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Background: Niemann-Pick type C (NPC) is a treatable genetic disorder, mainly characterized by neurological dysfunction and liver damage. Its diagnosis is based on an invasive test which requires a skin biopsy to demonstrate the cholesterol accumulation in culture fibroblasts of affected patients. In the last years, new biomarkers have been investigated aiming to facilitate the diagnosis and screening of NPC; two of these possible candidates are the oxysterol cholestane-3β,5α,6β-triol (triol), a product of non-enzymatic oxidation of cholesterol, and the enzyme chitotriosidase (CT), a fully active chitinase (EC-3.2.1.14) synthesized by activated macrophages. Methods: In this work, we investigated the potential use of the combined analysis of triol levels and CT activity for diagnosis, screening and monitoring of NPC in Brazilian patients, correlating with the results of Filipin staining and genetic analysis. We studied 122 untreated individuals with clinical suspicion of NPC who were separated in two groups according their concentrations of triol (higher or lower than the cutoff value of 100 ng/mL). We also analyzed blood samples from 5 patients with previous diagnosis of NPC who were under treatment with miglustat. Results:The results of this work demonstrated that patients with higher concentrations of triol (group A) also presented a high activity of CT and most of them had also a positive Filipin test. Two patients of this group presented an inconclusive Filipin test, being one eventually diagnosed as NPC by molecular investigation and the other eventually diagnosed as Niemann-Pick type A or B (NPA/B) by the low acid sphingomyelinase activity presented. Three patients with high triol concentrations who had a negative result in the Filipin test presented low activities of acid sphingomyelinase, being diagnosed as NPA/B. On the other hand, triol concentrations were normal in NPC patients treated with miglustat, although CT activity in these individuals remained abnormal. In the patients with triol lower than 100 ng/mL (group B), most presented a normal activity of CT. No patient of this group had a positive Filipin test and the few patients with inconclusive Filipin test did not present pathogenic mutations in the NPC1 or NPC2 genes. Conclusions: In conclusion, our data demonstrated that the combined analysis of triol and CT is quite sensitive and specific for the identification of NPC patients. Although the number of analysis in NPC patients treated with miglustat was small, the data indicate that the measurement of triol could also be potentially useful for treatment monitoring.

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Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is an inborn error of metabolism characterized by the accumulation of glycosaminoglycans (GAG) in lysosomes. Enzyme replacement therapy (ERT) can reduce GAG storage, ameliorate symptoms, and slow disease progression. Oxidative damages may contribute to the MPS II pathophysiology, and treatment with ERT might reduce the effects of oxidative stress. We evaluated levels of DNA damage (including oxidative damage) and chromosome damage in leukocytes of long-term-treated MPS II patients, by applying the buccal micronucleus cytome assay. We observed that, despite long-term ERT, MPS II patients had higher levels of DNA damage and higher frequencies of micronuclei and nuclear buds than did control. These genetic damages are presumably due to oxidation: we also observed increased levels of oxidized guanine species in MPS II patients. Therapy adjuvant to ERT should be considered, in order to decrease oxidative damage and cytogenetic alterations.

Background: Interest in screening methods for lysosomal storage diseases (LSDs) has increased in recent years, since early diagnosis and treatment are essential to prevent or attenuate the onset of symptoms and the complications of these diseases. In the current work, we evaluated the performance of tandem mass spectrometry (MS/MS) for the detection of some LSDs, aiming the future use of this methodology for the screening of these disorders. Methods: Standard curves and quality control dried blood spots were assayed to evaluate the precision, linearity, and accuracy. A total of 150 controls were grouped according to age and subjected to measurement of lysosomal enzymes deficient in Niemann-Pick A/B, Krabbe, Gaucher, Fabry, Pompe, and Mucopolysaccharidosis type I diseases. Samples from 59 affected patients with a diagnosis of LSDs previously confirmed by fluorimetric methods were analyzed. Results: Data from standard calibration demonstrated good linearity and accuracy and the intra-and interassay precisions varied from 1.17% to 11.60% and 5.39% to 31.24%, respectively. Except for galactocerebrosidase and a-L-iduronidase, enzyme activities were significantly higher in newborns compared to children and adult controls. Affected patients presented enzymatic activities significantly lower compared to all control participants. Conclusion: Our results show that MS/MS is a promising methodology, suitable for the screening of LSDs, but accurate diagnoses will depend on its correlation with other biochemical and/or molecular analyses.