BIOCHEMISTRY

1

• BIOCHEMISTRY
• 285 PHL
• Introduction
• Blood Glucose

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Blood

• Blood is vascular tissue that circulates in the
  closed system of blood vessels
• Functions
• Transportation
• Regulation of acid-base balance
• Regulation of body temperature
• Protection against infections
• Coagulation

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Blood Composition
Blood
Plasma
Formed Elements
Water Solids Diffusible - Anabolic -
Catabolic Non- diffusible
RBCs WBCs Platelets
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Types of Samples

• Whole blood
• Liquid (plasma) cells (RBCs, WBCs, platelets)
• Plasma
• Water solids (e.g. glucose, urea, albumin,
  fibrinogen)
• No cells
• Serum
• Serum plasma clotting factors

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Preparation of sample Plasma

• Plasma

anticoagulant
Transfer the clear supernatant to specimen tube
Add venous blood
Mix then centrifuge
cells
Cent. tube
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Preparation of sample Serum

• Serum

Venous Blood
Transfer the clear supernatant to specimen tube
Allow blood to clot (20min.)
Remove the clot and centrifuge
cells
Cent. tube
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Centrifuging tubes
Centrifugator
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Plasma vs. Serum
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Anticoagulants

• Definition
• Anticoagulants are chemicals which prevent blood
  clotting
• Types
• 1- Heparin
• MOA
• Prevents conversion of prothrombin to thrombin
• Advantage less interference with chemical tests
• Disadvantage high cost

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Anticoagulants cont.

• 2-EDTA (ethylene diamine tetraacetic acid)
• MOA
• Binds to calcium
• Advantage prevents platelets clumping
• 3-Oxalates (Na, K, Li, or NH4 salts)
• MOA
• Form insoluble complex with calcium
• Disadvantage interfere with lactate
  dehydrogenase
• N.B Na, K salts should not be used in
  electrolytes determination

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Anticoagulants cont.

• 4-Citrate
• e.g. trisodium citrate
• Used in ESR
• 5-Na fluoride (enzyme poison)
• Used in blood sugar determination b/c it inhibits
  glycolysis
• N.B it inhibits urease enz., therefore it should
  not be used in urea determination

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Deproteinization

• Purposes
• 1- To precipitate protein use the ppt. in
  plasma protein determination e.g. albumin
• 2- Proteins have UV absorption and could
  interfere with tests
• 3- Proteins are colloids which make the solution
  turbid difficult to read
• 4- Determination of non-protein nitrogen glucose

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Deproteinization Agents

• Acids
• 1- Trichloroacetic acid
• 2- Tungestic acid
• MOA ? pH, proteins become cations ppt as
  insoluble salts of acids
• Bases
• 1- Zinc hydroxide
• 2- Cu, Ba, Cd hydroxide
• MOA ? pH, proteins become anions ppt as
  insoluble salts of heavy metals
• Organic substances e.g. ethanol or ether
• MOA remove water from protein mol.

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Determination of Blood Glucose (BG)

• BG is determined by 2 methods
• 1- Oxidation method ( enzymatic method)
• Principle
• -Glucose O2 H2O Glucose oxidase Gluconic acid
  H2O2
• -H2O2 phenol amino-4-antipyrine Peroxidase
  Quinoneimine H2O

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Determination of Blood Glucose Oxidation Method

• Procedure

Sample name
Stand. name
Serum
0.1 ml
0.1 ml
Stand.
1 ml
1 ml
Reagent
Mix , then incubate at 37C for 10 min. Read the
absorbance of sample stand. at ? 505
nm against blank (reagent)
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Calculation

• Glugose (mg/dl) Sample absorbance x conc of
  standard
• Stand. Absorbance
• Normal level
• Fasting 75 -110 mg/dl (3.9 -6 mmol/L)

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Interpretation of the results

• If BG gt110 mg/dl HYPERGLYCEMIA
• Causes
• 1-Diabetis mellitus
• 2-Acromegaly
• 3-Acute stress
• 4-Adrenal hyperactivity (Cushing's syndrome)
• 5- Hyperthyroidism
• 6- Pancreatic cancer or pancreatitis
• 7- Drugs e.g. corticosteroids

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Interpretation of the results cont.

• If BG lt70 mg/dl HYPOGLYCEMIA
• Causes
• 1- Insulin overdose
• 2- Hypothyroidism hypopituitarism
• 3- Adrenal insufficiency (Addison's disease)
• 4- Liver diseases
• 5- Starvation

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Renal glucose threshold

• Definition
• -The blood glucose concentration at which the
  kidneys start to excrete glucose into the urine
• -BG level of 180 mg/dl is called Renal Glucose
  Threshold
• Glucoseurea
• Appearance of glucose in urine (occurs when BG
  conc. gt 180 mg/dl)

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Renal glucose threshold