Takehisa Fujiwaki

Application of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in tissues from sphingolipidosis patients

Sphingolipidosis is due to defects in enzymes involved in hydrolysis of sphingolipids. We analyzed sphingolipids in tissues from patients with sphingolipidosis, including Farber disease (FD, acid ceramidase deficiency), Gaucher disease (GD), Niemann-Pick disease type C (NPDC), and GM1-gangliosidosis (GM1G), using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS). Crude lipids were extracted from about 100 mg wet weight of autopsied tissues, including liver, spleen, cerebrum or cerebellum. After mild alkaline treatment, a sphingolipid fraction was prepared from the crude lipids and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) In FD liver both the ceramide/sphingomyelin and ceramide/monohexosylceramide ratios were significantly high; (b) in both liver and spleen from a GD patient, the glucosylceramide/sphingomyelin ratio was raised; (c) in liver from a NPDC patient, the monohexosylceramide/sphingomyelin ratio was markedly low, suggesting an increase of sphingomyelin; and (d) in all tissues examined in the GM1G patient, GM1-gangliosides or asialo-GM1-gangliosides, that are undetectable in a normal control, were increased. In conclusion, sphingolipids in human tissues could be directly determined by DE MALDI-TOF-MS, with only a small amount of specimens. This method will be useful for the diagnosis and biochemical evaluation of sphingolipidosis patients.

Quantitative evaluation of sphingomyelin and glucosylceramide using matrix-assisted laser desorption ionization time-of-flight mass spectrometry with sphingosylphosphorylcholine as an internal standard. Practical application to tissues from patients with Niemann-Pick disease types A and C, and Gaucher disease.

Niemann-Pick disease types A and C, and Gaucher disease are glycolipid storage disorders characterized by the systemic deposition of glycosphingolipids, i.e., sphingomyelin in Niemann-Pick disease types A and C tissues and glucosylceramide in Gaucher disease ones, respectively. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we analyzed the sphingolipids in liver and spleen specimens from patients with Niemann-Pick disease types A and C, and Gaucher disease. Crude lipids were extracted from tissue containing 5mg protein with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by MALDI-TOF/MS. The results were as follows: (a) ion peaks with m/z values corresponding to different sphingomyelin and ceramide monohexoside (CMH) species were clearly detected. (b) With sphingosylphosphorylcholine as the internal standard for quantification of sphingomyelin and CMH, the relative peak heights of sphingomyelin and CMH were calculated and plotted versus their contents. The relative peak heights of sphingomyelin and CMH showed linearity between 50 and 1500 ng sphingomyelin content, and between 5 and 150 ng CMH content, respectively. (c) Quantitative analysis revealed the accumulation of sphingomyelin in the liver and spleen specimens from the patients with Niemann-Pick disease types A and C. Striking accumulation of CMH was also detected in the liver and spleen specimens from the patients with Gaucher disease. This investigation indicated that accumulated sphingomyelin and CMH in small amounts of tissues from sphingolipidosis patients can be detected quantatively with the MALDI-TOF/MS method. This method will be useful not only for the diagnosis but also for biochemical pathophysiology evaluation of patients with various sphingolipidosis.

Application of delayed extraction–matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in pericardial fluid, peritoneal fluid and serum from Gaucher disease patients

Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE-MALDI-TOF-MS), we analyzed sphingolipids in pericardial fluid, peritoneal fluid, and serum from two patients with Gaucher disease. Crude lipids were extracted from 1 ml each of pericardial fluid, peritoneal fluid, and serum with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE-MALDI-TOF-MS. The results were as follows: (a) in all the specimens, peaks of ceramide monohexoside and sphingomyelin were detected in both the controls and Gaucher disease patients; (b) in pericardial fluid, peritoneal fluid, and serum, the ceramide monohexoside/sphingomyelin ratio was increased in the Gaucher disease patients compared with in the controls. It was indicated that the accumulation of ceramide monohexoside in such samples from Gaucher disease patients can be easily detected with this DE-MALDI-TOF-MS method.

Application of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry for analysis of sphingolipids in cultured skin fibroblasts from sphingolipidosis patients

Sphingolipidoses are caused by defects of enzymes involved in the hydrolysis of sphingolipids. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS), we analyzed sphingolipids in cultured skin fibroblasts from patients with sphingolipidoses, including: (a) Farber disease (FD, acid ceramidase deficiency); (b) Gaucher disease (GD); (c) Niemann-Pick disease type C (NPDC); and (d) GM1-gangliosidosis (GM1G). Crude lipids were extracted from about 50 mg wet weight of cultured skin fibroblasts. After mild alkaline treatment, a sphingolipid fraction was prepared from the crude lipids and analyzed by DE MALDI-TOF-MS. The results were as follows: (a) in fibroblasts from the FD patient, the ceramide/sphingomyelin and ceramide/monohexosylceramide ratios were both significantly high; (b) in the GD patient, the glucosylceramide/sphingomyelin ratio was increased; on the other hand; (c) in the NPDC patient, the monohexosylceramide/sphingomyelin ratio was within normal range; and (d) in the GM1G patient, no specific data were obtained. Sphingolipids in cultured fibroblasts can be evaluated by DE MALDI-TOF-MS, whereas GM1-ganglioside or its asialo derivatives are not detectable. With this DE MALDI-TOF-MS method, ceramide or monohexosylceramide accumulating in cultured fibroblasts from cases of sphingolipidoses, such as FD and GD, respectively, can be easily detected.

Neonatal lupus erythematosus associated with maternal mixed connective tissue disease

Neonatal lupus erythematosus (NLE) is characterized by typical clinical features and the presence of maternal autoantibodies. The major clinical manifestations in neonates are cardiac, dermatologic, and hepatic. A previous paper reported the association of maternal Sjogren’s syndrome, systemic lupus erythematosus and NLE. 1 In the present report we describe a case of NLE born to a mother with mixed connective tissue disease (MCTD).

Substitutions of Three Amino Acids in Human Heart/Muscle Type Carnitine Palmitoyltransferase I Caused by Single Nucleotide Polymorphisms

Heart/muscle type carnitine palmitoyltransferase I (M-CPTI) catalyzes the rate-limiting step of mitochondrial long-chain fatty acid (LCFA) oxidation in muscle and adipose tissue. Three replacements of nucleotides resulting in missense mutations of I66V, S427C, and E531K were observed in the M-CPTI gene of patients showing abnormal fatty acid metabolism. These nucleotide replacements were found to be common single nucleotide polymorphisms (SNPs) of this gene and not specific to patients. The question of whether these missense mutations caused by SNPs alter the functional properties of M-CPTI remains unanswered. Thus, we examined whether these missense mutations are associated with any changes in the enzymatic properties of M-CPTI. None of these mutations was found to cause remarkable alteration of its enzymatic properties. Based on the comparison of amino acid sequences of M-CPTI among different animal species, the roles of these amino acids in the enzyme are discussed.

Slowly progressive sleep apnea in late‐onset central hypoventilation syndrome

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Acute gastric mucosal lesions caused by Helicobacter pylori initial infection

Helicobacter pylori infection is highly prevalent in asymptomatic children. Initial infection probably occurs at an early age and its prevalence increases with age. 1 There is strong evidence for an association between H. pylori infection and gastritis, a duodenal ulcer disease in children, but the clinical features of an initial infection need further evaluation. 1 We present here the case of a 7-year-old boy with acute gastric mucosal lesions (AGML) caused by H. pylori infection. This case is suggestive for the comprehension of the clinical features of an initial infection and the natural history of H. pylori .

Postnatal remission of ocular, auditory, and somatic findings in Stickler syndrome

Stickler syndrome is a relatively common, dominantly inherited skeletal dysplasia. In general, intrafamilial variations tend to be less conspicuous while interfamilial variations are significant. The clinical hallmarks include ocular abnormalities, sensorineural hearing impairment, mid-face hypoplasia, micrognathia with a cleft palate, and generalized skeletal alterations. Ocular abnormalities comprise highly congenital myopia, vitreoretinal degeneration, retinal detachment, and cataracts, which worsen with age and often lead to blindness. Hearing impairment is generally mild, but occasionally requires a hearing aid. Skeletal alterations are classifiable as a mild form of spondyloepiphyseal dysplasia, resulting in premature degenerative joint disease. The radiological hallmark is metaphyseal broadening of the long bones, which may cause joint restriction. Stickler syndrome is genetically heterogeneous. The causative genes that have been identified to date include COL2A1 , COL11A1 , and COL11A2 , which are responsible for Stickler syndrome type I (STL1), Stickler syndrome type II (STL2), and Stickler syndrome type III (STL3), respectively. Craniofacial abnormalities and bone changes are indistinguishable among these subtypes. Ocular manifestations are severe in STL1, mild in STL2, and absent in STL3, whereas hearing impairment is mild in STL1, and severe in STL2 and 3. We encountered an affected woman and her affected son with a COL2A1 mutation. In the affected son, congenital cataracts, retinal degeneration, and hearing impairment regressed with age. The prenatal growth failure was also significantly caught-up during infancy. The distinctive clinical course is reported here.