This study aimed to investigate and improve the usefulness of the 48‐hour BH₄ loading test and to assess genotype for BH₄ responsiveness prediction, using the new definition of BH₄ responsiveness from the European guidelines, as well as an amended definition.

Applying the definition of the European guidelines (≥100% increase in natural protein tolerance) and an amended definition (≥100% increase in natural protein tolerance or tolerating a safe natural protein intake) to a previous dataset, we first assessed the positive predictive value (PPV) of the 48‐hour BH₄ loading test using a cutoff value of 30%. Then, we tried to improve this PPV by using different cutoff values and separate time points. Last, using the BIOPKU database, we compared predicted BH₄ responsiveness (according to genotype) and genotypic phenotype values (GPVs) in BH₄‐responsive and BH₄‐unresponsive patients.

The PPV of the 48‐hour loading test was 50.0% using the definition of the European guidelines, and 69.4% when applying the amended definition of BH₄ responsiveness. Higher cutoff values led to a higher PPV, but resulted in an increase in false‐negative tests. Parameters for genotype overlapped between BH₄‐responsive and BH₄‐unresponsive patients, although BH₄ responsiveness was not observed in patients with a GPV below 2.4.

The 48‐hour BH₄ loading test is not as useful as previously considered and cannot be improved easily, whereas genotype seems mainly helpful in excluding BH₄ responsiveness. Overall, the definition of BH₄ responsiveness and BH₄ responsiveness testing require further attention.