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The unprecedented rise in global ambient temperatures in the last decade has significantly impacted human health, yet how heat exposure affects the development of sarcopenia remains enigmatic. Here, we demonstrate that chronic heat exposure induces skeletal muscle volume loss, leading to muscle strength and functional decline in mice. The microbiota composition of heat‐exposed mice was analyzed using 16S ribosomal DNA analysis. Liquid chromatography‐mass spectrometry (LC–MS) was used to explore the effects of heat exposure on the blood metabolome and to further analyze the correlation between blood metabolism and gut microbiota. Transplantation of microbiota from heat‐exposed mice to germ‐free mice was sufficient to increase adverse effects on skeletal muscle function in the host. Mechanistically, using an untargeted metabolomics strategy, we reveal that altered gut microbiota due to high temperatures is associated with elevated serum levels of homocitrulline. Homocitrulline causes mitochondrial dysfunction in myocytes by exacerbating ferroptosis levels. And Nrf2 activator (Oltipraz) supplementation alleviates muscle atrophy and dysfunction induced by heat exposure. Our findings reveal the detrimental effects of heat exposure on muscle function and provide new strategies for treating sarcopenia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aim: Multiple sclerosis (MS) is a demyelinating disease of central nervous system. Myelin basic protein (MBP) is the major protein in the structure of the myelin sheath, and the abnormal autoimmune response to MBP is related to the demiyelinization in MS. This autoimmune response is thought to be related to extensive post-translational modifications that may occur in the primary structure of MBP. Considering the role of post-translational modifications of MBP in the pathogenesis of MS, this study aimed to investigate the role of carbamylation in the pathogenesis of MS by measuring Hcit levels in patients with MS. Materials and Methods: This study included 80 patients with MS according to McDonald criteria by clinicians, and 60 healthy volunteers. Patients were divided into 2 groups as relapsing-remitting multiple sclerosis (RRMS) (n=44) and secondary progressive MS (SPMS) (n=36) according to Expanded Disability Status Scale (EDSS) score evaluated by clinicians. Serum homocitrulline and lysine levels were measured with validated tandem mass spectrometric method. Results: Serum Hcit levels in patients with MS were statistically significantly higher than the healthy controls. Comparison of MS subgroups according to Hcit levels showed that serum Hcit levels were higher in patients with SPMS than in patients with RRMS. Serum Hcit levels were positively correlated with EDSS and disease duration. Conclusion: Serum Hcit levels were significantly elevated in patients with MS. Moreover, there was a correlation between serum Hcit levels and disease activity and duration of disease. [ABSTRACT FROM AUTHOR]

Objective: Peripheral arterial disease (PAH) is a chronic, progressive disease characterized by arterial stenosis or occlusion. Atherosclerosis is the most common cause of PAH (>90%). Carbamylation is one of the post-translational modification mechanisms of proteins and has been identified as a new risk factor for atherosclerosis. The most common carbamylation product known is homocitrulline. Our aim in this study was to contribute to the elucidation of the role of homocitrulline in the diagnosis of PAH. Methods: 70 patients with PAH and 65 individuals without PAH were included in the study. Serum homocitrulline and lysine levels were measured by AB Sciex API 3200 (Applied Biosystems/MDS Sciex) liquid chromatography-tandem mass spectrometry (LC-MS/ MS) device. Various hematological and biochemical parameters of the patients were measured in Beckman Coulter LH 780 and Beckman Coulter AU 5800 (Beckman Coulter, Brea, USA) autoanalyzers, and C-reactive protein (CRP) levels were measured in IMMAGE 800 (Beckman Coulter, Brea, USA) device, respectively. Results: Serum homocitrulline concentrations of patients with PAH were statistically significantly higher than the control group (p<0.001). Receiver Operating Characteristic (ROC) analysis showed that the optimal serum homocitrulline cut-off value was 165.1 µmol / mol lysine (p<0.001) (sensitivity, 71.4% and specificity, 86.7%) for PAH. The area under curve (AUC) value was 0.873 (95% confidence interval: 0.804- 0.925). There was a positive correlation between serum homocitrulline and urea, CRP, the neutrophillymphocyte ratio (NLR) and the platelet-lymphocyte ratio (PLR) levels. Conclusion: Our findings show that homocitrulline levels are significantly elevated in patients with PAH, and homocitrulline may be a useful marker in the early diagnosis of PAH. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • We developed a method to quantify 48 disease-related amino acids within 20 min. • Only 2.5 μL plasma was required for the analysis. • Age-specific reference values were established for 42 amino acids. • Patients with different types of urea cycle disorders were diagnosed by the method. • New findings related to pathogenesis of urea cycle disorders were obtained. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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-ΔΨ), 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 ΔΨ 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. [ABSTRACT FROM AUTHOR]

Background: 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). Methods: 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. Results: 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 (p<0.05) in CRF group compared to ARF group. The receiver operating characteristic curve analysis showed that HCit concentrations <289 μmol/mol Lys were predictive of ARF (Sensitivity: 83%, Specificity: 72%, AUC: 0.856). Conclusions: Our results demonstrate that HCit is a promising biomarker for distinguishing between ARF and CRF patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The aim of the study was to describe automated immunoassays for autoantibodies to homocitrulline or citrulline containing telopeptides of type I and II collagen in various disease categories in an early arthritis series. Methods: Serum samples were collected from 142 patients over 16 years of age with newly diagnosed inflammatory joint disease. All samples were analyzed with an automated inhibition chemiluminescence immunoassay (CLIA) using four different peptide pairs, each consisting of a biotinylated antigen and an inhibiting peptide. Assays were performed with an IDS-iSYS analyzer Autoantibodies binding to homocitrulline and citrulline containing C-telopeptides of type I (HTELO-I, TELO-I) and type II collagens (HTELO-II, TELO-II) were analyzed. Results: The mean ratio of HTELO-I inhibition in seropositive and seronegative rheumatoid arthritis (RA) was 3.07 (95% CI 1.41-11.60), p = 0.003, and in seropositive and seronegative undifferentiated arthritis (UA) 4.90 (1.85-14.49), p < 0.001. The respective mean ratios in seropositive and seronegative RA and UA were in TELO-I 8.72 (3.68-58.01), p < 0.001 and 3.13 (1.49-6.16), p = 0.008, in HTELO-II 7.57 (3.18-56.60), p < 0.001 and 2.97 (1.23-6.69), p = 0.037, and in TELO-II 3.01 (1.30-9.51), p = 0.002 and 3.64 (1.86-7.65), p = 0.008. In reactive arthritis, ankylosing spondylitis, psoriatic arthritis and unspecified spondyloarthritis the inhibition levels were similar to those observed in seronegative RA or UA. Conclusions: Autoantibodies binding to homocitrulline or citrulline containing telopeptides of type I and II collagen did not differ significantly. They were highest among patients with seropositive disease and they differentiated seropositive and seronegative arthritis. [ABSTRACT FROM AUTHOR]

Objective. Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), interleukin (IL)-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated. Methods. Patients ( n = 105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. Disease activity score (DAS-28) was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine (Cl-Tyr), homocitrulline (Hcit), IL-8, and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum. Results. DAS-28 and CRP levels were not significantly different between groups. MPO activity, and MPO, Cl-Tyr, and Hcit levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. Conclusions. MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and Hcit in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations. [ABSTRACT FROM AUTHOR]

Highlights: [•] The reactions contributing to protein structure and material properties of wheat gluten/glycerol films with aqueous ammonia/salicylic acid or urea were investigated. [•] Increased protein cross-linking reactions resulted in higher material strength and tensile modulus. [•] In gluten films with aqueous ammonia/salicylic acid or urea disulfide cross-links were the most important. [•] Lanthionine formation contributed significantly to the protein network in films with aqueous ammonia/salicylic acid. [•] The reactions in the gluten films with aqueous ammonia/salicylic acid or urea clearly impacted their molecular and mesoscale protein structures. [Copyright &y& Elsevier]

Background: Antibodies binding to citrullinated proteins are a frequent finding in rheumatoid arthritis patients and may precede the onset of clinical symptoms several years. The antibodies are a predisposing factor for bone erosions but their origin is unknown. In this study we analyze in detail the levels of protein bound citrulline and homocitrulline in several tissue samples of a single erosive arthritic surgery patient. Methods: Serum antibodies binding to CCP, MCV and citrulline- or homocitrulline-containing type I and II collagen carboxytelopeptides were measured. Tissue samples of a single RA patient, taken in two separate operations performed with two-year time span were hydrolyzed and analyzed for citrulline and homocitrulline content by HPLC. Results: Protein-bound citrulline and homocitrulline were found in several joint tissues of a RA patient with ACPApositive erosive disease. The amount of homocitrulline stayed relatively constant between the different tissues. The amount of citrulline in erosive tissue was 3-times higher than in non-erosive tissue in the first operation. In the samples of the second operation 3-4-times higher mean amounts of citrulline were found in two out of the six tissues investigated. Conclusions: Homocitrulline is present in rheumatoid nodule together with citrulline. There is more variation in the amount of citrulline than in the amount of homocitrulline between the tissues. The tissue sample containing the most citrulline was the most erosive. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice ( n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids ( p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples. [ABSTRACT FROM AUTHOR]

Abstract: Hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by a defect in the mitochondrial ornithine transporter, leading to accumulation of ornithine (Orn), homocitrulline (Hcit) and ammonia. Progressive neurological regression whose pathogenesis is not well established is common in this disease. The present work investigated the in vivo effects of intracerebroventricular administration of Orn and Hcit on important parameters of oxidative stress and energy metabolism in cerebral cortex from young rats. Orn and Hcit significantly increased thiobarbituric acid-reactive substances values and carbonyl formation, indicators of lipid and protein oxidative damage, respectively. Furthermore, N-acetylcysteine and the combination of the free radical scavengers ascorbic acid plus α-tocopherol attenuated the lipid oxidation and totally prevented the protein oxidative damage provoked by Orn and Hcit, suggesting that reactive species were involved in these effects. Hcit, but not Orn administration, also decreased glutathione concentrations, as well as the activity of catalase and glutathione peroxidase, indicating that Hcit provokes a reduction of brain antioxidant defenses. As regards to the parameters of energy metabolism, we verified that Orn and Hcit significantly inhibited the citric acid cycle function (inhibition of CO2 synthesis from [1-14C] acetate), the aerobic glycolytic pathway (reduced CO2 production from [U-14C] glucose) and complex I–III activity of the respiratory chain. Hcit also inhibited the activity of aconitase, an enzyme very susceptible to free radical attack. Taken together, our data indicate that mitochondrial homeostasis is disturbed by Orn and especially by Hcit. It is presumed that the impairment of brain bioenergetics and the oxidative damage induced by these metabolites may possibly contribute to the brain deterioration and neurological symptoms affecting patients with HHH syndrome. [Copyright &y& Elsevier]

Abstract: Ornithine 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 vitro effects of ornithine and homocitrulline on important parameters of oxidative stress in cerebral cortex from young rats. Ornithine significantly increased chemiluminescence and thiobarbituric acid-reactive substances levels, indicators of lipid peroxidation, while homocitrulline only augmented chemiluminescence values. Furthermore, ornithine-induced increase of thiobarbituric acid-reactive substances levels was attenuated (melatonin and reduced glutathione) or totally prevented (α-tocopherol) by free radical scavengers, suggesting that reactive species were involved in the lipid oxidative damage. We also observed that ornithine and homocitrulline significantly decreased the tissue antioxidant defenses, determined by reduced glutathione concentrations, the major non-enzymatic antioxidant defense found in the brain. Homocitrulline reduction of glutathione levels was completely prevented by melatonin and α-tocopherol, whereas ornithine-induced decrease of glutathione levels was only attenuated by these free radical scavengers. Ornithine and homocitrulline also induced protein oxidative damage, increasing carbonyl formation and sulfhydryl oxidation. In contrast, these amino acids did not affect nitric oxide production, indicating that nitrogen reactive species were not implicated in the lipid and oxidative damage provoked by ornithine and homocitrulline. Therefore, it is presumed that the major metabolites accumulating in hyperornithinemia–hyperammonemia–homocitrullinuria syndrome elicit oxidative stress and that this pathomechanism may possibly be involved in the brain damage found in patients affected by this disorder. [Copyright &y& Elsevier]

Abstract: Tissue accumulation of ornithine (Orn), homocitrulline (Hcit), ammonia and orotic acid (Oro) is the biochemical hallmark of patients affected by hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome, a disorder clinically characterized by neurological symptoms, whose pathophysiology is practically unknown. In the present study, we investigated the in vitro effect of Orn, Hcit and Oro on important parameters of energy metabolism in brain of 30-day-old Wistar rats since mitochondrial abnormalities have been observed in the affected patients. We first verified that Orn and Hcit significantly inhibited the citric acid cycle (inhibition of CO2 synthesis from [1-14C] acetate, as well as aconitase and α-ketoglutarate dehydrogenase activities), the aerobic glycolytic pathway (reduced CO2 production from [U-14C] glucose) and moderately the electron transfer flow (inhibitory effect on complex I–III). Hcit, but not Orn, was also able to significantly inhibit the mitochondrial creatine kinase activity. Furthermore, this inhibition was prevented by GSH, suggesting a possible role of reactive species oxidizing critical thiol groups of the enzyme. In contrast, the other enzyme activities of the citric acid cycle and of the electron transfer chain, as well as synaptic Na+,K+-ATPase were not altered by either Orn or Hcit at concentrations as high as 5.0 mM. Similarly, Oro did not interfere with any of the tested parameters. Taken together, these data strongly indicate that Orn and Hcit compromise brain energy metabolism homeostasis and Hcit also interferes with cellular ATP transfer and buffering. It is therefore suggested that Orn and especially Hcit may be involved in the neurological damage found in patients affected by HHH syndrome. [Copyright &y& Elsevier]

A liquid chromatography tandem mass spectrometric method is described for the analysis of homocitrulline in human urine, a key metabolite in the differential diagnosis of hyperammonemia, hyperornithinemia, homocitrullinuria (HHH) syndrome. Urine samples were prepared by mere five-fold dilution with a mixture of internal standards (2H2-citrulline and 2H3-creatinine) used for the simultaneous quantification of creatinine. Analytes were separated on a cyano column and eluted isocratically within seven min. Detection was achieved by monitoring transitions of 190 > 84 and 190 > 127 for homocitrulline, 178 > 115 for 2H2-citrulline, 114 > 44 for creatinine and 117 > 47 for 2H3-creatinine. Calibration curves were linear up to 100 micromol/L. Intraday ( n = 7) and interday ( n = 6) variations were less than 10%. In urine samples from three siblings confirmed to have HHH syndrome, homocitrulline levels were at 13.3 (74), 21.1 (50) and 108.2 (103) mmol/mol creatinine (micromol/L). Control values were 0–9 mmol/mol creatinine ( n = 120). The current method solves specificity issues in homocitrulline determination often encountered with some ninhydrin-based systems (coelution with methionine) and some o-phthalaldehyde-based ones (coelution with taurine), and presents an attractive alternative with a relatively high throughput. [ABSTRACT FROM AUTHOR]