Milliplex map human high sensitivity t cell magnetic bead panel immunoassay

Fasting plasma glucose (FPG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels were measured by the enzymatic method on a Roche/Hitachi cobas c system (Sydney Adventist Hospital pathology laboratory). Low density lipoprotein cholesterol (LDL-C) levels were calculated by the Friedewald equation [19 (link)]. Plasma C—reactive protein (CRP) levels were quantified by Immunoturbidimetric assay on a Roche/Hitachi cobas c system (Sydney Adventist Hospital pathology laboratory). Plasma tumour necrosis factor- α (TNF-α), interleukin-1β (IL-1β) and IL-6 levels were measured using the MILLIPLEX^® MAP Human High Sensitivity T Cell Magnetic Bead Panel immunoassay (Merck KGaA, Darmstadt, Germany). Plasma reactive oxygen species (in the form of hydroperoxides), and total antioxidant capacity, were measured indirectly using the FORT (Free Oxygen Radical Test) and FORD (Free Oxygen Radicals Defence) colorimetric assays (CR3000, Callegari S.r.l., Catellani Group, Parma, Italy),as previously described [20 ].

The measurement of plasma urea and calculation of eGFR were performed by the Sydney Adventist Hospital pathology laboratory using methods well established for clinical laboratories. Briefly, fasting plasma urea levels were determined by the enzymatic method on a Roche/Hitachi cobas c system. Estimated Glomerular filtration rate (eGFR) was calculated using the simplified Modification of Diet in Renal Disease study equation: GFR (ml/min/1.73m²) = 186 × (serum creatinine level [mg/dl])-1.154 × (age)-0.203× [0.742, if female] × [1.212, if black] [23 (link), 24 ].
Plasma levels of NAD⁺ and its metabolites [NADP⁺, cyclic ADP ribose (cADPR), nicotinamide (NAM), N-methylnicotinamide (MeNAM)] were measured by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), as previously described [25 ]. LC/MS/MS was carried out using a UPLC-MSD assembly consisting of an Accela UPLC pump, Accela AS injector, and a TSQ Vantage bench-top mass spectrometer (ThermoFisher Scientific, Waltham, US).
Plasma interleukin-6 (IL-6) levels were quantitated using the MILLIPLEX® MAP Human High Sensitivity T Cell Magnetic Bead Panel immunoassay (Merck KGaA, Darmstadt, Germany).
Plasma Kynurenine (Kyn) and Tryptophan (Trp) levels were measured by high-performance liquid chromatography (Shimadzu LC-10AVP system, equipped with SPD- 10A UV detector, Kyoto, Japan), as previously described [26 (link)].