The role of fatty acids in patients with Behçet's disease and their association with thrombosis.

Behçet's disease (BD) is a systemic disease with unknown etiopathogenesis and varying disease presentations. Fatty acids (FA) are essential biological compounds that are involved in complex metabolic pathways. They may contribute to inflammation and endothelial dysfunction by participating in many signaling pathways. Increased FAs levels are associated with an increased risk for various diseases. This study aimed to determine the relationship between FA, BD, and thrombotic complications. A total of 97 patients were recruited from the rheumatology department of a single center as a case-control study. The participants were divided into three groups: 36 patients with BD with thrombosis (Group 1), 24 patients with BD without thrombosis (Group 2), and 37 age- and sex-matched controls (Group 3). The analysis of 37 different FA with carbon numbers in the range of (4:0) and (24:1) in the samples were analyzed and compared between groups. Myristic acid (MA), methyl eicosatrienoate, and stearic acid (STA) levels were found to be significantly higher in BD with thrombosis than in BD without thrombosis, and palmitic acid (PA) levels were significantly higher in BD with thrombosis than in healthy individuals. MA was found to be a significant marker for differentiating between thrombotic BD. PA and STA are important markers for detecting thrombotic BD. In BD, lipotoxicity created by FA, such as PA, STA, and MA, plays a role as an inducer of inflammation and thrombosis through various mechanisms.

Altered kynurenine pathway metabolism and association with disease activity in patients with systemic lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease accompanied by increased release of proinflammatory cytokines that are known to activate the indoleamine 2,3-dioxygenase (IDO-1) enzyme, which catalyzes the rate-limiting step of the kynurenine pathway (KP). This study aimed to measure KP metabolite levels in patients with SLE and investigate the relationship between disease activity, clinical findings, and KP. The study included 100 patients with SLE and 100 healthy controls. Serum tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3HAA), 3-hydroxykynurenine (3HK), quinolinic acid (QA) concentrations were measured with tandem mass spectrometry. Serum KYN, KYNA, 3HAA, 3HK, and QA levels of the patients with SLE were significantly higher than the control group. Serum QA levels were elevated in patients with neurological involvement (four patients with peripheral neuropathy and two patients with mononeuropathy), serum KYN levels and KYN/TRP ratio increased in patients with joint involvement, and serum KYN, 3HK, and 3HAA levels and the KYN/TRP ratio were increased in patients with renal involvement. Moreover, KYN and KYN/TRP ratios were positively correlated with the disease activity score. These findings indicated that imbalances in KP metabolites may be associated with the pathogenesis, activation, and clinical manifestations of SLE.

Measurement of kynurenine pathway metabolites by tandem mass spectrometry.

Objectives: Recent studies have shown that derangements in kynurenine pathway metabolite levels are associated with various pathologies such as neurodegenerative diseases, schizophrenia, depression, bipolar disorder, rheumatoid arthritis, and cancer. Therefore, reliable, accurate, fast, and multiplex measurement methods for kynurenines have become increasingly important. This study aimed to validate a new mass spectrometric method for analyzing tryptophan metabolites. Methods: A tandem mass spectrometric method, including protein precipitation and evaporation steps, was developed to measure serum levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Samples were separated using a Phenomenex Luna C18 reversed-phase column. The kynurenine pathway metabolites were detected by tandem mass spectrometry. The developed method was validated according to Clinical & Laboratory Standards Institute (CLSI) guidelines and applied to hemodialysis samples. Results: The developed method was linear at the concentrations of 48.8 - 25,000, 0.98 - 500, 1.2-5000, 1.2-5000, and 0.98-250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, respectively. The imprecisions were less than 12 %. The median serum concentrations of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid were 10530, 1100, 218, 17.6, and 25.4 ng/mL in pre-dialysis blood samples, respectively. They were 4560, 664, 135, 7.4, and 12.8 ng/mL in post-dialysis blood samples, respectively. Conclusions: A fast, simple, cost-effective, accurate, robust, and validated tandem mass spectrometric method was developed, and the method was successfully used for the quantitation of kynurenine pathway metabolite concentrations in hemodialysis patients.

Effect of cigarette smoking on serum methylarginine and α-klotho levels.

BACKGROUND AND AIMS: Smoking causes many diseases such as cardiovascular, lung diseases, stroke and premature aging. However, the role of smoking in the pathogenesis of these diseases is unclear. Increasing evidence suggests that methylarginine pathway metabolites and α-klotho may be strong markers for pathologies such as premature aging, endothelial dysfunction, and oxidant damage. Therefore, the study aimed to measure the serum levels of arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), N-monomethyl-l-arginine (L-NMMA), and α-klotho levels in smokers. METHODS AND RESULTS: This case-control analytical study included 65 smokers and 71 non-smokers. Sociodemographic characteristics, routine biochemistry parameters, Framingham risk scores and Fagerström Nicotine Dependence Test (FTND) were recorded. Serum methylarginine and α-klotho levels were analyzed by tandem mass spectrometry and enzyme-linked immunosorbent assay (ELISA), respectively. Serum ADMA (p < 0.001), L-NMMA (p = 0.024), SDMA (p < 0.001) levels of smokers were higher than non-smokers, and serum α-klotho (p < 0.001) and arginine levels (p < 0.001) were lower. There was a positive correlation between serum ADMA levels with FNDT, age and pack/year in smokers, while there was a negative correlation between klotho levels and age. A positive correlation was found between serum ADMA levels, Framingham risk score and age in non-smokers. CONCLUSION: Smoking is related to premature aging and is a strong risk factor for various diseases such as cardiovascular, inflammatory, and renal diseases. Elevated serum methylarginine and decreased serum klotho levels were found in smokers. Therefore, our findings suggest that smoking may be involved in the pathogenesis of these diseases by affecting α-klotho and methylarginine-related pathways.

Impaired kynurenine metabolism in patients with primary Sjögren's syndrome.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is an autoinflammatory disease characterized by inflammation of the exocrine glands. Elevated inflammation causes an increase in kynurenine pathway (KP) metabolite levels by activating indoleamine 2,3-dioxygenase (IDO). The aim of this study was to measure serum KP metabolite concentrations in patients with pSS and to evaluate the relationship between these metabolites with disease activity score and clinical manifestations. DESIGN & METHODS: A total of 80 patients with pSS and 80 healthy controls were enrolled in this study. Serum tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3HAA), 3-hydroxykynurenine (3HK), quinolinic acid (QUIN) concentrations were quantified with liquid chromatography with tandem mass spectrometry (LC-MS/MS). Demographic characteristics, clinical manifestations and disease activity score (ESSDAI) of the participants were recorded. RESULTS: The serum level of KYN and QUIN were significantly higher in patients with pSS with low and moderate activity compared those healthy controls, while the serum level of TRP, KYNA/KYN and 3HK/KYN were lower. In addition, the significant difference for the serum level of KYNA was only in patients with moderate activity from healthy controls, and the difference was higher in favor of pSS patients. Moreover, the KYN/TRP levels were significantly increased with disease activity. The ESSDAI score was positively correlated with KYN/TRP ratio, but negatively correlated with KYNA/KYN ratio. CONCLUSIONS: These findings indicated that KP metabolites may play a role in the etiopathogenesis, activation and progression of pSS.

Inflammation and Bone Turnover Markers in Adult Obesity.

Obesity is a condition of abnormally increased body fat resulting from increased energy intake relative to energy expenditure. Excess body weight is a risk factor for many somatic and psychological disorders, including cardiovascular disease, type 2 diabetes mellitus, osteoarthritis, and cancer types. Bone metabolism, bone turnover, and mineral content are altered in severe obesity. This review will focus on the relationship between inflammation and bone biomarkers in adult obesity.

Elevated serum levels of kynurenine pathway metabolites in patients with Behçet disease.

Behçet disease (BD) is an inflammatory, multisystemic vasculitis of unknown etiopathogenesis. However, innate and adaptive immune system involvement and immune-mediated networks play a vital role in the inflammatory cascade. Indoleamine 2,3-dioxygenase 1 (IDO1) is activated in chronic inflammatory states and catalyzes the first and rate-limiting step of tryptophan (TRP) metabolism along the kynurenine pathway (KP). The study aimed to measure KP metabolites levels in patients with BD and investigate the relationship between disease activity and clinical findings with these metabolites. The study included 120 patients with BD and 120 healthy volunteers. Serum TRP, kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3HAA), 3-hydroxykynurenine (3HK), and quinolinic acid (QUIN) levels were measured with the tandem mass spectrometric method. Demographic data, clinical manifestations, and disease activity score (BDCAF) were recorded. Serum KYN, KYNA, 3HK, 3HAA, QUIN levels, and KYN/TRP ratio were higher (p < 0.05) in patients with BD compared to the control group, while TRP levels were lower (p < 0.05). KYN/TRP ratio and QUIN levels were significantly higher in the presence of neuro-Behçet, while serum KYN levels were significantly higher in the presence of arthritis (p < 0.05). In addition, serum QUIN levels were significantly higher in the presence of thrombosis (p < 0.05). BDCAF score positively correlated with KYN/TRP ratio. Our findings showed that serum KP metabolite levels were elevated in patients with BD, and there is a relationship between these metabolites with disease activity, clinical findings, and inflammatory burden.

New steps in acute coronary syndrome and antihyperlipidemic treatment: Determination of statins and metabolites by liquid chromatography tandem mass spectrometry.

Our aim in this study was to develop a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of atorvastatin, rosuvastatin and their major metabolites, and furthermore to evaluate patients' adherence to statin therapy and to investigate the effect of statin therapy on various hematological and biochemical parameters. A simple protein precipitation was performed for the extraction of analytes and the extracted samples were injected directly. The levels of drugs and their metabolites were measured by the validated method in a total of 210 patients diagnosed with unstable angina pectoris (USAP), ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction (NSTEMI). Various biochemical and hematological parameters were measured. The linearity ranges for atorvastatin and rosuvastatin were 1.22-2,500 and 0.97-2000 ng/ml, respectively. The inter-assay coefficient of variation for all analytes was ≤ 6%. In patients diagnosed with USAP, STEMI and NSTEMI, treatment compliance rates were 22.1, 23.5 and 41.2% for atorvastatin and 36.1, 40.2 and 67.1% for rosuvastatin, respectively. An economical, simple and reliable measurement method has been developed. Our findings support the poor patient compliance with statin therapy in the included population. It was observed that 6 months of statin treatment caused slight changes in biochemical and hematological parameters.

Developing a robust, fast and reliable measurement method for the analysis of methylarginine derivatives and related metabolites.

BACKGROUND: Nitric oxide (NO) plays an important role in endothelial homeostasis. Asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA) and symmetric dimethyl arginine (SDMA), which are derivatives of methylarginine, directly or indirectly reduce NO production. Therefore, these metabolites are an important risk factor for various diseases, including cardiovascular diseases. Numerous methods have been developed for the measurement of methylarginine derivatives, but various difficulties have been encountered. This study aimed to develop a reliable, fast and cost-effective method for the analysis and measurement of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, citrulline, homoarginine, ornithine), and to validate this method according to Clinical and Laboratory Standards Institute (CLSI) protocols. METHODS: For the analysis of ADMA, SDMA, L-NMMA, arginine, homoarginine, citrulline, ornithine, 200 Âµl of serum were precipitated with methanol, and subsequently derivatized with a butanol solution containing 5% acetyl chloride. Butyl derivatives were separated using a C18 reverse phase column with a 5 min run time. Detection of analytes was achieved by utilising the specific fragmentation patterns identified through tandem mass spectrometry. RESULTS: The method was linear for ADMA, SDMA, L-NMMA, ornithine, arginine, homoarginine and citrulline in the ranges of 0.023-6.0, 0.021-5.5, 0.019-5.0, 0.015-250, 0.015-250, 0.019-5 and 0.015-250 µM, respectively. The inter-assay CV% values for all analytes was less than 9.8%. CONCLUSIONS: Data obtained from method validation studies shows that the developed method is highly sensitive, precise and accurate. Short analysis time, cost-effectiveness, and multiplexed analysis of these metabolites, with the same pretreatment steps, are the main advantages of the method.

Effects of hydroxychloroquine and its metabolites in patients with connective tissue diseases.

Hydroxychloroquine has attracted attention in the treatment of COVID-19. Many conflicting findings have been reported regarding the efficacy and safety of this drug, which has been used safely in the rheumatological diseases for years. However, these studies lacked measurement methods that allow accurate assessment of hydroxychloroquine and its metabolite levels. The aim of this study was to measure hydroxychloroquine and its metabolite levels in whole blood samples of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogren's syndrome (SS) and scleroderma (Scl) by a robust, simple and accurate validated tandem mass spectrometric method, and to investigate the relationship between these levels with drug-related adverse effects and disease activity scores. The validated LC-MS/MS method was applied to measure blood hydroxychloroquine and its metabolite levels of patients with RA, SLE, SS, Scl. Various haematological and biochemical parameters were measured with Beckman-Coulter AU 5800 and Beckman Coulter LH 780 analyzers, respectively. QTc intervals were calculated with Bazett's formula, and the patients were followed up by clinicians in terms of clinical findings and adverse effects. Hydroxychloroquine levels of patients were similar to previous studies. There was a negative correlation between disease activity scores and hydroxychloroquine levels, while the highest correlation was between QTc interval, creatinine and GFR levels with desethylchloroquine. Bidetylchloroquine had the highest correlation with RBC count and liver function tests. Our findings showed that hydroxychloroquine and its metabolite levels were associated with disease activity scores, renal, hepatic function, QTc prolongation, and hematological parameters.

Altered kynurenine pathway metabolism in patients with ankylosing spondylitis.

BACKGROUND: Various studies reported that increased proinflammatory cytokines in patients with ankylosing spondylitis (AS). Proinflammatory cytokines can affect the expression of various kynurenine pathway enzymes and therefore lead to metabolic changes that can affect the inflammatory response and immunity. Our aim was to measure serum levels of kynurenine pathway metabolites in patients with AS. METHODS: The study included 85 patients with AS and 50 healthy volunteers. Serum tryptophan, kynurenine, kynurenic acid, 3-hydroxyanthranilic acid, 3-hydroxykynurenine, quinolinic acid concentrations were measured with tandem mass spectrometry. In addition, participants were divided into four groups according to the treatment regimen: TNF-α inhibitor group, conventional therapy group, control group and newly diagnosed AS group. These groups were compared in terms of kynurenine pathways metabolites, interleukin 6 (IL-6), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels. RESULTS: Serum tryptophan, kynurenic acid, 3-hydroxykynurenine levels were significantly decreased (p < 0.05) in both AS groups compared to the control group, while the levels of kynurenine, quinolinic acid, CRP, ESR, and IL-6 were higher (p < 0.05). The Kynurenine/Tryptophan ratio and CRP levels of the conventional therapy and anti-TNF therapy group were significantly lower than the newly diagnosed AS patients (p < 0.05). CONCLUSION: As a result of our study, we found that altered kynurenine pathway metabolism in patients with AS. Conventional therapy and anti-TNF-α therapy are effective in reducing the Kynurenine/Tryptophan ratio and CRP levels, although the effect of both treatments on other metabolites appears to be limited.

Determination of serum carbamazepine and its metabolite by validated tandem mass spectrometric method and the effect of carbamazepine on various hematological and biochemical parameters.

OBJECTIVES: The study aimed to develop a validated LC-MS / MS method for the measurement of carbamazepine and carbamazepine-10,11-epoxide (CBZE) levels, to compare the carbamazepine concentrations measured by AbSciex API 3200 LC-MS/MS and Beckman Coulter Emit® 2000 immunoassay and to investigate the effect of carbamazepine concentrations on various hematological and biochemical parameters. METHODS: For the measurement of carbamazepine and CBZE levels, a validated LC-MS / MS method was developed. Serum carbamazepine levels of patients were measured by AbSciex API 3200 LC-MS / MS and Beckman Coulter Emit® 2000 immunoassay. Biochemical, hematological parameters, and hormone levels were measured by Beckman-Coulter AU 5800 (Beckman Coulter, Brea, USA), Beckman Coulter LH 780 (Beckman Coulter, Miami, FL, USA), and Cobas 6000 (Roche Diagnostics, Germany) analyzers, respectively. RESULTS: The imprecision values for all analytes were less than 7.0 %. The correlation coefficient between the methods was 0.981 (95 % confidence interval: 0.975 to 0.985). Linear regression analysis demonstrated highly significant associations of carbamazepine concentrations with albumin (B = -0.300, p = 0.040), calcium (B = -0.262, p = 0.004), phosphorus (B = -0.241, p = 0.022), WBC (B = -0.288, p = <0.001), PLT (B = -0.236, p = 0.003), RBC (B = -0.257, p = 0.001), NEU% (B = -0.289, p = <0.001), LYM% (B = -0.268, p = 0.001), D vitamini (B = -0.344, p = 0.006) levels. CONCLUSIONS: A robust, rapid, and simple method has been developed. Our study revealed that carbamazepine and its metabolite have a significant correlation and likely impact on bone metabolism, blood cell counts, serum protein, albumin levels, and electrolyte concentrations.

Evaluation of serum interleukin-6 (IL-6), IL-13, and IL-17 levels and computed tomography finding in interstitial lung disease associated with connective tissue disease patients.

OBJECTIVE: Interstitial lung disease (ILD) is one of the most severe complications which is associated with connective tissue disease (CTD) and causes to morbidity and mortality. So, we aimed to determine serum levels of interleukin-6 (IL-6), IL-13, and IL-17, to investigate whether these cytokines are related to CTD-ILD, and to find their possible contribution to determining the prognosis of the disease. METHODS: A total of 150 participants, 80 patients diagnosed with CTD-ILD (mean age, 58.21 ± 12.36) and 70 healthy controls (mean age, 57.07 ± 9.60) were recruited from the rheumatology department between January 2016 and June 2019 in the study. High-resolution computed tomography (HRCT) findings were scored as similarly to previous studies. Serum IL-6, IL 13, and IL-17 levels were measured by ELISA test kits. RESULTS: The levels of IL-6, IL-13, and IL-17 in CTD patients were significantly higher than the healthy individuals (p < 001), but the HRCT score's relation were not determined. IL-6 was associated with disease duration and disease activity scores of DAS28, ESDAII, and dSSc. There was a significant relation between dSSc, HCRT fibrosis, and total score.CRP, hemoglobin, and platelets were associated with the HRCT inflammation pattern. CONCLUSION: At the study, it has been observed that serum IL-13, IL-6 and IL-17 levels are increased in patients with CTD-ILD. Besides, IL-6 was associated with disease activity scores of DAS28, ESDAII, and dSSc. Also, HRCT fibrosis score is associated with dSSc. Further and comprehensive studies are needed to understand better the complex intersection of lung disease with systemic autoimmunity. Key Points • Serum IL-13, IL-6, and IL-17 levels are increased in patients with CTD-ILD. • IL-6 was associated with disease activity scores of DAS28, ESDAII, and diffuse skin involvement. • HRCT fibrosis score is associated with diffuse skin involvement in patients with SSc-ILD. • HRCT inflammation score is associated with PAH.

Can altered colostrum miRNA expression profile after cesarean delivery be a risk factor for autoimmune diseases?

PROBLEM: The cesarean section (CS) rate has increased significantly in North America, Western Europe, and Latin America. However, it has been reported that the incidence of inflammatory and autoimmune diseases such as asthma and type 1 diabetes increased in parallel with CS in these countries. Our aim was to investigate the expression level of miRNAs associated with inflammatory response and autoimmune diseases in colostrum samples and contribute to elucidating the role of CS in the pathogenesis of immune system-related diseases. METHOD OF STUDY: Colostrum samples were taken from voluntary mothers who had 40 normal and 50 cesarean births. miRNAs were extracted from colostrums and detected to miRNA expression profiling (eighty-four miRNAs) by quantitative real-time PCR with the Fluidigm integrated microfluidic circuit technology. RESULTS: There was a statistically significant change in the expression levels of 17 miRNAs in the colostrums of mothers who had normal and cesarean delivery (p < .05), and all of miRNAs were upregulated in the colostrums of mothers who have had cesarean delivery. CONCLUSION: Our best knowledge is that the study we conducted was the first to investigate the effect of delivery method (CS or normal) on the miRNA profile of colostrum. Cesarean delivery is a potential risk factor for inflammatory and immune system-related diseases in children due to dysregulation in miRNA expression.

Development and validation of a sensitive, fast and simple LC-MS / MS method for the quantitation of favipiravir in human serum.

Favipiravir is a broad-spectrum inhibitor of viral RNA polymerase. It is currently used as a possible treatment for coronavirus disease 2019 (COVID-19). Pre-clinical or clinical trials of favipiravir require robust, sensitive, and accurate bioanalytical methods for quantitation of favipiravir levels. Recently, several studies have been reported about developing a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring favipiravir levels. However, these methods were validated predominantly for plasma samples, electrospray ionization was operated only in negative or positive mode, and clinical application of these methods has not been applied for patients with COVID-19. This study aimed was to develop a validated LC-MS/MS method for the measurement of favipiravir levels in positive and negative electrospray ionization mode and to perform a pilot study in patients with COVID-19 receiving favipiravir to demonstrate the applicability of this method in biological samples. Simple protein precipitation was used for the extraction of favipiravir from the desired matrix. Favipiravir levels were quantitated using MS / MS with an electrospray ionization source in positive and negative multiple reaction monitoring (MRM) mode. The chromatographic detection was performed on a reverse-phase Phenomenex C18 column (50 mm × 4.6 mm, 5 µm, 100 Å) with gradient elution using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase. The method was linear over the concentration ranges of 0.048-50 µg/mL (in negative ionization mode) and 0.062-50 µg/mL (in positive ionization mode) with a correlation coefficient (r2) better than 0.998. The total run time was 3.5 min. The intra-assay and inter-assay %CV values were less than 7.2% and 8.0%, respectively. A simple, rapid and robust LC-MS / MS method was developed for the measurement of favipiravir and validation studies were performed. The validated method was successfully applied for drug level measurement in COVID-19 patients receiving favipiravir.