A metabolomic study to identify new globotriaosylceramide-related biomarkers in the plasma of Fabry disease patients.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A, which results in the progressive accumulation of glycosphingolipids. In addition to the two biomarkers, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), which are routinely used for detection and high-risk screening of Fabry disease patients, novel urinary Gb3-related isoforms/analogues as well as newly defined lyso-Gb3 analogues in plasma and urine from Fabry patients have recently been described by our group. The aim of this study was to extend our recent analyses to identify and evaluate new potential Gb3-related biomarkers in the plasma of untreated male Fabry disease patients using a mass spectrometry metabolomic approach. A multivariate statistical analysis revealed five Gb3-related novel biomarkers in the plasma of male Fabry patients. Three of these new biomarkers correspond to Gb3, which has an extra double bond on the sphingosine with C16:0, C18:0, and C22:1 fatty acid chains. The fourth biomarker corresponds to a mixture of two structural isomers, the first with a d16:1 sphingosine and a C16:0 fatty acid and the second with a d18:1 sphingosine and a C14:0 fatty acid. To our knowledge, it is the first time that a Gb3 analogue with a d16:1 sphingosine moiety has been reported. In addition, this Gb3 analogue was also present in its methylated form. These biomarkers are part of a metabolic profile that may provide insight into the pathophysiology of Fabry disease.

The identification of new biomarkers for identifying and monitoring kidney disease and their translation into a rapid mass spectrometry-based test: evidence of presymptomatic kidney disease in pediatric Fabry and type-I diabetic patients.

Using label-free quantative proteomics, we have identified 2 potential protein biomarkers that indicate presymptomatic kidney disease in the urine of pediatric patients with type-I diabetes and Fabry disease (n = 20). Prosaposin and GM2 activator protein (GM2AP) were observed to be elevated in the urine of these patient groups compared to age- and sex-matched controls. These findings were validated by development of a rapid MRM-based tandem mass spectrometry test. Prosaposin was observed to be both significantly elevated in the urine of patients with Fabry disease compared to controls (p = 0.02) and reduced after 12 months enzyme replacement therapy (ERT, p = 0.01). Similarly, GM2AP concentrations were observed to be significantly higher compared to controls in the diabetic group (p = 0.049) and the pretreatment Fabry group (p = 0.003). In addition, this observed to be reduced significantly in the Fabry group following 12 months of ERT (p = 0.01). The process of detection of the biomarkers, development into a test and implications for monitoring patients and treatment are discussed.

Urine analysis of glucose tetrasaccharide by HPLC; a useful marker for the investigation of patients with Pompe and other glycogen storage diseases.

A high performance liquid chromatography method, adapted from an established urinary sugars method, has been developed for the analysis of a tetraglucose oligomer (Glc(4)) in urine. Pompe disease results from defects in the activity of lysosomal acid α-glucosidase (GAA) with patients typically excreting increased amounts of Glc(4). Rapid determination of GAA in dried blood spots is now possible. However, enzymatic analysis is unable to discriminate between patients with Pompe disease and those individuals harbouring pseudo deficiency mutations. This method was able to quantify Glc(4) levels in all patients analysed with an established diagnosis of Pompe disease, and all controls analysed had Glc(4) levels below the limit of detection for this method. Importantly the method was able to discriminate between an individual known to harbour a pseudo Pompe mutation and patients with Pompe disease, providing a useful supporting test to enzymatic analysis. Sequential measurement of urinary Glc(4) has been proposed to monitor the effects of enzyme replacement therapy (ERT). We observed a clear decrease in Glc(4) levels following commencement of treatment in three patients studied. Additionally, raised levels of Glc(4) were observed in patients with glycogen storage disease (GSD) type Ia and type III suggesting that this method may have applications in other GSDs.

HPLC of basic drugs using non-aqueous ionic eluents: evaluation of a 3 mum strong cation-exchange material.

HPLC columns packed with 3 microm particle size HPLC Technology Techsphere SCX (propylsulfonic acid-modified) silica offer considerable advantages over 5 microm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 microm column was not only more efficient for a given column length compared with 5 microm materials, but also elution times were less, a phenomenon observed in reversed-phase systems. The high efficiencies and excellent peak shapes obtained with the 3 microm SCX-modified packing together with the relatively low back-pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required.