Special Tests Chemistry

Glucose Bilirubin Ketones

NegativeNegative

Trace (100 mg/dL)Trace (100 mg/dL)

+ (250 mg/dL)+ (250 mg/dL)

++ (500 mg/dL)++ (500 mg/dL)

+++ (1000 mg/dL)+++ (1000 mg/dL)

++++ (2000+ mg/dL)++++ (2000+ mg/dL)

Glucose Dipstick:

Glucose + 2 H2O + O2 --->Gluconic Acid + 2 H2O2

Glucose Oxidase

3 H2O2 + KI ---> KIO3 + 3 H2O

Horseradish Peroxidase

Chemical Principle

Read at 30 secondsRR: Negative

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.

Uses and Limitations of Urine Glucose Detection

• 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

• 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

Glucose Clinical Significance

• 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

Nondiabetic Glycosuria

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

pregnancy

Renal Glycosuria

• 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

GlucoseReagent Strip Reactions

Glucose oxidaseGlucose + O2 (air) → gluconic acid + H2O2

PeroxidaseH2O2 + chromogen → oxidized colored

chromogen + H2O

Glucose Reagent Strip Reactions

• Chromogens used– 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%

GlucoseReagent Strip

• 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

GlucoseReaction Interference

• 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)

Glucose and other Reducing SugarsCopper Reduction Test (Clinitest)

HeatCuSO4 (cupric sulfide) + reducing substance -----

Alkali Cu2O (cuprous oxide) + oxidized substance → color

(blue/green to orange/red)

Copper Reduction Test

• 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

Clinitest Procedure

• 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

Reducing Substances

Ketones Metabolism

• 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

• 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

KetonesClinical Significance

• Ketonuria unrelated to diabetes– Inadequate intake/absorption of carbohydrates– Vomiting– Weight loss– Eating disorders– Frequent strenuous exercise

KetonesClinical Significance (cont’d)

• Primary reagent: sodium nitroprusside– (Nitroferricyanide)

• Measure primarily acetoacetic acid– Assumes the presence of β-hydroxybutyrate and

acetone• Acetoacetic acid (alkaline) + nitroprusside →

purple color

KetonesReagent Strip Reactions

KetonesReagent 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)

acetoacetate (and acetone) + sodium nitroprusside Alkaline

+ (glycine) ——————> purple color

Ketones Reagent Strip Reactions

• 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

KetonesReaction Interference

• Not a urine confirmatory test• Tablet = sodium nitroprusside, glycine, disodium

phosphate, lactose (gives better color)

Acetest

NegativeNegative

+ (weak)+ (weak)

++ (moderate)++ (moderate)

+++ (strong)+++ (strong)

Bilirubin Dipstick:

Bilirubin + Diazo salt ---------> AzobilirubinAcidic

Chemical Principle

Read at 30 secondsRR: Negative

• 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

• 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 (cont’d)

• 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 Clinical Significance

• 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

BilirubinReagent Strip Reactions

acidbilirubin glucuronide + *diazonium salt-------- azodye

(tan or pink to violet)

* diazonium salt- (2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate)

Bilirubin Reagent Strip Reactions

• 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 Reaction Interference

• 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

Bilirubin Ictotest