Blood aliquots were obtained from the left ventricle during the exsanguination, chilled on ice, and centrifuged for 10 min at 3500 rpm at 4 °C. After the centrifuge, the plasma fraction was stored at −80 °C. Plasma anti-ds-DNA antibody activity was quantified through a mouse Anti-dsDNA IgG ELISA Kit (Alpha Diagnostic International, San Antonio, TX, USA) as per the manufacturer’s instructions [37 (link)]. Combur Test strips (Roche Diagnostics, Mannheim, Germany) were utilized to determine proteinuria, depositing a drop of instant urine on top of the reactive strip and observing color changes and comparing them with the color guide provided by the manufacturer.
TMAO and TMA level determination in plasma was carried out by stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry on an AB SCIEX 5000 triple quadrupole mass spectrometer interfaced with a Shimadzu high-performance liquid chromatography system using a silica column (4.69250 mm, 5 lm Luna Silica; Regis) at a flow rate of 0.8 mL/min. The separation was carried out as described previously [38 (link)].
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