Chemical Analysis Lecture Week #9

CHEMICAL ANALYSIS Lect week 9 Urine chemical parameters (determined with dipsticks) Protein abnormalities in the chem. characteristics of urine: glucosuria, proteinuria, ketonuria, bilirubinuria, etc Glucose Ketones Bilirubin Occult blood – detects erythrocytes, hemoglobin and myoglobin Urobilinogen pH – acid, neutral, alkaline Nitrite Leukocytes Dry Reagent Chemistry U ses reagent strips or “dip sticks” Chemicals + color indicator are impregnated into each test pad The color reaction reported as negative (-), trace, 1+, 2+, 3+ or 4+, mg/dL, or in mmol/L Dipstick Analysis C olors on the strip are compared to manufacturer’s chart. Excess urine is tapped off. Do not allow mixing of reagents between pads. D o not touch test pads! ** follow manufacturer’s directions regarding WHEN colors should be read . PROTEIN Most of plasma protein filtered by glomerulus is reabsorbed by renal tubules. U rine normally contains a very small amount of protein not detectable by routine screening methods. Small amounts may be in voided urine or samples from bladder expression (protein = from secretions). Reagent test strips yield a 1+ to 4+ range (0 – 2000 mg/dL) S trips most sensitive for albumin and not reliable for detection of globulins False-positive results can occur highly concentrated alkaline urine technical error (urine left on test strip too long before reading) Protein in dilute urine is more significant than that in concentrated urine. Proteinuria occurs with: Kidney disease (glomerular and tubular disease) Hemorrhage into the urinary tract Urinary tract inflammation Shock Urine protein-to-creatinine ratio (UPC) Can be done at external lab In-clinic tests also available P art of monitoring cats & dogs with chronic kidney disease (International Renal Interest Society; IRIS) Should NOT be done when urinary tract is inflamed (increased protein negates validity of test) GLUCOSE Glucose filtered by glomeruli and reabsorption completed by proximal tubules when serum concentrations are < 10 mmol/L (dog), < 15 mmol/L (cat), and < 5.5 mmol/L (cow). Glucosuria occurs above these renal threshold values. Glucose is not found in urine normally Reagent strips use glucose oxidase test (specific for glucose). May be inhibited by ascorbic acid (vitamin C) and low temp (sample from fridge). Ensure that test strips have been stored properly and are not expired (false negative results). H yperglycemia / glucosuria may be caused by Stress or excitement IV glucose Drugs (corticosteroids, ketamine) Postprandial Persistent hyperglycemia / glucosuria may indicate – i.e diabetes mellitus, hyperadrenocorticism (Cushing’s syndrome) KETONES Produced during fatty acid metabolism (source of energy) F iltered by glomeruli & reabsorbed by proximal convoluted tubules Ketones include acetone, acetoacetic acid and beta-hydroxybutyric acids Excessive ketones can be toxic (CNS depression and acidosis = ketoacidosis) Standard reagent strip and tablets use nitroprusside reaction and are able to detect acetoacetate and acetone but not beta-hydroxybutyrate Beta-hydroxybutyrate is 1st ketone produced in ketosis Ketonemia results in ketonuria Milk Production – milk is tested directly for beta-hydroxybutyrate levels Ketometer available for blood ketones Ketonemia and Ketonuria occur in: Ketosis of cattle and pregnancy toxemia of ewes; hypoglycemia (high glucose demands) results in increased fat metabolism Diabetic ketoacidosis (uncontrolled diabetes mellitus leads to increased fat metabolism) Starvation and long-term anorexia (results in increased fat metabolism) BILLRUBIN Bilirubin results from the breakdown of heme and is a bile pigment Unconjugated bilirubin is bound to serum albumin and cannot pass into the glomerular filtrate and not found in urine Conjugated bilirubin passes into glomerular filtrate (urine) not reabsorbed by the renal tubules Tests include: Reagent strips – reliable in cats but may have false-positive or false-negative results in dogs Reagent tablets ( Ictotest ) – more sensitive Bilirubin is oxidized in light. E xcessive delay in urinalysis > false-negative result Dogs – tubular cells can conjugate bilirubin from absorbed hemoglobin = slight bilirubinuria normal Cats – any bilirubinuria is significant Bilirubinuria can be caused by obstruction of bile flow and regurgitation of conjugated bilirubin into blood stream or hemolytic anemia. UROBILINOGEN F ormed in the intestine by bacterial action on conjugated bilirubin. A bsorbed from the intestine and recycled through liver. S mall amount passes through glomerulus into urine. Not stable in urine , difficult to detect ( test pads not reliable ) BLOOD Reagent strip and tablets –do not distinguish between causes of blood in urine. M icroscopic eval, other tests (creatine kinase) , patient history and physical exam needed . Test pad for blood –not accurate differentiating hemoglobin, myoglobin and intact RBCs. Pink to red cloudy urine that usually clears on centrifugation RBCs in sediment No anemia or muscle disease Evidence of urinary or reproductive tract bleeding Red to brown urine that does not clear on centrifugation No RBCs in sediment Reddish discoloration of plasma No muscle disease Indicates intravascular hemolysis Brown urine that does not clear on centrifugation No RBCs in sediment No anemia Indicates muscle disease: confirm by determining serum concentration of creatine kinase (muscle enzyme) H ematuria : urine red and cloudy . C entrifugation > red cell pellet and supernatant normal. H emoglobinuria, supernatant remains red / brown after centrifugation and no erythrocyte pellet . Fresh plasma/serum that is red usually = hemoglobinemia > hemoglobinuria. Clear + normalcoloured plasma or serum = myoglobinuria. Confirm with e levated serum creatine kinase (muscle enzyme) levels + history muscle exertion PH pH is used to assess acid-base balance along with other parameters Acidic urine = pH < 7.0 Alkaline urine = pH > 7.0 pH is an expression of the hydrogen ion concentration Fresh urine required Urine becomes alkaline on standing because of loss of CO2 and/or conversion of urea to ammonia if urease-producing bacteria are in the urine Test methods : Reagent strips pH range is 5.5 – 8.5 Diet affects pH High-protein diets : Lower pH (6 – 7) (meat and milk in carnivores and nursing animals) Herbivores Higher pH (7 - 8.5) Bacterial cystitis affect on urine pH : Urease-producing bacteria and urine retention usually produce an alkaline urine pH because of bacterial degradation of urea to ammonia Urine pH affects the types of crystals/uroliths that may form in urine NITRITES Nitrite (conversion of nitrate in urine) not a reliable test in animals : false-negative results Instead send urine for bacteriological exam and culture & sensitivity or examine sediment for presence of bacteria (often see phagocytized bacteria in air-dried stained smear of sediment) LEUKOCYTES Leukocyte test pad (not reliable) detects leukocyte esterase (in WBCs except lymphocytes) False-positive results in cats commonly False-negative results in dogs commonly Evaluate fresh urine for presence of leukocytes: How would this be done and what type of cells would we be looking for? Test pads that are unreliable • Nitrite • Leukocytes • Urobilinogen • Specific gravity