Development of a kit for urine collection on filter paper as an alternative for Pompe disease screening and monitoring by LC-HRMS.

Pompe disease (PD) is an inborn error of metabolism caused by α-glucosidase acid enzyme deficiency. It significantly impacts patients' health and life quality and may lead to death in the first few years of life. Among the well-established diagnostic methods, urinary glucose tetrasaccharide (Glc4) screening by high performance-liquid chromatography has been helpful in monitoring Glc4 levels in patients on enzyme replacement therapy, demonstrating therapy efficacy. However, the specimen shipping process from a sample collecting location to a specialized laboratory for monitoring the Glc4 is costly and presents preanalytical challenges. In this work, we developed a filter paper based-urine collection kit to facilitate specimen shipment, and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) analysis to determine Glc4 and creatinine in dried urine on filter paper. The LC-HRMS was based on a combination of targeted and untargeted screening on the same specimen injection and was successfully developed and validated. Bland-Altman statistics revealed a good relationship between dried and liquid urine samples and Glc4 and creatinine. Glc4 and other metabolites in dried urine showed stability for at least 7 days at 4 and 22 °C, and 3 days at 50 °C. The stability of the analytes and the efficiency of the kit were tested simulating real conditions by sending it by post. After two days in transit without refrigeration, the stability of compounds was maintained, showing the reliability of the urine collection kit and analysis method to determine the PD biomarker Glc4.

Combined targeted and untargeted high-resolution mass spectrometry analyses to investigate metabolic alterations in pompe disease.

INTRODUCTION: Pompe disease is a rare, lysosomal disorder, characterized by intra-lysosomal glycogen accumulation due to an impaired function of α-glucosidase enzyme. The laboratory testing for Pompe is usually performed by enzyme activity, genetic test, or urine glucose tetrasaccharide (Glc4) screening by HPLC. Despite being a good preliminary marker, the Glc4 is not specific for Pompe. OBJECTIVE: The purpose of the present study was to develop a simple methodology using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for targeted quantitative analysis of Glc4 combined with untargeted metabolic profiling in a single analytical run to search for complementary biomarkers in Pompe disease. METHODS: We collected 21 urine specimens from 13 Pompe disease patients and compared their metabolic signatures with 21 control specimens. RESULTS: Multivariate statistical analyses on the untargeted profiling data revealed Glc4, creatine, sorbitol/mannitol, L-phenylalanine, N-acetyl-4-aminobutanal, N-acetyl-L-aspartic acid, and 2-aminobenzoic acid as significantly altered in Pompe disease. This panel of metabolites increased sample class prediction (Pompe disease versus control) compared with a single biomarker. CONCLUSION: This study has demonstrated the potential of combined acquisition methods in LC-HRMS for Pompe disease investigation, allowing for routine determination of an established biomarker and discovery of complementary candidate biomarkers that may increase diagnostic accuracy, or improve the risk stratification of patients with disparate clinical phenotypes.

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.

Clinical, biochemical and genetic characteristics of MOGS-CDG: a rare congenital disorder of glycosylation.

PURPOSE: To summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS. METHODS: Phenotypic characterisation was performed through an international and multicentre collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG. RESULTS: Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurological, immunological and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis was not consistently abnormal. CONCLUSION: The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry can be used as a reliable biochemical test for screening and confirmation of disease.

L-carnitine supplementation in the recovery of plasma L-carnitine in patients with heart failure submitted to coronary artery bypass grafting.

Coronary artery bypass grafting reduces plasma L-carnitine and may impair the production of myocardial energy. L-carnitine supplementation may elevate plasma L-carnitine and increase cardiac mechanical efficiency. The objective of this study was to verify the recovery of preoperative plasma LC in patients with heart failure undergoing coronary artery bypass grafting supplemented with a daily oral dose of 50 mg / kg. Volunteers with ischemic heart failure who underwent surgery were randomized into a supplemented group (A-received 50 mg / kg L-carnitine) or placebo group (B) for 60 days. Supplementation was started on the third postoperative day. The spectrophotometric enzymatic method was used to quantify plasma L-carnitine. In the preoperative period, both groups had plasma L-carnitine adequate to the reference range (18.9-71.1 µM). On the second postoperative day, there was a reduction in plasma L-carnitine in groups A (17.4%) and B (14.4%). In the comparison between the groups, plasma L-carnitine was higher in group A than B in 10º (p = 0.024), 30º (p = 0.001), and 60º postoperative day (p = 0.008). Supplementation of L-carnitine at a daily oral dose of 50 mg / kg in patients with heart failure undergoing coronary artery bypass grafting may recover preoperative plasma L-carnitine within 10 days.

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.

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.

Fumaric aciduria: an overview and the first Brazilian case report.

Fumaric aciduria is a rare metabolic disease, with 40 cases reported so far. Fumarase deficiency leads mainly to brain abnormalities, developmental delay, and great accumulation of fumaric acid in urine. This work presents the first case of fumaric aciduria described in Brazil, which presented with some interesting clinical and biochemical findings such as colpocephaly, hepatic alterations, and marked metabolic acidosis since birth. Common findings were ventriculomegaly, hypotonia, and microcephaly. Biochemically, besides the high urinary fumaric acid excretion, atypical elevation of plasma citrulline, tyrosine and methionine levels were also observed. In order to show all features and variants of fumaric aciduria, literature data of 40 patients was reviewed and compared with the case reported here. Findings in all these patients demonstrate that this disorder does not yet have its phenotype completely defined; it is important that more patients be described.

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.

Smith-Lemli-Opitz syndrome: clinical and biochemical findings in Brazilian patients

Smith-Lemli-Opitz syndrome (SLOS) or RSH syndrome comprises multiple congenital anomalies and mental retardation. The underlying defect is a deficiency in the activity of delta7-sterol reductase, which decreases cholesterol and increases 7-dehydrocholesterol (7-DHC) levels. Our aim was to identify and evaluate the frequency of SLOS manifestations in a group of Brazilian patients. Based on our own data and those reported previously, we present a simple method that allows the estimation of probabilities favoring the diagnosis of SLOS. We evaluated 30 patients clinically and determined their plasma levels of cholesterol and 7-dehydrocholesterol. In 11 patients, the diagnosis was confirmed by ultraviolet spectrophotometry (UV). Of 19 patients with normal laboratory results, 17 showed a high probability favoring the diagnosis of SLOS. The most significant signs and symptoms observed in over 2/3 of the biochemically confirmed cases were mental retardation (10/11), delayed neuropsychomotor development (10/11), syndactyly of 2nd/3rd toes (10/11), and craniofacial anomalies including microcephaly (11/11), incompletely rotated ears (8/11), palpebral ptosis (10/11), anteverted nostrils (10/11), and micrognathia (9/11). Genital anomalies were found in all male patients (6/6).