Screening for Urine Inborn Errors of Metabolism

Overview and Purpose

• Park's Textbook of Preventive and Social Medicine
• Harper's Illustrated Biochemistry, 32nd Ed.

Neonatal Blood Spot Screening (Guthrie Test)

• A disc from the DBS paper is placed on an agar plate seeded with a phenylalanine-requiring strain of Bacillus subtilis, along with a competitive inhibitor (beta-thienylalanine) calibrated to block bacterial growth at normal blood phenylalanine levels.
• If phenylalanine is elevated (as in phenylketonuria), it overcomes the inhibitor and bacteria form visible colonies.
• The same DBS method can test for PKU, galactosaemia, and maple syrup urine disease (MSUD).

• Harper's Illustrated Biochemistry, 32nd Ed., p. 585
• Park's Textbook of Preventive and Social Medicine

Urine Chemical Spot Tests

• Ferric chloride test: Detects phenylpyruvic acid (PKU) and other keto acids; colour varies by metabolite.
• DNPH (2,4-dinitrophenylhydrazine) test: Detects keto acids (positive in PKU, MSUD, tyrosinemia, organic acidaemias).
• Benedict's reaction: Detects reducing sugars in urine (galactose in galactosaemia, fructose in fructosuria, glucose).
• Nitroprusside reaction: Detects sulfhydryl groups - positive in homocystinuria and cystinuria.
• Adams and Victor's Principles of Neurology, 12th Ed.

Conditions Commonly Screened

• Adams and Victor's Principles of Neurology, 12th Ed.

Paper Chromatography for Urine IEM Screening

Principle

The Retardation Factor (Rf)

Rf = Ds / Df

• Ds = distance traveled by the compound from the point of application
• Df = distance traveled by the solvent front in the same time

Separation in planar chromatography: compound B is more retained (lower Rf) than compound A. Df = distance of solvent front; Ds = distance of analyte.

• Tietz Textbook of Laboratory Medicine, 7th Ed.

Application to Urine Sugars and Metabolites

• Urine is applied as a spot and subjected to ascending or descending chromatography on paper.
• After migration, the paper is developed with dinitrosalicylic acid (or other reagents like ninhydrin for amino acids) to produce coloured bands.
• Each sugar (glucose, galactose, fructose, lactose, etc.) has a characteristic Rf value in a given solvent system.
• Presumptive identification is made by comparing the unknown's Rf to known reference compounds run simultaneously on the same sheet.
• If Rf values match, detection properties (colour of bands, response to reagent) are compared for further confirmation.
• When paper chromatography is performed infrequently, it is simple, adequate, and requires little actual working time. When frequent separations are needed, thin-layer chromatography (TLC) is preferred for its shorter run time.
• One common setup uses a 6 x 18-inch Pyrex jar with a tightly fitting cover.

• Ninhydrin reagent is used to detect amino acids, producing characteristic purple/yellow spots.
• Two-dimensional paper chromatography (running the chromatogram in two directions with two different solvents) improves resolution when many amino acids are present.
• Abnormal patterns of amino acid spots indicate specific conditions (e.g., elevated phenylalanine in PKU, elevated leucine/isoleucine/valine in MSUD).
• Tietz Textbook of Laboratory Medicine, 7th Ed.

Paper vs. Thin-Layer Chromatography

Modern Supersession

• Tandem mass spectrometry (MS-MS): Simultaneously screens dozens of metabolites from a single DBS in minutes, detecting amino acidopathies, organic acidaemias, and fatty acid oxidation disorders.
• HPLC amino acid analysis: Provides quantitative amino acid profiles.
• Enzymatic assays: Specific for individual enzyme deficiencies (e.g., galactose-1-phosphate uridyltransferase for galactosaemia).
• Harper's Illustrated Biochemistry, 32nd Ed.; Thompson & Thompson Genetics and Genomics in Medicine, 9th Ed.