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Eva Skillgate,1,2 Oscar Javier Pico-Espinosa,1 Johan Hallqvist,3 Tony Bohman,1 Lena W Holm4 1Unit of Cardiovascular Epidemiology, Musculoskeletal and Sports Injury Epidemiology Center, Institute of Environmental Medicine, Karolinska Institutet, 2Naprapathögskolan - Scandinavian College of Naprapathic Manual Medicine, Stockholm, 3Department of Public Health and Caring Sciences, Uppsala University, Uppsala, 4Unit of Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Background: The role of healthy lifestyle behavior (HLB) in terms of physical activity, alcohol intake, smoking, and diet put together has not yet been explored for the risk of low back pain (LBP) and neck pain (NP). Our aim was to study if an HLB is protective against the onset of long duration troublesome LBP and NP in men and women. Methods: Two cohorts from the Stockholm Public Health Cohort, free from LBP (n=12,483) and NP (n=10,539), respectively, in 2006, were surveyed with questionnaires. Baseline information about physical activity, alcohol intake, diet, and smoking were dichotomized into being healthy/not healthy and combined in a categorical variable according to the number of healthy behaviors present. Binomial regression analyses were used to evaluate the role of HLB for the outcomes 4 years later.Results: When men with three or four healthy lifestyles were compared to men with none or one, the risk ratio (RR) of LBP was 0.63 (95% confidence interval [CI]: 0.39–1.02). The corresponding RR for LBP in women was 0.86 (95% CI: 0.56–1.32). When men with three or four healthy lifestyles were compared to men with none or one, the RR for NP was 1.13 (95% CI: 0.74–1.71). The corresponding RR for NP in women was 0.52 (95% CI: 0.35–0.77). Conclusion: An HLB seems to be protective for long duration troublesome LBP in men, and for long duration troublesome NP in women. Keywords: neck pain, low back pain, lifestyle, physical activity, smoking, alcohol, healthy diet, prevention, musculoskeletal

Abstract Background Neck pain is very common but the occurrence of bothersome neck pain is not well described. Therefore our objective was to report on the prevalence and incidence of, as well as the rate of recovery from, bothersome neck pain in men and women of different ages in the general population. Methods We used data from a recently conducted population-based cohort study, comprising 23,794 individuals in Stockholm County, Sweden. Study participants were surveyed with a self-administered questionnaire in 2002/2003 and 2007, and information on episodes of neck pain was gathered at baseline and at follow-up. We then measured bothersome neck pain in 2005 and 2006 retrospectively in 2007 using the follow-up questionnaire. Results The one-year prevalence of bothersome neck pain for at least seven consecutive days was 25% (95% confidence interval (CI): 24–25) among women and 16% (95% CI: 15–16) among men, peaking in individuals aged 30–59 years. The one-year incidence proportion of bothersome neck pain was 7% (95% CI: 6–7) among women, and 4% (95% CI: 4–5) among men. Women recovered more infrequently than men. The one-year incidence proportion of recovery (of at least one year duration) was 11% (95% CI: 10–12) among women and 14% (95% CI: 12–16) among men. Conclusion Bothersome neck pain is most common in middle-aged individuals. Women are more likely than men to have and to develop bothersome neck pain, and less likely to recover from such pain. Younger men and women have a higher incidence, but recover more often from bothersome neck pain than older individuals.

Abstract Background Type 2 diabetes is associated with low socioeconomic position (SEP) in high-income countries. Despite the important role of SEP in the development of many diseases, no socioeconomic indicator was included in the Comparative Risk Assessment (CRA) module of the Global Burden of Disease study. We therefore aimed to illustrate an example by estimating the burden of type 2 diabetes in Sweden attributed to lower educational levels as a measure of SEP using the methods applied in the CRA. Methods To include lower educational levels as a risk factor for type 2 diabetes, we pooled relevant international data from a recent systematic review to measure the association between type 2 diabetes incidence and lower educational levels. We also collected data on the distribution of educational levels in the Swedish population using comparable criteria for educational levels as identified in the international literature. Population attributable fractions (PAF) were estimated and applied to the burden of diabetes estimates from the Swedish burden of disease database for men and women in the separate age groups (30-44, 45-59, 60-69, 70-79, and 80+ years). Results The PAF estimates showed that 17.2% of the diabetes burden in men and 20.1% of the burden in women were attributed to lower educational levels in Sweden when combining all age groups. The burden was, however, most pronounced in the older age groups (70-79 and 80+), where lower educational levels contributed to 22.5% to 24.5% of the diabetes burden in men and 27.8% to 32.6% in women. Conclusions There is a considerable burden of type 2 diabetes attributed to lower educational levels in Sweden, and socioeconomic indicators should be considered to be incorporated in the CRA.

Abstract Background Although illness is an important cause of sick leave, it has also been suggested that non-medical risk factors may influence this association. If such factors impact on the period of decision making, they should be considered as triggers. Yet, there is no empirical support available. The aim was to investigate whether recent exposure to work-related psychosocial events can trigger the decision to report sick when ill. Methods A case-crossover design was applied to 546 sick-leave spells, extracted from a Swedish cohort of 1 430 employees with a 3-12 month follow-up of new sick-leave spells. Exposure in a case period corresponding to an induction period of one or two days was compared with exposure during control periods sampled from workdays during a two-week period prior to sick leave for the same individual. This was done according to the matched-pair interval and the usual frequency approaches. Results are presented as odds ratios (OR) with 95% confidence intervals (CI). Results Most sick-leave spells happened in relation to acute, minor illnesses that substantially reduced work ability. The risk of taking sick leave was increased when individuals had recently been exposed to problems in their relationship with a superior (OR 3.63; CI 1.44-9.14) or colleagues (OR 4.68; CI 1.43-15.29). Individuals were also more inclined to report sick on days when they expected a very stressful work situation than on a day when they were not under such stress (OR 2.27; CI 1.40-3.70). Conclusions Exposure to problems in workplace relationships or a stressful work situation seems to be able to trigger reporting sick. Psychosocial work-environmental factors appear to have a short-term effect on individuals when deciding to report sick.

Abstract Background Sudden emotions may interfere with mechanisms for keeping balance among the elderly. The aim of this study is to analyse if emotional stress and specifically feelings of anger, sadness, worries, anxiety or stress, can trigger falls leading to hip or pelvic fracture among autonomous older people. Methods The study applied the case-crossover design and was based on data gathered by face to face interviews carried out in Stockholm between November 2004 and January 2006 at the emergency wards of two hospitals. Cases (n = 137) were defined as persons aged 65 and older admitted for at least one night due to a fall-related hip or pelvic fracture (ICD10: S72 or S32) and meeting a series of selection criteria. Results are presented as relative risks with 95% confidence intervals. Results There was an increased risk for fall and subsequent hip or pelvic fracture for up to one hour after emotional stress. For anger there was an increased relative risk of 12.2 (95% CI 2.7–54.7), for sadness of 5.7 (95% CI 1.1–28.7), and for stress 20.6 (95% CI 4.5–93.5) compared to periods with no such feelings. Conclusion Emotional stress seems to have the potential to trigger falls and subsequent hip or pelvic fracture among autonomous older people. Further studies are needed to clarify how robust the findings are – as the number of exposed cases is small – and the mechanisms behind them – presumably balance and vision impairment in stress situation.

Abstract Background In general men tend to drink more alcohol and experience more alcohol-related sickness, injuries and mortality than women. In this paper, the overall hypothesis was that increased gender similarity in the division of parental duties would lead to convergence in alcohol-related harm. The aim was to analyse whether the risk of alcohol harm differs between parents who fit a gender-stereotypical versus those with a less gender-stereotypical division of childcare and paid work. Methods The study sample was a retrospective registry-based cohort study of all Swedish couples who had their first child together in 1978 (N = 49,120). A less gender-stereotypical parenthood was indicated by paternity leave for fathers (1978–1979) and full-time work for mothers (1980). The outcome was inpatient care and/or death caused by alcohol psychosis, alcoholism, liver disease, or alcohol intoxication in the two decades following (1981–2001). Our main statistical method was multivariate logistic regression with odds ratios used to estimate relative risks. Results The main results show that fathers who took paternity leave had 18% lower risk of alcohol-related care and/or death than other fathers. Mothers who worked full-time about two years after having a child had 71% higher risk than mothers who were unemployed or worked part-time. Conclusion A less gender-stereotypical division of duties between parents in early parenthood may contribute to a long-term decreased gender disparity regarding risky alcohol consumption and alcohol-related harm. In order to know more about the causal direction however, future research has to consider subjects' drinking patterns in the years prior to parenthood.

Abstract Background High body mass index (BMI) and lack of physical activity have been recognized as important risk factors for coronary heart disease. The aim of the present study was to evaluate whether leisure-time physical activity compensates for the increased risk of acute myocardial infarction associated with overweight and obesity. Methods Data from the SHEEP (Stockholm Heart Epidemiology Program) study were used. The SHEEP study is a large Swedish population-based case-control study, comprising 1204 male and 550 female cases, and 1538 male and 777 female controls, conducted in Stockholm County, Sweden, during the period 1992–1994. Odds ratios (OR), together with 95 % confidence intervals (95% CI), were calculated using unconditional logistic regression, as estimates of the relative risks. Results Regular leisure-time physical activity was associated with a decreased risk of myocardial infarction among lean, normal-weight and overweight subjects, but not among obese subjects. Obese (BMI ≥ 30) and physically active persons had an almost twofold risk of myocardial infarction, compared with normal-weight and sedentary persons (OR 1.85, 95% CI 1.07–3.18). The results were similar for men and women. Conclusion While regular leisure-time physical activity seems to provide protection against myocardial infarction among lean, normal-weight and overweight subjects, this does not appear to be the case in obese subjects.

Parkinson's disease is increasingly prevalent. It progresses from the pre-motor stage (characterised by non-motor symptoms like REM sleep behaviour disorder), to the disabling motor stage. We need objective biomarkers for early/pre-motor disease stages to be able to intervene and slow the underlying neurodegenerative process. Here, we validate a targeted multiplexed mass spectrometry assay for blood samples from recently diagnosed motor Parkinson's patients (n = 99), pre-motor individuals with isolated REM sleep behaviour disorder (two cohorts: n = 18 and n = 54 longitudinally), and healthy controls (n = 36). Our machine-learning model accurately identifies all Parkinson patients and classifies 79% of the pre-motor individuals up to 7 years before motor onset by analysing the expression of eight proteins-Granulin precursor, Mannan-binding-lectin-serine-peptidase-2, Endoplasmatic-reticulum-chaperone-BiP, Prostaglaindin-H2-D-isomaerase, Interceullular-adhesion-molecule-1, Complement C3, Dickkopf-WNT-signalling pathway-inhibitor-3, and Plasma-protease-C1-inhibitor. Many of these biomarkers correlate with symptom severity. This specific blood panel indicates molecular events in early stages and could help identify at-risk participants for clinical trials aimed at slowing/preventing motor Parkinson's disease., (© 2024. The Author(s).)

Objectives: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies., Methods: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR ( E gene) were compared to RT-LAMP time-to-positive (TTP) ( NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein., Results: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R 2 0.57, p-value 0.002)., Conclusions: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

As disease-modifying therapies are now available for Alzheimer's disease (AD), accessible, accurate and affordable biomarkers to support diagnosis are urgently needed. We sought to develop a mass spectrometry-based urine test as a high-throughput screening tool for diagnosing AD. We collected urine from a discovery cohort (n = 11) of well-characterised individuals with AD (n = 6) and their asymptomatic, CSF biomarker-negative study partners (n = 5) and used untargeted proteomics for biomarker discovery. Protein biomarkers identified were taken forward to develop a high-throughput, multiplexed and targeted proteomic assay which was tested on an independent cohort (n = 21). The panel of proteins identified are known to be involved in AD pathogenesis. In comparing AD and controls, a panel of proteins including MIEN1, TNFB, VCAM1, REG1B and ABCA7 had a classification accuracy of 86%. These proteins have been previously implicated in AD pathogenesis. This suggests that urine-targeted mass spectrometry has potential utility as a diagnostic screening tool in AD.

Dried blood spots (DBSs) biomarkers are convenient for monitoring for specific lysosomal storage diseases (LSDs), but they could have relevance for other LSDs. To determine the specificity and utility of glycosphingolipidoses biomarkers against other LSDs, we applied a multiplexed lipid liquid chromatography tandem mass spectrometry assay to a DBS cohort of healthy controls ( n = 10) and Gaucher ( n = 4), Fabry ( n = 10), Pompe ( n = 2), mucopolysaccharidosis types I-VI ( n = 52), and Niemann-Pick disease type C (NPC) ( n = 5) patients. We observed no complete disease specificity for any of the markers tested. However, comparison among the different LSDs highlighted new applications and perspectives of the existing biomarkers. We observed elevations in glucosylceramide isoforms in the NPC and Gaucher patients relative to the controls. In NPC, there was a greater proportion of C24 isoforms, giving a specificity of 96-97% for NPC, higher than 92% for the NPC biomarker N-palmitoyl-O-phosphocholineserine ratio to lyso-sphingomyelin. We also observed significantly elevated levels of lyso-dihexosylceramide in Gaucher and Fabry disease as well as elevated lyso-globotriaosylceramide (Lyso-Gb3) in Gaucher disease and the neuronopathic forms of Mucopolysaccharidoses. In conclusion, DBS glucosylceramide isoform profiling has increased the specificity for the detection of NPC, thereby improving diagnostic accuracy. Low levels of lyso-lipids can be observed in other LSDs, which may have implications in their disease pathogenesis.

Objectives: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity., Methods: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study., Results: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95., Conclusions: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Integration of multiple datasets can greatly enhance bioanalytical studies, for example, by increasing power to discover and validate biomarkers. In liquid chromatography-mass spectrometry (LC-MS) metabolomics, it is especially hard to combine untargeted datasets since the majority of metabolomic features are not annotated and thus cannot be matched by chemical identity. Typically, the information available for each feature is retention time (RT), mass-to-charge ratio ( m / z ), and feature intensity (FI). Pairs of features from the same metabolite in separate datasets can exhibit small but significant differences, making matching very challenging. Current methods to address this issue are too simple or rely on assumptions that cannot be met in all cases. We present a method to find feature correspondence between two similar LC-MS metabolomics experiments or batches using only the features' RT, m / z , and FI. We demonstrate the method on both real and synthetic datasets, using six orthogonal validation strategies to gauge the matching quality. In our main example, 4953 features were uniquely matched, of which 585 (96.8%) of 604 manually annotated features were correct. In a second example, 2324 features could be uniquely matched, with 79 (90.8%) out of 87 annotated features correctly matched. Most of the missed annotated matches are between features that behave very differently from modeled inter-dataset shifts of RT, MZ, and FI. In a third example with simulated data with 4755 features per dataset, 99.6% of the matches were correct. Finally, the results of matching three other dataset pairs using our method are compared with a published alternative method, metabCombiner, showing the advantages of our approach. The method can be applied using M2S (Match 2 Sets), a free, open-source MATLAB toolbox, available at https://github.com/rjdossan/M2S.

Advanced age is the major risk factor for idiopathic Parkinson's disease (PD), but to date the biological relationship between PD and ageing remains elusive. Here we describe the rationale and the design of the H2020 funded project "PROPAG-AGEING", whose aim is to characterize the contribution of the ageing process to PD development. We summarize current evidences that support the existence of a continuum between ageing and PD and justify the use of a Geroscience approach to study PD. We focus in particular on the role of inflammaging, the chronic, low-grade inflammation characteristic of elderly physiology, which can propagate and transmit both locally and systemically. We then describe PROPAG-AGEING design, which is based on the multi-omic characterization of peripheral samples from clinically characterized drug-naïve and advanced PD, PD discordant twins, healthy controls and "super-controls", i.e. centenarians, who never showed clinical signs of motor disability, and their offspring. Omic results are then validated in a large number of samples, including in vitro models of dopaminergic neurons and healthy siblings of PD patients, who are at higher risk of developing PD, with the final aim of identifying the molecular perturbations that can deviate the trajectories of healthy ageing towards PD development., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

'Long Covid', or medical complications associated with post SARS-CoV-2 infection, is a significant post-viral complication that is being more and more commonly reported in patients. Therefore, there is an increasing need to understand the disease mechanisms, identify drug targets and inflammatory processes associated with a SARS-CoV-2 infection. To address this need, we created a targeted mass spectrometry based multiplexed panel of 96 immune response associated proteins. We applied the multiplex assay to a cohort of serum samples from asymptomatic and moderately affected patients. All patients had tested positive for a SARS-CoV-2 infection by PCR and were determined to be subsequently positive for antibodies. Even 40-60 days post-viral infection, we observed a significant remaining inflammatory response in all patients. Proteins that were still affected were associated with the anti-inflammatory response and mitochondrial stress. This indicates that biochemical and inflammatory pathways within the body can remain perturbed long after SARS-CoV-2 infections have subsided even in asymptomatic and moderately affected patients., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Doykov I et al.)

Importance: Mutations of the glucocerebrosidase gene, GBA1 (OMIM 606463), are the most important risk factor for Parkinson disease (PD). In vitro and in vivo studies have reported that ambroxol increases β-glucocerebrosidase (GCase) enzyme activity and reduces α-synuclein levels. These observations support a potential role for ambroxol therapy in modifying a relevant pathogenetic pathway in PD., Objective: To assess safety, tolerability, cerebrospinal fluid (CSF) penetration, and target engagement of ambroxol therapy with GCase in patients with PD with and without GBA1 mutations., Interventions: An escalating dose of oral ambroxol to 1.26 g per day., Design, Setting, and Participants: This single-center open-label noncontrolled clinical trial was conducted between January 11, 2017, and April 25, 2018, at the Leonard Wolfson Experimental Neuroscience Centre, a dedicated clinical research facility and part of the University College London Queen Square Institute of Neurology in London, United Kingdom. Participants were recruited from established databases at the Royal Free London Hospital and National Hospital for Neurology and Neurosurgery in London. Twenty-four patients with moderate PD were evaluated for eligibility, and 23 entered the study. Of those, 18 patients completed the study; 1 patient was excluded (failed lumbar puncture), and 4 patients withdrew (predominantly lumbar puncture-related complications). All data analyses were performed from November 1 to December 14, 2018., Main Outcomes and Measures: Primary outcomes at 186 days were the detection of ambroxol in the CSF and a change in CSF GCase activity., Results: Of the 18 participants (15 men [83.3%]; mean [SD] age, 60.2 [9.7] years) who completed the study, 17 (8 with GBA1 mutations and 9 without GBA1 mutations) were included in the primary analysis. Between days 0 and 186, a 156-ng/mL increase in the level of ambroxol in CSF (lower 95% confidence limit, 129 ng/mL; P < .001) was observed. The CSF GCase activity decreased by 19% (0.059 nmol/mL per hour; 95% CI, -0.115 to -0.002; P = .04). The ambroxol therapy was well tolerated, with no serious adverse events. An increase of 50 pg/mL (13%) in the CSF α-synuclein concentration (95% CI, 14-87; P = .01) and an increase of 88 ng/mol (35%) in the CSF GCase protein levels (95% CI, 40-137; P = .002) were observed. Mean (SD) scores on part 3 of the Movement Disorders Society Unified Parkinson Disease Rating Scale decreased (ie, improved) by 6.8 (7.1) points (95% CI, -10.4 to -3.1; P = .001). These changes were observed in patients with and without GBA1 mutations., Conclusions and Relevance: The study results suggest that ambroxol therapy was safe and well tolerated; CSF penetration and target engagement of ambroxol were achieved, and CSF α-synuclein levels were increased. Placebo-controlled clinical trials are needed to examine whether ambroxol therapy is associated with changes in the natural progression of PD., Trial Registration: ClinicalTrials.gov identifier: NCT02941822; EudraCT identifier: 2015-002571-24.

Background: Fabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker for monitoring treatment is plasma globotriaosylsphingosine (lyso-Gb3), but it is not informative about the underlying and developing disease pathology., Methods: We have created a urine proteomic assay containing a panel of biomarkers designed to measure disease-related pathology which include the inflammatory system, lysosome, heart, kidney, endothelium and cardiovascular system. Using a targeted proteomic-based approach, a series of 40 proteins for organ systems affected in Fabry disease were multiplexed into a single 10 min multiple reaction monitoring Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) assay and using only 1 mL of urine., Results: Six urinary proteins were elevated in the early-stage/asymptomatic Fabry group compared with controls including albumin, uromodulin, α1-antitrypsin, glycogen phosphorylase brain form, endothelial protein receptor C and intracellular adhesion molecule 1. Albumin demonstrated an increase in urine and could indicate presymptomatic disease. The only protein elevated in the early-stage/asymptomatic patients that continued to increase with progressive multiorgan involvement was glycogen phosphorylase brain form. Podocalyxin, fibroblast growth factor 23, cubulin and Alpha-1-Microglobulin/Bikunin Precursor (AMBP) were elevated only in disease groups involving kidney disease. Nephrin, a podocyte-specific protein, was elevated in all symptomatic groups. Prosaposin was increased in all symptomatic groups and showed greater specificity (p<0.025-0.0002) according to disease severity., Conclusion: This work indicates that protein biomarkers could be helpful and used in conjunction with plasma lyso-Gb3 for monitoring of therapy or disease progression in patients with Fabry disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The microbial metabolite Trimethylamine-N-oxide (TMAO) has been linked to adverse cardiovascular outcome and mortality in the general population., Objective: To assess the contribution of TMAO to inflammation and mortality in chronic kidney disease (CKD) patients ranging from mild-moderate to end-stage disease and 1) associations with glomerular filtration rate (GFR) 2) effect of dialysis and renal transplantation (Rtx) 3) association with inflammatory biomarkers and 4) its predictive value for all-cause mortality., Methods: Levels of metabolites were quantified by a novel liquid chromatography/tandem mass spectrometry-based method in fasting plasma samples from 80 controls and 179 CKD 3-5 patients. Comorbidities, nutritional status, biomarkers of inflammation and GFR were assessed., Results: GFR was the dominant variable affecting TMAO (β = -0.41; p<0.001), choline (β = -0.38; p<0.001), and betaine (β = 0.45; p<0.001) levels. A longitudinal study of 74 CKD 5 patients starting renal replacement therapy demonstrated that whereas dialysis treatment did not affect TMAO, Rtx reduced levels of TMAO to that of controls (p<0.001). Following Rtx choline and betaine levels continued to increase. In CKD 3-5, TMAO levels were associated with IL-6 (Rho = 0.42; p<0.0001), fibrinogen (Rho = 0.43; p<0.0001) and hsCRP (Rho = 0.17; p = 0.022). Higher TMAO levels were associated with an increased risk for all-cause mortality that remained significant after multivariate adjustment (HR 4.32, 95% CI 1.32-14.2; p = 0.016)., Conclusion: Elevated TMAO levels are strongly associated with degree of renal function in CKD and normalize after renal transplantation. TMAO levels correlates with increased systemic inflammation and is an independent predictor of mortality in CKD 3-5 patients.