Method for measurement of glucose concentrations

Background
It is essential that everyone keeps track of the amount of glucose that circulates around the body. In particular, people who suffer from Diabetes Mellitus or have a history of Diabetes in their family have to be more concerned with the amount of glucose in their body. There are two main bodily fluids, namely urine and blood that may be used to measure the glucose level in the body. However, measurements of glucose in the blood have been found to be more accurate than urine glucose tests. Monitoring of blood glucose is especially fundamental for diabetics who need to know whether their diabetes is under control. It is expressed as millimoles per litre (mmol/l) and the normal blood glucose level usually stays within the narrow limits throughout the day of 4 to 8mmol/l (Henry J.B. 2001). The blood glucose levels are expected be the highest after meals. Blood glucose levels which are above the normal levels for long periods of time usually are at high risk of developing serious complications that are associated with diabetes.
Blood glucose levels may be measured using different methods but they all require a drop of capillary blood. There are many home blood glucose level testing kits and they all have at least two things, a measuring device and a strip. The method for use is very simple and quick for it involves the application of a blood sample on a test trip and this is then inserted into a meter for reading.
However, these kits are known for their imprecision (Clark, 1962). Most of these kits use a chemical method which relies on the non-specific reducing property of glucose in a chemical reaction with a marker that changes colour when reduced. However, as blood contains other compounds such as urea etc which also have reducing properties, this procedure can result in erroneous readings. The more commonly used technique, involves the use of enzymes which are specific to glucose and which are less vulnerable to false positives/ negatives (Schrot R.J., 2007). The two most universally used enzymes for this are (1) glucose oxidase and (2) hexokinase.
Electronic blood glucose meters is another method used to measure blood glucose levels. However, being electronic, this is a more expensive method of measurement but results in more accurate readings. Similar to the chemical method, it also requires a drop of blood (usually of a smaller amount compared to the amount required for the chemical method) which is applied to a test strip that is connected to a digital meter. After a few moments, the blood glucose level is provided on the digital display of the meter.
Another more advanced device for measuring blood glucose levels involves the use of glucose sensing bio-implants. These are devices for measuring glucose levels which are surgically implanted into the body. These bio-implants may be introduced by minor surgical implantation of the sensors. The longevity of these sensors ranges from one year to more than five years and differs from product to product.
Accordingly, the most cost-effective and yet accurate method, involves the use of enzyme glucose oxidase which is highly specific and will not catalyse the oxidation of any other sugar (Sacher R.A., 2001). Glucose oxidase catalyses the following reaction:
Glucose + Oxygen → Gluconic acid and Hydrogen Peroxide
Neither of the products of the above reaction are detectable, but the hydrogen peroxide can be used to oxidize a dye, for example 4-aminophenazone in a reaction catalysed by the enzyme peroxide. This generates a magenta coloured molecule whose concentration can be determined using a spectrophotometer. This method is accurate, cheap and simple to use.

Sample Preparation
Red blood cells is to be lysed and the protein removed by addition of protein precipitant. This step is carried out as red blood cells have a high concentration of protein, in particular hemoglobin, which in general has low water content and consequently less dissolved glucose compared to serum of blood. Further, a high red blood cell count may result in excessive glycolysis and consequently a substantial reduction of glucose level. The red blood cells are thus lysed and the sample processed quickly for a more accurate measure of the amount of blood glucose.

Standard Solutions
In order to determine the concentration of the glucose in the sample (i.e. blood), the sample is compared with standard solutions of glucose with known concentrations. The standard solutions undergo the same treatment as the samples to ensure consistency and for experimental uniformity. The results (i.e. absorbance) are plotted against the concentration of glucose measured to obtain a standard curve.

Method

1. 0.1 cm3 of the sample is mixed with 2.9 cm3 protein precipitant to form 3 cm³ of mixture
2. 1 cm3 of the mix is transferred to a fresh test tube
3. cm3 of enzyme glucose oxidase reagent is added to the test tube and gently mixed
4. The test tube of mixture is left to incubate at 37°C for 15 minutes
5. The absorbance at 515nm is read using an appropriate blank.

Same method is carried out on a range of controls with varying concentrations to be used as reference in determining the concentration of the sample.

1. 9 cm3 of distilled water is added into 6 different test tubes (A-F)
2. 1 cm3 stock glucose of known concentration (for example 0.1 mg/dl) is added to the first tube (A) for a serial dilution
3. 1 cm3 of the solution obtained in step 2 is added to tube B
4. 1 cm3 of the solution obtained in step 3 (from step B) is added to tube C and this is repeated right up to tube F.
5. 0.1 cm3 of the solutions of each tube (A-F) are added into 6 fresh tubes (A’-F’)and 2.9 cm3 of protein precipitant is then added into each tube
6. 1 cm3 of the solution obtained in each tube A’-F’ from step 5 is similarly added into another 6 fresh tubes and labeled as A”-F”
7. 3 cm3 glucose oxidase reagent is then added into all A”-F” tubes
8. All the tubes are incubated at 37oC for 15 minutes
9. The absorbance read at 515 nm using an appropriate blank.
10. Steps 1 to 9 are repeated 3 times with different standards to obtain an average absorbance for the each concentration of glucose to reduce the amount of error in each concentration of glucose and the absorbance is plotted against the concentration to obtain a glucose standard curve.
11. The coefficient variation (CV) of this assay may be measured using the formula:

Mean/ Standard Deviation

1. The standard deviation may be computed by determining the difference of each reading data point from the mean, and squaring the result.
2. The standard deviation for each concentration of glucose may be averaged based on the three absorbance readings obtained and the CV calculated for each concentration of glucose and the average CV obtained for the whole assay.

By using the standard graph plotted, the concentration of glucose may be obtained by measuring the absorbance of any sample. To confirm if the results are correct, a similar method as explained above can be used to measure the actual absorbance of known concentrations of glucose and comparing these results with the results derived from the standard curve. For example
Once the various concentrations are obtained, steps 5 to 10 from the above method are repeated to find an average absorbance for each concentration and to reduce the inaccuracy. The absorbance reading obtained from the blood sample may then be used together with the graph to determine the approximate concentration of glucose in the blood.

Discussion
As mentioned above, this method is not 100% accurate and as such gives a rough gauge of the concentration of glucose in the blood sample. A more accurate measure of the glucose in the blood can be obtained using an electronic blood glucose meter. However, if the method of the present experiment is preferred, the results obtained may be made more accurate by increasing the number of references so that the measure of glucose concentration in the blood is more accurate to the decimal. Further, instead of lysing the blood cells, it may be more appropriate to centrifuge the whole blood and remove the blood cells, thus only measuring the blood glucose level in the serum. This may overcome the disadvantages attributed by the presence of the blood cells.