Urine Screening for Metabolic Disorders - PDF

Urine Screening for Metabolic Disorders Medical Laboratory Science Program College of Allied Medical Professions Lyceum of the Philippines University - Batangas  COPYRIGHT NOTICE This material has been reproduced and communicated to you or on behalf of...

Urine Screening for Metabolic Disorders Medical Laboratory Science Program College of Allied Medical Professions Lyceum of the Philippines University - Batangas  COPYRIGHT NOTICE This material has been reproduced and communicated to you or on behalf of Lyceum of the Philippines University- Batangas pursuant to PART IV: The Law of Copyright of Republic Act RA 8293 or the "Intellectual Property Code of the Philippines". The University does not authorize you to reproduce or communicate this material. The material may contain works that are subject to copyright protection under RA 8293. Any reproduction, and/or communication of the material by you may be subject to take legal action against such infringement. Do not remove this notice. This material is prepared by the Faculty of the Department of Medical Laboratory Science, LPU-Batangas College of Allied Medical Professions, solely for the use of students enrolled in MLS 303 for 1st semester, A.Y. 2024-2025. Please do not distribute without permission. Metabolic Disorders I. Amino Acid Disorders PKU, tyrosyluria, alkaptonuria, melanuria, MSUD, organic acidemias, indicanuria, cystinuria and cystinosis II. Porphyrin Disorders III. Mucopolysaccharide Disorders Hurler's, Hunter's, Sanfilippo syndromes IV.Purine Disorders V. Carbohydrate Disorders 10/14/20 24  Overflow VS. Renal Disorders  Two major causes of abnormal metabolic substance appearance in the urine: 1. Overflow Type  Results from disruption of the normal metabolic pathway that causes increased plasma concentration of the nonmetabolized substance  Causes: Inborn error of metabolism or organ malfunction  Increased amino acid in blood & urine 2. Renal Type  Due to malfunctions in the tubular reabsorption mechanisms  Normal amino acid in blood ; increased amino acid in urine 10/14/20 24 Metabolic Disorders  Conditions detected by routine urinalysis:  Color: Homogentisic acid, melanin, indican, porphyrins  Odor: Phenylketonuria, Maple syrup urine disease, isovaleric acidemia, cystinuria, cystinosis, homocystinuria  Crystals: Cystine, leucine, tyrosine, Lesch-nyhan disease 10/14/20 24 Metabolic Disorders  Newborn Screening ✓ Urine testing: traditionally done to detect and monitor newborns for IEMs ✓ Blood testing: modern (elevates more rapidly in blood) Detection: Tandem mass spectrophotometry (MS/MS) ✓ MSUD Most common inborn error of metabolism in the Philippines 6th and latest newborn screening marker 10/14/20 24 Newborn Screening Test MS/MS is capable of screening the infant blood sample for specific substances associated with particular IEMs. 10/14/20 24  Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders B. Branched-Chain Amino Acid Disorders C. Tryptophan Disorders D. Cystine Disorders 10/14/20 24  Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders 1. Phenylketonuria (PKU) Most well-known of the aminoacidurias, occurs in 1 of every 10,000 to 20,000 births Results in severe mental retardation if undetected Failure to inherit the gene to produce phenylalanine hydroxylase Increased phenyalalanine in the blood can be detected as early as 4 hours after birth & if cut-off value is lowered from 4mg/dL to 2 mg/dL Urine: increased keto-acids (phenylpyruvate) (responsible for the mousy odor of urine and 10/14/20 24 sweat) Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders 1. Phenylketonuria (PKU)  Fair complexion due to decreased production of tyrosine and its metabolite melanin  Once discovered, dietary changes that eliminate phenylalanine, a major constituent of milk prevents excessive buildup in serum to avoid damage to child’s mental capabilities  Many products with large amounts of phenylalanine such as aspartame now features warning labels  More girls than boys escape detection of PKU during early tests because of slower rises in blood phenylalanine levels 10/14/20 24  Amino Acid Disorders ❖ Tests for PKU: 1. Guthrie’s Test (Bacterial Inhibition):  Specimen: Blood from heel puncture  Uses culture medium streaked with Bacillus subtilis which also contains Beta-2-thienylalanine, an inhibitor of Bacillus subtilis  Presence of increased phenylalanine neutralizes Beta-2-thienylalanine in the media allowing the growth of B. subtilis  (+) GROWTH around paper strip (increased phenylalanine) 2. Ferric chloride Tube Test : permanent blue-green color (phenylpyruvic acid is present) 3. Phenistix reagent strips: contain ferric ammonium sulfate, magnesium sulfate, and cyclohexylsulfamic acid 10/14/20 (+) gray to gray-green color after 30 secs 24 10/14/20 24  Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders 2. Tyrosyluria Occurs as a consequence of tyrosinemia Urinary metabolites: p-hydroxyphenylpyruvic acid, p- hydroxyphenyllactic acid Transitory tyrosinemia: most frequently seen in premature infants In severe liver diseases, tyrosyluria may be accompanied by tyrosine and cystine crystals 10/14/20 24 Tyrosine Crystals 10/14/20 24 Leucine Crystal 10/14/20 24  Amino Acid Disorders Three types: Type 1: fumarylacetoacetate hydrolase (FAH) deficiency Generalized tubular disorder Progressive liver failure in infants Type 2: tyrosine aminotransferase deficiency corneal erosions lesions of the palms, fingers and soles of the feet Type 3: p-hydroxyphenylpyruvic acid dioxygenase deficiency Mental retardation if dietary restriction is not 10/14/20 24 implemented Amino Acid Disorders  Screening tests using MS/MS are available for tyrosinemia types 1, 2, and 3.  Tests for tyrosyluria: 1.Nitroso-naphthol: orange-red (primary screening test) 2.FeCl3 Tube Test: transient green (tyrosine present) 10/14/20 24  Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders 3. Melanuria Melanin: pigment responsible for dark color of hair, skin and eyes Albinism: deficient production of melanin Darkening of urine upon exposure to air ✓5,6-dihydroxyindole (colorless precursor) → melanogen → melanin (dark urine) 10/14/20 24 Amino Acid Disorders Tests for Melanuria 1. FeCl3 Tube Test : gray or black ppt 2. Sodium nitroprusside (rgt strip) : red ✓Acetone and creatinine also produces red color ✓Remedy: Add glacial acetic acid melanin will revert back to green-black color acetone turns purple creatinine turns amber 3. Ehrlich’s : red 10/14/20 24  Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders 4. Alkaptonuria  Failure to inherit the gene to produce the homogentisic acid oxidase leading to accumulation of homogentisic acid accumulates in the blood, tissues, and urine. Darkening of urine after alkalinization Urinary metabolite: homogentisic acid 10/14/20 24 Amino Acid Disorders A. Phenylalanine-Tyrosine Disorders 4. Alkaptonuria Brown-stained or black stained cloth diapers or reddish-stained disposable diapers In later life, brown pigment becomes deposited in the body tissues (particularly noticeable in the ears). Deposits in the cartilage eventually lead to arthritis. A high percentage of persons with alkaptonuria develop liver and cardiac 10/14/20 disorders 24 Amino Acid Disorders Tests for Alkaptonuria: 1.FeCl3 Tube Test : transient deep blue color 2.Clinitest : yellow ppt 3.Addition of alkali to urine: darkening 4.Addition of silver nitrate and ammonium hydroxide to urine: Black color 10/14/20 24 Amino Acid Disorders B. Branched-Chain Amino Acid Disorders  Two types: 1. Accumulation of one or more of EARLY amino acid degradation products: MSUD 2. Accumulation of LATE amino acid metabolites: Organic Acidemias 10/14/20 24  Amino Acid Disorders B. Branched-Chain Amino Acid Disorders 1. Maple Syrup Urine Disease (MSUD) Deficient enzyme: Branched-chain alpha-keto acid dehydrogenase Urinary metabolite: keto acids Accumulation of 3 AA in blood & urine: Leucine, Isoleucine, Valine 10/14/20 24  Amino Acid Disorders B. Branched-Chain Amino Acid Disorders 1. Maple Syrup Urine Disease (MSUD) Newborns: Failure to thrive clinical symptoms after approx.1 week Strong maple syrup odor due to rapid accumulation of ketoacids in urine Notify physician of unusual odor to prevent severe mental retardation and death 2,4-dinitrophenylhydrazine (DNPH) : yellow turbidity or precipitate 10/14/20 24  Amino Acid Disorders B. Branched-Chain AA Disorders 2. Organic Acidemias Generalized symptoms include early severe illness, vomiting, metabolic acidosis, hypoglycemia, ketonuria and increased serum ammonia Three types: Isovaleric: sweaty feet odor of urine caused by accumulation of isovalerylglycine due to deficiency in isovaleryl coenzyme A in leucine pathway Propionic Methylmalonic acidemia 10/14/20 24 Amino Acid Disorders B. Branched-Chain AA Disorders 2. Tests for Organic Acidemias No urine screening test for isovaleric and propionic acidurias P-nitroaniline test available for methylmalonic aciduria (+) result: EMERALD GREEN color 10/14/20 24  Amino Acid Disorders C. Tryptophan Disorders 1. Indicanuria Presence of blue urine upon exposure to air Indican (colorless → indigo blue(blue)) CAUSES (all leads to production of excess indole in the intestine) Intestinal obstruction Presence of abnormal bacteria Malabsorption syndromes Hartnup’s disease (Blue Diaper Syndrome) - also leads to Fanconi syndrome Ferric Chloride: Deep blue or violet color 10/14/20 24  Amino Acid Disorders C. Tryptophan Disorders 2. 5-Hydroxyindoleacetic Acid (5-HIAA) Increased 5-HIAA in the urine is secondary to argentaffinoma (increased serotonin production) Recommended specimen: 24-hour urine preserved using HCl or boric acid Consumption of 5-HIAA rich foods such as bananas, pineapples, and tomatoes contain high amounts of 5-HIAA are restricted before testing Medications (phenothiazines, acetanilids) are also withheld for 72 hours before specimen collection Addition of nitrous acid and 1-nitroso-2-naphthol : purple to black 10/14/20 24  Amino Acid Disorders D. Cystine Disorders 1. Cystinuria Two mode of inheritance: Defect in reabsorption of Cystine, Ornithine, Lysine, Arginine (COLA) - more prone to renal calculi formation (65% risk) Defect in reabsorption of Cystine and Lysine Cystine is least soluble and hence prone to precipitating in the urine and become 10/14/20 calculi 24 Amino Acid Disorders D. Cystine Disorders 2. Cystinosis  True Inborn error of metabolism  Two categories Nephropathic: subdivided into infantile (rapid) and late-onset (gradual) cystinosis Nonnephropathic: benign but may cause ocular disorders  Characterized by intracellular cystine crystal deposition within lysosomes due to membrane defect  Crystals may accumulate in the cornea, bone marrow, lymph nodes, internal organs  Fanconi’s syndrome may occur as a complication of cystinosis 10/14/20 24 Amino Acid Disorders Cyanide-Nitroprusside Test / Brand’s modification of the Legal nitroprusside reaction Cystine is reduced to cysteine by sodium cyanide which then reacts with nitroprusside to produce a red-purple color Homocystine and ketones also positive 10/14/20 24  Amino Acid Disorders D. Cystine Disorders 3. Homocystinuria Defects in the metabolism of homocystine result due to deficiency of the enzyme cystathionine β- synthase Clinical manifestations: Failure to thrive, cataracts, mental retardation, thromboembolic problems and death Cyanide-Nitroprusside: Cystine, Ketones and homocysteine react Silver Nitroprusside Test (only homocysteine react): 10/14/20 red-purple 24  Porphyrin Disorders  Porphyrins are the intermediate compounds in the production of heme.  Detected in urine: d-aminolevulinic acid (ALA), porphobilinogen, uroporphyrin (most soluble), coproporphyrin (less soluble)  Detected in feces: coproporphyrin, protoporphyrin recommended sample for detection: BILE  CDC recommends analysis of whole blood for the presence of free erythrocyte protoporphyrin (FEP) as a screening test for lead poisoning. 10/14/20 24 10/14/20 24 10/14/20 24 Porphyrin Disorders Two types: Acquired Porphyrias: Causes are lead poisoning, excessive alcohol intake, iron deficiency, chronic liver disease and renal disease Inherited (rare) Porphyrias: caused by failure to inherit the gene that produce the needed enzyme in the metabolic pathway An indication of the possible presence of porphyrinuria is the observation of a red or port wine color to the urine after exposure to air. 10/14/20 24 Porphyrin Disorders  Tests for porphyrinuria: 1. Ehrlichs reaction: ALA and porphobilinogen  ALA must be converted first to porphobilinogen by addition of acetylacetone 2. Fluorescence under UV (550-600nm): uroporphyrin, coproporphyrin and protoporphyrin  (+): violet, pink, red fluorescence  (-): faint blue  Acute intermittent porphyria: Increased porphobilinogen 10/14/20 24 Mucopolysaccharide Disorders Mucopolysaccharides / glycosaminoglycans: consist of protein core with numerous polysaccharide branches Substances excreted in urine include: Keratan sulfate Heparan sulfate Dermatan sulfate 10/14/20 24 Mucopolysaccharide Disorders Three best known mucopolysaccharidoses are: Hunter’s syndrome: less severe abnormal skeletal structure and severe mental retardation Hurler syndrome: accumulation of mucopolysaccharide in the cornea of the eye, more severe abnormal skeletal structure and severe mental retardation Sanfilippo’s syndrome (mental retardation) 10/14/20 24  Mucopolysaccharide Disorders  Tests: 1. Acid Albumin: (+) thick white turbidity  Grade turbidity on a scale of 0-4 after 30 mins 2. Cetyltrimethylammonium bromide (CTAB)  Grade turbidity on a scale of 0-4 after 5 mins 3. Metachromatic staining procedure: uses basic dyes to react with acidic mucopolysaccharides  (+)Blue spot that cannot be washed away by dilute acidified methanol soln 10/14/20 24 Purine Disorders Lesch-Nyhan syndrome: inherited purine metabolism disorder due to failure to inherit the gene to produce the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT) Characterized by massive excretion of uric acid crystals in the urine Clinical manifestations: severe motor defects, mental retardation, a tendency of self-destruction, gout and renal calculi Appears in the first 6-8 months of life manifested by orange sand in diapers 10/14/20 24  Carbohydrate Disorders  Increased urinary sugar (mellituria)  GALACTOSURIA (galactose in urine) : inability to convert the galactose to glucose resulting to accumulation of galactose in the bloodstream Deficient enzymes: galactose-1-phosphate uridyl transferase, galactokinase, UDP-galactose-4- epimerase Failure of the infant to thrive, liver disorders, cataract formation and mental retardation.  Other cause of mellituria: Lactosuria: seen in pregnancy and lactation Fructosuria: associated with parenteral feeding Pentosuria: ingestion of large amounts of fruit 10/14/20 24 Thank you and God bless! ☺ 10/14/20 24