Browsing by Subject "metabolomics"

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  • Holma, Paula (Helsingfors universitet, 2011)
    Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.
  • Hämäläinen, Kreetta (Helsingin yliopisto, 2021)
    Personalized medicine tailors therapies for the patient based on predicted risk factors. Some tools used for making predictions on the safety and efficacy of drugs are genetics and metabolomics. This thesis focuses on identifying biomarkers for the activity level of the drug transporter organic anion transporting polypep-tide 1B1 (OATP1B1) from data acquired from untargeted metabolite profiling. OATP1B1 transports various drugs, such as statins, from portal blood into the hepatocytes. OATP1B1 is a genetically polymorphic influx transporter, which is expressed in human hepatocytes. Statins are low-density lipoprotein cholesterol-lowering drugs, and decreased or poor OATP1B1 function has been shown to be associated with statin-induced myopathy. Based on genetic variability, individuals can be classified to those with normal, decreased or poor OATP1B1 function. These activity classes were employed to identify metabolomic biomarkers for OATP1B1. To find the most efficient way to predict the activity level and find the biomarkers that associate with the activity level, 5 different machine learning models were tested with a dataset that consisted of 356 fasting blood samples with 9152 metabolite features. The models included both a Random Forest regressor and a classifier, Gradient Boosted Decision Tree regressor and classifier, and a Deep Neural Network regressor. Hindrances specific for this type of data was the collinearity between the features and the large amount of features compared to the number of samples, which lead to issues in determining the important features of the neural network model. To adjust to this, the data was clustered according to their Spearman’s rank-order correlation ranks. Feature importances were calculated using two methods. In the case of neural network, the feature importances were calculated with permutation feature importance using mean squared error, and random forest and gradient boosted decision trees used gini impurity. The performance of each model was measured, and all classifiers had a poor ability to predict decreasead and poor function classes. All regressors performed very similarly to each other. Gradient boosted decision tree regressor performed the best by a slight margin, but random forest regressor and neural network regressor performed nearly as well. The best features from all three models were cross-referenced with the features found from y-aware PCA analysis. The y-aware PCA analysis indicated that 14 best features cover 95% of the explained variance, so 14 features were picked from each model and cross-referenced with each other. Cross-referencing highest scoring features reported by the best models found multiple features that showed up as important in many models.Taken together, machine learning methods provide powerful tools to identify potential biomarkers from untargeted metabolomics data.
  • Vogt, Susanne; Wahl, Simone; Kettunen, Johannes; Breitner, Susanne; Kastenmueller, Gabi; Gieger, Christian; Suhre, Karsten; Waldenberger, Melanie; Kratzsch, Juergen; Perola, Markus; Salomaa, Veikko; Blankenberg, Stefan; Zeller, Tanja; Soininen, Pasi; Kangas, Antti J.; Peters, Annette; Grallert, Harald; Ala-Korpela, Mika; Thorand, Barbara (2016)
    Background: Numerous observational studies have observed associations between vitamin D deficiency and cardiometabolic diseases, but these findings might be confounded by obesity. A characterization of the metabolic profile associated with serum 25-hydroxyvitamin D [25(OH)D] levels, in general and stratified by abdominal obesity, may help to untangle the relationship between vitamin D, obesity and cardiometabolic health. Methods: Serum metabolomics measurements were obtained from a nuclear magnetic resonance spectroscopy (NMR)- and a mass spectrometry (MS)-based platform. The discovery was conducted in 1726 participants of the population-based KORA-F4 study, in which the associations of the concentrations of 415 metabolites with 25(OH)D levels were assessed in linear models. The results were replicated in 6759 participants (NMR) and 609 (MS) participants, respectively, of the population-based FINRISK 1997 study. Results: Mean [standard deviation (SD)] 25(OH)D levels were 15.2 (7.5) ng/ml in KORA F4 and 13.8 (5.9) ng/ml in FINRISK 1997; 37 metabolites were associated with 25(OH) D in KORA F4 at P <0.05/415. Of these, 30 associations were replicated in FINRISK 1997 at P <0.05/37. Among these were constituents of (very) large very-low-density lipoprotein and small low-density lipoprotein subclasses and related measures like serum triglycerides as well as fatty acids and measures reflecting the degree of fatty acid saturation. The observed associations were independent of waist circumference and generally similar in abdominally obese and non-obese participants. Conclusions: Independently of abdominal obesity, higher 25(OH)D levels were associated with a metabolite profile characterized by lower concentrations of atherogenic lipids and a higher degree of fatty acid polyunsaturation. These results indicate that the relationship between vitamin D deficiency and cardiometabolic diseases is unlikely to merely reflect obesity-related pathomechanisms.
  • Eloranta, Katja; Cairo, Stefano; Liljeström, Emmi; Soini, Tea; Kyrönlahti, Antti; Judde, Jean-Gabriel; Wilson, David B.; Heikinheimo, Markku; Pihlajoki, Marjut (2020)
    Background:Hepatoblastoma (HB) is the most common pediatric liver malignancy. Despite advances in chemotherapeutic regimens and surgical techniques, the survival of patients with advanced HB remains poor, underscoring the need for new therapeutic approaches. Chloroquine (CQ), a drug used to treat malaria and rheumatologic diseases, has been shown to inhibit the growth and survival of various cancer types. We examined the antineoplastic activity of CQ in cell models of aggressive HB. Methods:Seven human HB cell models, all derived from chemoresistant tumors, were cultured as spheroids in the presence of relevant concentrations of CQ. Morphology, viability, and induction of apoptosis were assessed after 48 and 96 h of CQ treatment. Metabolomic analysis and RT-qPCR based Death Pathway Finder array were used to elucidate the molecular mechanisms underlying the CQ effect in a 2-dimensional cell culture format. Quantitative western blotting was performed to validate findings at the protein level. Results:CQ had a significant dose and time dependent effect on HB cell viability both in spheroids and in 2-dimensional cell cultures. Following CQ treatment HB spheroids exhibited increased caspase 3/7 activity indicating the induction of apoptotic cell death. Metabolomic profiling demonstrated significant decreases in the concentrations of NAD(+)and aspartate in CQ treated cells. In further investigations, oxidation of NAD(+)decreased as consequence of CQ treatment and NAD(+)/NADH balance shifted toward NADH. Aspartate supplementation rescued cells from CQ induced cell death. Additionally, downregulated expression of PARP1 and PARP2 was observed. Conclusions:CQ treatment inhibits cell survival in cell models of aggressive HB, presumably by perturbing NAD(+)levels, impairing aspartate bioavailability, and inhibiting PARP expression. CQ thus holds potential as a new agent in the management of HB.
  • Lamichhane, Santosh; Ahonen, Linda; Dyrlund, Thomas Sparholt; Dickens, Alex M.; Siljander, Heli; Hyöty, Heikki; Ilonen, Jorma; Toppari, Jorma; Veijola, Riitta; Hyötyläinen, Tuulia; Knip, Mikael; Oresic, Matej (2019)
    Previous studies suggest that children who progress to type 1 diabetes (T1D) later in life already have an altered serum lipid molecular profile at birth. Here, we compared cord blood lipidome across the three study groups: children who progressed to T1D (PT1D; n = 30), children who developed at least one islet autoantibody but did not progress to T1D during the follow-up (P1Ab; n = 33), and their age-matched controls (CTR; n = 38). We found that phospholipids, specifically sphingomyelins, were lower in T1D progressors when compared to P1Ab and the CTR. Cholesterol esters remained higher in PT1D when compared to other groups. A signature comprising five lipids was predictive of the risk of progression to T1D, with an area under the receiver operating characteristic curve (AUROC) of 0.83. Our findings provide further evidence that the lipidomic profiles of newborn infants who progress to T1D later in life are different from lipidomic profiles in P1Ab and CTR.
  • Wang, Qin; Wurtz, Peter; Auro, Kirsi; Morin-Papunen, Laure; Kangas, Antti J.; Soininen, Pasi; Tiainen, Mika; Tynkkynen, Tuulia; Joensuu, Anni; Havulinna, Aki S.; Aalto, Kristiina; Salmi, Marko; Blankenberg, Stefan; Zeller, Tanja; Viikari, Jorma; Kahonen, Mika; Lehtimaki, Terho; Salomaa, Veikko; Jalkanen, Sirpa; Jarvelin, Marjo-Riitta; Perola, Markus; Raitakari, Olli T.; Lawlor, Debbie A.; Kettunen, Johannes; Ala-Korpela, Mika (2016)
    Background: Hormonal contraception is commonly used worldwide, but its systemic effects across lipoprotein subclasses, fatty acids, circulating metabolites and cytokines remain poorly understood. Methods: A comprehensive molecular profile (75 metabolic measures and 37 cytokines) was measured for up to 5841 women (age range 24-49 years) from three population-based cohorts. Women using combined oral contraceptive pills (COCPs) or progestin-only contraceptives (POCs) were compared with those who did not use hormonal contraception. Metabolomics profiles were reassessed for 869 women after 6 years to uncover the metabolic effects of starting, stopping and persistently using hormonal contraception. Results: The comprehensive molecular profiling allowed multiple new findings on the metabolic associations with the use of COCPs. They were positively associated with lipoprotein subclasses, including all high-density lipoprotein (HDL) subclasses. The associations with fatty acids and amino acids were strong and variable in direction. COCP use was negatively associated with albumin and positively associated with creatinine and inflammatory markers, including glycoprotein acetyls and several growth factors and interleukins. Our findings also confirmed previous results e.g. for increased circulating triglycerides and HDL cholesterol. Starting COCPs caused similar metabolic changes to those observed cross-sectionally: the changes were maintained in consistent users and normalized in those who stopped using. In contrast, POCs were only weakly associated with metabolic and inflammatory markers. Results were consistent across all cohorts and for different COCP preparations and different types of POC delivery. Conclusions: Use of COCPs causes widespread metabolic and inflammatory effects. However, persistent use does not appear to accumulate the effects over time and the metabolic perturbations are reversed upon discontinuation. POCs have little effect on systemic metabolism and inflammation.
  • Määttä, Anne; Salminen, Aino; Pietiäinen, Milla; Leskelä, Jaakko; Palviainen, Teemu; Sattler, Wolfgang; Sinisalo, Juha; Salomaa, Veikko; Kaprio, Jaakko; Pussinen, Pirkko (2021)
    Our aim was to analyze whether endotoxemia, i.e. translocation of LPS to circulation, is reflected in the serum metabolic profile in a general population and in participants with cardiometabolic disorders. We investigated three Finnish cohorts separately and in a meta-analysis (n = 7178), namely population-based FINRISK97, FinnTwin16 consisting of young adult twins, and Parogene, a random cohort of cardiac patients. Endotoxemia was determined as serum LPS activity and metabolome by an NMR platform. Potential effects of body mass index (BMI), smoking, metabolic syndrome (MetS), and coronary heart disease (CHD) status were considered. Endotoxemia was directly associated with concentrations of VLDL, IDL, LDL, and small HDL lipoproteins, VLDL particle diameter, total fatty acids (FA), glycoprotein acetyls (GlycA), aromatic and branched-chain amino acids, and Glc, and inversely associated with concentration of large HDL, diameters of LDL and HDL, as well as unsaturation degree of FAs. Some of these disadvantageous associations were significantly stronger in smokers and subjects with high BMI, but did not differ between participants with different CHD status. In participants with MetS, however, the associations of endotoxemia with FA parameters and GlycA were particularly strong. The metabolic profile in endotoxemia appears highly adverse, involving several inflammatory characters and risk factors for cardiometabolic disorders.
  • Heinonen, Hanna-Riikka; Mehine, Miika; Mäkinen, Netta; Pasanen, Annukka; Pitkänen, Esa; Karhu, Auli; Sarvilinna, Nanna S.; Sjöberg, Jari; Heikinheimo, Oskari; Bützow, Ralf; Aaltonen, Lauri A.; Kaasinen, Eevi (2017)
    Background: Uterine leiomyomas can be classified into molecularly distinct subtypes according to their genetic triggers: MED12 mutations, HMGA2 upregulation, or inactivation of FH. The aim of this study was to identify metabolites and metabolic pathways that are dysregulated in different subtypes of leiomyomas. Methods: We performed global metabolomic profiling of 25 uterine leiomyomas and 17 corresponding myometrium specimens using liquid chromatography-tandem mass spectroscopy. Results: A total of 641 metabolites were detected. All leiomyomas displayed reduced homocarnosine and haeme metabolite levels. We identified a clearly distinct metabolomic profile for leiomyomas of the FH subtype, characterised by metabolic alterations in the tricarboxylic acid cycle and pentose phosphate pathways, and increased levels of multiple lipids and amino acids. Several metabolites were uniquely elevated in leiomyomas of the FH subtype, including N6-succinyladenosine and argininosuccinate, serving as potential biomarkers for FH deficiency. In contrast, leiomyomas of the MED12 subtype displayed reduced levels of vitamin A, multiple membrane lipids and amino acids, and dysregulation of vitamin C metabolism, a finding which was also compatible with gene expression data. Conclusions: The study reveals the metabolomic heterogeneity of leiomyomas and provides the requisite framework for strategies designed to target metabolic alterations promoting the growth of these prevalent tumours.
  • Ottka, Claudia; Vapalahti, Katariina; Määttä, Ann-Marie; Huuskonen, Nanna; Sarpanen, Sinikka; Jalkanen, Liisa; Lohi, Hannes (2021)
    Background The kidneys have many essential metabolic functions, and metabolic disturbances during decreased renal function have not been studied extensively. Objectives To identify metabolic changes in blood samples with increased serum creatinine concentration, indicating decreased glomerular filtration. Animals Clinical samples analyzed using a nuclear magnetic resonance (NMR) based metabolomics platform. The case group consisted of 23 samples with serum creatinine concentration >125 mu mol/L, and the control group of 873 samples with serum creatinine concentration within the reference interval. Methods Biomarker association with increased serum creatinine concentration was evaluated utilizing 3 statistical approaches: Wilcoxon rank-sum test, logistic regression analysis (false discovery rate (FDR)-corrected P-values), and random forest classification. Medians of the biomarkers were compared to reference intervals. A heatmap and box plots were used to represent the differences. Results All 3 statistical approaches identified similar analytes associated with increased serum creatinine concentrations. The percentages of citrate, tyrosine, branched-chain amino acids, valine, leucine, albumin, linoleic acid and the ratio of phenylalanine to tyrosine differed significantly using all statistical approaches, acetate differed using the Wilcoxon test and random forest, docosapentaenoic acid percentage only using logistic regression (P <.05), and alanine only using random forest. Conclusions and Clinical Importance We identified several metabolic changes associated with increased serum creatinine concentrations, including prospective diagnostic markers and therapeutic targets. Further research is needed to verify the association of these changes with the clinical state of the dog. The NMR metabolomics test is a promising tool for improving diagnostic testing and management of renal diseases in dogs.
  • Wurtz, Peter; Cook, Sarah; Wang, Qin; Tiainen, Mika; Tynkkynen, Tuulia; Kangas, Antti J.; Soininen, Pasi; Laitinen, Jaana; Viikari, Jorma; Kahonen, Mika; Lehtimaki, Terho; Perola, Markus; Blankenberg, Stefan; Zeller, Tanja; Mannisto, Satu; Salomaa, Veikko; Jarvelin, Marjo-Riitta; Raitakari, Olli T.; Ala-Korpela, Mika; Leon, David A. (2016)
    Background: High alcohol consumption is a major cause of morbidity, yet alcohol is associated with both favourable and adverse effects on cardiometabolic risk markers. We aimed to characterize the associations of usual alcohol consumption with a comprehensive systemic metabolite profile in young adults. Methods: Cross-sectional associations of alcohol intake with 86 metabolic measures were assessed for 9778 individuals from three population-based cohorts from Finland (age 24-45 years, 52% women). Metabolic changes associated with change in alcohol intake during 6-year follow-up were further examined for 1466 individuals. Alcohol intake was assessed by questionnaires. Circulating lipids, fatty acids and metabolites were quantified by high-throughput nuclear magnetic resonance metabolomics and biochemical assays. Results: Increased alcohol intake was associated with cardiometabolic risk markers across multiple metabolic pathways, including higher lipid concentrations in HDL subclasses and smaller LDL particle size, increased proportions of monounsaturated fatty acids and decreased proportion of omega-6 fatty acids, lower concentrations of glutamine and citrate (P<0.001 for 56 metabolic measures). Many metabolic biomarkers displayed U-shaped associations with alcohol consumption. Results were coherent for men and women, consistent across the three cohorts and similar if adjusting for body mass index, smoking and physical activity. The metabolic changes accompanying change in alcohol intake during follow-up resembled the cross-sectional association pattern (R-2 = 0.83, slope = 0.7260.04). Conclusions: Alcohol consumption is associated with a complex metabolic signature, including aberrations in multiple biomarkers for elevated cardiometabolic risk. The metabolic signature tracks with long-term changes in alcohol consumption. These results elucidate the double-edged effects of alcohol on cardiovascular risk.
  • Palviainen, Mari; Saari, Heikki; Kärkkäinen, Olli; Pekkinen, Jenna; Auriola, Seppo; Yliperttula, Marjo; Puhka, Maija; Hanhineva, Kati; Siljander, Pia R-M (2019)
    One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.
  • Värri, Miika; Niskanen, Leo; Tuomainen, Tomi-Pekka; Honkanen, Risto; Kröger, Heikki; Tuppurainen, Marjo T. (2020)
    Purpose: Atherosclerosis (AS) and osteoporosis (OP) are common causes of morbidity and mortality in postmenopausal women and are connected via an unknown mechanistic link. Metabolite profiling of blood samples may allow the identification of new biomarkers and pathways for this enigmatic association. Patients and Methods: We studied the difference in 148 metabolite levels from serum samples in postmenopausal women with AS and OP compared with those in healthy participants in this cross-sectional study. Quantitative AS was assessed by carotid artery intima-media thickness (cIMT) and carotid artery calcifications (CACs) by ultrasound, as well as OP by femoral neck (FN) bone mineral density (BMD) and 148 metabolic measures with high-throughput proton (H-1) nuclear magnetic resonance (NMR) in serum samples from 280 postmenopausal (PM) women. Subjects were a randomly selected subsample from the population-based Kuopio Osteoporosis Risk Factor and Prevention (OSTPRE) study. The final study population included the following groups: OP with CAC (n=16, group I), non-OP with no CAC (n=59, group II), high cIMT tertile with OP (n=11, group III) and low cIMT tertile without OP (n=48, group IV). Results: There were differences in several metabolite levels between groups I and II. The acetate level was lower in group I compared to that in group II (group I mean +/- SD: 0.033 +/- 0.0070; group II: 0.041 +/- 0.014, CI95%:, p=0.014). The result was similar with diacylglycerol (p=0.002), leucine (p=0.031), valine (p=0.022) and several very low-density lipoprotein (VLDL) metabolite levels, which were lower in group I compared to those in group II. However, no associations were found in adjusted analyses with total body (TB) fat mass (FM), age and statin use (p>0.05). Conclusion: Our novel study found differences in the metabolite profiling of altered amino acid and lipoprotein metabolism in participants with OP and AS compared with those in healthy women. The causative mechanisms remain unknown and further studies are needed.
  • Buzkova, Jana; Nikkanen, Joni; Ahola, Sofia; Hakonen, Anna H.; Sevastianova, Ksenia; Hovinen, Topi; Yki-Järvinen, Hannele; Pietiläinen, Kirsi H.; Lönnqvist, Tuula; Velagapudi, Vidya; Carroll, Christopher J.; Suomalainen, Anu (2018)
    Mitochondrial disorders (MDs) are inherited multi-organ diseases with variable phenotypes. Inclusion body myositis (IBM), a sporadic inflammatory muscle disease, also shows mitochondrial dysfunction. We investigated whether primary and secondary MDs modify metabolism to reveal pathogenic pathways and biomarkers. We investigated metabolomes of 25 mitochondrial myopathy or ataxias patients, 16 unaffected carriers, six IBM and 15 non-mitochondrial neuromuscular disease (NMD) patients and 30 matched controls. MD and IBM metabolomes clustered separately from controls and NMDs. MDs and IBM showed transsulfuration pathway changes; creatine and niacinamide depletion marked NMDs, IBM and infantile-onset spinocerebellar ataxia (IOSCA). Low blood and muscle arginine was specific for patients with m.3243A>G mutation. A four-metabolite blood multi-biomarker (sorbitol, alanine, myoinositol, cystathionine) distinguished primary MDs from others (76% sensitivity, 95% specificity). Our omics approach identified pathways currently used to treat NMDs and mitochondrial stroke-like episodes and proposes nicotinamide riboside in MDs and IBM, and creatine in IOSCA and IBM as novel treatment targets. The disease-specific metabolic fingerprints are valuable "multi-biomarkers" for diagnosis and promising tools for follow-up of disease progression and treatment effect.
  • Sliz, Eeva; Kettunen, Johannes; Holmes, Michael V.; Williams, Clare Oliver; Boachie, Charles; Wang, Qin; Maennikkoe, Minna; Sebert, Sylvain; Walters, Robin; Lin, Kuang; Millwood, Iona Y.; Clarke, Robert; Li, Liming; Rankin, Naomi; Welsh, Paul; Delles, Christian; Jukema, J. Wouter; Trompet, Stella; Ford, Ian; Perola, Markus; Salomaa, Veikko; Jaervelin, Marjo-Riitta; Chen, Zhengming; Lawlor, Debbie A.; Ala-Korpela, Mika; Danesh, John; Davey Smith, George; Sattar, Naveed; Butterworth, Adam; Würtz, Peter (2018)
    Background: Both statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors lower blood low-density lipoprotein cholesterol levels to reduce risk of cardiovascular events. To assess potential differences between metabolic effects of these 2 lipid-lowering therapies, we performed detailed lipid and metabolite profiling of a large randomized statin trial and compared the results with the effects of genetic inhibition of PCSK9, acting as a naturally occurring trial. Methods: Two hundred twenty-eight circulating metabolic measures were quantified by nuclear magnetic resonance spectroscopy, including lipoprotein subclass concentrations and their lipid composition, fatty acids, and amino acids, for 5359 individuals (2659 on treatment) in the PROSPER (Prospective Study of Pravastatin in the Elderly at Risk) trial at 6 months postrandomization. The corresponding metabolic measures were analyzed in 8 population cohorts (N=72 185) using PCSK9 rs11591147 as an unconfounded proxy to mimic the therapeutic effects of PCSK9 inhibitors. Results: Scaled to an equivalent lowering of low-density lipoprotein cholesterol, the effects of genetic inhibition of PCSK9 on 228 metabolic markers were generally consistent with those of statin therapy (R-2=0.88). Alterations in lipoprotein lipid composition and fatty acid distribution were similar. However, discrepancies were observed for very-low-density lipoprotein lipid measures. For instance, genetic inhibition of PCSK9 had weaker effects on lowering of very-low-density lipoprotein cholesterol compared with statin therapy (54% versus 77% reduction, relative to the lowering effect on low-density lipoprotein cholesterol; P=2x10(-7) for heterogeneity). Genetic inhibition of PCSK9 showed no significant effects on amino acids, ketones, or a marker of inflammation (GlycA), whereas statin treatment weakly lowered GlycA levels. Conclusions: Genetic inhibition of PCSK9 had similar metabolic effects to statin therapy on detailed lipid and metabolite profiles. However, PCSK9 inhibitors are predicted to have weaker effects on very-low-density lipoprotein lipids compared with statins for an equivalent lowering of low-density lipoprotein cholesterol, which potentially translate into smaller reductions in cardiovascular disease risk.
  • Puhka, Maija; Takatalo, Maarit; Nordberg, Maria-Elisa; Valkonen, Sami; Nandania, Jatin; Aatonen, Maria; Yliperttula, Marjo; Laitinen, Saara; Velagapudi, Vidya; Mirtti, Tuomas; Kallioniemi, Olli; Rannikko, Antti; Siljander, Pia R-M; Af Hallstrom, Taija Maria (2017)
    Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi) RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV-and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 10(10) EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p <0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.
  • Wurtz, Peter; Wang, Qin; Soininen, Pasi; Kangas, Antti J.; Fatemifar, Ghazaleh; Tynkkynen, Tuulia; Tiainen, Mika; Perola, Markus; Tillin, Therese; Hughes, Alun D.; Mantyselka, Pekka; Kahonen, Mika; Lehtimaki, Terho; Sattar, Naveed; Hingorani, Aroon D.; Casas, Juan-Pablo; Salomaa, Veikko; Kivimaki, Mika; Jarvelin, Marjo-Riitta; Smith, George Davey; Vanhala, Mauno; Lawlor, Debbie A.; Raitakari, Olli T.; Chaturvedi, Nish; Kettunen, Johannes; Ala-Korpela, Mika (2016)
    BACKGROUND Statins are first-line therapy for cardiovascular disease prevention, but their systemic effects across lipoprotein subclasses, fatty acids, and circulating metabolites remain incompletely characterized. OBJECTIVES This study sought to determine the molecular effects of statin therapy on multiple metabolic pathways. METHODS Metabolic profiles based on serum nuclear magnetic resonance metabolomics were quantified at 2 time points in 4 population-based cohorts from the United Kingdom and Finland (N = 5,590; 2.5 to 23.0 years of follow-up). Concentration changes in 80 lipid and metabolite measures during follow-up were compared between 716 individuals who started statin therapy and 4,874 persistent nonusers. To further understand the pharmacological effects of statins, we used Mendelian randomization to assess associations of a genetic variant known to mimic inhibition of HMG-CoA reductase (the intended drug target) with the same lipids and metabolites for 27,914 individuals from 8 population-based cohorts. RESULTS Starting statin therapy was associated with numerous lipoprotein and fatty acid changes, including substantial lowering of remnant cholesterol (80% relative to low-density lipoprotein cholesterol [LDL-C]), but only modest lowering of triglycerides (25% relative to LDL-C). Among fatty acids, omega-6 levels decreased the most (68% relative to LDL-C); other fatty acids were only modestly affected. No robust changes were observed for circulating amino acids, ketones, or glycolysis-related metabolites. The intricate metabolic changes associated with statin use closely matched the association pattern with rs12916 in the HMGCR gene (R-2 = 0.94, slope 1.00 +/- 0.03). CONCLUSIONS Statin use leads to extensive lipid changes beyond LDL-C and appears efficacious for lowering remnant cholesterol. Metabolomic profiling, however, suggested minimal effects on amino acids. The results exemplify how detailed metabolic characterization of genetic proxies for drug targets can inform indications, pleiotropic effects, and pharmacological mechanisms. (C) 2016 by the American College of Cardiology Foundation.
  • Tikkanen, Emmi; Minicocci, Ilenia; Hällfors, Jenni; Di Costanzo, Alessia; D'Erasmo, Laura; Poggiogalle, Eleonora; Donini, Lorenzo Maria; Wurtz, Peter; Jauhiainen, Matti; Olkkonen, Vesa M.; Arca, Marcello (2019)
    Objective- Loss-of-function (LOF) variants in the ANGPTL3 (angiopoietin-like protein 3) have been associated with low levels of plasma lipoproteins and decreased coronary artery disease risk. We aimed to determine detailed metabolic effects of genetically induced ANGPTL3 deficiency in fasting and postprandial state. Approach and Results- We studied individuals carrying S17X LOF mutation in ANGPTL3 (6 homozygous and 32 heterozygous carriers) and 38 noncarriers. Nuclear magnetic resonance metabolomics was used to quantify 225 circulating metabolic measures. We compared metabolic differences between LOF carriers and noncarriers in fasting state and after a high-fat meal. In fasting, ANGPTL3 deficiency was characterized by similar extent of reductions in LDL (low-density lipoprotein) cholesterol (0.74 SD units lower concentration per LOF allele [95% CI, 0.42-1.06]) as observed for many TRL (triglyceride-rich lipoprotein) measures, including VLDL (very-low-density lipoprotein) cholesterol (0.75 [95% CI, 0.45-1.05]). Within most lipoprotein subclasses, absolute levels of cholesterol were decreased more than triglycerides, resulting in the relative proportion of cholesterol being reduced within TRLs and their remnants. Further, beta-hydroxybutyrate was elevated (0.55 [95% CI, 0.21-0.89]). Homozygous ANGPTL3 LOF carriers showed essentially no postprandial increase in TRLs and fatty acids, without evidence for adverse compensatory metabolic effects. Conclusions- In addition to overall triglyceride- and LDL cholesterol-lowering effects, ANGPTL3 deficiency results in reduction of cholesterol proportion within TRLs and their remnants. Further, ANGPTL3 LOF carriers had elevated ketone body production, suggesting enhanced hepatic fatty acid beta-oxidation. The detailed metabolic profile in human knockouts of ANGPTL3 reinforces inactivation of ANGPTL3 as a promising therapeutic target for decreasing cardiovascular risk.
  • Ellul, Susan; Wake, Melissa; Clifford, Susan A.; Lange, Katherine; Würtz, Peter; Juonala, Markus; Dwyer, Terence; Carlin, John B.; Burgner, David P.; Saffery, Richard (2019)
    Objectives Nuclear magnetic resonance (NMR) metabolomics is high throughput and cost-effective, with the potential to improve the understanding of disease and risk. We examine the circulating metabolic profile by quantitative NMR metabolomics of a sample of Australian 11-12 year olds children and their parents, describe differences by age and sex, and explore the correlation of metabolites in parent-child dyads. Design The population-based cross-sectional Child Health CheckPoint study nested within the Longitudinal Study of Australian Children. Setting Blood samples collected from CheckPoint participants at assessment centres in seven Australian cities and eight regional towns; February 2015-March 2016. Participants 1180 children and 1325 parents provided a blood sample and had metabolomics data available. This included 1133 parent-child dyads (518 mother-daughter, 469 mother-son, 68 father-daughter and 78 father-son). Outcome measures 228 metabolic measures were obtained for each participant. We focused on 74 biomarkers including amino acid species, lipoprotein subclass measures, lipids, fatty acids, measures related to fatty acid saturation, and composite markers of inflammation and energy homeostasis. Results We identified differences in the concentration of specific metabolites between childhood and adulthood and in metabolic profiles in children and adults by sex. In general, metabolite concentrations were higher in adults than children and sex differences were larger in adults than in children. Positive correlations were observed for the majority of metabolites including isoleucine (CC 0.33, 95% CI 0.27 to 0.38), total cholesterol (CC 0.30, 95% CI 0.24 to 0.35) and omega 6 fatty acids (CC 0.28, 95% CI 0.23 to 0.34) in parent-child comparisons. Conclusions We describe the serum metabolite profiles from mid-childhood and adulthood in a population-based sample, together with a parent-child concordance. Differences in profiles by age and sex were observed. These data will be informative for investigation of the childhood origins of adult non-communicable diseases and for comparative studies in other populations.
  • Talman, Virpi; Teppo, Jaakko Sakari; Pöhö, Päivi Anneli; Movahedi, Parisa; Vaikkinen, Anu; Karhu, Suvi Tuuli; Trošt, Kajetan; Suvitaival, Tommi; Heikkonen, Jukka; Pahikkala, Tapio; Kotiaho, Ahti Antti Tapio; Kostiainen, Risto Kalervo; Varjosalo, Markku Tapio; Ruskoaho, Heikki Juhani (2018)
    Background The molecular mechanisms mediating postnatal loss of cardiac regeneration in mammals are not fully understood. We aimed to provide an integrated resource of mRNA, protein, and metabolite changes in the neonatal heart for identification of metabolism‐related mechanisms associated with cardiac regeneration. Methods and Results Methods and results Mouse ventricular tissue samples taken on postnatal day 1 (P01), P04, P09, and P23 were analyzed with RNA sequencing and global proteomics and metabolomics. Gene ontology analysis, KEGG pathway analysis, and fuzzy c‐means clustering were used to identify up‐ or downregulated biological processes and metabolic pathways on all 3 levels, and Ingenuity pathway analysis (Qiagen) was used to identify upstream regulators. Differential expression was observed for 8547 mRNAs and for 1199 of 2285 quantified proteins. Furthermore, 151 metabolites with significant changes were identified. Differentially regulated metabolic pathways include branched chain amino acid degradation (upregulated at P23), fatty acid metabolism (upregulated at P04 and P09; downregulated at P23) as well as the HMGCS (HMG‐CoA [hydroxymethylglutaryl‐coenzyme A] synthase)–mediated mevalonate pathway and ketogenesis (transiently activated). Pharmacological inhibition of HMGCS in primary neonatal cardiomyocytes reduced the percentage of BrdU‐positive cardiomyocytes, providing evidence that the mevalonate and ketogenesis routes may participate in regulating the cardiomyocyte cell cycle. Conclusions This study is the first systems‐level resource combining data from genomewide transcriptomics with global quantitative proteomics and untargeted metabolomics analyses in the mouse heart throughout the early postnatal period. These integrated data of molecular changes associated with the loss of cardiac regeneration may open up new possibilities for the development of regenerative therapies
  • Soderholm, Sandra; Fu, Yu; Gaelings, Lana; Belanov, Sergey; Yetukuri, Laxma; Berlinkov, Mikhail; Cheltsov, Anton V.; Anders, Simon; Aittokallio, Tero; Nyman, Tuula A.; Matikainen, Sampsa; Kainov, Denis E. (2016)
    Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV-host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV-host interactions, which might be more effective than the currently available anti-influenza therapeutics.