Urinary metabolite profiling and risk of progression of diabetic nephropathy in 2670 individuals with type 1 diabetes.

AIMS/HYPOTHESIS: This prospective, observational study examines associations between 51 urinary metabolites and risk of progression of diabetic nephropathy in individuals with type 1 diabetes by employing an automated NMR metabolomics technique suitable for large-scale urine sample collections. METHODS: We collected 24-h urine samples for 2670 individuals with type 1 diabetes from the Finnish Diabetic Nephropathy study and measured metabolite concentrations by NMR. Individuals were followed up for 9.0 ± 5.0 years until their first sign of progression of diabetic nephropathy, end-stage kidney disease or study end. Cox regressions were performed on the entire study population (overall progression), on 1999 individuals with normoalbuminuria and 347 individuals with macroalbuminuria at baseline. RESULTS: Seven urinary metabolites were associated with overall progression after adjustment for baseline albuminuria and chronic kidney disease stage (p < 8 × 10-4): leucine (HR 1.47 [95% CI 1.30, 1.66] per 1-SD creatinine-scaled metabolite concentration), valine (1.38 [1.22, 1.56]), isoleucine (1.33 [1.18, 1.50]), pseudouridine (1.25 [1.11, 1.42]), threonine (1.27 [1.11, 1.46]) and citrate (0.84 [0.75, 0.93]). 2-Hydroxyisobutyrate was associated with overall progression (1.30 [1.16, 1.45]) and also progression from normoalbuminuria (1.56 [1.25, 1.95]). Six amino acids and pyroglutamate were associated with progression from macroalbuminuria. CONCLUSIONS/INTERPRETATION: Branched-chain amino acids and other urinary metabolites were associated with the progression of diabetic nephropathy on top of baseline albuminuria and chronic kidney disease. We found differences in associations for overall progression and progression from normo- and macroalbuminuria. These novel discoveries illustrate the utility of analysing urinary metabolites in entire population cohorts.

Multivariate Genome-wide Association Analysis of a Cytokine Network Reveals Variants with Widespread Immune, Haematological, and Cardiometabolic Pleiotropy.

Cytokines are essential regulatory components of the immune system, and their aberrant levels have been linked to many disease states. Despite increasing evidence that cytokines operate in concert, many of the physiological interactions between cytokines, and the shared genetic architecture that underlies them, remain unknown. Here, we aimed to identify and characterize genetic variants with pleiotropic effects on cytokines. Using three population-based cohorts (n = 9,263), we performed multivariate genome-wide association studies (GWAS) for a correlation network of 11 circulating cytokines, then combined our results in meta-analysis. We identified a total of eight loci significantly associated with the cytokine network, of which two (PDGFRB and ABO) had not been detected previously. In addition, conditional analyses revealed a further four secondary signals at three known cytokine loci. Integration, through the use of Bayesian colocalization analysis, of publicly available GWAS summary statistics with the cytokine network associations revealed shared causal variants between the eight cytokine loci and other traits; in particular, cytokine network variants at the ABO, SERPINE2, and ZFPM2 loci showed pleiotropic effects on the production of immune-related proteins, on metabolic traits such as lipoprotein and lipid levels, on blood-cell-related traits such as platelet count, and on disease traits such as coronary artery disease and type 2 diabetes.

Plasma fatty acids and the risk of vascular disease and mortality outcomes in individuals with type 2 diabetes: results from the ADVANCE study.

AIMS/HYPOTHESIS: This biomarker study aimed to quantify the association of essential and other plasma fatty acid biomarkers with macrovascular disease, microvascular disease and death in individuals with type 2 diabetes. METHODS: A case-cohort study (N = 3576), including 654 macrovascular events, 341 microvascular events and 631 deaths during 5 years of (median) follow-up, was undertaken as a secondary analysis of the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified-Release Controlled Evaluation (ADVANCE) study (full details of the study design and primary endpoints of the ADVANCE trial and its case-cohort have been published previously). This current study considers new data: fatty acids measured from baseline plasma samples by proton NMR analysis. The fatty acids measured were n-3, docosahexaenoic acid (DHA), n-6, linoleic acid, and polyunsaturated, monounsaturated and saturated fatty acids. HRs were modelled per SD higher (percentage) fatty acid. C statistics and continuous net reclassification improvement were used to test the added value of fatty acids compared with traditional cardiovascular risk factors. RESULTS: After adjustment for traditional cardiovascular risk factors, an inverse association was observed for n-3 fatty acids and DHA with the risk of macrovascular events (HR [95% CI]: 0.87 [0.80, 0.95] and 0.88 [0.81, 0.96], respectively, per 1 SD higher percentage), and for n-3 fatty acids with the risk of death (HR 0.91 [95% CI 0.84, 0.99] per 1 SD higher percentage). Such associations were also evident when investigating absolute levels of fatty acids. There were no statistically significant associations between any fatty acids and microvascular disease after adjustment. However, there was limited improvement in the predictive ability of models when any fatty acid was added. CONCLUSIONS/INTERPRETATION: Plasma n-3 fatty acids and DHA were found to be inversely associated with macrovascular disease, while n-3 fatty acids were also inversely associated with death. These results support the cardioprotective effects of n-3 fatty acids and DHA and further merit testing the role of high-dose supplementation with n-3 fatty acids in individuals with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00145925. Graphical abstract.

NAFLD risk alleles in PNPLA3, TM6SF2, GCKR and LYPLAL1 show divergent metabolic effects.

Fatty liver has been associated with unfavourable metabolic changes in circulation. To provide insights in fatty liver-related metabolic deviations, we compared metabolic association profile of fatty liver versus metabolic association profiles of genotypes increasing the risk of non-alcoholic fatty liver disease (NAFLD). The cross-sectional associations of ultrasound-ascertained fatty liver with 123 metabolic measures were determined in 1810 (Nfatty liver = 338) individuals aged 34-49 years from The Cardiovascular Risk in Young Finns Study. The association profiles of NAFLD-risk alleles in PNPLA3, TM6SF2, GCKR, and LYPLAL1 with the corresponding metabolic measures were obtained from a publicly available metabolomics GWAS including up to 24 925 Europeans. The risk alleles showed different metabolic effects: PNPLA3 rs738409-G, the strongest genetic NAFLD risk factor, did not associate with metabolic changes. Metabolic effects of GCKR rs1260326-T were comparable in many respects to the fatty liver associations. Metabolic effects of LYPLAL1 rs12137855-C were similar, but statistically less robust, to the effects of GCKR rs1260326-T. TM6SF2 rs58542926-T displayed opposite metabolic effects when compared with the fatty liver associations. The metabolic effects of the risk alleles highlight heterogeneity of the molecular pathways leading to fatty liver and suggest that the fatty liver-related changes in the circulating lipids and metabolites may vary depending on the underlying pathophysiological mechanism. Despite the robust cross-sectional associations on population level, the present results showing neutral or cardioprotective metabolic effects for some of the NAFLD risk alleles advocate that hepatic lipid accumulation by itself may not increase the level of circulating lipids or other metabolites.

Genome-wide Association Study Identifies 27 Loci Influencing Concentrations of Circulating Cytokines and Growth Factors.

Circulating cytokines and growth factors are regulators of inflammation and have been implicated in autoimmune and metabolic diseases. In this genome-wide association study (GWAS) of up to 8,293 Finns we identified 27 genome-widely significant loci (p < 1.2 × 10-9) for one or more cytokines. Fifteen of the associated variants had expression quantitative trait loci in whole blood. We provide genetic instruments to clarify the causal roles of cytokine signaling and upstream inflammation in immune-related and other chronic diseases. We further link inflammatory markers with variants previously associated with autoimmune diseases such as Crohn disease, multiple sclerosis, and ulcerative colitis and hereby elucidate the molecular mechanisms underpinning these diseases and suggest potential drug targets.

Metabolomic Signature of Angiopoietin-Like Protein 3 Deficiency in Fasting and Postprandial State.

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, ß-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 ß-oxidation. The detailed metabolic profile in human knockouts of ANGPTL3 reinforces inactivation of ANGPTL3 as a promising therapeutic target for decreasing cardiovascular risk.

Circulating metabolites and the risk of type 2 diabetes: a prospective study of 11,896 young adults from four Finnish cohorts.

AIMS/HYPOTHESIS: Metabolomics technologies have identified numerous blood biomarkers for type 2 diabetes risk in case-control studies of middle-aged and older individuals. We aimed to validate existing and identify novel metabolic biomarkers predictive of future diabetes in large cohorts of young adults. METHODS: NMR metabolomics was used to quantify 229 circulating metabolic measures in 11,896 individuals from four Finnish observational cohorts (baseline age 24-45 years). Associations between baseline metabolites and risk of developing diabetes during 8-15 years of follow-up (392 incident cases) were adjusted for sex, age, BMI and fasting glucose. Prospective metabolite associations were also tested with fasting glucose, 2 h glucose and HOMA-IR at follow-up. RESULTS: Out of 229 metabolic measures, 113 were associated with incident type 2 diabetes in meta-analysis of the four cohorts (ORs per 1 SD: 0.59-1.50; p< 0.0009). Among the strongest biomarkers of diabetes risk were branched-chain and aromatic amino acids (OR 1.31-1.33) and triacylglycerol within VLDL particles (OR 1.33-1.50), as well as linoleic n-6 fatty acid (OR 0.75) and non-esterified cholesterol in large HDL particles (OR 0.59). The metabolic biomarkers were more strongly associated with deterioration in post-load glucose and insulin resistance than with future fasting hyperglycaemia. A multi-metabolite score comprised of phenylalanine, non-esterified cholesterol in large HDL and the ratio of cholesteryl ester to total lipid in large VLDL was associated with future diabetes risk (OR 10.1 comparing individuals in upper vs lower fifth of the multi-metabolite score) in one of the cohorts (mean age 31 years). CONCLUSIONS/INTERPRETATION: Metabolic biomarkers across multiple molecular pathways are already predictive of the long-term risk of diabetes in young adults. Comprehensive metabolic profiling may help to target preventive interventions for young asymptomatic individuals at increased risk.

Metabolomic consequences of genetic inhibition of PCSK9 compared with statin treatment.

Background: Both statins and PCSK9 inhibitors lower blood low-density lipoprotein cholesterol (LDL-C) levels to reduce risk of cardiovascular events. To assess potential differences between metabolic effects of these two 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: 228 circulating metabolic measures were quantified by nuclear magnetic resonance spectroscopy, including lipoprotein subclass concentrations and their lipid composition, fatty acids, and amino acids, for 5,359 individuals (2,659 on treatment) in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) trial at 6-months post-randomization. The corresponding metabolic measures were analyzed in eight 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 LDL-C, the effects of genetic inhibition of PCSK9 on 228 metabolic markers were generally consistent with those of statin therapy (R2=0.88). Alterations in lipoprotein lipid composition and fatty acid distribution were similar. However, discrepancies were observed for very-low-density lipoprotein (VLDL) lipid measures. For instance, genetic inhibition of PCSK9 had weaker effects on lowering of VLDL-cholesterol compared with statin therapy (54% vs. 77% reduction, relative to the lowering effect on LDL-C; 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 VLDL lipids compared with statins for an equivalent lowering of LDL-C, which potentially translate into smaller reductions in cardiovascular disease risk.

Investigating the effects of lycopene and green tea on the metabolome of men at risk of prostate cancer: The ProDiet randomised controlled trial.

Lycopene and green tea consumption have been observationally associated with reduced prostate cancer risk, but the underlying mechanisms have not been fully elucidated. We investigated the effect of factorial randomisation to a 6-month lycopene and green tea dietary advice or supplementation intervention on 159 serum metabolite measures in 128 men with raised PSA levels (but prostate cancer-free), analysed by intention-to-treat. The causal effects of metabolites modified by the intervention on prostate cancer risk were then assessed by Mendelian randomisation, using summary statistics from 44,825 prostate cancer cases and 27,904 controls. The systemic effects of lycopene and green tea supplementation on serum metabolic profile were comparable to the effects of the respective dietary advice interventions (R2 = 0.65 and 0.76 for lycopene and green tea respectively). Metabolites which were altered in response to lycopene supplementation were acetate [ß (standard deviation difference vs. placebo): 0.69; 95% CI = 0.24, 1.15; p = 0.003], valine (ß: -0.62; -1.03, -0.02; p = 0.004), pyruvate (ß: -0.56; -0.95, -0.16; p = 0.006) and docosahexaenoic acid (ß: -0.50; -085, -0.14; p = 0.006). Valine and diacylglycerol were lower in the lycopene dietary advice group (ß: -0.65; -1.04, -0.26; p = 0.001 and ß: -0.59; -1.01, -0.18; p = 0.006). A genetically instrumented SD increase in pyruvate increased the odds of prostate cancer by 1.29 (1.03, 1.62; p = 0.027). An intervention to increase lycopene intake altered the serum metabolome of men at risk of prostate cancer. Lycopene lowered levels of pyruvate, which our Mendelian randomisation analysis suggests may be causally related to reduced prostate cancer risk.

Circulating amino acids and the risk of macrovascular, microvascular and mortality outcomes in individuals with type 2 diabetes: results from the ADVANCE trial.

AIMS/HYPOTHESES: We aimed to quantify the association of individual circulating amino acids with macrovascular disease, microvascular disease and all-cause mortality in individuals with type 2 diabetes. METHODS: We performed a case-cohort study (N = 3587), including 655 macrovascular events, 342 microvascular events (new or worsening nephropathy or retinopathy) and 632 all-cause mortality events during follow-up, in a secondary analysis of the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) study. For this study, phenylalanine, isoleucine, glutamine, leucine, alanine, tyrosine, histidine and valine were measured in stored plasma samples by proton NMR metabolomics. Hazard ratios were modelled per SD increase in each amino acid. RESULTS: In models investigating associations and potential mechanisms, after adjusting for age, sex and randomised treatment, phenylalanine was positively, and histidine inversely, associated with macrovascular disease risk. These associations were attenuated to the null on further adjustment for extended classical risk factors (including eGFR and urinary albumin/creatinine ratio). After adjustment for extended classical risk factors, higher tyrosine and alanine levels were associated with decreased risk of microvascular disease (HR 0.78; 95% CI 0.67, 0.91 and HR 0.86; 95% CI 0.76, 0.98, respectively). Higher leucine (HR 0.79; 95% CI 0.69, 0.90), histidine (HR 0.89; 95% CI 0.81, 0.99) and valine (HR 0.79; 95% CI 0.70, 0.88) levels were associated with lower risk of mortality. Investigating the predictive ability of amino acids, addition of all amino acids to a risk score modestly improved classification of participants for macrovascular (continuous net reclassification index [NRI] +35.5%, p < 0.001) and microvascular events (continuous NRI +14.4%, p = 0.012). CONCLUSIONS/INTERPRETATION: We report distinct associations between circulating amino acids and risk of different major complications of diabetes. Low tyrosine appears to be a marker of microvascular risk in individuals with type 2 diabetes independently of fundamental markers of kidney function.

Effect of Dietary Counseling on a Comprehensive Metabolic Profile from Childhood to Adulthood.

OBJECTIVES: To study the effects of repeated, infancy-onset dietary counseling on a detailed metabolic profile. Effects of dietary saturated fat replacement on circulating concentrations of metabolic biomarkers still remain unknown. STUDY DESIGN: The Special Turku Coronary Risk Factor Intervention Project (STRIP) study is a longitudinal, randomized atherosclerosis prevention trial in which repeated dietary counseling aimed at reducing the proportion of saturated fat intake. Nuclear magnetic resonance metabolomics quantified circulating metabolites from serum samples assessed at age 9 (n = 554), 11 (n = 553), 13 (n = 508), 15 (n = 517), 17 (n = 457), and 19 (n = 417) years. RESULTS: The intervention reduced dietary intake of saturated fat (mean difference in daily percentage of total energy intake: -2.1 [95% CI -1.9 to -2.3]) and increased intake of polyunsaturated fat (0.6 [0.5-0.7]). The dietary counseling intervention led to greater serum proportions of polyunsaturated fatty acids (P < .001), with greater proportions of both circulating omega-3 (P = .02) and omega-6 (P < .001) fatty acids. The proportion of saturated fatty acids in serum was lower for both boys and girls in the intervention group (P < .001), whereas the serum proportion of monounsaturated fat was lower for boys in the intervention group only (P < .001). The intervention also reduced circulating intermediate-density lipoprotein and low-density lipoprotein lipid concentrations (P < .01). Dietary intervention effects on nonlipid biomarkers were minor except from greater concentrations of glutamine in the intervention group. CONCLUSIONS: Repeated dietary counseling from infancy to early adulthood yielded favorable effects on multiple circulating fatty acids and lipoprotein subclass lipids, particularly in boys. These molecular effects substantiate the beneficial role of saturated fat replacement on the metabolic risk profile. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00223600.

Metabolic profiling of fatty liver in young and middle-aged adults: Cross-sectional and prospective analyses of the Young Finns Study.

Nonalcoholic fatty liver is associated with obesity-related metabolic disturbances, but little is known about the metabolic perturbations preceding fatty liver disease. We performed comprehensive metabolic profiling to assess how circulating metabolites, such as lipoprotein lipids, fatty acids, amino acids, and glycolysis-related metabolites, reflect the presence of and future risk for fatty liver in young adults. Sixty-eight lipids and metabolites were quantified by nuclear magnetic resonance metabolomics in the population-based Young Finns Study from serum collected in 2001 (n = 1,575), 2007 (n = 1,509), and 2011 (n = 2,002). Fatty liver was diagnosed by ultrasound in 2011 when participants were aged 34-49 years (19% prevalence). Cross-sectional associations as well as 4-year and 10-year risks for fatty liver were assessed by logistic regression. Metabolites across multiple pathways were strongly associated with the presence of fatty liver (P < 0.0007 for 60 measures in age-adjusted and sex-adjusted cross-sectional analyses). The strongest direct associations were observed for extremely large very-low-density lipoprotein triglycerides (odds ratio [OR] = 4.86 per 1 standard deviation, 95% confidence interval 3.48-6.78), other very-low-density lipoprotein measures, and branched-chain amino acids (e.g., leucine OR = 2.94, 2.51-3.44). Strong inverse associations were observed for high-density lipoprotein measures, e.g., high-density lipoprotein size (OR = 0.36, 0.30-0.42) and several fatty acids including omega-6 (OR = 0.37, 0.32-0.42). The metabolic associations were attenuated but remained significant after adjusting for waist, physical activity, alcohol consumption, and smoking (P < 0.0007). Similar aberrations in the metabolic profile were observed already 10 years before fatty liver diagnosis. CONCLUSION: Circulating lipids, fatty acids, and amino acids reflect fatty liver independently of routine metabolic risk factors; these metabolic aberrations appear to precede the development of fatty liver in young adults. (Hepatology 2017;65:491-500).

Association of branched-chain amino acids and other circulating metabolites with risk of incident dementia and Alzheimer's disease: A prospective study in eight cohorts.

INTRODUCTION: Metabolite, lipid, and lipoprotein lipid profiling can provide novel insights into mechanisms underlying incident dementia and Alzheimer's disease. METHODS: We studied eight prospective cohorts with 22,623 participants profiled by nuclear magnetic resonance or mass spectrometry metabolomics. Four cohorts were used for discovery with replication undertaken in the other four to avoid false positives. For metabolites that survived replication, combined association results are presented. RESULTS: Over 246,698 person-years, 995 and 745 cases of incident dementia and Alzheimer's disease were detected, respectively. Three branched-chain amino acids (isoleucine, leucine, and valine), creatinine and two very low density lipoprotein (VLDL)-specific lipoprotein lipid subclasses were associated with lower dementia risk. One high density lipoprotein (HDL; the concentration of cholesterol esters relative to total lipids in large HDL) and one VLDL (total cholesterol to total lipids ratio in very large VLDL) lipoprotein lipid subclass was associated with increased dementia risk. Branched-chain amino acids were also associated with decreased Alzheimer's disease risk and the concentration of cholesterol esters relative to total lipids in large HDL with increased Alzheimer's disease risk. DISCUSSION: Further studies can clarify whether these molecules play a causal role in dementia pathogenesis or are merely markers of early pathology.

Quantitative Serum Nuclear Magnetic Resonance Metabolomics in Large-Scale Epidemiology: A Primer on -Omic Technologies.

Detailed metabolic profiling in large-scale epidemiologic studies has uncovered novel biomarkers for cardiometabolic diseases and clarified the molecular associations of established risk factors. A quantitative metabolomics platform based on nuclear magnetic resonance spectroscopy has found widespread use, already profiling over 400,000 blood samples. Over 200 metabolic measures are quantified per sample; in addition to many biomarkers routinely used in epidemiology, the method simultaneously provides fine-grained lipoprotein subclass profiling and quantification of circulating fatty acids, amino acids, gluconeogenesis-related metabolites, and many other molecules from multiple metabolic pathways. Here we focus on applications of magnetic resonance metabolomics for quantifying circulating biomarkers in large-scale epidemiology. We highlight the molecular characterization of risk factors, use of Mendelian randomization, and the key issues of study design and analyses of metabolic profiling for epidemiology. We also detail how integration of metabolic profiling data with genetics can enhance drug development. We discuss why quantitative metabolic profiling is becoming widespread in epidemiology and biobanking. Although large-scale applications of metabolic profiling are still novel, it seems likely that comprehensive biomarker data will contribute to etiologic understanding of various diseases and abilities to predict disease risks, with the potential to translate into multiple clinical settings.

Nuclear inelastic scattering and density functional theory studies of a one-dimensional spin crossover [Fe(1,2,4-triazole)2(1,2,4-triazolato)](BF4) molecular chain.

Nuclear inelastic scattering (NIS) experiments have been performed in order to study the vibrational dynamics of the low- and high-spin states of the polynuclear 1D spin crossover compound [Fe(1,2,4-triazole)2(1,2,4-triazolato)](BF4) (1). Density functional theory (DFT) calculations using the functional B3LYP* and the basis set CEP-31G for heptameric and nonameric models of the compound yielded the normal vibrations and electronic energies for high-spin and low-spin isomers of three models differing in the distribution of anionic trz- ligands and BF4- anions. On the basis of the obtained energies a structural model with a centrosymmetric Fe(trzH)4(trz-)2 coordination core of the mononuclear unit of the chain is proposed. The obtained distribution of the BF4- counteranions in the proposed structure is similar to that obtained on the basis of X-ray powder diffraction studies by Grossjean et al. (Eur. J. Inorg. Chem., 2013, 796). The NIS data of the system diluted to 10% Fe(ii) content in a 90% Zn(ii) matrix (compound (2)) show a characteristic change of the spectral pattern of the low-spin centres, compared to the low-spin phase of the parent Fe(ii) complex (1). DFT calculations reveal that this is caused by a change of the structure of the neighbours of the low-spin centres. The spectral pattern of the high-spin centres in (2) is within a good approximation identical to that of the high-spin Fe(ii) isomer of (1). The inspection of the molecular orbitals of the monomeric model systems of [Fe(trzH)4(trz-)2] and [Fe(trzH)6], together with calculations of spin transition energies, point towards the importance of an electrostatic effect caused by the negatively charged ligands. This results in the stabilisation of the low-spin state of the complex containing the anionic ligand and shortening of the Fe-N(trz-) compared to the Fe-N(trzH) bond in high-spin, but not in low-spin [Fe(trzH)4(trz-)2].

Distribution and medical impact of loss-of-function variants in the Finnish founder population.

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5-5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10⁻8) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10⁻¹¹7). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10⁻4), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.

Metabolite profiling and cardiovascular event risk: a prospective study of 3 population-based cohorts.

BACKGROUND: High-throughput profiling of circulating metabolites may improve cardiovascular risk prediction over established risk factors. METHODS AND RESULTS: We applied quantitative nuclear magnetic resonance metabolomics to identify the biomarkers for incident cardiovascular disease during long-term follow-up. Biomarker discovery was conducted in the National Finnish FINRISK study (n=7256; 800 events). Replication and incremental risk prediction was assessed in the Southall and Brent Revisited (SABRE) study (n=2622; 573 events) and British Women's Health and Heart Study (n=3563; 368 events). In targeted analyses of 68 lipids and metabolites, 33 measures were associated with incident cardiovascular events at P<0.0007 after adjusting for age, sex, blood pressure, smoking, diabetes mellitus, and medication. When further adjusting for routine lipids, 4 metabolites were associated with future cardiovascular events in meta-analyses: higher serum phenylalanine (hazard ratio per standard deviation, 1.18; 95% confidence interval, 1.12-1.24; P=4×10(-10)) and monounsaturated fatty acid levels (1.17; 1.11-1.24; P=1×10(-8)) were associated with increased cardiovascular risk, while higher omega-6 fatty acids (0.89; 0.84-0.94; P=6×10(-5)) and docosahexaenoic acid levels (0.90; 0.86-0.95; P=5×10(-5)) were associated with lower risk. A risk score incorporating these 4 biomarkers was derived in FINRISK. Risk prediction estimates were more accurate in the 2 validation cohorts (relative integrated discrimination improvement, 8.8% and 4.3%), albeit discrimination was not enhanced. Risk classification was particularly improved for persons in the 5% to 10% risk range (net reclassification, 27.1% and 15.5%). Biomarker associations were further corroborated with mass spectrometry in FINRISK (n=671) and the Framingham Offspring Study (n=2289). CONCLUSIONS: Metabolite profiling in large prospective cohorts identified phenylalanine, monounsaturated fatty acids, and polyunsaturated fatty acids as biomarkers for cardiovascular risk. This study substantiates the value of high-throughput metabolomics for biomarker discovery and improved risk assessment.

Metabolic profiling of pregnancy: cross-sectional and longitudinal evidence.

BACKGROUND: Pregnancy triggers well-known alterations in maternal glucose and lipid balance but its overall effects on systemic metabolism remain incompletely understood. METHODS: Detailed molecular profiles (87 metabolic measures and 37 cytokines) were measured for up to 4260 women (24-49 years, 322 pregnant) from three population-based cohorts in Finland. Circulating molecular concentrations in pregnant women were compared to those in non-pregnant women. Metabolic profiles were also reassessed for 583 women 6 years later to uncover the longitudinal metabolic changes in response to change in the pregnancy status. RESULTS: Compared to non-pregnant women, all lipoprotein subclasses and lipids were markedly increased in pregnant women. The most pronounced differences were observed for the intermediate-density, low-density and high-density lipoprotein triglyceride concentrations. Large differences were also seen for many fatty acids and amino acids. Pregnant women also had higher concentrations of low-grade inflammatory marker glycoprotein acetyls, higher concentrations of interleukin-18 and lower concentrations of interleukin-12p70. The changes in metabolic concentrations for women who were not pregnant at baseline but pregnant 6 years later (or vice versa) matched (or were mirror-images of) the cross-sectional association pattern. Cross-sectional results were consistent across the three cohorts and similar longitudinal changes were seen for 653 women in 4-year and 497 women in 10-year follow-up. For multiple metabolic measures, the changes increased in magnitude across the three trimesters. CONCLUSIONS: Pregnancy initiates substantial metabolic and inflammatory changes in the mothers. Comprehensive characterisation of normal pregnancy is important for gaining understanding of the key nutrients for fetal growth and development. These findings also provide a valuable molecular reference in relation to studies of adverse pregnancy outcomes.

Diabetes risk and amino acid profiles: cross-sectional and prospective analyses of ethnicity, amino acids and diabetes in a South Asian and European cohort from the SABRE (Southall And Brent REvisited) Study.

AIMS/HYPOTHESIS: South Asian individuals have an increased risk of diabetes compared with Europeans that is unexplained by obesity and traditional or established metabolic measures. Circulating amino acids (AAs) may provide additional explanatory insights. In a unique cohort of European and South Asian men, we compared cross-sectional associations between AAs, metabolic and obesity traits, and longitudinal associations with incident diabetes. METHODS: Nuclear magnetic spectroscopy was used to measure the baseline (1988-1991) levels of nine AAs in serum samples from a British population-based cohort of 1,279 European and 1,007 South Asian non-diabetic men aged 40-69 years. Follow-up was complete for 19 years in 801 European and 643 South Asian participants. RESULTS: The serum concentrations of isoleucine, phenylalanine, tyrosine and alanine were significantly higher in South Asian men, while cross-sectional correlations of AAs with glycaemia and insulin resistance were similar in the two ethnic groups. However, most AAs were less strongly correlated with measures of obesity in the South Asian participants. Diabetes developed in 227 (35%) South Asian and 113 (14%) European men. Stronger adverse associations were observed between branched chain and aromatic AAs and incident diabetes in South Asian men. Tyrosine was a particularly strong predictor of incident diabetes in South Asian individuals, even after adjustment for metabolic risk factors, including obesity and insulin resistance (adjusted OR for a 1 SD increment, 1.47, 95% CI 1.17,1.85, p = 0.001) compared with Europeans (OR 1.10, 0.87, 1.39, p = 0.4; p = 0.045 for ethnicity × tyrosine interaction). CONCLUSIONS/INTERPRETATION: Branched chain and aromatic AAs, particularly tyrosine, may be a focus for identifying novel aetiological mechanisms and potential treatment targets for diabetes in South Asian populations and may contribute to their excess risk of diabetes.

High birth weight is associated with obesity and increased carotid wall thickness in young adults: the cardiovascular risk in young Finns study.

OBJECTIVE: There is some evidence that people born with high birth weight may be at increased risk of cardiovascular disease in adulthood. Details of the underlying mechanisms remain unknown. We sought to determine whether people born large for gestational age have poor arterial health, increased adiposity, and a poor cardiovascular risk factor profile. APPROACH AND RESULTS: Carotid intima-media thickness, brachial flow-mediated dilatation, and cardiovascular risk factors were compared between young adults (24-45 years) born at term who were large for gestational age (birth weight >90th percentile; n=171), and a control group with normal birth weight (50-75th percentile; n=525), in the Cardiovascular Risk in Young Finns Study. Those born large for gestational age had higher body mass index throughout childhood, adolescence, and as young adults (26.4 kg/m(2) [SD 4.9], versus normal birth weight 25.1 kg/m(2) [SD 4.6]; P=0.002), and 2-fold greater risk of obesity. Other cardiovascular risk factors and arterial function did not differ; however, carotid intima-media thickness was increased in people born large for gestational age (0.60 mm [SD 0.09], versus normal birth weight 0.57 mm [SD 0.09]; P=0.003), independent of cardiovascular risk factors (P=0.001 after adjustment). Both obesity and high birth weight were independently associated with carotid intima-media thickness in a graded and additive fashion. CONCLUSIONS: Young adults born large for gestational age are more likely to be obese, yet have an otherwise healthy cardiovascular risk profile. Nonetheless, they have increased carotid intima-media thickness, a marker of subclinical atherosclerosis, consistent with an increased risk of cardiovascular disease.