A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.
ABSTRACT Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.
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ABSTRACT: The crude protein fraction of human duodenal contents catalyzed the formation of ceramide from sphingomyelin most efficiently at slightly alkaline pH in the presence of conjugated bile salts. Under optimal conditions 1 ml duodenal contents hydrolyzed up to 300 nmole sphingomyelin per h. The hydrolysis of ceramide to sphingosine and free fatty acids and the reverse reaction were also catalyzed. The pH optimum of this reaction was about 7.6. That the enzymes are of intestinal origin was indicated by the data obtained when sphingomyelin and ceramide were incubated with rat pancreatic juice, pancreas homogenate and homogenate of pig intestinal mucosa. The preparations all had low sphingomyelinase activity at a pH of about 5, but only the pig mucosa homogenate had significant hydrolytic activity against sphingomyelin at alkaline pH and against oleoyl-sphingosine at pH 7.6. The latter enzymic activities were enriched in a brush-border preparation of rat intestinal mucosa to the same extent as the alkaline phosphatase activity. The brush border may therefore be the main site of hydrolysis of dietary sphingomyelin.Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism. 01/1969;
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ABSTRACT: Severe deficiency of acid ceramidase activity (4-5% of normal) was demonstrated in cultured skin fibroblasts, leukocytes and plasma from a 1-year-old boy who was diagnosed as being affected with Farber disease. Determination of ceramidase activity in plasma was achieved by a highly sensitive assay employing a ceramide substrate containing radiolabeled C12 N-acyl moiety (N-lauryl). The enzyme activity in the parents' leukocytes and plasma was found to be reduced to 18-47% of the respective normal values, and that determined in a plasma specimen from a patient with I-cell disease was about 4 times elevated above the normal level.Clinical Genetics 08/1989; 36(1):38-42. · 3.94 Impact Factor
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ABSTRACT: We describe a patient with the biochemically established combination of Farber and Sandhoff disease. A 6-month-old girl of consanguineous Turkish parents presented with hoarseness, stridor, scattered skin nodules, painful swelling of hand joints and ankles, and cherry-red macular spots. Until the age of 2 years her motor and physical condition deteriorated distinctly, however her mental state remained unchanged. A biopsied skin nodule disclosed lysosomal inclusions within storage cells that were typical of Farber disease (curved tubular structures). However, other inclusions (e.g. zebra bodies) were also found. Biochemical findings included ceramide accumulation in skin nodules and cultured fibroblasts, impaired ceramide degradation on loading of cultured fibroblasts with radioactive sphingomyelin, profoundly decreased ceramidase activity in fibroblasts as well as total beta-hexosaminidase activity in fibroblasts and serum, absent hexosaminidase A and B bands on cellogel zymograms, increased urinary oligosaccharide excretion of the Sandhoff disease type, and a partial reduction of ceramidase and total beta-hexosaminidase activities in fibroblasts from her father. A diagnosis of combined Farber and Sandhoff disease was made. The effect of both enzyme deficiencies on the clinical manifestations in this patient and the genetic basis of this combination require further studies.European Journal of Pediatrics 05/1989; 148(6):558-62. · 1.91 Impact Factor