Background: Galacto-oligosaccharides (GOS) intake has been linked to health benefits via modulation of the gut microbiome. Milk, where the majority of lactose is enzymatically converted to GOS (called here Novel or “N milk”), retains milk’s nutritional value with reduced lactose and a high amount of prebiotic GOS. Objectives: The aim of this study was to investigate the effect of N milk on the gut microbiome and related changes in health-related biomarkers, complemented by ex vivo fermentation experiments. Methods: In a 2-arm crossover, double-blind, randomized controlled clinical trial, 26 healthy adults consumed either N milk (containing 9 g GOS and 1.7 g of lactose per serving) or lactose-free milk (control), for 2 wk with a 2-wk washout period. Stool and fasting blood samples were collected at the start and the end of the intervention periods. Gut microbiome was analyzed using shotgun metagenomics, and metabolites using both targeted and untargeted methods. In addition, we tested lactose-free milk, N milk, and GOS in ex vivo colonic fermentation to obtain insights into the bacterial processing of substrates. Results: N milk intake led to a 3-fold increase in median gut bifidobacteria (P < 0.0001) and significant increases in plasma acetate, octanoic acid, β-alanine, and nicotinamide (all P < 0.05). Untargeted plasma metabolomics revealed a shift in amino acid metabolism, with an increase in 3-indole propionate, accompanied by a decrease in 2 uremic toxins, p-cresol sulfate, and indoxyl-sulfate (P < 0.05 without false discovery rate adjustment). Ex vivo fermentation experiments supported the results of the clinical study, whereby N milk increased bifidobacteria accompanied by higher production of short-chain fatty acids and a shift in microbial tryptophan metabolism, and indicated unique effects of N milk compared with GOS. Conclusions: N milk resulted in a significant increase in gut bifidobacteria, along with changes in plasma metabolites previously associated with immune and metabolic health benefits.This study was registered at clinicaltrials.gov as NCT05207839.

This study explored the effects of different human milk oligosaccharides (HMOs), solely and in combination, on gut microbiota composition and metabolic activity (organic acid production), using anaerobic in vitro batch culture fermenters. The aim was to compare prebiotic effects of HMOs (2'FL, 3'FL, 3'SL, 6'SL, LNT, LNnT, and 1:1 ratio mixes of 2'FL/3'SL and 3'SL/LNT) in faecal samples from irritable bowel syndrome (IBS) donors and healthy controls, and to determine the best-performing HMO in IBS. Fluorescent in situ hybridisation coupled with flow cytometry was utilised to study microbiota changes in major colonic genera, and organic acid production was assessed by gas chromatography. IBS donors had different starting microbial profiles compared to healthy controls and lower levels of organic acids. In response to HMOs, there were alterations in both the control and IBS faecal microbiomes. In IBS donor fermenters, Bifidobacterium, Faecalibacterium, total bacterial numbers, and organic acid production significantly increased post-HMO intervention. When comparing the effect of HMO interventions on the microbiota and organic acid production, a mix of 3'SL/LNT HMOs may be the most promising intervention for IBS patients.

The global type 2 diabetes epidemic is a major health crisis. Although the microbiome has roles in the onset of insulin resistance (IR), low-grade inflammation and diabetes, the microbial compounds controlling these processes remain to be discovered. Here, we show that the microbial metabolite trimethylamine (TMA) decouples inflammation and IR from diet-induced obesity by inhibiting interleukin-1 receptor-associated kinase 4 (IRAK4), a central kinase in the Toll-like receptor pathway sensing danger signals. TMA blunts TLR4 signalling in primary human hepatocytes and peripheral blood monocytic cells and rescues mouse survival after lipopolysaccharide-induced septic shock. Genetic deletion and chemical inhibition of IRAK4 result in metabolic and immune improvements in high-fat diets. Remarkably, our results suggest that TMA—unlike its liver co-metabolite trimethylamine N-oxide, which is associated with cardiovascular disease—improves immune tone and glycemic control in diet-induced obesity. Altogether, this study supports the emerging role of the kinome in the microbial–mammalian chemical crosstalk. The microbial metabolite trimethylamine (TMA), the precursor of TMAO, which is associated with adverse cardiometabolic outcomes, is shown to have beneficial metabolic and anti-inflammatory effects in the host in the context of obesity.

The rising prevalence of obesity in Saudi Arabia is a major contributor to the nation's high levels of cardiometabolic diseases such as type 2 diabetes. To assess the impact of obesity on the diabetic metabolic phenotype presented in young Saudi Arabian adults, participants (n = 289, aged 18-40 years) were recruited and stratified into four groups: healthy weight (BMI 18.5-24.99 kg/m(2)) with (n = 57) and without diabetes (n = 58) or overweight/obese (BMI > 24.99 kg/m(2)) with (n = 102) and without diabetes (n = 72). Distinct plasma metabolic phenotypes associated with high BMI and diabetes were identified using nuclear magnetic resonance spectroscopy and ultraperformance liquid chromatography mass spectrometry. Increased plasma glucose and dysregulated lipoproteins were characteristics of obesity in individuals with and without diabetes, but the obesity-associated lipoprotein phenotype was partially masked in individuals with diabetes. Although there was little difference between diabetics and nondiabetics in the global plasma LDL cholesterol and phospholipid concentration, the distribution of lipoprotein particles was altered in diabetics with a shift toward denser and more atherogenic LDL5 and LDL6 particles, which was amplified in the presence of obesity. Further investigation is warranted in larger Middle Eastern populations to explore the dysregulation of metabolism driven by interactions between obesity and diabetes in young adults.

The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible.

Cow’s milk protein allergy (CMPA) is a prevalent food allergy among infants and young children. We conducted a randomized, multicenter intervention study involving 194 non-breastfed infants with CMPA until 12 months of age (clinical trial registration: NCT03085134). One exploratory objective was to assess the effects of a whey-based extensively hydrolyzed formula (EHF) supplemented with 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) on the fecal microbiome and metabolome in this population. Thus, fecal samples were collected at baseline, 1 and 3 months from enrollment, as well as at 12 months of age. Human milk oligosaccharides (HMO) supplementation led to the enrichment of bifidobacteria in the gut microbiome and delayed the shift of the microbiome composition toward an adult-like pattern. We identified specific HMO-mediated changes in fecal amino acid degradation and bile acid conjugation, particularly in infants commencing the HMO-supplemented formula before the age of three months. Thus, HMO supplementation partially corrected the dysbiosis commonly observed in infants with CMPA. Further investigation is necessary to determine the clinical significance of these findings in terms of a reduced incidence of respiratory infections and other potential health benefits.

IntroductionBifidobacterium longum subspecies infantis (B. infantis) may play a key role in infant gut development. This trial evaluated safety, tolerability, and efficacy of B. infantis LMG11588 supplementation.MethodsThis randomized, placebo-controlled, double-blind study conducted in the Philippines included healthy breastfed and/or formula-fed infants (14–21 days old) randomized for 8 weeks to a control group (CG; n = 77), or any of two B. infantis experimental groups (EGs): low (Lo-EG; 1*108 CFU/day; n = 75) or high dose (Hi-EG; 1.8*1010 CFU/day; n = 76). Primary endpoint was weight gain; secondary endpoints included stooling patterns, gastrointestinal symptoms, adverse events, fecal microbiome, biomarkers, pH, and organic acids.ResultsNon-inferiority in weight gain was demonstrated for Hi-EG and Lo-EG vs. CG. Overall, probiotic supplementation promoted mushy-soft stools, fewer regurgitation episodes, and increased fecal acetate production, which was more pronounced in the exclusively breastfed infants (EBF) and positively correlated with B. infantis abundance. In EBF, fecal pro-inflammatory cytokines (IL-1 beta, IL-8) were reduced. Strain-level metagenomic analysis allowed attributing the increased abundance of B. infantis in EGs versus CG, to LMG11588 probiotic colonization. Colonization by autochthonous B. infantis strains was similar between groups.DiscussionB. infantis LMG11588 supplementation was associated with normal infant growth, was safe and well-tolerated and promoted a Bifidobacterium-rich microbiota driven by B. infantis LMG11588 colonization without disturbing the natural dispersal of autochthonous B. infantis strains. In EBF, supplementation stimulated microbial metabolic activity and beneficially modulated enteric inflammation.

The gut microbiome has an important role in infant health and development. We characterized the fecal mi-crobiome and metabolome of 222 young children in Dhaka, Bangladesh during the first two years of life. A distinct Bifidobacterium longum clade expanded with introduction of solid foods and harbored enzymes for utilizing both breast milk and solid food substrates. The clade was highly prevalent in Bangladesh, present globally (at lower prevalence), and correlated with many other gut taxa and metabolites, indicating an impor-tant role in gut ecology. We also found that the B. longum clades and associated metabolites were implicated in childhood diarrhea and early growth, including positive associations between growth measures and B. longum subsp. infantis, indolelactate and N-acetylglutamate. Our data demonstrate geographic, cultural, seasonal, and ecological heterogeneity that should be accounted for when identifying microbiome factors implicated in and potentially benefiting infant development.

Fermentation is an ancient food preservation process, and fermented products have been traditionally consumed in different cultures worldwide over the years. The interplay between human gut microbiota, diet and host health is widely recognized. Diet is one of the main factors modulating gut microbiota potentially with beneficial effects on human health. Fermented dairy products have received much attention, but other sources of probiotic delivery through food received far less attention. In this research, a combination of in vitro tools mimicking colonic fermentation and the intestinal epithelium have been applied to study the effect of different pasteurized and non-pasteurized water kefir products on gut microbiota, epithelial barrier function and immunomodulation. Water kefir increased beneficial short-chain fatty acid production at the microbial level, reduced detrimental proteolytic fermentation compounds and increased Bifidobacterium genus abundance. The observed benefits are enhanced by pasteurization. Pasteurized products also had a significant effect at the host level, improving inflammation-induced intestinal epithelial barrier disruption and increasing IL-10 and IL-1β compared to the control condition. Our data support the potential health benefits of water kefir and demonstrate that pasteurization, performed to prolong shelf life and stability of the product, also enhanced these benefits.

The impact of metabolism upon the altered pathology of joint disease is rapidly becoming recognized as an important area of study. Synovial joint fluid is an attractive and representative biofluid of joint disease. A systemic review revealed little evidence of the metabolic stability of synovial joint fluid collection, handling or storage, despite recent reports characterizing the metabolic phenotype in joint disease. We aim to report the changes in small molecule detection within human synovial fluid (HSF) using nuclear magnetic resonance (NMR) spectroscopy at varying storage temperatures, durations and conditions. HSF was harvested by arthrocentesis from patients with isolated monoarthropathy or undergoing joint replacement (n = 30). Short-term storage (0-12h, 4 degrees C & 18 degrees C) and the effect of repeated freeze-thaw cycles (-80 degrees C to 18 degrees C) was assessed. Long-term storage was evaluated by early (-80 degrees C, <21days) and late analysis (-80 degrees C, 10-12 months). 1D NMR spectroscopy experiments, NOESYGPPRID and CPMG identified metabolites and semi-quantification was performed. Samples demonstrated broad stability to freeze-thaw cycling and refrigeration of <4 h. Short-term room temperature or refrigerated storage showed significant variation in 2-ketoisovalerate, valine, dimethylamine, succinate, 2-hydroxybutyrate, and acetaminophen glucuronide. Lipid and macromolecule detection was variable. Long-term storage demonstrated significant changes in: acetate, acetoacetate, creatine, N,N-dimethylglycine, dimethylsulfone, 3-hydroxybutyrate and succinate. Changeable metabolites during short-term storage appeared to be energy-synthesis intermediates. Most metabolites were stable for the first four hours at room temperature or refrigeration, with notable exceptions. We therefore recommend that HSF samples should be kept refrigerated for no more than 4 hours prior to freezing at -80 degrees C. Furthermore, storage of HSF samples for 10-12 months before analysis can affect the detection of selected metabolites. Crown Copyright (C) 2021 Published by Elsevier B.V. All rights reserved.

Aims: The diagnosis of joint infections is an inexact science using combinations of blood inflammatory markers and microscopy, culture, and sensitivity of synovial fluid (SF). There is potential for small molecule metabolites in infected SF to act as infection markers that could improve accuracy and speed of detection. The objective of this study was to use nuclear magnetic resonance (NMR) spectroscopy to identify small molecule differences between infected and noninfected human SF. Methods: In all, 16 SF samples (eight infected native and prosthetic joints plus eight noninfected joints requiring arthroplasty for end-stage osteoarthritis) were collected from patients. NMR spectroscopy was used to analyze the metabolites present in each sample. Principal component analysis and univariate statistical analysis were undertaken to investigate metabolic differences between the two groups. Results: A total of 16 metabolites were found in significantly different concentrations between the groups. Three were in higher relative concentrations (lipids, cholesterol, and N-acetylated molecules) and 13 in lower relative concentrations in the infected group (citrate, glycine, glycosaminoglycans, creatinine, histidine, lysine, formate, glucose, proline, valine, dimethylsulfone, mannose, and glutamine). Conclusion: Metabolites found in significantly greater concentrations in the infected cohort are markers of inflammation and infection. They play a role in lipid metabolism and the inflammatory response. Those found in significantly reduced concentrations were involved in carbohydrate metabolism, nucleoside metabolism, the glutamate metabolic pathway, increased oxidative stress in the diseased state, and reduced articular cartilage breakdown. This is the first study to demonstrate differences in the metabolic profile of infected and noninfected human SF, using a noninfected matched cohort, and may represent putative biomarkers that form the basis of new diagnostic tests for infected SF.

Abstract Background:Implant wear in joint replacements is influenced by the chemical and physical properties of human synovial fluid (HSF). In vitro testing for implant wear uses 25% weight bovine calf serum (25BCS) to substitute for HSF, due to similar rheology and total protein content. However, previous studies have shown differences in the macromolecular composition. We aimed to evaluate the differences in small molecule composition between fluids and correlate these differences with their effects on implant material wear.Methods:HSF was harvested from osteoarthritis patients undergoing primary knee replacement (n=14). Nuclear magnetic resonance (NMR) spectroscopy with linear regression modelling analysed the metabolites present in HSF and commercially sourced 25BCS and investigated the differences. Wear properties of the fluids were evaluated using a validated quantitative laboratory bench-test utilising a cobalt/chromium/molybdenum (CoCrMo) ball oscillating against a CoCrMo disc and analysing the resulting wear scar. The variation in metabolite levels in both HSF and 25BCS was correlated to the wear properties of the fluids.Results:Differences in the levels of metabolites, lipids, and glycosaminoglycans (GAG) were observed between HSF and 25BCS: significance was confirmed by O-PLS-DA (p<0.05). The wear of CoCrMo was found to strongly correlate with the macromolecules GAG and proteins that potentially bind to glucose and citrate. Conclusions:The small molecule concentration differences between the fluids questions the validity of 25BCS as a model for wear analysis. The demonstration of variable metabolites present in HSF which correlate with material wear has implications for implant failure and targeted therapeutic manipulation of these metabolites. Trial Registration:Ethical approval was granted by the NRES Committee London, Chelsea, UK on the 12th May 2015. The study was performed in accordance with the ethical standards in the 1964 Declaration of Helsinki.

Background: The metabolites associated with a Mediterranean-style (AMED) diet may give insight into why AMED is robustly associated with protection against CVD. Previous investigations seeking to identify these have been limited by the use of modest sample sizes and targeted assays. Methods: Using samples from the Multi-Ethnic Study of Atherosclerosis (MESA), we conducted untargeted 1 H NMR spectroscopy (600 MHz) with internal and external annotation on the sera of ~3,698 participants, ages 45-84, who were free from overt CVD. We included data on baseline dietary intake (self-reported), and all incident CVD events (excluding stroke) over a 10-year period. From >100,000 spectral features, 845 significant associations (P<2.2*10 -6 ) were identified via linear regression, and reduced to a putative list of 46 via elastic net regularized models. Hierarchical clustering identified 11 feature groups, from which cluster scores were constructed. The ‘mediate’ package in R used bootstrapping (1000 simulations) to partition the association between AMED and incident CVD into the effects of dietary intake on CVD which are mediated by the metabolomic cluster scores (the “indirect” / mediated effects) and those effects which are not (“direct effects”). All association analyses controlled for age, sex, ethnicity, data collection site and daily caloric intake. Results: AMED score was associated with reductions in the incidence of CVD (HR=0.95; 95% CI: 0.91 - 0.99; P=0.02). All metabolomic cluster scores were associated with AMED (all P<2*10 -06 ; Table 1 ), with 6 significantly associated with incident CVD (all P<2.0*10 -4 ) in the expected direction given their associations with AMED score. Four of these six significantly mediated the association of AMED score with incident CVD (P<0.05; Table 1 ). Conclusions: These preliminary data suggest that specific molecules, if replicated in other studies, hold promise to identify the underlying pathways by which an AMED diet offers protection against CVD.

AimsThe diagnosis of joint infections is an inexact science using combinations of blood inflammatory markers and microscopy, culture, and sensitivity of synovial fluid (SF). There is potential for small molecule metabolites in infected SF to act as infection markers that could improve accuracy and speed of detection. The objective of this study was to use nuclear magnetic resonance (NMR) spectroscopy to identify small molecule differences between infected and noninfected human SF.MethodsIn all, 16 SF samples (eight infected native and prosthetic joints plus eight noninfected joints requiring arthroplasty for end-stage osteoarthritis) were collected from patients. NMR spectroscopy was used to analyze the metabolites present in each sample. Principal component analysis and univariate statistical analysis were undertaken to investigate metabolic differences between the two groups.ResultsA total of 16 metabolites were found in significantly different concentrations between the groups. Three were in higher relative concentrations (lipids, cholesterol, and N-acetylated molecules) and 13 in lower relative concentrations in the infected group (citrate, glycine, glycosaminoglycans, creatinine, histidine, lysine, formate, glucose, proline, valine, dimethylsulfone, mannose, and glutamine).ConclusionMetabolites found in significantly greater concentrations in the infected cohort are markers of inflammation and infection. They play a role in lipid metabolism and the inflammatory response. Those found in significantly reduced concentrations were involved in carbohydrate metabolism, nucleoside metabolism, the glutamate metabolic pathway, increased oxidative stress in the diseased state, and reduced articular cartilage breakdown. This is the first study to demonstrate differences in the metabolic profile of infected and noninfected human SF, using a noninfected matched cohort, and may represent putative biomarkers that form the basis of new diagnostic tests for infected SF. Cite this article: Bone Joint Res 2021;10(1):85–95.

Metabolic profiling of biological samples provides important insights into multiple physiological and pathological processes but is hindered by a lack of automated annotation and standardized methods for structure elucidation of candidate disease biomarkers. Here we describe a system for identifying molecular species derived from nuclear magnetic resonance (NMR) spectroscopy-based metabolic phenotyping studies, with detailed information on sample preparation, data acquisition and data modeling. We provide eight different modular workflows to be followed in a recommended sequential order according to their level of difficulty. This multi-platform system involves the use of statistical spectroscopic tools such as Statistical Total Correlation Spectroscopy (STOCSY), Subset Optimization by Reference Matching (STORM) and Resolution-Enhanced (RED)-STORM to identify other signals in the NMR spectra relating to the same molecule. It also uses two-dimensional NMR spectroscopic analysis, separation and pre-concentration techniques, multiple hyphenated analytical platforms and data extraction from existing databases. The complete system, using all eight workflows, would take up to a month, as it includes multi-dimensional NMR experiments that require prolonged experiment times. However, easier identification cases using fewer steps would take 2 or 3 days. This approach to biomarker discovery is efficient and cost-effective and offers increased chemical space coverage of the metabolome, resulting in faster and more accurate assignment of NMR-generated biomarkers arising from metabolic phenotyping studies. It requires a basic understanding of MATLAB to use the statistical spectroscopic tools and analytical skills to perform solid phase extraction (SPE), liquid chromatography (LC) fraction collection, LC-NMR-mass spectroscopy and one-dimensional and two-dimensional NMR experiments.

Background Identifying associations between serum metabolites and visceral adipose tissue ( VAT ) could provide novel biomarkers of VAT and insights into the pathogenesis of obesity‐related diseases. We aimed to discover and replicate metabolites reflecting pathways related to VAT . Methods and Results Associations between fasting serum metabolites and VAT area (by computed tomography or magnetic resonance imaging) were assessed with cross‐sectional linear regression of individual‐level data from participants in MESA (Multi‐Ethnic Study of Atherosclerosis; discovery, N=1103) and the NEO (Netherlands Epidemiology of Obesity) study (replication, N=2537). Untargeted 1 H nuclear magnetic resonance metabolomics profiling of serum was performed in MESA, and metabolites were replicated in the NEO study using targeted 1 H nuclear magnetic resonance spectroscopy. A total of 30 590 metabolomic spectral variables were evaluated. After adjustment for age, sex, race/ethnicity, socioeconomic status, smoking, physical activity, glucose/lipid‐lowering medication, and body mass index, 2104 variables representing 24 nonlipid and 49 lipid/lipoprotein subclass metabolites remained significantly associated with VAT ( P =4.88×10 −20 –1.16×10 −3 ). These included conventional metabolites, amino acids, acetylglycoproteins, intermediates of glucose and hepatic metabolism, organic acids, and subclasses of apolipoproteins, cholesterol, phospholipids, and triglycerides. Metabolites mapped to 31 biochemical pathways, including amino acid substrate use/metabolism and glycolysis/gluconeogenesis. In the replication cohort, acetylglycoproteins, branched‐chain amino acids, lactate, glutamine (inversely), and atherogenic lipids remained associated with VAT ( P =1.90×10 −35 –8.46×10 −7 ), with most associations remaining after additional adjustment for surrogates of VAT (glucose level, waist circumference, and serum triglycerides), reflecting novel independent associations. Conclusions We identified and replicated a metabolite panel associated with VAT in 2 community‐based cohorts. These findings persisted after adjustment for body mass index and appear to define a metabolic signature of visceral adiposity.

Ioanna Tzoulaki *, Raphaële Castagné, Claire L. Boulangé, Ibrahim Karaman , Elena Chekmeneva, Evangelos Evangelou, Timothy M.D. Ebbels, Manuja R. Kaluarachchi, Marc Chadeau-Hyam, David Mosen, Abbas Dehghan, Alireza Moayyeri, Diana L. Santos Ferreira, Xiuqing Guo, Jerome I. Rotter, Kent D. Taylor, Maryam Kavousi, Paul S. de Vries, Benjamin Lehne, Marie Loh, Albert Hofman, Jeremy K. Nicholson, John Chambers, Christian Gieger, Elaine Holmes,

Objective: To perform a systematic review of the small molecule metabolism studies of osteoarthritis utilising nuclear magnetic resonance (NMR) or mass spectroscopy (MS) analysis (viz., metabolomics or metabonomics), thereby providing coherent conclusions and reference material for future study. Method: We applied PRISMA guidelines (PROSPERO 95068) with the following MESH terms: 1. "osteoarthritis" AND ("metabolic" OR "metabonomic" OR "metabolomic" OR "metabolism") 2. (" synovial fluid" OR "cartilage" OR "synovium" OR "serum" OR "plasma" OR "urine") AND ("NMR" or " Mass Spectroscopy"). Databases searched were "Medline" and "Embase". Studies were searched in English and excluded review articles not containing original research. Study outcomes were significant or notable metabolites, species (human or animal) and the NewcastleeOttawa Score. Results: In the 27 studies meeting the inclusion criteria, there was a shift towards anaerobic and fatty acid metabolism in OA disease, although whether this represents the inflammatory state remains unclear. Lipid structure and composition was altered within disease subclasses including phosphatidyl choline (PC) and the sphingomyelins. Macromolecular proteoglycan destruction was described, but the correlation to disease factors was not demonstrated. Collated results suggested arachidonate signalling pathways and androgen sex hormones as future metabolic pathways for investigation. Conclusion: Our meta-analysis demonstrates significant small molecule differences between sample types, between species (such as human and bovine), with potential OA biomarkers and targets for local or systemic therapies. Studies were limited by numbers and a lack of disease correlation. Future studies should use NMR and MS analysis to further investigate large population subgroups including inflammatory arthropathy, OA subclasses, age and joint differences. Crown Copyright (c) 2018 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. All rights reserved.

Nephrotic syndrome with idiopathic membranous nephropathy as a major contributor, is characterized by proteinuria, hypoalbuminemia and oedema. Diagnosis is based on renal biopsy and the condition is treated using immunosuppressive drugs; however nephrotic syndrome treatment efficacy varies among patients. Multi-omic urine analyses can discover new markers of nephrotic syndrome that can be used to develop personalized treatments. For proteomics, a protease inhibitor (PI) is sometimes added at sample collection to conserve proteins but its impact on urine metabolic phenotyping needs to be evaluated. Urine from controls (n = 4) and idiopathic membranous nephropathy (iMN) patients (n = 6) were collected with and without PI addition and analysed using 1H NMR spectroscopy and UPLC-MS. PI-related data features were observed in the 1H NMR spectra but their removal followed by a median fold change normalisation, eliminated the PI contribution. PI-related metabolites in UPLC-MS data had limited effect on metabolic patterns specific to iMN. When using an appropriate data processing pipeline, PI-containing urine samples are appropriate for 1H NMR and MS metabolic profiling of patients with nephrotic syndrome.

IMPORTANCE Chinese women have the highest rate of lung cancer among female never-smokers in the world, and the etiology is poorly understood. OBJECTIVE To assess the association between metabolomics and lung cancer risk among neversmoking women. DESIGN, SETTING, AND PARTICIPANTS This nested case-control study included 275 neversmoking female patients with lung cancer and 289 never-smoking cancer-free control participants from the prospective ShanghaiWomen's Health Study recruited from December 28, 1996, to May 23, 2000. Validated food frequency questionnaires were used for the collection of dietary information. Metabolomic analysis was conducted from November 13, 2015, to January 6, 2016. Data analysis was conducted from January 6, 2016, to November 29, 2018. EXPOSURES Untargeted ultra-high-performance liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance metabolomic profiles were characterized using prediagnosis urine samples. A total of 39 416metabolites were measured. MAIN OUTCOMES AND MEASURES Incident lung cancer. RESULTS Among the 564 women, those who developed lung cancer (275 participants; median [interquartile range] age, 61.0 [52-65] years) and those who did not develop lung cancer (289 participants; median [interquartile range] age, 62.0 [53-66] years) at follow-up (median [interquartile range] follow-up, 10.9 [9.0-11.7] years) were similar in terms of their secondhand smoke exposure, history of respiratory diseases, and body mass index. A peak metabolite, identified as 5-methyl-2-furoic acid, was significantly associated with lower lung cancer risk (odds ratio, 0.57 [95% CI, 0.46-0.72]; P <.001; false discovery rate = 0.039). Furthermore, this peakwasweakly correlated with self-reported dietary soy intake (. = 0.21; P <.001). Increasing tertiles of this metabolite were associated with lower lung cancer risk (in comparison with first tertile, odds ratio for second tertile, 0.52 [95% CI, 0.34-0.80]; and odds ratio for third tertile, 0.46 [95% CI, 0.30-0.70]), and the association was consistent across different histological subtypes and follow-up times. Additionally, metabolic pathway analysis found several systemic biological alterations that were associated with lung cancer risk, including 1-carbon metabolism, nucleotide metabolism, oxidative stress, and inflammation. CONCLUSIONS AND RELEVANCE This prospective study of the untargeted urinarymetabolome and lung cancer among never-smoking women in China provides support for the hypothesis that soy-based metabolites are associated with lower lung cancer risk in never-smoking women and suggests that biological processes linked to air pollution may be associated with higher lung cancer risk in this population.