Commission Regulation (EEC) No 000/90 (repealed)Show full title

7. GLUCOSE AND FRUCTOSE U.K.

1. DEFINITION U.K.

Glucose and fructose may be determined individually by an enzymatic method, with the sole aim of calculating the glucose/fructose ratio.

2. PRINCIPLE OF THE METHOD U.K.

Glucose and fructose are phosphorylated by adenosine triphosphate (ATP) during an enzymatic reaction catalysed by hexokinase (HK), and produce glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P):

glucose + ATP G6P + ADP

fructose + ATP F6P + ADP

The glucose 6-phosphate is first oxidized to gluconate 6-phosphate by nicotinamide adenine dinucleotide phosphate (NADP) in the presence of the enzyme glucose 6-phosphate dehydrogenase (G6PDH). The quantity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) produced corresponds to that of glucose 6-phosphate and thus to that of glucose.

G6P + NADP⁺ gluconate 6-phosphate + NADPH + H⁺

The reduced nicotinamide adenine dinucleotide phosphate is determined from its absorption at 340 nm.

At the end of this reaction, the fructose 6-phosphate is converted into glucose 6-phosphate by the action of phosphoglucose isomerase (PGI):

F6P G6P

The glucose 6-phosphate again reacts with the nicotinamide adenine dinucleotide phosphate to give gluconate 6-phosphate and reduced nicotinamide adenine dinucleotide phosphate, and the latter is then determined.

3. APPARATUS U.K.

• A spectrophotometer enabling measurements to be made at 340 nm, the wavelength at which absorption by NADPH is at a maximum. Absolute measurements are involved (i.e. calibration plots are not used but standardization is made using the extinction coefficient of NADPH), so that the wavelength scales of and absorbence values obtained from the apparatus must be checked.

  If not available, a spectrophotometer using a source with a discontinuous spectrum which enables measurements to be made at 334 nm or at 365 nm may be used.

• Glass cells with optical path lengths of 1 cm or single-use cells.

• Pipettes for use with enzymatic test solutions, 0,02, 0,05, 0,1, 0,2 ml.

4. REAGENTS U.K.

4.1. Solution 1: buffer solution (0,3 M triethanolamine, pH 7,6, 4 × 10⁻³ M in Mg²⁺): dissolve 11,2 g triethanolamine hydrochloride ((C₂H₅)₃N · HCl) and 0,2 g MgSO₄ · 7H₂O in 150 ml of doubly distilled water, add about 4 ml of 5 M sodium hydroxide (NaOH) solution to obtain a pH value of 7,6 and make up to 200 ml. U.K.

This buffer solution may be kept for four weeks at + 4 °C.

4.2. Solution 2: nicotinamide adenine dinucleotide phosphate solution (about 11,5 × 10⁻³ M): dissolve 50 mg disodium nicotinamide adenine dinucleotide phosphate in 5 ml of doubly distilled water. U.K.

This solution may be kept for four weeks at +4 °C.

4.3. Solution 3: adenosine 5′-triphosphate solution (about 81 × 10⁻³ M): dissolve 250 mg disodium adenosine 5′-triphosphate and 250 mg sodium hydrogencarbonate (NaHCO₃) in 5 ml of doubly distilled water. U.K.

This solution may be kept for four weeks at +4 °C.

4.4. Solution 4: hexokinase/glucose 6-phosphate dehydrogenase: mix 0,5 ml hexokinase (2 mg of protein/ml or 280 U/ml) with 0,5 ml glucose 6-phosphate dehydrogenase (1 mg of protein/ml). U.K.

This mixture may be kept for a year at about +4 °C.

4.5. Solution 5: phosphoglucose isomerase (2 mg of protein/ml or 700 U/ml). The suspension is used undiluted. U.K.

This may be kept for a year at about +4 °C.

Note: U.K.

All solutions used above are available commercially. U.K.

5. PROCEDURE U.K.

5.1. Preparation of sample U.K.

Depending on the estimated amount of glucose + fructose per litre, dilute the sample as follows:

Measurementat 340 and 334 nm	Measurementat 365 nm	Dilution with water	Dilution factor F
up to 0,4 g/l	0,8 g/l	—	—
up to 4,0 g/l	8,0 g/l	1 + 9	10
up to 10,0 g/l	20,0 g/l	1 + 24	25
up to 20,0 g/l	40,0 g/l	1 + 49	50
up to 40,0 g/l	80,0 g/l	1 + 99	100
above 40,0 g/l	80,0 g/l	1 + 999	1 000

5.2. Determination U.K.

With the spectrophotometer adjusted to the 340 nm wavelength, make measurements using air (no cell in the optical path) or water as reference.

Temperature between 20 and 25 °C.

Into two cells with 1 cm optical paths, place the following:

	Reference cell	Sample cell
Solution 1 (4.1) (taken to 20 °C)	2,5 ml	2,5 ml
Solution 2 (4.2)	0,1 ml	0,1 ml
Solution 3 (4.3)	0,1 ml	0,1 ml
Sample to be measured		0,2 ml
Doubly distilled water	0,2 ml

Mix, and after about three minutes read off the absorbence of the solutions (A ₁). Start the reaction by adding:

Solution 4 (4.4)

0,02 ml

0,02 ml

Mix; wait 15 minutes; read off the absorbence and check that the reaction has stopped after a further two minutes (A ₂).

Add immediately:

Solution 5 (4.5)

0,02 ml

0,02 ml

Mix; read off the absorbence after 10 minutes and check that the reaction has stopped after a further two minutes (A ₃).

Calculate the differences in the absorbences:

A ₂ − A ₁ corresponding to glucose,

A ₃ − A ₂ corresponding to fructose,

for the reference and sample cells.

Calculate the differences in absorbence for the reference cell (ΔA _R) and the sample cell (ΔA _S) and then obtain:

for glucose: ΔA _G = ΔA _S − ΔA _R

for fructose: ΔA _F = ΔA _S − ΔA _R

Note: U.K.

The time needed for the completion of enzyme activity may vary from one batch to another. The above value is given only for guidance and it is recommended that it be determined for each batch. U.K.

5.3. Expression of results U.K.

5.3.1. Calculation U.K.

The general formula for calculating the concentrations is:

where

V

=

volume of the test solution (ml)

ν

=

volume of the sample (ml)

M

=

molecular mass of the substance to be determined

d

=

optical path in the cell (cm)

ε

=

absorption coefficient of NADPH at 340 nm (= 6,3 mM⁻¹ × l × cm⁻¹)

and

V

=

2,92 ml for the determination of glucose

V

=

2,94 ml for the determination of fructose

ν

=

0,20 ml

M

=

180

d

=

1

so that:

For glucose: C (g/l) = 0,417 ΔA _G

For fructose: C (g/l) = 0,420 ΔA _F

If the sample was diluted during its preparation, multiply the result by the dilution factor F.

Note: U.K.

If the measurements are made at 334 or 365 nm, then the following expressions are obtained: U.K.

• measurement at 334 nm: ε = 6,2 (mmole⁻¹ × l × cm⁻¹)

  For glucose: C (g/l) = 0,425 ΔA_G

  For fructose: C (g/l) = 0,428 ΔA_F

• measurement at 365 nm: ε = 3,4 (mmole × l⁻¹ × cm⁻¹)

  For glucose: C (g/l) = 0,773 ΔA_G

  For fructose: C (g/l) = 0,778 ΔA_F

5.3.2. Repeatability (r) U.K.

r = 0,056 x_i

5.3.3. Reproducibility (R) U.K.

R

=

0,12 + 0,076 x_i

x_i

=

concentration of glucose or fructose in g/l.