6-oxo-PIP has been considered as a candidate biomarker for PDE, which is also generated during lysine metabolism, and might be also potentially applicable to newborn screening15–18. To further examine the suitability of 6-oxo-PIP as a possible new biomarker for PDE-ALDH7A1, we used LC-MS/MS to quantitatively assay these markers in 12 patients, including plasma, DBS, urine, and DUS, and compared them with epilepsy (non-PDE) controls, which were age matched as much as possible.
It could be seen from our results that the values of α-AASA-P6C and 6-oxo-PIP were above the reference range, while α-AAA and PA could be normal or slightly elevated. The levels of 6-oxo-PIP in urine in our study were comparable to those previously reported by Kuhara et al16., and similar values observed in plasma were comparable to those reported by Wempe et al15, which confirmed the reliability of our test. According to the report by Kuhara et al., the levels of 6-oxo-PIP elevated in all four patients in urine, and it was undetectable in more than 200 normal control samples of various ages16. In 2021, Engelke et al. found that the concentrations of 6-oxo-PIP in PDE-ALDH7A1 were significantly higher than non PDE children (without mention of normal or epilepsy children)17. However, considering that PDE is also an inherited epilepsy disease, we collected epilepsy patients (non-PDE) as control to analyze the specificity of 6-oxo-PIP. And the result showed that there was no overlap in the values of the two groups and the difference was significant, for all four samples. Similar difference appeared in the concentrations of α-AASA-P6C. Whereas, there was an evident overlap between PA and α-AAA, and the values of α-AAA between the two groups was no difference in DUS, giving it a low specificity. It followed that 6-oxo-PIP and α-AASA-P6C were specific when compared with other epilepsy patients and PDE-ALDH7A1. In addition, age correlation analysis suggested that 6-oxo-PIP was negatively correlated with age and duration of pyridoxine treatment in plasma and DBS. But even if the age was over 9 years old or after nearly 10 years of treatment, the concentrations of 6-oxo-PIP remained significantly above the reference range, which indicated that 6-oxo-PIP is a specific biomarker for PDE regardless of the age of the patient, even under pyridoxine treatment. But it also suggested that age and whether taking pyridoxine treatment or not should be taken into account when measuring 6-oxo-PIP.
Previous reports suggested that the concentrations of PA varied greatly among different sample types, and the extent of PA elevation in CSF exceeded that of plasma by a factor of 2.2 to 4.8.19 And PA levels could be normalized after many months to years successful treatment with pyridoxine or with age, whether in urine or plasma2, 20–22. Our study also showed that normal PA levels could be seen in almost a half of PDE-ALDH7A1 patients, and the content of PA was negatively correlated with age, pyridoxine dose, and the duration of treatment in DUS or DBS samples. Therefore, the content of PA in DUS or DBS could be decreased with the increase of age, and pyridoxine treatment, whether in urine or blood, that is to say, the concentrations of PA is affected by a variety of factors. Currently, there were few tests for α-AAA, the direct downstream metabolite of α-AASA, and only our previously study has shown that α-AAA values could be normal or elevated12. In this result, in addition to showing that the levels of α-AAA in PDE-ALDH7A1 patients could be in reference range, it was also negatively correlated with age in plasma, respectively. Therefore, α-AAA might be also subject to multiple factors, which might explain the inter-individual variation in the level of this indicator among PDE-ALDH7A1.
Although plasma and urine were the most commonly used specimens in patients, the noninvasive nature of urine made it to be the preferred sample type. In addition, DBS and DUS were preferred in sample selection due to the convenient to transport characteristic. However, the previous assay samples for the detection of lysine intermediate metabolites in PDE-ALDH7A1 patients were limited, mostly single plasma or urine, lacking sample selectivity studies 9, 20, 23. Therefore, we analyzed the correlation between various biomarkers in different samples of blood or urine. Among the traditional biomarkers, our data showed that the correlation between plasma and DBS was significantly positive for PA, but not for α-AAA which were positive correlated in urine and DUS. However, the concentrations of PA and α-AAA in PDE-ALDH7A1 patients might be in reference range or elevated. Although the values of α-AASA-P6C was positively correlated in plasma and DBS or urine and DUS, the difference between plasma and DBS was significant. But surprisingly, a positive correlation for plasma and DBS, or urine and DUS of 6-oxo-PIP was found simultaneously. And our results showed that the values of 6-oxo-PIP were elevated in all samples of PDE-ALDH7A1 patients, with no difference in values between plasma and DBS or urine and DUS. Therefore, the application of 6-oxo-PIP as the screening index was also high sufficient to recognize PDE, and the sample type could be selected according to the actual demand during detection. In addition, the values of 6-oxo-PIP in urine and DUS were much higher than those in plasma and urine. Given that DUS is non-invasive and easy to prepare with small amounts of urine, making it to be an ideal alternative sample, especially for newborns.
Stability at RT conditions is essential, as it is more convenient to rely on sampled to be collected, dried, and shipped at RT conditions in clinical work. As mentioned earlier, α-AASA-P6C was unstable and extremely prone to degradation13, 14, 20, 23, 24, which is also found in our results, where degradation of α-AASA-P6C could still be observed even at temperatures of 4°C and − 20°C. In addition, we found that the degradation rate of α-AASA-P6C was relatively slowed down in DUS, which also indicated that DUS samples were more suitable for the detection of it. Wempe et al. studied the stability of 6-oxo-PIP in urine and found that it was measurable preserved in RT for up to 4 months15. We tested the stability 6-oxo-PIP at multiple temperatures in various samples and the results suggested that 6-oxo-PIP was stable under RT in all kinds of samples, including plasma, DBS, urine, and DUS, not to mention at 4℃ and − 20℃. At this point, the 6-oxo-PIP compensates for this weakness of α-AASA-P6C’s instability.
It was well known that the cause of neurodevelopmental delay in PDE patients was the accumulation of lysine intermediate metabolites6. However, our comparison showed no difference in biomarker levels between patients with different degrees of developmental delay and those with normal development. In addition, there were two cases developing uncontrolled seizures while being treated with pyridoxine monotherapy because of secondary brain injury in our PDE cohort. And there was also no significant difference in the levels of biomarkers between the two patients compared with those of others whose seizures had been controlled. Since age and pyridoxine treatment are negatively correlated with the level of individual biomarkers, it is theoretically possible that the biomarkers might be higher in young and untreated PDE-ALDH7A1 patients. Thus, the above results of no difference might be related to the fact that all samples in this study were collected during pyridoxine maintenance treatment. In addition, given that accumulation in the brain is the root cause of the developmental impact, the relevance for clinical symptoms is still limited due to the lack of studies on cerebrospinal fluid levels. More recently, triple therapy in PDE patients has shown improvements in development, accompanied by the reduction in biomarkers levels9, 11, 25. However, it remains unclear which biomarker, including α-AASA-P6C, α-AAA, PA or 6-oxo-PIP, is most sensitive to triple therapy.