Biochemical Assay of Glucose Notes

Learning Objectives

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

Understand the principle of spectrophotometry and its applications in clinical diagnostics.
Perform the proper steps for the accurate estimation of a blood glucose sample, including sample preparation and handling.
Properly choose and use a micropipette to ensure accurate volume measurements.
Operate a spectrophotometer, including calibration and quality control measures.
Interpret the laboratory result in relation to the normal values of blood glucose levels and understand the clinical implications of abnormal values.

Methods for Measuring Blood Glucose

Two primary methods exist for measuring glucose in urine or plasma:

Reduction of an Alkaline Copper Reagent:
- Detects any reducing compound present, making it non-specific. This includes substances like uric acid, creatinine, and ascorbic acid.
- Results in a reddish precipitate due to the formation of cuprous oxide (Cu_2O).
- Reducing agents besides glucose interfere with the results, leading to overestimation of glucose levels. Benedict's reagent is a common example.
Reaction with the Enzyme Glucose Oxidase (using spectrophotometer):
- Specific for glucose estimation, as the enzyme glucose oxidase selectively oxidizes glucose.
- Produces a color, where the intensity is proportional to the glucose concentration. The reaction typically involves the oxidation of a colorless dye to a colored compound.
- Other reducing compounds like ascorbic acid (vitamin C) and other reducing sugars can reduce the color back to a colorless form, leading to underestimation of glucose levels. Proper controls and calibration are essential to minimize these interferences.

Spectrophotometry

Definition

A spectrophotometer is an instrument used to measure the amount of light absorbed by a sample at a specific wavelength. It provides quantitative data about the concentration of substances in a solution.

Principle

Any solution containing a compound that absorbs visible light in the range of 380-750 nm will appear colored to the eye. The color observed is complementary to the color of light absorbed.
Deeper color indicates higher concentration, as more molecules absorb more light. This relationship is described by Beer-Lambert Law: A = \epsilon \cdot b \cdot c, where A is absorbance, \epsilon is molar absorptivity, b is path length, and c is concentration.

Spectrophotometer Operation Principle

A chemical reaction between a specific reagent and the parameter of interest (e.g., glucose) in the test sample yields a colored product. This reaction should be specific and well-characterized.
The intensity of the colored product is proportional to the concentration of the parameter of interest. This relationship must be linear within a defined range to ensure accurate measurements.
The colored product absorbs light at a specific wavelength, which is selected to maximize sensitivity and minimize interference from other substances.

Preparatory Steps Before the Assay

Wash hands with alcohol gel to prevent contamination of the sample and reagents.
Put on gloves to protect yourself from potentially infectious materials and prevent contamination of the sample.
Check the name and age on the sample to ensure correct patient identification and appropriate reference ranges.
Check the expiry date of the kit to ensure the reagents are still valid and will produce accurate results.

Micropipettes

Importance

Adding precise volumes is crucial for optimal results, hence the use of micropipettes. Inaccurate pipetting can lead to significant errors in the assay.

Micro pipetting Steps

Choose/set the required volume correctly (if it is a variable micropipette). Ensure the micropipette is calibrated for the intended volume.
Fit the tip securely to prevent leakage and ensure accurate aspiration of the liquid.
Depress the plunger to the first stop and immerse the tip into the liquid. The first stop corresponds to the set volume.
Draw up the liquid by releasing the plunger back to the rest position slowly and smoothly to avoid air bubbles.
Check the liquid drawn up for air bubbles in the tip. If bubbles are present, discard the sample and repeat the process.
Expel all the liquid by placing the tip into the test tube and depressing the plunger to the second stop. The second stop ensures complete dispensing of the liquid.

Colorimetric Estimation of Blood Glucose by Glucose Oxidase Reaction

Principle

Glucose is oxidized by glucose oxidase enzyme, releasing hydrogen peroxide (H2O2). The reaction is highly specific for glucose.
Hydrogen peroxide is dissociated into water and an oxygen atom by peroxidase enzyme. This step amplifies the signal and allows for color development.
The liberated oxygen is captured by a dye, producing a red color. The intensity of the red color is directly proportional to the amount of glucose in the sample.
Any compound that reduces H2O2, like vitamin C, will convert H2O2 into H_2O and inhibit the production of the red color. Other reducing sugars can also reduce the dye back to a colorless form. This can lead to falsely low glucose readings.

Procedure

Label three dry test tubes: T (Test sample), St (Standard sample), and B (Blank).
Pipette 1000 µl of the reagent into each of the three test tubes. Ensure the reagent is thoroughly mixed before pipetting.
Pipette 10 µl of the test sample into the 'T' tube, 10 µl of the standard into the 'St' tube, and 10 µl of distilled water into the 'B' tube. Use a fresh tip for each sample to avoid cross-contamination.
Mix the contents of the tubes and let them stand for 5 minutes at room temperature. This incubation period allows the enzymatic reaction to proceed to completion.

Note: These quantities may vary with each kit. Always follow the supplied instructions carefully. Different kits may have variations in reagent concentrations and incubation times.

	Blank	Standard	Test Sample
Standard	-	10 µl	-
Test Sample	-	-	10 µl
Reagent	1000 µl	1000 µl	1000 µl

Mix and incubate for 5 minutes. Read the absorbance of the sample/standard against the reagent blank at a wavelength of 546 nm. This wavelength provides optimal absorbance for the colored product formed in the reaction.

Add the contents of the test tubes to the cuvettes in the following order:-
- Add the blank, then press the R button to reset the spectrophotometer to 0.0. This calibrates the instrument to account for any background absorbance.
- Discard any blank leftover to prevent contamination.
- Add the test sample, press the T button, and record the absorbance.

The Blank

A blank is a cuvette containing all the carrier solvents EXCEPT the parameter of interest. A separate blank is needed for every unique reaction mixture. It's used to adjust the zero of the spectrophotometer, so that only the absorbance of the parameter of interest is measured. This ensures that the absorbance reading is specific to the analyte being measured.

Standard Sample

A standard sample is a prepared sample of known concentration, identical to the parameter of interest (e.g., glucose), provided with the kit. It is processed in the EXACT same way as the test sample to obtain its absorbance. The standard is used to calibrate the assay and calculate the concentration of the unknown samples.

Calculation

A{test} = \epsilon C{test}

A{standard} = \epsilon C{standard}

C{test} = \frac{A{test}}{A{standard}} \times C{standard}

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

Blood Glucose Monitoring

Test	Normal	Prediabetes*	Diabetes
Fasting plasma glucose (FPG) level	70-99 mg/dL	100-125 mg/dL	≥126 mg/dL
2-hour plasma glucose (2h-PG) during an OGTT	< 140 mg/dL	140-199 mg/dL	≥ 200 mg/dL

Other Tests to Assess Blood Glucose Levels

HbA1c (Glycosylated Hemoglobin): Indicates how well diabetes has been controlled in the past 2-3 months by measuring the percentage of hemoglobin that is glycated. An HbA1c of 6.5% or higher indicates diabetes.
Oral Glucose Tolerance Test (OGTT): A medical test where glucose is given orally, and blood and urine samples are taken afterward to determine how quickly it is cleared from the blood. It is used to diagnose gestational diabetes and insulin resistance.
- How the OGTT is done:
  1. Patient should be fasting for at least 8 hours to ensure accurate baseline glucose levels.
  2. The subject sits quietly throughout the test to minimize variations in glucose metabolism.
  3. A fasting blood sample (time 0) is withdrawn to establish the baseline glucose level.
  4. Fasting urine sample is collected to check for the presence of glucose or ketones.
  5. 75 g anhydrous glucose is dissolved in 250-300 ml water and is ingested orally within 5 min. The glucose load challenges the body's ability to regulate blood sugar.
  6. Blood is withdrawn & urine is collected every 30 min for 2 hours to monitor glucose clearance.
- Precautions for OGTT:
  - AVOID eating from midnight to ensure a true fasting state.
  - AVOID carbohydrates restriction for several days before the test to ensure normal insulin sensitivity.
  - AVOID severe exercise as it can affect glucose metabolism.
  - AVOID smoking as nicotine can affect glucose levels.
  - AVOID drinking coffee as caffeine can interfere with glucose metabolism.
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 differentiates between type I and II diabetes.
  - Type 1 diabetes: low levels of insulin and C-peptide due to autoimmune destruction of pancreatic beta cells.
  - Type 2 diabetes: normal or elevated level of C-peptide due to insulin resistance and compensatory insulin secretion.

Sample Results Interpretation