Urine Test

Urine Metabolic Urine Metabolic Screening TestScreening Test

Experiment 13Experiment 13

IntroductionIntroduction

Inborn Errors of Metabolism Inborn Errors of Metabolism disorders characterized by disorders characterized by

disruption of enzymatic disruption of enzymatic function in a metabolic pathwayfunction in a metabolic pathway

CausesCauses

Failure to inherit the gene to produce a Failure to inherit the gene to produce a particular enzymeparticular enzyme• Inability of a reaction to occur at a sufficient Inability of a reaction to occur at a sufficient

rate rate • Metabolite accumulates in increased amountsMetabolite accumulates in increased amounts• Reaction products fail to formReaction products fail to form• Reaction do not take placeReaction do not take place

Defect in transport system which control Defect in transport system which control the passage of molecular across the the passage of molecular across the membranemembrane• Defects of absence of enzymes causing Defects of absence of enzymes causing

metabolic blocks metabolic blocks

Clinical ManifestationClinical Manifestation Mental retardationMental retardation Development delaysDevelopment delays SeizuresSeizures Unexplained metabolic acidosisUnexplained metabolic acidosis Jaundice Jaundice VomitingVomiting HepatomegalyHepatomegaly Abnormal facial and body featuresAbnormal facial and body features DeathDeath

Urine Metabolic Screening Urine Metabolic Screening TestTest battery of tests that is performed on urine battery of tests that is performed on urine

specimens to detect the possibility of a specimens to detect the possibility of a metabolic disordermetabolic disorder

not specific and are used only as screening not specific and are used only as screening teststests

the kidneys remove waste material, the kidneys remove waste material, minerals, fluids, and other substances from minerals, fluids, and other substances from the blood for elimination in the urinethe blood for elimination in the urine

urine can contain hundreds of different urine can contain hundreds of different bodily waste productsbodily waste products

diet, fluid intake, exercise, and kidney diet, fluid intake, exercise, and kidney function, affect what is in the urinefunction, affect what is in the urine

UrineUrine good specimen of choice for the detection

of most metabolic disturbances• accumulated metabolites “spill over”

Routine UrinalysisRoutine Urinalysis


Urine is formed from our kidneys and is an Urine is formed from our kidneys and is an ultrafiltrate of plasmaultrafiltrate of plasma

Our normal daily urine output is 1200 - 1500 Our normal daily urine output is 1200 - 1500 ml, while a range of 600 – 2000ml may still be ml, while a range of 600 – 2000ml may still be considered normalconsidered normal

Urine composition is of urea and other Urine composition is of urea and other organic chemicals dissolved in waterorganic chemicals dissolved in water

To note the physical characteristics of the To note the physical characteristics of the urine sampleurine sample

To perform a routine chemical To perform a routine chemical examination on the urine sample (using a examination on the urine sample (using a reagent strip)reagent strip)

To identify materials present in the urine To identify materials present in the urine through microscopic analysisthrough microscopic analysis

To be able to correlate physical, chemical To be able to correlate physical, chemical and microscopic observations and know and microscopic observations and know their clinical significancestheir clinical significances

ObjectivesObjectives

MethodologyMethodologyNote the COLOR and TRANSPARENCY

of the urine sample

Subject the urine for testing using the reagent strip.

Centrifuge for 5 minutes

Use the SEDIMENT for microscopic analysis

Fill a test tube almost to the top with the sample urine (3/4 will suffice)

EXPERIMENTA L RESULTS: EXPERIMENTA L RESULTS: Physical PropertiesPhysical PropertiesSubject 1COLOR : pale

yellowODOR: aromaticREACTION: 6.0TRANSPARENCY:

clearSPECIFIC GRAVITY:

1.010

Subject 2COLOR : yellowODOR: aromaticREACTION: 6.0TRANSPARENCY:

clearSPECIFIC GRAVITY:

1.025

EXPERIMENTA L RESULTS: EXPERIMENTA L RESULTS: Chemical TestsChemical TestsSubject 1PROTEIN: (-)SUGAR: (-)BLOOD: (-)BILIRUBIN: (-)UROBILINOGEN:

normalNITRITE: (-)KETONE: (-)LEUKOCYTE: (-)

Subject 2PROTEIN: (-)SUGAR: (-)BLOOD: (-)BILIRUBIN: (-)UROBILINOGEN:

normalNITRITE: (-)KETONE: (-)LEUKOCYTE: (-)

EXPERIMENTA L RESULTS: EXPERIMENTA L RESULTS: Microscopic ElementsMicroscopic ElementsSubject 1RBC: 0-1/hpfWBC : 0-1/hpfBacteria : rareAmorphous

Urates: fewMucus Threads:

fewCasts: NONE

Subject 2RBC: 0-2/hpfWBC : 0-1/hpfBacteria : rareAmorphous

Urates: occasional

Mucus Threads: occasional

Casts: NONE

PHYSICAL PHYSICAL PROPERTIES: ColorPROPERTIES: Color

Color Cause Clinical/Lab Relationship

Colorless Recent fluid Consumption

Common in random specimens

Yellow Diabetes Insipidus Inc 24 hr volume

Dark Yellow Conc. Specimen Normal after strenuous exercise or w/ 1st morning specimen

Amber Dehydration from fever and burns

Orange Bilirubin

Acriflavin

Phenazopyridine

Nitrofurantoin

Phenindione

Yellow foam

(-) bile, w/ green fluorescence

Drug used for UTI

Also for UTI

Orange in alkaline, colorless in acid

Yellow-Green

Yellow-Brown

Bilirubin oxidized to biliverdin

False negative for Bilirubin

PHYSICAL PROPERTIES: PHYSICAL PROPERTIES: ColorColor

Color Cause Clinical/Lab Relationship

Green Pseudomonas (+) urine culture

Blue-green Amitriptyline

Methocarbamol

Clorets

Indican

Methylene Blue

Phenol

Antidepressant

Muscle-relaxant, maybe green-brown

None

Bacterial Infection

Fistulas

When oxidized

Pink RBC’s

Hgb

Myoglobin

Porphyrins

(+) chem for blood, cloudy, seen micro.

(+) chem for blood, clear, IV hemolysis

(+) chem for blood, muscle damage

(-) chem for blood

Brown Oxid RBC’s

Methemoglobin

Homogentisic Acid

Dehydration from fever and burns

Denatured Hgb

Seen in alkaline urine after standing

PHYSICAL PHYSICAL PROPERTIES: OdorPROPERTIES: Odor

Odor Cause

Aromatic Normal

Foul, Ammoniacal Bacterial composition, UTI

Fruity, Sweet Ketones

Maple Syrup MSUD

Mousy Phenylketonuria

Rancid Tyrosinemia

Sweaty Feet Isovaleric acidemia

Cabbage Methionine malabsorption

Bleach Contamination w/ semen

PHYSICAL PROPERTIES: PHYSICAL PROPERTIES: TransparencyTransparency

Clarity Term

Clear No visible particulates, transparent

Hazy Few particulates, print easily seen

Cloudy Many particulates, print blurred

Turbid Print cannot be seen through urine

Milky Maybe precipitated or clotted

Chemical Chemical ExaminationExamination

pH: can range from 4.5-8.0pH: can range from 4.5-8.0 Specific Gravitiy: 1.015-1.025Specific Gravitiy: 1.015-1.025 Protein: negative or traceProtein: negative or trace Sugar: negative or traceSugar: negative or trace Blood: negativeBlood: negative Bilirubin: negativeBilirubin: negative Urobilinogen: negativeUrobilinogen: negative Nitrite: negativeNitrite: negative Ketone: negativeKetone: negative Leukocyte: negativeLeukocyte: negative

Microscopic Microscopic ExaminationExamination

Normal ValuesNormal ValuesRBC: 0-2/hpfRBC: 0-2/hpfWBC: 0-5/hpfWBC: 0-5/hpfBacteria: none or few Bacteria: none or few Mucus threads; none, rare, few Mucus threads; none, rare, few Crystals (Amophous Urates and Amorphous Crystals (Amophous Urates and Amorphous

Phosphates): rare, few, moderatePhosphates): rare, few, moderateCasts: (hyaline casts): 0-2/lpfCasts: (hyaline casts): 0-2/lpf others: noneothers: none

Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism

Ferric Chloride TestFerric Chloride Test


Tests for the presence of Tests for the presence of high levels high levels ofof phenylpyruvate phenylpyruvate in urine in urine (phenylketonuria)(phenylketonuria)

Detects compounds such as Detects compounds such as aromatic aromatic hydroxyl hydroxyl groups, groups, phenolsphenols and enols and enols

TransientTransient or or permanentpermanent coloration coloration (usually purple, green or blue) indicates the (usually purple, green or blue) indicates the presence of aromatic hydroxyl compounds, presence of aromatic hydroxyl compounds, phenols and enolsphenols and enols

Ferric Chloride Test : Ferric Chloride Test : ReactionReaction

Ferric Chloride TestFerric Chloride TestThe OH (hydroxy group) which is attached

directly to an aromatic nucleus (e.g.Benzene)is detected by the Ferric chloride

Phenols will typically yield dramatic purple,blue,red or green color as an indication of a positive test.

Aromatic acids will test as a beige-tan color.(+) Aliphatic acids

non-aromatic organic acids (Acetic acid)


Ferric ion forms a colored complex with Ferric ion forms a colored complex with phenylpyruvatephenylpyruvate: : blue green colorblue green color

blue-greenblue-green color indicates the presence of color indicates the presence of metabolites of phenylalaninemetabolites of phenylalanine

ketone phenylpyruvic acid ketone phenylpyruvic acid in the urine in the urine gives the disease its name & a green color in gives the disease its name & a green color in the ferric chloride testthe ferric chloride test

Ketone Phenylpyruvic Ketone Phenylpyruvic AcidAcid


Why is it no longer used?Why is it no longer used?

It is It is complexedcomplexed with other compounds, with other compounds, producing producing interferinginterfering colors colors

May May maskmask the the blue-greenblue-green color of the ferric color of the ferric complex with phenylpyruvatecomplex with phenylpyruvate

Relatively Relatively high concentrations high concentrations of of phenylpyruvatephenylpyruvate must be present in order for must be present in order for there to be appreciate development of the there to be appreciate development of the characteristic color change characteristic color change assay is assay is lessless sensitive, and more prone to misinterpretationsensitive, and more prone to misinterpretation

PhenylketonuriaPhenylketonuria Inherited disorder (autosomal recessive) of

phenylalanine metabolism causing severe mental retardation

Diagnosed by finding excess phenylpyruvic acid in serum and urine

Lack of enzyme phenylalanine hydroxylase causes accumulation of phenylalanine in plasma

Urine: “Mousy odor” Physical: Physical:

Eczema Fair coloring as a result of tyrosine

deficiency((pigmentation metabolite, pigmentation metabolite, melaninmelanin))

EnzymeEnzyme: Phenylalanine Hydroxylase: Phenylalanine Hydroxylase the enzyme locus is on chromosome arm 12q

PhenylketonuriaPhenylketonuria

Materials and Materials and MethodsMethods

1 ml FeCl3 reagent1 ml FeCl3 reagent 10 drops urine10 drops urine

• shakeshake• observe resultobserve result

PICTURES

GAB OLIVE

Conditions/Substances giving a Conditions/Substances giving a (+) FeCl(+) FeCl33 Test Test

Condition/SubstanceCondition/Substance ResultResultAcetoacetic AcidAcetoacetic Acid Red-brownRed-brown

Alkaptonuria (homogentisic Acid)Alkaptonuria (homogentisic Acid) Blue-green (transient)Blue-green (transient)

p-Aminosalicylic acidp-Aminosalicylic acid Purple-brownPurple-brown

BilirubinBilirubin Blue-greenBlue-green

HistidinemiaHistidinemia Blue-gray to greenBlue-gray to green

Lactic AcidosisLactic Acidosis GrayGray

MSUDMSUD Green to grayGreen to gray

MelaninMelanin Gray ppt to blackGray ppt to black

Conditions/Substances giving a Conditions/Substances giving a (+) FeCl(+) FeCl33 Test Test

Condition/SubstanceCondition/Substance ResultResultMethionine MalabsorptionMethionine Malabsorption Purple to red-brownPurple to red-brown

PhenothiazinesPhenothiazines Purple brownPurple brown

PhenylketonuriaPhenylketonuria Blue greenBlue green

Pyruvic acidPyruvic acid Deep yellowDeep yellow

SalicylatesSalicylates PurplePurple

TyrosinemiaTyrosinemia Green (fades rapidly)Green (fades rapidly)

Xanthurenic acidXanthurenic acid Dark green to brownDark green to brown

Experimental ResultsExperimental Results

Both urine samples (GAB&OLIVE): NEGATIVE


Benedict’s TestBenedict’s Test

Benedict’s TestBenedict’s Test

test for the presence of monosaccharides test for the presence of monosaccharides Glucose Glucose FructoseFructose

test for the presence of some disaccharidestest for the presence of some disaccharidesMaltose Maltose

test for the presence of aldehydestest for the presence of aldehydes

Benedict’s TestBenedict’s Test Benedict’s reagent can be used to test for Benedict’s reagent can be used to test for

presence of glucose in urinepresence of glucose in urine Indication of diabetesIndication of diabetes

Heating a Benedict’s solution mixed with Heating a Benedict’s solution mixed with monosaccharides will produce a reddish-monosaccharides will produce a reddish-orange colororange color

Benedict’s ReagentBenedict’s Reagent

Contains blue copper(II) sulfate (CuSO4) The copper oxide is insoluble in water and

so it precipitates Contains NaOH and tartaric acid Color of the final solution ranges from

green to brick red depending on how many copper(II) are present

Benedict’s ReagentBenedict’s Reagent

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

H + Cu2+

O

H + Cu2O(s)

RR RR

ResultsResults

No precipitate ----

Green a trace

Yellow +

Orange ++

Red +++

5-mL of Benedict’s reagent

8 drops of urine

Heat to boiling

Test tube

Add

MethodologyMethodology

Boil again

Color change

After 2 minutes

Results noted as:(-) --> BLUE

(+) --> GREEN to YELLOW(++) --> YELLOW to BROWN

(+++) --> BROWN to ORANGE(++++) --> ORANGE to RED


Positive sample

Experimental 1

(++++) (-)

Group 1Group 1

Positive sampleExperimental 2

Group 2Group 2

(++++) (+)

Benedict’s TestBenedict’s Test detect the presence of detect the presence of

reducing sugarsreducing sugars Reducing sugarsReducing sugars - - sugars sugars

with a free aldehyde or with a free aldehyde or ketone groupketone group

AldehydeAldehyde groups are oxidized groups are oxidized to carboxylic acidsto carboxylic acids

KetosesKetoses can also be reducing can also be reducing sugars because they can sugars because they can isomerize (tautomerisation) to isomerize (tautomerisation) to aldoses via an enediolaldoses via an enediol

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

red precipitate

aldehyde aldehyde carboxylate carboxylate

Benedict’s ReagentBenedict’s Reagent Deep-blue alkaline solution of copper sulfate, sodium Deep-blue alkaline solution of copper sulfate, sodium

hydroxide, and tartaric acidhydroxide, and tartaric acid The copper sulfate (CuSO4) reacts with electrons from The copper sulfate (CuSO4) reacts with electrons from

the aldehyde or ketone group of the reducing sugar to the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu2O), a red-brown precipitate. form cuprous oxide (Cu2O), a red-brown precipitate.

CuSO4 CuSO4 Cu Cu++++ + SO4 + SO4---- 2 Cu2 Cu++++ + Reducing Sugar + Reducing Sugar Cu Cu++

(electron donor)(electron donor) CuCu++ Cu2O Cu2O (precipitate)(precipitate)

The final color - how much of this precipitate was The final color - how much of this precipitate was formedformed

gives an indication of how much reducing sugar was gives an indication of how much reducing sugar was present. present.

Increasing amounts of reducing sugar

bluegreenyellow brownorangered

Reducing Reducing SubstanceSubstance

Clinical StateClinical State

DrugsDrugs Ascorbic acid, chloral hydrate, tetracyclines, sulfonamides, Ascorbic acid, chloral hydrate, tetracyclines, sulfonamides, chloramphenicolchloramphenicol

FructoseFructose Fructosemia, essential fructosuria, hereditary fructose Fructosemia, essential fructosuria, hereditary fructose intoleranceintolerance

GalactoseGalactose Galactosemia, classic and variant (galactokinase deficiency)Galactosemia, classic and variant (galactokinase deficiency)

GlucoseGlucose Diabetes mellitus, renal glycosuria, Fanconi’s Syndrome, Diabetes mellitus, renal glycosuria, Fanconi’s Syndrome, Wilson’s DiseaseWilson’s Disease

Homogentisic acidHomogentisic acid AlkaptonuriaAlkaptonuria

LactoseLactose Lactase deficiency, lactose intolerance, newbornLactase deficiency, lactose intolerance, newborn

Phenolic compoundPhenolic compound Phenylketonurias, tyrosinosisPhenylketonurias, tyrosinosis

XyloseXylose Excessive fruit intakeExcessive fruit intake

XyluloseXylulose PentosuriaPentosuria

Urinary Substances and Clinical Syndromes Associated Urinary Substances and Clinical Syndromes Associated with Reducing Substanceswith Reducing Substances

FructoseFructose can also act as a can also act as a reducing reducing sugarsugar Under basic conditions, the fructose Under basic conditions, the fructose

molecules can, essentially, have the molecules can, essentially, have the location of the carbonyl bond switched to location of the carbonyl bond switched to convert them into a glucose molecule. convert them into a glucose molecule.

GalactoseGalactose is another is another reducing sugarreducing sugar forms a "6-ring" when dissolved. forms a "6-ring" when dissolved. almost identical to glucose, the only almost identical to glucose, the only

exception being the position of the exception being the position of the hydroxyl group on carbon 4.hydroxyl group on carbon 4.

Lactose - Lactose - the left ring is a locked the left ring is a locked hemiacetal, but the other ring can hemiacetal, but the other ring can open. This is a open. This is a reducing sugar.reducing sugar.

XyloseXylose is an essential sugar is an essential sugar saccharide of the pentose class and saccharide of the pentose class and vital to cellular communication. It is vital to cellular communication. It is also a reducing sugar because of also a reducing sugar because of the available aldehyde group.the available aldehyde group.

XyluloseXylulose is a ketopentose. In is a ketopentose. In nature it occurs in the L- and D- nature it occurs in the L- and D- isomers. L-xylulose accumulates in isomers. L-xylulose accumulates in the urine of the urine of pentosuriapentosuria patients. patients. Since L-xylulose is a Since L-xylulose is a reducing reducing sugarsugar like D-glucose, pentosuria like D-glucose, pentosuria patients have been wrongly patients have been wrongly diagnosed in the past to be diagnosed in the past to be diabetic.diabetic.


Nitrosonaphthol TestNitrosonaphthol Test

Nitrosonaphtol TestNitrosonaphtol Test

Screening for inborn error of TYROSINE Screening for inborn error of TYROSINE metabolismmetabolism

TyrosineTyrosine

Nitrosonaphtol ReagentNitrosonaphtol Reagent 1-nitroso-2-naphthol1-nitroso-2-naphthol organic compoundorganic compound yellow-brown, crystallineyellow-brown, crystalline melting point: 107melting point: 107°C°C commonly used to determine cobaltcommonly used to determine cobalt moderately hazardousmoderately hazardous reacts with substituted phenolic reacts with substituted phenolic

compoundscompounds

Nitrosonaphtol TestNitrosonaphtol TestProcedure:Procedure:

In a test tube:In a test tube:

1 ml 2.63N HNO31 ml 2.63N HNO3

1 drop Sodium Nitrite1 drop Sodium Nitrite

0.10 ml nitrosonapthol reagent0.10 ml nitrosonapthol reagent

25 mins.25 mins.

Positive: Positive:

Orange-red colorOrange-red color

MIXMIX

Nitrosonaphtol TestNitrosonaphtol TestResultsResults

Experimental:Experimental: Standard:Standard:(-) result(-) result (+) result(+) result

DiscussionDiscussion MechanismMechanism

1-nitroso-2-naphthol reacts with tyrosine or 1-nitroso-2-naphthol reacts with tyrosine or its metabolites to produce a positive result.its metabolites to produce a positive result.

Reacts with:Reacts with:Tyrosine and its metabolitesTyrosine and its metabolites5-hydroxyindoles5-hydroxyindolesSubstituted guiacolsSubstituted guiacols

Nitrosonaphtol TestNitrosonaphtol TestFalse Positive ResultFalse Positive Result IV fluids containing homovanillic acidIV fluids containing homovanillic acid 5-HIAA5-HIAA N-acetyltyrosineN-acetyltyrosine

TyrosinosisTyrosinosis

Very rare hereditary disorder of Tyrosine Very rare hereditary disorder of Tyrosine metabolismmetabolism

Defective formation of p-Defective formation of p-hydroxyphenylpyruvic acid oxidase or of hydroxyphenylpyruvic acid oxidase or of tyrosine transaminasetyrosine transaminase

Enhanced urinary excretion of p-Enhanced urinary excretion of p-hydroxyphenylpyruvic acid and other hydroxyphenylpyruvic acid and other tyrosyl metabolitestyrosyl metabolites


Nitroprusside TestNitroprusside Test


Alternative Names:Acetone bodies; Ketones - serum; Ketone bodies – serum

Measures the amount of ketones in the blood. Any amount of detectable ketones is considered abnormal.

Used in the screening of cystinuria, homocystinuria and β-mercaptolactate cysteine disulfiduria

CystinuriaCystinuriaRenal transport defectRenal transport defectUrinary excretion of cystine is markedly Urinary excretion of cystine is markedly

increaseincreaseComplication: formation of cystine stonesComplication: formation of cystine stones

HomosytinuriaHomosytinuriaDefect in cystathione synthaseDefect in cystathione synthaseElevated levels of homocystinuria in blood and Elevated levels of homocystinuria in blood and

urineurine

A person may have these disorders if he tests A person may have these disorders if he tests positive positive

MethodologyMethodology5 mL of Urine5 mL of Urine

+2mL of 5% NaCN+2mL of 5% NaCN

+5 drops of Conc. NH+5 drops of Conc. NH44OHOH

Stand for 10 min.Stand for 10 min.

MixMix

+4 drops of Sodium Nitroprusside+4 drops of Sodium Nitroprusside

Observe Color ChangeObserve Color Change

MixMix

Experimental ResultsExperimental Results The solution produced was yellow. Hence,

the subject tested negative for the test.

How does a positive test How does a positive test come about?come about?

CystineCystinean oxidized, dimeric form of cysteinean oxidized, dimeric form of cysteine

Cystine is reduced to cysteine via Cystine is reduced to cysteine via cystine reductasecystine reductase

Cysteine has free sulfhydryl groupsCysteine has free sulfhydryl groups

In the Nitroprusside Reaction, the sulhydryl groups react with nitroprusside pink color

Cystine crystals are hexagonal and colorless. They become pink in the nitroprusside reaction.

Positive Test Result+ - pink++ - pinkish+++ - purple++++ - dark purple


Cetyltrimethylammonium Cetyltrimethylammonium Bromide Test (CAB)Bromide Test (CAB)

Cetyltrimethyammonium Cetyltrimethyammonium Bromide TestBromide Test

Turbidometric techniqueTurbidometric technique Uses quaternary ammonium compounds Uses quaternary ammonium compounds

e.g. CABe.g. CAB Used for both qualitative and quantitative Used for both qualitative and quantitative

determination of urinary determination of urinary mucopolysaccharides and mucopolysaccharides and glycosaminoglycans in various forms of glycosaminoglycans in various forms of mucopolysaccharidosesmucopolysaccharidoses

Mucopolysaccharidoses Mucopolysaccharidoses (MPS) (MPS) Constitute a large and heterogeneous Constitute a large and heterogeneous

subgroup among the lysosomal storage subgroup among the lysosomal storage diseases (LSD)diseases (LSD)

Caused by deficiency of specific enzymes, Caused by deficiency of specific enzymes, which are responsible for glycosaminoglycan which are responsible for glycosaminoglycan (GAG) breakdown during different steps of its (GAG) breakdown during different steps of its degradation pathway degradation pathway

Differential diagnosis is important for a Differential diagnosis is important for a correct prognosis, definition of management correct prognosis, definition of management strategies, genetic counseling, prenatal strategies, genetic counseling, prenatal diagnosis, and prediction of future cases in diagnosis, and prediction of future cases in the family the family

ObjectiveObjective

To determine the presence of urinary To determine the presence of urinary mucopolysaccharides and mucopolysaccharides and glycosaminoglycans in the urineglycosaminoglycans in the urine

ProcedureProcedure

5 ml of urine was placed in a test tube5 ml of urine was placed in a test tube

Urine was allowed to stand at room temperatureUrine was allowed to stand at room temperature

1 ml of CAB reagent was added1 ml of CAB reagent was added

Test tube was observed for 30 minutesTest tube was observed for 30 minutes

ResultsResults

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

DiscussionDiscussion CetyltrimethylammoniCetyltrimethylammoni

um bromide (CTAB)um bromide (CTAB) Is a Is a Quaternary Quaternary

ammonium salt ammonium salt An ammonium salt in An ammonium salt in

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

Quaternary ammonium compounds fix soluble Quaternary ammonium compounds fix soluble acid mucopolysaccharides (e.g., mucin) by acid mucopolysaccharides (e.g., mucin) by forming highly insoluble complexes forming highly insoluble complexes For example, an aqueous solution of heparin For example, an aqueous solution of heparin

sodium salt is mixed with the quaternary sodium salt is mixed with the quaternary ammonium salt ammonium salt

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

Lysosomal storage disorders are variably Lysosomal storage disorders are variably referred to as simply storage disorders, referred to as simply storage disorders, lipidoses, mucopolysaccharidoses, or lipidoses, mucopolysaccharidoses, or mucolipidosesmucolipidosesCaused by deficiencies of enzymes which are Caused by deficiencies of enzymes which are

involved in the degradation sequence of GAGsinvolved in the degradation sequence of GAGs Diagnosis is through urine testing for the Diagnosis is through urine testing for the

presence of increased amounts of GAGspresence of increased amounts of GAGs

Storage Disorder Clinical Featuresa Enzyme Deficiency

Current DiagnosticApproachb,c

Hurler’s 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.

α -L-Iduronidase

Quantitative enzyme assay.

Demonstration of excess urinary

mucopolysaccharide consisting of dermatan and

heparan sulfatase.

Scheie’s syndrome(MPS Ia)

A mild form of MPS I with corneal opacity; mild or absent mental

retardation; claw-hand deformity; aortic stenosis.

α -L-Iduronidase

Hunter’s syndrome(MPS II)

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

in the severe forms

Iduronate sulfatasesX-linked

Morquio’s disease(MPS IVa)

Pronounced skeletal anomalies with small stature (short-trunk dwarfism);

short neck; prominent lower ribs; odontoid anomalies; normal intellect.

Galactosamine-6-sulfate

sulfatase

Demonstration of keratin sulfate in urine. Due to

phenotype: go directly to quantitative enzyme assay.


Ninhydrin TestNinhydrin Test


CC99HH66OO44

Appearance: white pale yellow crystalsAppearance: white pale yellow crystalsa chemical used to detect a chemical used to detect ammoniaammonia or primary or primary

and secondary and secondary aminesaminesproduces a deep blue or purple colorationproduces a deep blue or purple colorationNinhydrin Reagent Solution:Ninhydrin Reagent Solution:

Ninhydrin: 0.35gNinhydrin: 0.35gethanol or acetone/butanol ethanol or acetone/butanol so-propanol: 100mlso-propanol: 100ml

Ninhydrin (Triketohydrindane hydrate)

IntroductionIntroductionfor detection and quantification of amino for detection and quantification of amino

acids in substancesacids in substancesfor experiment: in urinefor experiment: in urinecan be used qualitatively can be used qualitatively (e.g. for (e.g. for

chromatographic visualization) chromatographic visualization) or quantitatively or quantitatively (e.g. for peptide sequencing)(e.g. for peptide sequencing)

once used for once used for fingerprint detection fingerprint detection (forensics)(forensics): amines leftover from peptides & : amines leftover from peptides & proteins (terminal amines or lysine proteins (terminal amines or lysine residues) sloughed off in residues) sloughed off in fingerprints react with ninhydrinfingerprints react with ninhydrin

Observe color

Violet: alpha amino acid

Yellow: proline

1 ml Ninhydri

n reagent

3 drops of urine

Warm for 30 secs. in water bath

MethodologyMethodology

Experimental ResultsExperimental ResultsGroup 1: (-) colorlessGroup 2: (+) slightly bluish coloration

Reaction MechanismReaction Mechanism

ninhydrin (or triketohydrindene hydrate) ninhydrin (or triketohydrindene hydrate) is a strong oxidizing agentis a strong oxidizing agent

amines (including all physiological amines (including all physiological αα--amino acids) react with itamino acids) react with it

cause oxidative deamination of cause oxidative deamination of αα-amino -amino acid functionacid function

products: aldehyde, ammonia, carbon products: aldehyde, ammonia, carbon dioxide, hydrindantindioxide, hydrindantin

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

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

alpha-keto acid + NH3 aldehyde + CO2

ReactionsReactions

Reaction MechanismReaction Mechanismammonia can react with hydrindantin and ammonia can react with hydrindantin and

another molecule of ninhydrinanother molecule of ninhydrinyields a bluish-purple product (Ruhemann’s yields a bluish-purple product (Ruhemann’s

purple; can be measured at 570 nm)purple; can be measured at 570 nm)αα-imino acids, e.g. proline and -imino acids, e.g. proline and

hydroxyproline: bright yellow-orange (440 hydroxyproline: bright yellow-orange (440 nm absorbance)nm absorbance)

Indication of ResultsIndication of Resultsbluish-purple solution: presence of amino bluish-purple solution: presence of amino

acids in urineacids in urineyellow-orange: presence of prolineyellow-orange: presence of prolineamino aciduria is presentamino aciduria is present

Amino AciduriaAmino Aciduriaincreased levels of amino acid excretion in increased levels of amino acid excretion in

the urine the urine indicates possible inborn errors of indicates possible inborn errors of

metabolism caused by a specific enzyme metabolism caused by a specific enzyme deficiency (e.g. Hartnup’s disease, deficiency (e.g. Hartnup’s disease, Tyrosinemia)Tyrosinemia)


Millon’s TestMillon’s Test

Millon’s TestMillon’s Test Used for the detection of Used for the detection of

phenol in the solutionphenol in the solution Indicates the presence of Indicates the presence of

Tyrosine in the urineTyrosine in the urine Millon’s Reagent: solution of Millon’s Reagent: solution of

mercuric and mercurous ions mercuric and mercurous ions in nitric and nitrous acids in nitric and nitrous acids

(+) Result: Peach to Red (+) Result: Peach to Red colored solutioncolored solution

Nitration of Phenol group Nitration of Phenol group which leads to a complexation which leads to a complexation reaction with Hg (I) and Hg (II) reaction with Hg (I) and Hg (II) ionsions

MethodologyMethodologyAdd 1 drop of Millon’s reagent to 1 mL

urine Water bath for 15 minutes

TyrosyluriaTyrosyluriaaccumulation of excess tyrosine in the plasma accumulation of excess tyrosine in the plasma

(tyrosinemia) producing urinary overflow (tyrosinemia) producing urinary overflow two actions directly involved: 1) contain excess two actions directly involved: 1) contain excess

tyrosine 2) its degradation p-tyrosine 2) its degradation p-hydroxyphenylpyruvic and p-hydroxyphenyllactic hydroxyphenylpyruvic and p-hydroxyphenyllactic acidacid

most frequently seen is a transitory tyrosinemia most frequently seen is a transitory tyrosinemia in premature infants, caused by in premature infants, caused by underdevelopment of liver function underdevelopment of liver function

severe liver disease = tyrosyluriasevere liver disease = tyrosyluriaurine sediments may show tyrosine and leucine urine sediments may show tyrosine and leucine

crystalscrystalshereditary and metabolic disorders : liver and hereditary and metabolic disorders : liver and

renal diseaserenal disease

Paper Chromatography Paper Chromatography of Amino Acidsof Amino Acids

Paper Paper ChromatographyChromatographymethod for testing the purity of compounds method for testing the purity of compounds

and identifying substancesand identifying substancesuseful technique because it is relatively useful technique because it is relatively

quick and requires small quantities of quick and requires small quantities of materialmaterial

used for screening patients for possible used for screening patients for possible amino acid abnormalitiesamino acid abnormalities

Paper Paper ChromatographyChromatographysubstances are distributed between a substances are distributed between a

stationary phase and a mobile phasestationary phase and a mobile phasestandard mixtures of pure amino acids are standard mixtures of pure amino acids are

run at the same time as the unknown run at the same time as the unknown (urine sample)(urine sample)

positions of the spots are comparedpositions of the spots are comparedpreliminary preparation of urine samples: preliminary preparation of urine samples:

to remove salts and proteinsto remove salts and proteins

MethodologyMethodologySpot 6 – 7 x, Dry in between applicationsSpot 6 – 7 x, Dry in between applications

Spot as small as possible Spot as small as possible (too big spots – samples (too big spots – samples overlap & influence movement of solute) overlap & influence movement of solute)

Place in chromatographic jarPlace in chromatographic jar

Solvent front Solvent front top of paper top of paper

Air dryAir dry

Spray with Ninhydrin Spray with Ninhydrin

Place in hot plate until spots are visiblePlace in hot plate until spots are visible

Measure distance travelled by amino acidsMeasure distance travelled by amino acids

Solvent system: ETHANOL: NH4OH: HSolvent system: ETHANOL: NH4OH: H22OO

(8:1:1)(8:1:1)

Experimental Experimental ResultsResults

Protein Distance Travelled

Rf Value

Cysteine 1 cm 5.9

Glycine 5.1 cm 30

Proline 9.5 cm 55.9

Tyrosine 8.4 cm 49.4

Tryptophan 10 cm 58.8

Unknown 10.1 cm 59.4


Distance traveled by solvent = 17cmran off the paper: margins were disregarded

Unknown = Tryptophan

Urine Test

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