Blood glucose biochemistry lab report

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER: Mr NJOGU STEPHENE/DR OKUN
HANDING DATE: 20 /11/2015
SUBJECT: PRACTICAL REPPORT ON TEST FOR BLOOD GLUCOSE
ESTIMATION USING GLUCOSE OXIDASE/TRINDER´S METHOD.
11/20/2015
LANDOELVIS.O
©

2. Theory:
Glucose is a simple sugar which is a permanent and immediate primary source of energy to all
of the cells in our body. The glucose in blood is obtained from the food that you eat. This
glucose gets absorbed by intestines and distributed to all of the cells in body through
bloodstream and breaks it down for energy.
Body tries to maintain a constant supply of glucose for your cells by maintaining a constant
blood glucose concentration. The concentration of glucose in blood, expressed in mg/dl, is
defined by the term glycemia. The value of blood sugar in humans generally ranges from 70 -
100 mg/dl. Blood sugar levels are regulated by the hormones insulin and glucagon which act
antagonistically. These two hormones are secreted by the islet cells of the pancreas, and thus
are referred to as pancreatic endocrine hormones. When the blood glucose levels are high,
insulin hormone secreted which causing liver to convert more glucose molecules into glycogen
and when the blood glucose levels are low glucagon secreted and act on liver cells to promote
the breakdown of glycogen to glucose and increases the blood glucose concentrations.
Essentially blood glucose levels determine the time of secretion of these hormones.
The blood glucose level is easily changed under the influence of some external and internal
factors such as body composition, age, physical activity and sex. Diabetes is a disease related by
the abnormal metabolism of blood sugar and defective insulin production. So blood sugar levels
are an important parameter for the study of diabetes. The level of glucose circulating in blood
at a given time is called as blood glucose level. The blood glucose level varies at different time
on various part of the day. Hypoglycemia is a possible side effect of diabetes medications in
which blood glucose level drops below 70mg/dl. In people with diabetes, the body doesn't
produce enough insulin or respond to insulin properly. The result is that sugar builds up in the
blood stream, damaging the body's organs, blood vessels and nerves. This condition in which
too much sugar in the blood stream is called hyperglycemia.
The blood glucose analysis is ordered to measure the amount of blood at the time of sample
collection. It is used to detect both hyperglycemia and hypoglycemia and via helping the
diagnosis of diabetes. An ideal blood glucose estimation method should determine only
glucose. It is adaptable for both macro- and semi micro- techniques. Reagents are relatively
inexpensive and the method should require a minimum of time, techniques and apparatus, be
accurate and yield reproducible results. Glucose oxidase is an enzyme highly specific for glucose
and is not react with blood saccharides. So it has been employed for the estimation of blood
glucose.

3. Principle:
Glucose oxidase is an enzyme extracted from the growth medium of Aspergillus niger. Glucose
oxidase catalyse the oxidation of Beta D- glucose present in the plasma to D glucono -1 ,5 -
lactone with the formation of hydrogen peroxide; the lactone is then slowly hydrolysed to D-
gluconic acid. The hydrogen peroxide produced is then broken down to oxygen and water by a
peroxidase enzyme. Oxygen then react with an oxygen acceptor such as ortho toluidine which
itself converted to a coloured compound, the amount of which can be measured
colorimetrically.
(Trinder’s method )
-D-glucose Mutarotase -D-glucose
-D-glucose +H2O+O2
Glucoseoxidase D-gluconic acid+H2O2
Unfortunately, neither of the products absorbs light in the visible region. Therefore, the
production of hydrogen peroxide is coupled to a reaction catalyzed by HRP.
H2O2+ 4-aminophenazone+phenol Peroxidase Quinonemine +4 H2O
The intensity of the color formed is proportional to the glucose concentration in the sample.
The reaction product absorbs light maximally at λmax = 505 nm with a molar absorptivity ε =
1.27 x 104 L/mol-cm (M-1 cm -1).
CLINICAL SIGNIFICANCE
Glucose is a major source of energy for most cells of the body; insulin facilitates glucose entry
into the cells. Diabetes is a disease manifested by hyperglycemia; patients with diabetes
demonstrate an inability to produce insulin. Clinical diagnosis should not be made on a single
test result; it should integrate clinical and other laboratory data.
PREPARATION
Working reagent (WR):
Dissolve the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer.
Cap and mix gently to dissolve contents.
The reagent is stable 1 month after reconstitution in the refrigerator (2-8ºC) or 7 days at room
temperature (15-25ºC).
Signs of reagent deterioration:
- Presence of particles and turbidity.
- Blank absorbance (A) at 505 nm 0.10.
Requirements:
Samples:

4. -Blood samples
Whole blood
Serum
Plasma (with Ca.oxalates/NaF), which is the preferred sample
-Fresh urine by double void collection technique…….?
-CSF collected in sterile clean container and to be done immediately or centrifuged to
get cell free fluid.
Instrumentation:
-Photometer adjusted on wavelength 540 nm
-Cuvette (light path) 1 cm
-Water bath at 37 ºC
-Automatic pipettes, disposable test tubes , racks and disposable tips for the
dispensers.
PROCEDURE
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . 505 nm (490-550)
Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path
Temperature. . . . . . . . . . . . . . . . . . . 37ºC / 15-25ºC
2. Adjust the instrument to zero with distilled water.
3. Pipette 2mls of glucose oxidase into 5 tubes labeled: Blanck, standard, sample1, sample2
and sample3.
4. To the standard add 20µl of standard solution into tube labeled standard. And 20µl of sample
solutions into their respective marked tubes in step 2 above.
5. Mix and incubate for 10 min at 37ºC or 15-20 min at room temperature (15-25ºC).
6. Read the absorbance (A) of the samples and standard, against the Blank.
The colour is stable for at least 30 minutes.

5. OBSERVATION AND RESULTS
Solutions in test tubes Absorbance at 505nm Color change
Blanck 0.036 Colorless
Standard 0.279 Pink
Sample 1 0.137 Light pink
Sample 2 0.259 Pink
Sample 3 0.570 Conc: pink
CALCULATIONS
(A) Sample x 100 (Standard conc.) = mg/dL glucose in the sample
(A) Standard
Conversion factor: mg/dL x 0.0555= mmol/L.
Solutions in test tubes Glucose conc; in Mg/dl Glucose conc; in Mmol/L
Blanck 12.903 0.716
Sample 1 49.103 2.275
Sample 2 92.831 5.152
Sample 3 204.3 11.34
Normal Range:
Blood glucose… Fasting= 70 - 110 mg/dl (2.7 mmol/L – 6.4mmol/L) & 2 hrs. Postprandial
= 110 - 140 mg/dl( up to 8.3mmol/L)
Urine glucose .. < detectable limit (Nil)
CSF glucose ~ 60 - 90 mg/dl
Interpretation:
I -Hypoglycemia : The patient considered critically hypoglycemic if:
Whole Blood glucose level < 40mg/dl
Serum/Plasma glucose level < 45mg/dl
A- Well Fed State Hypoglycemia:
1- Excessive Insulin Release:
a. Reactive Hypoglycemia
b. Alimentary Hyperinsulinism

6. c. Leucine Hypersensitivity
2- Inherited Enzyme Defect:
a. Galactose -1- Phosphate
b. Fructose -1- Phosphate
3- Fed Status Functional Hypoglycemia:
B- Fasting Hypoglycemia:
1-Organic Hypoglycemia:
a-Pancreatic B-Cell disease/CA
b-Non-Pancreatic Tumors
c-Anterior Pituitary Hypo-function
d-Adrenocortical Hypo-function
e-Ingestion of Akee Fruit
2- Functional Fasting Hypoglycemia
a- On specific hepatic enzyme deficiency
1- Genetic Deficiency or Delayed Maturation
of Enzymes in Pre-
mature Babies
2- Glycogen Storage Disease
b- Induced by Exogenous Agents:
1-Alcohol Intake
2-Excessive Insulin Administration
3-Excessive Sulfonylurea Administration
II - Hyperglycemia :
- Diabetes Mellitus
- Hemochromatosis
- Hypokalemia
- Stress
- Pheochromocytoma
- Anesthesia
- Pregnancy

7. - Hyperthyroidism
- Cushing disease
- Hyperpituitarism (gigantism)
Discussion and conclusion:
Sample 3 in test tube 3 showed more color intensity than other samples, this give a picture/hint
that the sample have a high concentration of glucose even before optical density is
spectrophotometically determined, thus if a prompt decision need to be decided in patient
management, can help the attending doctor to take quick decision.
After taking absorbance of the 3 samples and the control sample and calculating glucose
concentration in each, sample 3 measured at 11.34Mmol/l, sample 2 measured at 5.25mmol/l
and sample 1 at 2.75mmol/l glucose concentration respectively. This actually confirmed that
that sample 3 is hyperglyceamic, and sample 2 is within normal range of random blood glucose
accepted level, while sample 1 is hypoglycemic. Error in values in this experiment can arise from
poor skill, impurities from apparatuses used, inaccurate measurements and inaccuracy in taking
optical density. If done accurately it can therefore appropriate patient management can be
arrived at based on these values by the attending doctor.

8. REFFERENCE:
1.http://www.worthington-biochem.com/GOP/default.html
2.https://www.caymanchem.com/pdfs/10009582.pdf
3.http://www.chem.uky.edu/courses/che226/Labs/090-Glucose_Kinetics.pdf
4.http://www.austincc.edu/mlt/chem/chemlab9carbohydrates.pdf
5.Kenyatta university-laboratory-practical-manual- for-MBCHB-Year 1-semester1.