URINE METABOLIC
SCREENING TEST
Laboratory Conference
-TLSS-
THETA LAMBDA SIGMA SIGMA
THE LAST SUB-SECT
Inborn errors of metabolism
• Disruption of enzymatic function in metabolic
pathway
– Enzyme deficiency
– Inability of a reaction to occur at a rate sufficient
to metabolism of a metabolite
– Metabolite accumulates
– Reaction products fail to form
Inborn errors of metabolism
• Defect in transport systems
– Defects, absence of enzymes or “carrier protein”
situated in membranes causing metabolic blocks.
Various clinical manifestations of these metabolic
consequences are the following:
• Mental retardation
• Developmental delays
• Seizures
• Metabolic acidosis (unexplained)
• Jaundice
• Vomiting
• Hepatomegaly
• Abnormal body odors
• Abnormal facial and body
features
• Death
• Urine – most often a good specimen
of choice for the detection of most
metabolic disturbances.
• Routine Urinalysis
• Chemical Tests
– Ferric chloride test
– Benedict’s test
– Nitrosonaphthol test
– Nitroprusside test
– Cetyltrimethylammonium bromide test
– Ninhydrin test
– Millon’s test
The aim of this laboratory session is to
introduce ourselves to some frequently done
screening tests for inborn error of metabolism
Urinalysis
• Urinalysis can reveal diseases that have gone
unnoticed because they do not produce
striking signs or symptoms.
• The most cost-effective device used to screen
urine is a paper or plastic dipstick.
Methodology
A sample of well-mixed urine is centrifuged
in a test tube at relatively low speed for 5
minutes
The supernatant is used for the dipstick
method and decanted.
The sediment is resuspended in the
remaining supernate by flicking the bottom
of the tube several times. A drop of
resuspended sediment is poured onto a
glass slide and coverslipped.
MACROSCOPIC URINALYSIS
• Normal, fresh urine is pale to dark yellow or
amber in color and clear.
• Turbidity or cloudiness may be caused by
excessive cellular material or protein in the
urine or may develop from crystallization or
precipitation of salts upon standing at room
temperature or in the refrigerator.
URINE DIPSTICK CHEMICAL
ANALYSIS
pH
• Normal value - no normal values, must
consider with other patient information.
• Dipstick range is at 4.5 - 9.0
• Dipstick reaction based on acid -base
indicators (methyl red and bromthymol blue)
Specific Gravity (sp gr)
• Specific gravity (which is directly proportional
to urine osmolality which measures solute
concentration) measures urine density, or the
ability of the kidney to concentrate or dilute
the urine over that of plasma.
• Specific gravity between 1.002 and 1.035 on a
random sample should be considered normal
if kidney function is normal.
Glucose
• Dipsticks employing the glucose oxidase
reaction for screening are specific for glucose
but can miss other reducing sugars such as
galactose and fructose. For this reason, most
newborn and infant urines are routinely
screened for reducing sugars by methods
other than glucose oxidase (such as the
Clinitest, a modified Benedict's copper
reduction test).
• Glucose filtered out by glomerulus
• Reabsorbed in proximal tubule by active
transport
• Normal concentration of glucose in blood:
• 1) Fasting – 60-110 mg/dl
• 2) After meal – 120-160 mg/dl
• Maximal reabsorptive capacity – 160 - 180
mg/dl
Renal threshold substances
• are those substances which are almost
completely reabsorbed by renal tubular cells
when their concentration in the plasma is
within normal limits, but are no longer totally
reabsorbed when plasma limits are exceeded.
They will then appear in the urine.
Interfering substances for glucose
• 1. Clinitest – ascorbic acid and drug
metabolites may give false positive.
• 2. Clinistix – ascorbic acid gives false negative;
bleach or peroxide may give false positive.
Protein
• Of all the chemistry tests, urinary protein is
the most indicative of renal disease.
• Only small amounts filtered through
glomerulus (low molecular weight proteins)
• Most reabsorbed through tubules
• Dipsticks detect protein by production of color
with an indicator dye, Bromphenol blue,
which is most sensitive to albumin but detects
globulins and Bence-Jones protein poorly.
• Precipitation by heat is a better
semiquantitative method, but overall, it is not
a highly sensitive test. The sulfosalicylic acid
test is a more sensitive precipitation test. It
can detect albumin, globulins, and Bence-
Jones protein at low concentrations.
Ketone Bodies
• Products of fat catabolism
• Three forms:
1) Acetone
2) Diacetic Acid (Acetoacetic) - both acetone and
beta hydrosybutyric acid are produced from
diacetic acid.
3) Beta hydroxybutyric Acid = majority formed
• Provides clue to early diagnosis of ketoacidosis
and diabetic coma.
• Frequent occurrence in juvenile diabetic.
• Pregnant diabetic – fetal death due to
ketoacidosis
Nitrite
• A positive nitrite test indicates that bacteria
may be present in significant numbers in
urine. Gram negative rods such as E. coli are
more likely to give a positive test.
Leukocyte Esterase
• A positive leukocyte esterase test results from
the presence of white blood cells either as
whole cells or as lysed cells.
Blood
• Indicative of glomerular bleeding.
• Significant if urine is bloody.
Bilirubin and Urobilinogen
• presence in urine may be the 1st indication of
liver disease
Urobilinogen
• Urobilinogen is a colourless product of
bilirubin reduction.
• It is formed in the intestines by bacterial
action.
• Some urobilinogen is reabsorbed, taken up by
the hepatocytes into the circulation and
excreted by the kidney.
• Urobilinogen is converted to the yellow
pigmented urobilin apparent in urine.
• The urobilinogen remaining in the intestine
(stercobilinogen) is oxidized to brown
stercobilin which gives the feces their
characteristic color.
Results
• V/A: JC’s urine
• RBC = 0-1 / lpf
• WBC = 0-1 / lpf
• Epithelial cells = few
• Mucus Threads = few
Results of physical properties
• Color: Light Yellow
• Odor: Odorless
• pH=6.5
• Transparency: Light Yellow
• Specific gravity = 1.010
• The rest of the dipstick chemical test =
negative
Screening Tests for Certain Inborn
Errors of Metabolism
FERRIC CHLORIDE TEST
• Test for phenols, enols and a number of other
compounds with moderately acidic OH groups
• They react with ferric chloride to give colored
complexes
• Depending on the structure of the phenol, the color
can vary from green to purple.
• This color change can be used to differentiate
alcohols from phenols
• There are compounds that are believed to be
phenols but which give (-) tests with aqueous ferric
chloride
FERRIC CHLORIDE TEST
• 3ArOH + FeCl3 —> Fe(OAr)3 + 3HCl
OH OFeCl 2
+ FeCl 3 + HCl
Ferric Chloride Test
Procedure
• Place 1 ml of Ferric Chloride reagent
in a clean test tube
• Add 10 drops (0.5 ml) urine.
• Mix well by shaking and observe
result.
Results
Control (sample urine) Dark yellow (-)
Experimental (with disease) Dark yellow (-)
Clinical Conditions and substances giving a positive
FECl3 Test.
Condition FECl3 Test
(Drug) Result
Acetoacetic acid Red-brown Melanin Gray precipitate to
Alkaptonuria Blue-green- black
(homogentensic transient Methionine Purple to red
acid) malabsorption brown
P-aminosalicylic Purple-brown Phenothiazines Purple brown
acid Phenylketonuria Blue-green
Bilirubin Blue-green Pyruvic acid Deep yellow
Histidinemia Blue-gray to Salicylates Purple
green
Tyrosinemia Green, fades
Lactic acidosis Gray rapidly
Maple syrup Green to gray Xanthurenic acid Dark green to
urine disease brown
Benedict’s Solution Experiment
-Introduction
-Materials and Procedure
-Results
Benedicts Reagent
• A blue colored solution used to test for the presence of
a reducing sugar such as glucose, fructose,
glyceraldehyde, lactose etc. (note that sucrose is not a
reducing sugar)
• It is prepared from NaHCO3, Na Citrate and CuSO4
(Cupric)
• The carbonyl (aldehyde or ketone) group will reduce
and change Cu (II)’s electron configuration to Cu (I) as
manifested by the brick red color change
• Common, easy access test for gestational diabetes and
other forms of excess circulating blood sugar
Materials and Procedure
• Comparison of pathologic and sample urine
for glucose
- 2 Test tubes (one for the sample and the
pathologic) filled with 5 ml of Benedict’s
solution, bring to a boil
- Add 8 drops in the test tube (one for each
source) and bring to a boil again for 2 minutes
at least
Results
• Sample: (+)- distinct blue green
upper portion
• Pathologic: (+++)- murky brown
to orange upper portion
• Expected: blue (sugar should not
be excreted by the kidney’s; it is
100% reabsorbed)
Nitrosonaphthol Test
PROCEDURE
Nitrosonaphthol Test
• Associated to disorders with a marked
alternation in tyrosine metabolism
• Gives a positive result (orange red solution)
in the presence of tyrosine or its metabolites,
including p-hydroxyphenylpyruvic acid, p-
hydroxyphenyllactic acid, and p-
hydroxyphenylacetic acid
• Tyrosine and similar para-substituted
phenols react with 1-nitroso-2-naphthol only in the
presence of nitric acid.
• Nitrous acid greatly affects the rate of the reaction
and produces orange-red solutions
Disorders to be considered on positive
result:
• Tyrosinosis
– an inherited disorder of tyrosine metabolism in
which an intermediate product,
parahydroxyphenyl pyruvic acid, appears in the
urine and gives it an abnormal reducing power
– characterized by enhanced urinary excretion of
certain metabolites upon ingestion of tyrosine
Tyrosinemia
• an error of metabolism, usually inborn, in
which the body can not effectively break
down the amino acid tyrosine
• Types
– Type I is the most severe form of this disorder
and is caused by a shortage of the enzyme
fumarylacetoacetate hydrolase, encoded by the
gene FAH found on chromosome number 15
– Type II is caused by a deficiency of the enzyme
tyrosine aminotransferase, encoded by the gene
TAT
– Type III is a rare disorder caused by a deficiency
of the enzyme 4-hydroxyphenylpyruvate
dioxygenase, encoded by the gene HPD
• Fructosemia
• Galactosemia
• Severe liver disease
Procedure
acid (HNO3)
• 1 mL of 2.63 N nitric
• 1 drop sodium nitrite (NaNO3)
• 0.10 mL nitrosonaphthol
• 0.15 mL urine
Positive result = orange-red color within 25 min
Results
• Normal: same color
as reagent , yellow
• Pathologic: orange
red solution
Nitroprusside Test
Nitroprusside Test
• Cyanide Nitroprusside Test
- Screening for genetic metabolic disorder
like cystinuria, homocystinuria and beta-
mercaptolactate cysteine disuliduria.
Nitroprusside Test
• Definition of Terms:
1. Cystinuria- an inborn error of amino acid transport that results in the
defective absorption by the kidneys of the amino acid called cystine.
2. Cystine- are organic compounds needed by the body to make proteins
and for many normal functions. When the kidneys don't absorb cystine,
this compound builds up in the urine. When the amount of cystine in the
urine exceeds its solubility, crystals form.
3. Homocysteinuria- hereditary disease characterized by a deficiency of
the enzyme serine dehydratase causing incompletely dislocated lenses
after the age of 10, thromboembolisms, and usually mental retardation.
4. beta-mercaptolactate cysteine disulfiduria -
Nitroprusside Test
• Procedure
1.) 5ml of urine on a tube then add 5gtts. of
conc.ammonium hydroxide
2.) Add 2ml of freshly prepared 5% Sodium
cyanide and mix well. Stand for 10mins.
3.) Add 4 gtts. Of freshly prepared sodium
nitroprusside and mix. Observe color change.
Nitroprusside Test
• Results:
+ - Pink
++ - Pinkish Pink
+++ - Purple
++++ - Dark Purple
Nitroprusside Test
Normal: yellow (-)
Pathologic:
dark purple (++++)
Nitroprusside Test
• False Positive: • False Negative:
penicillin bacterial contamination
captopril
penicillamine
mercaptopropionylglycine
acetoacetate
Cetyltrimethylammonium
Bromide Test (CAB)
What is CAB test?
• Turbidometric technique
• Cetyltriammonium bromide (CAB) is
quaternary ammonium compound
• Determine urinary mucopolysaccharides
(MPS) and glycosaminoglycans (GAG’s)
MUCOPOLYSACCHARIDE
• Also known as glycosaminoglycans
• Group of large compounds located
primarily in the connective tissue
• protein core with numerous
polysaccharide branches
Polysaccharides are relatively complex
carbohydrates
MUCOPOLYSACCHARIDE
disorders
• Inherited disorders in the metabolism,
prevent complete breakdown of the
polysaccharide portion of these
compounds
• Accumulate in the lysosomes of
connective tissue cells and in excreted in
the urine
Hurler's syndrome (gargoylism)
Type I H mucopolysaccharidosis
• deficiency of alpha-L iduronidase
• marked by, hepatosplenomegaly,
dwarfism and gargoyle-like faces
• excess mucopolysaccharides are
excreted in the urine
Scheie's syndrome
Type I S mucopolysaccharidosis
• alpha-L iduronidase defect
• high levels of chondroitin sulfate
B in the urine
• progressive corneal clouding,
coarsening of the facies, general
dysplasia of the skeleton
• Unique: normal or near normal
stature, little or no mental
retardation, and a normal life
span.
Morquio's syndrome
type IV mucopolysaccharidosis
• presence of keratan sulfate in the
urine
• short stature due to severe
deformity of the spine and the
thorax, long bones with irregular
epiphyses, enlarged joints, flaccid
ligaments, and a waddling gait
• Type A: deficiency of the enzyme
N-acetylgalactosamine-6-sulfate
sulfatase.
Type B: deficiency of the enzyme
beta-galactosidase
Hunter’s syndrome
Type II mucopolysaccharidosis
• Lysosomal storage disease
• deficient (or absent) enzyme,
iduronate-2-sulfatase (I2S)
• glycosaminoglycan
deposition
• growth delay, joint stiffness,
and coarsening of facial
features
Procedure of CAB test
sample pathologic
Add 1ml CAB reagent
5ml of urine
(room temp.)
Result ?
Scale 0-4
30mins
Results
• Normal: clear
• Pathologic: turbid grade 2
Cetyltrimethylammonium Bromide
Test
• Results and discussions
Mucopolysaccharide
• Glycosaminoglycans
• Long chain sugar molecules
Mucopolysaccharidoses
• Inability to breakdown mucopolysaccharides
• Lack or deficiency of enzyme
• Inherited
• Lack of gene
Hurler’s Syndrome
• Lysosomal alpha-L-iduronidase
• Most severe
Scheie’s Syndrome
• Lysosomal alpha-L-iduronidase
• Mildest
• Autosomal recessive
Morquio’s Syndrome
• Type A - galactosamine-6-sulfatse
• Type B – beta-galactosidase
• Autosomal recessive
Hunter’s Syndrome
• Iduronate sulfatase
• X-linked
Consequences
• Degeneration of organs
• Mental retardation
• Death
Cetyltrimethylammonium Bromide
• Quaternary ammonium compound
• Insoluble complex
• Non-specific
Confirmation
• Genetic testing
Ninhydrin Test
• Used to detect urinary amino acids
• Used for detection and quantization of amino
acids in chromatographic procedures
• Urine ammonia and antibiotics react with
ninhydrin
Ninhydrin is strong oxidizing
agent and causes the
oxidative deamination of
the α-amino function
• The products of the reaction
are the resulting aldehyde,
ammonia, carbon dioxide,
and hydrindantin, a reduced
derivative of ninhydrin.
• The ammonia produced can
react with the hydrindantin and
another molecule of ninhydrin
to yield a purple product
(Ruhemann’s Purple)
Place 1ml of ninhydrin reagent in a test
tube
Add 3 drops of urine
Warm for about 30 seconds in water
bath
Observe color
• Violet = amino acids Yellow = Proline
Millon’s Test
• Procedure:
– 1ml of urine and 1 drop of
Millon’s Reagent was placed
in a test tube.
– The test tube was then
heated.
• Result:
– A peach or orange color
indicates a positive test.
Millon’s Test
• A test specific for tyrosine.
• The phenol group of tyrosine is first nitrated by nitric
acid in the test solution.
• Then the nitrated tyrosine complexes mercury(I) and
mercury (II) ions in the solution to form either a red
precipitate or a red solution which are both positive
test.
Millon’s Test
• Some proteins containing tyrosine initially
forms white precipitate that turns red when
heated.
Paper Chromatography of Amino Acids
• Paper chromatography is an analytical technique for
separating and identifying mixtures that are or can be
colored, especially pigments. This can also be used in
secondary or primary schools in ink experiments. This method
has been largely replaced by thin layer chromatography,
however it is still a powerful teaching tool. Two-way paper
chromatography, also called two-dimensional
chromatography, involves using two solvents and rotating the
paper 90° in between. This is useful for separating complex
mixtures of similar compounds, for example, amino acids.
Paper Chromatography of Amino Acids
• After development, the
spots corresponding to
different compounds may
be located by their color,
ultraviolet light, ninhydrin
(Triketohydrindane hydrate)
or by treatment with iodine
vapors. The final
chromatogram can be
compared with other
known mixture
chromatograms to identify
sample mixes using the Rf
value in an experiment.
Calculation of Rf values
Measure the distance from the start line to the
solvent front and to the front of each spot.
For each spot, calculate the Rf value (Rf means
relative to front):
distance moved by spot
distance moved by solvent front
METHODOLOGY
1. Using a pencil, draw a line approximately 2 centimeters
from each of the edges of the filter paper. Randomly
assign one side as the baseline.
2. Divide the baseline to accommodate six spots and label
each spot with the corresponding amino acid standards
and unknown.
3. Spot each sample (amino acid and unknown) for at least
6-7 times. Allowing the spot to dry before repeating the
process
4. When the spots have dried, roll the paper with spotted
side facing out. Staple the ends and place the paper in a
chromatographic jar
METHODOLOGY
5. Allow the solvent to run through the paper until it has
reached the upper margin.
6. Remove the chromatogram from the jar and air dry.
7. Spray ninhydrin solution and place on top of a hot plate
until spots are visible.
8. Trace the outlines of the colored spots and measure the
distance traveled by each amino acid.
9. Compute for the Rf values and compare it to the unknown
sample.
Results and Discussion
Paper Chromatography
• Chromatography is a technique used to
separate mixtures of substances into their
individual components.
• Principle is based on the affinity of the
components to the stationary phase of the
system
• Paper Chromatography
– Stationary phase is a Whatman filter paper.
– Mobile phase is an 8:1:1 mixture of Ethanol:
NH4OH: H2O
Paper Chromatography
• Separation is achieved based on the solubility of
the components in the mobile phase.
• More soluble- migrates faster
- farther from baseline ( Rf)
Less soluble- migrates slower
- near the baseline ( Rf)
Paper Chromatography
• Retardation Factor (Rf) - ratio of the distance a compound
moves from the baseline to the distance of the solvent
front from the baseline
•
• As the solvent front passes the sample spots, the
compounds in each sample are carried along at a rate
which is characteristic of their functionality, size and
interaction with the cellulose matrix of the paper.
Results
NOTE:
Usually, there is no need to
measure the Rf values as
identification can be done visually.
By comparing both the position and
color of the spots in the mixture with
those of the known amino acids, the
identity of an unknown sample can
be revealed.
Rf values of Amino Acids
Amino Acid Distance Rf value Distance traveled
Traveled (cm) (computed) by solvent- 14cm
Cysteine 2.5 cm 0.18
Glycine 4.7cm 0.34
Phenylalanine 8.5cm 0.60
Proline 6.8cm 0.49 Answer:
Tyrosine N/a - Unknown 6 is
Phenylalanine.
Unknown 6 8 0.57
The Ninhydrin Test
• Amines (including α-amino acids) react with ninhydrin to give a
coloured product.
• It can be used qualitatively (e.g. for chromatographic visualisation)
or quantitatively (e.g. for peptide sequencing).
• The α-amino acids typically give a blue-purple product.
• Proline, a secondary amine, gives a yellow-orange product.
• The test is sensitive enough such that ninhydrin can be used for the
visualisation of fingerprints.