Venous blood was extracted and subjected to Glucose Oxidase-Peroxidase Test

1. GROUP II
BLOOD GLUCOSE
DETERMINATION
USING OXIDASE-PEROXIDASE
METHOD

2. INTRODUCTION
 Diagnosis of hyper/hypoglycemic patients
 Three etiological types of diabetes
 late 19th century- Frederick Banting and Charles Best experimented
with extracts from the pancreas to isolate insulin.
 According to the International Diabetes Federation (2019), there are
425 million people who are diagnosed with diabetes in the whole world
and there were 3,721,900 cases of diabetes in the Philippines as of
2017. It is the sixth leading cause of death in the country and the
number one cause of kidney failure, lower limb amputations, and adult
blindness (CDC, 2018).

3. MATERIALS
 Spectrophotometer
 Centrifuge
 10 ml test tubes
 Un hemolyzed serum
 EDTA tube
 O-toluidine reagent
 Glucose standard 2 mg%

4. Methodology
 Blood Collection
- About 3 ml of the patient’s blood was collected by venipuncture and
transferred to an ethylene diamine tetraacetate (EDTA) tube. The tube
was gently shaken for complete mixing. Then the blood sample was
centrifuged and only the blood plasma was separately collected. The
sample was incubated at 37oC for five minutes to resemble normal body
temperature. The plasma sample was placed in a test tube and the O-
toluidine reagent was added to the sample.

5.  Reading of Absorbance
- The spectrophotometer was set to zero using blank at 500 nm. The
absorbance of standard and sample were measured at 500 nm against
the blank. The blank, standard, and the sample consist of the following:
Blank 1 ml of Reagent
Standard 10 ul of glucose and 1 ml of reagent
Sample 10 ul of Sample and 1 ml of reagent

6. RESULT AND DISCUSSION

7. Results and Discussion
- The absorbance reading obtained for the blood sample was 1.013 nm
while absorbance of the standard was 0.896. Using the chemical
equation below, the true blood glucose concentration was calculated
and had a value of 113.058 mg/dL
**the patient has a normal level of glucose in her blood as the computed value lies within the normal
range

9. High Blood
Sugar
 Hyperthyroidism
 Pancreatitis
 Pancreatic cancer
 Pheochromocytoma
 Acromegaly
 Cushing’s
 Glucagonoma
Low Blood
Sugar
 Insulinoma
 Vigorous exercise
 Hypopituitarism

10. Hyperglycemia
 Is an increase in blood glucose concentration
 It is toxic to beta cell function and impairs insulin
 Causes: stress, severe infection, dehydration or
pregnancy, pancreatectomy, insulin deficiency or
abnormal insulin receptor
 FBS level: >126 mg/dL

11. Laboratory Findings (Hyperglycemia)
 Increase glucose in plasma and urine
 Increase urine specific gravity
 Ketones in serum and urine
 Electrolyte imbalance (decrease Na+, increase K+,
decrease HCO3)

12. Hypoglycemia
 It results from an imbalance between glucose
utilization and production
 It involves decreased glucose levels and can have
many causes
 A diagnosis of hypoglycemia should not be made
unless a patient meets the criteria of Whipple’s
Triad:
1. Low blood glucose concentration
2. Typical symptoms
3. Symptoms alleviated by glucose administration

13. Hypoglycemic values:
 A blood glucose level of:
65-70 mg/dL = glucagon and other glycemic
hormones are released into the circulation
<60 mg/dL =strongly suggest hypoglycemia
50-55 mg/dL =observable symptoms of
hypoglycemia appears

14. Symptoms of Hypoglycemia:
 Tremors
 Palpitations
 anxiety
 Diaphoresis
 Dizziness
 Tingling
 Blurred vision
 Confusion
 Behavioral changes

15. Diabetes Mellitus
 Is a group of metabolic disorders characterized by
hyperglycemia resulting from defects in insulin secretion,
insulin receptors or both
 Glucosuria occurs when the plasma glucose level exceeds
180 mg/dL with normal renal function
 Ketosis develops in DM from excessive synthesis of
acetyl-CoA, as the body attempts to obtain required
energy from stored fat in the absence of an adequate
supply of carbohydrate metabolites – the presence of
ketone bodies is a frequent finding in individuals with
severe, uncontrolled diabetes
 The entire process of ketosis can be reverted by insulin
administration

16. Classification of Diabetes Mellitus:
1. Type 1 Diabetes Mellitus- formerly known as :
Insulin Dependent Diabetes Mellitus
Juvenile Onset Diabetes Mellitus
Brittle Diabetes
Ketosis Prone Diabetes
2. Type 2 Diabetes Mellitus-formerly known as:
Non-insulin Dependent Diabetes
Mellitus
Adult Onset Diabetes Mellitus
Stable Diabetes
Receptor Deficient Diabetes Mellitus
3. Gestational Diabetes

17. Type 1 Diabetes Mellitus
 Is a result of cellular-mediated autoimmune
destruction of the Beta-cells of the pancreas
 Diabetic individuals have insulinopenia( absolute
insulin deficiency) due to loss of pancreatic beta-
cell
 Depends on insulin to sustain life and prevent
ketosis
 80-90% reduction in the volume of the beta-cells
is required to induce symptomatic Type 1 DM - it
is only after most of the beta-cells are destroyed
that hyperglycemia develops

18. Type 1 Diabetes Mellitus:
 Signs and symptoms:
polyuria
polydipsia
polyphagia
rapid weight loss
hyperventilation
mental confusion

19. Type 2 Diabetes Mellitus
 Is characterized by hyperglycemia due to an
individual’s resistance to insulin
 There is relative insulin deficiency
 It is associated with strong genetic predisposition
and not related to an autoimmune disease – it
has been described as “Geneticist’s Nightmare”
 It has milder symptoms as compared to Type 1
Diabetes Mellitus, however, untreated Type 2
DM will result to nonketotic hyperosmolar coma
due to overproduction of glucose accompanied
by severe dehydration, electrolyte imbalance and
increased BUN and Creatinine

20. Type 2 Diabetes Mellitus
 Risk Factors:
Obesity
Family history
Advanced age
Hypertension
Lack of exercise
Gestational Diabetes
Impaired glucose metabolism

21. Recommendation!
It is recommended that adults ages 45 and older
be screened for diabetes every 3 years, but
screening should be performed earlier and more
frequently if the individual is at high risk.

22. Comparison Between Type 1 DM and
Type 2 DM
Type 1 DM Type 2 DM
Pathogenesis B-cells destruction Insulin resistance
Incidence rate 5-10 % 90-95 %
Onset Any, most common to
childhood/teens
Any, most common in
advancing age
Risk factors Genetic, Autoimmune Genetic, Obesity, Sedentary
Lifestyle, Polycystic ovarian
syndrome, dyslipidemia and
hypertension
Pre-diabetes Autoantibodies (+) Autoantibodies (-)
Symptomatol
ogy
Symptoms develop
abruptly
Symptoms develop gradually
(some patients are
asymptomatic)
Ketosis Common; poorly
controlled
Rare
Medication Insulin absolute Oral agents

23. Diagnostic Criteria for Diabetes
Mellitus:
1. Random Blood Sugar >=200mg/dL
2. Fasting Blood Sugar >=126 mg/dL
3. 2-hr Post Glucose Load >=200 mg/dL
4. HbA1c >=6.5%

24. Gestational Diabetes Mellitus
 A disorder characterized by impaired ability to
metabolize carbohydrate usually caused by deficiency of
insulin, metabolic or hormonal changes
 It occurs during pregnancy and disappears after delivery
but, in some cases, returned years later
 It is a type of glucose intolerance with onset or first
recognition during pregnancy
 Screening should be performed between 24 and 28 weeks
of gestation
 The screening and diagnosis of GDM is by the
performance of a 2-hour OGTT using 75 g glucose load

25. Gestational Diabetes Mellitus
Diagnostic Criteria for GDM:
1. FBS >= 92mg/dL
2. 1-hour GCT >=180 mg/dL
3. 2-hour OGTT >=153 mg/dL
*GDM is diagnosed if one of the three criteria is met
*Infants born to diabetic mothers are at increased risk for
respiratory distress syndrome, hypocalcemia and
hyperbilirubinemia.
*GDM coverts to DM within 10 years in 30-40% of cases

26. Answers to Questions
1. Give the characteristic difference between the glucose
tolerance curve of a diabetic person and that of a normal
individual.
 The classic oral glucose tolerance test measures blood glucose levels
five times over a period of three hours. Some physicians simply take
a baseline blood sample followed by a sample two hours after
drinking the glucose solution. In a person without diabetes, the
glucose levels rise and then fall quickly. In someone with diabetes,
glucose levels rise higher than normal and fail to come back down as
fast.

27. 2. In the OGTT why should the subject have adequate
carbohydrate intake three days before the test is
due?
 3 days before OGTT, the subject must have high
carbohydrate diet (≥ 150g per day) to avoid false negative
results, such that, low carbohydrate intake may impair
results.

28. 3. What conditions contribute to the high incidence of
positive glucose test?
 Diabetes Mellitus, Corticosteroid use (induce
gluconeogenesis), insufficient fasting time, endocrine
disorders (ex. polycystic ovaries – insulin resistance)

29. 4. What is the principle involved in the blood glucose
determination used in this experiment?
 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.

30. 5. Give two examples of commercial glucose test kits,
the conversion factors in each case and the normal
glucose values given.
 Capillary Blood Glucose Test (CBG), Glucometer – generic
name
 Brand: Quality BG-102 blood glucose monitoring system
: Alphatrak control solution
 Normal values- FBS: </=92mg/dl, > 126mg/dl- overt
1st Hour: </= 180 mg/dl
2nd Hour: </=153 mg/dl, >200mg/dl- overt D