Del Campo, Ikn Dzaia Erika A.

Biochemistry Lab Finals

BSMLS 2C Reviewer No. 3

Experiment 7: Metabolism and Urinalysis

Specimen Collection

- First morning urine (most concentrated)

- Record collection time
- Type of specimen (should be clean catch)
- Analyzed urine within 2 hours of collection
- Must be free of debris or vaginal secretion
 Cannot collect sample during menstration
 Wash first the genital
 Must be less squamous epithelial cell
 Normal to secrete sperm cell (after sex, wet dreams)

Types of Analysis

1. Physical Examination
2. Chemical Analysis (Urine Dipstick)
3. Microscopic Examination (Automated Urinalysis)
4. Culture
5. Cytological Examination (Histopath, Cancer)

Physical Examination

1. Color
 Colorless - diluted urine
 Deep yellow - concentrated urine or riboflavin
 Yellow green - bilirubin, biliverdin
 Red - blood or hemoglobin
 Brownish-red - Acidified blood (Acute GN – glomerulonephritis)
 Brownish-black - homogentisic acid (Melanin)

2. Odor
 Ammonia-like - urea-splitting bacteria
 Foul, offensive - old specimen, pus or inflammation
 Sweet - glucose
 Fruity - ketones
 Maple syrup-like - maple syrup urine disease

Microscopic Examination

1. Odor
2. Turbidity (transparency or clarity)
 Typically cells or crystals (normal or abnormal)
 Cellular elements and bacteria will clear by centrifugation
 Crystals dissolved by a variety of methods (acid or base)
 Microscopic examination will determine which particle is present
F. BLOOD: GUAIAC TEST (OCCULT BLOOD TEST)

1. Add 1 mL of guaiac solution

2. Add 1 mL of “old oil of turpentine”
3. Add 0.5 mL of random, very young, geriatric and diabetic urine in 4 tubes.

 A blue-green ring at the interface

 Followed by a dark blue ring will appear in the point of contact of the two solutions
 Mix and will appear to blue if present

Why blood is not normally detected blood in urine?

In most cases, blood in the urine (called hematuria) is the first sign of bladder cancer. ... Blood in
the urine doesn't always mean you have bladder cancer. More often it's caused by other things like an
infection, benign (not cancer) tumors, stones in the kidney or bladder, or other benign kidney diseases.

Guaiac Test

 Also known as FOBT (Fecal Occult Blood Test)

a. Test: to detect hidden blood in the urine

b. Principle: Test is based on the oxidation of phenolic compounds present in the guaiac to guinones
reducing in production of the blue color.
When a fecal specimen containing occult blood is applied to the test paper, contact is
made between the hemoglobin and guaiac

c. Indicator: Blue color

 Fresh blood Red Color – problem in colon or hemorrhoids

 Black Color – problem with the stomach, black meaning added with enzymes or acids or viand
 Green Color – ice cream and ube
 Stool cover must not be tight and covered in a long time, might explode stool emits methane gas

G. ELECTROLYTES

a. Sodium ion: Flame Test

1. Dip a cleaned flame test wire in into each urine

 Wire must be cleaned with 3M HCL
2. Place wire to the flame
3. Record the color

b. Chloride ion: Silver nitrate test

1. Place 3 mL of urine (random, very young, geriatric and diabetic) in a tube

2. Add 5 drops of 3M HNO3
3. Add 5 drops of 0.1M AgNO3, Mix
4. Record color of precipitate

H. DIPSTICK TEST

1. Dip the strip in the 4 urine samples

2. Record and compare the result
 Reagent Strip

 10 parameter
 4 parameter (glucose, pH, specific gravity and protein

10 Parameter

10 Parameter Reaction

1. Glucose Double Sequential Enzyme Reaction

2. Bilirubin Diazo Reaction

3. Ketones Sodium nitroprusside reaction

4. Specific Gravity Change in dissociation constant of
polyelectrolyte
5. Blood Pseudoperoxidase
6. pH Double indicator reaction
 Methyl red
 Bromothymol blue
7. Protein Protein errors of indicators
8. Urobilinogen Erlich reaction (Multitics), Diazo Reaction
(Chem strip)
9. Nitrite Greiss Reaction

10. Leukocyte esterase Diazo Reaction

1) Glucose
a. Principle: Double Sequential Enzyme Reaction
 Uses 2 enzyme (Glucose oxidase and Peroxide)
 Only test which reacts to glucose that not in the other reducing sugars

b. Significance: Diabetes mellitus, renal glycosuria

c. Color positive and negative: P = Green, N = Blue

Renal Threshold
 Blood level of the tubular reabsorption stops
 160 – 180 mg/dL = blood glucose level reuse by the blood
 Above 180 mg/dL = glucose will excrete to the urine

2) Bilirubin
a. Principle: Diazo Reaction
b. Significance: Hemolysis and Liver damge
c. Color positive and negative: P = Orange, N = Yellow

 Unconjugated or Indirect bilirubin (B1)

 Conjugated or Direct bilirubin (B2)
 Increase B1 = Increase Urobilinogen

3) Ketones
a. Principle: Sodium nitroprusside reaction
b. Significance: Diabetic ketoacidosis, prolonged fasting
c. Color positive and negative: P = Violet, N = Yellow

 Common in diabetes mellitus (type 1) – lacks of insulin production

4) Specific gravity
a. Principle: Change in dissociation constant of polyelectrolyte
b. Significance: Diabeties
c. Color positive and negative: SG High or Low

 High SG, more concentrated

 Polyelectrolyte will be ionized release hydrogen ions in proportion to the number of
ions of urine
 More ions = more hydrogen ions in the urine
 Higher pH = more hydrogen ions
 Yellow urine = more acidic

5) Blood
a. Principle: Pseudoperoxidase
b. Significance:
 Hematuria (Nephritis) - with dots specimen
 Hemoglobinuria (Hemolysis) - without dots
 Myoglobinuria (Rhabdomyolysis)

c. Color positive and negative: P = Green, N = Yellow

6) pH
a. Principle: Double indicator reaction
1. Methyl red (pH 4-6) - Red to yellow
2. Bromothymol blue (pH 6-9) – Yellow to blue
b. Significance:
 Acidic (less than 4.5)
 Metabolic acidosis
 High-protein diet

 Alkaline (greater than 8)

 Renal tubular acidosis (75.7)

c. Color positive and negative:

7) Protein
a. Principle: Protein errors of indicators
 A change of color due to the presence of protein
b. Significance: Proteinuria (Pre-renal, renal, Post-renal), Nephrotic syndrome
c. Color positive and negative:

 Indicator (pH)
 Change in pH meaning it accept hydrogen ions
 More protein = acidic
 Less protein = basic
 Albumin – more amino acid and it is acidic

8) Urobilinogen
a. Principle: Erlich reaction (Multistix), Diazo Reaction (Chemstrip)
b. Significance:
 High
 Increase hepatic processing of bilirubin
 Low
 Bile obstruction

Urine Bilirubin Urine Urobilinogen

(Conjugated)
 1. Bile duct Increase Normal
obstruction
2. Liver Positive or negative Increase
damaged  Depends
what part of
the liver is
affected

3. Hemolytic Increase Normal

disease  No changes
since liver is
okay

9) Nitrite
a. Principle: Greiss Reaction
 Presence of bacteria
 Bacteria can reduce nitrate to nitrite
b. Significance: Gram negative bacteriuria
 If exposed too long in room temperature, glucose can be false positive. It will be
eaten by the bacteria as source of energy

10) Leukocyte esterase

a. Principle: Diazo Reaction
 Detect leukocyte – 5 types (neutrophils, basophils, eosinophils, monocytes, and
lymphocytes)
b. Significance: Pyuria, Acute inflammation, Renal calculus