A 3-day-old neonate born to consanguineous parents presents with poor feeding, lethargy, and a characteristic 'musty' or 'mousy' odour on the breath. Serum phenylalanine is markedly elevated at 1200 µmol/L (normal <120 µmol/L). Which investigation is most appropriate to confirm the diagnosis and differentiate classical phenylketonuria from variant forms?

Explanation

## Investigation of Choice: Plasma Amino Acid Chromatography **Key Point:** Plasma amino acid chromatography is the gold-standard confirmatory test for phenylketonuria (PKU) and allows differentiation of classical PKU from variant forms (benign hyperphenylalaninemia, PAH-deficient variants). ### Why Plasma Amino Acid Chromatography? 1. **Quantifies phenylalanine level precisely** — distinguishes classical PKU (>1200 µmol/L) from benign forms (<600 µmol/L) 2. **Detects secondary amino acid abnormalities** — in classical PKU, tyrosine is low (due to competitive inhibition of tyrosine hydroxylase), and other amino acids show characteristic patterns 3. **Differentiates PAH deficiency from cofactor defects** — tetrahydrofolate (BH~4~) deficiency shows a different amino acid profile (elevated homocysteine, low methionine) 4. **Guides management** — benign forms do not require dietary restriction; classical PKU requires strict phenylalanine-restricted diet ### Complementary Investigation **Urine organic acid analysis** performed alongside plasma amino acids: - Detects phenylpyruvic acid, phenyllactic acid, and phenylacetyl glutamine (pathognomonic for PKU) - Helps exclude other aminoacidopathies ### Diagnostic Algorithm ```mermaid flowchart TD A[Elevated serum phenylalanine on newborn screening]:::outcome --> B[Plasma amino acid chromatography + urine organic acids]:::action B --> C{Phenylalanine level & amino acid pattern?}:::decision C -->|>1200 µmol/L + low tyrosine| D[Classical PKU - PAH deficiency]:::outcome C -->|600-1200 µmol/L + normal tyrosine| E[Benign hyperphenylalaninemia]:::outcome C -->|Elevated homocysteine, low methionine| F[BH4 deficiency variant]:::outcome D --> G[Phenylalanine hydroxylase gene sequencing]:::action F --> H[BH4 loading test]:::action ``` **High-Yield:** The combination of **elevated plasma phenylalanine + low tyrosine + urinary phenylpyruvic acid** is diagnostic of classical PKU. Tyrosine is low because phenylalanine competitively inhibits tyrosine hydroxylase. **Clinical Pearl:** Early detection via newborn screening and immediate dietary intervention (phenylalanine-restricted diet with tyrosine supplementation) prevents intellectual disability, light skin pigmentation, eczema, and the characteristic 'mousy' odour.