Tbars assay

Enzymatic and colorimetric assays were used to measure serum levels of 3-hydroxybutyrate (Stanbio B-HB LiquiColor Assay, EKF Diagnostics), NEFA (NEFA-HR; ref. 2 (link); Wako Diagnostics), and TG (TR0100, MilliporeSigma). Tissue TG levels were measured as previously reported (32 (link)). Lipid peroxidation in liver tissue was measured with thiobarbituric acid reactive substances (TBARS) assay (Cayman Chemical) as directed by the manufacturer. Untargeted metabolomics from flash-frozen liver samples was performed by Metabolon Inc. Serum ALT activity was measured by ALT Activity Assay kit (MAK052, MilliporeSigma) as directed by the manufacturer. Hepatokine GDF15, FGF21, and Angplt3 serum levels were measured by Quantikine ELISA kits (R&D Systems, Inc).

LDL was isolated from human plasma obtained from DRK-Blutspendedienst NSTOB, Springe, Germany (All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Committee of the Hannover Medical School. All donors signed the informed consent) and oxidized with CuSO₄ as previously described^{38 (link),39 (link)}. oxLDL was characterized by the protein concentration measured by DC-protein assay (Biorad, Munchen, Germany) and lipid peroxidation measured by the TBARS assay (Cayman chemicals, Michigan, USA). oxLDL concentration of 0.43 µM/mg protein (~10 ug/mL protein), 0.86 µM/mg protein (~20 ug/mL protein) and 1 µM/mg protein (~25 ug/mL protein) was used. Stimulation of the osteoclast with oxLDL and inhibition of the several receptors (CD36, LOX-1, TLR-4 and SRA-1) were all done at the beginning of osteoclast differentiation i.e. when RANKL was added. CD36, LOX-1, TLR-4 and SR-A were inhibited by 10 µM of sulfosuccinyloleate (SSO) (Cayman chemicals, Michigan, USA), 250 µM of κ-Carrageenan (Sigma, Steinheim, Germany), 5 µM of CLI-095 (Invivo-Gen) and 100 µg/mL of dextran sulphate (Sigma, Steinheim, Germany) respectively.

IMS32 cells were seeded at a density of 9 × 10³ cells/cm² and maintained for 1 h under each condition described as Measurement of ROS production. Intracellular MDA levels were assessed under each condition by TBARS assay (Cayman Chemical Company, Ann Arbor, MI, USA) and a plate reader according to the manufacturer’s instructions.

Patients/controls maternal blood and umbilical blood cords sample were centrifuged at 700–1,000 x g for 10 min at 4°C and plasma was transferred to a clean tube and stored on ice. Thiobarbituric reactive substances (TBARS) assay, that measures malonyldialdeide (MDA) in samples, was performed as previously described [27 (link)] by using the TBARS assay (Cayman Chemical, Michigan, USA). The absorbance of supernatants was read at 530–540 nm, using a spectrometer (BS1000 Spectra Count, San Jose, CA, USA).

Tissues were homogenized with 10 volumes of RIPA buffer containing protease and phosphatase inhibitors (Sigma-Aldrich P2714 and P5726, respectively) and centrifuged at 1600 g for 10 minutes at 4°C. The supernatant was used undiluted in the TBARS assay (Cayman Chemical, Ann Arbor, USA) according to the manufacturer's instructions. Briefly, the sample supernatant was combined with thiobarbituric acid assay reagents and boiled for 1 hour. Cooled sample preparations were loaded onto a 96-well plate and the absorbance read at 535 nm in a microplate reader (Molecular Devices, San Jose, USA), and lipid peroxides were interpolated from a malondialdehyde standard curve.