1. BIOCHEMISTRY 285 PHL Introduction Blood Glucose

2. Blood Blood is vascular tissue that circulates in the closed system of blood vessels Functions: TransportationTransportation Regulation of acid-base balanceRegulation of acid-base balance Regulation of body temperatureRegulation of body temperature Protection against infectionsProtection against infections CoagulationCoagulation

3. Blood: Composition Blood Plasma Formed Elements RBCs WBCs Platelets Water Solids : Diffusible - Anabolic - Catabolic Non- diffusible

4. Types of Samples Whole bloodWhole blood Liquid (plasma)+ cells (RBCs, WBCs, platelets) PlasmaPlasma Water + solids (e.g. glucose, urea, albumin, fibrinogen) No cells SerumSerum Serum = plasma – clotting factors

5. Preparation of sample: Plasma Plasma Cent. tube anticoagulan t Add venous blood Mix then centrifuge Transfer the clear supernatant to specimen tube cells

6. Preparation of sample: Serum Serum Venous Blood Cent. tube Allow blood to clot (20min.) Remove the clot and centrifuge Transfer the clear supernatant to specimen tube cells

7. Centrifuging tubes Centrifugator

8. Plasma vs. Serum Plasma Plasma Serum Serum - Anticoagulant - Contains clotting factors - Advantage: time saving - No anticoagulant - Contains no clotting factors - Advantage: less interference

9. 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

10. 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

11. 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

12. 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

13. 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.

14. Determination of Blood Glucose (BG) BG is determined by 2 methods: 1- Oxidation method ( enzymatic method): Principle -Glucose +O 2 + H 2 O Glucose oxidase Gluconic acid + H 2 O 2 -H 2 O 2 + phenol + amino-4-antipyrine Peroxidase Quinoneimine +H 2 O

15. Determination of Blood Glucose: Oxidation Method Procedure Sample name Stand. name Serum Stand. Reagent 0.1 ml 1 ml Mix, then incubate at 37°C for 10 min. Read the absorbance of sample & stand. at λ= 505 nm against blank (reagent)

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

17. Interpretation of the results If BG >110 mg/dl HYPERGLYCEMIAIf BG >110 mg/dl HYPERGLYCEMIA Causes: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

18. Interpretation of the results: cont. If BG <70 mg/dl HYPOGLYCEMIA Causes: 1- Insulin overdose 2- Hypothyroidism & hypopituitarism 3- Adrenal insufficiency (Addison's disease) 4- Liver diseases 5- Starvation

19. 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. > 180 mg/dl)

20. Renal glucose threshold