Lesson 7 : Urinary Screening for Metabolic Disorder

Overflow Disorders

Results from disruption of normal metabolic pathway that causes increased plasma concentrations of the non metabolized substances

Inborn error of metabolism

The disruption of enzyme function caused by failure to inherit the gene to produce a particular enzyme.

Renal Disorders

Caused by malfunctions /problems in the renal tubular reabsorption mechanism

Newborn Screening test

Blood collected by heel puncture

• Performed by using tandem mass spectrophotometry

• Phenylketonuria

• Tyrosyluria

• Melanuria

• Alkaptonuria

Amino Acid Disorders

• Maple Syrup Urine Disease

• Organic Acidemias Review

Branched - chain Amino Acid Disorders

• Tryptophan Disorders

• Cysteine Disorders

2 major Groups of Metabolic Disorders

• Indicanuria

• 5-hydroxyindolacetic acid

What are the Tryptophan Disorders ?

• Cystinosis

• Homocystinuria

What are the Cysteine Disorders?

Phenylketonuria

• autosomal recessive inherited disorder

• Both sexes affected equally

Phenylalanine hydroxylase

Phenylketonuria is caused by the absence of what enzyme ?

• Responsible for converting phenylalanine to tyrosine

• Increase plasma phenylalanine and phenylpyruvic acid

What happens when there is Absence of phenylalanine hydroxylase ?

Mental retardation

Major Clinical finding of Phenylketonuria

• Seizures

• Hyperactivity

• Developmental delay

• Psychiatric disturbances

• Fair complexion

• Lighter hair and eyes

• Mousy/musty urine and sweat odor

Clinical and Laboratory Features of Phenylketonuria

Mousy/musty urine and sweat odor

What odor does phenylketonuria give?

Phenyl acetic acid

Mousy/musty urine and sweat odor Is due to the presence of ?

Ferric Chloride Tube Test

Screening test for Phenylketonuria

• screening test for phenyl pyruvic acid

Blue-green color

When ferric chloride Is added to urine containing phenyl pyruvic acid what color does it produce?

Tyrosinemia with tyrosyluria

Occurs when abnormal metabolism of tyrosine from the diet or from phenylalanine is abnormal

• Fine , silky crystals

• Scattered singly or aggregated

• Brown to black color

• Precipitate in acid

• Soluble in alkali

Characteristics of Tyrosine crystals in urine?

Transitory Hypertyrosinemia

Occur in low-birthweight and premature infants as a benign condition

• Asymptomatic

• Absence of liver or renal disease

• Elevated plasma tyrosine, and phenylalanine levels

• Elevated urinary tyrosine , p-hydroxyphenyllactic, and p-hydroxylphenylpyruvic acids

Clinical and laboratory features of Transitory Hypertyrosinemia

Type I Hereditary Tyrosinemia

• Autosomal recessive disorder

• Succinylacetoacetone and succinylacetone accumulate

• Fumarylacetoacetate hydrolase (Ia)

• Maleylacetoacetate isomerse (Ib)

Type I Hereditary Tyrosinemia is caused by defects in what enzymes?

• Liver failure

• Hepatoma

• Renal dysfunction

• Rickets

• Acute intermittent porphyria-like symptoms

• Phosphaturia

• Glycosuria

Clinical and Laboratory features of Type I Hereditary Tyrosinemia

Type II Tyrosinemia

• autosomal recessive disorder

• Other name is Richter-Hanhart syndrome

• Elevated urinary phenolic acids

• Erosions of the cornea, soles , and palms

Clinical and laboratory features of Type II Tyrosinemia

Tyrosine aminotransferase

Type II Tyrosinemia is caused by the deficiency of what enzyme ?

p-hydroxyphenylpyruvic acid dioxygenase

Type III Tyrosinemia is caused by the deficiency of what enzyme ?

• Intellectual Disability

• Seizures

• Elevated plasma tyrosine

• Elevated urinary phenolic acids

• Intermittent ataxia

Clinical and laboratory features of Type III Tyrosinemia

Mnemonic : “I SEE It”

Nitroso-Naphthol Test

• orange red

Screening Test for Type III Tyrosinemia

• what is the resulting color?

Melanuria

• Increased urinary melanin

• Serious finding that indicates proliferation of normal melanin-producing cells, producing malignant melanoma

5,6 - dihydroxyindole

Secreted by tumors which oxidizes to melanogen and then to melanin

Production of Melanin Thyroxine Epinephrine protein and tyrosine sulfate

What is the second metabolic pathway for tyrosine responsible for?

Alkaptonuria

Urine from patients with this condition darkens and will turn brown to black after becoming alkaline from standing at room temperature

homogentisic acid oxidase

Alkaptonuria is caused by deficiency what enzyme?

• accumulation of homogentisic acid in blood, tissues and urine

• Brown-black urine on standing / with alkaline pH

• Brown pigmentation in cartilage and connective tissues

• Liver disorders

• Cardiac disorders

Clinical and Laboratory features of Alkaptonuria

• Ferric Chloride Test (dark blue color)

• Homogentisic Acid Test (black color)

Screening tests for Alkaptonuria

Maple Syrup Urine Disease

• Autosomal recessive disorder

• Caused by the failure to inherit the gene for the enzyme necessary for oxidative decarboxylation of keto acids

Branched-chain a-ketoacid dehydrogenase complex

Maple Syrup Urine Disease is caused by the defect of what enzyme complex?

• Leucine

• Isoleucine

• Valine

Amino acids involved in Maple Syrup Urine Disease

• a-ketoisocaproate

• a-keto-beta-methylvalerate

• a-ketoisovalerate

without the branch chain alpha keto acid dehydrogenase the metabolism of leucine, isoleucine, and valine cannot proceed. Now this leads to the buildup of what keto acids?

• Urine with Maple Syrup odor

• Severe neonatal vomiting

• Seizures

• Stupors

• Irregular respirations

• Hypoglycemia

Clinical and Laboratory findings of Maple Syrup Urine Disease

Mnemonics : “USSSIH”

2-4-dinitrophyenylhydrazine test (DNPH)

Screening test for Maple Syrup Urine Disease which produces yellow turbidity or precipitate

• Isovaleric Acidemias

• Propionic Acidemias

• Methylmalonic Acidemia

Organic Acidemias

• Vomiting

• Metabolic acidosis

• Hypoglycemia

• Ketonuria

• Elevated serum ammonia

Organic Acidemias general clinical and laboratory findings

Isovaleric Acidemia

May be suspected when urine specimens and sometimes even the patient possesses a characteristic odor of sweaty feet.

• Isovaleryl coenzyme A dehydrogenase

Isovaleric Acidemia is due to a deficiency of what enzyme?

Sweaty feet urine odor

Characteristic finding of Isovaleric Acidemia which is caused by accumulation of isovalerylglycine

Isovaleryl glycine

The sweaty feet odor of Isovaleric Acidemia is caused by ?

Propionic and Methylmalonic Acidemia

Results from errors in the metabolic pathway converting isoleucine, valine, threonine, and methionine to succinyl coenzyme A

propionyl coenzyme A carboxylase

Propionic Acidemia is caused by the deficiency of what enzyme ?

Methylmalonyl coenzyme A mutase

Methylmalonyl Acidemia is caused by the Deficiency of what enzyme?

Indicanuria

Due to increased amounts of tryptophan that are ultimately converted to Indican.

Caused obstruction, presence of abnormal bacteria, malabsorption syndromes, Hartnup disease

• Blue diaper syndrome

Tryptophan

either reabsorbed for protein synthesis or converted by intestinal bacteria to indole and excreted in the feces

Tryptophan in Intestinal Disorder

excess tryptophan is converted to indole, which is then reabsorbed into the bloodstream.

Liver

Where is indole converted to indican?

Urine

How is indican excreted from the body?

oxidized to indigo blue, giving the urine a blue color

What happens to indican when exposed to air?

Colorless

What is the color of indican before oxidation?

5-hydroxyindoleacetic acid (5-HIAA)

What tryptophan metabolite increases in malignant (argentaffin) tumors? (Seretonin)

Tryptophan → 5-hydroxytryptophan → Serotonin → 5-HIAA

Through what pathway is 5-HIAA formed?

• Agrentaffin cells

• Platelets

• 5-HIAA

Normal Physiology of 5-Hydroxyindolacetic Acid

Agrentaffin cells

Produces serotonin from tryptophan

Platelets

Carry serotonin throughout the body

5-HIAA

Degradation product of serotonin for excretion

Carcinoid tumors involving agrentaffin cells

• Causes excess production of serotonin

• Causes elevated urinary 5-HIAA levels

Excretion of > 25mg/24h

An indication of agrentaffin cell tumors (normal : 2-8mg/24h)

Silver Nitroprusside Test

Screening test for 5-hydroxyindolacetic acid

• resulting color is purple-black

Cystinuria

Caused by the inability of the renal tubules to reabsorb cystine filtered by the glomerulus

Cyanide-Nitroprusside Test

Screening test for Cystinuria (red-purple)

Cystinosis

Caused by a defect in the lysosomal membranes that prevent the release of cystine into the cellular cytoplasm for metabolism

• Nephropathic

• Non-nephropathic

General categories of Cystinosis

Nephropathic Cystinosis

Involves renal impairment and is the more severe form of the disease .

• infantile

• late onset

Non-Nephropathic cystinosis

Does not affect the kidneys and is considered relatively benign

• Photophobia

• Renal failure

• Rickets

• Growth failure

• Aminoaciduria

• Glucosuria

Clinical and laboratory features of Cystinosis (Cysteine disorder)

Homocystinuria

Caused by the deficiency of cystathionine B-synthase

Cystathionine B-synthase

This enzyme is responsible for catalyzing the formation of cystathionine from homocysteine and serine.

• Failure to thrive

• Cataracts

• Intellectual disability

• Thromboembolic problems (strokes, myocardial infarction)

Increased homocysteine can cause?

Silver-Nitroprusside Test for Homocysteine

Screening test for Homocystinuria

Porphyrins

Intermediate compounds in the production of heme

Porphyrin Disorders

Caused by the blockage of a pathway (heme synthesis is blocked)

Porphyrias

disorder of porphyrin metabolism and can be classified as either inherited or acquired.

Red/port wine color urine

Indication of possible porphyrinuria

• Aminolevulinic acid (ALA)

• Porphobilinogen

• Uroporphyrin

Most soluble and readily appear in urine

Coproporphyrin

Less soluble but found in urine

Protoporphyrin

Not found in urine

Ehrlich Reaction

Screening test for Porphyrin disorders

p-dimethylaminobenzaldehyde

Ehrlich Reagent

Watson-Schwartz Test

Differentiation between the presence of urobilinogen and porphobilinogen

Hoesch Test

Specific for presence of porphobilinogen

Fluorescence under UV (550 - 600nm)

• Detects other porphyrins

• Mixes the sample with glacial acetic acid + ethyl acetate

Violet/pink/red

Positive color for Fluorescence under UV (550 - 600nm) that detects porphyrins

Faint blue

Negative color for Fluorescence under UV (550 - 600nm) that detects porphyrins

• High-pressure liquid chromatography

• Ion exchange chromatography

• Spectrometry methodologies

Currently Used Tests for Porphyrins

Urobilinogen

• Soluble in Chloroform

• Extracted into butanol

Porphobilinogen

Not soluble in both chloroform and butanol

Ehrlich-reactive compounds

• Not soluble in chloroform

• Extracted into butanol

Porphobilinogen and Urobilinogen

Both layers appear red

• Mucopolysaccharidoses

• Carbohydrate Disorders

Mucopolysaccharide Disorders