Biochemical Assay of Glucose Notes

Spectrophotometry and Blood Glucose Estimation

Learning Objectives

By the end of this session, the student will be able to:

1. Understand the principle of Spectrophotometry.

2. Perform the proper steps for estimation of a blood glucose sample.

3. Properly choose and use a micropipette.

4. Operate a spectrophotometer.

5. Interpret the laboratory result in relation to the normal values of blood glucose levels.

Measurement of Blood Glucose

Two methods can be used to measure urine or plasma glucose:

1. Reduction of an alkaline copper reagent: This method detects any reducing compound present and is therefore non-specific. A reddish precipitate indicates the presence of reducing compounds. Reducing agents other than glucose interfere with the results of glucose detection by methods depending on its reducing power.

   • This method primarily relies on the reducing properties of glucose in an alkaline solution. When glucose reduces copper ions, it forms cuprous oxide, leading to the formation of a reddish precipitate.

   • Because other reducing substances can also react, this method is less accurate in samples containing multiple reducing agents.

2. Reaction with the enzyme glucose oxidase (using spectrophotometer): This method is specific for glucose estimation. It produces a color, and the intensity of the color is proportional to the glucose concentration. Other reducing compounds, including ascorbic acid (vitamin C) and a variety of other reducing sugars, present in the sample will reduce the color back to the colorless form.

   • The glucose oxidase enzyme selectively oxidizes glucose, producing hydrogen peroxide as a byproduct. This hydrogen peroxide then reacts with a chromogenic substance, forming a colored compound.

   • The specificity of the enzyme ensures that only glucose is measured, providing more accurate results in complex biological samples.

Spectrophotometry

Spectrophotometer

An instrument which can measure the amount of light absorbed by the sample at any selected wavelength.

Any solution containing a compound that absorbs visible light in the range of 380-750 nm will appear colored to the eye.

The deeper the color, the more molecules there are and the more light is "absorbed".

Principle of Spectrophotometer Operation

1. The chemical reaction between the specific reagent and the parameter of interest (e.g., glucose) in the test sample will give a colored product.

2. The intensity of this colored product is proportional to the concentration of the parameter of interest in the test sample.

3. This colored product will absorb light of a specific wavelength.

   • The spectrophotometer measures the amount of light that passes through the sample and compares it to the amount of light that passes through a blank or reference sample.

   • By analyzing the absorption spectrum, the concentration of the substance can be determined using Beer-Lambert Law.

Preparatory Steps Before the Assay

1. Wash hands with alcohol gel.

   • This crucial step minimizes contamination and ensures the integrity of the sample.

2. Put on gloves.

   • Gloves prevent contamination and protect the personnel from potentially hazardous materials.

3. Check the name and age on the sample.

   • Verifying patient information is essential for accurate record-keeping and result interpretation.

4. Check the expiry date of the kit.

   • Using expired reagents may lead to inaccurate results.

Micropipettes

Micropipetting Steps

1. The required volume is correctly chosen/set (if it is a variable micropipette).

   • Selecting the appropriate volume is crucial for accuracy.

2. The tip is securely fitted.

   • An airtight seal prevents leakage and ensures accurate volume measurement.

3. The plunger is depressed to the first stop, and the tip is immersed into the liquid.

   • Depressing to the first stop ensures the correct volume of liquid is drawn into the tip.

4. The liquid is drawn up by releasing the plunger back to the rest position.

   • Slow and steady release prevents air bubble formation.

5. The liquid drawn up is checked for air bubbles in the tip.

   • Air bubbles can cause inaccurate measurements.

6. Expel all the liquid by placing the tip into the test tube receiving the liquid, then the plunger is depressed to the second stop.

   • Depressing to the second stop ensures all the liquid is expelled from the tip.

Colorimetric Estimation of Blood Glucose by Glucose Oxidase Reaction

Principle of the Glucose Oxidase Reaction

• Glucose is oxidized by glucose oxidase enzyme with liberation of hydrogen peroxide (H2O2).

• Hydrogen peroxide is dissociated to water and oxygen atom by peroxidase enzyme.

• The liberated oxygen is captured by a dye giving the red color.

Note: Production of H2O2 by glucose oxidase is the key compound which gives the red color, so any other compound that reduces H2O2 as vit.C (antioxidant) will convert H2O2 into H_2O and will not produce the red color. Also, other reducing sugars present in the sample will reduce the dye back to the colorless form.

Steps for the Assay

1. Step 1: Label 3 dry test tubes: T (Test sample), St (Standard sample), and B (Blank).

2. Step 2: Pipette 1000 µl of the reagent into the previous 3 test tubes.

3. Step 3: Pipette 10 µl of the given Test sample, Standard, and distilled water into their corresponding test tubes.

4. Step 4: Mix the contents of the tubes and let stand for 5 minutes at room temperature.

   • Consistent mixing ensures uniform distribution of reactants.

   • Incubation at room temperature allows for optimal enzyme activity.

N.B. These quantities may vary with each kit. You must follow the supplied instructions carefully!

Read the absorbance of sample/Standard against the reagent blank at wavelength 546 nm.

5. Step 5: Add the contents of the test tubes to the cuvettes in the following order:-

   • A- blank then press the R button to reset the spectrophotometer. Make sure that the absorbance reads 0.0. Put the blank back in its test tube.

     • This step is vital for accurate calibration.

   • B- Empty the cuvette on a piece of tissue paper to discard any blank leftover.

     • This ensures no cross-contamination between samples.

   • C- then add the test sample to be measured, press the T button and record the given absorbance on a piece of paper.

     • Recording ensures proper tracking of results for subsequent analysis.

Blank

So that ONLY the absorbance of the parameter of interest is measured.

This is done with a blank - a cuvette which contains all the carrier solvents EXCEPT the parameter of interest. A separate BLANK is needed for every unique reaction mixture. It is used to adjust the zero of the spectrophotometer.

Standard sample

• It is a prepared sample of known concentration identical to the parameter of interest (eg: glucose) provided with the kit.

• It is processed EXACTLY the same way as the parameter of interest to obtain its absorbance.

Equation for calculation of the concentration of test sample

A{test} = varepsilon C{test}

A{Standard} = varepsilon C{Standard}

C{test} = frac{A{test}}{A_{Standard}}

imes C_{Standard}

Concentration of blood glucose (mg/dl) =\frac{Absorbance \space of \space Test}{Absorbance \space of \space standard} \times Concentration \space of \space standard

Blood Glucose Monitoring

Other tests to assess blood glucose levels

1- HbA1c (Glycosylated hemoglobin)

It indicates how well diabetes has been controlled in the past 2-3 months.

2- Oral Glucose Tolerance Test OGTT

It is a medical test in which glucose is given orally then blood and urine samples are taken afterward to determine how quickly it is cleared from the blood.

How the OGTT is done?

1. Patient should be fasting for at least 8 hours.

2. The subject sits quietly throughout the test.

3. A fasting blood sample (time 0) is withdrawn.

4. Fasting urine sample is collected.

5. 75 g anhydrous glucose is dissolved in 250-300 ml water and is ingested orally within 5 min.

6. Blood is withdrawn & urine is collected every 30 min for 2 hours.

   • This procedure helps monitor glucose clearance over time.

Precautions for OGTT

AVOID

• Eating from midnight

• Carbohydrates restriction

• Severe exercise

• Smoking

• Drinking coffee

  • These factors can affect the accuracy of the test results.

3- C-peptide

The connecting peptide, or C-peptide, is a short polypeptide that connects insulin's A-chain to its B-chain in the proinsulin molecule.

C-peptide level

It differentiates between type I and II diabetes.

• A person whose pancreas does not make any insulin (type 1 diabetes) has a low level of insulin and C-peptide.

• A person with type 2 diabetes has a normal level of C-peptide.

Results of testing urine samples