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.

Treatment of mucopolysaccharidosis type II (Hunter syndrome) with idursulfase: the relevance of clinical trial end points.

The current treatment of mucopolysaccharidosis type II (MPS II, Hunter syndrome) is enzyme replacement therapy with recombinant idursulfase (Elaprase®). The efficacy of ERT was established based primarily on reduction in urine glycosaminoglycans:creatinine (GAG:Cr) ratio and improvement in a composite score of predicted forced vital capacity (FVC% predicted) and 6-min walk-test distance (6MWT). We retrospectively reviewed these parameters in 11 boys with MPS II treated with idursulfase between April 2007 (or the time of diagnosis) and February 2010. Some results were inconsistent with published trial data, and there was only a small number of analyzable results obtained for the FVC% predicted and 6MWT. A major drawback was the high prevalence of neurological involvement and young age of patients in the study cohort compared with the clinical trials. This study emphasizes the limitations of the current tools utilized to monitor ERT efficacy and MPS II disease burden in clinical practice.

Effect of denervation on the content of cardiac troponin-T and cardiac troponin-I in rat skeletal muscle.

Abnormal rhabdomyocyte expression of cardiac troponin-T (cTnT) was thought to interfere with the cTnT assay. cTnT isoforms have been shown to be transiently expressed in skeletal muscle during development and in response to muscle denervation. The effect of denervation and aging on cTnT and cardiac troponin-I (cTnI) content in fast and slow rat skeletal muscles was assessed quantitatively. Sections of the tibial nerve were transected from one hind limb of both young (n=12) and old (n=12) rats. Animals were sacrificed at 1, 2, and 4 weeks after the operation, and the extensor digitorum longus (EDL) and the soleus were removed from both the denervated and the contralateral control limb. There was no significant difference in cTnI content between the fast EDL and slow soleus muscles. The cTnT content was significantly higher in the soleus than the EDL muscle (p<0.001). These data, combined with data on other models in the literature, indicate that re-expression of cTnT and cTnI isoforms in adult skeletal muscle is unlikely and does not interfere with cTnT assays for assessment of cardiac damage.

Measurement of cardiac troponin I in striated muscle using three experimental methods.

BACKGROUND: Qualitative and quantitative measures of cardiac troponin I (cTnI) in striated muscle have been reported as part of diverse investigations. However, there is disparity in the literature regarding the findings of these analyses. The cTnI molecule can exist in phosphorylated, non-phosphorylated, reduced, non-reduced, complexed or non-complexed forms. Each of these forms can change the antigenicity of cTnI, resulting in different antibody-antigen interactions in different experimental formats, thereby giving rise to the disparities in the literature. METHODS: cTnI in heart and skeletal muscles were investigated by three techniques employing the same specific cTnI antibodies: the recently revised Dade-Behring Dimension RXL assay, immunoblotting and immunohistochemistry. RESULTS: cTnI was detected in heart muscle but not skeletal muscle using the quantitative assay and immunoblotting. Unexpectedly, using the same antibodies, cTnI was not immunolocalized to either heart or skeletal muscle. CONCLUSION: The antibody-cTnI interaction might be impeded on fixed immunohistochemistry sections. Our findings reflect those of a previous study, showing that cTnI was not detected in skeletal muscle extracts using a quantitative assay. The behaviour of cTnI antibodies varies depending on experimental design. Conclusions drawn from experiments using qualitative methods cannot necessarily be extrapolated to the quantitative assay and vice versa.