Special Tests Chemistry Glucose Bilirubin Ketones

Horseradish Peroxidase Glucose Dipstick: Chemical Principle Negative Trace (100 mg/dL) + (250 mg/dL) ++ (500 mg/dL) +++ (1000 mg/dL) ++++ (2000+ mg/dL) Glucose Oxidase Glucose + 2 H2O + O2 ---> Gluconic Acid + 2 H2O2 Horseradish Peroxidase 3 H2O2 + KI ---> KIO3 + 3 H2O Read at 30 seconds RR: Negative

Significance Limitations Other Tests Clinitest Uses and Limitations of Urine Glucose Detection Significance Diabetes mellitus. Renal glycosuria. Limitations False Neg: reducing agents, ketones. False Pos: oxidizing detergents Only measures glucose and not other sugars (Glucose Oxidase) Renal threshold must be passed in order for glucose to spill into the urine. Other Tests Clinitest CuSO4 test for reducing sugars.

Glucose Clinical significance Major screening test for diabetes mellitus Renal threshold is 160 to 180 mg/dL Higher blood sugar = glycosuria Gestational diabetes Placental hormones block action of insulin High fetal glucose stresses baby’s pancreas Result is fat baby Mother prone to type 2 diabetes

Glucose Clinical Significance Elevated blood glucose, diabetes mellitus Renal threshold is ~160 to 180 mg/dL Higher blood sugar = glycosuria Collection under controlled conditions Fasting specimen “Second” collection 2 h postprandial

Nondiabetic Glycosuria Hormonal disorders: pancreatitis, pancreatic cancer, acromegaly, Cushing’s syndrome, hyperthyroidism, pheochromocytoma Hormones: glucagon, epinephrine, cortisol, thyroxine, growth hormone oppose glucose Insulin: converts glucose to storage glycogen Hormones: glycogen back to glucose Epinephrine: inhibits insulin; seen with stress, cerebral trauma, and myocardial infarction

Renal Glycosuria Tubular reabsorption disorder End-stage renal disease Cystinosis Fanconi syndrome Temporary lowering of renal threshold in pregnancy

Glucose Reagent Strip Reactions Glucose oxidase reaction specific for glucose Glucose oxidase, peroxide, chromogen, buffer on test pad Double sequential enzyme reaction Glucose oxidase catalyzes a reaction between glucose and oxygen Produces gluconic acid and peroxide Peroxidase catalyzes the reaction between peroxide and chromogen to form an oxidized colored compound Direct proportion to the concentration of glucose

Glucose Reagent Strip Reactions Glucose oxidase Glucose + O2 (air) → gluconic acid + H2O2 Peroxidase H2O2 + chromogen → oxidized colored chromogen + H2O

Glucose Reagent Strip Chromogens used Reporting results Potassium iodide (green to brown) (Multistix) Tetramethylbenzidine (yellow to green) (Chemstrip) Reporting results Neg, trace, 1+, 2+, 3+, 4+ 100 mg/dL to 2 g/dL 0.1% to 2%

Glucose Reaction Interference False-positive: only peroxide, oxidizing detergents False-negative: enzymatic reaction interference Ascorbic acid and strong reducing agents High levels of ketones (unlikely) High specific gravity and low temperature *Greatest source of error is old specimens Subjecting the glucose to bacterial degradation

Glucose and other Reducing Sugars Copper Reduction Test (Clinitest) Reduction of copper sulfate to cuprous oxide with alkali and heat Clinitest tablets: copper sulfate, sodium carbonate, sodium citrate, sodium hydroxide Sodium citrate + NaOH = heat Sodium carbonate = CO2 blocks room air Reducing substance + CuSO4 Color change: negative blue (CuSO4) through green, yellow, and orange/red (Cu2O)

Copper Reduction Test Heat Alkali CuSO4 (cupric sulfide) + reducing substance ----- Alkali   Cu2O (cuprous oxide) + oxidized substance → color (blue/green to orange/red)

Clinitest Procedure Pass through High levels of reducing substance Color from blue through red back to green-brown: rapid reaction Repeat with two-drop procedure 10 drops water 2 drops urine Values up to 5 g/L versus 2 g/L Separate chart must be used Hygroscopic tablets: strong blue color and excess fizzing = deterioration

Reducing Substances Not a specific test for glucose Sensitivity: 200 mg/dL (lower) than strip Clinitest does not provide a confirmatory test for glucose Interference from reducing sugars Galactose, lactose, fructose, maltose, pentoses, ascorbic acid, cephalosporins Major use is quick screen for “inborn error of metabolism” in children up to 2 years old Newborn screening programs for galactosemia in all states

Ketones Metabolism

Ketones Three intermediate products of fat metabolism Acetone: 2% Acetoacetic acid: 20% β-hydroxybutyrate: 78% Appear in urine when body stores of fat must be metabolized to supply energy

Ketones Clinical Significance Increased fat metabolism = inability to metabolize carbohydrate Primary causes Diabetes mellitus Vomiting (loss of carbohydrates) Starvation, malabsorption, dieting (↓ intake) Ketonuria shows insulin deficiency Monitor diabetes Diabetic ketoacidosis = increased accumulation of ketones in the blood Electrolyte imbalance, dehydration, and diabetic coma

Ketones Clinical Significance (cont’d) Ketonuria unrelated to diabetes Inadequate intake/absorption of carbohydrates Vomiting Weight loss Eating disorders Frequent strenuous exercise

Ketones Reagent Strip Reactions Primary reagent: sodium nitroprusside (Nitroferricyanide) Measure primarily acetoacetic acid Assumes the presence of β-hydroxybutyrate and acetone Acetoacetic acid (alkaline) + nitroprusside → purple color

Ketones Reagent Strip Reactions (cont’d) Report qualitatively Negative Trace Small (1+) Moderate (2+) Large (3+) Semiquantitatively Negative Trace (5 mg/dL) Small (15 mg/dL) Moderate (40 mg/dL) Large (80 to 160 mg/dL)

Ketones Reagent Strip Reactions acetoacetate (and acetone) + sodium nitroprusside Alkaline + (glycine) ——————> purple color

Ketones Reaction Interference Levodopa in large dosage Medications containing sulfhydryl groups May produce atypical color reactions False-positive results from improperly timed readings Falsely decreased values in improperly preserved specimens Breakdown of acetoacetic acid by bacteria

Acetest Not a urine confirmatory test Tablet = sodium nitroprusside, glycine, disodium phosphate, lactose (gives better color)

Bilirubin Dipstick: Negative Chemical Principle + (weak) Acidic ++ (moderate) +++ (strong) Chemical Principle Acidic Bilirubin + Diazo salt ---------> Azobilirubin Read at 30 seconds RR: Negative

Bilirubin Urine bilirubin early indicator of liver disease Normal degradation product of hemoglobin RBCs destroyed by liver and spleen following 120-day life span Body recycles iron, protein Protoporphyrin is broken down into bilirubin Bilirubin is bound to albumin Kidneys cannot excrete Unconjugated bilirubin: water insoluble

Bilirubin (cont’d) Conjugated bilirubin: water soluble Unconjugated bilirubin to the liver Conjugated with glucuronic acid Forms conjugated bilirubin From liver to intestines Reduced to urobilinogen, stercobilinogen, and urobilin by intestinal bacteria Excreted in feces

Bilirubin Clinical Significance Conjugated bilirubin appears in urine with bile duct obstruction, liver disease or damage Obstruction: bilirubin backs up into circulation and is excreted in urine No urobilinogen is formed Hepatitis, cirrhosis: conjugated bilirubin leaks back into circulation from damaged liver; some bilirubin passes to intestine Hemolytic disease: increased unconjugated bilirubin, increased urobilinogen Unconjugated Bilirubin not water soluble Not found in Urine

Bilirubin Reagent Strip Reactions Principle is a diazo reaction Report: neg, small (1+), moderate (2+), large (3+) Colors may be difficult to interpret Easily influenced by other pigments present in the urine Atypical colors can be problem for automated readers

Bilirubin Reagent Strip Reactions acid bilirubin glucuronide + *diazonium salt-------- azodye (tan or pink to violet) * diazonium salt- (2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate)

Bilirubin Reaction Interference False-positive Urine pigments Pyridium (phenazopyridine) Drugs indican, iodine False-negative Old specimens (biliverdin does not react) Ascorbic acid >25 mg/dL Nitrite Combine with diazonium salt and block bilirubin reaction

Bilirubin Ictotest Confirmatory for bilirubin Tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric acid Use specified mat for test; mat keeps bilirubin on surface for reaction Positive reaction = blue-to-purple color Interfering substances are washed into the mat, and only bilirubin remains on the surface