Mass spectrometric quantification of glycogen to assess primary substrate accumulation in the Pompe mouse Maria Fuller ⇑ , Stephen Duplock, Christopher Turner, Philippa Davey, Doug A. Brooks, John J. Hopwood, Peter J. Meikle 1 Lysosomal Diseases Research Unit, SA Pathology at Women’s and Children’s Hospital, North Adelaide, SA 5006, Australia article info Article history: Received 2 December 2011 Received in revised form 13 December 2011 Accepted 14 December 2011 Available online 20 December 2011 Keywords: Mass spectrometry Glycogen Mouse Pompe disease abstract Glycogen storage in the a-glucosidase knockout (6neo/6neo) mouse recapitulates the biochemical defect that occurs in the human condition; as such, this mouse serves as a model for the inherited metabolic defi- ciency of lysosomal acid a-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chroma- tography/electrospray ionization–tandem mass spectrometry. The method was sensitive enough to mea- sure as little as 0.1 lg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduc- tion in response to experimental therapies being trialed in this model. Ó 2011 Elsevier Inc. All rights reserved. Pompe disease, also known as glycogen storage disorder type II, is a rare inherited metabolic myopathy caused by a deficiency of the lysosomal enzyme, acid a-glucosidase. Consequently, lyso- somal glycogen, the substrate for a-glucosidase, accumulates in af- fected cells primarily in skeletal and cardiac muscle [1]. Pompe disease can affect individuals at any age, and in all cases there is severe muscle pathology leading to progressive muscle weakness, which eventually impairs motor and respiratory function [2]. The considerable clinical heterogeneity can be explained in part by the occurrence of different mutations in the lysosomal acid a-glu- cosidase gene producing variable effects on the functional capacity of the mutant enzyme [3]. Enzyme replacement therapy is now available for patients with Pompe disease predicated on proof- of-principle enzyme replacement therapy in mice [4]. Although a reduction in glycogen storage coupled with improvements in mus- cle architecture has been reported, not all patients respond equally well [5]. Recent evidence suggests that the success of therapy is very much dependent on the condition of the patient before the commencement of therapy [6]. Of note is the high dose of enzyme required, which is more than 20 times the relative amount of en- zyme that is used for other lysosomal storage disorders [7,8]. The high dose of acid a-glucosidase required is related, at least in part, to the resistance of skeletal muscle to enzyme replacement. When Pompe mice, homozygous for disruption of the acid a-gluco- sidase gene (6 neo /6 neo ) [9], are treated with enzyme replacement, they have shown an uneven pattern of glycogen clearance in skele- tal muscle, with type I muscle fibers clearing effectively but not type II muscle fibers [10]. The differential clearance of glycogen in skele- tal muscle is attributed to a number of factors, including the bioavailability of replacement enzyme from the circulatory system [11], lysosomal rupture and the release of glycogen and lysosomal enzymes into the cytoplasm [12], and the presence of autophagic buildup in type II muscle [13]. More recently, the suppression of autophagy coupled with enzyme replacement therapy has been shown to effectively reduce glycogen levels in muscle fibers of the Pompe mouse to normal [14]. In any assessment of therapeutic approaches for Pompe disease, the ability to accurately quantify small amounts of glycogen is invaluable as a biochemical measure of efficacy. This is particularly important when evaluating the effect of therapy on residual stores of glycogen and, in particular, cell types where the amount of gly- cogen is likely small and the biological sample is limited. Compar- isons of methods to measure glycogen in tissues have previously been reported but suffer from the requirement of relatively large 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.12.026 ⇑ Corresponding author. Fax: +61 8 8161 7100. E-mail address: maria.fuller@adelaide.edu.au (M. Fuller). 1 Current address: Baker IDI Heart and Diabetes Institute, Melbourne, VIC 3004, Australia. Analytical Biochemistry 421 (2012) 759–763 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio