Glucose Estimation

Introduction to ESTIMATION OF GLUCOSE

 1. BACKGROUND: Estimation of glucose in blood has become a routine practice in clinical biochemistry for screening of metabolic disorder like diabetes mellitus. Abnormalities of blood sugar level reflects disturbance in carbohydrate metabolism.

 2. LEARNING OBJECTIVES: At the end of this practical student should be able to;

  • Estimate the serum glucose.
  • Explain the principle and procedure of glucose.
  • Interpret the results obtained and correlate with the clinical finding
  • Explain the role of glucose in health & disease.
  • Principle of glucose estimation:
    • In quantitative estimation of blood sugar either whole blood, plasma or serum can be used. Glucose oxidase is an enzyme found in the growth medium of pencillium notatum. In this method glucose is oxidized by glucose oxidase to gluconic acid with the gluconolactone as intermediate. The H2O2 which is also formed is broken down to water and oxygen by a peroxidase, in presence of an oxygen acceptor which is converted to pink coloured compound. The overall reaction is as follows:
      • Glucose + O2 + H2O   Glucose oxidase     Gluconic acid + H2O2
      • H2O2 + 4AAP + 4HBA        Peroxidase        Quinoneimine dye + 2H2O
      • 4AAP: 4-Aminoantipyrine
      • 4HBA: 4-Hydroxy benzoic acid
  • The intensity of the pink colour formed is proportional to the glucose concentration and can be measured photometrically between 500-540nm.
  • Sample Collection:
    • Serum, plasma, CSF or urine. The stability of glucose in specimens may be reduced by bacterial contamination and by glycolysis. Serum should be separated from the cells as soon as possible to prevent glycolysis. The addition of sodium fluoride 10mg/ml of blood is recommended to inhibit glycolysis. Commonly use anticoagulants with concentrations not causing interference are:
      • Oxalate  –   20mg/ml blood
      • EDTA    –   10mg/ml blood                     
      • Citrate  –    30mg/ml blood
      • Heparin    2mg/ml blood
  • Glucose is stable in serum or plasma for 4 hours at 30°C and 24 hours at 2-8°C Storage. 

Reagents required: 

  • PROCEDURE

    Glucose estimation procedure: Take 3 test tubes and mark them as ‘blank’, ‘standard’& ‘test’ Flow sheet

Mix well and incubate for 15 min at 37°C, read the absorbance against blank at 505nm.

OBSERVATION:

  • Glucose:  
    • OD of Blank-
    • OD of Test-
    • OD of Standard-

CALCULATION:

INTERPRETATION:

  • Glucose Interpretation:
    • Glucose is one of the important risk factors for Hyperglycemia, Hypoglycemia.

NORMAL RANGE:

  • Glucose normal range:
    • Fasting              70-110mg/dl
    • Post prandial     90-170mg/dl
    • Random             ≥200mg/ml
  • Explain the role of glucose in health & disease.
    • Blood Glucose Levels and Diabetes
      • Your blood sugar level normally rises after you eat.
      • Then it dips a few hours later as insulin moves glucose into your cells.
      • Between meals, your blood sugar should be less than 100 milligrams per deciliter (mg/dl).
      • This is called your fasting blood sugar level.

    Without enough insulin, glucose can’t move into the cells.The blood glucose level stays high. A level over 200 mg/dl 2 hours after a meal or over 125 mg/dl fasting is high blood glucose, called hyperglycemia Too much glucose in your bloodstream for a long period of time can damage the vessels that carry oxygen-rich blood to your organs. High blood sugar can increase your risk for:

  • ➢Heart disease, heart attack, and stroke
  • ➢Kidney disease
  • ➢Nerve damage
  • ➢Eye disease called retinopathy
  • People with diabetes need to test their blood sugar  often. Exercise, diet, and medicine can help keep blood glucose in a healthy range and prevent these complications.

Diabetic keto-acidosis 

  • The term Diabetes Mellitus id derived from the Greek word Dia= through and Baitein= to go, Diabetes literally means ‘Pass through’ and mellitus=sugar. 
  • In this disease the weight loss is observed and glucose comes out through urine as if ‘the body mass passes through urine’.
  • Diabetes Mellitus is a metabolic disease due to absolute or relative Insulin deficiency. It’s a common metabolic syndrome where 1/5 person suffer from after the age of 50, having a morbidity and mortality rate as well.
  • Insulin deficiency leads to improper uptake of glucose inside the cell, causing increased blood glucose level onwards. So as high amount of glucose is present in the blood plasma, cells still starve for glucose inside.
  • Diabetes can be classified into 2 subtypes- type-I and type-II. Where type-I is insulin dependent and type-II is insulin independent.
  • Diabetic Keto-acidosis is a metabolic complication, found mostly in type-I diabetes mellitus, where ketone bodies get synthesized in high concentration.
  • Normal concentration of ketone bodies in serum is < 1mg/dL but in diabetes, this synthesis process exceeds the capacity of extra-hepatic tissues to utilize and starts appearing in serum cases Ketonemia.
  • Excretion through urine also increases called Ketonuria, and as a whole called as keto-acidosis.

Causes of keto-acidosis

  1. In diabetes:  In type-I diabetes, diabetic keto-acidosis (DKA) combined with hyperglycemia, glucosuria,ketonuria. Here glucose is plenty in the circulation but due to insulin deficiency cells cannot utilize it (as insulin dependent transporters are not working). To generate ATPs cells start utilizing fatty acids instead of glucose causes lipolysis and more and more fatty acids get released into the circulation.
  • Oxidation of fatty acid increases acetyl Co-A pool to enter into TCA cycle, which further forms ketone bodies in a bypass process of ketogenesis. 

2. In starvation:  In starvation dietary supply of glucose is decreased. Excess Oxaloacetate channeled to gluconeogenesis and lipolysis takes place to produce Acetyl co-A for TCA cycle, causes generation of ketone bodies.

Metabolism or Mode of action in presence of Insulin

  • Hormone Insulin enhances the uptake of glucose in liver, playing the central role in blood glucose regulation. It is produced in ß-cell of pancreas works via GluT-2 transporter also helps in phosphorylation of glucose by glucokinase, which promotes glycolysis and TCA.
  • In presence of Insulin, Break-down of Glucose takes place as well as Glycogenesis is also triggered where Glucogenolysis and Neoglucogenesis is suppressed.
Glucose uptake in presence of insulin via glut-2 and glut-4.
  • Insulin has an important role in fatty acid metabolism also, where in presence of insulin, Fatty acid synthesis is taking place and break-down of fatty acid is suppressed.
Fatty acid metabolism in presence of Insulin.

 Glucagon opposes the action of Insulin

  • Glucagon is produced by -cell of pancreas; found to work exactly opposite to insulin, its secretion gets stimulated by Hypoglycemia.
  • It stimulates gycogenolysis by activating phosphorylase and lipolysis by activating lipase to produce Acetyl Co-A.
  • In absence/deficieny of Insulin, glucagon gets activated and excess amount of Acetyl Co-A further forms Ketone bodies in circulation, promoting Keto-acidosis.
Insulin and Glucagon mediated acidosis in DKA

Other Biochemistry Notes

Biochemistry of Proteins

Carbohydrate Metabolism

VITAMIN PYRIDOXINE AND BIOTIN

Vitamin A

VITAMIN D

Pyrimidine metabolism

Purine Metabolism

Cardiac Biomarkers

. Blood Metabolism (Heme synthesis and breakdown)

Haemoglobinopathies

DNA Repair

Regulation of gene expression

DNA Replication

Transcription

Protein synthesis and genetic code

Detoxification (Xenobiotics)

Liver Function Test

Transamination Deamination & Urea Cycle

METABOLISM OF INDIVIDUAL AMINO ACIDS

Hydroxy amino acids

ACIDIC AMINO ACIDS (Aspartate and Glutamate)

Inborn Error Of Phenyl alanine, Tyrosine Metabolism

Electron Transport Chain & Biological Oxidation

RENAL FUNCTION TEST

Biochemistry Of Sodium, Potassium & Chloride

Congenital Kidney Diseases

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