1. Special Tests Chemistry
Glucose Bilirubin Ketones

2. 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

3. 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.

4. 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

5. 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

6. 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

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

8. 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

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

10. 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%

11. 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

12. 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)

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

14. 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

15. 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

16. Ketones Metabolism

17. 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

18. 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

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

20. 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

21. 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)

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

23. 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

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

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

26. 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

27. 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

28. 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

29. 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

30. 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)

31. 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

32. 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