A 6-month-old infant from Delhi presents with poor feeding, hypotonia, and seizures. Serum ammonia is 250 µmol/L. Plasma amino acid profile shows elevated citrulline (150 µmol/L; normal 20–50) and low arginine. Urine orotic acid is normal. Which investigation is most appropriate to confirm the diagnosis?

## Diagnostic Approach to Argininosuccinate Synthetase (ASS) Deficiency ### Clinical Presentation The infant has hyperammonemia with a **distinctive plasma amino acid pattern**: - **Elevated citrulline** (150 µmol/L) — accumulates because it cannot be converted to argininosuccinate - **Low arginine** — consumed in ammonia detoxification, not regenerated - **Normal urine orotic acid** — rules out OTC deficiency This profile is **pathognomonic for ASS deficiency** (argininosuccinate synthetase deficiency), also called **Citrullinemia Type I**. ### Why Plasma & Urine Argininosuccinate is the Investigation of Choice **Key Point:** Argininosuccinate is the **direct substrate of ASS** and accumulates dramatically in ASS deficiency: - **Plasma argininosuccinate** — elevated 10–100× normal - **Urine argininosuccinate** — massively elevated, often crystallizes in urine (pathognomonic) **High-Yield:** The combination of **elevated plasma citrulline + elevated urine argininosuccinate = ASS deficiency**. This is the most specific and rapid confirmatory test. ### Differential Diagnosis of Hyperammonemia with Elevated Citrulline | Disorder | Plasma Citrulline | Urine Orotic Acid | Urine Argininosuccinate | Plasma Arginine | |----------|-------------------|-------------------|-------------------------|------------------| | **ASS deficiency (Citrullinemia I)** | ↑↑↑ (very high) | Normal | ↑↑↑ (pathognomonic) | ↓ (low) | | **ASL deficiency (Citrullinemia II)** | ↑ (mildly elevated) | Normal | ↑ (elevated) | ↓ (low) | | **OTC deficiency** | ↓ (low) | ↑↑↑ (very high) | Normal | ↓ (low) | | **ARG1 deficiency** | Normal | Normal | Normal | ↑↑↑ (very high) | **Clinical Pearl:** Argininosuccinate crystals in urine are **virtually diagnostic of ASS deficiency** — a finding that can be noted on routine urinalysis. ### Mechanism ASS catalyzes: Citrulline + Aspartate → Argininosuccinate. When ASS is deficient: - Citrulline accumulates (cannot be converted) - Argininosuccinate cannot be formed (substrate backs up) - Arginine is depleted (not regenerated from argininosuccinate) - Ammonia detoxification fails