A 3-day-old neonate born to non-consanguineous parents presents with poor feeding, vomiting, and lethargy. Routine newborn screening shows elevated plasma phenylalanine (>20 mg/dL). Which investigation is most appropriate to confirm the diagnosis of classical phenylketonuria (PKU)?

## Diagnosis of Classical Phenylketonuria ### Clinical Context The neonate presents with elevated plasma phenylalanine (>20 mg/dL) on newborn screening. Confirmation of classical PKU requires distinguishing PAH deficiency from other causes of hyperphenylalaninemia (e.g., BH4 cofactor defects). ### Investigation of Choice: Urine Organic Acid Chromatography **Key Point:** Urine organic acid chromatography (urine metabolite analysis) is the most appropriate confirmatory investigation for classical PKU in clinical and NEET PG practice. It detects the characteristic accumulation of phenylalanine metabolites — **phenylpyruvate**, **phenyllactate**, and **phenylacetate** — which are pathognomonic of PAH deficiency. **High-Yield:** In classical PKU, blocked conversion of phenylalanine → tyrosine (due to PAH deficiency) shunts phenylalanine into alternative transamination pathways, producing these organic acids in urine. Their detection confirms the diagnosis non-invasively. ### Why Urine Organic Acid Chromatography? | Feature | Significance | |---------|-------------| | Non-invasive | Urine sample; safe in neonates | | Pathognomonic metabolites | Phenylpyruvate, phenyllactate, phenylacetate confirm PAH deficiency | | Differentiates PKU from BH4 defects | BH4 defects do NOT produce these organic acids | | Clinically practical | Standard confirmatory test in NEET PG / clinical practice | ### Why Not the Other Options? - **A) Plasma tyrosine level:** Low tyrosine is seen in PKU (since PAH converts Phe → Tyr), but it is not specific enough to confirm the diagnosis. - **C) Dihydrofolate reductase activity in RBCs:** This tests for BH4 recycling defects (hyperphenylalaninemia type III), not classical PKU. - **D) PAH enzyme activity in liver biopsy:** While theoretically the "gold standard," liver biopsy is invasive, impractical, and NOT used in routine clinical or NEET PG practice for confirming PKU. Genetic testing (PAH gene mutation analysis) has replaced it where direct enzyme assessment is needed. **Clinical Pearl:** The classic "mousy/musty odor" of urine in PKU is due to **phenylacetate** — the same compound detected on urine organic acid chromatography. The ferric chloride test (green color with phenylpyruvate) is a bedside screening correlate of this finding. **Mnemonic:** **PKU = Phenylpyruvate, Ketoacids in Urine** — urine organic acid chromatography detects these confirmatory metabolites. [cite: Harper's Illustrated Biochemistry 31e, Ch 29; Robbins 10e Ch 5]