The measurement of urinary Δ¹-piperideine-6-carboxylate, the alter ego of α-aminoadipic semialdehyde, in Antiquitin deficiency

Abstract

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.

Fig. 1

Spontaneous equilibrium of P6C and α-AASA

Fig. 2

Mass fragmentograms of the LC-MS/MS measurement of P6C; in the left panel: control urine sample (concentration 0.02 mmol/mol creatinine), and in the right panel: PDS patient urine sample (concentration 2.05 mmol/mol creatinine). IS represents the signal of [²H₉]pipecolic acid serving as internal standard

Fig. 3

P6C and α-AASA levels versus protein intake (grams protein/kg body weight). The Spearman correlation between P6C and protein intake was 0.33; P = 0.003 (Fig. 3a). The Spearman correlation between α-AASA and protein intake was 0.33; P = 0.004 (Fig 3b)

Fig. 4

P6C levels in reference population; the insert displays P6C levels in children from 0 to 12 months of age

Fig. 5

P6C levels in patients with proven PDS using a logarithmic Y-axis presentation

Fig. 6

Relationship between α-AASA and P6C levels in urine samples from PDS patients; spearman correlation is 0.78; p < 0.001