#AI Reads Urine# Quantifying New Biomarkers in Urine & Plasma for Pyridoxine-Dependent Epilepsy: Urine's Higher Translational Value

This paper developed and validated a method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify two new biomarkers, 2S,6S- and 2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine2-carboxylic acid (6-oxoPIP), in human urine and plasma for diagnosing and monitoring pyridoxine-dependent epilepsy (PDE), overcoming challenges of previous biomarkers.

### Comparison and translational potential of blood and urine biomarkers

#### Comparison of detection methods and parameters

- **Difficulty in sample processing and detection**: For urine biomarker detection, a 10µL urine sample is combined with a specific mixture and can be directly injected. For blood biomarker detection, a 10µL plasma sample needs to be mixed with a solution containing 50% methanol, followed by vortexing, centrifugation, and then the supernatant is taken for injection, which is relatively more complicated.

- **Linear range**: In urine, the linear range of 2 - OPP is 0 - 15µmol/L, and that of 6 - oxoPIP is 0 - 25µmol/L. In plasma, the linear range of 2 - OPP is 0 - 15µmol/L, and that of 6 - oxoPIP is 0 - 25µmol/L. The linear ranges of the two are similar.

- **Limit of detection (LOD) and limit of quantification (LOQ)**: In urine, the LOD of 2 - OPP is 13nmol/L, and the LOQ is 40nmol/L. In plasma, the LOD of 2 - OPP is 30nmol/L, and the LOQ is 90nmol/L. The LOD and LOQ of 2 - OPP in urine are lower, indicating higher sensitivity. In urine, the LOD of 6 - oxoPIP is 18nmol/L, and the LOQ is 55nmol/L. In plasma, the LOD of 6 - oxoPIP is 11nmol/L, and the LOQ is 32nmol/L. The LOD of 6 - oxoPIP in plasma is lower, but the LOQ in urine is also relatively low, and the difference between them is not significant.

- **Precision and accuracy**: The intra - day and inter - day precision (coefficient of variation, CV%) and accuracy (percentage difference from the nominal concentration) of 2 - OPP and 6 - oxoPIP in urine and plasma are all good and the values are similar, indicating that they perform equally well in this regard.

- **Stability**: The two biomarkers are stable in urine and plasma at + 4°C and - 20°C for one month and at room temperature for 24 hours, with comparable stability.

- **Matrix effect and extraction recovery**: The matrix effect and extraction recovery of both biomarkers in urine and plasma are within the acceptable range (± 15%), showing similar performance.

#### Comparison in clinical application

- **Levels in healthy population**: In the urine of healthy individuals, the concentration of 2 - OPP is ≤ 16.0µmol/mol Creu, and that of 6 - oxoPIP is ≤ 727.6µmol/mol Creu. In plasma, the concentrations of 2 - OPP and 6 - oxoPIP are below the LOQ. The background level of biomarkers in urine is relatively higher.

- **Levels in patients**: In the urine of PDE patients, the levels of 2 - OPP and 6 - oxoPIP are significantly increased. For example, in patient 3, the concentration of 2 - OPP in urine reaches 2885.30µmol/mol Creu, and that of 6 - oxoPIP reaches 1974.20µmol/mol Creu. In plasma, for patient 1 (treated), 2 - OPP is 0.20µmol/L, and 6 - oxoPIP is 10.50µmol/L. For patient 2 (treated and late - onset), 2 - OPP is 0.09µmol/L, and 6 - oxoPIP is 0.50µmol/L. The variation range of biomarker levels in urine is larger, making it easier to detect differences.

#### Conclusion

Overall, the urine biomarkers have lower LOD and LOQ for 2 - OPP detection and a larger variation range of biomarker levels in patients' urine, which is more conducive to disease diagnosis. Therefore, urine biomarkers are relatively more suitable for translational application. However, the detection of plasma biomarkers also has its value. For example, the LOD of 6 - oxoPIP in plasma is also relatively low, and it can also provide important diagnostic information in some cases. The two can complement each other for the diagnosis and monitoring of PDE.

The original publication link: https://www.sciencedirect.com/science/article/abs/pii/S0009898124023647

Youhe Gao

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