Note Elimination of glucose contamination from adipocyte glycogen extracts Patricia M. Nunes, a Euge ´nia Carvalho a and John G. Jones a,b, * a Center for Neurosciences and Cell Biology, Department of Zoology, University of Coimbra, 3004-517 Coimbra, Portugal b NMR Research Unit, Department of Biochemistry, Faculty of Sciences and Technology, Apartado 3126, University of Coimbra, 3001-401 Coimbra, Portugal Received 5 December 2007; received in revised form 19 February 2008; accepted 1 April 2008 Available online 7 April 2008 Abstract—Glycogen was quantified in rat adipocytes by isolation using conventional KOH digestion and ethanol precipitation, fol- lowed by hydrolysis and spectrophotometric assay of the glucose product. A concentration of 0.193 ± 0.020 lmol glucosyl units/ 10 6 cells was recorded. When this procedure was modified by including a 4 h incubation with glucose oxidase prior to glycogen hydrolysis, the glycogen concentration was found to be 0.055 ± 0.008 lmol glucosyl units/10 6 cells. Therefore in adipocytes, con- ventional glycogen assays give substantial overestimates due to incomplete removal of glucose during glycogen isolation. Contam- inant glucose can be scavenged in a simple manner by incubation with glucose oxidase prior to glycogen hydrolysis. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Adipocyte; Glycogen extraction; Glucose oxidase; Amyloglucosidase In addition to liver and muscle, a wide range of other tissues can synthesize and mobilize glycogen including brain, chondrocytes, and adipocytes. 1–3 Compared to liver and muscle, the concentrations of glycogen in these tissues are typically much smaller. There is evidence that these small glycogen pools may play a key role as a sub- strate buffer to counter the effects of fluctuating glucose supply and demand in neurons 3 and possibly other cell types. As a result, there is renewed interest in the precise quantification of glycogen in these tissues. The Achilles heel of the conventional glycogen assay method is the separation of glycogen from cell or medium glucose. Since glycogen is typically quantified in terms of glucose units following amyloglucosidase hydrolysis, incomplete removal of glucose during purification will result in an overestimate of glycogen. The potential for this error is increased when the glycogen concentrations are low relative to glucose. The contaminant glucose can be selectively removed from the isolated glycogen prepara- tion by the addition of glucose oxidase. This enzyme requires no cofactors or substrates to convert glucose to gluconate, only oxygen. Glucose oxidase also functions well in the same buffer medium used for the subsequent amyloglucosidase hydrolysis of glycogen to glucose hence there is no need for buffer adjustment or replacement. Given the amount of glycogen present in adipocyte preparations (0.1 lmol), a minor amount of glucose (>10 nmol) would result in a significant contamination. Therefore, all reagents and extraction steps were exam- ined for the presence or generation of glucose. The amyloglucosidase enzyme preparation did not contain detectable amounts of glucose as assayed by 1 H NMR and enzymatic assay. When the KOH extraction proce- dure was performed on solutions containing 7.5 mg of commercial glycogen (42 lmol of glucose equivalents or 400 times the amount of glycogen in a typical prep- aration), glucose was not detected by enzymatic assay and 1 H NMR analysis showed only signals from glyco- gen. Therefore, the glycogen extraction procedure per se does not result in the appearance of glucose. The glucose 0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2008.04.004 Abbreviations: KR, Krebs Ringer * Corresponding author. Tel.: +351 239 824 531; fax: +351 239 853 607; e-mail: john.jones@netc.pt Available online at www.sciencedirect.com Carbohydrate Research 343 (2008) 1486–1489