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Abnormal Constituents Urine.pptx
- 1. By Dr .Y. Venkatamani 2ndYear PG MD Biochemistry Gandhi Medical College Secunderabad ABNORMAL CONSTITUENTS OF URINE
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- 3. Indications For Urinalysis •Suspected renal diseases like glomerulonephritis, nephrotic syndrome, pyelonephritis, and renal failure. • Detection of urinary tract infection. • Detection and management of metabolic disorders like diabetes mellitus. • Differential diagnosis of jaundice. • Detection and management of plasma cell dyscrasias. • Diagnosis of pregnancy • For certain hormones like Catecholamines, 17-Hydroxy steroids, urinary free cortisol • Urinary VMA to detect excess of epinephrine and nor epinephrine To detect various conditions like lipuria, chyluria, choluria, Haemoglobinuria, Myoglobinuria, Detection of drugs, poisoning
- 4. The chemical examinationis carried out for the following substances :- • Proteins • Glucose • Ketones • Bilirubin • Bile salts • Urobilinogen • Blood • Hemoglobin • Myoglobin • Nitrite or leukocyte esterase Abnormal constituents of Urine
- 5. New Born Screeningfor Inborn Errors of Metabolism • Phenylketonuria • Tyrosyluria • Alcaptonuria • Melanuria • Branched Chain Amino Acid Disorders Maple Syrup urine disese Organic Acidemias • Tryptophan disorders – hartnup’s disease • 5-Hydroxyindoleacetic acid - (5-HIAA) • Cystine disorders • Homocystinuria • Porphyrin disorders • Mucopolysaccharide disorders • Purine disorders • Carbohydrate disorders
- 6. Abnormal Constituents ofUrine Substances which are not present in easily detectable quantities in urine of healthy individuals Solution A Physical Characteristics • Appearance – Transparent , Clear • Colour – colour less, • Odour –fruity odour
- 7. Test for ReducingSugars Benedict’s test In alkaline medium, sugars undergo tautomerization to give endiols forms which are powerful reducing agents. They reduce cupric ions to red cuprous oxide Benedict’s Reagent :- CuSO4, NO2CO3, Sodium Chloride Various colors of the precipitate depends upon the concentration of sugar in urine Color Sugar content (Grams %) Blue Nil Green 0.1 -0.5 g% Yellow 0.5 - 1.0 g% Orange 1 -1.5 g% Red 1.5 - 2 g% Brick Red > 2 g%
- 8. Test for ReducingSugars Benedict’s test Interpretation: from carbohydrates Glycosuria (>180 mg/dl) Causes : - Renal Glycosuria , pregnancy, heavy metal poisoning , renal tubular necrosis, alimentary Glycosuria Diabetes mellitus false + ve Hyperthyroidism Vit C Hyperpituitarism Gluconic acid Hyperadrenalism Homogentisic acid Stress Drug salicylates gets oxidized Severe infection
- 9. Test for ReducingSugars Experiment Observation Inference Take 5 ml of Benedict’s reagent and boil it for 1 min then add 8 drops urine. Again boil it for 2 min Colour varies from green to brick red depending on concentration of urinary sugar Indicates presence of reducing sugar in urine Benedict’s test
- 11. Test for KetoneBodies Rothera Test Principle :- In alkaline medium, Nitroprusside reacts with ketone to form purple coloured complex in presence of Ammonia . It is given by Acetone & acetoacetate but not by B-hydroxy butyric acid Acetone , acetoacetate , B hydoxy butyric acid - normal <1mg/1 day ketonemia, Ketonuria, Ketosis Ketonuria Causes :- Uncontrolled DM , Starvation High fat diet heavy exercise Toxemia pregnancy recurrent vomiting
- 12. Test for KetoneBodies Experiment Observation Inference Take 5 ml of urine in Test tube. Add solid Ammonium Sulphate until it is saturated the 2 drops of Sodium Nitroprusside. Add 1 ml of liquor ammonium along sides of test tube Purple colour ring is seen at junction Indicates ketone bodies Rothera Test
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- 14. Test for Urine HeatCoagulation test Principle: - By heating denaturation of proteins occurs denaturation causes loss of bioactivity of proteins due to unfolding of Protein tertiary structure Isoelectric PH (PI) of Albumin – 5.4 Globulin – 5.5 Caesin -4.6 Urine Protein composition Total Proteins – 150 g/day Albumin – 15 mg Tamm- Horsfall protein– 70 mg Mucopolysaccharides-15mg Blood group related antigens – 35 mg Immunoglobulin - 5 mg Rest hormones and enzymes - 10 mg Bence Jones Proteins - Light chain IG excreted in urine of multiple myeloma patients . It coagulates 40-600C, upon further heating turbidity appears on cooling it reappears Solution B
- 15. Test for Urine HeatCoagulation test Experiment Observation Inference Fill 3/4 of test tube with clear urine. If urine is turbid filter if before performing. Heat the upper 1/3rd of test tube. Add few drops of 1/3 acetic acid to the solution White Coagulum is formed Indicates presence of protein is urine Solution B
- 17. Proteinuria Presence of proteinin urine is called proteinuria Causes – Increased glomerular permeability Reduced tubular reabsorption Increased secretion of proteins Physiological – - Exercise, Postural, Pregnancy, exposure to cold Pathological - Pre-renal Renal dehydration Nephrotic syndrome Burns Renal failure Toxemia pregnancy Chronic / Acute GMN Fever Pyelonephritis TB of Kidney
- 18. Types of Proteinuria TubularProteinuria (< 1gm /day) - Wilson’s disease inherited - Interstitial Nephritis - Heavy metal poisoning Mechanism - Inc. permeability to LMW proteins, Reduced tubular function Over flow Proteinuria (> 15 -30 gm /day) • Multiple Myeloma Leukemia • Lymphoma • Production is more • Glomerular Proteinuria (> 30gm/day) - HTN • Nephrotic Syndrome - Diabetes Mellitus • Glomerular nephritis - Inc. permeability of HMNU proteins
- 19. Tests for Blood BenzidineTest Principle : - Peroxides in RBC splits H2O2 to produce nascent O2 which reacts with Benzidine in acidic medium to give bluish green colour complex • Hematuria (blood) • Hemoglobinuria (RBC) • Myoglobinuria Applied Aspect – Hematuria Causes of 1) Gross Hematuria 2) Microscopic Hematuria Renal stones sickle cell anemia Acute glomerulonephritis Pyelonephritis kidney disease malignancy Coagulation disorders Malaria Malignant hypertension Trauma Hemolytic Anemia TB
- 20. Tests for Blood BenzidineTest Experiment Observation Inference Take 2 ml of Benzidine solution. Add 2 ml of urine 1 ml of H2O2 Bluish green colour is seen Presence of blood in urine Solution C Physical characteristics • Appearance – transparent , clear • Colour –green • Odour – odourless
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- 22. Tests for BileSalts Hay’s Test Bile salts act as emulsifying agents which reduces the surface tension, hence sulphur powder sinks to bottom Interpretation Primary bile salts (liver) - Cholicacid, Chenodeoxycholicacid coagulation is by Glycine and Taurine Secondary Bile Salts (Intestine): Deoxy cholic acid , lithocholic acid Bile Salts : Na & K salts of taurocholic acid & glycocholate obstructive jaundices – Presence of Bile salts in urine Prehepatic – Na salts & pigments in urine Urine Pre Hepatic Hepatic Post Hepatic Bile Salts -ve - ve + ve Bile pigments - ve + ve + ve
- 23. Tests for BileSalts Experiment Observation Inference Take 5 ml of urine Sprinkle pinch of sulfur powder. Control – take 5 ml of water solution. Add pinch of sulfur powder Powder sinks Powder floats Presence of bile slats in urine Absence of bile salts in urine Hay’s Test
- 25. Tests for BilePigments Fouchet’s test Principle : Bile pigments are absorbed on BaSO4 precipitate which are oxidized by Fouchet’s reagent to biliverdin, & bili cyanin which are blue and green respectively . Variation of colour due to exposure to O2 Bile pigments : - Bilirubin, Biliverdin , Bilicyanin Diseases :- • Hepatic Causes : - Hepatitis (Viral, toxic) Cholesterol (intrahepatic) • Post Hepatic :- Obstruction gall stones, tumor of bile duct carcinoma of pancreas
- 26. Tests for BilePigments Experiment Observation Inference Take 5 ml of urine. Add 1 ml of MgSO4 Add l ml of BaSO4 Until white ppl. Is obtained. Filter the solution dry the residue and add 1-2 drops of Fouchet’s reagent to filter paper Series of yellow, blue, green colour are obtained Indicates presence of bile pigments Fouchet’s test
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- 29. Urea isthe highest(75%) non-protein nitrogen compound in the blood. Urea is the major excretory product of protein metabolism. It is formed in the liver from free ammonia generated during protein catabolism. The most important catabolic pathway for eliminating excess nitrogen in the human body. UREA (NH2CONH2):
- 30. Methods of ureaestimation MethodPrinciple Kinetic assay using glutamate dehydrogenas e method Ammonia reacts with α-ketoglutarate in presence of glutamate dehydrogenase and NADH. This results in the formation of L-glutamate and NAD+. Rate of decrease in concentration of NADH is monitored at 340nm Diacetyl monoxime method (DAM) Urea reacts with DAM under strong acidic condition in presence of ferric ions and thiosemicarbazide to give a pink colored complex. The intensity of pink color is a measure of the amount of urea in the sample, which is measured at 520nm. Nessler’s method Ammonia reacts with potas ium mercuric iodide (Nes ler’s reagent) to form yellow color, which is measured at 480nm. Berthelot reaction Ammonia reacts with sodium hypochlorite and sodium nitroprusside as a catalyst, in alkaline medium to produce blue colored complex, which is measured at620nm.
- 31. AIM: Toestimate the amountof urea in the given bloodsample. METHOD: Kinetic enzymatic method by glutamatedehydrogenase. PRINCIPLE: Ammonia is formed from urea by ureaseenzyme. Ammonia reacts with α-ketoglutarate in the presence of glutamate dehydrogenase and NADH. This α-ketoglutarate results in the formation of L-glutamate andNAD+.
- 32. The rate ofabsorbance (NADH) changing at 340nm is directly proportional to urea concentration in the serum Urea + 2H2O 2NH4 + + CO3 2- 2NH4 + + 2- oxoglutarate + NADH L-glutamate + NAD + + H2O Urea se Glutamate dehydroge nase
- 33. REAGENTS: • Tris buffer(pH – 7.8) – 9.6 mmol/L • ADP – 0.6 mmol/L • Urease – 16000 U/L • GLDH – 960 U/L • NADH – 0.25 mmol/L • α-ketoglutarate – 9 mmol/L • Urea standard – 50mg/dL R1 – tris buffer, α-ketoglutarate, Urease. R2 – Glutamate dehydrogenase, NADH.
- 34. WORKING REAGENTPREPARATION: The reagents(R1 and R2) are ready to use. The working reagent is prepared by gently mixing 4 parts of R1 with 1 part of R2. REAGENT STORAGE: Stored at 2-8oC. REAGENT STABILITY: The unopened reagents are stable till the expiry date stated on the bottle andkit label. The working reagent (4R1:1R2) is stable for 30 days at 2-8oC, when protected from contamination andlight. It is recommended to prepare fresh working solution before assay is performed.
- 35. SIGNS OF REAGENTDETERIORATION: •Presence of particles andturbidity. • Workingreagent absorbance<0.8 at 340nm. • Failure to recover control values within the acceptable range. SPECIMEN: Use unheamolytic serum, plasma orurine. • Do not use samples preserved with fluoride and heparinammonium salt as anti- coagulant. • Urine: dilute urine 1+100with distilled water and multiply results by 101.
- 36. PROCEDURE: Three test tubesare taken and labelled as standard(S), test(T), and blank(B). Mix well and aspirate standard followed by samples. Read the optical density at340nm. Reagents Standard (S) Test(T) Blank (B) Working reagent 1mL 1mL 1mL Standard 0.02mL - - Sample - 0.02mL - Distilled water - - 0.02mL
- 37. Calculation: Concentration of ureain mg/dl blood = ODT/ ODs X Concentration of Standard(mg/dl) Result:
- 38. AIM: Estimation of bloodurea. METHOD: Diacetyl monoxime method (DAM) PRINCIPLE: Urea reacts with diacetyl monoxime (CH3COCNOHCH3) or diacetyl (CH3COCOCH3) under strongly acidic condition in presence of ferric ions and thiosemicarbazide to give a pink colored complex. Proteins in blood do not interfere as they are precipitated with trichloroacetic acid (TCA). The intensity of the pink color is a measure of the amount of urea (NH2CONH2) present in blood .
- 39. REAGENTS: • 10% TCA •Diacetyl monoxime/ thiosemicarbazide reagent: Dissolve 1.56g diacetyl monoxime and41mg thiosemicarbazide in 250mL distilled water, store in brown bottle. • Phosphoric acid-sulfuric acid- ferric chloride reagent: Dissolve 324mg of anhydrous ferric chloride (FeCl3) in 10mL of 56% phosphoric acid. Add 1mL of this FeCl3 reagent to 1L of 20% sulfuric acid(H2SO4). • Diacetyl monoxime reagent: Mix equal volume of b and c. this is to be freshly prepared.
- 40. • Preservative diluentfor standard: Dissolve 40mg phenyl mercuric acetate in about 250mL water with heating. Transfer the solution into a measuring cylinder. Add 0.3mL concentrated H2SO4 and make up to 1L withwater. • Standard urea solution: 3mg urea in 100mL preservative diluent (0.03mg/mL). Procedure: Part I – Preparation of protein-free filtrate (PFF) from blood. • In a dry test tube, take 3.4mL distilled water, 0.1mL blood, and 1.5mL 10%TCA(dilution of blood = 1 in 50). • Mix, keep for 10 min, and filter in a dry test tube to obtain a clear solution of PFF.
- 41. Part II –Use PFF for blood urea estimation. • Label three test tubes as T(test), B(blank), and S(standard) Mix and keep the tubes in the boiling water bath for 15 min, cool, and read the optical density(OD) using colorimeter with a green filter of wavelength 520nm. Reagents T B S Protein-free filtrate 1mL - - Standard ureasolution - - 1mL Distilled water - 1mL - Diacetyl monoxime reagent 5mL 5mL 5mL
- 42. CALCULATION: Concentration of ureain mg/dl in blood: = ODT – ODB / ODs – ODB X Concentration of Standard/ Volume of sample X 100mg% = ODT – ODB / ODs – ODB X 0.03/0.02 X 100 mg%s = ODT – ODB / ODs – ODB X 150 mg%. RESULT:
- 43. UNIT CONVERSIONS: • Itis sometimes necessary to convert results expressed in terms of blood urea to blood urea nitrogen and vice versa or results expressed in units of millimoles of urea/L to mg of urea nitrogen and vice versa. • The conversion factors are: • mg urea nitrogen/dL x 2.14 = mgurea/dL. • mg urea/dL x 0.467 = mg urea nitrogen/dL • mg urea nitrogen/dL x 0.357 = nmol ofurea/L Factors are derived on the basis of molecular weight for urea (60D) and the nitrogen content in urea (28g/mol). INTERPRETATION: The normal range of blood urea level is 15-40mg/dl. • Physiological variations can be seen with age, gender and food(dietary intake of proteins).
- 44. Normal ranges forserum(blood) samples For diluted urine sample, urea is calculated for 24 hours. Normal range: 26-43g/day (0.43-0.72mol/24hrs) Age mg/dl mmol/l Adults Women <50yrs 13-40 2.6 – 6.7 Women >50yrs 21-43 3.5 – 7.2 Men <50yrs 19-45 3.2 – 7.3 Men >50yrs 18-55 3.0 – 9.2 Children 1 –3 yrs 11- 36 1.8 – 6.0 4 –13 yrs 15- 36 2.5 – 6.0 14 –19 yrs 18- 45 2.9 – 7.5
- 45. An abnormal urealevel may indicate the following tabulatedconditions Type Cause Note High urea (High urea concentrationin plasma is called (azotemia) Pre-renal Congestive heart failure •Dehydration •High protein diet •Increased protein catabolism Caused by reduced renal blood flow, less blood is delivered to kidney, then less urea is filtered. Renal Renal failure Post-renal Urinary tract obstruction. Low urea Low protein intake (Starvation, anorexia) •Liver disease •Pregnancy
- 46. BUN (Blood ureanitrogen): • It is the measure of amount of urea nitrogen in blood. • BUN is an indicator of renal function. • The normal range is 6-20mg/dL. Causes High urea in urine • too much protein in thediet • too much protein breakdown in the body Low urea in urine • malnutrition • too little protein in thediet • kidney issues
- 47. Clinical Application: Measurement ofurea is used to : • Evaluate renalfunction • Assess hydration status • Determine nitrogenbalance • Aid inthe diagnosis of renal diseases • Verify adequacy of dialysis • Check a person's protein balance
- 48. CLINICAL SIGNIFICANCE: • Theblood urea concentration in normal individual is 15-40mg%. • While urea concentration increases in the blood with the loss of renal function, it is neither a specific nor sensitive indicator of renal impairment. • Serum urea is also dependent on nonrenal factors, such as protein intake, hydration state, and renal perfusion. • Severe diarrhea, vomiting, and excessive fluid loss decreases rate of glomerular filtration, thereby increasing blood urealevels.
- 49. • Lower urinarytract obstruction and pathology resulting in decreased glomerular filtration also leads to elevated blood urea levels. • In renal pathology like chronic acute glomerulonephritis, nephrosis, malignant hypertension, chronic pyelonephritis and damage to the kidney tissues due to mercury poisoning or calcium deposition due to hyperthyroidism and hypervitaminosis (vit-D), blood urea levels are higher than normal. • Postrenal conditions like enlargement of prostate gland, stones in the urinary tract, or tumor of the bladder also leads to increased urealevels.