Development of an analytical method for serum P-Cresylsulphate and Indoxylsulphate in patients with Chronic Kidney Disease

Marco Bagnati1, Diletta Duranti1, Matteo Basile1, Matteo Vidali1, Ilaria Crespi1, Nadia Atzeni1, Marina Albertario1, Gabriele Tornotti1, Carlotta Ferraris1, Filomena Panza2, Chiara Ralli2, Ennio Duranti2, Umberto

Dianzani1, Giorgio Bellomo1

1 Clinical Chemistry Unit, Maggiore della Carità Hosp ital, Novara; 2 UOC Nephrology and Dialysis Unit, Hospital of Arezz o

Introduction and Aim

P-Cresylsulphate (PCS) and Indoxylsulphate (IXS) are toxins derived from gutbacterial transformation of proteins (Figure 1) [1]. Recent evidences suggest thatPCS and IXS may contribute to the development of kidney disease and theiraccumulation has been shown to be associated with cardiovascular mortality inchronic kidney disease (CKD) [2]. In collaboration with an industrial Partner (B.S.N.Srl Biological Sales Network, Castelleone, Cremona, Italy), we developed andvalidated an analytical method for measuring total (protein bound and unbound)and free (unbound) serum PCS and IXS in patients with Chronic Kidney Disease(CKD).

Methods

For total and free PCS and IXS fractions, respectively, 25µL of serum or 25µL ofultrafiltrated serum (cut-off 30kDa) were mixed with 100µL of a solution ofMetaphosphoric acid 5% and internal standard (PCS-D4). After centrifugation, 10µLof supernatant were diluted to 1mL with mobile phase (Figure 2). Samples werethen analysed using an AB SCIEX Triple Quad™ 6500 LC-MS/MS system (column:Zorbax Eclipse Plus C18, ID 2.1x50mm, 1.8µm; flow: 0.35mL/min; T 40°C; inj vol:1µL; MRM: PCS 187.0>107.0, IXS 212.0>80.0, PCS-D4 191.0>111.0; negativeionization) (Figure 3).In our study, 26 patients with CKD stage 2-5 and 20 controls were investigated.

Results

The validated method displayed as LLOD 0.06µg/mL (PCS) and 0.09µg/mL (IXS),as LLOQ 0.20µg/mL (PCS) and 0.31µg/mL (IXS), linearity 1-100µg/mL (PCS) and1.25-125µg/mL (IXS), recovery 92-118% (PCS) and 93-117% (IXS); intra-assayprecision in the measuring range was between 0.57-1.97% and 0.80-1.83%respectively for total and free PCS, 4.80-7.80% and 3.42-9.71% for total and freeIXS; inter-assay precision was between 3.19-4.75% and 2.82-4.47% respectivelyfor total and free PCS, 6.15-7.31% and 7.84-9.06% for total and free IXS.Patients (n=26) with CKD displayed significantly higher free and total median PCSand IXS levels than healthy subjects (n=20) (free PCS: 0.62 vs 0.06µg/mL,p<0.0005; total PCS: 15.54 vs 2.28µg/mL, p<0.0005; free IXS: 0.16 vs 0.02µg/mL,p<0.0005; total IXS: 2.34 vs 0.59µg/mL, p<0.0005). Both higher free and total PCSand IXS levels were associated with higher stages of CKD (Figure 4). No correlationwas found between IXS and PCS levels and creatinine concentration.

Conclusions

We confirm that free and total PCS and IXS levels are increased in CKD patients,possibly be regarded as valid toxicity markers for the progression of CKD.

References

[1] R. Vanholder, E. Schepers, A. Plentik, E.V. Nagler; J Am Soc Nephrol, 25 (2014),pp 1897-1907.

[2] B.K. Meijers, B. Bammens, B. DeMoor, K. Verbeke, Y. Vanrenterghem, et al.;Kidney Int, 73 (2008), pp 1174-1180.

Figure 1. Structure of PCS and IXS.

Figure 2. Sample preparation.

Figure 3. Calibration curves of PCS (upper panel) and IXS (lower panel).

Figure 4. Association between PCS and IXS levels and CKD stage.

Wednesday, 15:30Exhibit HallPoster #9b