MO571: Novel Insights Into Uremic Toxins in CKD: Rapid Detection of Microbiota-Derived Indoxyl Sulfate in CKD Patients
Authors: Caggiano Gianvito , Pesce Francesco , Antonio Colabufo Nicola , Colangiulo Santina , Amodio Loredana , Gesualdo Loreto 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Emerging evidence suggest that uremic toxins are involved in the progression of chronic kidney disease (CKD). Indoxyl Sulfate (IxS) is one of the most studied since resulted as strong predictor of cardiovascular disease (CVD) and linked with the prognosis of patients with CKD. Indeed, blood levels of IxS increase during CKD stages reaching a maximum in patients with end-stage renal disease (ESRD). Additionally, hemodialysis is able to eliminate only the blood-free fraction as the IxS albumin bond hides its removal. As a result, patients on dialysis show the highest IxS levels resulting in worsening condition, enhancement of CVD-related complication. Thus, IxS rapid quantification could offers clinicians an alternative method for monitoring CKD progression and address the dialysis effectiveness. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is commonly used for IxS detection and require dedicated equipment and skills, long analysis times and costly analytical instruments. To address these issues, the development of a simple and rapid analytical method for IxS detection is needed. Here, we present a study focused on the development and validation of a rapid and simple method of IxS determination based on colorimetric array-based analysis exploitable in clinical practice for CKD patients.

METHOD
A total of 20 blood samples (10 healthy people and 10 CKD patients) were used to assess the feasibility of our colorimetric assay. IxS from serum sample was quantified at time of inclusion by LC-MS/MS. Next, from the same samples, we used a derivatization reaction for splitting the serum IxS into indigo-blue. This method enables the rapid quantification of IxS-derived indigo by a smart spectrophotometer at λ = 420–450 nm. Analyte concentration was instantly calculated from absorbance/concentration calibration curve of indigo-blue. Finally, a correlation between the two methods were carried out for the colorimetric analysis validation.

RESULTS
To accurately determine concentrations of IxS in the low µM range for practical diagnostic purposes, a wide linear calibration curve of was successfully established. The calibration curve ranged from ‘0 µM to 330 µM’ for indigo blue. The regression equation between absorbance and concentration was y = 0.0003 x + 0.0192, and the correlation coefficient (r2) was 0.9941. The feasibility was confirmed by detecting IxS in serum samples from healthy people and CKD patient. A positive correlation was observed between IxS quantified by LC-MS/MS and IxS-derived indigo quantified by spectrophotometry (P &lt; 0 001, r = 0.088). Furthermore, the results of colorimetric analysis were compared with those obtained using LC–MS/MS, and no significant difference was found between them (Fig. 2). This result indicated that the colorimetric detection of IxS is comparable to LC–MS/MS, but it is simpler and faster for CKD monitoring in clinical practice.

CONCLUSION
The aim of our study was to develop and validate a quantitative method for the rapid estimation of serum IxS employable to monitor CKD progression in clinical setting, identify patient at risk and help further dialysis therapies aimed to lowering blood uremic toxins. Conventionally, LC-MS/MS is the most used diagnostic tool for the IxS quantification due to high sensitivity and accuracy. However, the great rigorousness analysis together with the requirement of real-time results hinders its employment in the clinical practice. Our results provide a low-cost, easy-to-operate, label-free, rapid and sensitive method for IxS determination for application in tracking IxS in blood and studying CKD progression.