Carboxymethylethanolamine, a Biomarker of Phospholipid Modification during the Maillard Reaction in Vivo

Abstract

N⑀ -(Carboxymethyl)lysine (CML) is a stable chemical modification of proteins formed from both carbohydrates and lipids during autoxidation reactions. We hypothesized that carboxymethyl lipids such as (carboxymethyl)phosphatidylethanolamine (carboxymethyl-PE) would also be formed in these reactions, and we therefore developed a gas chromatography-mass spectrometry assay for quantification of carboxymethylethanolamine (CME) following hydrolysis of phospholipids. In vitro, CME was formed during glycation of dioleoyl-PE under air and from linoleoylpalmitoyl-PE, but not from dioleoyl-PE, in the absence of glucose. In vivo, CME was detected in lipid extracts of red blood cell membranes, ϳ0.14 mmol of CME/mol of ethanolamine, from control and diabetic subjects, (n ‫؍‬ 22, p > 0.5). Levels of CML in erythrocyte membrane proteins were ϳ0.2 mmol/mol of lysine for both control and diabetic subjects (p > 0.5). For this group of diabetic subjects there was no indication of increased oxidative modification of either lipid or protein components of red cell membranes. CME was also detected in fasting urine at 2-3 nmol/mg of creatinine in control and diabetic subjects (p ‫؍‬ 0.085). CME inhibited detection of advanced glycation end product (AGE)-modified protein in a competitive enzyme-linked immunosorbent assay using an anti-AGE antibody previously shown to recognize CML, suggesting that carboxymethyl-PE may be a component of AGE lipids detected in AGE low density lipoprotein. Measurement of levels of CME in blood, tissues, and urine should be useful for assessing oxidative damage to membrane lipids during aging and in disease.The nonenzymatic reaction of blood glucose with body proteins (glycation) followed by browning and oxidation reactions of glycated proteins leads to cumulative chemical modifications of tissue proteins throughout the body. These chemical changes, collectively termed the Maillard reaction, are considered to cause a gradual deterioration in the structure and function of tissue proteins and to contribute to the pathophysiology of normal aging (1-3). Further, the Maillard reaction is accelerated during hyperglycemia in diabetes, yielding advanced glycation end products (AGEs) 1 thought to be involved in the pathogenesis of diabetic complications (4 -6). Among Maillard reaction products identified thus far in tissue proteins, concentrations of pentosidine (3, 7) and N ⑀ -(carboxymethyl)lysine (CML) (7) are known to increase in human skin collagen with age, and age-adjusted concentrations of both are increased in skin collagen in diabetes (7). Moreover, there is a strong relationship between levels of these products in collagen and the status of diabetic complications (8 -10). Both CML and pentosidine require oxidative conditions for their formation, hence their description as glycoxidation products (11). Recently we showed that CML can also be formed during the reaction of autoxidizing polyunsaturated fatty acids (PUFA) with proteins (12), so that its precise biochemical origin is uncertain. The formation of ...

LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma

“…The data suggest that hyperglycemia in diabetic patients does not affect AGE-PE concentrations in erythrocytes and plasma, whereas the Amadori-PE concentration was markedly increased under hyperglycemic conditions. Similar to the present results, Requena et al ( 12 ) reported that no differences were observed in erythrocyte CM-PE levels between healthy and diabetic subjects. The reason given was that the diabetic patients participating in that study were free of complications ( 12 ).…”

“…For the occurrence of AGE of PE in vivo, there have been at least three confl icting reports (12)(13)(14). Requena et al ( 12 ) and Pamplona et al ( 13 ) detected hydrolysis product of CM-PE in the acid hydrolysates of human erythrocyte and mammalian mitochondrial phospholipids using GC-MS.…”

Inflammatory mediators are induced by dietary glycotoxins, a major risk factor for diabetic angiopathy

“…Thus, it is conceivable that the frequent intake of dietary § Lin, J., Alt, A., Liersch, J., Reinhard, G. B., Brownlee AGE promotes a sustained low-grade inf lammatory state. In this sense, other markers of immune response such as cytokines and adhesion molecules are inducible by AGEs via reactive oxygen species production and nuclear factor B activation (7)(8)(9)(10)(11)(12)(13)(14)44), properties which are also exhibited by dietary AGEs (25). These reactive molecules (ranging between 12 and 22 million AGE units in a ''healthy meal''), which are ''seeding'' the systemic circulation regularly, could, together with hyperglycemia, contribute to the subtle inf lammatory state associated with diabetes (23,(41)(42)(43)45).…”

“…The nature of pathogenic AGEs is not known as yet (10)(11)(12), and multiple derivatives other than those of MG or CML moieties have yet to be identified. Immunoreagents to MG derivatives and CML-like products, however, have presented invaluable means for animal and clinical correlations (10,11,24,25).…”

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…2A). The nitrogen of the adjacent monomer unit then engages in a second cyclization through a six-membered transition state to form diketopiperazine 2, the dimer of a known metabolite (hydroxyethyl glycine) of the Maillard reaction (44). Although slow at low pH, this rearrangement is exceptionally fast and efficient at pH 7.4; homooligomers degrade with a half-life of 2 min (SI Appendix, Fig.…”