Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center Amsterdam, Amsterdam, The Netherlands.
J Inherit Metab Dis. 2012 Sep;35(5):909-16. doi: 10.1007/s10545-011-9443-0. Epub 2012 Jan 17.
The assessment of urinary α-aminoadipic semialdehyde (α-AASA) has become the diagnostic laboratory test for pyridoxine dependent seizures (PDS). α-AASA is in spontaneous equilibrium with its cyclic form Δ(1)-piperideine-6-carboxylate (P6C); a molecule with a heterocyclic ring structure. Ongoing diagnostic screening and monitoring revealed that in some individuals with milder ALDH7A1 variants, and patients co-treated with a lysine restricted diet, α-AASA was only modestly increased. This prompted us to investigate the diagnostic power and added value of the assessment of urinary P6C compared to α-AASA. Urine samples were diluted to a creatinine content of 0.1 mmol/L, followed by the addition of 0.01 nmol [(2)H(9)]pipecolic acid as internal standard (IS) and 5 μL was injected onto a Waters C(18) T3 HPLC column. Chromatography was performed using water/methanol 97/3 (v/v) including 0.03 % formic acid by volume with a flow rate of 150 μL/min and detection was accomplished in the multiple reaction monitoring mode: P6C m/z 128.1 > 82.1; [(2)H(9)]pipecolic acid m/z 139.1 > 93.1. Due to the dualistic nature of α-AASA/P6C, and the lack of a proper internal standard, the method is semi quantitative. The intra-assay CVs (n = 10) for two urine samples of proven PDS patients with only modest P6C increases were 4.7% and 8.1%, whereas their inter-assay CVs (n = 10) were 16 and 18% respectively. In all 40 urine samples from 35 individuals with proven PDS, we detected increased levels of P6C. Therefore, we conclude that the diagnostic power of the assessments of urinary P6C and α-AASA is comparable.
尿中α-氨基己二酸半醛（α-AASA）的测定已成为吡哆醇依赖癫痫（PDS）的诊断实验室检测指标。α-AASA 与它的环状形式 Δ(1)-哌啶-6-羧酸（P6C）处于自发平衡状态；P6C 是一种具有杂环结构的分子。正在进行的诊断性筛查和监测显示，在一些 ALDH7A1 变异较轻的个体以及联合赖氨酸限制饮食治疗的患者中，α-AASA 仅略有增加。这促使我们研究与 α-AASA 相比，尿中 P6C 的测定在诊断中的作用和附加价值。将尿样稀释至肌酐含量为 0.1mmol/L，然后加入 0.01nmol[（2）H9]哌啶酸作为内标（IS），并注入 5μL 到 Waters C18T3HPLC 柱上。采用体积比为 97/3（v/v）的水/甲醇（含 0.03%甲酸）作为流动相，流速为 150μL/min 进行色谱分离，检测采用多重反应监测模式：P6C m/z 128.1>82.1；[（2）H9]哌啶酸 m/z 139.1>93.1。由于 α-AASA/P6C 的双重性质以及缺乏合适的内标，该方法为半定量方法。两个 P6C 略有增加的确诊 PDS 患者尿液样本的批内 CV（n=10）分别为 4.7%和 8.1%，而其批间 CV（n=10）分别为 16%和 18%。在 35 名确诊 PDS 患者的 40 个尿液样本中，我们均检测到 P6C 水平升高。因此，我们得出结论，尿中 P6C 和 α-AASA 的测定在诊断能力上相当。
