Practical Biochemistry 2024-2025 PDF

First year Practical Biochemistry By STAFF MEMBERS OF Biochemistry 2024-2025  PRACTICAL BIOCHEMISTRY Index No. Title Page 1 Lab safety...

First year Practical Biochemistry By STAFF MEMBERS OF Biochemistry 2024-2025  PRACTICAL BIOCHEMISTRY Index No. Title Page 1 Lab safety 3 2 Blood & Plasma & Serum 11 3 Electrophoresis 21 4 Clinical enzymology 26 5 Urine analysis: physical properties 35 6 Urine analysis: chemical properties 41 7 Non-protein nitrogenous compounds 44 8 Blood glucose & Diabetes mellitus 50 9 DNA extraction 60 10 PCR 64 11 Urine sheet 68 12 Signature 70 MEDICAL BIOCHEMISTRY DEPARTMENT 2  PRACTICAL BIOCHEMISTRY Lab safety ❖ Basic practices in medical biochemistry laboratories: - Practice proper dressing of lab coat, gloves, protective googles, IDs. - Identify different signs in lab and apply its indication. - Identify methods of elimination of hazards material. - Describe the centrifuge and its function. - List Different types of centrifuges. - Explain ultracentrifugation. - Identify and handle lab glassware and perform micro pipetting. ❖ Remember: - The lab is a place for serious work! Careless behavior may endanger yourself and others and will not be tolerated! - Safe working protects: 1) You 2) Other lab workers 3) Cleaners 4) Visitors 5) Your work ❖ Safety Rules for Medical Biochemistry Students includes: A. Appearance and attitude in the lab: While working in the lab, the following instructions should be followed: - You must wear your clean white coat - Place books and personal items in the cabinet - Only laboratory manuals are permitted into the working area - Listen to your instructor carefully and stand in your proper position in the lab. - Read the instruction guide ahead of performing the experiment. MEDICAL BIOCHEMISTRY DEPARTMENT 3  PRACTICAL BIOCHEMISTRY - Eating and drinking are not allowed in the lab - Put all food and drinks in your bag. - Do not sit on lab benches - side talks are not allowed in the lab. At the end of the lab period: - Exit the lab in an orderly manner. - No running. - No pushing. B. General work procedure: - When in doubt – ASK!!! DO NOT GUESS!!!! - You should wear approved safety googles or safety glasses when instructed in the lab. - Wear gloves if you will come in contact with blood specimens. - Use only clean & dry glassware and never use broken glassware. - Check glassware for cracks. - Use the flame with extreme caution and keep your head and clothing away from the flame. - Never carry flammable or hot and dangerous equipment or chemicals through the lab. - Handle hot glassware with holder or tongs. - Always point test tubes away from yourself and others when heating. - Never taste anything or touch chemicals with hand. - Never pour used reagents back into the bottle. - Keep lids on bottles and containers when not in use. - Inform the instructor immediately about any non properly working equipment or any difficulty. MEDICAL BIOCHEMISTRY DEPARTMENT 4  PRACTICAL BIOCHEMISTRY C. Hygiene practice: Keep hands away from face, eyes and clothes during lab. Always clean the laboratory area before leaving - Keep table-tops clean. - Return all equipment to its original location before leaving the lab. - Clean all spills immediately. D. Emergency procedure: - In case of accident as an acid or alkali burns wash the affected area immediately with plenty of running water. - In case of eye injury flush eyes immediately with plenty of water for at least 15 minutes. - In case of Fainting provide fresh air and keep the head lower than the rest of the body. - Inform the supervisor immediately about any problem or accident in the lab. MEDICAL BIOCHEMISTRY DEPARTMENT 5  PRACTICAL BIOCHEMISTRY ❖ Signs of hazards in biochemical lab MEDICAL BIOCHEMISTRY DEPARTMENT 6  PRACTICAL BIOCHEMISTRY MEDICAL BIOCHEMISTRY DEPARTMENT 7  PRACTICAL BIOCHEMISTRY ❖ Laboratory Instruments and Apparatuses A. Instruments for Liquid Volume Measurements: - Instruments used for volume measurement depends on the accuracy and volumes needed. The smaller the diameter of the instrument the more accurate it would be e.g., pipettes. o Pipettes - A pipette measures and delivers exact volumes of liquids. - used to transfer small-volumes of liquids e.g. (1ml-10ml) - Automatic pipette: most accurate of all, used to transfer micro- volumes of liquids e.g. (1μl-1000μl) MEDICAL BIOCHEMISTRY DEPARTMENT 8  PRACTICAL BIOCHEMISTRY o Graduated Cylinders - A graduated cylinder is used to measure different volumes of liquids. B. Instruments For Transfer, Mixing, Boiling And storage of Chemicals o Test tubes - Used for performing chemical experiments and reactions in lab e.g. Holding liquid samples for heating, dissolution, centrifugation, and others. o Reagents and Chemicals Bottles - Used for: Storage of different chemicals, reagents, and even strong corrosives. So, they should always carry an informative label. C. Instruments used For handling of Chemicals: o Pipette pump - It is inserted into the end of the glass pipette to help the delivery of liquid without suction by mouth. o Spatula : - It resembles teaspoons. It resists corrosions. It is used to handle solid powdered chemicals. MEDICAL BIOCHEMISTRY DEPARTMENT 9  PRACTICAL BIOCHEMISTRY o Test tube Rack - Test tube racks are for holding and organizing test tubes on the laboratory counter. - Plastic racks may melt in contact with very hot test tubes. o Test Tube Holder - A test tube holder is useful for holding a test tube which is too hot to handle MEDICAL BIOCHEMISTRY DEPARTMENT 10  PRACTICAL BIOCHEMISTRY Blood & Plasma & Serum ❖ Some definitions - Blood: a red colored body fluid composed of blood cells suspended in blood plasma. - Plasma: a straw-colored body fluid consisting of proteins, glucose, clotting factors, minerals, wastes; the body’s protein and osmotic reserve. - Serum: plasma without fibrinogen or other protein clotting factors. ❖ Components of Blood: 1- Plasma (55-60%): a) Water (91%) b) Solid (9%): i. Inorganic components (Na, K, Ca, Mg, Cl, HCO3, HPO4) ii. Organic components: - plasma proteins 6-8% - hormones e.g., cortisol and thyroxine - vitamins, lipids, - blood gases e.g., O2and CO2 - other organic metabolites e.g., glucose, amino acids, urea, uric acid, etc. 2- Cellular Components (40-45%): RBCs, WBCs & platelets ❖ Components of Plasma 1- Water 90% 2- Plasma Proteins 6-8% 3- Electrolytes (Na-Cl) 1% 4- Other Components: a) Nutrients (glucose - amino acid) b) Hormones (cortisol - thyroxine) c) Wastes (urea) d) Blood gases (CO2-O2) MEDICAL BIOCHEMISTRY DEPARTMENT 11  PRACTICAL BIOCHEMISTRY ❖ Components of Serum - It is the blood plasma without clotting factors - Serum = plasma - clotting factors. ❖ Difference Between Serum and Plasma: - Serum: is the same as plasma except it does not contain clotting factors such as fibrin, mainly used in chemistry lab and serology. - Plasma: contains clotting factors. So, serum and plasma all have the same contents of electrolytes, enzymes, proteins, hormones except clotting factors. MEDICAL BIOCHEMISTRY DEPARTMENT 12  PRACTICAL BIOCHEMISTRY ❖ Methods of Separation of Plasma and Serum: Separate plasma from whole blood 1- The blood is mixed with an appropriate amount of anti-coagulants like heparin, oxalate or EDTA. 2- This preparation should be mixed immediately and thoroughly to avoid clotting. 3- Then solution is centrifuged for 5-10 min at 2000-2500 rpm. 4- Supernatant fluid is then separated and then labeled properly. 5- NB: - The RBCs which weight more than the other components, will settle to the bottom - The plasma will stay on top - The WBCs & platelets will remain suspended between the plasma & the RBCs. Separate serum from whole blood Serum could be obtained by either : - Collecting the blood by a tube that do not contain any anticoagulant (red cap) then centrifuge the blood - by centrifuging the plasma at 5000 rpm MEDICAL BIOCHEMISTRY DEPARTMENT 13  PRACTICAL BIOCHEMISTRY ❖ Protein concentration in different body fluid: Plasma proteins - Total protein content of normal plasma is 6-8 g/100 ml. - The plasma proteins consist of: Albumin (3.5 – 5 g/dl) Globulins (2.5 – 3.5 g/dl) Fibrinogen (200 – 400 mg/dl) - Almost all plasma proteins are synthesized in liver (except immunoglobulin) MEDICAL BIOCHEMISTRY DEPARTMENT 14  PRACTICAL BIOCHEMISTRY Albumin: globulin ratio (A/G ratio) - The albumin: globulin ratio is usually between 1.2 : 1 or 1.5 : 1 - This ratio is inverted in: 1. Liver diseases (due to decreased albumin synthesis) 2. Kidney diseases (due to loss of more albumin than globulins as albumin have smaller molecular weight). MEDICAL BIOCHEMISTRY DEPARTMENT 15  PRACTICAL BIOCHEMISTRY CSF - CSF protein levels of 15-45 mg/dl accepted as the ‘normal’ reference range - Infants have significantly higher CSF protein levels than older children and adults - For term infants and for preterm infants the upper levels were 150 mg/dL and 170 mg/dL - CSF protein concentration fall rapidly from birth to 6 months of age (40 mg/dL) - Plateaued between 3 and 10 years (32 mg/dL) - Then rose slightly from 10-16 years (41 mg/dL) Human breast milk Calories 172 Fat 11 gm Protein 3 gm. Carbohydrate 17gm MEDICAL BIOCHEMISTRY DEPARTMENT 16  PRACTICAL BIOCHEMISTRY Protein in urine - Normally, urine protein excretion is 150 mg/24 hours. - Albuminuria: Urinary albumin excretion > 30 mg. /24hour Types of albuminuria 1. Normo-albuminuria: < 30 mg/24hours. 2. Micro- albuminuria: the range in between urinary excretion of albumin 30 to 300 mg/24hours. 3. Macro- albuminuria: > 300 mg /24hours Causes of Proteinuria - Physiological: physical effort - Pathological: 1. Renal according to origin: Glomerular Tubular Glomerular and tubular. 2. Post renal e.g., stone or tumor in urinary bladder 3. Prerenal e.g., increase plasma concentration of low molecular weight protein (increase concentration in urine) MEDICAL BIOCHEMISTRY DEPARTMENT 17  PRACTICAL BIOCHEMISTRY Methods of detection of proteinuria 1- Qualitative method 2- Quantitative method. -Heat coagulation test. -24 hour urine collection -Nitric acid test. -Protein / creatinine ratio -Sulphocycilic test. -Biuret test. -Urine dipstick test. 1- Heat Coagulation Test: Method : -take 5ml of urine in test tube. -Heat upper part of tube for 2-5 minutes. Observation -In case of coagulation this means presence of protein in urine. 2- Nitric Acid Test: Method: take 3ml concentrated nitric acid and 2ml urine to form a separate layer Observation: appearance of white ring at the junction between two layers indicate presence of protein in urine. MEDICAL BIOCHEMISTRY DEPARTMENT 18  PRACTICAL BIOCHEMISTRY 3- Sulphosalicylic acid test Method: Take 5ml. Of urine in a test tube, add 1ml. of 25 % sulphosalicylic acid. Observation: Appearance of white precipitation indicates presence of protein in urine. 4- Biuret test: Method: Take 3ml of the sample in a test tube; add 1ml of sodium hydroxide and add 5 drops of copper sulphate. Observation: If the color changes from blue to purple the sample contains protein. 5- Urine dipstick - Offers a qualitative assessment of urinary protein excretion. - Primarily detect albuminuria - Less sensitive for other forms of proteinuria (LMW proteins, Bence Jones protein , gamma globulins) MEDICAL BIOCHEMISTRY DEPARTMENT 19  PRACTICAL BIOCHEMISTRY 24 Hour Urine Collection: - You should collect every drop of urine during each 24 hour. - Begin the urine collection in the morning after you wake up after you have emptied your bladder for the first time. - Urinate for the first time and flush it down the toilet. - Note the exact time (7 am). - You will begin the urine collection at this time. - Collect every drop of urine during the day and night in empty collection bottle. - Store bottle in the room temperature. - Finish by collecting the first urine passed at the next morning, adding it to the collection bottle. - This should be within ten minutes before or after time of the first morning void on the first Day (6:50 am or 7:10 am). Protein/Creatinine ratio - Alternative method: measurement of the total protein/creatinine ratio (mg/mg) on a spot urine sample. - Best performed on a first morning voided urine specimen to eliminate the possibility of orthostatic (postural) proteinuria. - Normal protein excretion ratios: < 0.5 in children < 2 years old < 0.2 in children > 2 years old - A ratio >2 suggests nephrotic range proteinuria. MEDICAL BIOCHEMISTRY DEPARTMENT 20  PRACTICAL BIOCHEMISTRY Electrophoresis ❖ Definition: Electrophoresis is the migration of charged particles such as proteins nucleic acids when an electric current is passed through them. ❖ Clinical Applications of Electrophoresis: 1. Plasma protein Electrophoresis. 2. lipoprotein Analysis. 3. Determination of serum protein phenotypes & micro heterogeneities e.g. α1 antitrypsin deficiency. Multiple myeloma. 4. Diagnosis of hemoglobinopathies. 5. Cerebrospinal fluid analysis. 6. Urine analysis (determination of GNs) ❖ Principles of Separation: According to charge: When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. According to size: the smaller run faster than the large one. ❖ Instruments and Reagents Power supply Tank (buffer, Supporting media) Detection & quantification Paper electrophoresis apparatus MEDICAL BIOCHEMISTRY DEPARTMENT 21  PRACTICAL BIOCHEMISTRY Plasma Protein Distribution ❖ Serum protein electrophoresis Is performed at alkaline pH Proteins are negatively charged, so they migrate towards the anode Intensity of bands (% of total protein) - Interpretation of results require knowledge of common patterns. - You should always interpret according to the normal pattern MEDICAL BIOCHEMISTRY DEPARTMENT 22  PRACTICAL BIOCHEMISTRY o Normal pattern: Determination of plasma proteins fraction - You are provided with the total protein concentration: 4.5 g/dl. - From the given data calculate the concentration of each plasma protein fraction. - Finally, interpret your results relative to normal pattern. The use of protein electrophoresis in diagnostics of diseases - Electrophoretic pattern is constant under physiological conditions (intensity of bands) - Spectrum of plasma proteins changes under various diseases ( their ratio ) - Evaluation of electrophoretic pattern Bands or peaks MEDICAL BIOCHEMISTRY DEPARTMENT 23  PRACTICAL BIOCHEMISTRY Abnormalities of Plasma Protein Electrophoresis o 𝖺-1 Antitrypsin Deficiency - 𝖺1-antitrypsin deficiency appears as either a decrease or complete absence of the 𝖺1 band depending on whether the condition is heterozygous or homozygous. o Nephrotic Syndrome - The nephrotic syndrome pattern is characterized by decrease in all protein fractions except 𝖺-2. - 𝖺-2 Macroglobulin is retained by the kidney owing to its large molecular size MEDICAL BIOCHEMISTRY DEPARTMENT 24  PRACTICAL BIOCHEMISTRY o Monoclonal Gammopathy Pattern - The large spike in the gamma region is indicative of an increase in one immunoglobulin class typically seen in monoclonal gammopathies such as multiple myeloma. o Cirrhosis - The typical electrophoresis pattern of liver cirrhosis shows a merging of the β and γ fraction, known as the β-γ bridge o Hypogammaglobulinemia MEDICAL BIOCHEMISTRY DEPARTMENT 25  PRACTICAL BIOCHEMISTRY Clinical Enzymology ❖ Enzyme Def: Biological catalysts which increase reaction rates Properties: All are proteins- so liable to denaturation Specific to substrates Partly specific to tissues Assay by measure of rate of specific reaction catalyzed by that enzyme ❖ Circulatory system enzymes Functional serum enzyme - Responsible for reaction taking place in blood e.g., clotting enzymes Non-Functional serum enzymes - Do not have their function in blood but they are present because of wear and tear of the tissue IN CONSTANT level. ❖ Clinical use of enzymes: Injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream. Elevated enzyme levels are often indicators of tissue problems and are used in the diagnosis of diseases. Enzyme activities in the body fluids are altered by pathological processes so, its measurement is used for disease investigation. MEDICAL BIOCHEMISTRY DEPARTMENT 26  PRACTICAL BIOCHEMISTRY ❖ Enzymes routinely measured Name of the enzyme Present in Aspartate Amino transferase (AST) or Liver and Heart Serum glutamate-oxaloacetate transaminase (SGOT) Alanine Amino transferase (ALT) or Liver and Heart Serum glutamate-pyruvate transaminase (SGPT) Alkaline Phosphatase (ALP) Bone, intestine, and other tissues Creatine kinase (CK) Muscle including cardiac muscle Lactate Dehydrogenase (LDH) Heart, liver, muscle, RBC Alanine aminotransferase (ALT) Reaction: - ketoglutarate + L-alanine ALT L- glutamate + pyruvate Distribution: Widely distributed, although the largest amounts found in the liver, smaller amounts occur in the heart. More specific for liver disease than AST. Aspartate aminotransferase (AST) Reaction: -ketoglutarate + L-aspartate AST L-glutamate + oxaloacetate Distribution: This enzyme is widely distributed in the body. Main sources: Heart, liver, skeletal muscle, and kidney. Causes of serum AST levels: - Liver diseases: Hepatitis, hepatic necrosis, cholestasis - Cardiac disease: Myocardial Infarction. - Diseases of skeletal muscle: Crush injury, trauma, myopathy. - From Erythrocytes: Hemolysis MEDICAL BIOCHEMISTRY DEPARTMENT 27  PRACTICAL BIOCHEMISTRY Alkaline phosphatase (ALP) Distribution: Widely distributed, high concentrations in intestines, liver, bone, spleen, placenta and kidney. The main sources of serum ALP are the hepatobiliary tree and bone disorders. Decreased levels are found in the inherited condition. Causes of increased serum alkaline phosphatase enzyme activity: Physiological: Bone disease: Hepatobiliary disease: -Infancy - during healing of - Cholestasis -Puberty (active fractures -Hepatitis growth) -Hyperparathyroidism - Cirrhosis - Osteomalacia -Pregnancy (3rd - rickets trimester) - Paget’s disease of bone - Intestinal isoenzymes - Osteomyelitis Isoenzymes ❖ Isoenzymes Def: Different forms of the same enzyme that catalyze the same reaction differ in aa sequence, affinity to substrate or other properties & tissue producing them Properties: Two or more polypeptide chains. Different polypeptide chains are products of same gene. Differ in AA sequence and physical properties. Are tissue specific. MEDICAL BIOCHEMISTRY DEPARTMENT 28  PRACTICAL BIOCHEMISTRY ❖ Diagram illustrating the origin of isoenzymes Lactate dehydrogenas Reaction: Convert Lactate Pyruvate and vice versa It is an enzyme formed of: - 4 subunits (Tetramer) 2 H and 2 M - Five isoenzymes are present in blood in low levels - They are combined in different ways to give active enzyme Lactate dehydrogenase (Tetramer) Five isoenzymes are present in blood LDH1 LDH2 LDH3 LDH4 LDH5 (HHHH) (HHHM) (HHMM) (HMMM) (MMMM) specific for specific for specific for specific for specific for HEART RBCs BRAIN LIVER MUSCLE MEDICAL BIOCHEMISTRY DEPARTMENT 29  PRACTICAL BIOCHEMISTRY Lactate dehydrogenase in MI: when we measure LDH we will find that: - Increased total LDH due to increase LDH-1 isoenzyme. Creatine kinase (CK) Reaction: Convert Creatine + ATP creatine phosphate + ADP It is an enzyme formed of : - 2 subunits (dimmer) B & M - Three isoforms are present: CK 1 CK2 CK3 (BB) (MB) (MM) specific for specific for specific for Brain Heart Skeletal muscle In myocardial infarction: - there is elevation of total CK due to marked elevation of CK-2 (MB). IN MYOCARDIAL INFARCTION Both of CK2 & LDH1 isoenzymes are increased CK2 rises within 4-8 hours after the onset of pain reaches peak within 24 hours. Returns to normal level after two-three days. LDH1 returns to normal level after a long period CK2 is used for early detection of MI while LDH1 for follow up of MI MEDICAL BIOCHEMISTRY DEPARTMENT 30  PRACTICAL BIOCHEMISTRY Clinical enzymology in liver diseases ❖ Liver Diseases Hepatic necrosis Hepatitis Cholestasis Jaundice ❖ Liver Enzymes (ALT, AST, ALP) Hepatocellular Injury In Cholestasis very high ALT/AST Higher values of ALP due to  mild /moderate elevation of ALP synthesis NB: mild/moderate elevation of AST ALT is more specific than AST. /ALT ❖ Levels of enzymes in diseases involving liver damage In viral hepatitis: - rapid rise in transaminases (AST & ALT) in serum occurs even before bilirubin rise is seen MEDICAL BIOCHEMISTRY DEPARTMENT 31  PRACTICAL BIOCHEMISTRY Clinical enzymology in Myocardial infarction ❖ Case scenario A 66-year-old smoker male complaining of crushing-like chest pain radiating to the left shoulder & arm. He is hypertensive. A diagnosis of myocardial infraction was made after doing ECG, ECHO, & cardiac biomarkers. ❖ Definition: Myocardial infarction (MI) occurs when blood flow stops to a part of the heart causing damage & death of heart muscles. ❖ Causes of MI MI occur due to coronary artery occlusion ❖ Risk factors OF MI High blood pressure Smoking Diabetes Lack of exercise Obesity High blood cholesterol ❖ Investigations of MI ECG Echocardiography Cholesterol measurement Cardiac biomarkers MEDICAL BIOCHEMISTRY DEPARTMENT 32  PRACTICAL BIOCHEMISTRY Cardiac biomarkers Definition: - Are biomarkers used for diagnosis and follow up of MI. Mechanism: - Death of cardiomyocytes abnormal release of these biomarkers which preset inside the cardiomyocytes into circulation abnormal increased levels in blood. They include: - Lactate dehydrogenase. - Creatine kinase. - Aspartate aminotransferase (AST) - Cardiac troponins Ck2 is used for early diagnosis of MI LDH is used for follow up OF MI MEDICAL BIOCHEMISTRY DEPARTMENT 33  PRACTICAL BIOCHEMISTRY Enzyme Assays ❖ Enzyme Assays: Def: Are laboratory measuring for enzymatic activity. All enzyme assays measure either the consumption of substrate or production of product over time. Different enzymes require different estimation methods depending on the type of reaction catalyzed, the nature of S and P or coenzyme Methods 1-Flourescence method 2-Spectrophotometric 3-Sampling method 4- Electrode method 5- Manometric method 6-Polarimetric method MEDICAL BIOCHEMISTRY DEPARTMENT 34  PRACTICAL BIOCHEMISTRY Urine Analysis Methods of urine collection ❖ Types of specimens: 1. Random specimen: it is subject to variable dilution 2. First morning specimen: The first morning specimen is the preferred specimen for routine urine examination, a clean-voided midstream specimen should be obtained. 3. Postprandial specimen: obtained 2 hr. after a meal; it contains proteins and glucose. 4. Afternoon specimen: Urobilinogen is best evaluated in a specimen obtained between 2 and 4 pm.. 5. Day specimen: The patient collect urine from 8 am to 8 pm 6. Night specimen: The patient collect urine from 8 pm to 8 am 7. Twenty-four-hours specimen: The patient collect urine from 8 am to 8 am. MEDICAL BIOCHEMISTRY DEPARTMENT 35  PRACTICAL BIOCHEMISTRY Physical properties of urine ❖ (1) Volume: Def: Measure exact amount of urine in a calibrated cylinder Normal values: - Adults: 1-1.8 l/24 hr. - The ratio of night to day excretion is usually 1:2 or 1:3. Abnormalities: A. Increased volume (polyuria): Def: The amount is increased over 2 liters/day Causes: 1) Physiological: excessive fluid intake. 2) Pathological: − Diuretics, during absorption of exudates and edema. − Chronic kidney diseases. − Diabetes insipidus (low specific gravity). − Diabetes mellitus (high specific gravity). B. Decreased volume (oliguria): Def: The amount of urine is decreased less than ½ liters/24hr Causes: 1) Physiological: dehydration due to diminished water intake or increased water loss. 2) Pathological: − Renal insufficiency − cardiac and hepatic insufficiency (edema and effusion) − Nephrosis − burns, hemorrhage, and shock MEDICAL BIOCHEMISTRY DEPARTMENT 36  PRACTICAL BIOCHEMISTRY Anuria Urinary retention Def - Complete suppression of urine blockage of the urinary outflow tract OR (LUT) by: - stones - Amount Less than 100ml/day - tumors - or an enlarged prostate kidneys indicates diminished renal secretory functioning, activity at least in the early stages. bladder empty Full ❖ (2) Color: Normally: - pale yellow to deep yellow depending on the degree of urine concentration as following Diluted urine: straw color Concentrated urine: deep orange - The color is due to mixture of pigments: Urochrome, Urobilin: Uroerythrin and porphyrin. Causes of change in urine color: 1- Pale yellow, colorless, or green urine: - Chronic renal diseases, D.I., D.M., severe iron deficiency 2- Deep Yellow urine: - Carotene concentrated urine as dehydration - food color as riboflavin. 3- Green and blue green: - Jaundice due to bile pigments 4- Red urine: - Hemoglobinuria - Beets - some drugs as rifampicin 5- Black urine: - Alkaptonuria - iron and phenol poisoning MEDICAL BIOCHEMISTRY DEPARTMENT 37  PRACTICAL BIOCHEMISTRY ❖ (3) Odor: Normal: - fresh urine is slightly aromatic due to non-volatile organic acids. - On standing: ammonical odor develops due to bacterial action on urea. Abnormal odor: 1- Acetone odor (Fruity odor): D.M., starvation, and dehydration 2- Ammoniacal odor: Bacterial decomposition 3- Maple syrup odor: Maple syrup urine disease 4- Offensive odor: Presence of pus and bacterial overgrowth 5- Some foods like garlic. 6- Some Drugs: methanol. 7- Mousey urine: Phenylketonuria ❖ (4) Reaction or pH of urine: Normally: - urine pH is slightly acidic pH 6.8 due to acid phosphates (true acidity). Abnormal odor: Causes of acidic urine Causes of alkaline urine Physiological: high protein diet. Physiological: vegetables and fruit Pathological: Pathological: 1- Respiratory and metabolic acidosis 1- Respiratory and metabolic alkalosis 2- Acid producing bacteria 2- Alkali producing bacteria 3- Drugs used in treatment of calcium 3- Drugs used in treatment of calcium carbonate stones oxalate and uric acid stones. MEDICAL BIOCHEMISTRY DEPARTMENT 38  PRACTICAL BIOCHEMISTRY ❖ (5) Specific gravity Def: - It is the measure of the concentration of the solutes that reflect the concentrating and diluting power of the kidney. Normally: - it ranges from 1.010 –1.035. - It measures the total solids in urine e.g. NaCl, urea and glucose. Measurement of specific gravity: - Specific gravity is measured by urinometer. - The procedure: Mix the urine well and allow it to stand in room temperature, float the instrument in the urine sample, read the bottom meniscus. Variation of the specific gravity - Normally in newborn baby: 1.015-1.018. - Adult random sample 1.001-1.025. Increase of specific gravity: values over 1.020 indicates - decrease fluid intake, fever, DM, x-ray contrast media, intravenous albumin, or urinary preservatives. Decrease of specific gravity: values of less than 1.009 indicates: - increase of fluid intake, hypothermia, Diabetes insipidus, progressive renal failure, or sickle cell anemia. MEDICAL BIOCHEMISTRY DEPARTMENT 39  PRACTICAL BIOCHEMISTRY ❖ (6) Aspect: Normally freshly voided urine is clear. Turbidity is due to the following: 1- Crystal: - phosphates and carbonates present in alkaline urine - Urates and oxalate present in acidic urine - dissolved is produced by 12% hydrochloric acid. 2- Pus (pyuria), blood, and epithelial cells: - About 200 white blood cells/mm3 produce turbidity - about 500 red blood cells/mm3 produce turbidity. 3- Bacteria: UTI 4- Fat (lipuria): - The presence of fat gives a milky appearance. 5- Chyle (Chyluria): - due to parasitic (filarial) or non-parasitic (as in thoracic duct obstruction, trauma, and tumor. MEDICAL BIOCHEMISTRY DEPARTMENT 40  PRACTICAL BIOCHEMISTRY Chemical examination of the urine ❖ (1) Reducing sugars in urine: Normally: - very small amounts of reducing sugars are present in urine which are not detected by reagents used, however in some conditions the following reducing sugars are excreted in urine: 1. Glucose in glycosuria 2. Galactose in galactosemia 3. Lactose in pregnancy and lactation. 4. Fructose after ingestion of big amounts of fruits. Glucosuria: 1. Diabetic glucosuria. 2. Non-diabetic glucosuria: A-Glycosuria with hyperglycemia: B- Glycosuria without hyperglycemia: 1- Hyperthyoidism. 1. Renal Glycosuria. 2-Increased intracranial pressure. 2. Alimentary glycosuria. 3-Emotional disturbances. 3. Glycosuria of pregnancy. Detection of sugars in urine: Benedict test: - Aim: test positive for reducing sugar, they act in alkaline media at high temperature - Steps: 1. Put 2 ml of Benedicts solution in test tube then add 0.5 ml urine. 2. Boil over a flame for 2-3 minutes. - Result: Appearance of green, yellow, red, or brown precipitate indicates positive result (presence of a reducing sugar). MEDICAL BIOCHEMISTRY DEPARTMENT 41  PRACTICAL BIOCHEMISTRY ❖ (2) Ketone bodies in urine: Ketone bodies: - Acetone, acetoacetic acid and B-hydroxybutyric acid are collectively known as ketone bodies. - Ketone bodies are obtained as the intermediate products in the oxidation of fatty acids and are oxidized to carbon dioxide and water under normal conditions. - Normally no ketone body in urine Causes of ketosis 1- Starvation. 2- Diabetes Mellitus. 3- Low carbohydrate and high fat diet. Detection of keton bodies in urine: Rothera test: - Steps : Saturate 1 ml of urine with Rothera`s powder. - Observation and result: Appearance of purple (permanganate calomel red) color indicates presence of ketone bodies in urine. MEDICAL BIOCHEMISTRY DEPARTMENT 42  PRACTICAL BIOCHEMISTRY ❖ (3) Urinary Proteins: Causes of proteinuria: A. Physiological causes: 1. Orthostatic (prolonged standing). 2. Severe muscular exercise. 3. Prolonged exposure to cold. 4. Pregnancy. B. Pathological causes: I-Pre-renal causes: II-Renal causes: III-Post-renal causes: -Fevers and trauma. - Nephritis as: - inflammation of the -Intraabdominal tumors. Glomerulonephritis urinary tract below the -Drugs and chemical poisoning. pyelonephritis. kidney as in cystitis. -Bence Jones protein in multiple myeloma. -Nephrosis. - tumors of the urinary -Myoglobin in crush injury. tract below the kidney -Hemoglobin in hemolytic diseases (all causes of hematuria). -Heart failure. Bence Jones protein: - An abnormal globulin that appears in multiple myeloma (Bone marrow cancer), leukemia and lymphosarcoma. - Urine is characterized by a triphasic reaction : 1. Clotting when heated to 60◦c, 2. redissolved at 100◦C 3. and reclothing on cooling. Detection of proteins in urine: Heat coagulation test: - Steps : Take 5 ml of urine in test tube. Heat upper part of the tube for 2-5 min. - Result: If solution coagulable ----- > Albumin or globulin present MEDICAL BIOCHEMISTRY DEPARTMENT 43  PRACTICAL BIOCHEMISTRY Non-protein nitrogenous compounds ❖ Non-protein nitrogenous compounds (NPN): Definition: - they are a group of high-protein feedstuffs which contain nitrogen in a form other than peptides or proteins. NPN compounds are: - Urea, Ammonia, Uric acid, Creatinine and Amino acid ❖ Urea: Definition: Urea (H2N-CO-NH2) is the main end product of protein catabolism. Urea formation is the pathway through which the liver can convert toxic ammonia into non-toxic urea Site of urea formation: 1. Liver is the only site for urea formation. 2. Then urea is transported in the blood to the kidney to be excreted in urine. Plasma urea: Level of urea in plasma is 20-40 mg/dl. Diagnostic importance of plasma urea determination: - Measurement of plasma urea is one of the kidney function tests. - In kidney diseases as in renal failure, kidney fails to excrete urea → High blood urea concentration (uremia). Urine urea: Normal urine urea : 20-40 g/day It constitutes about half of the total urinary solid. It represents about 18-19% of the total urinary nitrogen. It is the end product of protein catabolism. MEDICAL BIOCHEMISTRY DEPARTMENT 44  PRACTICAL BIOCHEMISTRY Causes of increased urinary urea: - Increase protein catabolism as in D.M., fever and hyperthyroidism. Causes of decreased urinary urea: - Liver disease, Renal failure Detection of urea in urine: Diacetyl monoxime test - Steps: 1. Take 1 ml of urine (urea) + 1 ml diacetyl monoxime reagent + 0.5 ml H2SO4 2. Boil in water bath for 2-5 min - Observation & result: Appearance of Orange yellow or reddish brown color indicates present of urea in urine ❖ Ammonia: Blood ammonia: Ammonia is a toxic substance especially to the central nervous system. Any ammonia formed in the peripheral tissue must be moved to the liver to be converted into urea. this maintains ammonia at low level in circulating blood. Blood contains traces of ammonia: 10- 80 ug/dL. Source of ammonia: 1. Trans-deamination of amino acids. 2. Glutamine: The kidneys form ammonia from glutamine by glutaminase enzyme. Most of this ammonia is used in regulation of Acid base balance. 3. Purines and pyrimidines metabolism. 4. In intestine: ammonia is produced by the action of bacterial enzymes on: a. Dietary amino acids. b. Urea secreted into the intestine. MEDICAL BIOCHEMISTRY DEPARTMENT 45  PRACTICAL BIOCHEMISTRY Fate of ammonia: 1. Formation of non-essential amino acids 2. Formation of urea 3. Excretion in urine 4. Formation of glutamine: By Glutamine synthetase Glutamine functions: -Regulation of acid base balance -Removes the toxic effect of ammonia -Source of N3 & N9 of purine base -Detoxication Ammonia in urine: Normally there is very little ammonia in the freshly voided urine, it is about 2.5-4.5% of the total urinary nitrogen and in average 0.7g. is excreted per day. It increases in acidosis as in D.M. and high protein diet. It decreased in alkalosis. ❖ Uric acid Serum uric acid: Normal serum uric acid: - Male: 3-7 mg/dL - Female: 2-6 mg/dl Exceeding uric acid level = Hyperuricemia Gout (hyperuricemia) Definition: - It is a metabolic disease characterized by hyperuricemia. Gout occurs mainly in males MEDICAL BIOCHEMISTRY DEPARTMENT 46  PRACTICAL BIOCHEMISTRY Causes: - Under-excretion of uric Acid - Over-production of uric Acid Clinical picture : o Arise from low solubility of uric acid in body fluids, so insoluble sodium Urate crystals precipitate into joints, kidneys, and soft tissues. o Joints: - Deposition of sodium urate crystals in joints, tendons, and surrounding tissues - Inflammatory process with infiltration of granulocytes that phagocytose urate crystals - This process generates oxygen metabolites that damage tissues with the release of lysosomal enzymes that evoke an inflammatory response - Deposition of needle shaped urate crystals in joints causing sever inflammation (gouty arthritis). o Kidney: uric acid stones o Soft tissue: - nodular masses of sodium urate crystals (tophi) may be deposited, under the skin. MEDICAL BIOCHEMISTRY DEPARTMENT 47  PRACTICAL BIOCHEMISTRY Uric acid in urine: Uric Acid is produced from degradation (catabolism) of purines (It is the end product of purine catabolism). Humans excrete uric acid in urine (About 0.7-1g of uric acid is excreted per day). Its excretion increases in metabolic gout. ❖ Creatinine: Serum creatinine Creatinine: - It is break down product of creatine phosphate in muscle - Creatinine is cleared from the body by the kidney, so serum creatinine is an important indicator of kidney function - The amount of creatinine excreted through urine by an adult individual is about 1.2-1.7 g per24 hrs. Normal serum Creatinine: - 0.7-1.2 mg/dL for men - 0.5 -1 mg/dL for women Urine Excretion: Creatinine is the excretory form of creatine. - In males: 1.5 gm / day - In females: 1.0 gm/ day. - The excretion of creatinine depends on the muscle bulk of the individual, not affected by diet. MEDICAL BIOCHEMISTRY DEPARTMENT 48  PRACTICAL BIOCHEMISTRY ❖ Amino acids: Amino acids in Urine: Amount: About 150-200mg/day.9 Cause of aminoaciduria: - Liver damage due to failure deamination of amino acids - Defect in renal tubular reabsorption of amino acids MEDICAL BIOCHEMISTRY DEPARTMENT 49  PRACTICAL BIOCHEMISTRY Blood glucose & Diabetes Mellitus Blood glucose ❖ Blood glucose level: The normal fasting blood concentration of glucose: 65-110 mg/dl. One hour after carbohydrate meal: 120-150mg/dl. Two hours after carbohydrate meal: 65-140mg/dl. ❖ Sources of blood glucose: Dietary carbohydrate: - absorbed glucose following digestion of carbohydrate. Liver glycogen: - through glycogenolysis. Liver glycogen can supply body with glucose for about 18 hrs. of fasting. Amino acids and other metabolites: - Through gluconeogenesis liver and kidney can convert these substrates into glucose. ❖ Regulation of blood glucose level: Hormonal regulation: A. Insulin hormone: - It is secreted by beta cells of islets of Langerhans of pancreas. It is the only hormone which reduces blood glucose level through the following: 1- Transfer of glucose inside the cells. 2- Stimulation of glucose oxidation. 3- Stimulation of glycogen storage (glycogenesis) 4- Stimulation of conversion of glucose into fat (lipogenesis). 5- Inhibition of glycogen breakdown (glycogenolysis) 6- Inhibition of conversion of amino acids and other metabolites into glucose (gluconeogenesis) MEDICAL BIOCHEMISTRY DEPARTMENT 50  PRACTICAL BIOCHEMISTRY B. Anti-insulin hormones: - They oppose the metabolic actions of insulin. - They include Glucagon, catecholamine, glucocorticoids, and growth hormone. 1. Glucagon: - it is hormone secreted by alpha cells of islets of Langerhans of pancreas. - it increases blood glucose level through: ↑gluconeogenesis. ↑glycogenolysis 2. Catecholamines (epinephrine & norepinephrine) - They are secreted from suprarenal medulla - They Stimulate glycogenolysis 3. Glucocorticoids as cortisol: - They are secreted from suprarenal cortex - They stimulate gluconeogensis. 4. Growth hormone: - secreted from anterior pituitary gland - Inhibition of glucose uptake by cells. - Block the insulin action at cell membrane. Hepatic Regulation (Role of Liver): - Liver regulates the blood glucose level according to the fed state: During Feeding State: - It stimulates oxidation of glucose by activating the enzymes of glycolysis, also it stimulates glycogenesis and lipogenesis. During Fasting: - more than 90% of glucose is derived from liver gluconeogenesis and glycogenolysis. MEDICAL BIOCHEMISTRY DEPARTMENT 51  PRACTICAL BIOCHEMISTRY Renal Regulation: - Circulating blood glucose is filtered in glomerular filtrate and reabsorbed again within limits (average 180 mg/dl) which called Renal Threshold. This saves blood glucose from loss in urine Low Renal Threshold: - glucose appears in urine even blood glucose is low (100mg/dl) - it may be due to defect in the absorption of glucose from renal tubule - it appears in 25% of pregnant women. High Renal Threshold: - where glucose appears in urine in concentration above 180 mg/dl - occurs in elderly and in renal damages. ❖ Hypoglycemia Definition: - ↓ Blood glucose below 40 mg/ dl. - it’s more dangerous than hyperglycemia because brain depend on glucose Symptoms: - Confusion, dizziness. - Tremors, weakness, tachycardia. - If not managed lead to coma. ❖ Hyperglycemia Definition: ↑ blood glucose above normal. - Fasting: more than 140 mg/ dl. - 1 H after meal: more than 200 mg/ dl. Causes: 1. ↓ Insulin as in: a) Diabetes mellitus. b) Surgical removal of pancreas. 2. ↑ Anti Insulin hormone: a) Adrenaline: Stress, emotion. b) Cortisone: as drug. c) Thyroid: Hyperthyroidism d) Growth hormone: Gigantism. MEDICAL BIOCHEMISTRY DEPARTMENT 52  PRACTICAL BIOCHEMISTRY Diabetes Mellitus ❖ Introduction Diabetes is a chronic metabolic disorder characterized by hyperglycemia due to lack of or resistance to insulin. Diabetes can be classified into two groups: - Type I diabetes being insulin dependent (IDDM), - Type II insulin independent (NIDDM). - Both type 1 and type 2 cause hyperglycemia, which in turn cause endothelial dysfunction. ❖ Clinical presentation: The 3P’s of diabetes: - Polyuria: increased frequency of urination and passing abnormally large amounts of urine mostly as a result of increased renal activity due to the increased need to filter excess sugar in the bloodstream. - Polydipsia: excessive thirst and increased water intake most likely because of frequent urination. - Polyphagia: excessive hunger or increased appetite. The classic symptoms of untreated diabetes are: - Polyuria, Thirst (polydipsia), weight loss. Several other non-specific signs and symptoms may also occur, including: - Fatigue - blurred vision - Have more infections than usual - Genital itchiness due to candida infection. - Have sores that heal slowly - Numb hands or feet About half of affected individuals may also be asymptomatic. MEDICAL BIOCHEMISTRY DEPARTMENT 53  PRACTICAL BIOCHEMISTRY ❖ Complications: Diabetic ketoacidosis Definition: - Diabetic ketoacidosis is a medical emergency due to excessive production of ketone bodies that occurs most commonly in type 1 but may also occur in type 2 if it has been longstanding or if the individual has significant β-cell dysfunction. Signs and symptoms including: - nausea, vomiting, abdominal pain - the smell of acetone in the breath - deep breathing known as Kussmaul breathing. - In severe cases; decreased level of consciousness. Hypoglycemia Hypoglycemia is a recognized complication of insulin treatment used in diabetes. An acute presentation can include: - Mild symptoms such as: sweating, trembling, and palpitation. - to more serious effects including impaired cognition, confusion, seizures, coma, and rarely death. MEDICAL BIOCHEMISTRY DEPARTMENT 54  PRACTICAL BIOCHEMISTRY ❖ Comparison between Type I and Type II diabetes mellitus: Type 1 (IDDM) Type 2 (NIDDM) Age of onset Usually during childhood or Frequently after age 35 puberty (young) (old). Juvenile onset Adult onset Defect or β-cells destruction Inability of β cells to deficiency (No insulin) produce appropriate Insulin deficiency quantities of insulin or insulin resistance Plasma insulin Low to absent Normal to high Cause Autoimmune or genetic Obesity or aging Nutritional status Frequently undernourished Obesity usually present at time of onset of (thin) disease ketosis common rare Oral hypoglycemic No response responsive drugs TTT with insulin Always necessary Usually not required ❖ Diagnosis of DM: 1. Measurement of fasting plasma glucose level (8-10 hours after last meal): a) 65-110 mg/dl: normal blood glucose. b) >126 mg/dl: DM. 2. Measurement of 2-hour postprandial plasma glucose level: a) 65-140 mg/dl: normal. b) >200 mg/dl on two different occasions: D.M. 3. Random Plasma Glucose Test: - A random plasma glucose concentration of 200 mg/dl or higher in the presence of symptoms indicates diabetes. 4. Glucose Tolerance Test (GTT) 5. Glycosylated Hb (Hb A1C) MEDICAL BIOCHEMISTRY DEPARTMENT 55  PRACTICAL BIOCHEMISTRY Glucose Tolerance Test (GTT) Definition: - It is the ability of the body to utilize carbohydrates. - It is a laboratory method to check how the body breaks down (metabolizes) blood sugar, and how quickly it is cleared from the blood. Types of glucose tolerance test: - Standard Oral glucose tolerance test. - I/V Glucose tolerance test. - Mini Glucose tolerance test. Indications of glucose tolerance test: - In asymptomatic persons with glucosuria. - Persons with + ve family history of DM. - Pregnant women at risk of DM (Test is done at 24-28 week of gestation). An overview of glucose tolerance test - Initially fasting blood glucose is estimated. - A loading dose of glucose is given. - The blood glucose levels are estimated at regular intervals after the glucose load. - In conditions of insulin deficiency, blood glucose levels get elevated due to impaired utilization of glucose. Procedure of oral GTT: - At 8 a.m. the fasting blood and urine samples are collected. These are called zero samples. - A loading dose of 75 gm anhydrous glucose dissolved in 250 ml of water is given to the patient. - Then, blood and urine samples are collected at half hourly intervals of the next two and a half or three hours. - Glucose is estimated in all the blood samples. - Urine is analyzed for the presence of glucose. MEDICAL BIOCHEMISTRY DEPARTMENT 56  PRACTICAL BIOCHEMISTRY Glucose Tolerance Curve: - A curve is plotted with the blood glucose levels on the vertical axis against the time of collection on the horizontal axis. - The curve so obtained is called glucose tolerance curve. - Types of curves of OGTT: 1- Normal curve. 2- Curve of IGT. 3- Curve of DM. Normal curve Diabetic curve MEDICAL BIOCHEMISTRY DEPARTMENT 57  PRACTICAL BIOCHEMISTRY Mini or Modern GTT - According to current WHO recommendations, in the mini or modern glucose tolerance test, only two samples are collected, fasting (zero hour) and 2-hour post glucose load. - Urine samples are also collected during the same time. - The diagnosis is made from the variations observed in these results. Criteria for diagnosis of DM Glucose conc. (mg/dl) Fasting 2 hours After Meal Normal 65-110 mg/dl 65-140 mg/dl Impaired glucose tolerance 110-126 mg/dl >140-200 mg/dl D.M > 126 mg/dl >200 mg/dl MEDICAL BIOCHEMISTRY DEPARTMENT 58  PRACTICAL BIOCHEMISTRY Glycosylated Hb (Hb A1C) Hemoglobin A is the major hemoglobin in adults (97%) Glycosylated Hb is a non-enzymatic reaction between the Hb and glucose Normal Hb A1C = 4-6 %. In diabetic person above 6.5 % and may reach 12% or more of total HB A. Advantages: Its level in red cell is directly proportional to blood glucose concentration over 2-3 months (life span of RBCs) so this test can be used as index of diabetic control over 2-3 months. Disadvantage: Hence people with hemolytic anemia and sickle cell disease are not suitable for this test. MEDICAL BIOCHEMISTRY DEPARTMENT 59  PRACTICAL BIOCHEMISTRY DNA Extraction ❖ Definition: - DNA extraction is the removal of deoxyribonucleic acid (DNA) from the cells in which it normally resides. ❖ Sources of DNA: - Any living tissues ❖ Method of Extraction: There are 3 parts of the cell you must go through to remove the DNA. Barriers to overcome - BARRIER 1: Cell Wall (only in plant) - BARRIER 2: Cell membrane - BARRIER 3: Nuclear membrane o Barrier 1 – Cell wall Extraction Procedure: Crushing Activity function: These breaks open the cell wall (LYSE), cell membrane, and the nuclear envelope to allow the DNA to be released. MEDICAL BIOCHEMISTRY DEPARTMENT 60  PRACTICAL BIOCHEMISTRY o Barrier 2 – Cell Membrane & Barrier 3- Nuclear Membrane Extraction Procedure: Extraction buffer It contains detergent, salt and EDTA Activity function: - Breakdown the fat and proteins that make up the cell and nuclear membrane. - Detergent is used to dissolve the cell membrane (made of phospholipids) - Salt is used to stabilize DNA (stabilizes the negatively charged phosphate groups and allows the DNA strands to clump together). - EDTA to chelate Mg to inhibit nuclease enzyme and stabilize DNA o Last step Extraction Procedure: Ice Cold Alcohol Activity function: - DNA does not dissolve in alcohol. (It is soluble in water). - The addition of alcohol causes the DNA to precipitate. - Ethanol is less dense than water, so it floats on top. - All the proteins we broke up will sink to the bottom; the DNA will float on top. ❖ DNA Extraction Protocol o Materials Required - Source of DNA (tomato). - Extraction buffer: Salt (NaCl). EDTA (To chelate Mg to inhibit nuclease). Dishwashing Soap (Fairy, Pril, etc.) - Alcohol: 95-100% Ethyl Alcohol. MEDICAL BIOCHEMISTRY DEPARTMENT 61  PRACTICAL BIOCHEMISTRY o Steps Step (1): Crushing - Place the tomato in a plate - cut the tomato in with knife into to form "cells solution to begin the process Step (2): Extraction Buffer - Add about 2 (tablespoons) of (extraction buffer), swirl to mix. - Let the mixture sit for 5-10 minutes. Step (3): Filtration of cells solution - by nylon gauze and funnel into your test tube (to get fibers-free solution). Step (4): Add ice cold 90% alcohol. - Tilt your test tube and slowly pour alcohol into the tube. - Pour it down the side so that it forms a layer on top of the tomato mixture. - Pour until you have about the same amount of alcohol in the tube as tomato mixture. Step (5): Extracting DNA - DNA will rise into the alcohol layer from the tomato layer. - let the preparation sit for 15 minutes. Step (6): Pick the DNA - from the top of your test tube by a Pasteur pipette. - DNA is a long, stringy molecule that likes to clump together. o To increase the yield of DNA Allow more time for each step to complete. Using ice-cold alcohol Make sure that you started with enough DNA MEDICAL BIOCHEMISTRY DEPARTMENT 62  PRACTICAL BIOCHEMISTRY ❖ Practical Applications of DNA Technology: 1. Medicine: - Diagnosis of infectious diseases and genetic disorders - Human gene therapy: currently aimed at fighting heart disease and cancer - Pharmaceutical products: growth hormones, insulin, vaccines 2. Forensics: - DNA fingerprints: diagnose paternity and crime field. 3. Environmental: - Genetically engineered organism: Able to extract heavy metals from soil as mercury (toxic to living organisms) - Sewage treatment - Research: engineering organisms to degrade chlorinated hydrocarbons & other toxic chemicals. 4. Agricultural: - Transgenic organisms: ✓ Increased productivity. ✓ Pest resistance, disease resistance. ✓ “pharm “animals ✓ "Golden rice" – enriched with beta-carotene. MEDICAL BIOCHEMISTRY DEPARTMENT 63  PRACTICAL BIOCHEMISTRY PCR ❖ PCR A method of in vitro cloning Allows amplification of specific DNA molecules (fragments) in vitro through cycles of enzymatic DNA synthesis The most popular and widely used technique in all fields of biological studies probably. ❖ Advantages: 1. Simple 2. Powerful Sensitive, specific and reliable 3. Fast ❖ Principle To amplify a lot of double stranded DNA molecules (fragments) with same (identical) size and sequence by enzymatic method and cycling condition. ❖ Components Chemical components - Target DNA - DNA polymerase - Primers - dNTPs DNA template - DNA containing region to be sequenced. Primers - 2 sets of primers - 20-30 nucleotides long - Synthetically produced - Complimentary to the 3’ ends of target DNA - Not complimentary to each other MEDICAL BIOCHEMISTRY DEPARTMENT 64  PRACTICAL BIOCHEMISTRY Enzyme - Should be thermostable polymerases usually Taq Polymerase. - Stable at T0 up to 950 C - High processivity - Taq Pol has 5’-3’ exo only, no proofreading ❖ Steps (thermal cycle) 1. Denaturation of ds DNA template: Heat 95oC 2. Annealing of primers: temperature is lowered (Tm) and primers anneal to target sequences. 3. Extension of ds DNA molecules: at 72oC Taq DNA polymerase catalyzes primer extension as complementary nucleotides are incorporated. End of the 1st PCR cycle results in two copies of target sequence. [Target Amplification] MEDICAL BIOCHEMISTRY DEPARTMENT 65  PRACTICAL BIOCHEMISTRY ❖ Types of PCR Conventional PCR. Multiplex PCR. Nested PCR. Reverse Transcriptase-PCR Quantitative RT PCR. Hot start PCR Multiplex-PCR: It is a special type of the PCR used for detection of multiple pathogens by using multiple primer sets each one targets a particular pathogen. Uses: - This permits the simultaneous analysis of multiple targets in a single sample. RT-PCR (Reverse Transcription PCR) Used to reverse-transcribe and amplify RNA to cDNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions may be combined in a tube. Uses: - Detection of RNA virus like (HCV). - Detection of other M.O. through targeting of their Ribosomal RNA. MEDICAL BIOCHEMISTRY DEPARTMENT 66  PRACTICAL BIOCHEMISTRY ❖ PCR Applications: PCR in genetic engineering - These cloned DNA fragments can then be inserted into the target organism, including microorganisms, plants, or animals, using vectors such as bacteria and viruses. Diagnosis of genetic disease - using tissue samples from the chorionic villus; amniotic fluid; or the small quantities of foetal DNA Detection of infection in the environment - PCR is used to detect the spread of infectious disease within an animal or human population. - PCR can also be used to detect bacterial and viral DNA in the environment, for example looking at pathogens in water supplies. Detection of mutations Detection and diagnosis of infectious disease - PCR can detect infectious disease before appearance of antibodies, so allowing treatment to start much earlier. Classification of organisms. Evolutionary studies: - PCR in research PCR can be used in analysis of gene expression. Genomic studies / Genotyping - compare the genotype of two organisms and identify the difference between them. Forensic Medicine and Genetic Fingerprints - PCR is very important for the identification of criminal. - The DNA fingerprinting technique is used in forensic science. - A single molecule of DNA (stains of blood, hair etc.) is enough for amplification. MEDICAL BIOCHEMISTRY DEPARTMENT 67  PRACTICAL BIOCHEMISTRY Urine sheet ❖ Physical properties: 1. Volume: (sample) Normally 1- 1.5 L/day 2. Color: yellow 3. Odor: Aromatic odor 4. Reaction: acidic 5. Specific gravity: Normally 1015-1025 6. Aspect: Clear ❖ Chemical properties: 1. Reducing sugar (Benedict test) - Test: 2 ml Benedict + 1 ml solution Heat directly on flame for 2 min. - Result: Appearance of green, yellow, brown ppt. Means positive result 2. Ketone bodies – Acetone (Rothera test) - Test: Put some of Rothera powder on filter paper, Put ½ ml of urine on it - Result: Appearance of violet color means acetone in urine 3. Urinary protein (Albumin) (Heat coagulation test) - Test: Put 5 ml of urine in tube then heat upper half of urine for 2-3 min. - Result: Appearance of white coagulation on surface of tube means presence of urinary protein (Albumin) 4. Urea - Test: 1 ml urine + 1 ml diacetyl monoxime + ½ ml sulfuric acid then Boil on water bath for 3 min. - Result: Appearance of deep yellow color means urea in urine MEDICAL BIOCHEMISTRY DEPARTMENT 68  PRACTICAL BIOCHEMISTRY 5. Phosphate - Test: Put 2 ml of urine in tube + 1 ml conc sulfuric acid + 3 ml ammonium molybdate - Result: Appearance of yellow Canarian color means phosphate in urine 6. Calcium - Test: 2 ml urine + 2 ml ammonium oxalate - Result: Appearance of white milky color means calcium in urine 7. Bile salts, bile pigment, urobilinogen: ✓ Hay sulfur test - Test: put 2 ml urine in test tube then put some sulfur powder on the surface with slight shake - Result: If powder remain on surface means negative ✓ Erlich test (urobilinogen) - Test :1 ml urine + 1 ml Erlich - Result: deep red color means urobilinogen. MEDICAL BIOCHEMISTRY DEPARTMENT 69  PRACTICAL BIOCHEMISTRY El-Minia University Date: / / 2024 Faculty of Medicine Practical exam Biochemistry Department Student name Group No. Unknown No. Urine Analysis Report Physical properties: Volume Odor Color Reaction Specific gravity Aspect Chemical properties: Reducing Sugars Ketone bodies Calcium Urea Phosphate Urinary proteins Bile Pigments Bile salts. Urobilinogen Comment MEDICAL BIOCHEMISTRY DEPARTMENT 70  PRACTICAL BIOCHEMISTRY Subject Date Signature 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. MEDICAL BIOCHEMISTRY DEPARTMENT 71