Urine Screening for Metabolic Disorders PDF

ADI Analysis of urine and other body fluids URINE SCREENING FOR METABOLIC DISORDERS Urine is the end product of body metabolism. Major Disorders of CHON and CHO Metabolism Protein and carbohydrate metabolism. Associate...

ADI Analysis of urine and other body fluids URINE SCREENING FOR METABOLIC DISORDERS Urine is the end product of body metabolism. Major Disorders of CHON and CHO Metabolism Protein and carbohydrate metabolism. Associated with Abnormal Urinary Constituents Inborn error of metabolism, disease, toxic Classified as to Functional Defect substances. Inherited Metabolic Renal Abnormal metabolic constituents or conditions Phenylketonuria Tyrosinemia Hartnup detected in routine urinalysis Disease Tyrosinemia Melanuria Cystinuria Alkaptonuria Indicanuria MSUD 5- Hydroxyindole Acetic Acid Organic acidemias Porphyria Cystinosis Porphyria Mucopolysaccharidoses Melituria (Galactosuria) Lesch- Nyhan Disease Amino Acid Disorder: PTAMOICC Phenylketonuria (PKU) Tyrosinuria Alkaptonuria Melanuria MSUD Organic acidemias Indicanuria Cystinuria Cystinosis I. PHENYLALANINE- TYROSINE DISORDERS PHENYLKETONURIA (PKU) Associated with a disorder in Phenylalanine- Tyrosine metabolic pathway (assignment) Failure to inherit the gene to produce phenylalanine hydroxylase enzyme. Color: PHIM PKU: 1 in every 10,000 – 20,000 births Homogentisic acid Detected as early as 4 hrs after birth. Melanin NV: lowered from 4mg/dL to 2 mg/dL Indican Most well- known of the aminoacidurias Porphyrin May cause mental retardation Odor: PHIMCC Newborn screening should be done to all Phenylketonuria newborns before discharge from the MSUD hospital. Isovaleric acidemia Early catch of PKU: dietary restriction of Cystinuria phenylalanine Cystinosis Urine testing – follow up procedure in Homocystinuria questionable diagnostic cases. Crystal: CLLT Bacterial inhibition using Bacillus subtilis Cystine streaked in culture media – most well- known blood test for PKU. Leucine Tyrosine Lesch- Nyhan Disease ADI Guthrie Procedure : Developed by Robert ALKAPTONURIA Guthrie. Failure to inherit the gene that produces the enzyme Blood from heelstick is placed and absorbed HOMOGENTISIC ACID OXIDASE into a filter paper circle and placed into a culture Urine darkens after standing at room media streaked with B. subtilis organism. temperature If phenylalanine is present in increased level, it In the absence of the enzyme phenylalanine- will counteract the action of beta-2- tyrosine, pathway is not completed and thienylalanine, an inhibitor of B. subtilis Homogentisic acid accumulates in the blood,.Positive result – growth around the paper disk. tissues and urine. increase level of Phenylalanine Observe for: Brown or black stained cloth diaper Other method for Phenylalanine: Reddish stained disposable diaper Automated technique -measures the Observe for: fluorescence of phenylalanine when heated in In later life – brown pigment deposited in the presence of Ninhydrin and L-leucyl-L- the ear and cartilages leading to arthritis alanine or Glycyl-L-leucine Persons with alkaptonuria develop liver and Test for Phenylpyruvic acid cardiac disorder Procedure: Ferric chloride tube test Place 1mL of urine in a tube Screening tests for Alkaptonuria: Slowly add five drops of 10% Ferric chloride Ferric chloride test: transient deep blue color Positive result: permanent blue-green color Benedict’s test – yellow precipitate Add alkali to freshly voided urine – dark color TYROSYLURIA ascorbic acid- interfering substance Inherited or due to metabolic defect Add silver nitrate and ammonium hydroxide – Tyrosinemia – accumulation of excess tyrosine in the dark urine color. plasma Spectrophotometry – quantitative measurement of Tyrosiluria - Urinary overflow of tyrosine homogentisic acid Degradation products of tyrosine that maybe Chromatography present in the urine: 1. p-hydroxyphenylpyruvic acid MELANURIA 2. P-hydroxyphenyllactic acid Melanin – responsible for dark color of hair, skin and eyes Maybe seen in: Metabolism of melanin follows the same (1) Premature infants due to underdeveloped liver pathway function. Deficient production leads to ALBINISM Tyrosinemia may be confused with PKU due Elevated urine melanin to positive Ferric chloride test but gives a Urine darkens after exposure to air or over green color that fades rapidly. (Transient proliferation of melanocytes (melanin- tyrosinemia) producing cells) causing malignant melanoma (2) Acquired liver disease – Tyrosiluria Malignant Melanoma: a tumor that secrets 5, (3) Hereditary disorder- absence of the enzyme 6- dihydroxyindole which oxidizes to tyrosine transaminase may lead to fatal condition melanogen then to melanin. resulting to liver and renal disease TEST FOR MELANIN Tyrosine and Leucine crystals seen in both cases 1. Ferric chloride test – gray or black precipitate 2. Na Nitroprusside (nitroferricyanide) (ACETONE Screening test for tyrosine and its metabolites: REAGENT STRIP): red color Nitroso-Naphthol Test (procedure assignment) Interference: red color from ACETONE and Nitroso-Naphthol Test for Tyrosine CREATININE 1. Place five drops of urine in a tube. Add glacial acetic acid 2. Add 1 mL of 2.63N nitric acid. Melanin – revert to green- black color 3. Add one drop of 21.5% sodium nitrite. Acetone – turns purple 4. Add 0.1 mL 1-nitroso-2-napthol. Creatinine – becomes amber 5. Mix. *Differentiate between Melanin and Homogentisic 6. Wait 5 minutes. acid 7. Observe for an orange-red color, indicating tyrosine metabolites. Positive result: Orange- red color ADI II. BRANCHED- CHAIN AMINO ACID ✓ Chromatography: Screening test for ✓ Positive ketonuria in newborn ISOVALERYLGLYCINE significant finding in Branched Chain AA Disorders MAPLE SYRUP URINE DISEASE (MSUD) PROPIONIC AND METHYLMALONIC rare disease ACIDEMIAS failure to thrive after 1 week error in metabolic pathway converting Isoleucine, inborn error of metabolism involving 3 amino Valine, Threonine, and Methionine to succinyl acids coenzyme A 1. Leucine p-Nitroaniline test 2. Isoleucine Screening test for Methylmalonic aciduria 3. Valine ▪ emerald green color Metabolic pathway: transamination III. TRYPTOPHAN DISORDER 3 amino acid keto acids Pathway (assignment) liver INCREASED urinary excretion of Indican and 5- 3 keto acids hydroxyindoleacetic acid (5-HIAA) 1. a-ketoisovaleric INDICANURIA 2. a-ketoisocaproic presence of indican in urine 3. B-methylvaleric o see pathway (assignment) Failure to inherit the gene to produce Colorless - Indican in urine oxydative decarboxylation of the 3- keto acids Exposure to air – oxidized to INDIGO BLUE. result to accumulation in the blood and urine. TEST FOR INDICAN Suspect if the urine has a STRONG MAPLE o Ferric chloride test: deep blue or violet color SYRUP ODOR – resulting from rapid 5- Hyroxyindoleacetic acid accumulation of ketoacids in the urine. - see pathway (assignment) Early detection and reporting can prevent Production of serotonin (carried by platelets MENTAL RETARDATION and DEATH. throughout the body) by Argentaffin cells in the DETECTION ON THE 11TH DAY with intestine – stimulation of smooth muscles dietary restriction and monitoring can lead to NV of 5-HIAA : 2-8 mg daily excretion better prognosis. Argentaffin cell tumor : 160 – 628 mg/24 hrs Screening test Test for 5-HIAA: 2,4-dinitrophenylhydrazine (DNPH) + urine = yellow nitrous acid + 1-nitroso-2-naphthol + urine = purple to turbidity or precipitate (+ keto acids) black color Interfering substance: Ampicillin (large dose) use random, first AM urine or 24hr urine sample preserved w/ HCL or boric acid DNPH not specific for MSUD Foods containing serotonin: Banana, pineapple and positive in PKU tomatoes. Positive in urine with ketones Restrict patients from these food prior to test ✓ Confirmatory test: chromatography Interfering substances: medication like phenothiazine, acetanilids. – 72 hrs restriction ORGANIC ACIDEMIAS 3 most commonly encountered organic acidemias HARTNUP DISEASE 1. Isovaleric acidemia “BLUE DIAPER SYNDROME” 2. Propionic acidemia Affects intestinal reabsorption of tryptophan and renal 3. Methylmalonic acidemia tubular reabsorption of amino acids Generalized symptoms of Organic Acidemia Good prognosis if w/ proper diet supplementation with 1. Early severe illness Niacin 2. Vomiting 3. Metabolic acidosis - hypoglycemia - ketonuria - increased serum ammonia ISOVALERIC ACIDEMIA SWEATY FEET ODOR Accumulation of ISOVALERYLGLYCINE due to deficiency of Isovaleryl coenzyme A in the Leucine pathway ADI IV. CYSTINE DISORDER PORPHYRINURIA 2 TYPES red or port wine urine CYSTINURIA – defect in the renal tubular transport of amino acids Most common Porphyrias CYSTINOSIS – in born error of metabolism Porphyria Elevated Clinical Laboratory Compound Symptom Testing Acute ALA Neurologic/ Intermittent CYSTINURIA Intermittent Porphobilinogen Psychiatric Elevated AA CYSTINE in urine due to inability of Porphyria renal tubules to reabsorb cystine filtered by the Porphyria Uroporphyrin Photosensitivity Urine glomerulus. Cutanea Tarda fluorescence 65% persons with Cystinuria develop urinary calculi Congenital Uroporphyrin Photosensitivity Urine or feces early in life. Erythropoietic Coproporphyrin fluorescence Screening Test Porphyria Chromatography Variegate Coproporphyrin Photosensitivity Bile or feces Cyanide– Nitroprusside test = red-purple color Porphyria / Neurologic fluorescence False positive: Presence of ketones and homocystine Erythropoietic Protoporphyrin Photosensitivity Bile or feces Protoporphyria fluorescence CYSTINOSIS (IEM) Lead Poisoning ALA Neurologic Urine in born error of metabolism (aminolevulinic porphobilinogen incomplete metabolism of cystine result to acid) Ehrlich’s reaction deposition in cornea, bone marrow, lymph nodes, Protoporphyrin Blood FEP internal organs. Specimens used to detect specific disorder: Lead to renal failure 1. Urine – ALA (a-aminolevulinic acid), Lab findings: PGPL porphobilinogen, uroporphyrin (3 soluble) Polyuria - coproporphyrin (less soluble) Generalized aminoaciduria 2. Feces – coproporphyrin and protoporphyrin Positive test– reducing substances 3. Bile– more acceptable specimen for coproporphyrin Lacks urinary concentration and protoporphyrin to avoid iron interference HOMOCYSTINURIA: FCMTD 4. Blood – free erythrocyte protoporphyrin (FEP) defect in metabolism screening for lead poisoning Failure to thrive Ehrlich’s reaction Cataracts ALA Mental retardation Porphobilinogen Thromboembolism Acetyl acetone + Urine ALA to Porphobilinogen Death Fluorescence technique Confirmatory test: Silver Nitroprusside test Uses UV light from 550 to 600 nm range Use fresh urine sample Negative: faint blue fluorescence Positive: violet, pink, red depends on porphyrin V. PORPHYRIN DISORDER concentration in urine PORPHYRIAS Collective term for disorders of porphyrin metabolism VI. MUCOPOLYSACCHARIDE DISORDER PORPHYRINS (uroporphyrin, coproporphyrin and Mucopolysaccharides protoporphyrin) are important components of heme Glycosaminoglycans synthesis. compounds located in the connective tissues Blockage or disruption at any level can cause disorder can lead to severe mental retardation accumulation of products prior to interruption. Screening test: Acquired- from erythrocytic and hepatic malfunction 1. Acid-albumin test – white turbidity after 30min or exposure to toxic agents. 2. Cetyltriethylammonium bromide (CTAB) turbidity examples: test – white turbidity after 5 min lead poisoning Grade in the scale of 0 - 4 excessive alcohol exposure 3. Metachromatic staining spot test – blue spot iron deficiency liver and renal disease Inherited more rare than acquired failure to inherit the gene that produces enzyme needed in the pathway. Note: Suspect Congenital Porphyria – red discoloration in the infant’s diaper ADI VII. PURINE DISORDER Lesch- Nyhan Disease a disorder of purine metabolism resulting in massive excretion of URIC ACID CRYSTAL. failure to inherit the gene to produce the enzyme Hypoxanthine Guanine Phosphoribosyltransferase – accumulation of Uric Acid throughout the body Effects of purine disorder: SMSGR Severe motor defects Mental retardation Self- destruction Gout Renal calculi Normal development for the first 6 to 8 months Orange sand in diaper – uric acid crystals VIII. CHO DISORDER Melituria increased urinary sugar Screening tests for reducing sugars: Benedict’s test Clinitest copper reduction test Positive Copper reduction test + negative reagent strip glucose oxidase test = CHO metabolism disorder Galactosemia: LICS infant failure to thrive liver disorder cataracts severe mental retardation Galactosuria inability to metabolize galactose to glucose Note: remove lactose (precursor of galactose) from diet Lactosuria- may be seen during pregnancy and lactation. Fructosuria- is associated with parenteral feeding and pentosuria with ingestion of large amounts of fruit. Pentosuria - Chromatography should be done in the presence of nonglucose- reducing sugars in pediatric patients Lactose screening test (assign procedure) – brick red precipitate Fructose screening test (assign procedure)– red precipitate