Validation study to compare effects of processing protocols on measured Nɛ-(carboxymethyl)lysine and Nɛ-(carboxyethyl)lysine in blood

Abstract

Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured Nε-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, Nε-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.

High-throughput quantification of carboxymethyl lysine in serum and plasma using high-resolution accurate mass Orbitrap mass spectrometry

“…CML concentrations were also found to be similar in plasma versus serum samples in a previous study. 4…”

“…A high-throughput 96-well deep-well plate method of sample preparation based on previously published methods was developed. 1,4–6 Sodium tetraborate (Sigma, Dorset, UK), sodium borohydride (Alfa Aesar, Lancashire, UK), trichloroacetic acid (Sigma, Dorset, UK), hydrochloric acid (Sigma, Dorset, UK) were used for sample preparation. Plasma samples were defrosted for 90 min and centrifuged at 20,000 × g for 5 min.…”

“…1,2 Increased CML concentrations are associated with cardiovascular disease, diabetic nephropathy and retinopathy, chronic kidney disease and others. 2–4 CML has been proposed as a potential biomarker of cardiovascular disease; however, conflicting results have been found. 4–6 Enzyme linked immunosorbent assays (ELISAs) are available but suffer from steric hindrance of the antigen and interference from endogenous anti-AGE antibodies.…”

“…2–4 CML has been proposed as a potential biomarker of cardiovascular disease; however, conflicting results have been found. 4–6 Enzyme linked immunosorbent assays (ELISAs) are available but suffer from steric hindrance of the antigen and interference from endogenous anti-AGE antibodies. 1,2 Isotope dilution mass spectrometry (IDMS) methods are available for quantification of protein-bound CML, and samples require chemical reduction, protein denaturation, hydrolysis and drying prior to IDMS analysis.…”

Advanced Glycation End Products Evolution after Pancreas‐Kidney Transplantation: Plasmatic and Cutaneous Assessments

“…AGE measurement can in fact be made in plasma [ 26 – 30 ], in urine [ 26 ], or in tissues, skin being the most often used tissue [ 31 , 32 ]. Among several compounds already studied, CML is the best characterized AGE [ 29 ] and the most consistently assessed one in plasma analysis [ 26 – 30 ]. Higher plasmatic CML levels correlated with higher thickening rate of the glomerular basement membrane [ 26 ], increased arterial stiffness [ 27 ], increased coronary artery calcification [ 28 ], and higher incidence of fatal and nonfatal CV events [ 30 ] in diabetic patients; they even correlate with CV events in elderly nondiabetics subjects [ 33 ].…”