*Biochemical Analysis*

The determination of homocysteic acid (HCA) was performed as recently described in detail [36] with minor modifications, as the method was originally validated for the analysis of human serum and urine. Briefly, HCA was determined in murine serum and urine using a combination of protein precipitation and solid phase extraction for sample preparation followed by an LC–MS/MS analysis using a combination of a HILIC separation and tandem mass spectrometry. Samples were processed as previously described [36] by adding formic acid followed by protein precipitation using cooled acetonitrile. Samples were vortexed, centrifuged and loaded onto conditioned tables (Strata X AW SPE columns (33 μm, 30 mg / 1 mL, Phenomenex, Ascha ffenburg, Germany) using the automated sample preparation system Extrahera (Biotage, Uppsala, Sweden). After washing the cartridges using water, methanol and a mixture of acetonitrile and aqueous ammonium hydroxide solution, HCA was eluted using two times a mixture of methanol and aqueous ammonium hydroxide solution. The eluate was dried and reconstituted by adding ammonium acetate solution and acetonitrile separately. Afterwards, the samples were injected into the LC-MS/MS system. The LC-MS/MS system consisted of a triple quadrupole mass spectrometer QTRAP 6500+ (Sciex, Darmstadt, Germany) equipped with a Turbo Ion Spray source operated in negative electrospray ionization mode and an Agilent 1290 Infinity LC-system with binary HPLC pump, column oven and autosampler (Agilent, Waldbronn, Germany). The chromatographic separation was performed using a Luna 3 μm HILIC 200 Å 100 × 2 mm column in combination with a KrudKatcher in-line filter (both Phenomenex, Ascha ffenburg, Germany). Data acquisition was done using Analyst Software 1.6.3 and quantification was performed with MultiQuant Software 3.0.2 (both Sciex, Darmstadt, Germany), employing the internal standard method. Calibration curves were calculated by linear regression with 1/x weighting. Acceptance criteria and quality assurance measures have been applied as previously described [36].

The determination of homocysteine (HCys) was performed using protein precipitation in combination with LC–MS/MS. Briefly, 20 μL of serum or urine was pipetted to a polypropylene tube and 20 μL of 15 mg/mL aqueous TCEP-solution (tris(2-carboxyethyl)phosphine), 40 μL IS working solution (500 ng/mL HCys-d4 in methanolic TCEP solution, 1 mg/mL) and 40 μL methanolic TCEP solution, 1 mg/mL were added. Afterwards, samples were vortexed, centrifuged, transferred into another polypropylene tube, and dried using nitrogen. The dried samples were reconstituted using 50 μL of water containing 10 mM ammonium acetate bu ffer and 10 mM acetic acid, centrifuged again and injected into the LC-MS/MS system. The same LC-MS/MS-system and acceptance criteria as described for HCA were used. However, positive electrospray ionization mode was applied and a Luna Omega 1.6 μm Polar C18 100 × 2.1 mm column in combination with a respective pre column (both Phenomenex, Ascha ffenburg, Germany) was used.
