Comparative CKD risk prediction using homocitrulline and carbamylated albumin: two circulating markers of protein carbamylation.

BACKGROUND: Protein carbamylation, a post-translational protein modification primarily driven by urea, independently associates with adverse clinical outcomes in patients with CKD. Biomarkers used to quantify carbamylation burden have mainly included carbamylated albumin (C-Alb) and homocitrulline (HCit, carbamylated lysine). In this study, we aimed to compare the prognostic utility of these two markers in order to facilitate comparisons of existing studies employing either marker alone, and to inform future carbamylation studies. METHODS: Both serum C-Alb and free HCit levels were assayed from the same timepoint in 1632 individuals with CKD stages 2-4 enrolled in the prospective Chronic Renal Insufficiency Cohort (CRIC) study. Adjusted Cox proportional hazard models were used to assess risks for the outcomes of death (primary) and end stage kidney disease (ESKD) using each marker. C-statistics, net reclassification improvement, and integrated discrimination improvement were used to compare the prognostic value of each marker. RESULTS: Participant demographics included mean (SD) age 59 (11) years; 702 (43%) females; 700 (43%) white. C-Alb and HCit levels were positively correlated with one another (Pearson correlation coefficient 0.64). Higher C-Alb and HCit levels showed similar increased risk of death (e.g., the adjusted hazard ratio [HR] for death in the 4th carbamylation quartile compared to the 1st was 1.90 (95% confidence interval [CI] 1.35-2.66) for C-Alb, and 1.89 [1.27-2.81] for HCit; and on a continuous scale, the adjusted HR for death using C-Alb was 1.24 [1.11 to 1.39] per standard deviation increase, and 1.27 [1.10-1.46] using HCit). Both biomarkers also had similar HRs for ESKD. The C-statistics were similar when adding each carbamylation biomarker to base models (e.g., for mortality models, the C-statistic was 0.725 [0.707-0.743] with C-Alb and 0.725 [0.707-0.743] with HCit, both compared to a base model 0.723). Similarities were also observed for the net reclassification improvement and integrated discrimination improvement metrics. CONCLUSIONS: C-Alb and HCit had similar performance across multiple prognostic assessments. The markers appear readily comparable in CKD epidemiological studies.

Accumulation of Carbamylation-Derived Products in Aneurysmal Aorta.

INTRODUCTION: Carbamylation is a nonenzymatic post-translational modification of proteins characterized by the binding of isocyanic acid to amino groups of proteins, which leads to the alteration of their properties. An increase in serum carbamylation-derived products, including homocitrulline (HCit), has been shown to be associated with the development of cardiovascular diseases. METHODS: HCit was quantified by LC-MS/MS within extracts of aneurysmal and control human aortas. A mouse model of aortic aneurysm (ApoE-/- mice perfused with angiotensin II and fed with sodium cyanate) was used to evaluate the role of carbamylation in aneurysm development. RESULTS: HCit quantification showed a greater heterogeneity of values in aneurysmal aortas in comparison with control ones. At the maximum diameter of dilation, HCit values were significantly higher (+94%, p < 0.05) compared with less dilated areas. No differences were observed according to aneurysm size or when comparing ruptured and unruptured aneurysms. No significant effect of carbamylation on aneurysm development was observed using the animal model. CONCLUSIONS: These results evidenced the accumulation of HCit within aneurysmal aortas but do not allow concluding about the exact participation of protein carbamylation in the development of human abdominal aortic aneurysms.

Comparison of homocitrulline and carbamylated albumin as biomarkers of carbamylation reactions in hemodialyzed patients.

To describe the association between levels of homocitrulline (HCit) and the degree of albumin carbamylation in a cohort of hemodialyzed patients. Plasma total and protein-bound HCit concentrations in samples from hemodialyzed patients included in NICOREN trial were determined by LC-MS/MS at baseline and after 24 weeks of treatment with either sevelamer or nicotinamide. HCit concentrations at all timepoints and in both groups were positively and significantly correlated with the degree of albumin carbamylation. Plasma concentrations of total HCit, protein-bound HCit and carbamylated albumin did not decrease after 24 weeks of treatment with either sevelamer or nicotinamide. The present results demonstrate that plasma total and protein-bound HCit concentrations were closely associated with albumin carbamylation in hemodialyzed patients. Therefore, total and protein-bound HCit concentrations might be valuable biomarkers of the overall intensity of protein carbamylation in this context. Given the less complex and time-consuming analytical methods required, these markers should be favored in future clinical studies of carbamylation reaction.

High expression level of homocitrulline is correlated with seborrheic keratosis and skin aging

Abstract Backgroud Homocitrulline (Hcit), is involved in the pathological processes of some diseases. However, the role and function of Hcit (CBL) in human skin remains largely obscure. Objective To investigate the correlation of the level of Hcit in seborrheic keratosis, skin aging, and its clinical significance. Methods Immunohistochemistry was used to analyze the level of Hcit in skin lesions of seborrheic keratosis (SK), unaffected skin (distant 0.5 centimeters from SK lesion), and normal skin of healthy subjects in the control group. ELISA test was used to detect the serum level of CBL in SK patients and healthy subjects of different ages. Results Hcit was mainly localized in the nucleus of epidermal cells. In healthy control skin, the expression of Hcit increased with age and showed a positive correlation with age (the correlation coefficient was 0.806, p = 0.0002). The expressional level of Hcit in SK lesions was higher than that in healthy control skin (Z = −3.703, p = 0.0002). The serum level of CBL in healthy subjects and in SK patients increased with age (the correlation coefficient were 0.5763, p = 0.0032; 0.682, p = 0.004. respectively). The serum level of CBL in SK patients was higher than that in healthy subjects (Z = −2.19, p = 0.030). Study limitations The small serum sample size in the study. Conclusion The high expressional level of Hcit is correlated with seborrheic keratosis and skin aging. HCit may be one of the potential biomarkers of skin aging.

Altered ureido protein modification profiles in seminal plasma extracellular vesicles of non-normozoospermic men.

Introduction: Extracellular vesicles (EVs) have been recognized as key players in numerous physiological functions. These vesicles alter their compositions attuned to the health and disease states of the organism. In men, significant changes in the proteomic composition(s) of seminal plasma EVs (sEVs) have already been found to be related to infertility. Methods: Methods: In this study, we analyze the posttranslational configuration of sEV proteomes from normozoospermic (NZ) men and non-normozoospermic (non-NZ) men diagnosed with teratozoospermia and/or asthenozoospermia by unbiased, discovery-driven proteomics and advanced bioinformatics, specifically focusing on citrulline (Cit) and homocitrulline (hCit) posttranscriptional residues, both considered product of ureido protein modifications. Results and discussion: Significant increase in the proteome-wide cumulative presence of hCit together with downregulation of Cit in specific proteins related to decisive molecular functions have been encountered in sEVs of non-NZ subjects. These findings identify novel culprits with a higher chance of affecting fundamental aspects of sperm functional quality and define potential specific diagnostic and prognostic non-invasive markers for male infertility.

Measurement of Homocitrulline, A Carbamylation-derived Product, in Serum and Tissues by LC-MS/MS.

Carbamylation corresponds to the nonenzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest in evaluating the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaching to the ε-NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This article describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on preanalytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample pretreatment for the quantification of homocitrulline by LC-MS/MS Alternate Protocol: Preanalytical steps for the quantification of homocitrulline in tissue samples Basic Protocol 2: LC-MS/MS quantification of homocitrulline Basic Protocol 3: LC-MS/MS quantification of lysine in hydrolysates.

Serum carboxymethyllysine concentration is associated with erosive hand osteoarthritis.

OBJECTIVE: Carboxymethyllysine (CML) and homocitrulline (HCit) are the products of two non-enzymatic post-translational modifications of protein, a process related to age. We investigated whether serum CML and HCit concentrations were associated with hand osteoarthritis (HOA), especially erosive HOA. DESIGN: Serum CML and HCit were measured by using liquid chromatography coupled with tandem mass spectrometry at inclusion in 386 patients included in the DIGItal Cohort Design (DIGICOD) cohort. We investigated whether serum CML and/or HCit concentrations were associated with erosive HOA or with HOA clinical and radiological features. Moreover, we compared the tissular concentrations of CML and HCit in OA and non-OA cartilage from proximal interphalangeal and metacarpo-phalangeal (MCP) joints from human cadaveric donors. RESULTS: Median (IQR) serum CML concentration was lower in patients with erosive HOA than those with non-erosive HOA (178.7 [157.1-208.8] vs 194.7 [168.9-217.1] µmol/mol Lys, P = 0.002), but median HCit concentration did not differ between the groups (193.9 [162.9-232.0] vs 193.9 [155.9-224.6] µmol/mol Lys). Cartilage HCit and CML concentrations were not correlated with clinical features. Serum CML concentration was higher in OA than non-OA MCPs (7.0 vs 4.0 mmol/mol Lys, P = 0.01). CONCLUSIONS: Serum CML concentration was lower in erosive HOA than non-erosive HOA, and cartilage CML concentration was higher in OA than non-OA cartilage. These results encourage further studies to test whether serum CML could be a new prognostic biomarker in HOA.

High expression level of homocitrulline is correlated with seborrheic keratosis and skin aging.

BACKGROUD: Homocitrulline (Hcit), is involved in the pathological processes of some diseases. However, the role and function of Hcit (CBL) in human skin remains largely obscure. OBJECTIVE: To investigate the correlation of the level of Hcit in seborrheic keratosis, skin aging, and its clinical significance. METHODS: Immunohistochemistry was used to analyze the level of Hcit in skin lesions of seborrheic keratosis (SK), unaffected skin (distant 0.5 centimeters from SK lesion), and normal skin of healthy subjects in the control group. ELISA test was used to detect the serum level of CBL in SK patients and healthy subjects of different ages. RESULTS: Hcit was mainly localized in the nucleus of epidermal cells. In healthy control skin, the expression of Hcit increased with age and showed a positive correlation with age (the correlation coefficient was 0.806, p = 0.0002). The expressional level of Hcit in SK lesions was higher than that in healthy control skin (Z = -3.703, p = 0.0002). The serum level of CBL in healthy subjects and in SK patients increased with age (the correlation coefficient were 0.5763, p = 0.0032; 0.682, p = 0.004. respectively). The serum level of CBL in SK patients was higher than that in healthy subjects (Z = -2.19, p = 0.030). STUDY LIMITATIONS: The small serum sample size in the study. CONCLUSION: The high expressional level of Hcit is correlated with seborrheic keratosis and skin aging. HCit may be one of the potential biomarkers of skin aging.

Hyperargininemia Due to Arginase 1 Deficiency: Variability in Clinical and Biochemical Presentations in Malaysian children.

Objective: Hyperargininemia due to Arginase 1 deficiency is a rare inborn error of the urea cycle with an incidence estimated at 1:950 000. It has typical severe and progressive abnormal neurological features with biochemical findings of hyperargininemia and hyperexcretion of orotic acid. The aim of our study is to review the clinical and biochemical presentations of 4 children diagnosed with Arginase 1 deficiency in Malaysia and compare with the literature review. Design and Methods: We retrospectively reviewed the medical records of 4 patients with molecularly confirmed Arginase 1 deficiency. Patients were identified from a selective high-risk screening of 51 682 symptomatic patients from January 2006 to December 2020. Results: Our patients exhibited heterogeneous clinical presentations with acute and progressive neurological abnormalities and varying degrees of plasma arginine and urine orotic acid excretions. Interestingly, an unusual hyperexcretion of homocitrulline was found in 3 patients. Conclusions: Hyperargininemia due to Arginase 1 deficiency can present acutely and hyperexcretion of homocitrulline can be an additional biochemical feature of Arginase 1 deficiency.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta.

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor" apoA-I forms via the nonenzymatic CNO- pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Serum metabolomic profiling reveals an increase in homocitrulline in Chinese patients with nonalcoholic fatty liver disease: a retrospective study.

BACKGROUNDS: Nonalcoholic fatty liver disease (NAFLD) has multiple causes, is triggered by individual genetic susceptibility, environmental factors, and metabolic disturbances, and may be triggered by acquired metabolic stress. The metabolic profiles of NAFLD show significant ethnic differences, and the metabolic characteristics of NAFLD in Chinese individuals are unclear. Our study aimed to identify the metabolites and pathways associated with NAFLD in a Chinese cohort. METHODS: One hundred participants, including 50 NAFLD patients and 50 healthy controls, were enrolled in this retrospective observational study at Jinling Hospital in Nanjing; serum samples were collected from the patients and healthy subjects. The metabolome was determined in all samples by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). Univariate and multivariate statistical analyses were used to compare the metabolic profiles between the two groups. RESULTS: The comparison indicated that the levels of 89 metabolites were different between the two groups. The glycerophospholipid family of metabolites was the most abundant family of metabolites that demonstrated significant differences. L-acetylcarnitine, L-homocitrulline, and glutamic acid were the top three metabolites ranked by VIP score and had favorable effective functions for diagnosis. Moreover, pathway enrichment analysis suggested 14 potentially different metabolic pathways between NAFLD patients and healthy controls based on their impact value. Biological modules involved in the lipid and carbohydrate metabolism had the highest relevance to the conditions of NAFLD. Glycerophospholipid metabolism had the strongest associations with the conditions of NAFLD. CONCLUSIONS: Our data suggest that the serum metabolic profiles of NAFLD patients and healthy controls are different. L-Homocitrulline was remarkably increased in NAFLD patients.

[Autoantibodies and the autoreactive immune response : There is more to ACPA than ACPA]. / Autoantikörper und die autoreaktive Immunantwort : ACPA sind mehr als nur ACPA.

The immune response against posttranslationally modified (PTM) antigens, in particular the generation of anti-citrullinated protein antibodies (ACPA), is a very specific hallmark of rheumatoid arthritis. The factors that initiate this immune response and the triggers that stimulate the transition from asymptomatic autoimmunity to autoimmune disease are so far unknown. Genetic risk factors and the maturation of the ACPA response prior to the onset of arthritis indicate an important role for helper T cells in this process. Antigens that trigger this process, however, remain to be defined. Notably, recent data demonstrate that ACPA do not only recognize citrullinated protein antigens. Other posttranslational protein modifications such as homocitrulline and acetyllysine are also recognized. This cross-reactivity towards different PTM antigens was found for various monoclonal ACPA and broadens the spectrum of antigens that can stimulate and activate ACPA-expressing B cells. Also, it suggests that such B cells could receive help from autoreactive but also from non-autoreactive T cells. This review summarizes these recent findings and provides insight into their potential relevance for the disease rheumatoid arthritis.

Antibodies against carbamylated vimentin exist in systemic lupus erythematosus and correlate with disease activity.

OBJECTIVES: Antibodies against carbamylated protein (anti-CarP) were found to be a promising marker to evaluate joint damage and disease activity in patients with rheumatoid arthritis (RA). However, whether anti-CarP antibodies were present in systemic lupus erythematosus (SLE) remained ambiguous. We have therefore undertaken this study to assess the levels of serum anti-CarP antibodies and to evaluate their clinical value in SLE. METHODS: Serum levels of antibodies against carbamylated-vimentin (anti-Carp) were measured by enzyme immunosorbent assay in 100 patients with SLE, 76 with RA, 17 with primary Sjögren syndrome (pSS), and 68 healthy controls. Data analyses between anti-Carp antibodies and other laboratory measures were performed using SPSS 24 software for Windows. RESULTS: The levels of serum anti-CarP antibodies in patients with SLE were significantly higher than those in healthy controls. In addition, anti-CarP antibodies were present in SLE patients lacking the disease-specific antibodies, including anti-Smith-negative patients (24.4%, 21/86), anti-dsDNA-negative patients (29.3%, 12/41), anti-nucleosome-negative patients (21.4%, 9/42), and antiribosomal P protein antibody-negative patients (23.7%, 18/76). There were significant differences between the anti-CarP-positive and anti-CarP-negative SLE patients in clinical and laboratory features, such as age, erythrocyte sedimentation rate (ESR), C-reactive protein, rheumatoid factor, third-generation cyclic citrullinated peptide (CCP3), anticardiolipin, D-dipolymer, complement 3, immunoglobulin G (IgG), red blood cell count (RBC) and hemoglobin. After adjusting for age and disease duration, the high levels of anti-CarP antibodies were still correlated with low RBC, hemoglobin and high ESR, IgG and CCP3. Active SLE patients demonstrated higher anti-CarP IgG than inactive patients. Moreover, the levels of anti-CarP were significantly higher in SLE patients with arthralgia and/or arthritis than in those without joint involvement. CONCLUSIONS: Anti-CarP antibodies were present in SLE patients and associated with the disease severity. These might provide a potential supplement to other specific autoantibodies for diagnosis of SLE and serve as a promising marker for measuring joint damage in the disease.

Specificity of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. The majority of individuals with RA are positive for the disease-specific anti-citrullinated protein antibodies (ACPAs). These antibodies are primarily of cross-reactive nature, hence, the true autoantigen to ACPA remains unidentified. In this study, we analyzed the reactivity of RA sera to several post-translationally modified epitopes, in order to further characterize the specific nature of ACPAs by immunoassays. Substituting citrulline with other amino acids, e.g., D-citrulline, homo-citrulline and methyl-arginine illustrated that ACPAs are utmost specific for citrullinated targets. Collectively, these findings support that ACPAs and citrullinated targets are specific for RA, making citrulline-containing peptide targets the most effective assays for detection of ACPAs.

Proteasome-dependent degradation of intracellular carbamylated proteins.

Carbamylation, which corresponds to the binding of isocyanic acid to the amino groups of proteins, is a nonenzymatic post-translational modification responsible for alterations of protein structural and functional properties. Tissue accumulation of carbamylation-derived products and their role in pathological processes such as atherosclerosis or chronic renal failure have been previously documented. However, few studies have focused on the carbamylation of intracellular proteins and their subsequent role in cellular aging. This study aimed to determine the extent of intracellular protein carbamylation, its impact on cell functions and the ability of cells to degrade these modified proteins. Fibroblasts were incubated with cyanate or urea and the carbamylation level was evaluated by immunostaining and homocitrulline quantification. The results showed that carbamylated proteins accumulated intracellularly and that all proteins were susceptible. The presence of intracellular carbamylated proteins did not modify cell proliferation or type I collagen synthesis nor did it induce cell senescence, but it significantly decreased cell motility. Fibroblasts were able to degrade carbamylated proteins through the ubiquitin-proteasome system. In conclusion, intracellular proteins are susceptible to carbamylation but their accumulation does not seem to deeply affect cell function, owing largely to their elimination by the ubiquitin-proteasome system.

Simultaneous quantification of 48 plasma amino acids by liquid chromatography-tandem mass spectrometry to investigate urea cycle disorders.

Urea cycle disorders (UCD) are inborn errors of ammonia detoxification in which early diagnosis and treatment are critical to prevent metabolic emergencies. Unfortunately, the diagnosis was often and pronounced delayed. To improve diagnosis, we developed herein a liquid chromatography-tandem mass spectrometry method to investigate the disturbance of amino acid profile caused by UCD. The method enabled absolute quantification of 48 amino acids (AAs) within 20 min. Only 2.5 µL plasma was required for the analysis. The lower limits of quantification for most AAs were 0.01 µmol/L. Method accuracies ranged from 89.9% to 113.4%. The within- and between-run coefficients of variation were 0.8-7.7% and 2.6-14.5%, respectively. With this method, age-specific reference values were established for 42 AAs by analyzing 150 samples from normal controls, and patients with different subtypes of UCD were successfully distinguished. The data of patients revealed that UCD not only disturbed the metabolism of urea cycle AAs and induced accumulation of ammonia detoxification AAs, but also interfered the metabolism of some nervous system related AAs, such as pipecolic acid and N-acetylaspartic acid. This data may provide new insight into pathogenesis for UCD.

Post-translational modification-derived products are associated with frailty status in elderly subjects.

Background Identifying frail elderly subjects is of paramount importance in order to conduct a tailored care. The characterization of frailty status is currently based on the collection of clinical data and on the use of various tools such as Fried's criteria, which constitutes a difficult and time-consuming process. Up to now, no biological markers have been described as reliable tools for frailty characterization. We tested the hypothesis that a link between frailty and protein molecular aging existed. This study aimed therefore at determining whether post-translational modification derived products (PTMDPs), recognized as biomarkers of protein aging, were associated with frailty status in elderly subjects. Methods Frailty status was determined according to Fried's criteria in 250 elderly patients (>65 years old) hospitalized in a short-term care unit. Serum concentrations of protein-bound PTMDPs, including carboxymethyllysine (CML), pentosidine, methylglyoxal-hydroimidazolone-1 and homocitrulline (HCit), were determined by liquid chromatography coupled with tandem mass spectrometry, and tissue content of advanced glycation end-products was assessed by skin autofluorescence (SAF) measurement. Associations between PTMDPs and frailty status were analyzed using logistic regression models. Results Frail patients had significantly (p<0.01) higher CML, HCit, and SAF values compared to non-frail and pre-frail subjects. By multivariate analysis, only HCit concentrations and SAF values remained associated with frailty status (p=0.016 and p=0.002, respectively), independently of age, comorbidities, renal function, C-reactive protein and albumin concentrations. Conclusions HCit and SAF are significantly associated with frailty status in elderly subjects. This study suggests that PTMDPs constitute promising biomarkers for identifying frail patients and guiding personalized patient care.

Measurement of Homocitrulline, A Carbamylation-derived Product, in Serum and Tissues by LC-MS/MS.

Carbamylation corresponds to the non-enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaches to the ε-NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This unit describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on pre-analytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2018 by John Wiley & Sons, Inc.

Protein Carbamylation: Chemistry, Pathophysiological Involvement, and Biomarkers.

Protein carbamylation refers to a nonenzymatic modification, which consists in the binding of isocyanic acid on protein functional groups. This reaction is responsible for the alteration in structural and functional properties of proteins, which participate in their molecular aging. Protein molecular aging is now considered a molecular substratum for the development of chronic and inflammatory diseases, including atherosclerosis, chronic kidney disease, or rheumatoid arthritis. As a consequence, carbamylation-derived products have been proposed as interesting biomarkers in various pathological contexts and appropriate analytical methods have been developed for their quantification in biological fluids. The purpose of this review is (i) to describe the biochemical bases of the carbamylation reaction, (ii) to explain how it contributes to protein molecular aging, (iii) to provide evidence of its involvement in aging and chronic diseases, and (iv) to list the available biomarkers of carbamylation process and the related analytical methods.

Metabolomic Alterations Associated with Cause of CKD.

BACKGROUND AND OBJECTIVES: Causes of CKD differ in prognosis and treatment. Metabolomic indicators of CKD cause may provide clues regarding the different physiologic processes underlying CKD development and progression. DESIGN, SETTING, PARTICIPANTS & MEASUREMENTS: Metabolites were quantified from serum samples of participants in the Modification of Diet in Renal Disease (MDRD) Study, a randomized controlled trial of dietary protein restriction and BP control, using untargeted reverse phase ultraperformance liquid chromatography tandem mass spectrometry quantification. Known, nondrug metabolites (n=687) were log-transformed and analyzed to discover associations with CKD cause (polycystic kidney disease, glomerular disease, and other cause). Discovery was performed in Study B, a substudy of MDRD with low GFR (n=166), and replication was performed in Study A, a substudy of MDRD with higher GFR (n=423). RESULTS: Overall in MDRD, average participant age was 51 years and 61% were men. In the discovery study (Study B), 29% of participants had polycystic kidney disease, 28% had glomerular disease, and 43% had CKD of another cause; in the replication study (Study A), the percentages were 28%, 24%, and 48%, respectively. In the discovery analysis, adjusted for demographics, randomization group, body mass index, hypertensive medications, measured GFR, log-transformed proteinuria, and estimated protein intake, seven metabolites (16-hydroxypalmitate, kynurenate, homovanillate sulfate, N2,N2-dimethylguanosine, hippurate, homocitrulline, and 1,5-anhydroglucitol) were associated with CKD cause after correction for multiple comparisons (P<0.0008). Five of these metabolite associations (16-hydroxypalmitate, kynurenate, homovanillate sulfate, N2,N2-dimethylguanosine, and hippurate) were replicated in Study A (P<0.007), with all replicated metabolites exhibiting higher levels in polycystic kidney disease and lower levels in glomerular disease compared with CKD of other causes. CONCLUSIONS: Metabolomic profiling identified several metabolites strongly associated with cause of CKD.