Experiment 13

Urine Metabolic Screening Test

Introduction
Inborn Errors of Metabolism

y disorders characterized by disruption of

enzymatic function in a metabolic pathway

Causes
 Failure to inherit the gene to produce a particular

enzyme
Inability of a reaction to occur at a sufficient rate Metabolite accumulates in increased amounts Reaction products fail to form Reaction do not take place

 Defect in transport system which control the passage of

molecular across the membrane

Defects of absence of enzymes causing metabolic blocks

Clinical Manifestation
 Mental retardation  Development delays  Seizures  Unexplained metabolic acidosis  Jaundice  Vomiting  Hepatomegaly  Abnormal facial and body features  Death

Urine Metabolic Screening Test

 battery of tests that is performed on urine specimens to

detect the possibility of a metabolic disorder  not specific and are used only as screening tests

y the kidneys remove waste material, minerals, fluids, and

other substances from the blood for elimination in the urine y urine can contain hundreds of different bodily waste products y diet, fluid intake, exercise, and kidney function, affect what is in the urine

Urine
 good specimen of choice for the detection of most

metabolic disturbances
accumulated metabolites spill over

Routine Urinalysis

Introduction
y Urine is formed from our kidneys and is an ultrafiltrate of

plasma y Our normal daily urine output is 1200 - 1500 ml, while a range of 600 2000ml may still be considered normal y Urine composition is of urea and other organic chemicals dissolved in water

Objectives
y To note the physical characteristics of the urine sample y To perform a routine chemical examination on the urine

sample (using a reagent strip) y To identify materials present in the urine through microscopic analysis y To be able to correlate physical, chemical and microscopic observations and know their clinical significances

Methodology
Note the COLOR and TRANSPARENCY of the urine sample

Fill a test tube almost to the top with the sample urine (3/4 will suffice) Subject the urine for testing using the reagent strip. Centrifuge for 5 minutes

Use the SEDIMENT for microscopic analysis

EXPERIMENTA L RESULTS: Physical Properties

Subject 1 COLOR : pale yellow ODOR: aromatic REACTION: 6.0 TRANSPARENCY: clear SPECIFIC GRAVITY: 1.010 Subject 2 COLOR : yellow ODOR: aromatic REACTION: 6.0 TRANSPARENCY: clear SPECIFIC GRAVITY: 1.025

EXPERIMENTA L RESULTS: Chemical Tests

Subject 1 PROTEIN: (-) SUGAR: (-) BLOOD: (-) BILIRUBIN: (-) UROBILINOGEN: normal NITRITE: (-) KETONE: (-) LEUKOCYTE: (-) Subject 2 PROTEIN: (-) SUGAR: (-) BLOOD: (-) BILIRUBIN: (-) UROBILINOGEN: normal NITRITE: (-) KETONE: (-) LEUKOCYTE: (-)

EXPERIMENTA L RESULTS: Microscopic Elements

Subject 1 RBC: 0-1/hpf WBC : 0-1/hpf Bacteria : rare Amorphous Urates: few Mucus Threads: few Casts: NONE Subject 2 RBC: 0-2/hpf WBC : 0-1/hpf Bacteria : rare Amorphous Urates: occasional Mucus Threads: occasional Casts: NONE

PHYSICAL PROPERTIES: Color

Color Colorless Yellow Dark Yellow Amber Orange Bilirubin Acriflavin Phenazopyridine Nitrofurantoin Phenindione Bilirubin oxidized to biliverdin Cause Recent fluid Consumption Diabetes Insipidus Conc. Specimen Clinical/Lab Relationship Common in random specimens Inc 24 hr volume Normal after strenuous exercise or w/ 1st morning specimen Dehydration from fever and burns Yellow foam (-) bile, w/ green fluorescence Drug used for UTI Also for UTI Orange in alkaline, colorless in acid False negative for Bilirubin

Yellow-Green Yellow-Brown

PHYSICAL PROPERTIES: Color

Color Green Blue-green Cause Pseudomonas Amitriptyline Methocarbamol Clorets Indican Methylene Blue Phenol RBCs Hgb Myoglobin Porphyrins Oxid RBCs Methemoglobin Homogentisic Acid Clinical/Lab Relationship (+) urine culture Antidepressant Muscle-relaxant, maybe green-brown None Bacterial Infection Fistulas When oxidized (+) chem for blood, cloudy, seen micro. (+) chem for blood, clear, IV hemolysis (+) chem for blood, muscle damage (-) chem for blood Dehydration from fever and burns Denatured Hgb Seen in alkaline urine after standing

Pink

Brown

PHYSICAL PROPERTIES: Odor

Odor Aromatic Foul, Ammoniacal Fruity, Sweet Maple Syrup Mousy Rancid Sweaty Feet Cabbage Bleach Cause Normal Bacterial composition, UTI Ketones MSUD Phenylketonuria Tyrosinemia Isovaleric acidemia Methionine malabsorption Contamination w/ semen

PHYSICAL PROPERTIES: Transparency

Clarity Clear Hazy Cloudy Turbid Milky Term No visible particulates, transparent Few particulates, print easily seen Many particulates, print blurred Print cannot be seen through urine Maybe precipitated or clotted

Chemical Examination
y y y y y y y y y y

pH: can range from 4.5-8.0 4.5Specific Gravitiy: 1.015-1.025 1.015Protein: negative or trace Sugar: negative or trace Blood: negative Bilirubin: negative Urobilinogen: negative Nitrite: negative Ketone: negative Leukocyte: negative

Microscopic Examination
Normal Values RBC: 0-2/hpf 0WBC: 0-5/hpf 0Bacteria: none or few Mucus threads; none, rare, few Crystals (Amophous Urates and Amorphous Phosphates): rare, few, moderate Casts: (hyaline casts): 0-2/lpf 0others: none

Ferric Chloride Test

Screening Tests for Certain Inborn Errors of Metabolism

Ferric Chloride Test

y Tests for the presence of high levels of phenylpyruvate

in urine (phenylketonuria) (phenylketonuria)

y Detects compounds such as aromatic hydroxyl groups,

phenols and enols

y Transient or permanent coloration (usually purple, green

or blue) indicates the presence of aromatic hydroxyl compounds, phenols and enols

Ferric Chloride Test : Reaction

Ferric Chloride Test

y The OH (hydroxy group) which is attached directly to an

aromatic nucleus (e.g.Benzene)is detected by the Ferric chloride y Phenols will typically yield dramatic purple,blue,red or green color as an indication of a positive test.
y Aromatic acids will test as a beige-tan color. y (+) Aliphatic acids
y non-aromatic organic acids (Acetic acid)

Ferric Chloride Test

y Ferric ion forms a colored complex with phenylpyruvate: phenylpyruvate:

blue green color

y blue-green color indicates the presence of metabolites of blue-

phenylalanine
y ketone phenylpyruvic acid in the urine gives the disease its

name & a green color in the ferric chloride test

Ketone Phenylpyruvic Acid

Ferric Chloride Test

y Why is it no longer used? used?
y It is complexed with other compounds, producing

interfering colors
y May mask the blue-green color of the ferric complex with blue-

phenylpyruvate
y Relatively high concentrations of phenylpyruvate must be

present in order for there to be appreciate development of the characteristic color change assay is less sensitive, and more prone to misinterpretation

Phenylketonuria
y Inherited disorder (autosomal recessive) of phenylalanine metabolism

causing severe mental retardation y Diagnosed by finding excess phenylpyruvic acid in serum and urine y Lack of enzyme phenylalanine hydroxylase causes accumulation of phenylalanine in plasma
y Urine: Mousy odor y Physical:
y Eczema y Fair coloring as a result of tyrosine deficiency(pigmentation metabolite, (

melanin) melanin)
y Enzyme: Phenylalanine Hydroxylase Enzyme:
y the enzyme locus is on chromosome arm 12q

Phenylketonuria

Materials and Methods

1 ml FeCl3 reagent 10 drops urine

shake observe result

PICTURES

y GAB

y OLIVE

Conditions/Substances giving a (+) FeCl3 Test

Condition/Substance
Acetoacetic Acid

Result
Red-brown Red-

Alkaptonuria (homogentisic Acid) Blue-green (transient) Bluep-Aminosalicylic acid Purple-brown PurpleBilirubin Histidinemia Lactic Acidosis MSUD Melanin Blue-green BlueBlue-gray to green BlueGray Green to gray Gray ppt to black

Conditions/Substances giving a (+) FeCl3 Test

Condition/Substance
Methionine Malabsorption Phenothiazines Phenylketonuria Pyruvic acid Salicylates Tyrosinemia Xanthurenic acid

Result
Purple to red-brown redPurple brown Blue green Deep yellow Purple Green (fades rapidly) Dark green to brown

Experimental Results
y Both urine samples (GAB&OLIVE): NEGATIVE

Benedicts Test
Screening Tests for Certain Inborn Errors of Metabolism

Benedicts Test
y test for the presence of monosaccharides
y Glucose y Fructose

y test for the presence of some disaccharides

y Maltose

y test for the presence of aldehydes

Benedicts Test
y Benedicts reagent can be used to test for presence of glucose

in urine
y Indication of diabetes

y Heating a Benedicts solution mixed with monosaccharides

will produce a reddish-orange color reddish-

Benedicts Reagent
y Contains blue copper(II) sulfate (CuSO4) y The copper oxide is insoluble in water and so it precipitates y Contains NaOH and tartaric acid y Color of the final solution ranges from green to brick red

depending on how many copper(II) are present

Benedicts Reagent
y Carbohydrates that contain aldehydes or a-hydroxymethyl

ketones can be oxidized by Cu(II)ions are classified as reducing sugars. They reduce the Cu(II) to Cu(I).

O R H + Cu2+ R

O H + Cu2O(s)

Results
No precipitate Green Yellow Orange Red ---a trace + ++ +++

Methodology
5-mL of Benedicts reagent Test tube

Heat to boiling Add

8 drops of urine

Boil again After 2 minutes Color change

Results noted as: (-) --> BLUE (+) --> GREEN to YELLOW (++) --> YELLOW to BROWN (+++) --> BROWN to ORANGE (++++) --> ORANGE to RED

Experimental Results
Group 1
Positive sample Experimental 1

(++++)

(-)

Group 2

Positive sample

Experimental 2

(++++)

(+)

Benedicts Test
y detect the presence of reducing sugars y Reducing sugars - sugars with a free

aldehyde or ketone group

y Aldehyde groups are oxidized to

carboxylic acids y Ketoses can also be reducing sugars because they can isomerize (tautomerisation) to aldoses via an enediol tautomerisation)

y They are classified as reducing sugars since they reduce the Cu2+ to Cu+ which forms as a red precipitate, copper (I) oxide.

red precipitate aldehyde carboxylate

Benedicts Reagent
y Deep-blue alkaline solution of copper sulfate, sodium hydroxide, and Deep-

tartaric acid y The copper sulfate (CuSO4) reacts with electrons from the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu2O), a redredbrown precipitate.

CuSO4 Cu++ + SO4-2 Cu++ + Reducing Sugar Cu+ (electron donor) Cu+ Cu2O (precipitate)
y The final color - how much of this precipitate was formed y gives an indication of how much reducing sugar was present.
Increasing amounts of reducing sugar

bluegreenyellow brownorangered

Urinary Substances and Clinical Syndromes Associated with Reducing Substances

Reducing Substance Drugs Fructose Galactose Glucose Homogentisic acid Lactose Phenolic compound Xylose Xylulose Clinical State Ascorbic acid, chloral hydrate, tetracyclines, sulfonamides, chloramphenicol Fructosemia, essential fructosuria, hereditary fructose intolerance Galactosemia, classic and variant (galactokinase deficiency) Diabetes mellitus, renal glycosuria, Fanconis Syndrome, Wilsons Disease Alkaptonuria Lactase deficiency, lactose intolerance, newborn Phenylketonurias, tyrosinosis Excessive fruit intake Pentosuria

y Fructose can also act as a reducing sugar y Under basic conditions, the fructose molecules can, essentially, have the location of the carbonyl bond switched to convert them into a glucose molecule. y Galactose is another reducing sugar y forms a "6-ring" when dissolved. "6y almost identical to glucose, the only exception being the position of the hydroxyl group on carbon 4. y Lactose - the left ring is a locked hemiacetal, but the

other ring can open. This is a reducing sugar.

y Xylose is an essential sugar saccharide of the

pentose class and vital to cellular communication. It is also a reducing sugar because of the available aldehyde group. y Xylulose is a ketopentose. In nature it occurs in the L- and D- isomers. L-xylulose LDLaccumulates in the urine of pentosuria patients. Since L-xylulose is a reducing sugar Llike D-glucose, pentosuria patients have been Dwrongly diagnosed in the past to be diabetic.

Nitrosonaphthol Test
Screening Tests for Certain Inborn Errors of Metabolism

Nitrosonaphtol Test
y Screening for inborn error of TYROSINE metabolism

Tyrosine

Nitrosonaphtol Reagent
y 1-nitroso-2-naphthol nitrosoy organic compound y yellow-brown, crystalline yellowy melting point: 107C point: y commonly used to determine cobalt y moderately hazardous y reacts with substituted phenolic compounds

Nitrosonaphtol Test
Procedure:
In a test tube: 1 ml 2.63N HNO3 1 drop Sodium Nitrite 0.10 ml nitrosonapthol reagent

MIX

25 mins. Positive: OrangeOrange-red color

Nitrosonaphtol Test
Results

Experimental: (-) result

Standard: (+) result

Discussion
y Mechanism

1-nitroso-2-naphthol reacts with tyrosine or its metabolites to nitrosoproduce a positive result. result. y Reacts with: with:
y Tyrosine and its metabolites y 5-hydroxyindoles y Substituted guiacols

Nitrosonaphtol Test
False Positive Result y IV fluids containing homovanillic acid y 5-HIAA y N-acetyltyrosine

Tyrosinosis
y Very rare hereditary disorder of Tyrosine metabolism y Defective formation of p-hydroxyphenylpyruvic acid p-

oxidase or of tyrosine transaminase y Enhanced urinary excretion of p-hydroxyphenylpyruvic pacid and other tyrosyl metabolites

Nitroprusside Test
Screening Tests for Certain Inborn Errors of Metabolism

Introduction
y Alternative Names:Acetone bodies; Ketones -

serum; Ketone bodies serum

y Measures the amount of ketones in the blood.

Any amount of detectable ketones is considered abnormal.

y Used in the screening of cystinuria, homocystinuria

and -mercaptolactate cysteine disulfiduria

y Cystinuria
y Renal transport defect y Urinary excretion of cystine is markedly increase y Complication: formation of cystine stones

y Homosytinuria
y Defect in cystathione synthase y Elevated levels of homocystinuria in blood and urine

y A person may have these disorders if he tests positive

Methodology
5 mL of Urine

+5 drops of Conc. NH4OH Mix +2mL of 5% NaCN Stand for 10 min. +4 drops of Sodium Nitroprusside Mix Observe Color Change

Experimental Results
y The solution produced was yellow. Hence, the subject tested

negative for the test.

How does a positive test come about?

y Cystine y an oxidized, dimeric form of cysteine y Cystine is reduced to cysteine via cystine reductase y Cysteine has free sulfhydryl groups

In the Nitroprusside Reaction, the sulhydryl groups react with nitroprusside pink color y Cystine crystals are hexagonal and colorless. They become pink in the nitroprusside reaction.

y Positive Test Result

+ - pink ++ - pinkish +++ - purple ++++ - dark purple

Cetyltrimethylammonium Bromide Test (CAB)

Screening Tests for Certain Inborn Errors of Metabolism

Cetyltrimethyammonium Bromide Test

y Turbidometric technique y Uses quaternary ammonium compounds e.g. CAB y Used for both qualitative and quantitative determination of

urinary mucopolysaccharides and glycosaminoglycans in various forms of mucopolysaccharidoses

Mucopolysaccharidoses (MPS)
y Constitute a large and heterogeneous subgroup among

the lysosomal storage diseases (LSD) y Caused by deficiency of specific enzymes, which are responsible for glycosaminoglycan (GAG) breakdown during different steps of its degradation pathway y Differential diagnosis is important for a correct prognosis, definition of management strategies, genetic counseling, prenatal diagnosis, and prediction of future cases in the family

Objective
y To determine the presence of urinary mucopolysaccharides

and glycosaminoglycans in the urine

Procedure
5 ml of urine was placed in a test tube

Urine was allowed to stand at room temperature

1 ml of CAB reagent was added

Test tube was observed for 30 minutes

Results
y Team 1: no turbidity observed (0) y Team 2: positive turbidity (1)

Discussion
y Cetyltrimethylammonium

bromide (CTAB)
y Is a Quaternary ammonium

salt
y An ammonium salt in which all

four groups attached to the nitrogen atom of the ammonium ion are hydrocarbon groups

y Quaternary ammonium compounds fix soluble acid

mucopolysaccharides (e.g., mucin) by forming highly insoluble complexes

y For example, an aqueous solution of heparin sodium salt is mixed

with the quaternary ammonium salt y Consequently, heparin in the heparin complex is a mucopolysaccharide chain with negative sulfate groups in it associated with the positive quaternary ammonium groups

y Lysosomal storage disorders are variably referred to as simply

storage disorders, lipidoses, mucopolysaccharidoses, or mucolipidoses

y Caused by deficiencies of enzymes which are involved in the

degradation sequence of GAGs

y Diagnosis is through urine testing for the presence of increased

amounts of GAGs

Storage Disorder

Clinical Featuresa

Enzyme Deficiency

Current Diagnostic Approachb,c

Hurlers syndrome (MPS I)

Progressive mental and physical debilitation beginning at age 1; corneal opacities; coarse facies; gingival hyperplasia; dysostosis multiplex; stiff joints (claw-hands); dwarfing; organomegaly.

-L-Iduronidase

Scheies syndrome (MPS Ia)

A mild form of MPS I with corneal opacity; mild or absent mental retardation; claw-hand deformity; aortic stenosis.

-L-Iduronidase

Quantitative enzyme assay. Demonstration of excess urinary mucopolysaccharide consisting of dermatan and heparan sulfatase.

Hunters syndrome (MPS II)

Dysostosis multiplex essentially the same as in MPS I; mental retardation in the severe forms

Iduronate sulfatases X-linked

Morquios disease (MPS IVa)

Pronounced skeletal anomalies with small stature (short-trunk dwarfism); short neck; prominent lower ribs; odontoid anomalies; normal intellect.

Galactosamine-6sulfate sulfatase

Demonstration of keratin sulfate in urine. Due to phenotype: go directly to quantitative enzyme assay.

Ninhydrin Test
Screening Tests for Certain Inborn Errors of Metabolism

Introduction
Ninhydrin (Triketohydrindane hydrate)
y C 9H 6O 4 y Appearance: white pale yellow crystals y a chemical used to detect ammonia or primary and secondary

amines y produces a deep blue or purple coloration Ninhydrin Reagent Solution:

Ninhydrin: 0.35g ethanol or acetone/butanol so-propanol: 100ml so-

Introduction
y for detection and quantification of amino acids in substances y for experiment: in urine y can be used qualitatively (e.g. for chromatographic visualization) or quantitatively (e.g. for peptide sequencing) y once used for fingerprint detection (forensics): amines (forensics):

leftover from peptides & proteins (terminal amines or lysine residues) sloughed off in fingerprints react with ninhydrin

Methodology
1 ml Ninhydrin reagent 3 drops of urine

Warm for 30 secs. in water bath Observe color Violet: alpha amino acid Yellow: proline

Experimental Results
y Group 1: (-) colorless y Group 2: (+) slightly bluish coloration

Reaction Mechanism

y ninhydrin (or triketohydrindene hydrate) is a strong

oxidizing agent y amines (including all physiological -amino acids) react with it y cause oxidative deamination of -amino acid function y products: aldehyde, ammonia, carbon dioxide, hydrindantin

Reactions
alpha-amino acid + ninhydrin reduced ninhydrin + alpha-amino acid + H2O

alpha-amino acid + H2O alpha-keto acid + NH3

alpha-keto acid +NH3 aldehyde + CO2

Reaction Mechanism
y ammonia can react with hydrindantin and another molecule

of ninhydrin y yields a bluish-purple product (Ruhemanns purple; can be bluishmeasured at 570 nm) y -imino acids, e.g. proline and hydroxyproline: bright yellowyellow-orange (440 nm absorbance)

Indication of Results
y bluish-purple solution: presence of amino acids in urine bluishy yellow-orange: presence of proline yellowy amino aciduria is present

Amino Aciduria
y increased levels of amino acid excretion in the urine y indicates possible inborn errors of metabolism caused by a

specific enzyme deficiency (e.g. Hartnups disease, Tyrosinemia)

Millons Test
Screening Tests for Certain Inborn Errors of Metabolism

Millons Test
y Used for the detection of phenol in the y y

y y

solution Indicates the presence of Tyrosine in the urine Millons Reagent: solution of mercuric and mercurous ions in nitric and nitrous acids (+) Result: Peach to Red colored solution Nitration of Phenol group which leads to a complexation reaction with Hg (I) and Hg (II) ions

Methodology
y Add 1 drop of Millons reagent to 1 mL urine y Water bath for 15 minutes

Tyrosyluria
y accumulation of excess tyrosine in the plasma (tyrosinemia) y

y y y y

producing urinary overflow two actions directly involved: 1) contain excess tyrosine 2) its degradation p-hydroxyphenylpyruvic and ppphydroxyphenyllactic acid most frequently seen is a transitory tyrosinemia in premature infants, caused by underdevelopment of liver function severe liver disease = tyrosyluria urine sediments may show tyrosine and leucine crystals hereditary and metabolic disorders : liver and renal disease

Paper Chromatography of Amino Acids

Paper Chromatography
y method for testing the purity of compounds and identifying

substances y useful technique because it is relatively quick and requires small quantities of material y used for screening patients for possible amino acid abnormalities

Paper Chromatography
y substances are distributed between a stationary phase and a

mobile phase y standard mixtures of pure amino acids are run at the same time as the unknown (urine sample) y positions of the spots are compared y preliminary preparation of urine samples: to remove salts and proteins

Methodology
Spot 6 7 x, Dry in between applications Spot as small as possible (too big spots samples overlap & influence movement of solute)

Place in chromatographic jar Solvent front top of paper

Air dry Spray with Ninhydrin Place in hot plate until spots are visible Measure distance travelled by amino acids

y Solvent system: ETHANOL: NH4OH: H2O

(8:1:1)

Experimental Results

Experimental Results
Protein Cysteine Glycine Proline Tyrosine Tryptophan Unknown Distance Travelled 1 cm 5.1 cm 9.5 cm 8.4 cm 10 cm 10.1 cm Rf Value 5.9 30 55.9 49.4 58.8 59.4

Experimental Results
y Distance traveled by solvent = 17cm
y ran off the paper: margins were disregarded

y Unknown = Tryptophan