Urinalysis and other Body Fluids - Screening for Metabolic Disorders

o ANALYSIS OF URINE AND OTHER BODY FLUIDS URINE SCREENING FOR METABOLIC DISORDERS o OUTLINE • • Introduction Urine Screening for Metabolic Disorders o Overflow versus Renal Disorders o Phenylalanine/Tyrosine Metabolic Pathway o Amino Acid Disorders (Aminoacidurias) o Porphyrin Disorders o Mucopolysaccharide Disorders o Purine Disorders o Carbohydrate Disorders INTRODUCTION • o o o URINE SCREENING FOR METABOLIC DISORDERS OVERFLOW VERSUS RENAL DISORDERS • Kidney disease have a direct effect on the urinalysis results. The same could be said about certain metabolic disorders. The presence of metabolic disorders may have an influence on the chemical constituents of the urine. NEWBORN SCREENING • • Checks newborn for 50 rare or common metabolic disorders (US) o PH - 18-19 Tests for expanded Includes blood spot screening, hearing screening, and pulse oximetry Fill each circle one at a time until five circles are completely filled. Five completely filled blood spots gives the state lab enough blood to test for all of the genetic disorders. After finishing collecting the blood drops, follow your normal procedures for care of the heel stick site. After taking care of the infant, put the collection kit on a dry, clean, flat, non-absorbent surface. Use a drying rack for several kits. Let the blood spots dry for a minimum of three hours. • Overflow o Disruption of a normal metabolic pathways o Increased plasma concentrations of the nonmetabolized substances (preservatives) o Overrides reabsorption ability of renal tubules o Inherited lack of specific enzyme for protein fat, or carbohydrate metabolism--inborn error of metabolism Renal o Malfunctions in the tubular reabsorption mechanism DISORDERS CLASSIFIED BY DEFECT SPECIMEN COLLECTION • • Supplies o Specimen collection kit o Heel warmer o Alcohol pads/cotton balls o Gloves o Heel lancet (1mm x 2.5 mm) o Sterile gauze o Aftercare supplies Procedure o Check identity before starting collection o Warm the heel using heel warmer or warm cloth. Warming increase blood supply. o Clean hands and put on gloves. o Heel puncture should be made on the sole of the foot. Use safe areas to prevent damage to tendons, nerves. Do use previous area/puncture site. o Clean skin and let skin air dry. o With the heel lancet, Stick the heel with the slight angle. Wipe away the first drop with a sterile gauze. Allow a second large blood for to drop. This is done because the first blood drop may contain tissue fluids. o Either side of paper can be used but apply drops to only one side of the paper. o Lightly touch the blood drop to the filter paper. Let the blood soak through the paper. If the first drop of blood doesn't completely fill the circle, quickly express another blood drop and continue to fill the same circle. o To help the blood flow, you may apply a very gentle intermittent pressure to the area surrounding the puncture site. However, do not milk the heel or the puncture site. o Do not go back to partially filled circles. The specimen is not acceptable for testing if it is oversaturated, layered, or clotted. PHENYLALANINE/TYROSINE METABOLIC PATHWAY • • • • • • Current state-mandated screening for as many as 30 or more inborn errors of metabolism (IEM) Phenylalanine - protein Urine tests are primarily for follow-up Disorders can cause abnormal urinalysis results Heel stick blood test are used for testing o Performed before infant leaves hospital o But >24h o Metabolites appear first in the blood Analyze by tandem mass spectrophotometry, MS/MS o • • Tyrosine can be converted to melanin (skin pigmentation) o Glutathione composed of 4 amino acid molecules (antioxidants) ▪ Potent antioxidant ▪ Normally produced by the body in right quantities BRANCHED CHAIN AMINO ACID DISORDERS • • AMINO ACID DISORDERS (AMINOACIDURIAS) PHENYLALANINE-TYROSINE DISORDERS • • • • Phenylketonuria, tyrosyluria, alkaptonuria Phenylketonuria o 1 in 10,000 to 20,000 births o Autosomal recessive; heterozygous normal o Eliminate phenylalanine from diet (milk) o Damage to child’s mental capacity; mental retardation o Alternate pathways as child matures o Avoid ↑ phenylalanine foods (aspartame) o Phenylalanine hydroxylase is missing o Urine test ▪ Urine and 5% ferric chloride produces a permanent green-blue color Tyrosyluria/Tyrosinemia o Metabolic defects ▪ Premature transient tyrosinemia • Underdevelopment of liver function ▪ Acquired severe liver disease o Hereditary defects ▪ Type 1: enzyme deficiency is fumarylacetoacetate acid hydrolase; renal tubular disease and liver failure in infants ▪ Type 2: enzyme deficiency is tyrosine aminotransferase; corneal erosion and lesions on hands and feet ▪ Type 3: enzyme deficiency is phydroxyphenylpyruvate oxidase; mental retardation if no dietary restrictions (milk) o Screening tests ▪ Screening tests using MS/MS are available for tyrosinemia types 1, 2, and 3 Melanuria o Second pathway for tyrosine ▪ Melanin, thyroxine, epinephrine, protein, and tyrosine sulfate Melanin ▪ Pigment for dark hair, skin ▪ Defect causes albinism ▪ Increased production = malignant melanoma (cancer of melanocyte) ▪ 5,6-dihydroxyindole • Dark urine from oxidation of melanogen to melanin Alkaptonuria o Enzyme deficiency is caused by a failure to inherit gene to produce the enzyme homogentisic acid oxidase o Third major defect in the phenylalanine-tyrosine pathway o Black alkaline urine, possible black-stained diapers o Manifests later in life with brown pigment deposits in tissues o Urine: blue with ferric chloride, yellow precipitate with Clinitest, black with silver nitrate and ammonium hydroxide’ quantitative tests available • • • Amino acids with a methyl group that branches from the main aliphatic carbon chain Two groups o Maple syrup urine (MSUD); early degradation products accumulate o Organic acidemias: accumulation of organic acids further down in pathway Ketonuria in a newborn Maple syrup urine disease (MSUD) o Inborn error of metabolism, autosomal recessive o Amino acids involved are leucine, isoleucine, and valine o 1-week failure to thrive is noticed o Urine: strong odor of maple syrup, and thick and dark appearance o Dietary regulation by day 11 shows good outcomes o Positive urine ketones o Screening test 2,4-dinitrophenylhydrazine produces yellow precipitate turbidity o 2,4-dinitrophenylhydrazine (DNPH) Test 1. Place 1 mL of urine in a tube 2. Add 10 drops of 0.2% of 2,4-DNPH in 2N HCl 3. Wait 10 minutes 4. Observe for yellow or white precipitate (yellow, +) Organic acidemias o Early: severe vomiting, metabolic acidosis, hypoglycemia, ketonuria o Isovaleric, propionic, methylmalonic acidemias o Isovaleric: “sweaty feet odor” from patient ▪ Deficiency of isovaleryl coenzyme A o Propionic and methylmalonic: no conversion of valine, threonine, methylmalonate to succinyl coenzyme A o Isovaleric, propionic, and methylmalonic acidemias can be detected by newborn screening programs using MS/MS o TRYPTOPHAN DISORDERS • Increase urinary excretion of the metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA) • • • Indicanuria o Tryptophan enters intestine, is reabsorbed or is converted to indole by bacteria, and leaves in the feces o Intestinal disorders and Hartnup disease cause increase tryptophan conversion to indole o Increased indole reabsorbed, excrete by kidney on its way to the liver o Exposure of urine to air = indigo blue o Hartnup disease: blue diaper syndrome ▪ Inherited disorder affects intestinal reabsorption of indole and renal tubular absorption= Fanconi syndrome ▪ Requires dietary supplements: niacin 5-Hydroxyindoleacetic Acid (5-HIAA) o Tryptophan produces serotonin o Serotonin from tryptophan is produced by the intestinal argentaffin cells and is carried in the body to the muscles by platelets o Excess excreted in the urine as 5-HIAA o Argentaffin (enterochromaffin) cell tumors = ↑↑ 5-HIAA o in urine from excess serotonin produced o Urine test ▪ Nitrous acid and -nitroso-2-naphtol produce purple to black color ▪ Normal 2-8 mg/day, >25 mg/day in disease ▪ Can perform test on random specimens ▪ Patient instructions • No bananas, pineapples, tomatoes, phenothiazines, and acetanilids for 72 hours • 24-hour urine samples must be preserved with HCl or boric acid CYSTINE DISORDERS • • Two different disorders; both have noticeable odor of sulfur Cystinuria o Inherited disorder affecting renal reabsorption o Two modes of inheritance: (1) only cystine and lysine are not reabsorbed; (2) cystine, lysine, arginine, and ornithine are not reabsorbed o Increased calculi formation early in life for both modes o Approximately 65% of the people in whom all four amino acids are affected can be expected to produce calculi early in life o Cystine is the least soluble accounting for cystine crystals • Cystine is also the only amino acid found during the analysis of calculi from these patients o Urine screening test: cyanide-nitroprusside ▪ Na cyanide reduces cystine, and nitroprusside produces a red purple color if excess cystine is present ▪ False-positives: ketonuria, homocystinuria o Cyanide-Nitroprusside Test 1. Place 3 mL of urine in a tube. 2. Add 2 mL sodium cyanide. 3. Wait 10 minutes. 4. Add five drops 5% sodium nitroprusside. 5. Observe for red-purple color. Cystinosis o Genuine IEM o Ranging from a severe fatal disorder developed in infancy to a benign form appearing in adulthood o Two categories ▪ Nephropathic • Infantile • Later life ▪ Non-nephropathic o Defect in lysosomal membranes prevents release of cystine into cytoplasm for metabolism = crystalline cystine deposits in body o Deposits: cornea, bone marrow, lymph nodes, organs o Renal tubules are affected by deposits causing Fanconi syndrome, which is not inherited o Infantile: rapid progression to renal failure o Late-onset: gradual progression to renal failure o Non-nephropathic: benign, some ocular problems o Laboratory: aminoaciduria, reducing substances, cystine crystals o Renal transplants and cystine-depleting medications Homocystinuria o Defect in metabolism of methionine, producing increased methionine in body o Failure to thrive, cataracts, mental retardation, thromboemboli, death o Requires changes in diet o Additional screening with silver nitrate instead of sodium cyanide o Included in newborn screening programs performed using MS/MS testing o Silver Nitroprusside Test 1. Place 1 mL of urine in a tube. 2. Add two drops concentrated NH4OH. 3. Add 0.5 mL 5% silver nitrate. 4. Wait 10 minutes. 5. Add five drops sodium nitroprusside. 6. Observe for red-purple color. o • PORPHYRIN DISORDERS • • • • • • • • • • • • • • • Intermediate compounds in the production of heme Primary porphyrins: uroporphyrin, coproporphyrin, protoporphyrin Precursors: α-aminolevulinic acid (ALA) and porphobilinogen Detection of pathway disruptions in urine, blood, bile, and feces Urine: ALA, porphobilinogen, urobilinogen Feces/bile: coproporphyrin, protoporphyrin Blood: free erythrocyte protoporphyrin (FEP) for lead poisoning Collectively termed porphyrias Inherited: gene in metabolic pathway is missing Classified by clinical symptoms as neurologic/psychiatric, cutaneous/photosensitivity, or both Acquired (more common): lead poisoning, alcoholism, iron deficiency, chronic liver and renal disease Urine: port wine color after air exposure, also seen on diapers Ehrlich reaction: only for ALA and porphobilinogen o Convert ALA to porphobilinogen by adding acetyl acetone o Watson-Schwartz test o Ehrlich reaction now included on the Multistix urobilinogen pad Fluorescence under ultraviolet light 550- to 600-nm range is used for other porphyrins; extract into glacial acetic acid and ethyl acetate; does not distinguish o Violet, pink, red, based on concentration Watson-Schwartz Differentiation Test 1. To each tube add: Tube 1 Tube 2 2 mL urine 2 mL urine 2 mL chloroform 2 mL butanol 4 mL sodium acetate 4 mL sodium acetate 2. Observe the color of the layers. Interpretation: Watson-Schwartz Test 1. Label two tubes #1 and #2. 2. To each tube add: Tube 1 Tube 2 2 mL urine 2 mL urine 2 mL chloroform 2 mL butanol 4 mL sodium acetate 4 mL sodium acetate 3. Vigorously shake both tubes. 4. Place in a rack for layers to settle. 5. Observe both tubes for red color in the layers o Interpretation: ▪ Tube 1 Upper layer = urine; if colorless = porphobilinogen or Ehrlich-reactive compounds. Bottom layer = chloroform; if red = urobilinogen. If both layers are red, re-extract the urine layer from tube 1. Place 2 mL of urine layer from Tubes 1 and 2 mL chloroform and 4 mL sodium acetate into a new tube. Repeat procedure. Interpretation: Upper layer—urine colorless Bottom layer—chloroform—red = excess urobilinogen Both layers red = porphobilinogen and urobilinogen ▪ Tube 2 Upper layer = butanol. If red = urobilinogen or Ehrlich-reactive compounds Bottom layer = urine If colorless = porphobilinogen MUCOPOLYSACCHARIDE DISORDERS • • • • • • Inherited disorders preventing the metabolism of glycosaminoglycans in the connective tissue Incompletely metabolized polysaccharides accumulate in connective tissue Substances found in urine are dermatan sulfate, keratan sulfate, and heparin sulfate Mucopolysaccharidoses o Hurler syndrome: abnormal skeletal structure, severe mental retardation, and corneal damage o Hunter syndrome: abnormal skeletal structure, severe mental retardation, inherited as a sex-linked recessive trait, rare in females o Sanfilippo syndrome: mental retardation Bone marrow transplantation and gene therapy Urinary Screening Tests o Acid-albumin and cetyltrimethylammonium bromide turbidity tests ▪ Thick, white precipitate o o o Metachromatic staining procedures ▪ Positive urine produces a blue color that cannot be washed away with dilute acidified methanol Cetyltrimethylammonium Bromide (CTAB) Test 1. Place 5 mL of urine in a tube. 2. Add 1 mL 5% CTAB in citrate buffer. 3. Read turbidity in 5 minutes Mucopolysaccharide Paper test 1. Dip filter paper into 0.59% azure A dye in 2% acetic acid. 2. Dry. 3. Add one drop of urine to paper. 4. Wash with 1 mL acetic acid + 200 mL methanol diluted to a liter. 5. Observe for blue color. PURINE DISORDERS • Lesch-Nyhan disease o Inherited sex-linked recessive o Massive excretion of uric acid crystals o Motor defects, mental retardation, self-destruction, gout, renal calculi o Normal development 6 to 8 months o Orange sand in diaper o Be alert for increased uric acid crystals in pediatric patients CARBOHYDRATE DISORDERS • • • • • • • • Termed melituria; frequently due to inherited disorder No problems except for galactosuria Three enzymes: most important is galactose-1-phosphate uridyl transferase (GALT) also included in MS/MS screens Failure to thrive, severe mental retardation, cataracts, liver disorders Remove lactose from diet Included in newborn testing of RBCs Clinitest positive Other Meliturias o Also glycosuria o Lactosuria ▪ Pregnancy and lactation o Fructosuria ▪ Parenteral feeding ▪ Resorcinol screening test o Pentosuria ▪ Ingestion of large amounts of fruit