Urine Test
Transcript of Urine Test
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Urine Metabolic Urine Metabolic Screening TestScreening Test
Experiment 13Experiment 13
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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
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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
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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
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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
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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
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UrineUrine good specimen of choice for the detection
of most metabolic disturbances• accumulated metabolites “spill over”
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Routine UrinalysisRoutine Urinalysis
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IntroductionIntroduction
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
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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
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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)
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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
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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: (-)
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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
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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
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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
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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
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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
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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
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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
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Ferric Chloride TestFerric Chloride Test
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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
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Ferric Chloride Test : Ferric Chloride Test : ReactionReaction
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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)
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Ferric Chloride TestFerric Chloride Test
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
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Ketone Phenylpyruvic Ketone Phenylpyruvic AcidAcid
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Ferric Chloride TestFerric Chloride Test
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
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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
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PhenylketonuriaPhenylketonuria
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Materials and Materials and MethodsMethods
1 ml FeCl3 reagent1 ml FeCl3 reagent 10 drops urine10 drops urine
• shakeshake• observe resultobserve result
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PICTURES
GAB OLIVE
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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
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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
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Experimental ResultsExperimental Results
Both urine samples (GAB&OLIVE): NEGATIVE
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Benedict’s TestBenedict’s Test
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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
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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
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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
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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
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ResultsResults
No precipitate ----
Green a trace
Yellow +
Orange ++
Red +++
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5-mL of Benedict’s reagent
8 drops of urine
Heat to boiling
Test tube
Add
MethodologyMethodology
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Boil again
Color change
After 2 minutes
Results noted as:(-) --> BLUE
(+) --> GREEN to YELLOW(++) --> YELLOW to BROWN
(+++) --> BROWN to ORANGE(++++) --> ORANGE to RED
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Experimental ResultsExperimental Results
Positive sample
Experimental 1
(++++) (-)
Group 1Group 1
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Positive sampleExperimental 2
Group 2Group 2
(++++) (+)
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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
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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
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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
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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
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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.
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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.
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Nitrosonaphthol TestNitrosonaphthol Test
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Nitrosonaphtol TestNitrosonaphtol Test
Screening for inborn error of TYROSINE Screening for inborn error of TYROSINE metabolismmetabolism
TyrosineTyrosine
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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
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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
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Nitrosonaphtol TestNitrosonaphtol TestResultsResults
Experimental:Experimental: Standard:Standard:(-) result(-) result (+) result(+) result
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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
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Nitrosonaphtol TestNitrosonaphtol TestFalse Positive ResultFalse Positive Result IV fluids containing homovanillic acidIV fluids containing homovanillic acid 5-HIAA5-HIAA N-acetyltyrosineN-acetyltyrosine
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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
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Nitroprusside TestNitroprusside Test
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IntroductionIntroduction
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
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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
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A person may have these disorders if he tests A person may have these disorders if he tests positive positive
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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
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Experimental ResultsExperimental Results The solution produced was yellow. Hence,
the subject tested negative for the test.
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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
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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.
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Positive Test Result+ - pink++ - pinkish+++ - purple++++ - dark purple
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Cetyltrimethylammonium Cetyltrimethylammonium Bromide Test (CAB)Bromide Test (CAB)
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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
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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
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ObjectiveObjective
To determine the presence of urinary To determine the presence of urinary mucopolysaccharides and mucopolysaccharides and glycosaminoglycans in the urineglycosaminoglycans in the urine
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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
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ResultsResults
Team 1: no turbidity observed (0)Team 1: no turbidity observed (0) Team 2: positive turbidity (1)Team 2: positive turbidity (1)
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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
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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
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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
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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.
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Ninhydrin TestNinhydrin Test
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IntroductionIntroduction
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)
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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
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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
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Experimental ResultsExperimental ResultsGroup 1: (-) colorlessGroup 2: (+) slightly bluish coloration
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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
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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
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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)
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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
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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)
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Screening Tests for Certain Screening Tests for Certain Inborn Errors of MetabolismInborn Errors of Metabolism
Millon’s TestMillon’s Test
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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
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MethodologyMethodologyAdd 1 drop of Millon’s reagent to 1 mL
urine Water bath for 15 minutes
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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
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Paper Chromatography Paper Chromatography of Amino Acidsof Amino Acids
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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
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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
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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
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Solvent system: ETHANOL: NH4OH: HSolvent system: ETHANOL: NH4OH: H22OO
(8:1:1)(8:1:1)
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Experimental ResultsExperimental Results
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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
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Experimental ResultsExperimental Results
Distance traveled by solvent = 17cmran off the paper: margins were disregarded
Unknown = Tryptophan