A patient has elevated phenylalanine levels (40 mg/dL), but phenylalanine hydroxylase enzyme levels are normal. Which cofactor deficiency is most likely responsible for this condition?

Correct Answer: C. Tetrahydrobiopterin (BH4)

Phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine, and this reaction requires tetrahydrobiopterin (BH₄) as an essential cofactor. The question presents a critical clinical scenario: elevated phenylalanine with normal PAH enzyme levels. This rules out classic phenylketonuria (PKU), where the enzyme itself is deficient or mutated. Instead, the elevated phenylalanine indicates the enzyme cannot function properly despite being present—the defect lies in the cofactor. BH₄ is a pteridine cofactor synthesized from GTP via a multi-step pathway. Deficiency of BH₄ (either from impaired synthesis or increased degradation) results in atypical PKU or malignant hyperphenylalaninemia. These patients present with elevated phenylalanine but normal PAH protein levels and activity when measured in vitro with supplemental BH₄. This is a critical distinction from classic PKU. In Indian clinical practice, BH₄ supplementation (sapropterin) is now recognized as a therapeutic option for BH₄-responsive PKU variants. The cofactor is also essential for tyrosine hydroxylase and tryptophan hydroxylase, explaining why BH₄ deficiency causes not only hyperphenylalaninemia but also neurological manifestations (low dopamine, serotonin) and progressive neurodegeneration if untreated.

Why the other options are wrong

A. Thiamine — Thiamine (vitamin B₁) is a cofactor for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase—enzymes in carbohydrate metabolism and the pentose phosphate pathway. It has no role in phenylalanine hydroxylation. Thiamine deficiency causes Wernicke-Korsakoff syndrome and beriberi, not hyperphenylalaninemia. This is a distractor exploiting knowledge of B-vitamin deficiencies. B. Tetrahydrofolate — Tetrahydrofolate (THF) is a one-carbon carrier cofactor essential for nucleotide synthesis and methylation reactions. It is not involved in phenylalanine hydroxylase catalysis. THF deficiency causes megaloblastic anemia and neurological symptoms, not elevated phenylalanine. This option exploits confusion between different pteridine-based cofactors (THF vs. BH₄). D. Pyridoxine — Pyridoxal phosphate (active form of vitamin B₆) is a cofactor for aminotransferases, decarboxylases, and other amino acid metabolism enzymes, but not for phenylalanine hydroxylase. Pyridoxine deficiency causes peripheral neuropathy, seizures, and anemia—not selective hyperphenylalaninemia. This is a trap using a common B-vitamin cofactor unrelated to this specific enzyme.

High-Yield Facts

Mnemonics

PAH's Cofactor Rule Phenylalanine Alanine Hydroxylase needs Biopterin (BH₄). When enzyme is normal but substrate is high → think cofactor, not enzyme mutation. Atypical PKU Clue Normal enzyme + High phenylalanine = Cofactor problem. Classic PKU = Mutant enzyme. Atypical PKU = Missing BH₄ (the 'helper').

NBE Trap

NBE pairs "elevated phenylalanine" with "enzyme deficiency" to lure students into selecting classic PKU answers. The key discriminator is "normal enzyme levels"—this immediately shifts the diagnosis from enzyme mutation to cofactor deficiency, a concept many students conflate.

Clinical Pearl

In Indian newborn screening programs, infants with elevated phenylalanine on initial screening but normal PAH enzyme activity on enzyme assay should be investigated for BH₄ deficiency—early sapropterin therapy can prevent the severe neurological sequelae (dystonia, seizures, developmental delay) seen in untreated atypical PKU.

_Reference: Harper Biochemistry Ch. 31 (Amino Acid Metabolism); KD Tripathi Pharmacology Ch. 59 (Vitamins and Cofactors)_