsuren, Yuichi Mushimoto, Seiji Yamaguchi, Effect of heat stress and bezafibrate on mitochondrial beta-oxidation: Comparison between cultured cells from normal and mitochondrial fatty acid oxidation disorder children using in vitro probe acylcarnitine profiling assay, BRAIN & DEVELOPMENT, 10.1016/j.braindev.2009.06.001, Vol.32, No.5, pp.362-370, 2010.05, Hyperpyrexia occasionally triggers acute life-threatening encephalopathy-like illnesses, including influenza-associated encephalopathy (IAE) in childhood, and can be responsible for impaired fatty acid beta-oxidation (FAO). In this regard, patients with impaired FAO may be more susceptible to febrile episodes. The effects of heat stress and a hypolipidemic drug, bezafibrate, on mitochondrial FAO were investigated using cultured cells from children with FAO disorders and from normal controls, using an in vitro probe acylcarnitine (AC) profiling assay. Fibroblasts were incubated in medium loaded with unlabelled palmitic acid for 96 h at 37 and 41 degrees C, with or without bezafibrate. AC profiles in culture medium were analyzed by electrospray ionization tandem mass spectrometry. Heat stress, introduced by 41 degrees C, significantly increased acetylcarnitine (C2) but slightly decreased the other acylcarnitines (ACs) in controls and medium-chain acyl-CoA dehydrogenase (MCAD)-deficient cells. On the other hand, in very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient cells, accumulation of long-chain ACs were enhanced at 41 degrees C, compared with that at 37 degrees C. In contrast, bezafibrate decreased long-chain ACs with significant increase of C2 in both control and VLCAD-deficient cells at 37 degrees C. These data suggest that heat stress specifically inhibits long-chain FAO, whereas bezafibrate recovers the impaired FAO. Our approach is a simple and promising strategy to evaluate the effects of heat stress or therapeutic drugs on mitochondrial FAO. (c) 2009 Elsevier B.V. All rights reserved..
||S. Yamaguchi, H. Li, J. Purevsuren, Y. Mushimoto, H. Kobayashi, Y. Hasegawa, S. Fukuda, EFFECT OF HEAT STRESS AND BEZAFIBRATE ON MITOCHONDRIAL FATTY ACID OXIDATION (FAO) IN FAO DISORDERS: EVALUATION BY IN VITRO PROBE ACYLCARNITINE ASSAY, JOURNAL OF INHERITED METABOLIC DISEASE, Vol.33, p.S57, 2010.08.
||Yuichi Mushimoto, Seiji Fukuda, Yuki Hasegawa, Hironori Kobayashi, Jamiyan Purevsuren, Hong Li, Takeshi Taketani, Seiji Yamaguchi, Clinical and molecular investigation of 19 Japanese cases of glutaric acidemia type 1, MOLECULAR GENETICS AND METABOLISM, 10.1016/j.ymgme.2010.11.159, Vol.102, No.3, pp.343-348, 2011.03, Glutaric acidemia type 1 (GA1) is a metabolic disease caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Untreated patients mostly develop severe striatal degeneration. More than 200 mutations have been reported in the GCDH gene, and common R402W and IVS10-2A > C were found in Caucasian and Chinese/Taiwanese, respectively. However, in Japan, genetic mutations have only been reported in a few cases. Herein, we report the clinical and molecular basis of GA1 in 19 Japanese patients, including six previously reported patients. All cases showed high urinary glutaric acid excretion. Eleven patients were severely impaired (three patients died), three had mild impairment, and five showed normal development. Four of 5 patients that developed normally were detected in the presymptomatic stage by neonatal or sibling screening. Nineteen mutations in 26 alleles were identified, and eight of them (89 or 90delC, Y155C, IVS4+2T C, G244S, Q352X, G354A, K361E, and 1144-1145delGC) were novel. S305L (12.1%, 4/34 alleles) was found in several cases, suggesting that this mutation is a common mutation. In contrast, R402W was not identified and IVS10-2A>C was only found in one allele, suggesting that Japanese patients with GA1 show allelic heterogeneity and have a different genetic background to patients from other countries. One of a pair of sisters with the same mutations (M339V/S305L) lacking residual activity was severely retarded, whereas the older girl remains asymptomatic at 22 years of age, indicating that genotype does not necessarily predict GA1 phenotype. We consistently found that there was no association between genotype and phenotype. However, children with mild impairment were diagnosed and treated earlier than severely impaired cases (4.7 +/- 2.5 months (range: 2-8 months) vs. 11.6 +/- 12.7 months (range: 4-51 months)). Our results suggest that early detection and treatment but not genotype are associated with better patient outcome, reinforcing the importance of neonatal screening. (C) 2010 Elsevier Inc. All rights reserved..
||Takashi Hamajima, Yuichi Mushimoto, Hironori Kobayashi, Yoshiro Saito, Kaxumichi Onigata, Novel compound heterozygous mutations in the SBP2 gene: characteristic clinical manifestations and the implications of GH and triiodothyronine in longitudinal bone growth and maturation (vol 166, pg 757, 2012), EUROPEAN JOURNAL OF ENDOCRINOLOGY, 10.1530/EJE-11-0812e, Vol.166, No.5, p.957, 2012.05.
||S. Yamaguchi, J. Purevsuren, K. Yamada, T. Takahashi, Y. Mushimoto, H. Kobayashi, Y. Hasegawa, M. Takayanagi, S. Fukuda, INTRACELLULAR ACYLCARNITINE PROFILING USING IN VITRO PROBE ASSAY AT VARIOUS C0 CONCENTRATIONS SELECTIVELY IDENTIFIES CPT-1 DEFICIENCY AND PRIMARY CARNITINE DEFICIENCY, JOURNAL OF INHERITED METABOLIC DISEASE, Vol.35, p.S69, 2012.09.