Determination of Glucose in Drinks

Table of Contents

Introduction
Methods
Results
Conclusions
About Bibby Scientific

Introduction

The rate of an enzyme reaction is based on substrate concentration hence this can be used for determining the amount of substrate present. While measuring such a reaction, it is important to determine the amount of product produced or the disappearance of substrate consumed. This cannot be done directly with many assays hence the reaction may be combined with a second enzyme that can convert one of the products into a measurable substance. The coupling of reactions to dehydrogenases that use nicotinamide adenine dinucleotide (NAD⁺) or the reduced form, NADH, as coenzymes is one such example. It is possible to readily measure NADH in a spectrophotometer at 340nm or by fluorimetry with excitation at 340nm and emission at 460nm. Several enzyme reactions are used as analytical tools.

In this article, the use of the Glucose (HK) Assay kit from Sigma (product code GAHK-20) is demonstrated to determine the amount of glucose in three different types of drink.

The kit works on the following principle:

Firstly, glucose is phosphorylated by hexokinase while reacting with ATP. The obtained glucose-6-phosphate is oxidized to 6-phosphogluconate in the presence of NAD⁺ in a reaction catalysed by glucose-6-phosphate dehydrogenase(G6PDH).

An equimolar amount of NAD⁺ is reduced to NADH during this oxidation. Hence the reaction can be monitored by determining the increase in absorbance at 340nm and this increase is directly proportional to the original glucose concentration.

Methods

Based on the manufacturer’s instructions, the Glucose (HK) Assay Reagent was reconstituted in 20ml deionised water. The three drinks tested include:

• Lucozade Energy Original (GlaxoSmithKline plc, UK),
• Pressed Apple Juice (Wm Morrison Supermarkets plc, UK) and a
• sweet white dessert wine

The procedure followed was:

• Lucozade Energy was diluted 1 in 100 and the apple juice and wine were diluted 1 in 50 with deionised water to bring them to 0.05 to 5mg glucose/ml.
• 100µl of these solutions were added to 1.0ml of the Glucose Assay Reagent in a cuvette and incubated at room temperature for 15 minutes.
• A sample blank comprising 100µl of sample and 1.0ml of water and a reagent blank consisting of 1.0ml of Glucose Assay Reagent and 100µl of water were also prepared.
• The absorbance at 340nm was measured after 15 minutes against a deionised water blank.
• The cuvettes were measured in four different spectrophotometers for comparison: models 6300, 6315, Genova and 6505.

Results

It is possible to calculate the concentration of glucose in the samples using the millimolar extinction coefficient for NADH at 340nm using the equation shown below as given in the manufacturers' instructions.

The glucose concentration determined in each of the drinks with this calculation is shown in Table 1.

Table 1. Measured glucose content of three different drinks.

Lucozade Energy is commercially marketed as a high energy drink that offers an energy boost after intense physical activity. This is shown in the high glucose level that offers a ready source of carbohydrate that can be easily metabolised. According to the bottle label the drink contains 21.8g sugars per 250ml which equates to 87.2mg/ml. It was found that the glucose measured by the assay was only half of this hence the drink contains sugars other than glucose.

The sugar content of apple juice consists mainly of glucose, fructose and sucrose and the profile of each of these depends strongly on the cultivator and region in which the fruit is grown. Apple glucose content can range from less than 10mg/ml to greater than 30mg/ml. The glucose content of the juice measured in this experiment (16mg/ml) is within limits.

The main fermentable sugars in grape juice are glucose and fructose. Any sugars remaining in the wine at the end of fermentation are termed as residual sugars and the amount of these defines the level of sweetness of the wine.

Residual sugar concentration is expressed in g/l and includes all reducing sugars including glucose, fructose and other unfermentable sugars such as pentoses. The assay in this experiment measured only the residual glucose present in the wine and this was determined to be around 22g/l. Since the rate of fermentation of glucose is faster than fructose this signifies only a fraction of the total residual sugar in the wine sample. Wines from around 18g/l up to 45g/l are classified as medium sweet and those with more than 45g/l are classified as sweet. The wine in this experiment was a sweet white dessert wine so the results are in agreement of this classification.

Conclusions

One of the many assays that can be combined with dehydrogenases that use NAD⁺ or NADH as coenzymes is glucose phosphorylation by hexokinase. Dehydrogenases that use these coenzymes or the phosphorylated forms NADP⁺ and NADPH can also be assayed directly by measuring the changes in absorbance at 340nm. As NADH has a well defined extinction coefficient there is no need for using standards thus avoiding the need of constructing a calibration curve, as with other types of colorimetric assay. All of the Jenway spectrophotometers used gave comparable results and are therefore suitable for these types of assays.

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