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.

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.

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.

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.

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.

The effect of cholesterol-lowering agents on hepatic and plasma cholesterol in Niemann-Pick disease type C.

Niemann-Pick disease type C (NP-C) is a neurovisceral lipidosis characterized by defective intracellular trafficking of cholesterol and lysosomal accumulation of unesterified cholesterol, believed to be an offending metabolite. We studied the effect of cholesterol-lowering agents on hepatic and plasma cholesterol levels in NP-C by randomly assigning 25 patients with NP-C to one of five treatment regimens containing different combinations of cholestyramine, lovastatin, nicotinic acid, or dimethyl sulfoxide (DMSO). Unesterified cholesterol content was measured in liver biopsies before and after 4 months' treatment. All drug regimens except DMSO alone reduced hepatic and plasma cholesterol levels. Toxicity was limited and did not prevent any patient from completing the study. The combination of cholestyramine, lovastatin, and nicotinic acid lowered cholesterol levels in liver and blood with minimal side effects. A controlled clinical study will be necessary to determine if this regimen influences the rate of neurologic progression.

Sea blue histiocytosis in a patient with chronic non-neuropathic Niemann-Pick disease.

A patient with Niemann-Pick disease is reported together with family studies. Her liver and bone marrow were shown to be infiltrated with sea blue histiocytes. Other organs, spleen and lung, were presumably also involved but histological proof was not obtained. Enzyme assay of leucocytes, lymphocytes, and cultured skin fibroblasts showed the patient to be deficient in sphingomyelinase activity. In fibroblasts, activity was 5% of normal while for the parents activity was about 50% of normal. The expected partial deficiency was not found using leucocytes or lymphocytes from the parents. Heat stability studies on fresh fibroblast extracts from the propositus indicated that residual sphingomyelinase activity was slightly more labile than that of the controls. It seems clear that chronic Niemann-Pick disease without neurological involvement is associated with sea blue histiocytosis.

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.

Niemann-Pick disease in association with homozygous hemoglobin E: a case report.

The first case of Niemann-Pick disease Type A in a Thai infant was reported. The patient also had abnormal hemoglobin E. The diagnosis was based on the clinical features, bone marrow findings and sphingomyelinase levels in the culture of skin fibroblasts. The autosomal recessive mode of inheritance was confirmed in this case.

[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.

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.

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.

[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.