Carbs: Enzymatic & Cupric Ion Reduction Methods

Hexokinase Methods

Based on a coupled enzyme assay that uses HK and glucose-

6-phosphate dehydrogenase (G6PD)

Hexokinase Method

is considered the reference (gold standard) method for measuring glucose in clinical laboratories

Hexokinase Method

The method is based on two enzyme reactions that result in the formation of NADPH, a compound that absorbs light at 340 nm. The amount of NADPH formed is directly

proportional to the glucose concentration.

Glucose Oxidase Method

An enzymatic colorimetric test

Glucose Oxidase Method

Hydrogen peroxide reacts with a color reagent or chromogen such as O-dianisidine or aminoantipyrine or phenol to produce a colored product. The intensity of color is directly proportional to the amount of glucose in the sample. It can either be pink, red, or blue depending on the reagents used.

Glucose Dehydrogenase Method

This method uses the enzyme glucose dehydrogenase or

GDH, which oxidizes glucose to gluconolactone. During

this reaction, a coenzyme NAD is reduced to NADH, and

the resulting product can be measured either

colorimetrically or electrochemically.

Glucose Dehydrogenase Method

The NADH formed absorbs light at 340 nanometers.

Folin-Wu Method

Measures: All reducing substances in blood (not only glucose)

Phosphomolybdic acid

Reagent that binds with Cu⁺ in Folin-Wu Method.

Phosphomolybdenum blue

End product of Folin-Wu Method.

Folin-Wu Method

Not specific for glucose — other reducing substances (like uric acid, creatinine, etc.) can interfere

Nelson-Somogyi Method

Measures: True glucose

Arsenomolybdic acid

Reagent that binds with Cu⁺ in Nelson-Somogyi Method

Arsenomolybdenum blue

End product of Nelson-Somogyi Method

Neocuproine

Reagent that binds with Cu⁺ in Neocuproine Method

Cuprous–neocuproine complex

End product of Neocuproine Method

Yellow to yellow-orange

End color of Neocuproine Method

Folin-Wu Method

Used historically as a reference method before

enzymatic methods became available

Neocuproine Method

More stable and sensitive color reaction

Hagedorn-Jensen Method

Glucose reduces yellow ferricyanide (Fe³⁺) to colorless ferrocyanide (Fe²⁺).

Hagedorn-Jensen Method

Type of Colorimetry: Inverse colorimetry — the less yellow the

solution becomes; the more glucose is present

Ferrocyanide

End product of Hagedorn-Jensen Method

Colorless

End color change of Hagedorn-Jensen Method

Hagedorn-Jensen Method

Measurement: Decrease in yellow color intensity is inversely

proportional to glucose concentration.

Hexokinase

is an enzyme that catalyzes the phosphorylation of glucose

1. glucose-6-phosphate

2. ADP

1st Step: Phosphorylation of Glucose

o Glucose reacts with ATP to form __ and

__ with the enzyme hexokinase

y6-phosphogluconate

2nd Step: Oxidation of Glucose-6-Phosphate

o Glucose-6-phosphate is oxidized or converted into __ via glucose-6-phosphate

dehydrogenase, producing NADPH in the process.

NADPH absorbs light at 340 nanometers, and the increase

in the absorbance at this wavelength is directly

proportional to the glucose concentration

Glucose oxidase

catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide (H 2 O 2 ).

1. gluconic acid

2. hydrogen peroxide

1st step: Oxidation of glucose

o Glucose reacts with oxygen to form __ and

__ with the use of the enzyme glucose

oxidase.

1. O-dianisidine

2. aminoantipyrine

3. phenol

2nd: Color Formation: pink/red/ blue

o Hydrogen peroxide reacts with a color reagent or

chromogen such as __ or __ or

__ to produce a colored product. The intensity of color

is directly proportional to the amount of glucose in the

sample. It can either be pink, red, or blue depending on the

reagents used.

Glucose dehydrogenase

catalyzes the oxidation of glucose to gluconolacctone with concomitant reduction of NAD + to NADH