Sunde, R. B., Thorsen, J., Kim, M., Schoos, A. M., Stokholm, J., Bonnelykke, K., Bisgaard, H. and Chawes, B. (2024) Bacterial colonisation of the airway in neonates and risk of asthma and allergy until age 18 years

BACKGROUND: We previously showed an association between neonatal bacterial airway colonisation and increased risk of persistent wheeze/asthma until age 5 years. Here, we study the association with persistent wheeze/asthma and allergy-related traits until age 18 years. METHODS: We investigated the association between airway colonisation with Streptococcus pneumoniae, Moraxella catarrhalis and/or Haemophilus influenzae in 1-month-old neonates from the COPSAC(2000) mother-child cohort and the development of persistent wheeze/asthma and allergy-related traits longitudinally until age 18 years using generalised estimating equations. Replication was sought in the similarly designed COPSAC(2010) cohort of 700 children. RESULTS: Neonatal airway colonisation was present in 66 (21%) out of 319 children and was associated with a 4-fold increased risk of persistent wheeze/asthma (adjusted OR 4.01 (95% CI 1.76-9.12); p<0.001) until age 7 years, but not from age 7 to 18 years. Replication in the COPSAC(2010) cohort showed similar results using 16S data. Colonisation was associated with an increased number of exacerbations (adjusted incidence rate ratio 3.20 (95% CI 1.38-7.44); p<0.01) until age 7 years, but not from age 7 to 18 years. Colonisation was associated with increased levels of blood eosinophils (adjusted geometric mean ratio 1.24 (95% CI 1.06-1.44); p<0.01) and tumour necrosis factor (TNF)-alpha (adjusted geometric mean ratio 1.09 (95% CI 1.02-1.16); p=0.01) until age 12 years. There were no associations with lung function, bronchial reactivity, fractional exhaled nitric oxide, allergic sensitisation, total IgE or atopic dermatitis up to age 18 years. CONCLUSIONS: Neonatal airway colonisation was associated with early-onset persistent wheeze/asthma, exacerbations, elevated blood eosinophils and elevated TNF-alpha in blood, most prominent in early childhood, thereafter diminishing and no longer evident by age 18 years.

auzour, D., Scholey, A., White, D. J., Cohen, N. J., Cassidy, A., Gillings, R., Irvine, M. A., Kay, C. D., Kim, M., King, R., Legido-Quigley, C., Potter, J. F., Schwarb, H. and Minihane, A. M. (2023) A combined DHA-rich fish oil and cocoa flavanols intervention does not improve cognition or brain structure in older adults with memory complaints: results from the CANN randomized, controlled parallel-design study

BACKGROUND: There is evidence that both omega-3 long-chain polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) and cocoa flavanols can improve cognitive performance in both healthy individuals and in those with memory complaints. However, their combined effect is unknown. OBJECTIVES: To investigate the combined effect of EPA/DHA and cocoa flavanols (OM3FLAV) on cognitive performance and brain structures in older adults with memory complaints. METHODS: A randomized placebo-controlled trial of DHA-rich fish oil (providing 1.1 g/d DHA and 0.4 g/d EPA) and a flavanol-rich dark chocolate (providing 500 mg/d flavan-3-ols) was conducted in 259 older adults with either subjective cognitive impairment or mild cognitive impairment. Participants underwent assessment at baseline, 3 mo, and 12 mo. The primary outcome was the number of false-positives on a picture recognition task from the Cognitive Drug Research computerized assessment battery. Secondary outcomes included other cognition and mood outcomes, plasma lipids, brain-derived neurotrophic factor (BDNF), and glucose levels. A subset of 110 participants underwent structural neuroimaging at baseline and at 12 mo. RESULTS: 197 participants completed the study. The combined intervention had no significant effect on any cognitive outcomes, with the exception of reaction time variability (P = 0.007), alertness (P < 0.001), and executive function (P < 0.001), with a decline in function observed in the OM3FLAV group (118.6 [SD 25.3] at baseline versus 113.3 [SD 25.4] at 12 mo for executive function) relative to the control, and an associated decrease in cortical volume (P = 0.039). Compared with the control group, OM3FLAV increased plasma HDL, total cholesterol ratio (P < 0.001), and glucose (P = 0.008) and reduced TG concentrations (P < 0.001) by 3 mo, which were sustained to 12 mo, with no effect on BDNF. Changes in plasma EPA and DHA and urinary flavonoid metabolite concentrations confirmed compliance to the intervention. CONCLUSIONS: These results suggest that cosupplementation with omega-3 PUFAs and cocoa flavanols for 12 mo does not improve cognitive outcomes in those with cognitive impairment. This trial was registered at clinicaltrials.gov as NCT02525198.

Thiele, M., Suvitaival, T., Trost, K., Kim, M., de Zawadzki, A., Kjaergaard, M., Rasmussen, D. N., Lindvig, K. P., Israelsen, M., Detlefsen, S., Andersen, P., Juel, H. B., Nielsen, T., Georgiou, S., Filippa, V., Kuhn, M., Nishijima, S., Moitinho-Silva, L. (2023) Sphingolipids Are Depleted in Alcohol-Related Liver Fibrosis

BACKGROUND & AIMS: Alcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in alcohol-related liver disease (ALD) is unclear. In this biopsy-controlled, prospective, observational study, we characterized the liver and plasma lipidomes in patients with early ALD. METHODS: We performed mass spectrometry-based lipidomics of paired liver and plasma samples from 315 ALD patients, and of plasma from 51 matched healthy controls. We associated lipid levels to histological fibrosis, inflammation and steatosis with correction for multiple testing and adjustment for confounders. We further investigated sphingolipid regulation by qPCR sequencing of miRNA, prediction of liver-related events, and tested causality with Mendelian randomization. RESULTS: We detected 198 lipids in the liver and 236 lipids in the circulation from 18 lipid classes. Most sphingolipids (sphingomyelins and ceramides) and phosphocholines were co-downregulated in both liver and plasma, where lower abundance correlated with higher fibrosis stage. Sphingomyelins showed the most pronounced negative correlation to fibrosis, mirrored by negative correlations in both liver and plasma with hepatic inflammation. Reduced sphingomyelins furthermore predicted future liver-related events. This seemed to be characteristic of 'pure ALD', as sphingomyelin levels were higher in patients with concomitant metabolic syndrome and ALD/NAFLD overlap. Mendelian randomization in FinnGen and UK Biobanks indicated ALD as the cause of low sphingomyelins, while alcohol use disorder did not correlate with genetic susceptibility to low sphingomyelin levels. CONCLUSION: Alcohol-related liver fibrosis is characterized by selective and progressive lipid depletion in liver and blood, particularly sphingomyelins, which also associates with progression to liver-related events.

Slieker, R. C., Donnelly, L. A., Akalestou, E., Lopez-Noriega, L., Melhem, R., Gunes, A., Abou Azar, F., Efanov, A., Georgiadou, E., Muniangi-Muhitu, H., Sheikh, M., Giordano, G. N., Akerlund, M., Ahlqvist, E., Ali, A., Banasik, K., Brunak, S., Barovic, M (2023) Identification of biomarkers for glycaemic deterioration in type 2 diabetes

We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Prince, N., Kelly, R. S., Chu, S. H., Kachroo, P., Chen, Y., Mendez, K. M., Begum, S., Bisgaard, H., Bonnelykke, K., Kim, M., Levy, O., Litonjua, A. A., Wheelock, C. E., Weiss, S. T., Chawes, B. L. and Lasky-Su, J. A. (2023) Elevated third trimester corticosteroid levels are associated with fewer offspring infections

Respiratory infections are a leading cause of morbidity and mortality in early life, and recurrent infections increase the risk of developing chronic diseases. The maternal environment during pregnancy can impact offspring health, but the factors leading to increased infection proneness have not been well characterized during this period. Steroids have been implicated in respiratory health outcomes and may similarly influence infection susceptibility. Our objective was to describe relationships between maternal steroid levels and offspring infection proneness. Using adjusted Poisson regression models, we evaluated associations between sixteen androgenic and corticosteroid metabolites during pregnancy and offspring respiratory infection incidence across two pre-birth cohorts (N = 774 in VDAART and N = 729 in COPSAC). Steroid metabolites were measured in plasma samples from pregnant mothers across all trimesters of pregnancy by ultrahigh-performance-liquid-chromatography/mass-spectrometry. We conducted further inquiry into associations of steroids with related respiratory outcomes: asthma and lung function spirometry. Higher plasma corticosteroid levels in the third trimester of pregnancy were associated with lower incidence of offspring respiratory infections (P = 4.45 x 10(-7) to 0.002) and improved lung function metrics (P = 0.020-0.036). Elevated maternal androgens were generally associated with increased offspring respiratory infections and worse lung function, with some associations demonstrating nominal significance at P < 0.05, but these trends were inconsistent across individual androgens. Increased maternal plasma corticosteroid levels in the late second and third trimesters were associated with lower infections and better lung function in offspring, which may represent a potential avenue for intervention through corticosteroid supplementation in late pregnancy to reduce offspring respiratory infection susceptibility in early life.Clinical Trial Registry information: VDAART and COPSAC were originally conducted as clinical trials; VDAART: ClinicalTrials.gov identifier NCT00920621; COPSAC: ClinicalTrials.gov identifier NCT00798226.

Kim, M., Brustad, N., Ali, M., Gurdeniz, G., Arendt, M., Litonjua, A. A., Wheelock, C. E., Kelly, R. S., Chen, Y., Prince, N., Guo, F., Zhou, X., Stokholm, J., Bonnelykke, K., Weiss, S. T., Bisgaard, H., Lasky-Su, J. and Chawes, B. (2023) Maternal vitamin D-related metabolome and offspring risk of asthma outcomes

BACKGROUND: Gestational vitamin D deficiency is implicated in development of respiratory diseases in offspring, but the mechanism underlying this relationship is unknown. OBJECTIVE: We sought to study the link between gestational vitamin D exposure and childhood asthma phenotypes using maternal blood metabolomics profiling. METHODS: Untargeted blood metabolic profiles were acquired using liquid chromatography-mass spectrometry at 1 week postpartum from 672 women in the Copenhagen Prospective Studies on Asthma in Childhood(2010) (COPSAC(2010)) mother-child cohort and at pregnancy weeks 32 to 38 from 779 women in the Vitamin D Antenatal Asthma Reduction Trial (VDAART) mother-child cohort. In COPSAC(2010), we employed multivariate models and pathway enrichment analysis to identify metabolites and pathways associated with gestational vitamin D blood levels and investigated their relationship with development of asthma phenotypes in early childhood. The findings were validated in VDAART and in cellular models. RESULTS: In COPSAC(2010), higher vitamin D blood levels at 1 week postpartum were associated with distinct maternal metabolome perturbations with significant enrichment of the sphingomyelin pathway (P < .01). This vitamin D-related maternal metabolic profile at 1 week postpartum containing 46 metabolites was associated with decreased risk of recurrent wheeze (hazard ratio [HR] = 0.92 [95% CI 0.86-0.98], P = .01) and wheeze exacerbations (HR = 0.90 [95% CI 0.84-0.97], P = .01) at ages 0 to 3 years. The same metabolic profile was similarly associated with decreased risk of asthma/wheeze at ages 0 to 3 in VDAART (odds ratio = 0.92 [95% CI 0.85-0.99], P = .04). Human bronchial epithelial cells treated with high-dose vitamin D(3) showed an increased cytoplasmic sphingolipid level (P < .01). CONCLUSIONS: This exploratory metabolomics study in 2 independent birth cohorts demonstrates that the beneficial effect of higher gestational vitamin D exposure on offspring respiratory health is characterized by specific maternal metabolic alterations during pregnancy, which involves the sphingomyelin pathway.

Gurdeniz, G., Kim, M., Brustad, N., Ernst, M., Russo, F., Stokholm, J., Bonnelykke, K., Hougaard, D., Rasmussen, M., Cohen, A. and Chawes, B. (2023) Furan fatty acid metabolite in newborns predicts risk of asthma

BACKGROUND: Intake of fish-oil and fatty fish during pregnancy has been shown to reduce the risk of childhood asthma but biomarkers of such intake are lacking. OBJECTIVE: To establish biomarkers of prenatal fish-oil exposure from newborn dry blood spot metabolomics profiles and assess their relevance for childhood asthma risk stratification. METHODS: The Danish COPSAC(2010) mother-child cohort was utilized to investigate the effect of a double-blinded randomized controlled trial of fish-oil supplementation during pregnancy on dry blood spot liquid-chromatography mass spectrometry-based metabolomics profiles of 677 newborns. We thereafter investigated the association between fish-oil associated biomarkers in the newborn and development of asthma-related outcomes. Replication was sought in the independent observational COPSAC(2000) cohort with 387 newborn metabolomics profiles. RESULTS: The newborn metabolomics profiles differed between children in the fish-oil vs. placebo group in COPSAC(2010) (area under the receiver operator curve = 0.94 +/- 0.03, p < .001). The fish-oil metabolomics profile and the top biomarker, 3-carboxy-4-methyl-5-propyl-2-furan propanoic acid (CMPF) were both associated with a decreased risk of asthma by age 6 years (HR = 0.89, p = .002 and HR = 0.67, p = .005, respectively). In COPSAC(2000) , newborn CMPF level was also inversely associated with asthma risk by age 6 years (HR = 0.69, p = .01). Troublesome lung symptoms and common infections in the first 3 years were also inversely associated with newborn CMPF levels in both cohorts. CONCLUSIONS: Newborn children's blood levels of the furan fatty acid metabolite CMPF reflect fish-oil and fatty fish intake during pregnancy and are associated with a lower risk of asthma across two cohorts, which could aid newborn screening for childhood asthma.

Elingaard-Larsen, L. O., Villumsen, S. O., Justesen, L., Thuesen, A. C. B., Kim, M., Ali, M., Danielsen, E. R., Legido-Quigley, C., van Hall, G., Hansen, T., Ahluwalia, T. S., Vaag, A. A. and Brons, C. (2023) Circulating Metabolomic and Lipidomic Signatures Identify a Type 2 Diabetes Risk Profile in Low-Birth-Weight Men with Non-Alcoholic Fatty Liver Disease

The extent to which increased liver fat content influences differences in circulating metabolites and/or lipids between low-birth-weight (LBW) individuals, at increased risk of type 2 diabetes (T2D), and normal-birth-weight (NBW) controls is unknown. The objective of the study was to perform untargeted serum metabolomics and lipidomics analyses in 26 healthy, non-obese early-middle-aged LBW men, including five men with screen-detected and previously unrecognized non-alcoholic fatty liver disease (NAFLD), compared with 22 age- and BMI-matched NBW men (controls). While four metabolites (out of 65) and fifteen lipids (out of 279) differentiated the 26 LBW men from the 22 NBW controls (p </= 0.05), subgroup analyses of the LBW men with and without NAFLD revealed more pronounced differences, with 11 metabolites and 56 lipids differentiating (p </= 0.05) the groups. The differences in the LBW men with NAFLD included increased levels of ornithine and tyrosine (P(FDR) </= 0.1), as well as of triglycerides and phosphatidylcholines with shorter carbon-chain lengths and fewer double bonds. Pathway and network analyses demonstrated downregulation of transfer RNA (tRNA) charging, altered urea cycling, insulin resistance, and an increased risk of T2D in the LBW men with NAFLD. Our findings highlight the importance of increased liver fat in the pathogenesis of T2D in LBW individuals.

Chen, Y., Checa, A., Zhang, P., Huang, M., Kelly, R. S., Kim, M., Chen, Y. S., Lee-Sarwar, K. A., Prince, N., Mendez, K. M., Begum, S., Kachroo, P., Chu, S. H., Stokholm, J., Bonnelykke, K., Litonjua, A. A., Bisgaard, H., Weiss, S. T., Chawes, B. L., Whee (2023) Sphingolipid classes and the interrelationship with pediatric asthma and asthma risk factors

BACKGROUND: While dysregulated sphingolipid metabolism has been associated with risk of childhood asthma, the specific sphingolipid classes and/or mechanisms driving this relationship remain unclear. We aimed to understand the multifaceted role between sphingolipids and other established asthma risk factors that complicate this relationship. METHODS: We performed targeted LC-MS/MS-based quantification of 77 sphingolipids in plasma from 997 children aged 6 years from two independent cohorts (VDAART and COPSAC(2010) ). We examined associations of circulatory sphingolipids with childhood asthma, lung function, and three asthma risk factors: functional SNPs in ORMDL3, low vitamin D levels, and reduced gut microbial maturity. Given racial differences between these cohorts, association analyses were performed separately and then meta-analyzed together. RESULTS: We observed elevations in circulatory sphingolipids with asthma phenotypes and risk factors; however, there were differential associations of sphingolipid classes with clinical outcomes and/or risk factors. While elevations from metabolites involved in ceramide recycling and catabolic pathways were associated with asthma and worse lung function [meta p-value range: 1.863E-04 to 2.24E-3], increased ceramide levels were associated with asthma risk factors [meta p-value range: 7.75E-5 to .013], but not asthma. Further investigation identified that some ceramides acted as mediators while some interacted with risk factors in the associations with asthma outcomes. CONCLUSION: This study demonstrates the differential role that sphingolipid subclasses may play in asthma and its risk factors. While overall elevations in sphingolipids appeared to be deleterious overall; elevations in ceramides were uniquely associated with increases in asthma risk factors only; while elevations in asthma phenotypes were associated with recycling sphingolipids. Modification of asthma risk factors may play an important role in regulating sphingolipid homeostasis via ceramides to affect asthma. Further function work may validate the observed associations.

Brustad, N., Olarini, A., Kim, M., Chen, L., Ali, M., Wang, T., Cohen, A. S., Ernst, M., Hougaard, D., Schoos, A. M., Stokholm, J., Bonnelykke, K., Lasky-Su, J., Rasmussen, M. A. and Chawes, B. (2023) Diet-associated vertically transferred metabolites and risk of asthma, allergy, eczema, and infections in early childhood

BACKGROUND: Evidence suggests maternal pregnancy dietary intake and nutrition in the early postnatal period to be of importance for the newborn child's health. However, studies investigating diet-related metabolites transferred from mother to child on disease risk in childhood are lacking. We sought to investigate the influence of vertically transferred metabolites on risk of atopic diseases and infections during preschool age. METHODS: In the Danish population-based COPSAC(2010) mother-child cohort, information on 10 diet-related vertically transferred metabolites from metabolomics profiles of dried blood spots (DBS) at age 2-3 days was analyzed in relation to the risk of childhood asthma, allergy, eczema, and infections using principal component and single metabolite analyses. RESULTS: In 678 children with DBS measurements, a coffee-related metabolite profile reflected by principal component 1 was inversely associated with risk of asthma (odds ratio (95% CI) 0.78 (0.64; 0.95), p = .014) and eczema at age 6 years (0.79 (0.65; 0.97), p = .022). Furthermore, increasing stachydrine (fruit-related), 3-carboxy-4-methyl-5-propyl-2-furanpropanoate (fish-related), and ergothioneine (fruit-, green vegetables-, and fish-related) levels were all significantly associated with reduced risks of infections at age 0-3 years (p < .05). CONCLUSION: This study demonstrates associations between pregnancy diet-related vertically transferred metabolites measured in children in early life and risk of atopic diseases and infections in childhood. The specific metabolites associated with a reduced disease risk in children may contribute to the characterization of a healthy nutritional profile in pregnancy using a metabolomics-based unbiased tool for predicting childhood health.

Brustad, N., Kim, M., Skov, F., Schoos, A. M., Stokholm, J., Bonnelykke, K. and Chawes, B. L. (2023) 25-hydroxyvitamin D and risk of atopic diseases and infections in early childhood
Zawadzki, A., Thiele, M., Suvitaival, T., Wretlind, A., Kim, M., Ali, M., Bjerre, A. F., Stahr, K., Mattila, I., Hansen, T., Krag, A. and Legido-Quigley, C. (2022) High-Throughput UHPLC-MS to Screen Metabolites in Feces for Gut Metabolic Health

Feces are the product of our diets and have been linked to diseases of the gut, including Chron's disease and metabolic diseases such as diabetes. For screening metabolites in heterogeneous samples such as feces, it is necessary to use fast and reproducible analytical methods that maximize metabolite detection. As sample preparation is crucial to obtain high quality data in MS-based clinical metabolomics, we developed a novel, efficient and robust method for preparing fecal samples for analysis with a focus in reducing aliquoting and detecting both polar and non-polar metabolites. Fecal samples (n = 475) from patients with alcohol-related liver disease and healthy controls were prepared according to the proposed method and analyzed in an UHPLC-QQQ targeted platform in order to obtain a quantitative profile of compounds that impact liver-gut axis metabolism. MS analyses of the prepared fecal samples have shown reproducibility and coverage of n = 28 metabolites, mostly comprising bile acids and amino acids. We report metabolite-wise relative standard deviation (RSD) in quality control samples, inter-day repeatability, LOD (limit of detection), LOQ (limit of quantification), range of linearity and method recovery. The average concentrations for 135 healthy participants are reported here for clinical applications. Our high-throughput method provides a novel tool for investigating gut-liver axis metabolism in liver-related diseases using a noninvasive collected sample.

Prince, N., Kim, M., Kelly, R. S., Diray-Arce, J., Bonnelykke, K., Chawes, B. L., Huang, M., Levy, O., Litonjua, A. A., Stokholm, J., Wheelock, C. E., Bisgaard, H., Weiss, S. T. and Lasky-Su, J. A. (2022) Reduced Steroid Metabolites Identify Infection-Prone Children in Two Independent Pre-Birth Cohorts

Recurrent respiratory infections are a leading cause of morbidity and mortality in early life, but there is no broadly accepted means to identify infection-prone children during this highly vulnerable period. In this study, we investigated associations between steroid metabolites and incident respiratory infections in two pre-birth cohorts to identify novel metabolomic signatures of early infection proneness. Children from the Vitamin D Antenatal Asthma Reduction Trial and the Copenhagen Prospective Studies on Asthma in Childhood were included, and profiling was performed on plasma samples collected at ages 1 and 6 years. Both cohorts recorded incidence of lower respiratory infections, upper respiratory infections, ear infections, and colds. Poisson regression analysis assessed the associations between 18 steroid metabolites and the total number of respiratory infections that occurred in offspring during follow-up. We found that steroid metabolites across androgenic, corticosteroid, pregnenolone, and progestin classes were reduced in children that suffered more infections, and these patterns persisted at age 6 years, generally reflecting consistency in direction of effect and significance. Our analysis suggested steroid metabolite measurement may be useful in screening for infection proneness during this critical developmental period. Future studies should clinically evaluate their potential utility as a clinical screening tool.

Olarini, A., Ernst, M., Gurdeniz, G., Kim, M., Brustad, N., Bonnelykke, K., Cohen, A., Hougaard, D., Lasky-Su, J., Bisgaard, H., Chawes, B. and Rasmussen, M. A. (2022) Vertical Transfer of Metabolites Detectable from Newborn's Dried Blood Spot Samples Using UPLC-MS: A Chemometric Study

The pregnancy period and first days of a newborn's life is an important time window to ensure a healthy development of the baby. This is also the time when the mother and her baby are exposed to the same environmental conditions and intake of nutrients, which can be determined by assessing the blood metabolome. For this purpose, dried blood spots (DBS) of newborns are a valuable sampling technique to characterize what happens during this important mother-child time window. We used metabolomics profiles from DBS of newborns (age 2-3 days) and maternal plasma samples at gestation week 24 and postpartum week 1 from n=664 mother-child pairs of the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC2010) cohort, to study the vertical mother-child transfer of metabolites. Further, we investigated how persistent the metabolites are from the newborn and up to 6 months, 18 months, and 6 years of age. Two hundred seventy two metabolites from UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) analysis of DBS and maternal plasma were analyzed using correlation analysis. A total of 11 metabolites exhibited evidence of transfer (R>0.3), including tryptophan betaine, ergothioneine, cotinine, theobromine, paraxanthine, and N6-methyllysine. Of these, 7 were also found to show persistence in their levels in the child from birth to age 6 years. In conclusion, this study documents vertical transfer of environmental and food-derived metabolites from mother to child and tracking of those metabolites through childhood, which may be of importance for the child's later health and disease.

Gurdeniz, G., Ernst, M., Rago, D., Kim, M., Courraud, J., Stokholm, J., Bonnelykke, K., Bjorkbom, A., Trivedi, U., Sorensen, S. J., Brix, S., Hougaard, D., Rasmussen, M., Cohen, A. S., Bisgaard, H. and Chawes, B. (2022) Neonatal metabolome of caesarean section and risk of childhood asthma

BACKGROUND: Birth by caesarean section is linked to an increased risk of developing asthma, but the underlying mechanisms are unclear. OBJECTIVE: To elucidate the link between birth by caesarean section and asthma using newborn metabolomic profiles and integrating early-life gut microbiome data and cord blood immunology. METHODS: We investigated the influence of caesarean section on liquid chromatography mass spectrometry metabolomic profiles of dried blood spots from newborns of the two independent Copenhagen Prospective Studies on Asthma in Childhood cohorts, i.e. COPSAC2010 (n=677) and COPSAC2000 (n=387). We assessed the associations between the caesarean section metabolic profile, gut microbiome data and frequency of cord blood regulatory T-cells (Tregs) at 1 week of age. RESULTS: In COPSAC2010, a partial least square discriminant analysis model showed that children born by caesarean section versus natural delivery had different metabolic profiles (area under the curve (AUC)=0.77, p=2.2x10(-16)), which was replicated in COPSAC2000 (AUC=0.66, p=1.2x10(-5)). The metabolic profile of caesarean section was significantly associated with an increased risk of asthma at school age in both COPSAC2010 (p=0.03) and COPSAC2000 (p=0.005). Caesarean section was associated with lower abundance of tryptophan, bile acid and phenylalanine metabolites, indicative of a perturbed gut microbiota. Furthermore, gut bacteria dominating after natural delivery, i.e. Bifidobacterium and Bacteroides were correlated with caesarean section-discriminative microbial metabolites, suggesting maternal microbial transmission during birth regulating the newborn's metabolism. Finally, the caesarean section metabolic profile was associated with frequency of cord blood Tregs. CONCLUSIONS: These findings propose that caesarean section programmes the risk of childhood asthma through perturbed immune responses and gut microbial colonisation patterns reflected in the blood metabolome at birth.

Green, R., Lord, J., Xu, J., Maddock, J., Kim, M., Dobson, R., Legido-Quigley, C., Wong, A., Richards, M. and Proitsi, P. (2022) Metabolic correlates of late midlife cognitive outcomes: findings from the 1946 British Birth Cohort

Investigating associations between metabolites and late midlife cognitive function could reveal potential markers and mechanisms relevant to early dementia. Here, we systematically explored the metabolic correlates of cognitive outcomes measured across the seventh decade of life, while untangling influencing life course factors. Using levels of 1019 metabolites profiled by liquid chromatography-mass spectrometry (age 60-64), we evaluated relationships between metabolites and cognitive outcomes in the British 1946 Birth Cohort (N = 1740). We additionally conducted pathway and network analyses to allow for greater insight into potential mechanisms, and sequentially adjusted for life course factors across four models, including sex and blood collection (Model 1), Model 1 + body mass index and lipid medication (Model 2), Model 2 + social factors and childhood cognition (Model 3) and Model 3 + lifestyle influences (Model 4). After adjusting for multiple tests, 155 metabolites, 10 pathways and 5 network modules were associated with cognitive outcomes. Of the 155, 35 metabolites were highly connected in their network module (termed 'hub' metabolites), presenting as promising marker candidates. Notably, we report relationships between a module comprised of acylcarnitines and processing speed which remained robust to life course adjustment, revealing palmitoylcarnitine (C16) as a hub (Model 4: beta = -0.10, 95% confidence interval = -0.15 to -0.052, P = 5.99 x 10(-5)). Most associations were sensitive to adjustment for social factors and childhood cognition; in the final model, four metabolites remained after multiple testing correction, and 80 at P < 0.05. Two modules demonstrated associations that were partly or largely attenuated by life course factors: one enriched in modified nucleosides and amino acids (overall attenuation = 39.2-55.5%), and another in vitamin A and C metabolites (overall attenuation = 68.6-92.6%). Our other findings, including a module enriched in sphingolipid pathways, were entirely explained by life course factors, particularly childhood cognition and education. Using a large birth cohort study with information across the life course, we highlighted potential metabolic mechanisms associated with cognitive function in late midlife, suggesting marker candidates and life course relationships for further study.

Xu, J., Green, R., Kim, M., Lord, J., Ebshiana, A., Westwood, S., Baird, A. L., Nevado-Holgado, A. J., Shi, L., Hye, A., Snowden, S. G., Bos, I., Vos, S. J. B., Vandenberghe, R., Teunissen, C. E., Kate, M. T., Scheltens, P., Gabel, S., Meersmans, K., Blin (2021) Sex-Specific Metabolic Pathways Were Associated with Alzheimer's Disease (AD) Endophenotypes in the European Medical Information Framework for AD Multimodal Biomarker Discovery Cohort

BACKGROUND: physiological differences between males and females could contribute to the development of Alzheimer's Disease (AD). Here, we examined metabolic pathways that may lead to precision medicine initiatives. METHODS: We explored whether sex modifies the association of 540 plasma metabolites with AD endophenotypes including diagnosis, cerebrospinal fluid (CSF) biomarkers, brain imaging, and cognition using regression analyses for 695 participants (377 females), followed by sex-specific pathway overrepresentation analyses, APOE epsilon4 stratification and assessment of metabolites' discriminatory performance in AD. RESULTS: In females with AD, vanillylmandelate (tyrosine pathway) was increased and tryptophan betaine (tryptophan pathway) was decreased. The inclusion of these two metabolites (area under curve (AUC) = 0.83, standard error (SE) = 0.029) to a baseline model (covariates + CSF biomarkers, AUC = 0.92, SE = 0.019) resulted in a significantly higher AUC of 0.96 (SE = 0.012). Kynurenate was decreased in males with AD (AUC = 0.679, SE = 0.046). CONCLUSIONS: metabolic sex-specific differences were reported, covering neurotransmission and inflammation pathways with AD endophenotypes. Two metabolites, in pathways related to dopamine and serotonin, were associated to females, paving the way to personalised treatment.

Slieker, R. C., Donnelly, L. A., Fitipaldi, H., Bouland, G. A., Giordano, G. N., Akerlund, M., Gerl, M. J., Ahlqvist, E., Ali, A., Dragan, I., Festa, A., Hansen, M. K., Mansour Aly, D., Kim, M., Kuznetsov, D., Mehl, F., Klose, C., Simons, K., Pavo, I., Pu (2021) Replication and cross-validation of type 2 diabetes subtypes based on clinical variables: an IMI-RHAPSODY study

AIMS/HYPOTHESIS: Five clusters based on clinical characteristics have been suggested as diabetes subtypes: one autoimmune and four subtypes of type 2 diabetes. In the current study we replicate and cross-validate these type 2 diabetes clusters in three large cohorts using variables readily measured in the clinic. METHODS: In three independent cohorts, in total 15,940 individuals were clustered based on age, BMI, HbA(1c), random or fasting C-peptide, and HDL-cholesterol. Clusters were cross-validated against the original clusters based on HOMA measures. In addition, between cohorts, clusters were cross-validated by re-assigning people based on each cohort's cluster centres. Finally, we compared the time to insulin requirement for each cluster. RESULTS: Five distinct type 2 diabetes clusters were identified and mapped back to the original four All New Diabetics in Scania (ANDIS) clusters. Using C-peptide and HDL-cholesterol instead of HOMA2-B and HOMA2-IR, three of the clusters mapped with high sensitivity (80.6-90.7%) to the previously identified severe insulin-deficient diabetes (SIDD), severe insulin-resistant diabetes (SIRD) and mild obesity-related diabetes (MOD) clusters. The previously described ANDIS mild age-related diabetes (MARD) cluster could be mapped to the two milder groups in our study: one characterised by high HDL-cholesterol (mild diabetes with high HDL-cholesterol [MDH] cluster), and the other not having any extreme characteristic (mild diabetes [MD]). When these two milder groups were combined, they mapped well to the previously labelled MARD cluster (sensitivity 79.1%). In the cross-validation between cohorts, particularly the SIDD and MDH clusters cross-validated well, with sensitivities ranging from 73.3% to 97.1%. SIRD and MD showed a lower sensitivity, ranging from 36.1% to 92.3%, where individuals shifted from SIRD to MD and vice versa. People belonging to the SIDD cluster showed the fastest progression towards insulin requirement, while the MDH cluster showed the slowest progression. CONCLUSIONS/INTERPRETATION: Clusters based on C-peptide instead of HOMA2 measures resemble those based on HOMA2 measures, especially for SIDD, SIRD and MOD. By adding HDL-cholesterol, the MARD cluster based upon HOMA2 measures resulted in the current clustering into two clusters, with one cluster having high HDL levels. Cross-validation between cohorts showed generally a good resemblance between cohorts. Together, our results show that the clustering based on clinical variables readily measured in the clinic (age, HbA(1c), HDL-cholesterol, BMI and C-peptide) results in informative clusters that are representative of the original ANDIS clusters and stable across cohorts. Adding HDL-cholesterol to the clustering resulted in the identification of a cluster with very slow glycaemic deterioration.

Slieker, R. C., Donnelly, L. A., Fitipaldi, H., Bouland, G. A., Giordano, G. N., Akerlund, M., Gerl, M. J., Ahlqvist, E., Ali, A., Dragan, I., Elders, P., Festa, A., Hansen, M. K., van der Heijden, A. A., Mansour Aly, D., Kim, M., Kuznetsov, D., Mehl, F., (2021) Distinct Molecular Signatures of Clinical Clusters in People With Type 2 Diabetes: An IMI-RHAPSODY Study

Type 2 diabetes is a multifactorial disease with multiple underlying aetiologies. To address this heterogeneity, investigators of a previous study clustered people with diabetes according to five diabetes subtypes. The aim of the current study is to investigate the etiology of these clusters by comparing their molecular signatures. In three independent cohorts, in total 15,940 individuals were clustered based on five clinical characteristics. In a subset, genetic (N = 12,828), metabolomic (N = 2,945), lipidomic (N = 2,593), and proteomic (N = 1,170) data were obtained in plasma. For each data type, each cluster was compared with the other four clusters as the reference. The insulin-resistant cluster showed the most distinct molecular signature, with higher branched-chain amino acid, diacylglycerol, and triacylglycerol levels and aberrant protein levels in plasma were enriched for proteins in the intracellular PI3K/Akt pathway. The obese cluster showed higher levels of cytokines. The mild diabetes cluster with high HDL showed the most beneficial molecular profile with effects opposite of those seen in the insulin-resistant cluster. This study shows that clustering people with type 2 diabetes can identify underlying molecular mechanisms related to pancreatic islets, liver, and adipose tissue metabolism. This provides novel biological insights into the diverse aetiological processes that would not be evident when type 2 diabetes is viewed as a homogeneous disease.

Israelsen, M., Kim, M., Suvitaival, T., Madsen, B. S., Hansen, C. D., Torp, N., Trost, K., Thiele, M., Hansen, T., Legido-Quigley, C., Krag, A. and MicrobLiver, Consortium (2021) Comprehensive lipidomics reveals phenotypic differences in hepatic lipid turnover in ALD and NAFLD during alcohol intoxication

BACKGROUND & AIMS: In experimental models, alcohol induces acute changes in lipid metabolism that cause hepatocyte lipoapoptosis and inflammation. Here we study human hepatic lipid turnover during controlled alcohol intoxication. METHODS: We studied 39 participants with 3 distinct hepatic phenotypes: alcohol-related liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), and healthy controls. Alcohol was administrated via nasogastric tube over 30 min. Hepatic and systemic venous blood was sampled simultaneously at 3 time points: baseline, 60, and 180 min after alcohol intervention. Liver biopsies were sampled 240 min after alcohol intervention. We used ultra-high performance liquid chromatography mass spectrometry to measure levels of more than 250 lipid species from the blood and liver samples. RESULTS: After alcohol intervention, the levels of blood free fatty acid (FFA) and lysophosphatidylcholine (LPC) decreased, while triglyceride (TG) increased. FFA was the only lipid class to decrease in NAFLD after alcohol intervention, whereas LPC and FFA decreased and TG increased after intervention in ALD and healthy controls. Fatty acid chain uptake preference in FFAs and LPCs were oleic acid, linoleic acid, arachidonic acid, and docosahexaenoic acid. Hepatic venous blood FFA and LPC levels were lower when compared with systemic venous blood levels throughout the intervention. After alcohol intoxication, liver lipidome in ALD was similar to that in NAFLD. CONCLUSIONS: Alcohol intoxication induces rapid changes in circulating lipids including hepatic turnaround from FFA and LPC, potentially leading to lipoapoptosis and steatohepatitis. TG clearance was suppressed in NAFLD, possibly explaining why alcohol and NAFLD are synergistic risk factors for disease progression. These effects may be central to the pathogenesis of ALD. CLINICAL TRIALS REGISTRATION: The study is registered at Clinicaltrials.gov (NCT03018990). LAY SUMMARY: We report that alcohol induces hepatic extraction of free unsaturated fatty acids and lysophosphatidylcholines, hepatotoxic lipids which have not been previously associated with alcohol-induced liver injury. We also found that individuals with non-alcoholic fatty liver disease have reduced lipid turnover during alcohol intoxication when compared with people with alcohol-related fatty liver disease. This may explain why alcohol is particularly more harmful in people with non-alcoholic fatty liver and why elevated BMI and alcohol have a synergistic effect on the risk of liver-related death.

Coughlan, G., Larsen, R., Kim, M., White, D., Gillings, R., Irvine, M., Scholey, A., Cohen, N., Legido-Quigley, C., Hornberger, M. and Minihane, A. M. (2021) APOE ε4 alters associations between docosahexaenoic acid and preclinical markers of Alzheimer's disease

Docosahexaenoic acid is the main long-chain omega-3 polyunsaturated fatty acids in the brain and accounts for 30-40% of fatty acids in the grey matter of the human cortex. Although the influence of docosahexaenoic acid on memory function is widely researched, its association with brain volumes is under investigated and its association with spatial navigation is virtually unknown. This is despite the fact that spatial navigation deficits are a new cognitive fingerprint for symptomatic and asymptomatic Alzheimer's disease. We investigated the cross-sectional relationship between docosahexaenoic acid levels and the major structural and cognitive markers of preclinical Alzheimer's disease, namely hippocampal volume, entorhinal volume and spatial navigation ability. Fifty-three cognitively normal adults underwent volumetric magnetic resonance imaging, measurements of serum docosahexaenoic acid (DHA, including lysophosphatidylcholine DHA) and APOE ε4 genotyping. Relative regional brain volumes were calculated and linear regression models were fitted to examine DHA associations with brain volume. APOE genotype modulated serum DHA associations with entorhinal cortex volume and hippocampal volume. Linear models showed that greater serum DHA was associated with increased entorhinal cortex volume, but not hippocampal volume, in non APOΕ ε4 carriers. APOE also interacted with serum lysophosphatidylcholine DHA to predict hippocampal volume. After testing interactions between DHA and APOE on brain volume, we investigated whether DHA and APOE interact to predict spatial navigation performance on a novel virtual reality diagnostic test for Alzheimer's disease in an independent population of APOE genotyped adults (n = 46). APOE genotype modulated DHA associations with spatial navigation performance, showing that DHA was inversely associated with path integration in APOE ε4 carriers only. This exploratory analysis suggests that interventions aiming to increase DHA blood levels to protect against cognitive decline should consider APOE ε4 carrier status. Future work should focus on replicating our initial findings and establishing whether a specific dose of supplementary DHA, at a particular time in the preclinical disease course can have a positive impact on Alzheimer's disease progression in APOE ε4 carriers.

Al-Sari, N., Schmidt, S., Suvitaival, T., Kim, M., Trost, K., Ranjan, A. G., Christensen, M. B., Overgaard, A. J., Pociot, F., Norgaard, K. and Legido-Quigley, C. (2021) Changes in the lipidome in type 1 diabetes following low carbohydrate diet: Post-hoc analysis of a randomized crossover trial

AIMS: Lipid metabolism might be compromised in type 1 diabetes, and the understanding of lipid physiology is critically important. This study aimed to compare the change in plasma lipid concentrations during carbohydrate dietary changes in individuals with type 1 diabetes and identify links to early-stage dyslipidaemia. We hypothesized that (1) the lipidomic profiles after ingesting low or high carbohydrate diet for 12 weeks would be different; and (2) specific annotated lipid species could have significant associations with metabolic outcomes. METHODS: Ten adults with type 1 diabetes (mean +/- SD: age 43.6 +/- 13.8 years, diabetes duration 24.5 +/- 13.4 years, BMI 24.9 +/- 2.1 kg/m(2), HbA(1c) 57.6 +/- 2.6 mmol/mol) using insulin pumps participated in a randomized 2-period crossover study with a 12-week intervention period of low carbohydrate diet (< 100 g carbohydrates/day) or high carbohydrate diet (> 250 g carbohydrates/day), respectively, separated by a 12-week washout period. A large-scale non-targeted lipidomics was performed with mass spectrometry in fasting plasma samples obtained before and after each diet intervention. Longitudinal lipid levels were analysed using linear mixed-effects models. RESULTS: In total, 289 lipid species were identified from 14 major lipid classes. Comparing the two diets, 11 lipid species belonging to sphingomyelins, phosphatidylcholines and LPC(O-16:0) were changed. All the 11 lipid species were significantly elevated during low carbohydrate diet. Two lipid species were most differentiated between diets, namely SM(d36:1) (beta +/- SE: 1.44 +/- 0.28, FDR = 0.010) and PC(P-36:4)/PC(O-36:5) (beta +/- SE: 1.34 +/- 0.25, FDR = 0.009) species. Polyunsaturated PC(35:4) was inversely associated with BMI and positively associated with HDL cholesterol (p < .001). CONCLUSION: Lipidome-wide outcome analysis of a randomized crossover trial of individuals with type 1 diabetes following a low carbohydrate diet showed an increase in sphingomyelins and phosphatidylcholines which are thought to reduce dyslipidaemia. The polyunsaturated phosphatidylcholine 35:4 was inversely associated with BMI and positively associated with HDL cholesterol (p < .001). Results from this study warrant for more investigation on the long-term effect of single lipid species in type 1 diabetes.

Xu, J., Bankov, G., Kim, M., Wretlind, A., Lord, J., Green, R., Hodges, A., Hye, A., Aarsland, D., Velayudhan, L., Dobson, R. J. B., Proitsi, P., Legido-Quigley, C. and AddNeuroMed, Consortium (2020) Integrated lipidomics and proteomics network analysis highlights lipid and immunity pathways associated with Alzheimer's disease

BACKGROUND: There is an urgent need to understand the pathways and processes underlying Alzheimer's disease (AD) for early diagnosis and development of effective treatments. This study was aimed to investigate Alzheimer's dementia using an unsupervised lipid, protein and gene multi-omics integrative approach. METHODS: A lipidomics dataset comprising 185 AD patients, 40 mild cognitive impairment (MCI) individuals and 185 controls, and two proteomics datasets (295 AD, 159 MCI and 197 controls) were used for weighted gene co-expression network analyses (WGCNA). Correlations of modules created within each modality with clinical AD diagnosis, brain atrophy measures and disease progression, as well as their correlations with each other, were analyzed. Gene ontology enrichment analysis was employed to examine the biological processes and molecular and cellular functions of protein modules associated with AD phenotypes. Lipid species were annotated in the lipid modules associated with AD phenotypes. The associations between established AD risk loci and the lipid/protein modules that showed high correlation with AD phenotypes were also explored. RESULTS: Five of the 20 identified lipid modules and five of the 17 identified protein modules were correlated with clinical AD diagnosis, brain atrophy measures and disease progression. The lipid modules comprising phospholipids, triglycerides, sphingolipids and cholesterol esters were correlated with AD risk loci involved in immune response and lipid metabolism. The five protein modules involved in positive regulation of cytokine production, neutrophil-mediated immunity, and humoral immune responses were correlated with AD risk loci involved in immune and complement systems and in lipid metabolism (the APOE epsilon4 genotype). CONCLUSIONS: Modules of tightly regulated lipids and proteins, drivers in lipid homeostasis and innate immunity, are strongly associated with AD phenotypes.

Stamate, D., Kim, M., Proitsi, P., Westwood, S., Baird, A., Nevado-Holgado, A., Hye, A., Bos, I., Vos, S. J. B., Vandenberghe, R., Teunissen, C. E., Kate, M. T., Scheltens, P., Gabel, S., Meersmans, K., Blin, O., Richardson, J., De Roeck, E., Engelborghs, (2019) A metabolite-based machine learning approach to diagnose Alzheimer-type dementia in blood: Results from the European Medical Information Framework for Alzheimer disease biomarker discovery cohort

INTRODUCTION: Machine learning (ML) may harbor the potential to capture the metabolic complexity in Alzheimer Disease (AD). Here we set out to test the performance of metabolites in blood to categorize AD when compared to CSF biomarkers. METHODS: This study analyzed samples from 242 cognitively normal (CN) people and 115 with AD-type dementia utilizing plasma metabolites (n = 883). Deep Learning (DL), Extreme Gradient Boosting (XGBoost) and Random Forest (RF) were used to differentiate AD from CN. These models were internally validated using Nested Cross Validation (NCV). RESULTS: On the test data, DL produced the AUC of 0.85 (0.80-0.89), XGBoost produced 0.88 (0.86-0.89) and RF produced 0.85 (0.83-0.87). By comparison, CSF measures of amyloid, p-tau and t-tau (together with age and gender) produced with XGBoost the AUC values of 0.78, 0.83 and 0.87, respectively. DISCUSSION: This study showed that plasma metabolites have the potential to match the AUC of well-established AD CSF biomarkers in a relatively small cohort. Further studies in independent cohorts are needed to validate whether this specific panel of blood metabolites can separate AD from controls, and how specific it is for AD as compared with other neurodegenerative disorders.

Kim, M., Snowden, S., Suvitaival, T., Ali, A., Merkler, D. J., Ahmad, T., Westwood, S., Baird, A., Proitsi, P., Nevado-Holgado, A., Hye, A., Bos, I., Vos, S., Vandenberghe, R., Teunissen, C., Ten Kate, M., Scheltens, P., Gabel, S., Meersmans, K., Blin, O. (2019) Primary fatty amides in plasma associated with brain amyloid burden, hippocampal volume, and memory in the European Medical Information Framework for Alzheimer's Disease biomarker discovery cohort

INTRODUCTION: A critical and as-yet unmet need in Alzheimer's disease (AD) is the discovery of peripheral small molecule biomarkers. Given that brain pathology precedes clinical symptom onset, we set out to test whether metabolites in blood associated with pathology as indexed by cerebrospinal fluid (CSF) AD biomarkers. METHODS: This study analyzed 593 plasma samples selected from the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery study, of individuals who were cognitively healthy (n = 242), had mild cognitive impairment (n = 236), or had AD-type dementia (n = 115). Logistic regressions were carried out between plasma metabolites (n = 883) and CSF markers, magnetic resonance imaging, cognition, and clinical diagnosis. RESULTS: Eight metabolites were associated with amyloid beta and one with t-tau in CSF, these were primary fatty acid amides (PFAMs), lipokines, and amino acids. From these, PFAMs, glutamate, and aspartate also associated with hippocampal volume and memory. DISCUSSION: PFAMs have been found increased and associated with amyloid beta burden in CSF and clinical measures.

Kim, Min and Legido-Quigley, Cristina (2018) Small molecule biomarkers in Alzheimer’s disease
Bajaj, J. S., Kakiyama, G., Cox, I. J., Nittono, H., Takei, H., White, M., Fagan, A., Gavis, E. A., Heuman, D. M., Gilles, H. C., Hylemon, P., Taylor-Robinson, S. D., Legido-Quigley, C., Kim, M., Xu, J., Williams, R., Sikaroodi, M., Pandak, W. M. and Gill (2018) Alterations in gut microbial function following liver transplant

Liver transplantation (LT) improves daily function and ameliorates gut microbial composition. However, the effect of LT on microbial functionality, which can be related to overall patient benefit, is unclear and could affect the post-LT course. The aims were to determine the effect of LT on gut microbial functionality focusing on endotoxemia, bile acid (BA), ammonia metabolism, and lipidomics. We enrolled outpatient patients with cirrhosis on the LT list and followed them until 6 months after LT. Microbiota composition (Shannon diversity and individual taxa) and function analysis (serum endotoxin, urinary metabolomics and serum lipidomics, and stool BA profile) and cognitive tests were performed at both visits. We enrolled 40 patients (age, 56 +/- 7 years; mean Model for End-Stage Liver Disease score, 22.6). They received LT 6 +/- 3 months after enrollment and were re-evaluated 7 +/- 3 months after LT with a stable course. A significant improvement in cognition with increase in microbial diversity, increase in autochthonous and decrease in potentially pathogenic taxa, and reduced endotoxemia were seen after LT compared with baseline. Stool BAs increased significantly after LT, and there was evidence of greater bacterial action (higher secondary, oxo and iso-BAs) after LT although the levels of conjugated BAs remained similar. There was a reduced serum ammonia and corresponding rise in urinary phenylacetylglutamine after LT. There was an increase in urinary trimethylamine-N-oxide, which was correlated with specific changes in serum lipids related to cell membrane products. The ultimate post-LT lipidomic profile appeared beneficial compared with the profile before LT. In conclusion, LT improves gut microbiota diversity and dysbiosis, which is accompanied by favorable changes in gut microbial functionality corresponding to BAs, ammonia, endotoxemia, lipidomic, and metabolomic profiles. Liver Transplantation 24 752-761 2018 AASLD.

Proitsi, P., Kim, M., Whiley, L., Simmons, A., Sattlecker, M., Velayudhan, L., Lupton, M. K., Soininen, H., Kloszewska, I., Mecocci, P., Tsolaki, M., Vellas, B., Lovestone, S., Powell, J. F., Dobson, R. J. and Legido-Quigley, C. (2017) Association of blood lipids with Alzheimer's disease: A comprehensive lipidomics analysis

INTRODUCTION: The aim of this study was to (1) replicate previous associations between six blood lipids and Alzheimer's disease (AD) (Proitsi et al 2015) and (2) identify novel associations between lipids, clinical AD diagnosis, disease progression and brain atrophy (left/right hippocampus/entorhinal cortex). METHODS: We performed untargeted lipidomic analysis on 148 AD and 152 elderly control plasma samples and used univariate and multivariate analysis methods. RESULTS: We replicated our previous lipids associations and reported novel associations between lipids molecules and all phenotypes. A combination of 24 molecules classified AD patients with >70% accuracy in a test and a validation data set, and we identified lipid signatures that predicted disease progression (R(2) = 0.10, test data set) and brain atrophy (R(2) >/= 0.14, all test data sets except left entorhinal cortex). We putatively identified a number of metabolic features including cholesteryl esters/triglycerides and phosphatidylcholines. DISCUSSION: Blood lipids are promising AD biomarkers that may lead to new treatment strategies.

Kim, M., Nevado-Holgado, A., Whiley, L., Snowden, S. G., Soininen, H., Kloszewska, I., Mecocci, P., Tsolaki, M., Vellas, B., Thambisetty, M., Dobson, R. J. B., Powell, J. F., Lupton, M. K., Simmons, A., Velayudhan, L., Lovestone, S., Proitsi, P. and Legid (2017) Association between Plasma Ceramides and Phosphatidylcholines and Hippocampal Brain Volume in Late Onset Alzheimer's Disease

Lipids such as ceramides and phosphatidylcholines (PC) have been found altered in the plasma of Alzheimer's disease (AD) patients in a number of discovery studies. For this reason, the levels of 6 ceramides and 3 PCs, with different fatty acid length and saturation levels, were measured in the plasma from 412 participants (AD n = 205, Control n = 207) using mass spectrometry coupled with ultra-performance liquid chromatography. After this, associations with AD status, brain atrophy, and age-related effects were studied. In the plasma of AD participants, cross-sectional analysis revealed elevated levels of three ceramides (Cer16:0 p < 0.01, Cer18:0 p < 0.01, Cer24:1 p < 0.05). In addition, two PCs in AD plasma (PC36:5 p < 0.05, PC38:6 p < 0.05) were found to be depleted compared to the control group, with PC36:5 also associating with hippocampal atrophy (p < 0.01). Age-specific analysis further revealed that levels of Cer16:0, Cer18:0, and Cer20:0 were associated with hippocampal atrophy only in younger participants (age < 75, p < 0.05), while all 3 PCs did so in the older participants (age > 75, p < 0.05). PC36:5 was associated with AD status in the younger group (p < 0.01), while PC38:6 and 40:6 did so in the older group (p < 0.05). In this study, elevated ceramides and depleted PCs were found in the plasma from 205 AD volunteers. Our findings also suggest that dysregulation in PC and ceramide metabolism could be occurring in different stages of AD progression.

Voyle, N., Kim, M., Proitsi, P., Ashton, N. J., Baird, A. L., Bazenet, C., Hye, A., Westwood, S., Chung, R., Ward, M., Rabinovici, G. D., Lovestone, S., Breen, G., Legido-Quigley, C., Dobson, R. J., Kiddle, S. J. and Alzheimer's Disease Neuroimaging, Init (2016) Blood metabolite markers of neocortical amyloid-beta burden: discovery and enrichment using candidate proteins

We believe this is the first study to investigate associations between blood metabolites and neocortical amyloid burden (NAB) in the search for a blood-based biomarker for Alzheimer's disease (AD). Further, we present the first multi-modal analysis of blood markers in this field. We used blood plasma samples from 91 subjects enrolled in the University of California, San Francisco Alzheimer's Disease Research Centre. Non-targeted metabolomic analysis was used to look for associations with NAB using both single and multiple metabolic feature models. Five metabolic features identified subjects with high NAB, with 72% accuracy. We were able to putatively identify four metabolites from this panel and improve the model further by adding fibrinogen gamma chain protein measures (accuracy=79%). One of the five metabolic features was studied in the Alzheimer's Disease Neuroimaging Initiative cohort, but results were inconclusive. If replicated in larger, independent studies, these metabolic features and proteins could form the basis of a blood test with potential for enrichment of amyloid pathology in anti-amyloid trials.

Simpson, B. N., Kim, M., Chuang, Y. F., Beason-Held, L., Kitner-Triolo, M., Kraut, M., Lirette, S. T., Windham, B. G., Griswold, M. E., Legido-Quigley, C. and Thambisetty, M. (2016) Blood metabolite markers of cognitive performance and brain function in aging

We recently showed that Alzheimer's disease patients have lower plasma concentrations of the phosphatidylcholines (PC16:0/20:5; PC16:0/22:6; and PC18:0/22:6) relative to healthy controls. We now extend these findings by examining associations between plasma concentrations of these PCs with cognition and brain function (measured by regional resting state cerebral blood flow; rCBF) in non-demented older individuals. Within the Baltimore Longitudinal Study of Aging neuroimaging substudy, participants underwent cognitive assessments and brain (15)O-water positron emission tomography. Plasma phosphatidylcholines concentrations (PC16:0/20:5, PC16:0/22:6, and PC18:0/22:6), cognition (California Verbal Learning Test (CVLT), Trail Making Test A&B, the Mini-Mental State Examination, Benton Visual Retention, Card Rotation, and Fluencies-Category and Letter), and rCBF were assessed. Lower plasma phosphatidylcholine concentrations were associated with lower baseline memory performance (CVLT long delay recall task-PC16:0/20:5: -2.17-1.39-0.60 p = 0.001 (beta with 95% confidence interval subscripts)) and lower rCBF in several brain regions including those associated with memory performance and higher order cognitive processes. Our findings suggest that lower plasma concentrations of PC16:0/20:5, PC16:0/22:6, and PC18:0/22:6 are associated with poorer memory performance as well as widespread decreases in brain function during aging. Dysregulation of peripheral phosphatidylcholine metabolism may therefore be a common feature of both Alzheimer's disease and age-associated differences in cognition.

Casanova, R., Varma, S., Simpson, B., Kim, M., An, Y., Saldana, S., Riveros, C., Moscato, P., Griswold, M., Sonntag, D., Wahrheit, J., Klavins, K., Jonsson, P. V., Eiriksdottir, G., Aspelund, T., Launer, L. J., Gudnason, V., Legido Quigley, C. and Thambis (2016) Blood metabolite markers of preclinical Alzheimer's disease in two longitudinally followed cohorts of older individuals

INTRODUCTION: Recently, quantitative metabolomics identified a panel of 10 plasma lipids that were highly predictive of conversion to Alzheimer's disease (AD) in cognitively normal older individuals (n = 28, area under the curve [AUC] = 0.92, sensitivity/specificity of 90%/90%). METHODS: Quantitative targeted metabolomics in serum using an identical method as in the index study. RESULTS: We failed to replicate these findings in a substantially larger study from two independent cohorts-the Baltimore Longitudinal Study of Aging ([BLSA], n = 93, AUC = 0.642, sensitivity/specificity of 51.6%/65.7%) and the Age, Gene/Environment Susceptibility-Reykjavik Study ([AGES-RS], n = 100, AUC = 0.395, sensitivity/specificity of 47.0%/36.0%). In analyses applying machine learning methods to all 187 metabolite concentrations assayed, we find a modest signal in the BLSA with distinct metabolites associated with the preclinical and symptomatic stages of AD, whereas the same methods gave poor classification accuracies in the AGES-RS samples. DISCUSSION: We believe that ours is the largest blood biomarker study of preclinical AD to date. These findings underscore the importance of large-scale independent validation of index findings from biomarker studies with relatively small sample sizes.

Xu, J., Casas-Ferreira, A. M., Ma, Y., Sen, A., Kim, M., Proitsi, P., Shkodra, M., Tena, M., Srinivasan, P., Heaton, N., Jassem, W. and Legido-Quigley, C. (2015) Lipidomics comparing DCD and DBD liver allografts uncovers lysophospholipids elevated in recipients undergoing early allograft dysfunction

Finding specific biomarkers of liver damage in clinical evaluations could increase the pool of available organs for transplantation. Lipids are key regulators in cell necrosis and hence this study hypothesised that lipid levels could be altered in organs suffering severe ischemia. Matched pre- and post-transplant biopsies from donation after circulatory death (DCD, n = 36, mean warm ischemia time = 2 min) and donation after brain death (DBD, n = 76, warm ischemia time = none) were collected. Lipidomic discovery and multivariate analysis (MVA) were applied. Afterwards, univariate analysis and clinical associations were conducted for selected lipids differentiating between these two groups. MVA grouped DCD vs. DBD (p = 6.20 x 10(-12)) and 12 phospholipids were selected for intact lipid measurements. Two lysophosphatidylcholines, LysoPC (16:0) and LysoPC (18:0), showed higher levels in DCD at pre-transplantation (q < 0.01). Lysophosphatidylcholines were associated with aspartate aminotransferase (AST) 14-day post-transplantation (q < 0.05) and were more abundant in recipients undergoing early allograft dysfunction (EAD) (p < 0.05). A receiver-operating characteristics (ROC) curve combining both lipid levels predicted EAD with 82% accuracy. These findings suggest that LysoPC (16:0) and LysoPC (18:0) might have a role in signalling liver tissue damage due to warm ischemia before transplantation.

Proitsi, P., Kim, M., Whiley, L., Pritchard, M., Leung, R., Soininen, H., Kloszewska, I., Mecocci, P., Tsolaki, M., Vellas, B., Sham, P., Lovestone, S., Powell, J. F., Dobson, R. J. and Legido-Quigley, C. (2015) Plasma lipidomics analysis finds long chain cholesteryl esters to be associated with Alzheimer's disease

There is an urgent need for the identification of Alzheimer's disease (AD) biomarkers. Studies have now suggested the promising use of associations with blood metabolites as functional intermediate phenotypes in biomedical and pharmaceutical research. The aim of this study was to use lipidomics to identify a battery of plasma metabolite molecules that could predict AD patients from controls. We performed a comprehensive untargeted lipidomic analysis, using ultra-performance liquid chromatography/mass spectrometry on plasma samples from 35 AD patients, 40 elderly controls and 48 individuals with mild cognitive impairment (MCI) and used multivariate analysis methods to identify metabolites associated with AD status. A combination of 10 metabolites could discriminate AD patients from controls with 79.2% accuracy (81.8% sensitivity, 76.9% specificity and an area under curve of 0.792) in a novel test set. Six of the metabolites were identified as long chain cholesteryl esters (ChEs) and were reduced in AD (ChE 32:0, odds ratio (OR)=0.237, 95% confidence interval (CI)=0.10-0.48, P=4.19E-04; ChE 34:0, OR=0.152, 95% CI=0.05-0.37, P=2.90E-04; ChE 34:6, OR=0.126, 95% CI=0.03-0.35, P=5.40E-04; ChE 32:4, OR=0.056, 95% CI=0.01-0.24, P=6.56E-04 and ChE 33:6, OR=0.205, 95% CI=0.06-0.50, P=2.21E-03, per (log2) metabolite unit). The levels of these metabolites followed the trend control>MCI>AD. We, additionally, found no association between cholesterol, the precursor of ChE and AD. This study identified new ChE molecules, involved in cholesterol metabolism, implicated in AD, which may help identify new therapeutic targets; although, these findings need to be replicated in larger well-phenotyped cohorts.

Chen, Y., A. Checa, P. Zhang, M. Huang, R. S. Kelly, M. Kim, Y.-C. S. Chen, K. A. Lee-Sarwar, N. Prince, K. M. Mendez, S. Begum, P. Kachroo, S. H. Chu, J. Stokholm, K. Bønnelykke, A. A. Litonjua, H. Bisgaard, S. T. Weiss, B. L. Chawes, C. E. Wheelock and (2024) Sphingolipid classes and the interrelationship with pediatric asthma and asthma risk factors.

Background While dysregulated sphingolipid metabolism has been associated with risk of childhood asthma, the specific sphingolipid classes and/or mechanisms driving this relationship remain unclear. We aimed to understand the multifaceted role between sphingolipids and other established asthma risk factors that complicate this relationship. Methods We performed targeted LC–MS/MS-based quantification of 77 sphingolipids in plasma from 997 children aged 6 years from two independent cohorts (VDAART and COPSAC2010). We examined associations of circulatory sphingolipids with childhood asthma, lung function, and three asthma risk factors: functional SNPs in ORMDL3, low vitamin D levels, and reduced gut microbial maturity. Given racial differences between these cohorts, association analyses were performed separately and then meta-analyzed together. Results We observed elevations in circulatory sphingolipids with asthma phenotypes and risk factors; however, there were differential associations of sphingolipid classes with clinical outcomes and/or risk factors. While elevations from metabolites involved in ceramide recycling and catabolic pathways were associated with asthma and worse lung function [meta p-value range: 1.863E-04 to 2.24E-3], increased ceramide levels were associated with asthma risk factors [meta p-value range: 7.75E-5 to .013], but not asthma. Further investigation identified that some ceramides acted as mediators while some interacted with risk factors in the associations with asthma outcomes. Conclusion This study demonstrates the differential role that sphingolipid subclasses may play in asthma and its risk factors. While overall elevations in sphingolipids appeared to be deleterious overall; elevations in ceramides were uniquely associated with increases in asthma risk factors only; while elevations in asthma phenotypes were associated with recycling sphingolipids. Modification of asthma risk factors may play an important role in regulating sphingolipid homeostasis via ceramides to affect asthma. Further function work may validate the observed associations.

Kim, M., P. E. Nørrisgaard, N. Vahman, O. N. F. Cexus, P. A. Townsend, J. Stokholm, K. Bønnelykke, B. Chawes and N. Brustad (2024) Maternal Diet Associates with Offspring Bone Mineralization, Fracture Risk and Enamel Defects in Childhood and Influences the Prenatal Effect of High-Dose Vitamin D Supplementation

We previously demonstrated a beneficial effect of high-dose vitamin D in pregnancy on offspring bone and dental health. Here, we investigated the effect of maternal dietary patterns during pregnancy on the risk of bone fractures, bone mineralization and enamel defects until age 6 years in the offspring. Further, the influence of diet on the effect of high-dose vitamin D was analyzed in the COPSAC2010 mother–child cohort including 623 mother–child pairs. A weighted network analysis on FFQs revealed three specific maternal dietary patterns that associated (Bonferroni p < 0.05) with both offspring bone and dental health. The effect of prenatal high-dose (2800 IU/day) vs. standard-dose (400 IU/day) vitamin D on offspring bone mineral content (adjusted mean difference (aMD): 33.29 g, 95% CI: 14.48–52.09, p < 0.001), bone mineral density (aMD: 0.02 g/cm2 (0.01–0.04), p < 0.001), fracture risk (adjusted incidence rate ratio: 0.36 (0.16–0.84), p = 0.02), and enamel defects in primary (adjusted odds ratio (aOR): 0.13 (0.03–0.58), p < 0.01) and permanent molars (aOR: 0.25; (0.10–0.63), p < 0.01) was most pronounced when mothers had lower intake of fruit, vegetables, meat, eggs, sweets, whole grain, offal and fish. This study suggests that prenatal dietary patterns influence offspring bone and dental development, and should be considered in order to obtain the full benefits of vitamin D to enhance personalized supplementation strategy.