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Quantitative Estimation of Blood Glucose

The document outlines the estimation of blood glucose levels, emphasizing its importance in diagnosing and monitoring diabetes mellitus. It details two main methods for glucose estimation: enzymatic and reduction methods, with a focus on Trinder’s method for its specificity. The document also provides guidelines for specimen collection, procedure, calculation, interpretation of results, and clinical conditions related to hyperglycemia and hypoglycemia.

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24 views6 pages

Quantitative Estimation of Blood Glucose

The document outlines the estimation of blood glucose levels, emphasizing its importance in diagnosing and monitoring diabetes mellitus. It details two main methods for glucose estimation: enzymatic and reduction methods, with a focus on Trinder’s method for its specificity. The document also provides guidelines for specimen collection, procedure, calculation, interpretation of results, and clinical conditions related to hyperglycemia and hypoglycemia.

Uploaded by

33.nehapriya.mbbs2024gmckmr
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Estimation of blood glucose

Date: 23/04/2024 (2-4pm)

Aim
● To estimation blood sugar level

Introduction
● The term ‘blood sugar’ includes all hexoses even though the main hexose present
in blood is glucose. Hence, effectively blood sugar means blood glucose.

● Blood glucose level is an important indicator of body function and is actively


maintained in normal range.

● It is an essential nutrient for the brain and RBCs.

● Abnormalities of blood glucose level indicate disturbance of carbohydrate


metabolism.

● Diabetes mellitus is the commonest disease of carbohydrate metabolism.

● The estimation of blood glucose is routinely done for the diagnosis and follow up
of diabetes mellitus patients.
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Method
● Broadly 2 types of methods are available:

○ 1) Enzymatic methods

○ 2) Reduction methods

● 1) Enzymatic methods:

These are specific methods in which enzymes specifically act only on glucose.

The enzymatic reaction of glucose is coupled with a colored reaction whose


intensity is proportional to glucose.

Commonly used enzymatic methods are

a) Glucose oxidase - Peroxidase method (Trinder’s method)


b) Hexokinase method

● 2) Reduction methods:

These are based on the reducing property of glucose.

In hot alkaline medium sugars reduces metal ions like copper (Cu) and
ferricyanide ions.

This reduction is coupled with a reaction to give a colored product whose


intensity is proportional to glucose level.

Several methods based on this principal are available as follows

a) King and Asatoor method


b) Nelson somogyi method
c) Folin Wu method
3

● These methods are relatively non-specific because many other substances


present in blood other than glucose, also cause reduction leading to higher
results.
● Such non glucose reducing substances are
a) Glutathione
b) Vitamin C
c) Uric acid
d) Glucuronic acid
e) Threonine etc.

Estimation of Glucose by Trinder’s method


● Principle:

Glucose in the sample is oxidised to yield gluconic acid and hydrogen


peroxide in the presence of glucose oxidase.

The enzyme peroxidase catalyses the oxidative coupling of 4-amino


antipyrine with phenol to yield colored quinonemine complex, with
absorbance proportional to the concentration of glucose in the sample.

Glucose oxidase
Glucose + O2 + H2O ------------------------------> Gluconic acid + H2O2

Peroxidase
H2O2 + 4-HBA + 4-AAP ----------------------------> Quinonemine Dye + 2H2O

● 4-AAP : 4-Aminoantipyrine
● 4-HBA : 4-Hydroxy Benzoic acid

● Reagent composition:
a) Glucose oxidase
b) Peroxidase
c) 4-Amino antipyrine
4

d) 4-Hydroxy Benzoic acid


e) Phosphate buffer
f) Glucose standard (100 mg/dL)

● Specimen collection and handling:


● Use unhemolytic serum, plasma (Heparin, EDTA) or Urine.
● Stability after addition of a glycolytic inhibitor (Fluoride, Monoiodoacetate,
Mannose)
○ 2 days at 20-250C
○ 7 days at 4-80C
● Discard contaminated specimens.

● Procedure:

Pipette into tubes marked Blank Standard Test

Reagent 1000 microL 1000 microL 1000 microL

Distilled water 10 microL – –

Standard – 10 microL –

Test – – 10 microL

Mix well and incubate for 15 minutes at 37oC (Room temperature).

Read the absorbance of standard and each test tube against reagent blank at
505 nm (500-540 nm) or 505/670 nm on Bichromatic analyzer.

● Calculation:

𝐺𝑙𝑢𝑐𝑜𝑠𝑒 (𝑚𝑔/𝑑𝐿) = 𝐴𝑏𝑠. 𝑜𝑓 𝑡𝑒𝑠𝑡/𝐴𝑏𝑠. 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑥 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (𝑚𝑔/𝑑𝐿)

● Interpretation:

Serum:
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Glucose fasting

● Cord : 45 – 96 mg/dl
● Newborn, 1 Day : 40 – 60 mg/dl
● Newborn, >1 Day : 50 – 80 mg/dl
● Child : 60 – 100 mg/dl
● Adult : 74 – 100 mg/dl
● > 60 years : 82 – 115 mg/dl
● > 90 years : 75 – 121 mg/dl

Glucose 2 hours postprandial : < 120 mg/dl

Urine : 1 – 15 mg/dl

● It is recommended that each laboratory verify this range or drives reference


interval for the population it serves.

Clinical conditions:

Hyperglycemia : Increased blood glucose levels, observed in following


conditions

Physiological :

● After high carbohydrate diet


● Emotional stress, Anger, Anxiety
● Pregnancy

Pathological :
● Diabetes Mellitus
● Hyperactivity of thyroid, adrenal & pituitary glands
● Diseases of pancreas like – Acute Pancreatitis & Pancreatic carcinoma
● Sepsis
● Moderate hyperglycemia is seen in increased intracranial pressure present
in Meningitis , Encephalitis, Hemorrhages, Intracranial tumors etc.
● Drugs - Steroids, O.C Pills, Estrogen.
Hypoglycemia:
● Decreased blood glucose levels (< 60 mg/dl) , observed in following
conditions.
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Physiological
● During starvation
● After severe exercise
Pathological
● Due to excess Insulin - Excess dose of Insulin, no food intake after Insulin
dose, Tumors of Pancreas (Insulinoma).
● Glycogen storage diseases (Von-Gierke’s disease).
● Hypoactivity of Thyroid, Adrenal & Pituitary glands.

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