Epidemiological studies have shown that elevated plasma advanced glycation endproducts levels are associated with disease conditions such as diabetes, kidney disease and heart disease(Reference Semba, Nicklett and Ferrucci1), thus AGE have been subsequently used as a marker for the progression of disease states(Reference Vlassara and Palace2). CML is the best characterised AGE. Some investigators have previously employed a protocol to stabilise CML at the time of blood collection. However, it has not been shown whether this additional protocol prevents artefactual CML and/or CEL formation, and including additional steps in handling of blood samples at collection inevitably increases the costs associated with CML analysis, and reduces the likelihood of such samples being collected in large population studies. The aim of this validation study was to compare measured CML and CEL in blood samples collected under various processing conditions.
The investigation examined the effect on the level of CML and CEL in pooled samples (n 10) of (i) different blood collection tubes (clotted (serum), EDTA and lithium heparin (both plasma)), (ii) the addition of a stabilising cocktail solution (0.2 mM indomethacin, 5 mM butylated hydroxytoulene and 10 mM diethylenetriaminepentaacetic acid), (iii) use of fresh and stored (1 d and 1 week, at 4°C) stability cocktail, (iv) storing samples at 4 and 21°C for 1, 2, 4, 8 and 24 h, prior to blood centrifugation and/or addition of stabilising cocktail solution, and (v) effect of one and two freeze thaw cycles. All samples were reduced, precipitated, hydrolysed and underwent solid-phase extraction using a C18 cartridge. Extracts were analysed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with the use of isotopically labelled internal standards and by reference to an external standard calibration curve(Reference Teerlink, Barto and Ten Brink3). Samples were extracted in triplicate (n 3). The inter-sample variability was <7%, and the intra and inter assay coefficient of variability were 5.1 and 5.7%, respectively.
The table shows the effect of time before centrifugation on CML and CEL in EDTA plasma samples, stored at room temperature.
There was no significant effect of any of the different experimental conditions tested (ANOVA). Additional processing of blood at collection does not enhance measured CML and CEL. This indicates that specific collection protocols are not required for measuring CML and CEL, thus indicating that many samples currently stored for epidemiological studies could be used for CML and CEL analyses.
