Biochemical Genetics Laboratory



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Glycogen Storage Disease Laboratory

Urine Glucose Tetrasaccharide (Hex4) Biomarker Assay

Test Order Forms and Information

Detailed Background Information and Clinical Utility

Glucose Polymers
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.

Glucose Tetrasaccharides
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.

Glc4 has been found to be elevated in a number of pathological conditions including GSD II (GAA deficiency), GSD III (debranching enzyme deficiency), GSD VI (liver phosphorylase deficiency), certain malignancies, acute pancreatitis and muscular diseases including Duchene’s muscular dystrophy. It is thus a potential biomarker for these conditions. Studies suggest that Glc4 excretion in the urine in GSD II reflects the degree of glycogen accumulation in the patient and may therefore be useful for monitoring treatments.
Maltotetraose Glca1-4Glca1-4Glca1-4Glc, M4 is another hexose tetrasaccharide found in plasma, and to some extent in urine. It is an intermediate product of amylase digestion of glucose polymers. Studies in our laboratory suggest Glc4 is the predominant (>90%) hexose tetrasaccharide (Hex4) in urine from controls and patients with Pompe disease, whereas both M4 and Glc4 are present in varying proportions in plasma.

Urine is the sample of choice for monitoring and diagnosis as Glc4 is more concentrated in the urine and hence urine has a higher sensitivity for Pompe disease and analysis in urine is less expensive to perform.

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

The urine Hex4 assay is a biomarker assay for glycogen storage in skeletal and cardiac muscle. It can be used to aid in the diagnosis of Pompe disease and to monitor patients on enzyme replacement therapy.
  • As a diagnostic tool, urinary Hex4 is similar to plasma creatine kinase (CK) levels in that elevations reflect muscle damage, but it also provides additional information with respect to muscle glycogen storage. As with CK, urine Hex4 elevations can be caused by a number of disease states or conditions that affect muscle integrity. When used in combination with the dried blood spot assay for acid alpha-glucosidase (GAA) activity, it can provide valuable information with regards to the severity of glycogen storage, and hence the severity of the disease in a patient who is found to be GAA deficient. It has close to 100% sensitivity for the infantile Pompe disease and approximately 64% sensitivity for the adult-onset form. We recommend that the urine Hex4 assay be done in addition to CK levels for patients in which Pompe disease (early and late-onset forms) is suspected.

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).