Test Order Forms and Information
Glucose exists in several polymeric forms. Amylose, a component of starch, consists of glucose residues linked in linear chains by alpha-1-4 bonds. Amylopectin, a second component of starch, and glycogen, the glucose storage compound found in liver, kidney and muscle, consist of glucose residues linked by alpha-1-4 and alpha-1-6 bonds. The alpha-1-6 bonds create branch points in these glucose polymers. Glycogen is normally degraded in vivo by two pathways. The cytosolic pathway involves the action of phosphorylase, an exoglycosidase, which degrades glycogen from the nonreducing end by hydrolyzing the alpha-1-4 bonds of terminal glucose residues. When only four glucose residues remain before a branch point, debranching enzyme removes the three outer-most residues and places them at the end of another branch in alpha-1-4 linkage. The remaining branch-point glucose residue is hydrolyzed by the amylo-1,6-glucosidase activity of the debranching enzyme. Glycogen may also be degraded in lysosomes by acid alpha-glucosidase, that hydrolyzes both alpha-1-4 and alpha-1-6 bonds. Amylose and amylopectin are partially digested in the gastrointestinal tract by amylase, generating oligosaccharides of varying lengths, some of which are limit dextrins (containing alpha1-6 linkages). These oligosaccharides may be absorbed by the gut and/or further degraded to smaller oligosaccharides and glucose by the brush border enzymes, sucrase-isomaltose and glycoamylase-maltase.
The specific hexose tetrasaccharide, Glca1-6Glca1-4Glca1-4Glc, (Glc4) is a limit dextrin produced by the amylolytic digestion of glucose polymers that contain alpha-1-6 bonds and is found in the urine and plasma of normal individuals. Its excretion may be moderately increased by ingestion of starch, glycogen (meat) and physical activity. In addition to the dietary origin, evidence suggests Glc4 may also be derived from glycogen released into the circulation from tissues (due to tissue damage/turnover) and acted upon by amylase and alpha-glucosidase activity (such as the ubiquitous glucosidase C) in the circulation.
Principle of Assay
The total urinary hexose tetrasaccharide (Hex4) fraction is analyzed as butyl 4-aminobenzoate (BAB) derivatives using stable isotope dilution-electrospray ionization-tandem mass spectrometry (MS/MS) with multiple reaction monitoring (MRM). Hex4 concentrations are measured relative to creatinine. Urine is mixed with a [13C6]-labeled hexose tetrasaccharide internal standard and incubated with the derivatization reagent. Excess reagent and salts in the derivatization mixture are removed by solid phase extraction (SPE). Derivatized oligosaccharides are eluted from the SPE cartridge and dried and reconstituted in matrix. During analysis by MS/MS the hexose tetrasaccharide fraction is concentrated by elution through a narrow bore short C18 column (note: components within the hexose tetrasaccharide fraction are not separated). The Na+-adducts of urinary Hex4 and the internal standard are detected by multiple reaction monitoring. The intensity ratio of Hex4 to the internal standard is converted to a concentration by means of a calibration curve.
Clinical Utility of Assay
As a monitoring tool, urine Hex4 can be used as an indirect measure of the degree of skeletal muscle glycogen clearance in patients with Pompe disease receiving enzyme replacement therapy. Studies have shown changes in Hex4 levels have correlated with the clinical response to treatment in patients with infantile Pompe disease (An et al, 2005 Mol. Genet. Metab. 85:247-254).