Neopterin

@article{pmid40435173,

title = {Insight into the metabolic shifts in Graves' hyperthyroidism: a study of acylcarnitine and lipid profiles},

author = {Mikael Thomassen Neset and Arve Ulvik and Kristian Løvås and Johnny Laupsa-Borge and Lars Ertesvåg Breivik and Ann-Elin Meling Stokland and Bjørn Gunnar Nedrebø and Eyvind Rødahl and Eystein Husebye and Grethe Åstrøm Ueland and Hans Olav Ueland},

doi = {10.1530/ETJ-25-0004},

issn = {2235-0802},

year  = {2025},

date = {2025-06-01},

journal = {Eur Thyroid J},

volume = {14},

number = {3},

abstract = {OBJECTIVE: Graves' disease increases metabolism and leads to net lipid degradation. Circulating acylcarnitines reflect lipid metabolism and the state of fatty acid oxidation in individuals. The aim of this study was to explore the lipid and acylcarnitine profiles in patients with Graves' disease.nnMETHODS: Seven lipids/apolipoproteins and 23 acylcarnitines were analysed in 100 newly diagnosed Graves' disease patients and validated in another 51 patients. Both groups were age- and sex-matched with healthy subjects.nnRESULTS: The hyperthyroid Graves' disease patients (n = 88) from the main cohort (78 females, median age 42 (17-67) years) had significantly (P < 0.05) higher levels of medium- and long-chain acylcarnitines, and lower levels of short-chain acylcarnitines, compared with healthy subjects. Factor analysis showed that medium- and long-chain acylcarnitines explained most of the differences between the two groups. Serum levels of the five lipids/apolipoproteins were significantly lower in the hyperthyroid Graves' disease patients compared with healthy subjects. Patients (n = 21) treated with antithyroid drugs for 6 weeks had acylcarnitine levels closer to healthy subjects, compared with 79 treatment-naïve hyperthyroid patients. The main findings were confirmed in the validation group.nnCONCLUSION: Increased levels of medium- and long-chain acylcarnitines in patients with newly diagnosed Graves' disease may reflect accelerated catabolism. Lower levels of short-chain acylcarnitines point to Graves' disease being a catabolic condition, with a shift in energy source from carbohydrates to fat.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39922470,

title = {Longitudinal associations of dietary intake with fatigue in colorectal cancer survivors up to 1 year post-treatment, and the potential mediating role of the kynurenine pathway},

author = {Daniëlle D B Holthuijsen and Judith J M Rijnhart and Martijn J L Bours and Eline H van Roekel and Per M Ueland and Stéphanie O Breukink and Maryska L G Janssen-Heijnen and Joop L Konsten and Eric T P Keulen and Adrian McCann and Stefanie Brezina and Biljana Gigic and Cornelia M Ulrich and Matty P Weijenberg and Simone J P M Eussen},

doi = {10.1016/j.bbi.2025.02.003},

issn = {1090-2139},

year  = {2025},

date = {2025-05-01},

journal = {Brain Behav Immun},

volume = {126},

pages = {144--159},

abstract = {INTRODUCTION: A healthy diet may help to reduce cancer-related fatigue, but evidence is limited and mechanisms remain unclear. Both diet and fatigue following colorectal cancer (CRC) have been linked to metabolites (kynurenines) of the kynurenine pathway (KP). We investigated longitudinal associations between dietary intake and fatigue, and the potential mediating role of the KP, in CRC survivors up to 1 year post-treatment.nnMETHODS: Measurements at 6 weeks, 6 months, and 1 year post-treatment were performed in 209 stage I-III CRC survivors. Diet was assessed by 7-day food records. Plasma kynurenines were analyzed using LC-MS/MS. Fatigue, including subjective fatigue, was assessed using validated questionnaires. To analyse longitudinal associations between diet and fatigue and to explore potential mediation by the KP, we used confounder-adjusted multilevel parallel-multiple mediator models with all kynurenines included simultaneously, and simple mediator models with established KP ratios to estimate total (c: diet-fatigue), direct (c': diet-fatigue, while controlling for mediators), metabolite-specific indirect (ab: diet-metabolite-fatigue), and total indirect (ab: diet-metabolites-fatigue) effects.nnRESULTS: Higher intake of total carbohydrates and mono- and disaccharides was longitudinally associated with more subjective fatigue, while higher intake of plant protein, total fat, and unsaturated fats was associated with less subjective fatigue (c). Most associations remained statistically significant after controlling for KP metabolites, except for mono- and disaccharides (c'). All kynurenines simultaneously did not mediate longitudinal associations between diet and subjective fatigue (ab). The kynurenic acid-to-quinolinic acid (KA/QA) ratio significantly mediated associations of intakes of carbohydrate, mono- and disaccharides, alcohol, magnesium, and zinc with subjective fatigue, whereas the HKr significantly mediated the association between polysaccharide intake and subjective fatigue (ab).nnCONCLUSION: Our findings suggest that carbohydrate intake is associated with greater fatigue, while protein and fat intake are associated with lower fatigue in CRC survivors up to 1 year post-treatment. While all KP metabolites simultaneously did not significantly mediate associations between diet and fatigue in our population, the KA/QA ratio and HKr were significant mediators in several diet-fatigue associations. These results should be repeated in larger observational studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40178293,

title = {Exercise training restores longevity-associated tryptophan metabolite 3-hydroxyanthranilic acid levels in middle-aged adults},

author = {Niklas Joisten and Marcel Reuter and Friederike Rosenberger and Andreas Venhorst and Marie Kupjetz and David Walzik and Alexander Schenk and Adrian McCann and Per Magne Ueland and Tim Meyer and Philipp Zimmer},

doi = {10.1111/apha.70041},

issn = {1748-1716},

year  = {2025},

date = {2025-05-01},

journal = {Acta Physiol (Oxf)},

volume = {241},

number = {5},

pages = {e70041},

abstract = {AIM: Recent pre-clinical evidence suggests that the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) and the related enzyme activity along the kynurenine metabolic pathway (KP) are associated with lifespan extension. We aimed to translate these findings into humans and expose exercise training as a potential non-pharmacological intervention to modulate this metabolic hub.nnMETHODS: To explore whether recent pre-clinical findings might also be of relevance for humans, we analyzed the evolutionary conservation of KYNU and HAAO, the two core KP enzymes associated with 3-HAA. In a cross-sectional analysis of young-to-middle-aged adults (N = 84), we examined potential associations of serum 3-HAA and its precursor anthranilic acid with age. We then investigated whether 26 weeks of endurance exercise (increasing intensity (INC) during the intervention period (n = 17) vs. conventional moderate continuous training (CON) matched for energy expenditure (n = 17)) impacted 3-HAA levels, related metabolic ratios, and other KP metabolites.nnRESULTS: We demonstrate that the core KP enzymes associated with 3-HAA are evolutionarily conserved in humans. Serum 3-HAA and its precursor anthranilic acid were consistently associated with age in young-to-middle-aged adults. Both exercise modes tested induced an increase in 3-HAA levels of 134% (p < 0.001) and 85% (p < 0.001) compared with baseline, respectively, without a significant time*group interaction effect.nnCONCLUSION: We translate the association between systemic 3-HAA levels and age from animal models into humans and highlight longer-term exercise training as an efficient strategy to boost systemic 3-HAA levels in middle-aged adults. Our findings open promising research avenues concerning the mediating role of 3-HAA in training adaptations, health, and longevity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40356307,

title = {Plasma levels of immune system activation markers Neopterin and Kynurenine-to-Tryptophan Ratio, and oral health among community-dwelling adults in Norway: a population-based, cohort study},

author = {Sarala Banjara and Ellen Berggreen and Jannicke Igland and Anne-Kristine Åstrøm and Øivind Midttun and Dagmar Bunæs and Gerhard Sulo},

doi = {10.2340/aos.v84.43535},

issn = {1502-3850},

year  = {2025},

date = {2025-05-01},

journal = {Acta Odontol Scand},

volume = {84},

pages = {218--225},

abstract = {OBJECTIVE: Periodontitis is a condition characterised by inflammation. Neopterin and kynurenine-to-tryptophan ratio (KTR) are markers of immune system activation in response to inflammation whose elevated levels are linked to higher incidence and poorer prognosis of various systemic diseases. Their potential association with oral health remains underexplored. The aim of this study was to prospectively investigate the associations between these biomarkers and periodontal health status among community-dwelling adults in Hordaland County, Norway.nnMATERIALS AND METHODS: Neopterin and KTR were measured in 1,298 participants of the Hordaland Health Study, 1997-1999. Information on oral health indicators was obtained from the 'Hordaland-Oral Health Survey', 2020-2022. Ordinal logistic regression and negative binomial regression were used to explore the association between biomarkers and periodontitis, tooth loss, and current inflammation (extend of sites with pocket depth ≥4mm and bleeding on probing) and odds ratios (OR) and incidence rate ratios (IRR), along with respective 95% confidence intervals (CI) were reported.nnRESULTS: No association was found between biomarker levels and periodontitis [neopterin: OR = 0.96, 95% CI: 0.69-1.33 for fourth (Q4) vs. first quartile (Q1); KTR: OR = 0.85, 95% CI: 0.61-1.18 for Q4 vs. Q1], tooth loss [neopterin: IRR = 1.00, 95% CI: 0.94-1.06 for Q4 vs. Q1; KTR: IRR = 0.97, 95% CI: 0.91-1.03 for Q4 vs. Q1) or extend of inflammation [neopterin: OR = 0.87, 95% CI: 0.70-1.09 for Q4 vs. Q1; KTR: OR = 0.98, 95% CI: 0.78-1.23 for Q4 vs. Q1].nnCONCLUSION: Plasma levels of neopterin and KTR were not prospectively associated with periodontal health and number of missing teeth.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39961686,

title = {Metabolic profiles and malnutrition in hospitalized adults: A metabolomic cohort study},

author = {Marte A Trollebø and Randi J Tangvik and Eli Skeie and Ottar Nygård and Tomas M L Eagan and Adrian McCann and Jutta Dierkes},

doi = {10.1002/jpen.2728},

issn = {1941-2444},

year  = {2025},

date = {2025-04-01},

journal = {JPEN J Parenter Enteral Nutr},

volume = {49},

number = {3},

pages = {365--372},

abstract = {BACKGROUND: Malnutrition or risk of malnutrition is present in about one-third of patients admitted to Western hospitals and is identified by either screening for malnutrition or further nutrition assessment. To date, there are no commonly accepted biomarkers of malnutrition, which could expedite screening efforts, ease diagnosis, and hasten treatment. We aimed to investigate whether metabolomics could identify markers associated with malnutrition in hospitalized patients and performed a retrospective metabolomic cohort study in this patients' group.nnMETHODS: The study population included adult patients hospitalized in a medical unit. Malnutrition was identified by the second step of the Global Leadership Initiative on Malnutrition criteria independently of the outcome of the screening step (nutritional risk screening 2002). Amino acids were determined by targeted metabolomics using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry. Logistic regression analyses with Benjamini-Hochberg procedure to reduce false discovery rate were used to identify biomarkers associated with malnutrition.nnRESULTS: In total, 218 patients were included in the final analysis, with 62 patients having a diagnosis of malnutrition. In crude analyses, 11 metabolites were associated with malnutrition, but further adjustment attenuated the associations. After multiple adjustment, neopterin and cystatin C were positively associated with malnutrition, whereas His, Cys, and kynurenine to tryptophan ratio were negatively associated.nnCONCLUSION: The observed associations require confirmation in a replication cohort before they can be recommended as biomarkers of malnutrition.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40058591,

title = {Inflammatory markers after supplementation with marine n-3 or plant n-6 PUFAs: A randomized double-blind crossover study},

author = {Elise Grytten and Johnny Laupsa-Borge and Kaya Cetin and Pavol Bohov and Jan Erik Nordrehaug and Jon Skorve and Rolf K Berge and Elin Strand and Bodil Bjørndal and Ottar K Nygård and Espen Rostrup and Gunnar Mellgren and Simon N Dankel},

doi = {10.1016/j.jlr.2025.100770},

issn = {1539-7262},

year  = {2025},

date = {2025-04-01},

journal = {J Lipid Res},

volume = {66},

number = {4},

pages = {100770},

abstract = {Omega-3 (n-3) (e.g., EPA/DHA) and omega-6 (n-6) (e.g., linoleic acid [LA]) FAs are suggested to have opposite effects on inflammation, but results are inconsistent and direct comparisons of n-3 and n-6 are lacking. In a double-blind, randomized, and crossover study, females (n = 16) and males (n = 23) aged 30-70 years with abdominal obesity were supplemented with 3-4 g/d EPA/DHA (fish oil) or 15-20 g/d LA (safflower oil) for 7 weeks, with a 9-week washout phase. Cytokines and chemokines (multiplex assay), acute-phase proteins (MALDI-TOF mass spectrometry), endothelial function (vascular reaction index), blood pressure, FA composition (red blood cell membranes/serum/adipose tissue, GC-MS/MS), and adipose gene expression (microarrays, quantitative PCR) were measured. While significant differences between treatments in relative change scores were found for systolic blood pressure (n-3 vs. n-6: -1.81% vs. 2.61%, P = 0.003), no differences between n-3 and n-6 were found for any circulatory inflammatory markers. However, compared with baseline, n-3 was followed by reductions in circulating TNF (-24.9%, P < 0.001), regulated upon activation, normal T cell expressed and secreted (-12.1%, P < 0.001), and macrophage inflammatory protein 1-beta (-12.5%, P = 0.014), and n-6 by lowered TNF (-18.8%, P < 0.001), regulated upon activation, normal T cell expressed and secreted (-7.37%, P = 0.027), monocyte chemoattractant protein-1 (-7.81%, P = 0.020), and macrophage inflammatory protein 1-beta (-14.2%, P = 0.010). Adipose tissue showed significant treatment differences in weight percent of EPA (n-3 vs. n-6: 50.2%∗ vs. -1.38%, P < 0.001, ∗: significant within-treatment change score), DHA (16.0%∗ vs. -3.67%, P < 0.001), and LA (-0.033 vs. 4.91%∗, P < 0.001). Adipose transcriptomics revealed overall downregulation of genes related to inflammatory processes after n-3 and upregulation after n-6, partly correlating with changes in circulatory markers. These data point to tissue-specific proinflammatory effects of high n-6 intake, but a net systemic anti-inflammatory effect as for n-3.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40287426,

title = {Parkinson's disease is characterized by vitamin B6-dependent inflammatory kynurenine pathway dysfunction},

author = {Edward N Wilson and Jacob Umans and Michelle S Swarovski and Paras S Minhas and Justin H Mendiola and Øivind Midttun and Arve Ulvik and Marian Shahid-Besanti and Patricia Linortner and Siddhita D Mhatre and Qian Wang and Divya Channappa and Nicole K Corso and Lu Tian and Carolyn A Fredericks and Geoffrey A Kerchner and Edward D Plowey and Brenna Cholerton and Per M Ueland and Cyrus P Zabetian and Nora E Gray and Joseph F Quinn and Thomas J Montine and Sharon J Sha and Frank M Longo and David A Wolk and Alice Chen-Plotkin and Victor W Henderson and Tony Wyss-Coray and Anthony D Wagner and Elizabeth C Mormino and Nima Aghaeepour and Kathleen L Poston and Katrin I Andreasson},

doi = {10.1038/s41531-025-00964-7},

issn = {2373-8057},

year  = {2025},

date = {2025-04-01},

journal = {NPJ Parkinsons Dis},

volume = {11},

number = {1},

pages = {96},

abstract = {Recent studies demonstrate that Parkinson's disease (PD) is associated with dysregulated metabolic flux through the kynurenine pathway (KP), in which tryptophan is converted to kynurenine (KYN), and KYN is subsequently metabolized to neuroactive compounds quinolinic acid (QA) and kynurenic acid (KA). Here, we used mass-spectrometry to compare blood and cerebral spinal fluid (CSF) KP metabolites between 158 unimpaired older adults and 177 participants with PD. We found increased neuroexcitatory QA/KA ratio in both plasma and CSF of PD participants associated with peripheral and cerebral inflammation and vitamin B deficiency. Furthermore, increased QA tracked with CSF tau, CSF soluble TREM2 (sTREM2) and severity of both motor and non-motor PD clinical symptoms. Finally, PD patient subgroups with distinct KP profiles displayed distinct PD clinical features. These data validate the KP as a site of brain and periphery crosstalk, integrating B-vitamin status, inflammation and metabolism to ultimately influence PD clinical manifestation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39549999,

title = {The l-Arginine pathway may act as a mediator in the association between impaired one-carbon metabolism and hypertension},

author = {Carla Ramos-Rodríguez and Alejandra Rojas-Gomez and Luis A Santos-Calderón and Santiago Ceruelo and Lídia Ríos and Per M Ueland and Joan D Fernandez-Ballart and Albert Salas-Huetos and Michelle M Murphy},

doi = {10.1016/j.biochi.2024.11.006},

issn = {1638-6183},

year  = {2025},

date = {2025-03-01},

journal = {Biochimie},

volume = {230},

pages = {86--94},

abstract = {Elevated fasting plasma total homocysteine (tHcy) and the methylenetetrahydrofolate reductase C677T polymorphism (rs1801133) have been associated with hypertension. Whether the l-Arginine pathway is involved, is unclear. We aimed to investigate whether the association between tHcy, the rs1801133 polymorphism and hypertension involves the l-Arginine pathway. THcy, plasma folate and cobalamin, erythrocyte glutathionine reductase activation coefficient, rs1801133 genotype, plasma l-Arginine, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were determined in a cross-sectional study of 788 adults (aged 18 to 75), randomly selected from 2 town population registers. Participants participated in a medical checkup and provided a fasting blood sample. Associations between tHcy, rs1801133 genotype and l-Arginine pathway metabolites were assessed by multiple linear regression analysis and whether the tHcy and rs1801133 genotype are associated with hypertension via the l-Arginine pathway was investigated using mediation analysis. tHcy was positively associated with ADMA (B = 0.003; SE = 0.001; P < 0.001) and SDMA (B = 0.007; SE = 0.002; P < 0.001) and negatively associated with the l-Arginine/ADMA (B = -1.140; SE = 0.451; P < 0.05) and ADMA/SDMA (B = -0.006; SE = 0.003; P < 0.05) ratios. The MTHFR 677 CT vs CC genotype was negatively associated with ADMA (B = -0.013; SE = 0.007; P < 0.05) and with SDMA (B = -0.029; SE = 0.013; P < 0.05) in participants under 50 years. Each standard deviation increase (37.6) in the l-Arginine/ADMA ratio was associated with reduced hypertension risk (OR [95%CI], 0.6 [0.5, 0.8]). Mediation analysis showed that tHcy and ADMA were mediators in the association between the rs1801133 TT vs CC genotypes and hypertension. Our results support the l-Arginine pathway as a mediator in the association of impaired One-Carbon metabolism and hypertension.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39906357,

title = {Elevated plasma trimethyllysine is associated with incident atrial fibrillation},

author = {Mads M Svenningsson and Gard Ft Svingen and Per M Ueland and Gerhard Sulo and Espen Ø Bjørnestad and Eva R Pedersen and Indu Dhar and Dennis W Nilsen and Ottar Nygård},

doi = {10.1016/j.ajpc.2025.100932},

issn = {2666-6677},

year  = {2025},

date = {2025-03-01},

journal = {Am J Prev Cardiol},

volume = {21},

pages = {100932},

abstract = {BACKGROUND/AIM: Trimethyllysine (TML) is a methylated amino acid, which is linked to epigenetic regulation and can serve as a precursor of trimethylamine-N-oxide (TMAO). TMAO is a microbiota-derived metabolite and a potential risk factor of cardiovascular disease. TML has recently been linked to atherosclerosis, acute myocardial infarction and prevalent atrial fibrillation (AF). However, any association between circulating TML and incident AF has not yet been reported and was the aim of the current study in a large community based cohort.nnMETHODS: Information regarding AF was obtained by linking patient data to national health registries. Risk associations were explored by logistic regression. Potential improvements in risk reclassification were calculated by the continuous net reclassification index (NRI˃0) and the Receiver Operating Curve Area Under the Curve (ROC-AUC).nnRESULTS: At baseline 3117 patients were included. During a median (25th-75th percentile) follow-up of 10.8 (9.4 - 11.2) years, 492 patients (15.8 %) developed AF. Higher plasma TML was associated with incident AF per 1 SD log-transformed TML (OR (95 % CI) 1.30 (1.16-1.46)  < 0.01). Further analyses also showed an increase in NRI>0 (95 % CI) of 0.24 (0.14-0.33)  < 0.001 and ROC-AUC (95 % CI) of 0.013 (0.004-0.022)  = 0.006.nnCONCLUSION: TML was associated with, and improved risk classification of, new-onset AF in this large cohort of community dwelling adults. Our results motivate further studies on the association between TML and cardiac arrhythmias.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39308420,

title = {Circulating tryptophan-kynurenine pathway metabolites are associated with all-cause mortality among patients with stage I-III colorectal cancer},

author = {Victoria Damerell and Niels Klaassen-Dekker and Stefanie Brezina and Jennifer Ose and Arve Ulvik and Eline H van Roekel and Andreana N Holowatyj and Andreas Baierl and Jürgen Böhm and Martijn J L Bours and Hermann Brenner and Johannes H W de Wilt and William M Grady and Nina Habermann and Michael Hoffmeister and Pekka Keski-Rahkonen and Tengda Lin and Peter Schirmacher and Petra Schrotz-King and Alexis B Ulrich and Fränzel J B van Duijnhoven and Christy A Warby and David Shibata and Adetunji T Toriola and Jane C Figueiredo and Erin M Siegel and Christopher I Li and Andrea Gsur and Ellen Kampman and Martin Schneider and Per M Ueland and Matty P Weijenberg and Cornelia M Ulrich and Dieuwertje E Kok and Biljana Gigic and },

doi = {10.1002/ijc.35183},

issn = {1097-0215},

year  = {2025},

date = {2025-02-01},

journal = {Int J Cancer},

volume = {156},

number = {3},

pages = {552--565},

abstract = {Alterations within the tryptophan-kynurenine metabolic pathway have been linked to the etiology of colorectal cancer (CRC), but the relevance of this pathway for prognostic outcomes in CRC patients needs further elucidation. Therefore, we investigated associations between circulating concentrations of tryptophan-kynurenine pathway metabolites and all-cause mortality among CRC patients. This study utilizes data from 2102 stage I-III CRC patients participating in six prospective cohorts involved in the international FOCUS Consortium. Preoperative circulating concentrations of tryptophan, kynurenine, kynurenic acid (KA), 3-hydroxykynurenine (HK), xanthurenic acid (XA), 3-hydroxyanthranilic acid (HAA), anthranilic acid (AA), picolinic acid (PA), and quinolinic acid (QA) were measured by liquid chromatography-tandem mass spectrometry. Using Cox proportional hazards regression, we examined associations of above-mentioned metabolites with all-cause mortality, adjusted for potential confounders. During a median follow-up of 3.2 years (interquartile range: 2.2-4.9), 290 patients (13.8%) deceased. Higher blood concentrations of tryptophan, XA, and PA were associated with a lower risk of all-cause mortality (per doubling in concentrations: tryptophan: HR = 0.56; 95%CI:0.41,0.76, XA: HR = 0.74; 95%CI:0.64,0.85, PA: HR = 0.76; 95%CI:0.64,0.92), while higher concentrations of HK and QA were associated with an increased risk of death (per doubling in concentrations: HK: HR = 1.80; 95%CI:1.47,2.21, QA: HR = 1.31; 95%CI:1.05,1.63). A higher kynurenine-to-tryptophan ratio, a marker of cell-mediated immune activation, was associated with an increased risk of death (per doubling: HR = 2.07; 95%CI:1.52,2.83). In conclusion, tryptophan-kynurenine pathway metabolites may be prognostic markers of survival in CRC patients.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39764583,

title = {Mitochondrial dysfunction and lipid alterations in primary sclerosing cholangitis},

author = {Guri Fossdal and Peder Braadland and Johannes Roksund Hov and Eystein Sverre Husebye and Trine Folseraas and Per Magne Ueland and Arve Ulvik and Tom Hemming Karlsen and Rolf Kristian Berge and Mette Vesterhus},

doi = {10.1080/00365521.2024.2447521},

issn = {1502-7708},

year  = {2025},

date = {2025-02-01},

journal = {Scand J Gastroenterol},

volume = {60},

number = {2},

pages = {165--173},

abstract = {OBJECTIVES: Indications of mitochondrial dysfunction are commonly seen in liver diseases, but data are scarce in primary sclerosing cholangitis (PSC). Analyzing circulating and liver-resident molecules indirectly reflecting mitochondrial dysfunction, we aimed to comprehensively characterize this deficit in PSC, and whether this was PSC specific or associated with cholestasis.nnMATERIALS AND METHODS: We retrospectively included plasma from 191 non-transplant patients with large-duct PSC and 100 healthy controls and explanted liver tissue extracts from 24 PSC patients and 18 non-cholestatic liver disease controls. Using mass spectroscopy, we profiled lipids and fatty acids, carnitine, acylcarnitines, and metabolites in the tryptophan-kynurenine-nicotinamide pathway.nnRESULTS: Hierarchal clustering of fatty acid levels identified patients with PSC and healthy controls as separate clusters. Compared to healthy controls, PSC patients had increased levels of monounsaturated fatty acids (MUFA) and palmitate (C16:0) in plasma, but reduced levels of long-chain saturated fatty acids (SFAs). These findings were more pronounced in PSC patients with cholestasis. Several n-3 polyunsaturated fatty acids were elevated in PSC but not associated with cholestasis. Acylcarnitine ratios C2/C5 and C2/C3 were elevated while C2/C16 was reduced in PSC, indicating impaired mitochondrial fatty acid oxidation of medium-long chained fatty acids. Levels of intermediates in the tryptophan-kynurenine pathway indicated impaired NAD biosynthesis, suggesting impaired energy supply to mitochondria in PSC.nnCONCLUSIONS: We found that mitochondrial dysfunction was prominent in PSC and associated with increasing cholestasis. Whether this is merely a marker of liver disease and severity, or an underlying driver and potential therapeutic target in PSC remains to be explored.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39500416,

title = {Acute exercise boosts NAD metabolism of human peripheral blood mononuclear cells},

author = {David Walzik and Niklas Joisten and Alexander Schenk and Sina Trebing and Kirill Schaaf and Alan J Metcalfe and Polyxeni Spiliopoulou and Johanna Hiefner and Adrian McCann and Carsten Watzl and Per Magne Ueland and Sebastian Gehlert and Anna Worthmann and Charles Brenner and Philipp Zimmer},

doi = {10.1016/j.bbi.2024.11.004},

issn = {1090-2139},

year  = {2025},

date = {2025-01-01},

journal = {Brain Behav Immun},

volume = {123},

pages = {1011--1023},

abstract = {Nicotinamide adenine dinucleotide (NAD) coenzymes are the central electron carriers in biological energy metabolism. Low NAD levels are proposed as a hallmark of ageing and several diseases, which has given rise to therapeutic strategies that aim to tackle these conditions by boosting NAD levels. As a lifestyle factor with preventive and therapeutic effects, exercise increases NAD levels across various tissues, but so far human trials are mostly focused on skeletal muscle. Given that immune cells are mobilized and redistributed in response to acute exercise, we conducted two complementary trials to test the hypothesis that a single exercise session alters NAD metabolism of peripheral blood mononuclear cells (PBMCs). In a randomized crossover trial (DRKS00017686) with 24 young adults (12 female) we show that acute exercise increases gene expression and protein abundance of several key NAD metabolism enzymes with high conformity between high-intensity interval training (HIIT) and moderate-intensity continuous training (MICT). In a longitudinal exercise trial (DRKS00029105) with 12 young adults (6 female) we confirm these results and reveal that - similar to skeletal muscle - NAD salvage is pivotal for PBMCs in response to exercise. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD salvage pathway, displayed a pronounced increase in gene expression during exercise, which was accompanied by elevated intracellular NAD levels and reduced serum levels of the NAD precursor nicotinamide. These results demonstrate that acute exercise triggers NAD biosynthesis of human PBMCs with potential implications for immunometabolism, immune effector function, and immunological exercise adaptions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid40889707,

title = {Alterations in one-carbon metabolism in metabolic dysfunction associated steatotic liver disease may be modified by semaglutide},

author = {Mikkel Parsberg Werge and Adrian McCann and Elias Badal Rashu and Shi Min Lam and Liv Eline Hetland and Mira Thing and Puria Nabilou and Anders Ellekaer Junker and Jenny Norlin and Sanne Veidal and Dorte Holst and Anne Bugge and Birgitte Martine Viuff and Henning Hvid and Kristian M Bendtsen and Gianluca Mazzoni and Lea Moerch Harder and Mogens Vyberg and Reza Serizawa and Flemming Bendtsen and Per Magne Ueland and Elisabeth Douglas Galsgaard and Nicolai J Wewer Albrechtsen and Lise Lotte Gluud},

doi = {10.1016/j.aohep.2025.102107},

issn = {1665-2681},

year  = {2025},

date = {2025-01-01},

journal = {Ann Hepatol},

volume = {30},

number = {2},

pages = {102107},

abstract = {INTRODUCTION AND OBJECTIVES: Disruptions in one-carbon metabolism (OCM) have been linked to cardiometabolic diseases. We evaluated alterations in OCM metabolites and enzymes and the impact of semaglutide in MASLD.nnMATERIALS AND METHODS: Using targeted metabolomics and bulk-transcriptomics, we analyzed components of OCM in plasma samples and liver biopsies from MASLD patients (n = 100 with F0-F4 fibrosis, 51 % type 2 diabetes) and healthy controls (n = 50). Untargeted metabolomics and transcriptomics were used to analyze plasma and liver specimens from mice with diet-induced obesity and steatohepatitis (DIO-MASH) treated with vehicle or semaglutide compared with chow for 12 weeks.nnRESULTS: In patients with MASLD vs healthy controls, changes in the concentrations of folate and pyridoxal 5'-phosphate (vitamin B6), betaine, serine, and glycine suggested alteration to the activity of the transsulfuration pathway as well as the methionine and folate cycles. Increased concentrations of metabolites related to enhanced homocysteine conversion and elevated glutamate were also observed. These findings were supported by corresponding alterations in the activity of key enzymes. Similar patterns were identified in comparisons of patients with significant vs no/mild fibrosis and in DIO-MASH mice vs chow. Treatment with semaglutide led to reductions in body weight, hepatic inflammation, and fibrosis and largely reversed the metabolic and gene expression changes observed in DIO-MASH mice.nnCONCLUSIONS: MASLD may be linked to alterations in key metabolites and enzymes involved in OCM. The changes in humans were replicated in a MASLD mouse model, and reversal was found after treatment with semaglutide.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid41140571,

title = {Prevalent low Mediterranean diet adherence and low folate status in a Spanish Km 0 Mediterranean coast population},

author = {Ailende Eigbefoh-Addeh and Albert Salas-Huetos and Carla Ramos-Rodríguez and Santiago Ceruelo and Lídia Ríos and Per M Ueland and Klaus Meyer and Joan D Fernandez-Ballart and Michelle M Murphy},

doi = {10.1016/j.crfs.2025.101217},

issn = {2665-9271},

year  = {2025},

date = {2025-01-01},

journal = {Curr Res Food Sci},

volume = {11},

pages = {101217},

abstract = {BACKGROUND: Folate, vitamin B, riboflavin, and vitamin B, play interrelated roles in one-carbon metabolism, vital for several physiological processes.nnOBJECTIVE: To assess intake and status of these B-vitamins in an adult Mediterranean population unexposed to mandatory food fortification or B-vitamin supplementation, and to examine the associations with Mediterranean diet adherence (MDA).nnMETHODS: A representative sample of adults aged 18-77 (n = 740) provided fasting blood samples and 3-day dietary records. MDA was assessed using the Trichopoulou score. Plasma folate, red blood cell folate (RBCF) and plasma vitamin B were measured via microbiological assays. Riboflavin and vitamin B status were determined by erythrocyte glutathionine reductase (EGRAC) and aspartate aminotransferase activation coefficients (EASTAC), respectively.nnRESULTS: Intake below average requirement was observed in 39.5 % (for folate), 6.5 % (for vitamin B), 20.1 % (for riboflavin), and 19.6 % (for vitamin B) of the participants. Plasma folate <7 nmol/L, plasma B < 148 pmol/L, RBCF <340 nmol/L, EGRAC ≥1.4, and EASTAC ≥1.6 occurred in 18.6 %, 1.6 %, 1.5 %, 35.9 %, and 56.0 % of participants, respectively. Overall, 82.64 % of participants had low MDA (score <6). Participants in the highest MDA quartile had higher folate intake (354.5 vs. 214.9 μg/day;  < 0.001), plasma folate (14.5 vs. 10.0 nmol/L;  < 0.001), and RBCF (895.7 vs. 692.8 nmol/L;  < 0.001) compared to those in the lowest quartile (score ≤2). Nevertheless, 15.5 % of high-MDA individuals did not meet the folate average requirements, and 8.5 % were folate deficient.nnCONCLUSIONS: High vs low MDA was associated with better folate intake and status but this was not true for the other vitamins. Only 17.4 % of the population had high MDA and folate deficiency still occurred in this group. A fortification policy may be required, to prevent folate deficiency in the population.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39326699,

title = {Folate and cobalamin status, indicators, modulators, interactions, and reference ranges from early pregnancy until birth: the Reus-Tarragona birth cohort study},

author = {Luis A Santos-Calderón and Pere Cavallé-Busquets and Carla Ramos-Rodríguez and Carme Grifoll and Alejandra Rojas-Gómez and Mónica Ballesteros and Per M Ueland and Michelle M Murphy},

doi = {10.1016/j.ajcnut.2024.09.015},

issn = {1938-3207},

year  = {2024},

date = {2024-11-01},

journal = {Am J Clin Nutr},

volume = {120},

number = {5},

pages = {1269--1283},

abstract = {BACKGROUND: Folate and cobalamin status, although essential for pregnancy, are not routinely monitored in prenatal care.nnOBJECTIVES: To investigate folate and cobalamin status and determinants throughout pregnancy, in the absence of mandatory folic acid (FA) fortification.nnMETHODS: In a cohort study of 831 mothers recruited at <12 gestational weeks (GW), plasma folate, total homocysteine (tHcy), cobalamin, holotranscobalamin (holoTC), methylmalonic acid (MMA), red blood cell folate (RBCF), and the combined cobalamin status indicator (cB12) were determined at ≤12, 15, 24-27, 34 GW, labor and in the cord. Single nucleotide polymorphisms affecting folate and cobalamin status were determined. FA, cobalamin, micronutrient supplement use, and dietary folate and cobalamin intake (food frequency questionnaire) were recorded. Folate and cobalamin status predictors were assessed by multiple linear regression analysis.nnRESULTS: Only 36.1% of the participants took FA preconceptionally and 47.4% and 7.3% had suboptimal RBCF (<906 nmol/L) and plasma cobalamin status (≤221 pmol/L), respectively, at ≤12 GW. RBCF determinants included planned pregnancy, FA supplementation, plasma cobalamin, and methylenetetrahydrofolate (MTHFR) 677C>T genotype. Cobalamin supplementation was positively associated with plasma cobalamin and early holoTC. Smoking and BMI were inversely associated with plasma cobalamin and early holoTC, but none were associated with MMA. Only participants with the MTHFR 677TT genotype, exceeding FA supplement recommendations, improved their folate status (interaction term: B (95% CI):0.15 (0.01, 0.29), P = 0.032). Smoking was inversely associated with plasma cobalamin status in participants with the methionine synthase reductase (MTRR) 524CC genotype only (interaction term:0.07 (0.01, 0.04), P = 0.014). Mothers with low early pregnancy cobalamin status and also those with bigger newborns, had lower cobalamin status at labor.nnCONCLUSIONS: Suboptimal early pregnancy folate or cobalamin status affected 47.4% and 7.3% of the participants, respectively. The MTHFR 677TT genotype predicted folate status throughout pregnancy. Smoking and BMI were negatively associated with cobalamin status throughout pregnancy. Clinical Trial Registry number and website where it was obtained: NCT01778205. www.nnCLINICALTRIALS: gov.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39422147,

title = {Serum concentrations of lipids, ketones and acylcarnitines during the postprandial and fasting state: the Postprandial Metabolism (PoMet) study in healthy young adults},

author = {Åslaug Matre Anfinsen and Vilde Haugen Myklebust and Christina Osland Johannesen and Jacob Juel Christensen and Johnny Laupsa-Borge and Jutta Dierkes and Ottar Nygård and Adrian McCann and Hanne Rosendahl-Riise and Vegard Lysne},

doi = {10.1017/S0007114524001934},

issn = {1475-2662},

year  = {2024},

date = {2024-10-01},

journal = {Br J Nutr},

volume = {132},

number = {7},

pages = {851--861},

abstract = {To improve the interpretation and utilisation of blood lipids, ketones and acylcarnitine concentrations as biomarkers in clinical assessments, more information is needed on their dynamic alterations in response to dietary intake and fasting. The aim of this intervention study was to characterise the changes in serum lipid, ketone and acylcarnitine concentrations 24 h after a standardised breakfast meal. Thirty-four healthy subjects (eighteen males and sixteen females) aged 20-30 years were served a breakfast meal (∼500 kcal, 36 E% fat, 46 E% carbohydrates, 16 E% protein, 2E% fibre), after which they consumed only water for 24 h. Blood samples were drawn before and at thirteen standardised timepoints after the meal. Metabolite concentrations were plotted as a function of time since the completion of the breakfast meal. Results demonstrated that concentrations of HDL-cholesterol and LDL-cholesterol decreased until ∼2 h (-4 % for both), while TAG concentrations peaked at 3 h (+27 %). Acetoacetate and -hydroxybutyrate were highest 24 h after the meal (+433 and +633 %, respectively). Acetylcarnitine, butyrylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine and dodecanoylcarnitine reached the lowest values at 60 min (decreases ranging from -47 to -70 %), before increasing and peaking at 24 h after the meal (increases ranging from +86 to +120 %). Our findings suggest that distinguishing between fasting and non-fasting blood samples falls short of capturing the dynamics in lipid, ketone, carnitine and acylcarnitine concentrations. To enhance the utility of serum acylcarnitine analyses, we strongly recommend accounting for the specific time since the last meal at the time of blood sampling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38783597,

title = {Longitudinal associations of plasma kynurenines and ratios with fatigue and quality of life in colorectal cancer survivors up to 12 months post-treatment},

author = {Daniëlle D B Holthuijsen and Eline H van Roekel and Martijn J L Bours and Per M Ueland and Stéphanie O Breukink and Maryska L G Janssen-Heijnen and Eric T P Keulen and Stefanie Brezina and Biljana Gigic and Anita R Peoples and Cornelia M Ulrich and Arve Ulvik and Matty P Weijenberg and Simone J P M Eussen},

doi = {10.1002/ijc.34992},

issn = {1097-0215},

year  = {2024},

date = {2024-10-01},

journal = {Int J Cancer},

volume = {155},

number = {7},

pages = {1172--1190},

abstract = {Fatigue is prevalent in colorectal cancer (CRC) survivors, impacting their health-related quality of life (HRQoL). Inflammation-induced activation of the kynurenine pathway may play a role in cancer-related fatigue and HRQoL, but evidence is scarce. Therefore, we aimed to investigate longitudinal associations of plasma tryptophan, kynurenines, and ratios with fatigue and HRQoL in CRC survivors up to 12 months post-treatment. Repeated measurements at 6 weeks, 6 months, and 12 months post-treatment were performed in 249 stage I-III CRC survivors. Plasma tryptophan and eight kynurenines were analyzed using liquid chromatography-tandem mass spectrometry (LC/MS-MS). Fatigue and HRQoL outcomes were evaluated using validated questionnaires. Confounder-adjusted linear mixed models were conducted to analyze longitudinal associations, with false discovery rate (FDR) correction. Higher tryptophan (Trp), kynurenic acid (KA), and xanthurenic acid (XA) concentrations, as well as a higher kynurenic acid-to-quinolinic acid ratio (KA/QA), were associated with less fatigue and better functioning, while a higher kynurenine-to-tryptophan ratio (KTR) and 3-hydroxykynurenine ratio (HKr) were associated with more fatigue and worse functioning. Finally, higher KA and XA concentrations and a higher KA/QA ratio were associated with a higher overall HRQoL summary score, while a higher HKr was associated with a lower overall HRQoL summary score. In conclusion, we observed that tryptophan and several kynurenines were longitudinally associated with fatigue and HRQoL in CRC survivors up to 12 months post-treatment. Future research is needed to validate our findings and explore the potential of the kynurenine pathway as intervention target for reducing fatigue and enhancing HRQoL after CRC treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38783413,

title = {Maternal serum unmetabolized folic acid concentration following multivitamin and mineral supplementation with or without folic acid after 12 weeks gestation: A randomized controlled trial},

author = {Dian C Sulistyoningrum and Thomas R Sullivan and Monika Skubisz and Debra J Palmer and Simon Wood and Per Magne Ueland and Adrian McCann and Maria Makrides and Timothy J Green and Karen P Best},

doi = {10.1111/mcn.13668},

issn = {1740-8709},

year  = {2024},

date = {2024-10-01},

journal = {Matern Child Nutr},

volume = {20},

number = {4},

pages = {e13668},

abstract = {Pregnant women are advised to take folic acid (FA) supplements before conception and during the first trimester of pregnancy. Many women continue FA supplementation throughout pregnancy, and concerns have been raised about associations between excessive FA intake and adverse maternal and child health outcomes. Unmetabolized folic acid (UMFA) is found in serum after high FA intakes and is proposed as a biomarker for excessive FA intake. We aimed to determine if removing FA from prenatal micronutrient supplements after 12 weeks of pregnancy reduces serum UMFA concentrations at 36 weeks gestation. In this double-blind, randomized controlled trial conducted in South Australia, 103 women with a singleton pregnancy were randomly assigned at 12-16 weeks gestation to take a micronutrient supplement containing no FA or 800 µg/day FA from enrollment until 36 weeks gestation. Ninety women (0 µg/day FA n = 46; 800 µg/day FA n = 44) completed the study. Mean, UMFA concentration was lower in the women randomized to the 0 µg/day group compared to the 800 µg/day FA group, 0.6 ± 0.7 and 1.4 ± 2.7 nmol/L, respectively. The adjusted mean difference (95% CI) in UMFA between the groups was [-0.85 (-1.62, -0.08) nmol/L, p = 0.03]. Maternal serum and red blood cell folate concentrations were lower in the 0 µg/day FA group than in the 800 µg/day group (median 23.2 vs. 49.3 and 1335 vs. 1914 nmol/L, respectively; p < 0.001). Removing FA at 12-16 weeks gestation from prenatal micronutrient supplements reduced the concentration of UMFA at 36 weeks gestation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid39399688,

title = {Parkinson's disease is characterized by vitamin B6-dependent inflammatory kynurenine pathway dysfunction},

author = {Edward Wilson and Jacob Umans and Michelle Swarovski and Paras Minhas and Øivind Midttun and Arve Arve Ulvik and Marian Shahid-Besanti and Patricia Linortner and Siddhita Mhatre and Qian Wang and Divya Channappa and Nicole Corso and Lu Tian and Carolyn Fredericks and Geoffrey Kerchner and Edward Plowey and Brenna Cholerton and Per Ueland and Cyrus Zabetian and Nora Gray and Joseph Quinn and Thomas Montine and Sharon Sha and Frank Longo and David Wolk and Alice Chen-Plotkin and Victor Henderson and Tony Wyss-Coray and Anthony Wagner and Elizabeth Mormino and Nima Aghaeepour and Kathleen Poston and Katrin Andreasson},

doi = {10.21203/rs.3.rs-4980210/v1},

issn = {2693-5015},

year  = {2024},

date = {2024-09-01},

journal = {Res Sq},

abstract = {Parkinson's disease (PD) is a complex multisystem disorder clinically characterized by motor, non-motor, and premotor manifestations. Pathologically, PD involves neuronal loss in the substantia nigra, striatal dopamine deficiency, and accumulation of intracellular inclusions containing aggregates of α-synuclein. Recent studies demonstrate that PD is associated with dysregulated metabolic flux through the kynurenine pathway (KP), in which tryptophan is converted to kynurenine (KYN), and KYN is subsequently metabolized to neuroactive compounds quinolinic acid (QA) and kynurenic acid (KA). This multicenter study used highly sensitive liquid chromatography-tandem mass-spectrometry to compare blood and cerebral spinal fluid (CSF) KP metabolites between 158 unimpaired older adults and 177 participants with PD. Results indicate that increased neuroexcitatory QA/KA ratio in both plasma and CSF of PD participants associated with peripheral and cerebral inflammation and vitamin B6 deficiency. Furthermore, increased QA tracked with CSF tau and severity of both motor and non-motor PD clinical dysfunction. Importantly, plasma and CSF kynurenine metabolites classified PD participants with a high degree of accuracy (AUC = 0.897). Finally, analysis of metabolite data revealed subgroups with distinct KP profiles, and these were subsequently found to display distinct PD clinical features. Together, these data further support the hypothesis that the KP serves as a site of brain and periphery crosstalk, integrating B-vitamin status, inflammation and metabolism to ultimately influence PD clinical manifestation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid38749786,

title = {Influence of cardiorespiratory fitness on obesity-associated inflammation in women and men: The FATCOR study},

author = {Helga Midtbø and Arve Ulvik and Dana Cramariuc and Klaus Meyer and Per M Ueland and Hilde Halland and Eva Gerdts},

doi = {10.1016/j.numecd.2024.04.002},

issn = {1590-3729},

year  = {2024},

date = {2024-08-01},

journal = {Nutr Metab Cardiovasc Dis},

volume = {34},

number = {8},

pages = {1942--1949},

abstract = {BACKGROUND AND AIMS: Cardiorespiratory fitness has been postulated to lower chronic inflammation in obesity. We assessed sex-specific associations of inflammation with cardiorespiratory fitness in overweight and obese persons.nnMETHODS AND RESULTS: Peak oxygen uptake (VO) was measured by treadmill in 566 participants (age 48 ± 9 years, 60% women) with body mass index >27.0 kg/m in the FAT associated CardiOvasculaR dysfunction (FATCOR) study. Fitness was identified from age- and sex specific reference levels of VO. The inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), kynurenine:tryptophan ratio (KTR) and pyriodoxic acid ratio (PAr) were measured by mass spectrometry. In the total study population 63% had obesity and 74% were cardiorespiratory unfit. Unfit women had the highest fat percentage and the highest serum levels of CRP and SAA (p < 0.05). In multivariable linear regression analyses in women, higher CRP (β -0.15, p = 0.001), SAA (β -0.10, p = 0.03) and PAr (β -0.09, p = 0.03) were associated with lower VO after adjusting for confounders. In multivariable analyses in men, higher PAr (β -0.14, p = 0.02) was associated with lower VO. In multivariable analyses in obese women, higher CRP and PAr remained associated with lower VO (p < 0.05), while in obese men there was no significant association. When normalizing VO for fat-free mass (VO) higher CRP, SAA and PAr index were associated with lower VO in women, while only higher PAr index was associated with lower VO in men.nnCONCLUSION: The association of inflammation with lower cardiorespiratory fitness was more pronounced in women than men, in particular when obesity was present.nnCLINICAL TRIAL REGISTRATION: URL: http://www.nnCLINICALTRIALS: gov NCT02805478.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}