SUMMARY AND EXPLANATION

Glucose determinations have been proven useful in the diagnosis and management of diabetes mellitus. It is the major carbohydrate present in peripheral blood and its oxidation is the major source of cellular energy in the body. Elevated levels are associated with pancreatitis, diseases of the adrenal pituitary and thyroid systems, renal failure and liver disease. Low levels are associated with insulinoma, hypopituitarism, neoplasms or insulin induced hypoglycemia. (1)

Earlier methods for glucose determination such as Folin-Wu (2) and Somogyi-Nelson (3) were dependent on the reduction of heavy metals by the alkehyde group of glucose. These methods were subject to interference by carbohydrates other than glucose. The first enzymatic methods introduced in the 1940's such as the Trinder (4) method were based on using glucose oxidase to greatly improve specificity and ease of handling. This method, however, does require a colorimetric end-point and extended incubation time which makes it difficult for automatic instrumentation. In 1963, Slein (5) introduced an enzymatic method using the hexokinase reaction which can be completed in five minutes at room temperature. The EAGLE GLUCOSE (HEXOKINASE) PROCEDURE is based on a modification of the Slein method.

PRINCIPAL

Glucose is phosphorylated with adenosine triphosphate (ATP) in the reaction catalyzed by hexokinase (HK). The glucose-6-phosphate (G6P) product formed is then oxidized with the concomitant reduction of nicotinamede adenine dinucleotide (NAD) to NADH in the reaction catalyzed by glucose-6-phosphate-dehydrogenase (G6PDH). The formation of NADH causes an increase in absorbance at 340 nm directly proportional to glucose concentration.

Glucose + ATP HK G6P + ADP

G6P + NAD G6PDH 6-Phosphogluconate + NADH + H+

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No 2820 provides:

GLUCOSE (HEXOKINASE) REAGENT - (Cat. No. 2821)

REACTIVE INGREDIENTS:

After reconstitution: ATP - 1.0 mM; G6PDH - 3250 u/L; Hexokinase (yeast) - 1000 u/L; NAD - 1.0 mM; Tris Buffer to pH=7.5; stabilizer and non-reactive fillers added.

RECONSTITUTION:

Dissolve the reagent in the vial with the amount of D-H2O indicated on the vial label. Mix gently to dissolve. Do Not Shake.

PRECAUTIONS:

Do not ingest. Toxicity has not been established.

STORAGE AND STABILITY:

Store at 2-8ºC. Stable until expiration date if sealed tightly. After reconstitution, the reagent is stable for 30 days stored at 2-8ºC or for approximately two days stored at room temperature (18-25ºC). Prolonged exposure to room temperature will shorten usability of reagent even when stored 2-8ºC. For maximum stability, store reagent at 2-8ºC immediately after reconstitution.

DETERIORATION:

The reagent should be dry powder. Caking of the powder would indicate that moisture has entered the vial and the reagent should not be used. If the reconstituted reagent, when read at 340 nm vs water, has an absorbance of greater than 0.2 it should not be used.

GLUCOSE CALIBRATOR - (Cat. No. 2802)

REACTIVE INGREDIENTS:

100 mg/dL D-glucose. Preservative added.

PRECAUTIONS:

Do Not Ingest.

STORAGE AND STABILITY:

Store at 2-8ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The calibrator should be clear, colorless solution. Turbidity would indicate deterioration.

INSTRUMENTS

PRECAUTIONS:

Use a spectrophotometer or colorimeter calibrated at 340 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Use fresh, unhemolyzed serum. Plasma from tubes containing heparin, citrate, oxalate or EDTA may be used.

2. It is important that the serum or plasma be separated from the redblood cells as soon as possible after collection as glucose in whole blood decreases at a rate of 7% per hour.

3. Turbid or icteric sera require a sample blank.

4. 24 hour urine sample collected with preservative may be used.
 
 

SAMPLE STORAGE:

Serum glucose appears stable for 72 hours at 2-8ºC. Cerebrospinal fluid is frequently contaminated with bacteria and should be processed immediately to avoid falsely low results or stored frozen. Plasma with sodium flouride appears stable at room temperature for 24 hours.

ADDITIVES:

No special additives or preservatives are needed for serum, plasma or spinal fluid. Preserve a 24 hour urine sample with 5mL glacial acetic acid or 5g. sodium benzoate.

INTERFERING SUBSTANCES:

Young et al (6) have reviewed drug effects on glucose assays.

PROCEDURE

MATERIALS PROVIDED:

GLUCOSE (HEXOKINASE) REAGENT - (Cat. No. 2821) and GLUCOSE CALIBRATOR - (Cat. No. 2802).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.005 mL micropipettor

2. 1.0 mL pipettor

3. Accurate timer

4. Test tubes and rack

LIMITATIONS

Fructose will cause a positive interference with the hexokinase procedure, however, only small amounts are found in normal fasting serum. After ingestion of 2 g. sucrose/kg of body weight, serum fructose increases up to 8 - 10 mg/dL within one hour. Solutions administered during glucose tolerance testing should not contain fructose or fructose-containing saccharides (7).

EXPECTED VALUES

Serum / Plasma: 70 - 110 mg/dL

Spinal Fluid:

Infant: 60 - 80 mg/dL

Adult: 40 - 70 mg/dL

Urine (24 hour): 1 - 15 mg/dL

This range represents the 95% confidence interval from a clinically normal population (5). Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 600 mg/dL.

PRECISION:

Normal and abnormal control sera were assayed 10 times each to establish within run precision and for 10 working days to establish run to run precision.

WITHIN RUN

MEAN / STD. DEV. / %CV

Normal 99 / 0.70 / 0.8

Abnormal 304 / 2.94 / 1.0

RUN TO RUN

Normal 100 / 1.59 / 1.6

Abnormal 303 / 2.93 / 1.0

SPECIFICITY:

A comparison of this GLUCOSE (HEXOKINASE) PROCEDURE with another widely used commercial method showed a 99.9% correlation with a regression equation of y = 1.02 x -1.55

SENSITIVITY:

This procedure has a sensitivity of 0.5 mg/dL per 0.001 absorbance units.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd Ed., W.B. Saunders Co., Philadelphia, PA (1976), 243.

2. Folin, O., and Wu, H.J., J. Biol. Chem. 38 (1919), 81.

3. Nelson, N.J., Biol. Chem. 153 (1944), 375.

4. Trinder, P., J. Clin. Path. 22, 246 (1969).

5. Slein, M.W., Methods of Enzymatic Analysis, Academic Press, NY (1963).

6. Young, D.S., et al, Clin. Chem. 21, 304D (1975).

7. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd Ed., W.B. Saunders Co., Philadelphia, (1986), 787.