Human tissues were homogenized in ice-cold phosphate-buffered saline (PBS) and centrifuged, and supernatants were collected for Hcy quantification. Hcy concentrations were determined using an Axis Homocysteine Enzyme Immunoassay Kit (Axis-Shield). To assay metabolite levels, cells were harvested by PBS washing and denatured in pre-chilled 60% methanol (in ddH2O, pre-cooled at −80 °C for 1–2 h). Cell lysates were centrifuged (10,000 × g) at 4 °C for 5 min. Supernatants were vacuum-dried, re-dissolved in ddH2O, and subjected to ultrafiltration on a polyvinylidene fluoride low protein binding membrane (Millex-GV4 and Millex-HV4, Millipore). Metabolites were extracted and Hcy was analyzed using LC-MS. SAM and SAH levels were detected using a SAM & SAH ELISA Combo Kit (Cell Biolabs). 1-Methylnicotinamide was measured using a UHPLC-QTOF-MS System (Agilent Technologies, 1290 LC, 6550 MS) as described previously99 (link). Each assay was repeated in triplicate, and means were used for analysis.
Axis homocysteine enzyme immunoassay kit
Manufactured by Axis-Shield
The Axis Homocysteine Enzyme Immunoassay Kit is a laboratory test used to measure the level of homocysteine in a patient's blood sample. Homocysteine is an amino acid that can be used as a biomarker for various health conditions. The kit employs an enzyme immunoassay technique to quantitatively determine the concentration of homocysteine.
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Lab products found in correlation
Millex gv4, by Merck Group (2 mentions)
Millex hv4, by Merck Group (2 mentions)
Cobas e411 analyzer, by Roche (2 mentions)
Uhplc qtof ms system, by Agilent Technologies (1 mentions)
Sam sah elisa combo kit, by Cell Biolabs (1 mentions)
Elecsys folate 3, by Roche (1 mentions)
Proteasome activity assay kit, by Abcam (1 mentions)
4 protocols using axis homocysteine enzyme immunoassay kit
1
Homocysteine Analysis in Human Tissues
2
Plasma Biomarker Quantification Protocol
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EDTA-treated plasma samples were obtained, centrifuged immediately, and stored in a −80°C freezer until analysis for folate, homocysteine, and triglyceride content. Folate concentration was determined using the Elecsys Folate III competitive chemiluminescent immunoassay (Roche) by using a Cobas E411 Analyzer (Roche). Homocysteine concentration was quantified using the Axis Homocysteine Enzyme Immunoassay Kit (Axis-Shield; Norton, MA). TG, total cholesterol, HDL-C, LDL-C, apolipoprotein A1, apolipoprotein B, FFA, fasting blood glucose, and glycated albumin were analyzed using an automatic biochemical analyzer (Hitachi 7180; WAKO).
3
Quantification of Homocysteine and Metabolites in Heart Tissues
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Heart tissues were homogenized on ice-cold PBS (PBS) and centrifuged (10,000 x g) at 4°C for 15 min, and the supernatants were collected for Hcy quantification. Hcy concentration was determined using an Axis Homocysteine Enzyme Immunoassay Kit (Axis-Shield). HTL was assayed as the following: heart tissues were harvested by PBS washing followed by denaturing by pre-chilled 80% methanol (dissolved in ddH2O, precooled in −80°C). The lysate was centrifuged (10,000 x g) at 4°C for 10 min. The supernatant was vacuum dried, then re-dissolved in ddH2O and subjected to ultra-filtration on a polyvinylidene fluoride low protein binding membrane (Millex-GV4 and Millex-HV4, Millipore). The collected metabolites were extracted and the HTL was analyzed using liquid chromatography-mass spectrometry (LC-MS) as previously described.57 (link)
4
Measuring Metabolite Levels in Biological Samples
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Proteasomal activity was measured with the Proteasome Activity Assay Kit (Abcam, Cambridge, UK), as described in the online-only Data Supplement.
Metabolite Quantification in Human Plasma, Tissues, and Cultured Cells Folate concentration was detected by the Elecsys FOL III competitive chemiluminescent immunoassay (Roche, Shanghai, China) using a Cobas E411 Analyzer (Roche). Homocysteine concentration was quantified with the Axis Homocysteine Enzyme Immunoassay Kit (Axis-Shield, Norton, MA), and S-adenosylmethionine and S-adenosylhomocysteine were measured with the S-adenosylmethionine and S-adenosylhomocysteine ELISA Combo Kit (Cell Biolabs, San Diego, CA). Detailed information is provided in the online-only Data Supplement.
Metabolite Quantification in Human Plasma, Tissues, and Cultured Cells Folate concentration was detected by the Elecsys FOL III competitive chemiluminescent immunoassay (Roche, Shanghai, China) using a Cobas E411 Analyzer (Roche). Homocysteine concentration was quantified with the Axis Homocysteine Enzyme Immunoassay Kit (Axis-Shield, Norton, MA), and S-adenosylmethionine and S-adenosylhomocysteine were measured with the S-adenosylmethionine and S-adenosylhomocysteine ELISA Combo Kit (Cell Biolabs, San Diego, CA). Detailed information is provided in the online-only Data Supplement.
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