Untargeted LC-HRMS metabolomics reveals candidate biomarkers for mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses (MPSs) are inherited genetic diseases caused by an absence or deficiency of lysosomal enzymes responsible for catabolizing glycosaminoglycans (GAGs). Undiagnosed patients, or those without adequate treatment in early life, can be severely and irreversibly affected by the disease. In this study, we applied liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics to identify potential biomarkers for MPS disorders to better understand how MPS may affect the metabolome of patients. METHODS: Urine samples from 37 MPS patients (types I, II, III, IV, and VI; untreated and treated with enzyme replacement therapy (ERT)) and 38 controls were analyzed by LC-HRMS. Data were processed by an untargeted metabolomics workflow and submitted to multivariate statistical analyses to reveal significant differences between the MPS and control groups. RESULTS: A total of 12 increased metabolites common to all MPS types were identified. Dipeptides, amino acids and derivatives were increased in the MPS group compared to controls. N-acetylgalactosamines 4- or 6-sulfate, important constituents of GAGs, were also elevated in MPS patients, most prominently in those with MPS VI. Notably, treated patients exhibited lower levels of the aforementioned acylaminosugars than untreated patients in all MPS types. CONCLUSIONS: Untargeted metabolomics has enabled the detection of metabolites that could improve our understanding of MPS physiopathology. These potential biomarkers can be utilized in screening methods to support diagnosis and ERT monitoring.

Oral exfoliative cytology for the diagnosis of paracoccidoidomycosis in a patient with human immunodeficiency virus: a case report.

BACKGROUND: Paracoccidioidomycosis is not the most common fungal disease in patients infected with human immunodeficiency virus (HIV), except for endemic regions in Latin America countries. CASE: A 33-year-old man with HIV presented with mulberry-like lesions on the palate. The diagnosis was made by exfoliative cytology and Papanicolaou staining. Microscopic analysis revealed fungal structures with birefringent walls and exosporulation conferring an airplane radial motor appearance, or even bowel-like or goblet-like forms compatible with Paracoccidioides brasiliensis. CONCLUSION: This process spares the immunosuppressed patient from undergoing invasive biopsy procedures.

Early hematopoietic stem cell transplantation in a patient with severe mucopolysaccharidosis II: A 7 years follow-up.

Mucopolysaccharidosis type II (MPS II - Hunter syndrome) is an X-linked lysosomal storage disorder caused by a deficiency in the enzyme iduronate-2 sulfatase (I2S), leading to the accumulation of the glycosaminoglycans, affecting multiple organs and systems. Enzyme replacement therapy does not cross the blood brain barrier, limiting results in neurological forms of the disease. Another option of treatment for severe MPS, hematopoietic stem cell transplantation (HSCT) has become the treatment of choice for the severe form of MPS type I, since it can preserve neurocognition when performed early in the course of the disease. To date, only few studies have examined the long-term outcomes of HSCT in patients with MPS II. We describe the seven-year follow-up of a prenatally diagnosed MPS II boy with positive family history of severe MPS form, submitted to HSCT with umbilical cord blood cells at 70 days of age. Engraftment after 30 days revealed mixed chimerism with 79% donor cells; after 7 years engraftment remains at 80%. I2S activity 30 days post-transplant was low in plasma and normal in leukocytes and the same pattern is observed to date. At age 7 years growth charts are normal and he is very healthy, although mild signs of dysostosis multiplex are present, as well as hearing loss. The neuropsychological evaluation (Wechsler Intelligence Scale for Children - Fourth Edition - WISC-IV), disclosed an IQ of 47. Despite this low measured IQ, the patient continues to show improvements in cognitive, language and motor skills, being quite functional. We believe that HSCT is a therapeutic option for MPS II patients with the severe phenotype, as it could preserve neurocognition or even halt neurodegeneration, provided strict selection criteria are followed.

Systematic analysis of glycerol: colourimetric screening and gas chromatography-mass spectrometric confirmation.

Glycerol is a naturally occurring polyol in the human body, essential for several metabolic processes. It is widely used in the food, pharmaceutical, and medical industries and in clinical practice as a plasma volume expander (PVE). Athletes, however, may use glycerol to mask the presence of forbidden substances or to enhance performance, inclusively through hyperhydration achieved by glycerol ingestion with added fluid. These practices are considered doping, and are prohibited by the World Anti-Doping Agency (WADA). Therefore, glycerol was introduced in the prohibited list. Doping through glycerol ingestion can readily be identified by detection of elevated glycerol concentrations in urine. In this paper, a protocol for the fast detection of glycerol in urine is proposed. It consists of a previous visual colourimetric screening, followed by a quantitative/qualitative confirmation analysis by mass spectrometry. The screening procedure involves a reaction in which polyhydric alcohols are oxidized by periodate to formic acid and formaldehyde, which is detected by the addition of a fuchsin solution. For the subsequent qualitative/quantitative confirmation analysis, a gas chromatography-mass spectrometry based approach with a non-deuterated internal standard and a drying step of only 10 min is proposed. The linear correlation was demonstrated within WADA´s threshold range. The calculated RSD were 2.1% for within-day precision and 2.8% for between-day precision. The uncertainty estimation was calculated, and a value of 2.7% was obtained. The procedure may also be used for the analysis of other polyols in urine, as for example the PVE mannitol.

Determination of six pterins in urine by LC-MS/MS.

BACKGROUND: The present work describes an analytical method for urinary pterins by LC-MS/MS, with emphasis on the separation of 6- and 7-positional isomers of bio- and neopterins. RESULTS: Urine sample preparation consisted of oxidation by MnO(2), filtration and direct dilution in the mobile phase. The method was validated in urine spiked at five concentration levels with true triplicates of each level. Separation of the pterins, including the positional isomers, was achieved by employing a LUNA amino column. Six pterins were quantified (pterin, isoxanthopterin, 6-biopterin, 7-biopterin, 6-neopterin, 7-neopterin) and a linear behavior was observed; LOD varied from 7 to 360 pg/ml and correlation coefficients above 0.98 were obtained for all pterins. In addition, pterin levels were evaluated in 41 urine samples of healthy subjects, in ten urine samples of patients with classical phenylketonuria (PKU) and in one with atypical PKU. CONCLUSION: The proposed method allowed to identify, separate and quantify six pterins in urine, using a simple and rapid sample preparation. The atypical PKU was unequivocally differentiated from the classical form, demonstrating that this method could be very useful for characterization and follow-up of diseases.

Plasma volume expanders: use in medicine and detecting misuse in sports.

Plasma volume expanders comprise a heterogeneous group of substances used in medicine that are intravenously administered in cases of great blood loss owing to surgery or medical emergency. These substances, however, can also be used to artificially enhance performance of healthy athletes in sport activities, and to mask the presence of others substances. These practices are considered doping, and are therefore prohibited by the International Olympic Committee and the World Antidoping Agency. Consequently, drug testing procedures are essential. The present work provides an overview of plasma volume expanders, assembling pertinent data such as chemical characteristics, physiological aspects, adverse effects, doping and analytical detection methods, which are currently dispersed in the literature.

Screening for hydroxyethyl starch (HES) doping in sport.

The artificial colloid hydroxyethyl starch (HES) is among the most frequently used plasma volume expanders in the medical field. However, in 1998, its misuse by the athletic community was officially reported and since 2000, HES is prohibited by the International Olympic Committee (IOC). Therefore, several methods enabling the detection of HES in urine were developed, most based on gas chromatography-mass spectrometry (GC-MS). In the present work, a simple and low-cost screening method, intended to reduce the number of samples to be analysed by GC-MS, was developed. The method is based on the acid hydrolysis of HES and detection of the resulting glucose and hydroxyethyl glucose derivatives by Benedict's reaction (reduction of copper sulfate to brick-red cuprous oxide by glucose and/or derivatives). Samples considered suspect were submitted to GC-MS analysis for identification of HES. The method was successfully applied for screening of HES in 2627 urine samples from 1346 Brazilian soccer players and 1281 athletes from the Pan-American Games (Rio de Janeiro, 2007); 71 (2.7%) samples, considered suspect, were submitted to GC-MS, but no positive results were found. Moreover, a thin layer chromatography (TLC) method was adapted for visualisation of the characteristic band pattern of HES hydrolysis products. The results indicate that the methods are efficient and useful for the screening of HES in urine.

Detection of Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) infantum chagasi in Brazilian bats.

Although bats are one of the most abundant mammals in the new world and are present in virtually all ecosystems, including urban and peri-urban environments, few studies have investigated the role of these animals in the epidemiological chain of leishmaniosis. Here, we report a study of 683 bats captured in São Paulo county (southeastern from Brazil), which were screened by serology, parasitologic methods and polymerase chain reaction (PCR) for trypanosomatids. The indirect immunofluorescent antibody test demonstrated that 0.9% of bats react positively for leishmaniosis and PCR detected the presence of DNA of Leishmania (Leishmania) amazonensis in 18 bats and Leishmania (Leishmania) infantum chagasi in 3 specimens. These results indicate that further studies are necessary to evaluate the role of bats in maintenance of the Leishmania life cycle, especially in areas where these diseases are endemic.

Human and canine leptospirosis: serological data of São Paulo City, Brazil, 2000 to 2003.

Sera from humans and dogs were tested by microscopic agglutination test. Icterohaemorrhagiae was the predominant serogroup in both groups, associated with the infestation of Rattus norvegicus.

A survey on inborn errors of metabolism in 323 neonates from the state of Rio de Janeiro, Brazil.

Inborn errors of metabolism (IEM) are a relevant cause of morbidity and death among children, and neonates in particular. However, little is known about the prevalence of these disorders in Brazilian newborns. Our laboratory of IEM (LABEIM) at the Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), has been working on the diagnosis of IEM since 1988. Out of 3,300 patients (90% children), screened and evaluated from 1989 to 2,000 because of a high clinical suspicion of having an IEM, 323 (9.8%) were neonates. Patients came from different regions of the state of Rio de Janeiro, in which lives approximately 8.5% of the total Brazilian population. Chemical tests, various chromatographic techniques and enzyme assays were performed in urine, plasma and in some cases, cerebrospinal fluid (CSF). This study describes our laboratory and the experience with the 323 investigated neonates, among which 28 cases (8.7%) of IEM were identified and 18 (5.6%), strongly suspected. All these cases were related mainly to the metabolism of amino acids, organic acids, lysosomal enzymes and carbohydrates. Furthermore, data on population, community and health services are presented.