Significance: Human longevity is increasing worldwide because of the advances in scientific knowledge and patient care, which leads to the frequent development of age-related pathologies. Aging remains an elusive process associated with genetic and environmental features, and better understanding would promote sustained wellbeing. We show here for the first time, to our knowledge, that carbamylation, a nonenzymatic posttranslational modification of proteins characterized by the spontaneous binding of isocyanic acid mainly derived from urea, is highly associated with aging and life expectancy in three mammalian species. Carbamylation promotes molecular aging through alteration of protein functions, especially long-lived extracellular matrix proteins. Tissue accumulation of carbamylated proteins may be considered a general hallmark of aging, enabling us to establish a link between cumulative metabolic alterations and age-related complications.

BACKGROUND: Carbamylation is a posttranslational modification of proteins resulting from the nonenzymatic reaction between isocyanic acid and specific free functional groups. This reaction alters protein structural and functional properties and thus contributes to molecular ageing. Many studies have shown the involvement of carbamylated proteins in diseases, especially in chronic renal failure and atherosclerosis.CONTENT: In this review we describe the biochemical basis of the carbamylation process and its role in protein molecular ageing. We summarize the current evidence of protein carbamylation involvement in disease, identify available biomarkers of the carbamylation process and their related analytical methods, and discuss the practical relevance of these biomarkers.SUMMARY: Carbamylation-induced protein alterations are involved in the progression of various diseases, because carbamylation-derived products (CDPs) are bioactive compounds that trigger specific and inappropriate cellular responses. For instance, carbamylation may promote hormone and enzyme inactivation, and carbamylated proteins, as diverse as collagen or LDLs, induce characteristic biochemical events of atherosclerosis progression. CDPs are potential biomarkers to monitor diseases characterized by an increased rate of carbamylation (e.g., chronic renal failure and atherosclerosis). Different methods (e.g., liquid chromatography–tandem mass spectrometry and immunoassays) to measure specific carbamylated proteins or general markers of carbamylation, such as protein-bound homocitrulline, have been described. Their use in clinical practice must still be validated by appropriate clinical studies.

BACKGROUND: During their biological life, proteins are exposed in a cumulative fashion to irreversible nonenzymatic, late posttranslational modifications that are responsible for their molecular aging. It is now well established that these damaged proteins constitute a molecular substratum for many dysfunctions described in metabolic and age-related diseases, such as diabetes mellitus, renal insufficiency, atherosclerosis, or neurodegenerative diseases. Accordingly, the specific end products derived from these reactions are considered potentially useful biomarkers for these diseases. CONTENT: The aim of this review is to give an overview of nonenzymatic posttranslational modifications of proteins and their influence in vivo, take inventory of the analytical methods available for the measurement of posttranslational modification–derived products, and assess the potential contribution of new technologies for their clinical use as biological markers of protein molecular aging. SUMMARY: Despite their clinical relevance, biomarkers of posttranslational modifications of proteins have been studied only in the context of experimental clinical research, owing to the analytical complexity of their measurement. The recent implementation in clinical chemistry laboratories of mass spectrometry–based methods that provide higher specificity and sensitivity has facilitated the measurement of these compounds. These markers are not used currently by clinicians in routine practice, however, and many challenges, such as standardization, have to be confronted before these markers can be used as efficient tools in the detection and monitoring of long-term complications of metabolic and age-related diseases.

HbA_1c was discovered in the late 1960s and its use as marker of glycemic control has gradually increased over the course of the last four decades. Recognized as the gold standard of diabetic survey, this parameter was successfully implemented in clinical practice in the 1970s and 1980s and internationally standardized in the 1990s and 2000s. The use of standardized and well-controlled methods, with well-defined performance criteria, has recently opened new directions for HbA_1c use in patient care, e.g., for diabetes diagnosis. Many reports devoted to HbA_1c have been published in Clinical Chemistry and Laboratory Medicine (CCLM) journal. This review reminds the major steps of HbA_1c history, with a special emphasis on the contribution of CCLM in this field.

In living organisms, proteins are progressively modified by spontaneous non-enzymatic reactions generating many post-translational modification derived products (PTMDPs) which exert deleterious effects and may be considered endogenous toxins in diabetes mellitus and chronic renal failure. Non-enzymatic glycation, which refers to the spontaneous binding of reducing sugars to free amino groups, is increased in diabetes mellitus because of hyperglycemia and is amplified by oxidative processes ('glycoxidation'). Glycoxidation leads to the formation of 'advanced glycation end products' (AGEs), together with products of other oxidative pathways. AGEs alter tissue organization and cell-protein interactions, mainly in the case of long-lived extracellular matrix proteins, and interact with membrane receptors, among which RAGE (receptor of AGEs), a multiligand receptor which triggers intracellular signaling pathways stimulating inflammatory functions. Another major protein modification, carbamylation, is increased in chronic renal failure, which may occur during the course of diabetes mellitus. Carbamylation is due to the binding of isocyanic acid on the α-NH2 extremity of proteins or amino acids, or on ε-NH2 lysine groups, generating homocitrulline, a potential biomarker in atherosclerosis. Isocyanic acid is formed in vivo either by spontaneous dissociation of urea or by myeloperoxidase action on thiocyanate. Carbamylated proteins exhibit altered properties. For example, carbamylated collagen is unable to stimulate oxidative functions of polymorphonuclear neutrophils but increases matrix metalloproteinase-9 production by monocytes. Lipoprotein functions are altered by carbamylation and may contribute to atherogenesis. Thus, the numerous PTMDPs may be considered both hallmarks of protein damage in chronic diseases and endogenous toxins acting at the molecular and cellular levels.

Tissue aging is a complex phenomenon involving molecular aging of matrix proteins, which mainly results from their progressive alteration by nonenzymatic post-translational modifications (NEPTMs) such as glycation and carbamylation. These two reactions, which correspond to the binding of reactive metabolites (i.e. reducing sugars and urea-derived cyanate, respectively) on amino groups of proteins, occur during aging and are amplified in various chronic diseases such as diabetes mellitus or chronic renal disease (CKD). Since these reactions target the same functional groups, they can reciprocally compete for protein modification. Determining which NEPTM is predominant in tissues is necessary to better understand their role in the development of long-term complications of chronic diseases. For that purpose, two different murine models were used for reproducing such a competitive context: a CKD-diabetic mice model and a cyanate-consuming mice model. The competition has been evaluated by quantifying glycation and carbamylation products by LC-MS/MS in skin and aorta total extracts as well as in skin type I collagen. The results showed that the simultaneous enhancement of glycation and carbamylation reactions resulted in a decrease of the formation of glycation products (especially Amadori products) whereas the concentrations of homocitrulline, a carbamylation product, remained similar. These results, which have been obtained in both tissues and in purified skin type I collagen, suggest that carbamylation takes precedence over glycation for the modification of tissue proteins, but only in pathological conditions favouring these two NEPTMs. While glycation has been considered for a long time the predominant NEPTM of matrix proteins, carbamylation seems to also play an important role in tissue aging. The existence of competition between these NEPTMs must be taken into account to better understand the consequences of molecular aging of matrix proteins in tissue aging.