Practical of Biochemistry - Learning Materials

Practical of biochemistry first year of medicine *Colorimetric method reagent 20% Transmittance Photo- diodp • Serum = Plasma - Fibrinogen) Standard tube known conc. Sample tut f unknown Conc. Reagent will make color to Jetect the substance. Qualitative Spectrophotometer measure the absorbance of sample to detect Concentration) LED 400-700mm" monochromatis light 801 -Absorbaum of Spect photometer absorbanc Directly proportional Beer's law Quantita Sample conc. = AlSorbance of Sample Absorbence of Standard Standard X conc Concentrativ ① Estimon of serum protein Biuret method. Fmal level " 6-89/ looml Plasma albumin 60% Globulins 36 fibringen All plasma proteins are produced by liver except globulin is produced by reticuloendothelial system Q. All are glycoproteins except albumin pure proteinn. t/½= 20 days 9. A G ratio = reversed in viral hepatitis or infection 2 1 • Principle Colorimetryn Cu+2 + alkaline PN Biuret reagent - Cur bind to peptide Violet Color bond •Hypoproteinemia liver cirrhosis or failure 4% Glycoprotermalnutrition, malabsorption Protem losing enteropathy Nephrotic syndrome, 1055 of albumin in urine @090 oncotic Pressur •Hyper proteinemia-r-globulinn infectionr-globulin @Multiple myeloma (2) Estimation of serum albumin Bromocresol green or BCG) • Normal level 3.1-4.39/1 • martam oncotic pressure low mut, may be filtred Murimp • Transport of Ca², Cử², mg+? drugs. bilirubin, fatty acids. •Albumin- 30 mg 24 his →Ca, mga no tetany Abnormal distribution • Hypo albuminemia •Synthesis liver disease loss or degradation Nephrotic syndrome, Malnutrition (overhydration • principle 628 nm BCG •Hyper albuminemig • Rare •Dehydration, Shock "Hemoconcentration. albumin Practical of biochemistry first year of medicine ③ Estimation of serum Lascorbicacid - vitamin ca 9. Normal level 0.4 - 15 Stable in acidic PH, Jestyed by heat or alkaline PH. antioxidant • Derived from glucose found in adrenal medulla and WBC 700 nm PTA Phospho- tinge acid •Deficiency Scurvy jefect in collagen Cium bleeding loose teeth • anemia, osteoporosis • Jelay healing, hyperlipidemiq • 2 forms: L-ascorbic acid 15 active hydro ascorbic acid actiu • Toxicity: Increase oxalate Salts lead to Renal Homo- gentistic acid * Urine analysis: UA. Clear PHS.S-6.8 •PH is increved by bacteria il analyzed after one hour alkaline bacteria ghuth • Early morning Sampio collected. • Appearance Clear Cloudy@ Dau llovy or orange smoky red Brown red Black urine Normal Pus in urine Pyourian Bilirubin Rifampicin X or concentrated orine Hematuria RAC Hemoglobinuria Alkaptonuria - formic acid • milky urine Chyluria Flymphin Urine • Fresh red → beet eating • Normal urine color is amber yellow -800-2000m/ Urine volume = 1.5L/Jay 9. Polyuria 3000 ml/day • oliguria 400 ml/day @anuria 100ml/day Causes of Polyuria diluted water intake Dm, Diabetes insipidus. HOVT- dimetic intake Chronic renal failure • Causes of oliguria excessive Sweating Dehydration, Shock ③ Edema Acate renal damage Nephritis O specific gravity = 1.010-1.025 - Urime concentrations •Specific gravity • Diabetes mellitus & • Diuretics • Sweating specific gravity • water intake • Diabetes insipidus • Chronic renal failure Pain Hematuria albuminurid •PH reaction Diet reach in protein acidic Plt Diet reach in vegetable alkaline PH • Hematuria Blood in urine RBC in unive Acute nephritis or renal failure •Causes of →proteinuria, hematuria, oliguria 2 Benzidime test -react with Ho hematuria Stone blue to green color 8 PB Tumor Trauma Nemolysis hemoglobinuria * Urine Contain 4% of waste products called "NPN Non protein nitrogen • Urea •Creatining Nigh plasma Creatining indicate renal failure ADN Uric acid *Glucosulia-Glucose in urime when blood glucose exceeds 180 mg/dl renal threshold Benedict's test: Bene •tonuria Dm, Starvation Ketone bodies : acetone aceto acetate B-hydroxy butyrate G asilic Cuz no glucose Green + 0.59. J Yellow ++ 19 red 1-29 +++ Violet Rothera 's test QQQQQ •Diagnosis of obstructive jaundisp ↓ Bilirubin • Glucostix i another method to detect glucosuria Glucosp oxidase - Glucosuria - Benedict test is affected" by vit.c Phenylalani-p and lactose intestin Fouchet reagent Bile salts Green Hay Sulfur if they Sink Bile sal • Hemolysis Hemolytic Jaundice or Shlesinger test Urimp Turobilinogen Serum Calcium estimation Normal level 9-11 mg/01" 8.5-10.5 mg/01/ Hypercalcemia Hyper parathyrainin •Vit D foxicity •Thiazide lithium milk alkali syndrome, Paget's disease, Hypocalcemia vit.D Jeficiency rickets, osteomalaria- ✓ Renal failure • Hypo Parathyroidism, Pseudohypoparathyroidism malnutritin, malabsorption. • reagent: O-Cresol phthalein. ⑥ serum phosphate - intracellular anion, Urive buffer Normal level: adult: 12.6-4.5 mg1815 •children: 14-6 mg/dl Hyperphosphatemia ✓ Renal failure most comms Hypophosphatemia Hyper Parathyroidism •Hypo Parathyroidism • Hemolysis Po Fanconi Syndrome reabsorption • antacids intake faluminium insulin intake hydroxide Surgikal removal oncogenic by tumor • Malnutrikun • phosphate is Jetected by ammonium molybdate) • Knuri tone bodies Serum transaminases liver enzymer. ①aka + alanine < ALT Glue ②aka + aspartale AST Hoxaby Violet acetale ring *t/½ = 47 hours. Cytosol ALT AST •Normal levels ALT 10-SSIU cytosol mit AST-10-40 5.01 Diagn . Viral hef. titi, toxic hepatitis TALT. AST Carbon tetrachloride To times Diconolic hepatitis AST 2 AG = less than 300IU ALT 1 @myocardial infarction $AST=4-6-4-5 hours • Sume antibiotics elevate transaminases Extranotes:- - Urine analysis: Contains 4% of dissolved waste products Volume 1.5L PH5.5-7.5 Color: amber yellow due to Urachrome and Urobilin, Uroerthrin". Diet medicine and chemicals may alter the color. a) Physiological Causes: 1- Pale Urine yellow color" due to Tamount of water 2. Dark yellow in fasting and summer time or food dyes. B Pathological causes & 1- Colorless or faint Urine Polyuria e. Red Color Hematurian due to blood or HG... 3- Greenish or dark brown @obstructive Jaundicen 4- Dark yellow or orange fever. 5. Blackish color in alkaptonuria e Homogentisicaci! 6- Milky in chyluria or pyuria urine pus " ② Volume: Normal 800-12000 ml/day a) Polyuria" increased volume 2000ml/day" Physiological Polyurias ①Excessive fluid intake High Protein or salt intake ⅱ Pathological Polyuria: Diabetes mellitus @chronic renal failure Diabetes insipidus 6) oliguria Decreased urine volume of 500ml/day" Anuria 125 ml/day Physiological: less water intake or excessive Sweating. ②Pathological: ① Dehydration Vomiting diarrhea. I sweating. Renal ischemia: f blood supply. Shack, hemarchage and heart failure.. Renal disease: Acute renal failure. ① obstruction: Stone or prostate hyperplasia or Carcinoma ③ Urine appearance • clear and transparent a) Normal turbidity on standing Cloudy flocculen may be seen due to nucleo. of muco protein. 6) Abnormal turbidity: i. Pus and bacterial UTI ii. Hematuria lii. crystals: urate or oxalate ④ Urine reactions. PH=4.8-7.5" Usually acidic a) acidic Urine : i. High Protein intake.. li. Vitaminc iii. lactic acid Exercise" iv. Diabetic keto acidosis vi. Respiratory acidosis. v. lenkemia uric acid. b) Alkaline urine: i. metabolic alkalosis. ii. Infected urine @ Ammonia * Measurment of serum albumin. Albumin is glucose free pure proteins and represents 55.65% of plasma proteins.. Decrease of serum albumin Cause & Ca, Mge because these ions bound to albumin.. *Principle. Colorimetry 628 nm BCG Brom cresol green. conly bound to albumin tightly Color The absorbance is directly proportional to albumin concentration. - Normal range: 3.5-5.29/11 - Hyperalbuminemia: acute dehydration or Shock - Hypo albuminemia: Nephrotic syndrome • Urine loss ② Decrease Production liver cirrhosis Same causes of hypo Proteinemia safety in the laboratory : * General safety: ① laboratory coat, gloves @safety gaggles must be worn a not contact lenses Clothing shoes not sandals must be worn * Handling chemicals : ① Do not taste or ingest Avoid touching chemicals with your hands. ③ work in fume hood when reaction produce gases ④ No mouth pipetting Always add acids to water not water to acid to reduce the heat generates.. labels all flasks, test tubes. Kold reagent bottles. Dispose laboratory waste. ⑨ waft the fumes by your hands, do not put Your nose over the container to inhale the fume. • Electrical safety: ① your hands are dry. Unplug. all electrical equipments at the end of lab. Heating safety : ① let hotplates cool down before touching them never leave burner wrattended, use heat resistanci glassware like pyrex. ③move test tube around the flame when you heat it... laboratory supervision: No unauthorized experiment and no student may work alone Behavior in laboratory ① No food and beverages in the lab. @leave your book bags and backpacks out... No aggressive or harassing behaviour or Horseplay" * Glassware safety : any broken. chipped or cracked glassware should not be used and disposed. Before you leave a clean your beach and.. wash your hands". Emergency procedure. :- Rinse bag water ant safety shower any chemical spills before. it cause injury In Case of fire, use blanket and roll on the floor or ask some one to pull awater. •cord to shower. - wash minor cuts or burn by cold water. and report all incidents no matter how minor to the instructor.