Labsolutions version 5

Data analyses were performed by LabSolutions version 5.91 (Shimadzu Co.). Box plots were produced by Microsoft Excel 2016. Hierarchical clustering analysis (HCA) using the auto scaling peak area values was performed by the Ward method with a web-based statistical tool, MetaboAnalyst 4.0.^{24 (link))}

The creatinine concentration of each urine specimen was measured using a creatinine (urinary) Colorimetric Assay Kit (Cayman Chemicals) according to the manufacturer’s protocol. Data from SRM-MS were analyzed using LabSolutions version 5.91 (Shimadzu), and the integrated peak area of each metabolite was normalized to the IS and creatinine response. The normalized peak areas were statistically compared between pre- and post-tranilast administration using Prism 9 (GraphPad Software, San Diego, CA, USA), with statistical significance set at p < 0.05. Analysis was performed on 6 patients with urine specimens at 0, 4, 12, and 24 weeks (long-term study) and 17 patients with urine specimens only at 0 and 4 weeks (short-term study). For the long-term study, statistical significance was determined using Friedman’s test with Dunn’s post-hoc analysis to examine differences in metabolite levels pre- and post-tranilast administration. Correlations between urinary metabolite levels and other markers, such as BNP concentration and 8-OHdG, were evaluated using Spearman’s rank correlation coefficient. For the short-term study, the Wilcoxon signed-rank test was used to calculate statistical significance.

The Shimadzu “Lab Solutions^® version 5.72” software was used for peak area integration. Least square linear regression was used to generate calibration curve data. GraphPad prism 5.0 was used to generate plasma-time concentration curves. Liensinine plasma concentration versus the time data for each group of rats was analyzed using Drug and Statistics software (DAS, Version 3.0, Shanghai BioGuider Medicinal Technology, China).

The oligomeric state of the antidin sbAvd-7 was analyzed with size exclusion chromatography (SEC) using a liquid chromatography instrument (CBM-20A, Shimadzu Corporation) equipped with an autosampler (SIL-20A), UV-Vis (SDP-20A), and a fluorescence detector (RF-20Axs). The instrument was integrated with a static light scattering instrument (SLS, Zetasizer μV light scattering detector (Malvern Instruments Ltd.)) to determine molecular weight of the eluted proteins. The instrument was controlled using Lab Solutions Version 5.51 (Shimadzu Corporation) and OmniSEC 4.7 (Malvern Instruments Ltd.). Samples (~50 μg in 10–100 μl) were injected onto a Superdex200 Increase 5/150GL column (GE Healthcare) and equilibrated with the buffer the protein was dialyzed against (50 mM sodium phosphate, 650 mM NaCl, pH 7) with a flow rate of 0.1 ml/min at 20°C. Molecular weight determination was done by calculating a standard curve based on the elution volume of the molecular weight markers (CA, carbonic anhydrase 29 kDa; BSA, Bovine Serum Albumin 66 kDa; ADH, Alcohol Dehydrogenase 150 kDa; BA, ß-Amylase 200 kDa, Sigma-Aldrich), and alternatively, using the light-scattering intensity-based determination protocol involving BSA (monomeric peak) in SLS detector calibration using a Malvern microV detector and the OmniSEC software (Malvern Instruments Ltd.) (Fig 6).

Proteins were analysed using a liquid chromatography instrument (CBM-20A, Shimadzu Corporation, Kyoto, Japan) equipped with autosampler (SIL-20A), UV-VIS (SPD-20A) and fluorescence detector (RF-20Axs) as well as Zetasizer µV light scattering detector (Malvern Instruments Ltd, Worcestershire, UK) for molecular weight (static light scattering) and hydrodynamic size (dynamic light scattering) determination. The instrument was controlled using Lab Solutions Version 5.51 (Shimadzu Corporation) and OmniSEC 4.7 (Malvern Instruments Ltd.). Samples (70 µg in 40–100 µl) were injected on a Superdex75 5/150GL column (GE healthcare, Uppsala, Sweden) equilibrated with Na₂HPO₄/NaH₂PO₄, 650 mM NaCl, pH 7. Runs were executed with a flow rate of 0.25 ml/min at 12°C. Molecular weight determination was either done by calculating a standard curve of molecular weight markers (cytochrome C, 12.4 kDa; carbonic anhydrase, 29 kDa; ovalbumin, 44 kDa; BSA, 66 kDa, Sigma-Aldrich) or was based on the light-scattering intensity of the eluting protein, for which BSA was used for instrument calibration using the OmniSEC software (Malvern Instruments Ltd.).