Assay Principle

The GlycoMark enzymatic assay was developed by Nippon Kayaku, Inc. for use on automated open chemistry analyzers.

The assay can be performed using either serum or EDTA plasma. There are two reactions in the method. Reaction 1 is a sample pretreatment performed by the addition of glucokinase (GK) to convert glucose to glucose 6-phosphate in the presence of adenosine triphosphate (ATP), pyruvate kinase (PK) and phosphoenol pyruvate (PEP). The purpose of this reaction is to alter glucose so it can not react in the primary assay for 1,5-AG (reaction 2).

Reaction 2 uses pyranose oxidase (PROD) to oxidize the second hydroxyl of 1,5-anhydroglucitol. The amount of hydrogen peroxide generated in this reaction is directly related to serum 1,5-AG concentrations and is detected by colorimetry using peroxidase (POD). (Note: A schematic of these reactions in presented on the package inserts)

Specimen requirements

Either serum or EDTA plasma can be used in the GlycoMark assay. At the lowest limit of detection (0.49 +/- 2SD), 1,5-anhydroglucitol is stable at 4oC for 7 days, at 22oC for 5 days and at -80oC for 14 days. 1,5-AG can withstand three freeze-thaw cycles at -80oC.

Normal ranges

Studies have shown individuals of differing ages, genders and ethnic origins indicate that the only differences in GlycoMark normal values occur between genders. Nowatzke W. et al. report the United States reference intervals (nonparametric 2.5th to 97.5th percentiles) as 10.2-33.8 ug/ml (males) and 5.9-31.8 ug/ml (females). The GlycoMark package insert reports a reference interval of 10.7 -32.0 ug/ml (males) and 6.8-29.3 ug/ml (females).

Note: Each laboratory should determine its reference range for its own instrument.

Assay limitations

The GlycoMark evaluation studies by Nowatzke W. et al. indicate that there is very little interference from triglyceride, hemoglobin or bilirubin (<10% to concentrations of 12.6 mmol/l, 12.1 ug/ml and 911.4 ug/ml, respectively. The GlycoMark reagent package insert confirms these results and also notes that GlycoMark is unaffected by glucose to 1,000 mg/dL, maltose to 500 mg/dL, ascorbic acid to 25 mg/dL, uric acid to 20 mg/dL, creatinine to 10 mg/dL and urea to 20mg/dL.

Note: GlycoMark has a clear utility for monitoring post-prandial hyperglycemic and short (1-2 week moving window) term glycemia. It is most useful as an adjunct to other diabetes markers and should be interpreted in conjunction with these tests and comprehensive clinical review.

Adverse physical and drug effects

GlycoMark values may be lowered by persistent glucosurea or by oxyhyperglycemia after gastrectomy. I,5-AG is also lowered by pregnancy, renal failure (creatinine over 3 mg/dL), dialysis, cirrhosis, in patients unable to orally ingest food and those with abnormal glomerular filtration.

Some drugs may increase 1,5-AG. The package insert lists Polygala, Tenuifolia and Senega syrup. Anti-hyperglycemic drug therapy results in increased GlycoMark values; whereas steroids may decrease 1,5-AG.

1,5-AAG may also increase during intravenous hyperalimentation.

Specimen storage and shipment

Serum samples are stable at either room temperature or 2-8oC for up to one week. For longer storage, freezing is recommended. Shipment of samples should be on ice or, over long distances, on dry ice.

Kit description

Kit stability
The GlycoMark reagent, control and standard kits are stable when refrigerated (2-8oC) until the expiration date printed on the box. Usually there is a twelve month expiration date from the time of manufacture for the reagent and standard and eighteen months for the control set. Open reagent, control and standard vials are stable for 30 days (2-8oC).

Reagents Kit (NK-8300)
The reagent kit consists of two offset vials, R1 (20 ml) and R2 (10 ml), suitable for deployment on an open automated chemistry analyzer. Correlation coefficients are reported at >99 between lot numbers on the Hitachi 917.
Note: R1 and R2 contain sodium azide and proper precautions should be followed.

Controls Kit (NK-8330)
The Controls kit contains three 2ml vials of each control. There are a low (Control L) and a high control (Control H).

Standard Kit (NK-8320)
The Calibration Standard is at 50 ug/ml of 1,5-AG. The kit contains three 5 ml vials.
Other non-supplied items include a saline reagent blank (0.9% sodium chloride in deionized water).

Procedure

The following description of the assay is generally applicable to all automated open chemistry analyzers. Although Hitachi 917 GlycoMark procedure is summarized, the complete instrument parameters are available on the package insert.

The GlycoMark assay is kinetic. All reagents are placed undiluted directly on the analyzer. The reaction occurs at 37oC. At zero time, 4 ul of standard, control, reagent blank or serum or EDTA plasma sample is added to 120ul of R1. At 5 minutes, 60 ul of R2 is added and the absorbances at 546 nm (primary) and 700 nm (secondary) are immediately obtained. At 10 minutes, both absorbances are again determined. The absorbance at 700 nm is subtracted from that at 546 nm as a background correction for both the 5 and 10 minute readings. Finally, the 5 min corrected value is subtracted from the 10 minute value. The 1,5-anhydroglucitol concentrations are then determined from standard curve (usually linear) established from the kinetic change in absorbance of the reagent blank and the 50 ug/ml 1,5-AG standard.

Instrumentation

GlycoMark is adaptable to automated open chemistry analyzers and is currently being successfully run on the following instruments:

GlycoMark parameters are available for these instruments and may be available for other open chemistry analyzers. Please call GlycoMark technical support at 336-725-8181 for this information.

Yield expectations

Users of GlycoMark are reporting between 125 and 135 tests per kit depending upon their instrument and the number of control sets and 50 ug/ml standards they include in each run. When applied to batch runs with 125 samples the controls and standard can be maximized and therefore their contribution to cost per test is minimal. The controls and standards are also supplied in several vials so they are not limited by the 30-day open expiration period.