Chapter 2 28 The proton NMR spectra were deconvoluted using proprietary software as follows. The singlet peak emanating from four equivalent protons of the ethylene moiety in the MgEDTA complex (-N-CH2-CH2-N-) appearing at 2.66 ppm in the NMR spectrum was used for quantitation. As the Mg-EDTA NMR signal overlaps with a signal from circulating proteins, the deconvolution method included the protein signal encompassing approximately 50 Hz, and a 16 Hz wide region of the Mg-EDTA peak was integrated. The relation between Mg-EDTA signal area and Mg concentrations were established by standard addition experiments on dialyzed serum, and the conversion factor thus obtained was applied to transform Mg-EDTA signal areas to concentrations expressed in mmol/L. The Mg concentrations were standardized against a 25.0 mM solution of ACS Reagent Grade MgCl2.6H2O (MilliporeSigma, US). Defined amounts of the standard MgCl2 solution were spiked into dialyzed serum devoid of ionized Mg. Accuracy was ascertained through recovery experiments done from 0 to 4.0 mM Mg concentrations. Similar to other NMR assays on Vantera, the commercial assay would also involve running 2 levels of serum controls serving daily check on accuracy and guarding against drift with time. We tested for imprecision in the NMR-measured ionized Mg assay as per CLSI guidelines. Pooled samples with two varying concentrations of Mg (low and high) were tested to determine within-lab (inter-assay) precision. Roche modular total Mg assay and assay comparison The Roche Modular assay is a colorimetric end point assay that measures total Mg in a serum, heparin plasma or urine sample. The method is based on the reaction of Mg with xylidyl blue in an alkaline solution containing ethylene glycol-bis(β-aminoethyl ether)- N,N,N’,N’-tetraacetic acid (EGTA), which has a lower affinity for Mg, in order to mask the calcium in the sample. In the alkaline solution, Mg forms a purple complex with the xylidyl blue diazonium salt and the concentration of Mg is determined photometrically via the decrease in the xylidyl blue absorbance (505/600 nm). In order to understand the differences between the two assays, we compared values from the NMR-based ionized Mg assay with total Mg measured on a Roche Modular Analyzer (Roche Diagnostics, Mannheim, Germany) in 799 samples of appropriate specimen types from the second screening of the PREVEND cohort. The Roche Mg assay has an inter-assay coefficient of variation of 1.3%. Assessment of covariates Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Smoking status was defined as self-reported never smoker, former smoker or current
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