Development of a Diagnostic Screening Strategy for Niemann-Pick Diseases Based on Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analyses of N-Palmitoyl-O-phosphocholine-serine and Sphingosylphosphorylcholine.

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (method 1) and a validated LC-MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations. Nexera and API 5000 were used as LC-MS/MS systems. C18 columns with lengths of 10 and 50 mm were used for method 1 and 2, respectively. 2H3-Labeled PPCS and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 and 8 min were set for methods 1 and 2, respectively. In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC-MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

Metabolism of Non-Enzymatically Derived Oxysterols: Clues from sterol metabolic disorders.

Cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7ß-hydroxycholesterol (7ß-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3ß,5α,6ß-triol, 7-OC and 7ß-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3ß,5α,6ß-triol, 7-OC and 7ß-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3ß,5α,6ß-trihydroxycholanoic, 3ß-hydroxy-7-oxochol-5-enoic and 3ß,7ß-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma.

Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry.

BACKGROUND: There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life. OBJECTIVE: To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases. METHODS AND RESULTS: Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0). DISCUSSION: A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

Comprehensive Evaluation of Plasma 7-Ketocholesterol and Cholestan-3ß,5α,6ß-Triol in an Italian Cohort of Patients Affected by Niemann-Pick Disease due to NPC1 and SMPD1 Mutations.

Niemann-Pick C disease (NPCD) is a rare autosomal recessive neurovisceral disorder with a heterogeneous clinical presentation. Cholestan-3ß,5α,6ß-triol and 7-ketocholesterol have been proposed as biomarkers for the screening of NPCD. In this work, we assessed oxysterols levels in a cohort of Italian patients affected by NPCD and analyzed the obtained results in the context of the clinical, biochemical and molecular data. In addition, a group of patients affected by Niemann-Pick B disease (NPBD) were also analyzed. NPC patients presented levels of both oxysterols way above the cut off value, except for 5 siblings presenting the variant biochemical phenotype who displayed levels of 3ß,5α,6ß-triol below or just above the cut-off value; 2 of them presented also normal levels of 7-KC. Both oxysterols were extremely high in a patient presenting the neonatal systemic lethal phenotype. All NPB patients showed increased oxysterols levels. In conclusion, the reported LC-MS/MS assay provides a robust non-invasive screening tool for NPCD. However, false negative results can be obtained in patients expressing the variant biochemical phenotype. These data strengthen the concept that the results should always be interpreted in the context of the patients' clinical picture and filipin staining and/or genetic studies might still be undertaken in patients with normal levels of oxysterols if symptoms are highly suggestive of NPCD. Both oxysterols are significantly elevated in NPB patients; thus a differential diagnosis should always be performed in patients presenting isolated hepatosplenomegaly, a common clinical sign of both NPCD and NPBD.

Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.

Increased copper levels in in vitro and in vivo models of Niemann-Pick C disease.

Niemann-Pick type C disease (NPC) is a hereditary neurovisceral atypical lipid storage disorder produced by mutations in the NPC1 and NPC2 genes. The disease is characterized by unesterified cholesterol accumulation in late endosomal/lysosomal compartments and oxidative stress. The most affected tissues are the cerebellum and the liver. The lysotropic drug U18666A (U18) has been widely used as a pharmacological model to induce the NPC phenotype in several cell culture lines. It has already been reported that there is an increase in copper content in hepatoma Hu7 cells treated with U18. We confirmed this result with another human hepatoma cell line, HepG2, treated with U18 and supplemented with copper in the media. However, in mouse hippocampal primary cultures treated under similar conditions, we did not find alterations in copper content. We previously reported increased copper content in the liver of Npc1 (-/-) mice compared to control animals. Here, we extended the analysis to the copper content in the cerebella, the plasma and the bile of NPC1 deficient mice. We did not observe a significant change in copper content in the cerebella, whereas we found increased copper content in the plasma and decreased copper levels in the bile of Npc1(-/-) mice. Finally, we also evaluated the plasma content of ceruloplasmin, and we found an increase in this primary copper-binding protein in Npc1 (-/-) mice. These results indicate cell-type dependence of copper accumulation in NPC disease and suggest that copper transport imbalance may be relevant to the liver pathology observed in NPC disease.

[Comparison and clinical application of two methods for determination of plasma chitotriosidase activity].

OBJECTIVE: Chitotriosidase (CT) is a plasma biomarker for Gaucher disease (GD), the enzyme activity is usually markedly elevated in plasma of Gaucher patients, and it was reported that levels of plasma chitotriosidase activity was mildly-moderately increased in patients with Niemann-Pick disease (NPD). The aim of this study was to compare chitotriosidase activity using 4-methylumbelliferyl-ß-D-N, N', N″-triacetyl-chitotrioside (4MU-C3) with 4-methylumbelliferyl 4-deoxy-ß-D-chitobiose (4MU-4dC2) as substrates, and apply chitotriosidase activity measurement to help clinical determination of GD and NPD, and to monitor therapy in GD patients. METHOD: Plasma of 45 healthy individuals, 31 patients with GD and 9 patients with NPD type A/B was collected from outpatient clinics of the Department of Pediatric Endocrinologic, Genetic and Metabolic Diseases, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Plasma chitotriosidase activity was measured with the substrates 4MU-C3 and 4MU-4dC2 respectively. Determinations were based on the methods described by Hollak et al and Rodrigues et al. Meanwhile, common mutation dup24 of the human chitotriosidase gene was detected. RESULT: (1) Chitotriosidase activity when measured with 4MU-4dC2 gave higher values than 4MU-C3. In the healthy controls chitotriosidase activity was increased 3.7-fold when the 4MU-dC2 was used as substrate as compared with the 4MU-C3 (Z = -4.703, P < 0.001). In the untreated GD patients, the median value was increased 794-fold and 610-fold of the control subjects (Z = -3.823, P < 0.001) when the enzyme was measured with two substrates respectively. In the GD patients during therapy, chitotriosidase activity was increased 134-fold and 79-fold, and after changing therapeutic dose chitotriosidase activity was increased 215-fold and 118-fold of the controls (Z = -2.521, P < 0.05). In the NPD patients chitotriosidase activity was increased 8-fold and 14-fold of the controls (Z = -1.604, P = 0.109). (2) Consistent with the results of chitotriosidase activity, 30 of 85 (35.3%) individuals were homozygotes of dup24 mutation, which are completely chitotriosidase enzyme deficiency. Among GD patients with wild-type and heterozygotes for the dup24 mutation, chitotriosidase activity highly increased in the plasma compared with the controls. CONCLUSION: The use of 4MU-4dC2 as substrate makes chitotriosidase activity measurement more sensitive. The determination of plasma chitotriosidase activity is a useful tool to assist the clinical identification of Gaucher disease, and to monitor enzyme replacement therapy (ERT) of non-chitotriosidase deficient GD patients. Chitotriosidase activity determination has no value in the clinical identification of NPD.

Identification of novel biomarkers for Niemann-Pick disease using gene expression analysis of acid sphingomyelinase knockout mice.

Although several therapies are available or being developed for lysosomal storage disorders (LSDs), assessment of therapeutic efficacy is challenged by the lack of markers to assess disease progression and severity. This is particularly true for rare diseases such as LSDs, since natural history data from human populations are often lacking. Herein we describe the use of gene expression analysis in the acid sphingomyelinase-deficient mouse model (ASMKO) of Types A and B Niemann-Pick disease (NPD) to identify novel serum biomarkers. We used microarray and real-time PCR analyses to compare mRNA expression in ASMKO and normal mice in two important sites of pathology, lung and brain, and from these data identified and validated several potential biomarkers. The cytokine MIP-1alpha was markedly elevated in ASMKO mouse serum, and following enzyme replacement therapy (ERT) it was reduced to normal levels. Total iron levels were similarly elevated in ASMKO mice, reflective of the elevated ferritin light chain transcript, and decreased to normal after ERT. Serum growth hormone levels were also elevated in ASMKO mice and were reduced to normal after brain-directed gene therapy, but not ERT. These studies illustrate the value of gene expression analysis for the identification of biomarkers, and provide new insight into the pathobiology of NPD.

Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B.

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass ( approximately 50-100%). Analysis of LCAT kinetics parameters (V(max) and K(m)) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [(3)H]SM-labeled LpA-I and HDL(3). Furthermore, expression of mutant SMase (DeltaR608) in CHO cells revealed that DeltaR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.

ESI-MS quantitation of increased sphingomyelin in Niemann-Pick disease type B HDL.

HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for approximately 25-44% of total SM molecular species. Similarly, we observed an increase of approximately 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.

[Metabolic abnormalities with hematologic manifestation]. / Anomalii metabolice cu expresie hematologica.

These are unusual diseases in pediatric pathology. We present the morphologic aspects of peripheral blood smear and bone marrow smear of nine storage disease cases.

The natural history of type B Niemann-Pick disease: results from a 10-year longitudinal study.

OBJECTIVES: Type B Niemann-Pick disease (NPD-B) caused by acid sphingomyelinase deficiency is a rare, autosomal recessive, lysosomal storage disorder with a broad range of disease severity. The objectives of this study were to document the natural history of the disease in a large, clinically heterogeneous patient population that was followed for a period of 10 years and to determine how genotype influences phenotype. METHODS: Twenty-nine patients with NPD-B had serial evaluations at least 9 months apart. Organ volumes, hematologic indices, lipid concentrations, pulmonary function, and hepatic activity were studied, and individual phenotypic severity was compared with genotype. RESULTS: All patients with intact spleens had splenomegaly (mean value: 12.7 multiples of normal [MN]; range: 4.5-27.3 MN), and all but 1 had hepatomegaly (mean volume: 1.91 MN; range: 0.93-3.21 MN). At initial visit, 39% had thrombocytopenia and 3% had leukopenia. At final visit, the percentages increased to 54% and 34%, respectively. Mean annual decreases in platelet count and leukocyte count were 7 x 10(3) and 0.2 x 10(3) per mm3, respectively. The typical atherogenic lipid profile was worse in older patients. A total of 69% of patients had low diffusion capacity for carbon monoxide, and more than one third had low forced expiratory volume in 1 second, forced vital capacity, and forced expiratory volume in 1 second/forced vital capacity at initial visit. All measurements of pulmonary function showed a gradual deterioration over time. Liver dysfunction was characterized by stable elevation of hepatic transaminases and bilirubin. Homozygotes for DeltaR608, P323A, and P330R had milder disease than patients with all other genotypes. CONCLUSIONS: The natural history of NPD-B is characterized by hepatosplenomegaly with progressive hypersplenism, worsening atherogenic lipid profile, gradual deterioration in pulmonary function, and stable liver dysfunction.

Lipid abnormalities in children with types A and B Niemann Pick disease.

OBJECTIVE: To characterize the lipid profiles in patients with types A and B Niemann Pick disease (NPD) and determine if lipid abnormalities are associated with evidence of early cardiovascular disease or correlate with genotype. STUDY DESIGN: The study was a cross-sectional analysis of 10 patients with NPD type A and 30 patients with NPD type B that was carried out in the General Clinical Research Center. For each patient, fasting lipid profile and glucose, T4, height or length, weight, resting blood pressure, and acid sphingomyelinase deficiency genotype were measured. In type B patients, electrocardiograhic-gated helical computed tomography of the heart also was obtained. RESULTS: Lipid abnormalities included low (<35 mg/dL) high-density lipoprotein cholesterol in 100% of patients and hypertriglyceridemia and increased low-density lipoprotein cholesterol in 62% (25/40) and 67% (27/40) of patients, respectively. Coronary artery calcium scores were positive (>1.0) in 10 of 18 type B patients studied. There was no correlation of the Delta R608 genotype with a milder phenotype for the lipid abnormalities, as has been observed for a number of other NPD manifestations. CONCLUSIONS: Lipid abnormalities are part of the phenotype in types A and B NPD and may be associated with early atherosclerotic heart disease.

Growth restriction in children with type B Niemann-Pick disease.

OBJECTIVES: To compare growth of children with type B Niemann-Pick disease (NPD) with disease variables including genotype, organomegaly, bone age, and serum insulin-like growth factor-1 (IGF-1). STUDY DESIGN: A cross-sectional analysis of growth was performed in 23 children and adolescents with enzymatically and genotypically confirmed NPD. Liver and spleen volumes were measured by quantitative computed tomography and skeletal age by a wrist radiograph. RESULTS: The mean Z scores for height and weight were -1.24 (29th percentile) and -0.75 (34th percentile). The mean liver and spleen volumes were 2.06 and 13.46 times normal for weight, respectively. Skeletal age was delayed by an average of 2.5 years, and serum IGF-1 level was at or below the 2nd percentile in 8 of 12 patients. Short stature and low weight were significantly correlated with large organ volumes, delayed bone age, and low IGF-1 levels. In contrast to patients with other mutations, individuals homozygous for the DeltaR608 mutation had normal height and weight, markedly less hepatosplenomegaly and bone age delay, and normal IGF-1 levels. CONCLUSIONS: Abnormal linear growth and delayed skeletal maturation are common in children and adolescents with type B NPD; however, homozygosity for DeltaR608 is associated with normal growth.

A fluorescence-based, high-performance liquid chromatographic assay to determine acid sphingomyelinase activity and diagnose types A and B Niemann-Pick disease.

Acid sphingomyelinase (ASM; sphingomyelin phosphodiesterase, EC 3.1.4.12) is the lysosomal enzyme that hydrolyzes sphingomyelin (SPM) to phosphorylcholine and ceramide. An inherited deficiency of ASM activity results in Types A and B Niemann-Pick disease (NPD). In this study we report a new assay method to detect ASM activity and diagnose NPD using the fluorescent substrate BODIPY C12-SPM and reverse-phase high-performance liquid chromatography (HPLC). The reaction product, BODIPY C12-ceramide (B12Cer), could be clearly and efficiently separated from the substrate within 4 min using a reverse-phase column (Aquasil C18, Keystone Scientific). Femtomole quantities of B12Cer could be detected in as little as 1.0 micro l of human plasma, providing a sensitive measure of ASM activity. The mean ASM activity in human plasma from NPD patients (36 pmol/ml/h) was only 2.7% of that in normal plasma (1334 pmol/ml/h), confirming the specificity and diagnostic value of this new assay method. Importantly, the mean ASM activity in human plasma from NPD carriers (258.3 pmol/ml/h) also was significantly reduced (19.5% of normal). The ranges of ASM plasma activities in NPD patients (N=19), NPD carriers (N=11), and normal subjects (N=15) were 2.5-97.3, 108-551, and 1030-2124 pmol/ml/h, respectively. Based on these results, we suggest that this fluorescence-based HPLC assay method is a reliable, rapid, and highly sensitive technique to determine ASM activity and that plasma is a very reliable and simple source for the accurate diagnosis of NPD patients and carriers based on ASM activity.

A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.

Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C.

Niemann-Pick disease, type C, was diagnosed in a 3-month-old boy with hepatosplenomegaly, mild signs of cholestasis, hepatic inflammation and extramedullary erythropoiesis, together with chronic airway disease. He developed muscular hypotonia, psychomotor retardation, rickets, and signs of peripheral neuropathy. The patient was found to excrete abnormal amounts of unusual bile acids in urine at 3 and 5 months of age. These acids were shown to have a 3beta-hydroxy-Delta(5) structure and to carry an oxo or hydroxy group at C-7. They were sulfated at C-3 and nonamidated or conjugated with glycine or taurine at C-24. Part of the 7-hydroxy acids, presumably the 7beta-hydroxylated one, was also conjugated with N-acetylhexosamine, probably N-acetylglucosamine, at the 7-hydroxy group. Possible metabolic pathways for the formation of the 7-oxo and 7beta-hydroxycholenoic acids are discussed. Based on previous data concerning the effects of 3beta-hydroxy-Delta(5) bile acids on bile acid transport, it is suggested that the formation of such bile acids is responsible for the cholestasis in this patient.

An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins which can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase, and the resultant accumulation of sphingomyelin in cells and tissues. In this paper we report a new, enzyme-based method to quantify the levels of sphingomyelin in tissues and plasma of normal individuals and NPD patients. The method utilizes sphingomyelinase from Bacillus cereus to completely hydrolyze the sphingomyelin into ceramide. Quantification of the sphingomyelin-derived ceramide is accomplished using Escherichia coli diacylglycerol (DAG) kinase and [gamma-(32)P]ATP. The resulting [(32)P]ceramide is quantified using a phosphor-imager system following TLC separation. This procedure allowed quantification of sphingomyelin over a broad range from 10 pmol to 1 nmol. To validate this assay we quantified sphingomyelin in plasma and tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma was significantly elevated compared to that of normal mice (up to twofold). Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also were significantly elevated compared to normal individuals of the same age. Based on these results we propose that this new, enzyme-based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful tool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method provides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract.

Treatment of patients with Niemann-Pick type is using repeated amniotic epithelial cells implantation: correction of aggregation and coagulation abnormalities.

Variations of platelet aggregation and plasma levels of clotting factors V, IX, XI, and XII were studied in 5 patients with Niemann-Pick disease type Is in the course of a 3-year study of treatment with periodic subcutaneous infusions of amniotic epithelial cells. Before commencement of treatment, the concentrations of these factors were found to be abnormal in four of five patients. It was possible to complete the study protocol in only two patients. Platelet aggregation and plasma levels of V, IX, XI, and XII clotting factors had been determined before each epithelial amniotic cells implantation and after 24, 48, and 72 hours. In both patients the aggregation test and the plasma levels of coagulation factors V, IX, XI, and XII were below the normal values of reference. Results showed that the epithelial amniotic cells treatment normalized platelet aggregation after each implantation in the two studied patients, both in terms of intensity of response (increase in light transmission after addition of adenosine diphosphate up to 350%) and in terms of obtaining an irreversible aggregation with 3 and 8 microM of adenosine diphosphate. The data related to clotting factors showed an increase of these concentrations up to 60% and some of these concentrations normalized completely.