Capillary electrophoresis time of flight mass spectrometry

The metabolomics data quantified in our previous study [12 (link),15 (link)] were also used in this study. Briefly, saliva samples were collected between 8:00 a.m. and 12:00 p.m. Eating and drinking were not permitted for at least 1.5 h before the collection of saliva samples. Using oral hygiene products such as toothpaste and mouthwash was not permitted for at least 1.0 h before sample collection. Patients were asked to rinse their mouths with water immediately before saliva sample collection. On average, 400 μL of unstimulated whole saliva was collected. The metabolites in these saliva samples were quantified using capillary electrophoresis time-of-flight mass spectrometry (Agilent Technologies, Palo Alto, CA, USA).

Among 97 subjects, exploratory metabolome profiling analysis was performed using seven serum samples, including from two subjects in the VAKI group, two subjects in the infection subgroup, and three subjects in the HC subgroup (Table S1). To avoid interference with metabolomics profiling, we selectively included subjects without CKD history, previous malignancy, or chemotherapy by medical record review. The analysis was performed by Human Metabolome Technologies, Inc. (Yamagata, Japan) using capillary electrophoresis time-of-flight mass spectrometry with a fused silica capillary inner diameter of 50 μm × 80 cm (Agilent Technologies, Santa Clara, CA, USA) in two modes for cationic and anionic metabolites, as previously described [23 (link)]. Comparative analysis and hierarchical cluster analysis were performed using the relative peak area of putative metabolites. The Visualization and Analysis of Networks Containing Experimental Data (VANTED) software (VANTED version 2.5, www.vanted.org, accessed on 28 April 2021) was used for creating metabolic pathway maps based on peak profiles of putative metabolites.

Capillary electrophoresis mass spectrometry analysis was performed at the Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan, under the supervision of T.S. Cells were washed twice with 5% mannitol solution and covered with 700 μl of methanol containing 25 μM internal standards [20 μM each of methionine sulfone, 2-(N-morpholino)-ethanesulfonic acid and d-camphor-10-sulfonic acid] for 10 min. The resulting extracts were mixed with 200 μl of Milli-Q water and 400 μl of chloroform and centrifuged at 4600g for 15 min at 4°C. Then, 400 μl of the aqueous phase of the sample solution was subjected to ultrafiltration through a 5-kDa cutoff filter (Human Metabolome Technologies, Tsuruoka, Japan) to remove proteins. The filtrate was centrifugally concentrated and dissolved in 25 μl of Milli-Q water that contained reference compounds (200 μM each of 3-aminopyrrolidine and trimesic acid) immediately before metabolome analysis. The concentrations of all the charged metabolites were measured by capillary electrophoresis time-of-flight mass spectrometry (Agilent Technologies, Palo Alto, CA, USA) using the methods described previously (58 (link), 59 (link)). Analysis was performed using MasterHands Software (v2.13.0.8h, Keio University).