Search

Background and Aim: Although it is widely known that the total parenteral nutrition (TPN) used frequently in intensive care units has unwanted side effects, there is little known about how it interferes with the amino acid levels taken during the diagnosis of metabolic diseases. Amino acid can lead to inaccurate measurements with mass spectrometry due to its high molecular content of lipids and carbohydrates, which modifies the blood matrix. The purpose of this study was to emphasize the results of amino acid interference, measured with mass spectrometry, in patients administered with TPN. Case Presentation: Incorrect clinical interpretation resulted in the case of a pneumonia patient with false positive and negative blood amino acid levels caused by TPN infusion. The amino acid profile had been requested to rule out an amino acid metabolic defect in the two-year-old boy who arrived at the pediatric clinic complaining of respiratory distress, tachypnea and hypoxemia. He was monitored in the intensive care unit for further investigation. The personnel who had performed phlebotomy also carried out the sampling during the TPN infusion administration. This caused the amino acid results and an incorrect interpretation. The following deviation ratios were detected: phenylalanine 102%, leucine 86%, isoleucine 106%, GABA 200%, citrulline 238%, glutamine 178%, ornithine 216%, 1- methyl-l-histidine 1471%, serine 312%, alanine 163%, glycine 355%, homocitrulline and carnosine 444%. The amino acid blood level measurements taken for diagnosis and screening in suspected metabolic disease may lead to involuntary false low or elevated results in patients administered with TPN. Conclusion: This case demonstrates that TPN solutions affect the reference method of mass spectrometry measurement methods due to the concentration of ingredients. We suggest that inaccurate results can be avoided by carrying out the sampling prior to TPN infusion in patients whose plasma amino acid levels will be measured.

Carbamylation is the non-enzymatic reaction between isocyanic acid and macromolecules (mainly proteins) which results in carbamylation-derived products (CDPs) generation, wherein the macromolecules show altered structure and function. In this study, we examined the modifications caused in human serum albumin (HSA) upon interaction with potassium cyanate (KCNO). HSA was incubated with varying concentrations of KCNO for 6 h at 37 °C. The resultant product was characterized by biochemical and biophysical techniques. Among other changes, the carbamylated-HSA showed homocitrulline generation (LC-MS), increase in mass (DLS), and amyloidogenic aggregate formation (Congo red, SEM, TEM). The Gibb's free energy was calculated to be −2.91 to −3.95 kcal mol−1, suggesting that the binding was spontaneous and energetically favourable. The results indicate that in chronic kidney disease patients, elevated levels of isocyanic acid (formed from urea) may modify the albumin structure and lead to its conversion into amyloidogenic aggregates, thus accelerating kidney damage.

Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.

Background Ornithine transcarbamylase deficiency (OTCD) is pleomorphic congenital hyperammonemia, in which the prognosis of the patient is determined both by genotype and environmental factors. This study investigated the clinical and biochemical characteristics of OTCD patients with different prognosis. Method Of 35 OTCD patients, six males deceased at the first disease-onset, 17 males survived and had controllable ammonia levels after treatment, and 12 females survived through the first disease-onset but had intractable hyperammonemia and high mortality. Fasting blood samples from patients collected at three disease stages were used for the analysis of amino acid (AA) profile, acylcarnitine profile, and micronutrients. Differences in profiles between patients and healthy controls and within patient groups were studied. Results All OTCD patients had accumulation of glutamine, homocitrulline, lysine, glutamate, cystathionine, and pipecolic acid, as well as deficiency of citrulline, tryptophan, threonine, and carnitine. For male non-survivors, most other AAs and long-chain acylcarnitines were elevated at disease onset, of which the levels of creatine, N-acetylaspartic acid, and homoarginine were remarkably high. Male survivors and female patients had most other AAs at low to normal levels. Compared with male survivors, female patients had much lower protein-intolerance, as indicated by significantly lower levels of protein consumption indicators, including essential AAs, 1-methylhistidine, acylcarnitines et al., but high levels of ammonia. Female patients still had significantly higher levels of citrulline, homocitrulline, and citrulline/arginine compared to male survivors. Conclusion Unique profiles were observed in each group of OTCD patients, indicating specific physiological changes that happened to them.

Abnormal gut microbiota and blood metabolome profiles have been reported both in children and adults with uncomplicated type 1 diabetes as well as in adults with type 1 diabetes and advanced stages of diabetic nephropathy. In this study we aimed to investigate the gut microbiota and a panel of targeted plasma metabolites in individuals with type 1 diabetes of long duration without and with different levels of albuminuria. In a cross-sectional study we included 161 individuals with type 1 diabetes and 50 healthy control individuals. Individuals with type 1 diabetes were categorised into three groups according to historically measured albuminuria: (1) normoalbuminuria (

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.

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.

Aim and Objective:: To determine the mechanisms present in the etiopathogenesis of nasal polyposis. It is not clear whether amino acids contribute in a causal way to the development of the disease. Therefore, the aim of this study was to determine the plasma-free amino acid profile in patients with nasal polyposis and to compare the results with a healthy control group. Materials and Methods:: This was a prospective controlled study that took place in the Otolaryngology Department at the Harran University Faculty of Medicine between April 2017 and April 2018. Plasmafree amino acid profile levels were studied in serum samples taken from a patient group and a healthy control group. Patients who were diagnosed with bilateral diffuse nasal polyposis and were scheduled for surgical interventions were included in this study. Individuals whose age, gender, and body mass index values were compatible with that of the patient group and who did not have any health problems were included in the control group. All the participants whose levels of plasma-free amino acid were thought to be affected by one or more of the following factors were excluded from the study: smoking and alcohol use, allergic rhinitis presence, the presence of acute or chronic sinusitis, a history of endoscopic sinus surgery, unilateral nasal masses, a history of chronic drug use, systemic or topical steroid use in the last three months for any reason, and liver, kidney, hematological, cardiovascular, metabolic, neurological, or psychiatric disorders or malignancies. Results: In patients with nasal polyposis, 3-methyl histidine (3-MHIS: nasal polyposis group (ng) = 3.22 (1.92 – 6.07); control group (cg) = 1.21 (0.77 – 1.68); p = 0.001); arginine (arg: ng = 98.95 (70.81 – 117.75); cg = 75.10 (54.49 – 79.88); p = 0.005); asparagine (asn: ng = 79.84 (57.50 – 101.44); cg = 60.66 (46.39 – 74.62); p = 0.021); citrulline (cit: ng = 51.83 (43.81 – 59.78); cg = 38.33 (27.81 – 53.73); p = 0.038); cystine (cys: ng = 4.29 (2.43 – 6.66); cg = 2.41 (1.51 – 4.16); p = 0.019); glutamic acid (glu: ng = 234.86 (128.75 – 286.66); cg = 152.37 (122.51 – 188.34); p = 0.045); histidine (his: ng = 94.19 (79.34 – 113.99); cg = 74.80 (62.76 – 98.91); p = 0.018); lysine (lys: ng = 297.22 (206.55 – 371.25); cg = 179.50 (151.58 – 238.02); p = 0.001); ornithine (ng = 160.62 (128.36 – 189.32); cg = 115.91 (97.03 – 159.91); p = 0.019); serine (ser: ng = 195.15 (151.58 – 253.07); cg = 83.07 (67.44 – 92.44); p = 0.001); taurine (tau: ng = 74.69 (47.00 – 112.13); cg = 53.14 (33.57 – 67.31); p = 0.006); tryptophan (trp: ng = 52.31 (33.81 – 80.11); cg = 34.44 (25.94 – 43.07); p = 0.005), homocitrulline (ng = 1.75 (1.27 – 2.59); cg = 0.00 (0.00 – 0.53); p = 0.001); norvaline (ng = 6.90 (5.61 – 9.18); cg = 4.93 (3.74 – 7.13); p = 0.021); argininosuccinic acid (ng = 14.33 (10.06 – 25.65); cg = 12.22 (5.77 – 16.87) p = 0.046); and plasma concentrations were significantly higher than in the healthy control group (p Conclusion: In this study, plasma levels of 15 free amino acids were significantly higher in the nasal polyposis group than in the healthy control group. A plasma level of 1 free amino acid was found to be significantly lower in the nasal polyposis group compared to the healthy control group. Therefore, it is important to determine the possibility of using the information obtained to prevent the recurrence of the condition and to develop effective treatment strategies. This study may be a milestone for studies of this subject. However, this study needs to be confirmed by further studies conducted in a larger series.

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 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.

Rainbow trout and common carp are commercially important aquaculture fish species. To the best of our knowledge, the global composition of fish seminal plasma (SP) metabolites has never been studied. The aim of this study was to identify metabolites in carp and rainbow trout SP and to compare their relative abundances through the use of liquid chromatography-mass spectrometry (LC-MS)-based metabolic fingerprinting. Different solvents and sample/solvent ratios were tested for metabolite extraction. The largest number of metabolic features and the highest signal reproducibility were achieved using a 1:1 sample/acetone ratio for extraction. Accurate masses were searched against metabolite databases, and the identities of 48 and 40 metabolites for rainbow trout and carp SP, respectively, were confirmed using LC-MS/MS or through the standards. Most of the identified metabolites have not been previously described in fish SP. Several metabolites (amino acids, acylcarnitines, vitamins) were found in the SP of both species. However, differences between carp and rainbow trout SP metabolic fingerprints were observed. Certain metabolites appeared to be species-specific, e.g., propionic acid for carp and adipic and picolinic acids, homocitrulline and niacin for rainbow trout. Functional analysis revealed the involvement of SP metabolites in free radical scavenging, lipid and energy metabolism, molecular transport, cell death and survival, and inflammatory response. The identification of a high number of carp and rainbow trout seminal plasma metabolites provides new opportunities to develop novel biomarkers for sperm quality, optimise artificial reproduction and develop efficient cryopreservation procedures for these important aquaculture species.

We have deployed a multi-omics approach in large cohorts of patients with existing type 2 diabetes to identify biomarkers for disease progression across three molecular classes, metabolites, lipids and proteins. A Cox regression analysis for association with time to insulin requirement in 2,973 patients in the DCS, ANDIS and GoDARTS cohorts identified homocitrulline, isoleucine and 2-aminoadipic acid, as well as the bile acids glycocholic and taurocholic acids, as predictive of more rapid deterioration. Increased levels of eight triacylglycerol species, and lowered levels of the sphingomyelin SM 42:2;2 were also predictive of disease progression. Of ∼1,300 proteins examined in two cohorts, levels of GDF-15/MIC1, IL-18RA, CRELD1, NogoR, FAS, and ENPP7 were associated with faster progression, whilst SMAC/DIABLO, COTL1, SPOCK1 and HEMK2 predicted lower progression rates. Strikingly, identified proteins and lipids were also associated with diabetes incidence and prevalence in external replication cohorts. Implicating roles in disease compensation, NogoR/RTN4R improved glucose tolerance in high fat-fed mice and tended to improved insulin signalling in liver cells whilst IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. Conversely, high NogoR levels led to islet cell apoptosis. This comprehensive, multi-disciplinary approach thus identifies novel biomarkers with potential prognostic utility, provides evidence for new disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Background and aims Abnormalities in lipid metabolism, accumulation of uremic toxins and advanced glycation end products may contribute to worsening atherosclerosis. This study characterized the glycation and carbamoylation profile of serum albumin isolated from individuals with diabetic kidney disease and its influence on cholesterol efflux. Material and methods 49 patients with type 2 diabetes (T2DM) and different eGFR evaluated glycation and carbamoylation profile by measurement of carboxymethyl lysine (CML) and carbamoylated proteins (CBL) in plasma by ELISA, homocitrulline (HCit) in plasma by colorimetry. In the isolated albumins, we quantified CBL (ELISA) and total AGE and pentosidine by fluorescence. Macrophages were treated with albumin isolated, and 14C-Cholesterol efflux mediated by HDL2 or HDL3 was measured. Kruskal-Wallis test, Jonckheere-Terpstra test and Brunner's posttest were used for comparisons among groups. Results Determination of CML, HCit, CBL in plasma, as total AGE and pentosidine in albumins, did not differ between groups; however, CBL in the isolated albumins was higher in the more advanced stages of CKD (p = 0.0414). There was reduction in the 14C-cholesterol efflux after treatment for 18 h with albumin isolated from patients with eGFR Conclusions Albumins isolated from individuals with T2DM and eGFR

The high prevalence of hypertension contributes to an increased global burden of cardiovascular diseases. Calcium channel blockers (CCBs) and angiotensin type 1 receptor blockers (ARBs) are the most widely used antihypertensive drugs, and the effects of these drugs on serum metabolites remain unknown. Untargeted metabolomics has been proved to be a powerful approach for the detection of biomarkers and new compounds. In this study, we aimed to determine the changes in metabolites after single-drug therapy with a CCB or ARB in patients newly diagnosed with mild to moderate primary hypertension. We enrolled 33 patients and used an untargeted metabolomics approach to measure 625 metabolites associated with the response to a 4-week treatment of antihypertensive drugs. After screening based on P0.05, fold change1.2 or fold change0.83, and variable importance in projection1, 63 differential metabolites were collected. Four metabolic pathways-cysteine and methionine metabolism, phenylalanine metabolism, taurine and hypotaurine metabolism, and tyrosine metabolism-were identified in participants treated with ARBs. Only taurine and hypotaurine metabolism were identified in participants treated with CCBs. Furthermore, homocitrulline and glucosamine-6-phosphate were relevant to whether the blood pressure reduction achieved the target blood pressure (P0.05). Our study provides some evidence that changes in certain metabolites may be a potential marker for the dynamic monitoring of the protective effects and side effects of antihypertensive drugs.

Carbamylation is an ubiquitous process in which cyanate (OCN−) reacts with the N-terminal amino or e-amino moiety and generates α-carbamyl amino acids and e-carbamyl-lysine (homocitrulline). The process leads to irreversible changes in protein charge, structure and function. In this study, we have investigated the effect of carbamyl (generated from potassium cyanate) on human serum albumin (HSA) structure and function. The carbamylated-HSA (c-HSA) showed various modifications when examined by UV, fluorescence, FT-IR and far-UV CD spectroscopies. c-HSA exhibited hypochromicity, loss in α-helical content, changes in the amide I and amide II band, etc. Native-PAGE showed increase in the mobility of c-HSA compared to native-HSA. Aggregate(s) formation in c-HSA was detected by thioflavin T dye. The biochemical investigations carried out on c-HSA suggested increase in carbonyl content and decreased binding of TNBS (trinitrobenzenesulphonic acid) and Sakaguchi reagent. The attachment of the carbamyl moiety to HSA was confirmed from MALDI-TOF results. The functional defects in c-HSA were confirmed from the low binding of bilirubin. Taken together, carbamylation of albumin caused changes in the structural and functional properties of HSA. To the best of our knowledge, this is the first report on detailed biophysical characterization of carbamylated-HSA.

Reduced skeletal muscle density in older adults is associated with insulin resistance, decreased physical function, and an increased all-cause mortality risk. To elucidate mechanisms that may underlie the maintenance of skeletal muscle density, we conducted a secondary analysis of previously published muscle composition and serum metabolomic data in 73 older adults (average age, 78 y). Multivariable-adjusted linear regression was used to examine associations between 321 metabolites with muscle composition, defined as the ratio between normal density (NDM) with low density (LDM) thigh muscle cross sectional area (NDM/LDM). Sixty metabolites were significantly (p ≤ 0.05 and q < 0.30) associated with NDM/LDM. Decreased renal function and the immune response have been previously linked with reduced muscle density, but the mechanisms underlying these connections are less clear. Metabolites that were significantly associated with muscle composition were then tested for their association with circulating markers of renal function (blood urea nitrogen, creatinine, uric acid), and with the immune response (neutrophils/lymphocytes) and activation (kynurenine/tryptophan). 43 significant NDM/LDM metabolites (including urea) were co-associated with at least 1 marker of renal function; 23 of these metabolites have been previously identified as uremic solutes. The neutrophil/lymphocyte ratio was significantly associated with NDM/LDM (β ± SE: −0.3 ± 0.1, p = 0.01, q = 0.04). 35 significant NDM/LDM metabolites were co-associated with immune activation. Carbamylation (defined as homocitrulline/lysine) was identified as a pathway that may link renal function and immune activation with muscle composition, as 29 significant NDM/LDM metabolites were co-associated with homocitrulline/lysine, with at least 2 markers of renal function, and with kynurenine/tryptophan. When considering that elevated urea and uremic metabolites have been linked with an increased systemic microbial burden, that antimicrobial defense can be reduced in the presence of carbamylation, and that adipocytes can promote host defense, we propose the novel hypothesis that the age-related increase in adipogenesis within muscle may be a compensatory antimicrobial response to protect against an elevated microbial burden.

Objective.Antibodies that target citrullinated protein/peptide (ACPA) and homocitrullinated/carbamylated protein/peptide (AHCPA) are associated with rheumatoid arthritis (RA). The relationship between ACPA and AHCPA remains unclear. We examined the expression and cross-reactivity of these antibodies using citrulline- and homocitrulline-containing synthetic peptides, CitJED and HomoCitJED, respectively, which have equal numbers of citrulline or homocitrulline residues on the same peptide backbone.Methods.Serum from healthy subjects (n = 51) and patients with RA (n = 137), systemic lupus erythematosus (SLE; n = 37), and psoriatic arthritis (PsA; n = 37) were screened for IgG anti-CitJED and anti-HomoCitJED antibodies by ELISA. Cross-reactivity of these antibodies was examined by inhibition with various concentrations of CitJED and HomoCitJED.Results.Out of 137 patients with RA, antibodies to CitJED and HomoCitJED were detected in 69 (50%) and 78 (57%), respectively. Anti-CitJED and HomoCitJED antibodies were 77% concordant and their levels were strongly correlated [Spearman correlation coefficient (rs) = 0.6676]. Sera from 25/27 patients (93%) with RA were inhibited by both CitJED and HomoCitJED with equal or higher affinity for the cognate (homologous) peptide.Conclusion.Antibodies to CitJED and HomoCitJED frequently occurred in RA, but were not found in SLE or PsA, suggesting that these antibodies are specific to RA. Cross-reactivity between anti-HomoCitJED and anti-CitJED antibodies suggests that ACPA and AHCPA are derived from the same B cell population and both may contribute to the pathogenesis of RA.

Protein carbamoylation is a non-enzymatic post-translational modification that binds isocyanic acid, which can be derived from the dissociation of urea or from the myeloperoxidase-mediated catabolism of thiocyanate, to the free amino groups of a multitude of proteins. Although the term 'carbamoylation' is usually replaced by the term "carbamylation" in the literature, carbamylation refers to a different chemical reaction (the reversible interaction of CO2 with α and e-amino groups of proteins). Depending on the altered molecule (for example, collagen, erythropoietin, haemoglobin, low-density lipoprotein or high-density lipoprotein), carbamoylation can have different pathophysiological effects. Carbamoylated proteins have been linked to atherosclerosis, lipid metabolism, immune system dysfunction (such as inhibition of the classical complement pathway, inhibition of complement-dependent rituximab cytotoxicity, reduced oxidative neutrophil burst, and the formation of anti-carbamoylated protein antibodies) and renal fibrosis. In this Review, we discuss the carbamoylation process and evaluate the available biomarkers of carbamoylation (for example, homocitrulline, the percentage of carbamoylated albumin, carbamoylated haemoglobin, and carbamoylated low-density lipoprotein). We also discuss the relationship between carbamoylation and the occurrence of cardiovascular events and mortality in patients with chronic kidney disease and assess the effects of strategies to lower the carbamoylation load.

Objective: Anti-carbamylated protein antibodies (ACarPA) are associated with rheumatoid arthritis (RA) risk and severity and are primarily directed against fibrinogen. The lack of understanding of ACarPA reactivity has limited analysis of their immunopathogenic associations in RA. To address this shortcoming, we mapped ACarPA epitope reactivity in RA patient sera. Methods: Immunoblotting identified a patient serum with specific reactivity to carbamylated human fibrinogen β-chain. Liquid chromatography/mass spectrometry (LC/MS) identified sites of homocitrullines (carbamylated lysines) present in the human fibrinogen β-chain. The reactivity of an ACarPA+ cohort to specific peptides containing carbamylated lysines was determined by ELISA through direct binding (n=63) and by competition assays (n=40). Results: Serum from an RA patient with specific reactivity to carbamylated, but not citrullinated, fibrinogen β-chain was identified. LC/MS identified carbamylation of 9/34 lysines in the human fibrinogen β-chain. Mapping of immunoreactivity against tryptic peptide fragments demonstrated several candidate carbamylated epitopes that were confirmed by competition experiments. Peptides containing a homocitrulline at position 83 appeared to be an immunodominant epitope in some RA patient sera, with additional reactivity to peptides containing homocitrullines at positions 52, 264, 351, 367, and 374. Conclusion: ACarPA appear to preferentially target specific regions of the human fibrinogen β-chain that contain homocitrullines. Interestingly, humoral immunoreactivity appears relatively restricted in some patients, which may enable detection of specific relationships with disease phenotype. This article is protected by copyright. All rights reserved.

In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA.

Objectives Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA. Methods The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA. Results Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV. Conclusions Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease. Trial registration number ISRCTN36745608; EudraCT Number: 2006-003146-41.

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. METHODS Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histone-derived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). RESULTS The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for citrullinated peptides. CONCLUSION ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti-carbamylated and anti-citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

Rheumatoid arthritis (RA) is a prototype autoimmune disease, with the hallmark signs of synovial inflammation and the presence of autoantibodies. One of the most prominent examples of such autoantibodies are anti-citrullinated protein antibodies (ACPA), which are directed against a wide-array of citrullinated proteins. The immune response to citrullinated antigens is a dynamic response that expands before the onset of disease and generates antibodies that are extensively glycosylated in the variable domain. This feature of ACPAs is remarkable and might be involved in the breach of tolerance to citrullinated proteins as well as function as an additional biomarker to predict disease onset in subjects at risk. Next to ACPA, it has become clear that the autoantibody response in RA extends towards several other modified proteins, such as proteins modified by carbamylation or acetylation. Carbamylation leads to the formation of homocitrulline. Structurally, homocitrulline greatly resembles citrulline but is one methylene group longer. In contrast, acetylation is mediated by intracellular acetyltransferases and is structurally less related to citrulline or homo-citrulline. Although the presence of autoantibodies against these post-translationally modified proteins (Anti-Modified Protein Antibodies; AMPA) hallmark RA, at present, there is no conceptual framework explaining the concordant presence of different AMPA-responses in RA. In the context of this presentation, the latest insights into the development of humoral response against citrullinated-, carbamylated and acetylated proteins in relation to their role as biomarkers to predict the development of RA will be discussed. Disclosure of Interests: Rene Toes Grant/research support from: Sanofi

Background Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. Methods Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n = 108) just before (M0) and six months (M6) after the initiation of HD therapy. Results Mean HCit concentrations reached 1000 μmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6 months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r = 0.58) and carbamylated hemoglobin (r = 0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. Conclusion HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.

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.

Carbamylation corresponds to the non-enzymatic binding of isocyanic acid, mainly derived from urea decomposition, on amino groups of proteins, and participates in their molecular aging. This process is increased during chronic kidney disease (CKD) because of hyperuremia, and in other pathologies like atherosclerosis, where isocyanic may be formed from thiocyanate by myeloperoxidase in atheroma plates. Carbamylation triggers structural and functional modifications of proteins, thus impairing their biological roles and their interactions with cells. Much experimental evidence in vitro has shown the potential deleterious effects of carbamylated proteins on cell and tissue functions. Carbamylation-derived products (CDPs), and especially their major component homocitrulline, accumulate in organism in long half-life proteins, and may participate in the development of different complications of CKD, especially cardiovascular diseases, renal fibrosis, or nutritional and metabolic troubles. Recent clinical studies have confirmed the link between serum protein carbamylation and morbi-mortality in patients suffering from CKD or undergoing hemodialysis. Some CDPs could be used as biomarkers in these pathologies.

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.

Antibodies to proteins/peptides containing citrulline are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE). RA patients also generate antibodies to homocitrulline-containing proteins/peptides (also referred to as anti-carbamylated protein antibodies (Anti-CarP)). This study was undertaken to investigate the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express the human SE. C57BL/6 (B6) and DR4tg (on a B6 background) mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). Splenic T cell proliferation was evaluated by 3H-thymidine incorporation assay. Antibodies to homocitrullinated and citrullinated antigens were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with HomoCitJED and its citrullinated counterpart peptide, CitJED (the number of homocitrullines in HomoCitJED is equal to the number of citrullines in CitJED). HomoCitJED-immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to citrullinated antigens. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. In conclusion, DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice.

Background Autoimmunity in Rheumatoid arthritis (RA) patients is characterized by a spectrum of anti-modified protein antibodies (AMPA) directed against post-translationally modified (PTM) proteins. The best-known AMPA in RA are anti-citrullinated protein antibodies (ACPA). Much less is known about the occurrence and aetiology of other AMPA responses in RA such as autoantibodies directed to malondialdehyde-acetaldehyde (MAA) adducts, acetylated antigens, and carbamylated proteins. Anti-carbamylated protein (anti-CarP) autoantibodies recognize carbamylated proteins containing a homocitrulline, a PTM structurally similar to citrulline. With the presence of various AMPA responses in RA, PTM proteins have been implicated in the breach of autoreactive B-cell tolerance leading to the formation of these autoantibodies. At present it is unkown how AMPA are generated and how autoreactive B-cell responses against PTM proteins are induced. Objectives To investigate how PTM proteins, more specifically carbamylated proteins, could contribute to a breach of B-cell tolerance. Methods Serum reactivity towards five different carbamylated proteins was determined for 160 RA-patients and 40 healthy individuals. Anti-CarP antibody cross-reactivity was studied by inhibition experiments. Mass spectrometry was performed to identify carbamylated self-proteins in human rheumatic and osteoarthritic joint tissue. Mice were immunized with carbamylated- or non-modified foreign (OVA) and self-antigens (mouse Albumin). Sera of immunized mice and monoclonal antibodies were analyzed for antigen reactivity. Results We show that anti-CarP antibodies in RA are highly cross-reactive towards multiple carbamylated proteins, including modified self- as well as modified non-self proteins. Immunization with carbamylated foreign proteins (Ca-OVA) induced a strong anti-CarP response against both carbamylated foreign- and self-proteins. Similar to murine serum anti-CarP antibodies, murine monoclonal anti-CarP antibodies were highly specific and cross-reactive to multiple carbamylated (auto)antigens. Although citrulline greatly resembles homocitrulline residues in structure, murine anti-CarP antibodies differ in antigen recognition profile from ACPA as they are able to discriminate between citrullinated and homocitrullinated forms of the same protein. Interestingly, we were able to identify carbamylated-albumin, in RA synovial tissue, indicating that albumin is present in carbamylated form locally in the inflamed joint. Conclusions Self-reactive AMPA-responses can be induced by exposure to foreign proteins containing PTM. Our findings show that autoreactive B-cell responses against PTM self proteins can be induced by exposure to PTM foreign proteins and provide new insights on the breach of autoreactive B-cell tolerance by foreign proteins. Disclosure of Interest None declared

Rheumatoid arthritis (RA) is a prototype autoimmune disease, with the hallmark signs of synovial inflammation and the presence of autoantibodies. Of the different autoantibody systems present in RA, rheumatoid factors (RF) are probably the best studied. Their presence was first detected >70 years and it was in the late 1950s when it was realized that RF reacted to gamma globulins. Since the discovery of RF several other autoantibody systems have been discovered in RA, many of them directed against post-translationally modified protein antigens. The most prominent example of such autoantibodies are anti-citrullinated protein antibodies (ACPA), which are directed against a wide-array of citrullinated proteins. Now RF and ACPA determination are the two major diagnostic laboratory tests for RA and part of the EULAR and ACR criteria for RA. In the past few years, it has become clear that the autoantibody response present in RA extends towards several other modified proteins, such as proteins modified by acetylation or carbamylation. As all these auto-antibodies recognize Post-Translationally Modified (PTM) proteins, these antibodies are collectively called Anti-Modified Protein Antibodies (AMPA). In the context of this presentation, I will focus on the auto-antibody response against citrullinated, carbamylated and acetylated proteins. Carbamylation leads to the formation of homocitrulline. Structurally, homocitrulline greatly resembles citrulline but is one methylene group longer. Citrulline is generated when PAD enzymes modify the amino acid arginine. In contrast, the amino acid homocitrulline is generated by a chemical reaction in which cyanate reacts with the amino acid lysine. Arginine and lysine are located at different positions in the amino acid sequence of proteins, and therefore these modifications occur at different positions in proteins with different flanking amino acids. Intriguingly, although homocitrulline residues can also be recognised by auto-antibodies, these auto-antibodies often do not crossreact with citrulline. Acetylation is a process where acetyl groups are added to free amines of lysine residues by acetyl transferases. Acetylated lysine does not resemble citrulline but bears similarity to homocitrulline except at the side chain terminal amine, which is replaced by a methyl moiety. By now it is clear that AMPA consist of different auto-antibody families that are largely distinct, but that can also display a certain degree of cross-reactivity. Therefore, the notion is emerging that, although cross-reactivity exist, different classes of AMPAs are generally seen as distinct auto-antibody families that target different antigens, but intriguingly often co-occur. As the AMPA-responses in RA are often found together, it indicates that –somehow- AMPA-reactivity has a commonality that is currently not understood. Although, the reason why an immune response starts against PTM proteins is not known, it appears crucial to obtain understanding on the breach of tolerance towards PTM proteins as the immune response against these proteins has been intimately implicated in disease-pathogenesis. Understanding the full AMPA response, the triggers that drive AMPA production, their mutual crosstalk and the pathways by which AMPA and/or AMPA-expressing B cells possibly contribute to RA will be important for the development of curative interventions in RA. In the context of this presentation, some of these aspects will be discussed. Disclosure of Interest None declared

Summary Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during aging and is a risk factor for various diseases. The most common product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group has reacted with urea-derived cyanate. HCit has recently been detected in collagen; however, given that 15%–90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and in vivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys 933 , a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation.

Background Several investigators have reported discrepancies between the bromocresol-purple (BCP), bromocresol-green (BCG) and immunonephelometric (INP) assays in dialysis patients. This study compared the abovementioned assays and investigated whether hemodialysis itself or carbamylation of albumin is the cause for this discrepancy. Methods Samples obtained from hemodialysis patients were analyzed by BCP, BCG and INP. Furthermore, albumin was carbamylated in vitro using isocyanate. Isocyanate converts lysine to homocitrulline. Results No differences were observed between samples of the pre- and post-hemodialysis groups for BCP. In the control group, BCG averaged 6 g/L higher than INP. BCP did not statistically deviate from INP. In the dialysis group BCG averaged 5 g/L higher when compared to INP, whereas BCP averaged 2 g/L lower. BCP was affected by carbamylation of albumin. BCG and INP measurements were affected to a much lesser extent. Homocitrulline content of hydrolysates was increased in both the carbamylated albumin as well as in the dialysis population. Conclusion In a hemodialysis population albumin concentrations are not consistently estimated by both BCG and BCP methods. Relative to INP measurements BCG overestimates the albumin concentration (4–10 g/L), whereas BCP leads to an underestimation (0–4 g/L). Carbamylation of albumin is the main attributor to the discrepancy found with BCP.

Post-translational modifications such as citrullination and phosphorylation have been shown to provide novel target for cancer vaccines. Epitopes from modified proteins allow the generation of cytotoxic CD4 cells capable of targeting tumor. In this study we show that homocitrulline, an amino acid produced when lysine is post translationally modified by the process of carbamylation, can be a target for effective tumor immunity. Homocitrullinated epitopes from the cytoskeletal protein vimentin and the glycolytic enzyme aldolase were selected based upon MHC-II binding algorithms. Immunization of HLA transgenic mice with three homocitrullinated peptides, one from vimentin and two from aldolase A, induced potent IFNγ responses restricted via HLA-DR4, DR1 or DP4. Responses are specific to the homocitrullinated peptides and did not cross react with unmodified peptides. The responses have been characterized as CD4-mediated, Th1-type responses with minimal IL-10 or IL-17 production. In humans, we have also shown that healthy donors and cancer patients both have a CD4 T-cell repertoire recognizing these homocitrullinated peptides. In B16 melanoma tumor models, vaccination with homocitrullinated peptides increases survival in transgenic HLA-DR4 (survival 70%, p=0.0042), HHDII/DR1 (survival 50%, p Citation Format: Katherine W. Cook, Wei Xue, Peter Symonds, Mohamed Gijon, Sabaria Shah, Suha Atabani, Poonam Vaghela, Ruhul Choudhury, Rachael L. Metheringham, Ian Daniels, Victoria Brentville, Lindy Durrant. Post-translationally modified homocitrulline induced by MDSCs can be an effective antitumor target for CD4 T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B104.

Homoarginine is a novel biomarker for cardiovascular diseases. In the present large cohort study, we evaluate how homoarginine is linked to kidney function and examine the potential interaction of homoarginine and kidney function as predictors of cardiovascular outcomes.Serum homoarginine (mean: 2.41 ± 1.05 µmol/L), cystatin C and creatinine-based estimated GFR (eGFR, mean: 86.2 ± 23.0 mL/min per 1.73 m(2)) were measured in 3037 patients (mean age: 62.8 ± 10.6 years; 31.5% women) who were referred to coronary angiography.Homoarginine was positively associated with eGFR (age- and gender-adjusted partial correlation coefficient: 0.20, P0.001); using multiple regression analysis, eGFR emerged as an independent predictor of serum homoarginine (β = 0.10, SE 0.01, P0.001). Overall cardiovascular mortality was 18.5% (563 cardiovascular deaths) after 9.9 years. Multivariate Cox proportional hazard analysis revealed that compared with participants in the highest gender-specific homoarginine tertile, those in the lowest tertile were at increased risk of cardiovascular death [multivariate-adjusted HR 1.47; 95% confidence interval (95% CI) 1.15-1.87, P = 0.002]. After adjustment for confounders, both homoarginine and eGFR were associated independently with cardiovascular mortality, with a strong synergistic interaction (P for interaction 0.005). After stratifying the cohort into persons with eGFRs60 and ≥60 mL/min per 1.73 m(2), there was a stronger association between homoarginine and cardiovascular mortality in patients within eGFR below 60 (mean: 46.5 ± 12.0 mL/min per 1.73 m(2); HR per log SD increment of homoarginine 0.78; 95% CI 0.65-0.95, P = 0.013) compared to those with eGFR values ≥60 mL/min per 1.73 m(2). Subgroup analysis revealed that homoarginine is exclusively associated with death due to heart failure in subjects with eGFR values60 mL/min per 1.73 m(2) (HR per log SD 0.56; 95% CI 0.37-0.85; P = 0.006).Low homoarginine is strongly related to decreased kidney function, adverse cardiovascular events and death due to heart failure. The relationship between low homoarginine and adverse cardiovascular outcomes is most obvious when kidney function is impaired.

Wheat gluten/glycerol (WGG) films were extruded with aqueous ammonia/salicylic acid or urea to investigate the reactions contributing to their hexagonal close-packed protein structures and material properties. The addition of aqueous ammonia and salicylic acid increased the pH, which, in turn, increased the level of intermolecular disulfide and lanthionine cross-links in the WGG films. Increased protein cross-linking reactions resulted in higher material strength and tensile modulus. These cross-linking reactions and the resulting material properties were similar for WGG films with 7.5 and 10% aqueous ammonia. Added urea into WGG film partially degraded into cyanate and ammonium. Cyanate subsequently reacted with lysine and cysteine to -carbamyllysine and S-carbamylcysteine, respectively. Even though these reactions resulted in a more alkaline reaction environment, hereby favoring disulfide bond formation and decreasing protein extractability, they also prevented the involvement of cysteine and lysine in protein cross-linking. The alkylation of these reactive amino acids, together with the plasticizing effect of urea, led to lower material strength and modulus with increasing levels of urea. publisher: Elsevier articletitle: Crosslinks in wheat gluten films with hexagonal close-packed protein structures journaltitle: Industrial Crops and Products articlelink: http://dx.doi.org/10.1016/j.indcrop.2013.08.077 content_type: article copyright: Copyright © 2013 Published by Elsevier B.V. ispartof: Industrial Crops and Products vol:51 pages:229-235 status: published

Aims Cerebellar ataxia is commonly observed in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disorder biochemically characterized by ornithine (Orn), homocitrulline (Hcit) and ammonia accumulation. Since the pathophysiology of cerebellum damage in this disorder is still unknown, we investigated the effects of Hcit and Orn on important parameters of redox and energy homeostasis in cerebellum of young rats. Material and methods We determined thiobarbituric acid-reactive substance (TBA-RS) levels, carbonyl content, nitrate and nitrite production, hydrogen peroxide production, GSH concentrations, sulfhydryl content, as well as activities of respiratory chain complexes I–IV, creatine kinase, Na + ,K + -ATPase, aconitase and α-ketoglutarate dehydrogenase. Key findings Orn and Hcit significantly increased TBA-RS levels (lipid oxidation), that was totally prevented by melatonin and reduced glutathione (GSH). We also found that nitrate and nitrite production was not altered by any of the metabolites, in contrast to hydrogen peroxide production which was significantly enhanced by Hcit. Furthermore, GSH concentrations were significantly reduced by Orn and Hcit and sulfhydryl content by Orn, implying an impairment of antioxidant defenses. As regards energy metabolism, Orn and Hcit provoked a significant reduction of aconitase activity, without altering the other parameters. Furthermore, Orn-elicited reduction of aconitase activity was totally prevented by GSH, indicating that the critical groups of this enzyme were susceptible to oxidation caused by this amino acid. Significance Taken together, our data indicate that redox homeostasis is disturbed by the major metabolites accumulating in HHH syndrome and that this mechanism may be implicated in the ataxia and cerebellar abnormalities observed in this disorder.

Traditional risk factors fail to explain the increased risk for cardiovascular morbidity and mortality in ESRD. Cyanate, a reactive electrophilic species in equilibrium with urea, posttranslationally modifies proteins through a process called carbamylation, which promotes atherosclerosis. The plasma level of protein-bound homocitrulline (PBHCit), which results from carbamylation, predicts major adverse cardiac events in patients with normal renal function, but whether this relationship is similar in ESRD is unknown. We quantified serum PBHCit in a cohort of 347 patients undergoing maintenance hemodialysis with 5 years of follow-up. Kaplan-Meier analyses revealed a significant association between elevated PBHCit and death (log-rank P

Lysinuric protein intolerance (LPI; MIM 222700) is an inherited aminoaciduria with an autosomal recessive mode of inheritance. Biochemically, affected patients present with increased excretion of the cationic amino acids: lysine, arginine, and ornithine. We report the first case of LPI diagnosed in Malaysia presented with excessive excretion of homocitrulline. The patient was a 4-year-old male who presented with delayed milestones, recurrent diarrhea, and severe failure to thrive. He developed hyperammonemic coma following a forced protein-rich diet. Plasma amino acid analysis showed increased glutamine, alanine, and citrulline but decreased lysine, arginine and ornithine. Urine amino acids showed a marked excretion of lysine and ornithine together with a large peak of unknown metabolite which was subsequently identified as homocitrulline by tandem mass spectrometry. Molecular analysis confirmed a previously unreported homozygous mutation at exon 1 (235 G > A, p.Gly79Arg) in the SLC7A7 gene. This report demonstrates a novel mutation in the SLC7A7 gene in this rare inborn error of diamino acid metabolism. It also highlights the importance of early and efficient treatment of infections and dehydration in these patients. Conclusion: The diagnosis of LPI is usually not suspected by clinical findings alone, and specific laboratory investigations and molecular analysis are important to get a definitive diagnosis.

Background Cyanate has recently gained attention for its role in the pathogenesis of vascular injury. Nonetheless, the effect of cyanate on angiogenesis remains unclear. Methods and Results In this study, we demonstrated that oral administration of cyanate impaired blood perfusion recovery in a mouse hind‐limb ischemia model. A reduction in blood perfusion recovery at day 21 was observed in the ischemic tissue of cyanate‐treated mice. Likewise, there were fewer capillaries in the ischemic hind‐limb tissue of cyanate‐exposed mice. Our in vitro study showed that cyanate, together with its carbamylated products, inhibited the migration, proliferation, and tube‐formation abilities of endothelial cells. Further research revealed that cyanate regulated angiogenesis partly by interrupting the vascular endothelial growth factor receptor 2/phosphatidylinositol 3‐kinase/Akt pathway. The serum concentrations of homocitrulline, a marker of cyanate exposure, were determined in 117 patients with stable angina and chronic total occlusion. Consistent with the antiangiogenic role of cyanate, homocitrulline levels were increased in patients with poor coronary collateralization (n=58) compared with those with high collateralization (n=59; 21.09±13.08 versus 15.54±9.02 ng/mL, P =0.009). In addition, elevated homocitrulline concentration was a strong predictor of poor coronary collateral growth. Conclusions Impaired angiogenesis induced by cyanate might contribute to poor coronary collateral growth.

The specificities and cross-reactions of antibodies induced by citrulline- and homocitrulline-containing proteins may give implications on the role of citrulline- and homocitrulline-binding antibodies in the pathogenesis and progression of rheumatoid arthritis (RA). Here we use rabbits as an experimental model of antibody development in RA. Thirty-two animals were immunized with peptide antigens containing either homocitrulline or citrulline. The sera were tested for binding to CCP and MCV antigens and to peptide sequences related to carboxyterminal telopeptides of type I and II collagens and containing arginine, citrulline, or homocitrulline. The binding of CCP and MCV antigens to antisera against homocitrulline-containing immunogens could be inhibited by human serum albumin containing homocitrulline, whereas similar binding to sera against citrulline-containing immunogens was not inhibited. The antisera induced with citrulline-containing collagen telopeptides recognized type I collagen-related antigens in a sequence-specific manner, as antibody binding to both citrulline- and homocitrulline-containing peptides was inhibited by corresponding citrullinated and native peptides. In contrast, type II collagen-related peptides were recognized by the antisera in a ureido group-specific manner, as their binding to homocitrulline-containing peptide was inhibited by both citrulline- and homocitrulline-containing, but not native peptide. Binding of the citrullinated type II collagen peptide could only be inhibited by the similarly citrullinated peptide. In conclusion, antibodies induced with citrulline or homocitrulline-containing antigens bound antigens in a ureido group-specific manner, recognizing citrulline and homocitrulline also in other sequences than those used in the original immunization. In competitive situations the amino acid present in the immunization antigen was favored.

Ornithine, ammonia and homocitrulline are the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a genetic disorder characterized by neurological regression whose pathogenesis is still not understood. The present work investigated the in vivo effects of intracerebroventricular administration of ornithine and homocitrulline in the presence or absence of hyperammonemia induced by intraperitoneal urease treatment on a large spectrum of oxidative stress parameters in cerebral cortex from young rats in order to better understand the role of these metabolites on brain damage. Ornithine increased thiobarbituric acid-reactive substances (TBA-RS) levels and carbonyl formation and decreased total antioxidant status (TAS) levels. We also observed that the combination of hyperammonemia with ornithine resulted in significant decreases of sulfhydryl levels, reduced glutathione (GSH) concentrations and the activities of catalase (CAT) and glutathione peroxidase (GPx), highlighting a synergistic effect of ornithine and ammonia. Furthermore, homocitrulline caused increases of TBA-RS values and carbonyl formation, as well as decreases of GSH concentrations and GPx activity. Hcit with hyperammonemia (urease treatment) decreased TAS and CAT activity. We also showed that urease treatment per se was able to enhance TBA-RS levels. Finally, nitric oxide production was not altered by Orn and Hcit alone or in combination with hyperammonemia. Our data indicate that the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome provoke lipid and protein oxidative damage and a reduction of the antioxidant defenses in the brain. Therefore, it is presumed that oxidative stress may represent a relevant pathomechanism involved in the brain damage found in patients affected by this disease.

Background The number of newborns and the number of disorders detected by large-scale screening programs has increased dramatically in the last decade. Newborn screening is a multi-step process requiring confirmatory testing to establish and refine a diagnosis. Whereas screening cutoffs are established to detect all cases of a specific disease, confirmatory testing is performed against a backdrop of many co-morbid conditions and interpretation of results is often not straightforward. The goal of the current study was to define the range of amino acid concentrations encountered in normal, premature, and acutely ill newborns as an aid to the interpretation of follow-up testing for suspicious newborn screens. Materials and methods Residual plasma samples from 354 neonates (age 1–10 days) were utilized. 206 samples were from neonates with uncomplicated birth histories and prompt hospital discharge. 98 specimens were derived from a population of children receiving intensive care with diagnoses that included sepsis, respiratory insufficiency, cardiac malfunction/malformation and gastrointestinal complications. 50 samples were from infants born after 32 but before 37 full weeks gestation that were discharged following uneventful hospital courses. 25 amino acids were quantitated by LC-MS/MS and reference intervals determined by non-parametric statistical methods. Distributions were compared using Kruskal–Wallis analyses for independent samples. Results The distributions of multiple amino acids in premature and critically ill infants were significantly different than those observed in healthy newborns. Differing distributions were found for phenylalanine, the branched chain amino acids, methionine, glutamine, glutamate, arginine, lysine, α-aminoadipic acid, and β-aminoisobutyric acid. In most cases median values were increased and distributions more positively skewed in premature or ill newborns compared to healthy newborns. In addition, we report neonatal homocitrulline reference intervals for these newborn populations determined by an LC-MS/MS technique that is not confounded by methionine interference. Conclusion These data provide a basis for improved detection of genetic metabolic disorders in newborns, particularly those born prematurely or with a variety of critical co-morbid conditions.

BACKGROUNDCarbamylation 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.CONTENTIn 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.SUMMARYCarbamylation-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.

Nonenzymatic Post-Translational Modification Derived Products: New Biomarkers of Protein AgingDuring their biological life, proteins are exposed in a cumulative way to irreversible nonenzymatic post-translational modifications that are responsible for their molecular aging and generate specific by-products called »post-translational modification derived products« (PTMDPs). PTMDPs are involved in the pathogenesis of various diseases such as diabetes mellitus, renal insufficiency and atherosclerosis, and are potential biomarkers in clinical practice. Nonenzymatic glycation refers to the spontaneous binding of glucose and reducing sugars to free amino groups and is amplified by oxidative processes (referred to as »glycoxidation«). It generates many reactive by-products such as aldehydes and leads to the formation of »advanced glycation end products« (AGEs). AGEs accumulatein vivo, alter tissue organization and activate membrane receptors such as RAGE, which triggers inflammatory responses. Carbamylation is due to the binding of isocyanic acid, formedin vivoeither by spontaneous dissociation of urea or by action of myeloperoxidase on thiocyanate, and generates homocitrulline from lysine groups. Carbamylation leads to alteration of the structural and biological properties of proteins, and favors inflammation and atherosclerosis. PTMDPs may be assayed by different methods, among others LC-MS/MS or immuno assays, constitute a promising field of investigation in basic research and are potential major biomarkers in laboratory medicine.

The posttranslational modification of proteins has the potential to generate neoepitopes that may subsequently trigger immune responses. The carbamylation of lysine residues to form homocitrulline may be a key mechanism triggering inflammatory responses. We evaluated the role of carbamylation in triggering immune responses and report a new role for this process in the induction of arthritis. Immunization of mice with homocitrulline-containing peptides induced chemotaxis, T cell activation, and Ab production. The mice also developed erosive arthritis following intra-articular injection of peptides derived from homocitrulline and citrulline. Adoptive transfer of T and B cells from homocitrulline-immunized mice into normal recipients induced arthritis, whereas systemic injection of homocitrulline-specific Abs or intra-articular injection of homocitrulline-Ab/citrulline-peptide mixture did not. Thus, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of anti-homocitrulline Abs, is critical for the induction of autoimmune reactions against citrulline-derived peptides and provides a novel mechanism for the pathogenesis of arthritis.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by mutations in ORNT1 gene that encodes a mitochondrial ornithine transporter. It has variable clinical presentations with episodic hyperammonemia, liver dysfunction, and chronic neurological manifestations. In this work, we report the findings of HHH syndrome in 3 Saudi siblings. The 4-year-old proband presented with recurrent Reye-like episodes, hypotonia, and multiple stroke-like lesions on brain MRI. Biochemical and molecular analysis confirmed that she had HHH syndrome. She significantly improved on protein restriction and sodium benzoate. Her two older siblings have milder phenotypes with protein intolerance and learning problems. In comparison to their sister, their homocitrulline and orotic acid were only mildly elevated even before treatment. The three patients were homozygous for a novel mutation in ORNT1 with a Gly220Arg change. In view of the CNS lesions, which initially were felt to be suggestive of MELAS, we sequenced the entire mtDNA genome and no potential pathogenic mutations were detected. Analysis of ORNT2 did not provide explanation of the clinical and biochemical variability. This work presents a yet unreported CNS involvement pattern, notably multiple supratentorial stroke-like lesions in association with HHH syndrome. Moreover, it illustrates considerable clinical/biochemical correlation, and describes a novel mutation. We suggest including HHH syndrome in the differential diagnosis of patients found to have stroke-like lesions on brain MRI.

Carbamylation is a non-enzymatic post-translational modification of proteins characterized by the addition of isocyanic acid to amino groups. As isocyanic acid mainly originates from the spontaneous dissociation of urea, carbamylation rate is increased during renal failure. The aim of the study was to evaluate serum homocitrulline (HCit), which results from the carbamylation of ε-amino groups of lysine (Lys) residues, in acute renal failure (ARF) and to determine if it could be useful for differentiating acute from chronic renal failure (CRF).In total, 213 patients with renal failure referred to the nephrology department of the university hospital of Reims were included. Patients were classified into three groups: patients with ARF (ARF group, n=39), patients with CRF complicated with ARF (A/CRF group, n=29) and patients with CRF (CRF group, n=145). Serum HCit concentrations were measured by LC-MS/MS. Concentration kinetics of HCit and urea were studied in patients suffering from ARF. The HCit thresholds distinguishing ARF and CRF were investigated.HCit concentrations increased in ARF patients reaching a peak delayed compared to urea concentration peak. HCit concentrations were positively correlated with urea concentrations (r=0.51) and with the time elapsed since the estimated onset of ARF (r=0.57). Serum HCit concentrations were higher (pOur results demonstrate that HCit is a promising biomarker for distinguishing between ARF and CRF patients.

To the Editor: Ornithine aminotransferase (OAT) deficiency is a rare metabolic disorder characterized by gyrate atrophy of choroid and retina resulting in loss of all functional vision between fourth to sixth decades [1]. Rarely, neonates with OAT deficiency can present with hyperammonemia [2]. Here, a patient who was diagnosed as OAT deficiency with a rare presentation of symptomatic neonatal hyperammonemia is reported. Diagnosis was confirmed with a novel OAT gene mutation. A female patient, born at term from consanguineous parents developed vomiting and abnormal cycling movements at 18 d of age. Physical examination revealed reduced level of consciousness, increased deep tendon reflexes and hypertonicity. Her plasma ammonia level was measured as 280 μmol/L. She was treated with intravenous sodium benzoate and arginine and enteral feeding was discontinued. Plasma ammonia level fell to normal ranges within 4 h. Initial metabolic investigations revealed elevated plasma glutamine levels whereas plasma ornithine and citrulline levels were remarkably reduced (Table 1). Acylcarnitine profile and urinary organic acid analysis were unremarkable. Urinary orotic acid excretion was normal. Oral sodium benzoate and arginine supplementation were started as a maintenance therapy and patient was fed by a low-protein diet. In follow-up she had no further episodes of hyperammonemia. By 3 mo of age, a rise in plasma ornithine level was detected (Table 1). Urinary homocitrulline excretion was normal. Depending on increased ornithine levels accompanied by normal urinary homocitrulline excretion; diagnosis of ornithine aminotransferase (OAT) deficiency was suspected. Molecular analysis of OAT gene demonstrated homozygote mutation for IVS7-1G>A (c.901-1G>A) which has not been reported before (Fig. 1). Depending on OAT deficient mouse model, Wang et al. explained different physiologic functions of OAT enzyme at different developmental stages. In the neonatal period, the main role of the enzyme is ornithine synthesis and maintenance of urea cycle by converting glutamine to arginine, whereas in different developmental stages its role is ornithine degradation and deficiency of the enzyme causes hyperornithinemia and retinal toxicity [3]. In literature, there are a few reports about neonatal OAT deficiency [2–5]. As early diagnosis of disease may prevent the ophthalmological findings, OAT deficiency should be considered in differential diagnosis of neonatal hyperammonemia. * Tanyel Zubarioglu tanyel0554@yahoo.com

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an inborn error of metabolism caused by a defect in the transport of ornithine (Orn) into mitochondrial matrix leading to accumulation of Orn, homocitrulline (Hcit), and ammonia. Affected patients present a variable clinical symptomatology, frequently associated with cerebellar symptoms whose pathogenesis is poorly known. Although in vitro studies reported induction of oxidative stress by the metabolites accumulating in HHH syndrome, so far no report evaluated the in vivo effects of these compounds on redox homeostasis in cerebellum. Therefore, the present work was carried out to investigate the in vivo effects of intracerebellar administration of Orn and Hcit on antioxidant defenses (reduced glutathione concentrations and the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase), lipid oxidation (malondialdehyde concentrations), as well as on the activity of synaptic Na(+), K(+)-ATPase, an enzyme highly vulnerable to free radical attack, in the cerebellum of adolescent rats. Orn significantly increased malondialdehyde levels and the activities of all antioxidant enzymes, and reduced Na(+), K(+)-ATPase activity. In contrast, glutathione concentrations were not changed by Orn treatment. Furthermore, intracerebellar administration of Hcit was not able to alter any of these parameters. The present data show for the first time that Orn provokes in vivo lipid oxidative damage, activation of the enzymatic antioxidant defense system, and reduction of the activity of a crucial enzyme involved in neurotransmission. It is presumed that these pathomechanisms may contribute at least partly to explain the neuropathology of cerebellum abnormalities and the ataxia observed in patients with HHH syndrome.

Anti-citrullinated protein antibodies (ACPA) are important serological markers in the diagnosis of rheumatoid arthritis (RA) and are part of the recent disease classification criteria. However, there is a strong need for reliable markers for measuring and predicting joint damage and disease activity. Recently, antibodies directed against carbamylated antigens (anti-CarP antibodies) were identified. A total of 120 RA patients were tested for anti-CCP antibodies using different methods and for anti-CarP antibodies using carbamylated fetal calf serum according to the method described by Shi et al. Additionally, ACPA fine specificities (to three citrullinated peptides) were measured. Disease activity was assessed at baseline using the disease activity score 28 (DAS28) in 80 patients. For 40 RA patients, joint erosion score (JES) was established. The median JES was 14.1 with a standard deviation of 11.5. Anti-CarP antibodies were correlated with joint erosion score (ρ = 0.34, 95 % CI 0.03–0.59; p = 0.0332). No correlation between ACPA and joint erosion score was observed. No individual marker correlated with DAS28. When one ACPA peptide was combined with anti-CarP antibodies in a score (ACPA peptide 1 divided by anti-CarP), a statistically relevant correlation was found (p = 0.0264). In this small cohort, the presence of anti-CarP antibodies, but not ACPA correlate with joint erosion score. Anti-CarP antibodies combined with ACPA fine specificities correlated with DAS28. Therefore, anti-CarP antibodies might represent a promising marker to predict joint damage and disease activity in RA patients.