Serum metabolomics analysis reveals increased lipid catabolism in mildly hyperbilirubinemic Gilbert's syndrome individuals.

BACKGROUND: The protective role of mildly elevated bilirubin against CVD and diabetes mellitus type 2 (DMT2) is associated with a favorable lipid phenotype. As the mechanistic understanding of this protection in humans remains elusive, we aimed to assess the metabolomics profile of mildly hyperbilirubinemic (Gilbert's syndrome; GS) individuals especially targeting lipid catabolism. METHODS AND RESULTS: Using NMR serum metabolomics of 56 GS individuals and 56 age and gender-matched healthy controls, GS individuals demonstrated significantly greater concentrations of acetylcarnitine (+20%, p < 0.001) and the ketone bodies, 3-hydroxybutyric acid (+132%, p < 0.001), acetoacetic acid (+95%, p < 0.001) and acetone (+46%, p < 0.001). Metabolites associated with an increased mitochondrial lipid metabolism such as citrate (+15%, p < 0.001), anaplerotic amino acid intermediates and creatinine were significantly greater and creatine significantly reduced in GS individuals. Stimulators of lipid catabolism including AMPK (+59%, p < 0.001), pPPARα (+24%, p < 0.001) and T3 (+9%, p = 0.009) supported the metabolomics data while concomitantly blood glucose and insulin (-33%, p = 0.002) levels were significantly reduced. We further showed that the increased lipid catabolism partially mediates the favorable lipid phenotype (lower triglycerides) of GS individuals. Increased trimethylamine (+35%, p < 0.001) indicated changes in trimethylamine metabolism, an emerging predictor of metabolic health. CONCLUSION: We showed an enhanced lipid catabolism in mildly hyperbilirubinemic individuals, novel evidence as to why these individuals are leaner and protected against chronic metabolic diseases emphasizing bilirubin to be a promising future target in obese and dyslipidemia patients.

Improved Automated Quantification Algorithm (AQuA) and Its Application to NMR-Based Metabolomics of EDTA-Containing Plasma.

We have recently presented an Automated Quantification Algorithm (AQuA) and demonstrated its utility for rapid and accurate absolute metabolite quantification in 1H NMR spectra in which positions and line widths of signals were predicted from a constant metabolite spectral library. The AQuA quantifies based on one preselected signal per metabolite and employs library spectra to model interferences from other metabolite signals. However, for some types of spectra, the interspectral deviations of signal positions and line widths can be pronounced; hence, interferences cannot be modeled using a constant spectral library. We here address this issue and present an improved AQuA that handles interspectral deviations. The improved AQuA monitors and characterizes the appearance of specific signals in each spectrum and automatically adjusts the spectral library to model interferences accordingly. The performance of the improved AQuA was tested on a large data set from plasma samples collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant (n = 772). These spectra provided a suitable test system for the improved AQuA since EDTA signals (i) vary in intensity, position, and line width between spectra and (ii) interfere with many signals from plasma metabolites targeted for quantification (n = 54). Without the improvement, ca. 20 out of the 54 metabolites would have been overestimated. This included acetylcarnitine and ornithine, which are considered particularly difficult to quantify with 1H NMR in EDTA-containing plasma. Furthermore, the improved AQuA performed rapidly (<10 s for all spectra). We believe that the improved AQuA provides a basis for automated quantification in other data sets where specific signals show interspectral deviations.

Differences in metabolic profiles between the Burmese, the Maine coon and the Birman cat-Three breeds with varying risk for diabetes mellitus.

Feline diabetes mellitus shares many features with type 2 diabetes in people, regarding clinical presentation, physiology, and pathology. A breed predisposition for type 2 diabetes has been identified, with the Burmese breed at a fivefold increased risk of developing the condition compared to other purebred cats. We aimed to characterize the serum metabolome in cats (n = 63) using nuclear magnetic resonance metabolomics, and to compare the metabolite pattern of Burmese cats with that of two cat breeds of medium or low risk of diabetes, the Maine coon (MCO) and Birman cat, respectively. Serum concentrations of adiponectin, insulin and insulin-like growth factor-1 were also measured (n = 94). Burmese cats had higher insulin and lower adiponectin concentrations than MCO cats. Twenty one metabolites were discriminative between breeds using a multivariate statistical approach and 15 remained significant after adjustment for body weight and body condition score. Burmese cats had higher plasma levels of 2-hydroxybutyrate relative to MCO and Birman cats and increased concentrations of 2-oxoisocaproic acid, and tyrosine, and lower concentrations of dimethylglycine relative to MCO cats. The metabolic profile of MCO cats was characterized by high concentrations of arginine, asparagine, methionine, succinic acid and low levels of acetylcarnitine while Birman cats had the highest creatinine and the lowest taurine plasma levels, compared with MCO and Burmese. The pattern of metabolites in Burmese cats is similar to that in people with insulin resistance. In conclusion, the metabolic profile differed between healthy cats of three breeds. Detection of an abnormal metabolome might identify cats at risk of developing diabetes.

Metabolomics analysis reveals altered metabolites in lean compared with obese adolescents and additional metabolic shifts associated with hyperinsulinaemia and insulin resistance in obese adolescents: a cross-sectional study.

INTRODUCTION: Hyperinsulinaemia and insulin resistance (IR) are strongly associated with obesity and are forerunners of type 2 diabetes. Little is known about metabolic alterations separately associated with obesity, hyperinsulinaemia/IR and impaired glucose tolerance (IGT) in adolescents. OBJECTIVES: To identify metabolic alterations associated with obesity, hyperinsulinaemia/IR and hyperinsulinaemia/IR combined with IGT in obese adolescents. METHODS: 81 adolescents were stratified into four groups based on body mass index (lean vs. obese), insulin responses (normal insulin (NI) vs. high insulin (HI)) and glucose responses (normal glucose tolerance (NGT) vs. IGT) after an oral glucose tolerance test (OGTT). The groups comprised: (1) healthy lean with NI and NGT, (2) obese with NI and NGT, (3) obese with HI and NGT, and (4) obese with HI and IGT. Targeted nuclear magnetic resonance-based metabolomics analysis was performed on fasting and seven post-OGTT plasma samples, followed by univariate and multivariate statistical analyses. RESULTS: Two groups of metabolites were identified: (1) Metabolites associated with insulin response level: adolescents with HI (groups 3-4) had higher concentrations of branched-chain amino acids and tyrosine, and lower concentrations of serine, glycine, myo-inositol and dimethylsulfone, than adolescents with NI (groups 1-2). (2) Metabolites associated with obesity status: obese adolescents (groups 2-4) had higher concentrations of acetylcarnitine, alanine, pyruvate and glutamate, and lower concentrations of acetate, than lean adolescents (group 1). CONCLUSIONS: Obesity is associated with shifts in fat and energy metabolism. Hyperinsulinaemia/IR in obese adolescents is also associated with increased branched-chain and aromatic amino acids.

Glucose Appearance Rate Rather than the Blood Glucose Concentrations Explains Differences in Postprandial Insulin Responses between Wholemeal Rye and Refined Wheat Breads-Results from A Cross-Over Meal Study.

SCOPE: Ingestion of rye bread leads to lower postprandial plasma insulin concentrations than wheat bread ingestion, but most often not too different glucose profiles. The mechanism behind this discrepancy is still largely unknown. This study investigates whether glucose kinetics may explain the observed discrepancy. METHODS AND RESULTS: Nine healthy men participated in a crossover study, eating 50 g of available carbohydrates as either refined wheat (WB) or traditional wholemeal rye bread (WMR) during d-[6,6-2 H2 ]glucose infusion. Labeled glucose enrichment is measured by an HPLC-TOF-MS method. The calculated rate of glucose appearance (RaE) is significantly lower after ingestion of WMR during the initial 15 min postprandial period. Additionally, the 0-90 min RaE area under the curve (AUC) is significantly lower after ingestion of WMR, as is plasma gastric inhibitory polypeptide (GIP) at 60 and 90 min. Postprandial glycemic responses do not differ between the breads. Postprandial insulin is lower after ingestion of WMR at 45 and 60 min, as is the 0-90 min AUC. CONCLUSION: Ingestion of WMR elicits a lower rate of glucose appearance into the bloodstream compared with WB. This may explain the lower insulin response observed after rye bread ingestion, commonly known as the rye factor.

AQuA: An Automated Quantification Algorithm for High-Throughput NMR-Based Metabolomics and Its Application in Human Plasma.

A key limiting step for high-throughput NMR-based metabolomics is the lack of rapid and accurate tools for absolute quantification of many metabolites. We developed, implemented, and evaluated an algorithm, AQuA (Automated Quantification Algorithm), for targeted metabolite quantification from complex 1H NMR spectra. AQuA operates based on spectral data extracted from a library consisting of one standard calibration spectrum for each metabolite. It uses one preselected NMR signal per metabolite for determining absolute concentrations and does so by effectively accounting for interferences caused by other metabolites. AQuA was implemented and evaluated using experimental NMR spectra from human plasma. The accuracy of AQuA was tested and confirmed in comparison with a manual spectral fitting approach using the ChenomX software, in which 61 out of 67 metabolites quantified in 30 human plasma spectra showed a goodness-of-fit (r2) close to or exceeding 0.9 between the two approaches. In addition, three quality indicators generated by AQuA, namely, occurrence, interference, and positional deviation, were studied. These quality indicators permit evaluation of the results each time the algorithm is operated. The efficiency was tested and confirmed by implementing AQuA for quantification of 67 metabolites in a large data set comprising 1342 experimental spectra from human plasma, in which the whole computation took less than 1 s.

Metabolomics Approach To Evaluate a Baltic Sea Sourced Diet for Cultured Arctic Char (Salvelinus alpinus L.).

Aqua feeds traditionally rely on fishmeal as a protein source, which is costly and unsustainable. A new feed was formulated in the study with Baltic Sea sourced decontaminated fishmeal, Mytilus edulis and Saccharomyces cerevisiae, and given to Arctic char (Salvelinus alpinus) for ten months. The diet-induced changes on metabolic profile in fish plasma, liver, and muscle were studied relative to a fishmeal-based standard diet by using a 1H NMR-based metabolomics approach. Fish fed the test diet had higher content of betaine and lower levels of trimethylamine-N-oxide and aromatic amino acids in plasma or tissues, which were mainly caused by the diet. The metabolomics results are useful to understand the mechanism of lower body mass, smaller Fulton's condition factor, and a tendency of less lipid content observed in fish fed the test diet. Thus, modifications on the dietary levels of these compounds in the feed are needed to achieve better growth performance.

Metabolic changes in serum metabolome in response to a meal.

PURPOSE: The change in serum metabolic response from fasting state to postprandial state provides novel insights into the impact of a single meal on human metabolism. Therefore, this study explored changes in serum metabolite profile after a single meal. METHODS: Nineteen healthy postmenopausal women with normal glucose tolerance participated in the study. They received a meal consisting of refined wheat bread (50 g carbohydrates, 9 g protein, 4.2 g fat and 2.7 g dietary fibre), 40 g cucumber and 300 mL noncaloric orange drink. Blood samples were collected at fasting and five postprandial time points. Metabolic profile was measured by nuclear magnetic resonance and targeted liquid chromatography-mass spectrometry. Changes over time were assessed with multivariate models and ANOVA, with baseline as control. RESULTS: The metabolomic analyses demonstrated alterations in phospholipids, amino acids and their breakdown products, glycolytic products, acylcarnitines and ketone bodies after a single meal. More specifically, phosphatidylcholines, lysophosphatidylcholines and citrate displayed an overall declining pattern, while leucine, isoleucine, methionine and succinate increased initially but declined thereafter. A sharp decline in acylcarnitines and ketone bodies and increase in glycolytic products postprandially suggest a switch in the body's energy source from ß-oxidation to glycolysis. Moreover, individuals with relatively high postprandial insulin responses generated a higher postprandial leucine responses compared to participants with lower insulin responses. CONCLUSIONS: The study demonstrated complex changes from catabolic to anabolic metabolism after a meal and indicated that the extent of postprandial responses is different between individuals with high and low insulin response.

Nuclear anomalies in exfoliated buccal cells in healthy and diabetic individuals and the impact of a dietary intervention.

This study aimed to compare the frequencies of nuclear anomalies in buccal cells between diabetic and non-diabetic individuals and to assess the impact of a 'healthy diet'-a cornerstone in the treatment of diabetes. Seventy-six diabetic and 21 non-diabetic individuals participated in this parallel, randomised, intervention trial. All participants received information about the importance of a healthy diet, while participants randomly assigned to the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids (PUFA) per day for 8 weeks. Cytogenetic damage in buccal cells was assessed at baseline and after 8 weeks using the buccal micronucleus cytome assay. Micronucleus (MN) frequency at baseline was significantly higher in participants with diabetes (0.58±0.30‰) compared with non-diabetic individuals (0.28±0.29‰). Further analysis of baseline data revealed significantly higher MN levels in participants of the highest tertile of waist circumference (+40%), fasting plasma glucose (+55%), glycated haemoglobin (+41%) and cardiovascular disease risk (+39%) relative to participants of the lowest tertile. The dietary intervention had no effect on MN frequencies. Glycated haemoglobin and biomarkers reflecting cytokinetic defect or acute cell death were reduced in both the intervention and 'information only' groups. The results of this study suggest a strong impact of abdominal obesity and glucose metabolism on genomic stability. Similar effects on nuclear anomalies were observed in the 'information only' group and the intervention group receiving vegetables and PUFA-rich plant oil.

Anti-genotoxic potential of bilirubin in vivo: damage to DNA in hyperbilirubinemic human and animal models.

The bile pigment bilirubin is a known antioxidant and is associated with protection from cancer and cardiovascular disease (CVD) when present in too strong concentrations. Unconjugated bilirubin (UCB) might also possess anti-genotoxic potential by preventing oxidative damage to DNA. Moderately elevated bilirubin levels are found in individuals with Gilbert syndrome and more severe in the hyperbilirubinemic Gunn rat model. This study was therefore aimed to assess the levels of oxidative damage to DNA in Gilbert syndrome subjects and Gunn rats compared to matched controls. Seventy-six individuals (age- and sex-matched) were allocated into Gilbert syndrome (UCB ≥17.1 µmol/L; n = 38) or control groups (UCB < 17.1 µmol/L; n = 38). In addition, 40 Gunn rats were used to support the results of the human trial. Single-cell gel electrophoresis (SCGE) assay measuring standard conditions (strand breaks, apurinic/apyrimidinic sites) and formamidopyrimidine glycosylase (FPG)-sensitive sites was conducted in human peripheral blood mononuclear cells (PBMC) and rat PBMCs, colon, and hepatocytes. Furthermore, urinary 8-oxo-2'-deoxyguanosine (8oxodGuo, DNA oxidation) and 8-oxo-guanosine (8oxoGuo, RNA oxidation) were measured in humans. The Gilbert syndrome and Gunn rat groups had significantly higher UCB levels (P < 0.001) than the corresponding controls. No further differences in damage to DNA or RNA were detected between the two groups, except higher strand breaks (PBMCs) in Gunn rats when compared with controls. However, when demographic effects were analyzed, lower 8oxodGuo concentrations were detected in the human group with a BMI ≥25 kg/m(2) (1.70 ± 0.67 vs. 1.38 ± 0.43 nmol/mmol creatinine, P < 0.05), although this group showed lower UCB levels than normal weight subjects. This study suggests that the disease preventative effect of UCB is unrelated to DNA oxidation/strand breaks in human and animal models of hyperbilirubinaemia.

Haem catabolism: a novel modulator of inflammation in Gilbert's syndrome.

BACKGROUND: Moderately elevated unconjugated bilirubin concentrations protect against inflammatory diseases and are present in individuals with Gilbert's syndrome. This study examined the relationship between circulating haem oxygenase catabolites, unconjugated bilirubin, carboxy haemoglobin, iron and inflammatory parameters. MATERIALS AND METHODS: Seventy-six matched individuals were allocated to Gilbert's syndrome (GS) or control group (unconjugated bilirubin ≥ or < 17.1 µM). Iron, carboxy haemoglobin and high-sensitivity C-reactive protein were analysed using routine diagnostic tests. Unconjugated bilirubin and haem were analysed using high-performance liquid chromatography. The cytokines IL-1ß, TNF-α and IL-6 were assessed using high-sensitivity enzyme-linked immunosorbent assays. RESULTS: Gilbert's syndrome subjects had significantly greater levels of unconjugated bilirubin (P < 0.05), carboxy haemoglobin (P < 0.05), iron (P < 0.05), IL-1ß (P < 0.05), a significantly lower body mass index (P < 0.05) and IL-6 concentrations (P < 0.05) vs. controls. Regression analysis revealed that unconjugated bilirubin mainly explained IL-1ß results (16%), and body mass index+IL-6 predicted 26% of the variance in C-reactive protein concentrations. CONCLUSIONS: A positive relationship between unconjugated bilirubin and free plasma haem, iron and carboxy haemoglobin indicated a positive feedback loop of haem oxygenase induction possibly mediated by unconjugated bilirubin. Furthermore, reduced body mass index in Gilbert's syndrome individuals was linked to reduced inflammation status, which could be influenced by circulating haem oxygenase catabolites and contribute to reduced risk of noncommunicable diseases in this population.

Genome damage in peripheral blood lymphocytes of diabetic and non-diabetic individuals after intervention with vegetables and plant oil.

Recent studies suggest increased cancer risk in patients with type 2 diabetes mellitus (T2DM) compared with healthy individuals. The present study aims to assess whether T2DM is associated with increased genome instability and whether a healthy diet with natural foods can improve genome stability in peripheral blood lymphocytes (PBLs). Seventy-six diabetic and 21 non-diabetic individuals were randomly assigned to either an 'intervention' or an 'information only' group. All participants received information about the beneficial effects of a healthy diet, while subjects of the intervention group received additionally 300g of vegetables and 25ml of plant oil rich in polyunsaturated fatty acids per day for 8 weeks. Chromosomal damage was assessed using the cytokinesis-block micronucleus (MN) cytome assay. Levels of chromosomal damage did not differ between diabetic and non-diabetic individuals. However, diabetic individuals with MN frequency above the high 50th percentile had significantly higher levels of fasting plasma glucose, glycosylated haemoglobin and were at higher risk for cardiovascular disease (CVD), assessed by the Framingham general cardiovascular risk score. Non-diabetic individuals with MN frequency above the 50th percentile had significantly lower vitamin B12 levels. The intervention with vegetables and plant oil led to significant increases in folate, γ-tocopherol, α- and ß-carotene while vitamin B12 was significantly reduced. Levels of chromosomal damage were not altered, only apoptosis was slightly increased. The results suggest interactions between glycaemic control, CVD risk and genome stability in individuals with T2DM. However, a healthy diet does not improve genome damage in PBLs.

Vegetables and PUFA-rich plant oil reduce DNA strand breaks in individuals with type 2 diabetes.

SCOPE: Type 2 diabetes is a multifactorial disease associated with increased oxidative stress, which may lead to increased DNA damage. The aim of this study was to investigate the effect of a healthy diet on DNA oxidation in diabetics and nondiabetics. METHODS AND RESULTS: Seventy-six diabetic and 21 nondiabetic individuals participated in this study. All subjects received information about the benefits of a healthy diet, while subjects randomly assigned to the intervention group received additionally 300 g of vegetables and 25 mL PUFA-rich plant oil per day. DNA damage in mononuclear cells (Comet Assay), urinary excretion of 8-oxo-7-hydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and glycated hemoglobin (HbA1c) were measured at baseline, after 4, 8 (end of intervention), and 16 weeks. The intervention with vegetables and PUFA-rich oil led to a significant increase in plasma antioxidant concentrations. Diabetic individuals of the intervention group showed a significant reduction in HbA1c and DNA strand breaks. Levels of HbA1c were also improved in diabetics of the information group, but oxidative damage to DNA was not altered. Urinary 8-oxodG and 8-oxoGuo excretion remained unchanged in both groups. CONCLUSIONS: This study provides evidence that a healthy diet rich in antioxidants reduces levels of DNA strand breaks in diabetic individuals.

Vitamin and antioxidant rich diet increases MLH1 promoter DNA methylation in DMT2 subjects.

BACKGROUND: Oxidative stress may lead to an increased level of unrepaired cellular DNA damage, which is discussed as one risk for tumor initiation. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. One important gene in the MMR complex is the MutL homolog 1 (MLH1) gene. Since a diet rich in antioxidants has the potential to counteract harmful effects by reactive oxygen species (ROS), we investigated the impact of an antioxidant, folate, and vitamin rich diet on the epigenetic pattern of MLH1. These effects were analyzed in individuals with non-insulin depended diabetes mellitus type 2 (NIDDM2) and impaired fasting glucose (IFG). METHODS: In this post-hoc analysis of a randomized trial we analyzed DNA methylation of MLH1, MSH2, and MGMT at baseline and after 8 weeks of intervention, consisting of 300 g vegetables and 25 ml plant oil rich in polyunsaturated fatty acids per day. DNA methylation was quantified using combined bisulfite restriction enzyme analysis (COBRA) and pyrosequencing. MLH1 and DNMT1 mRNA expression were investigated by qRT-PCR. DNA damage was assessed by COMET assay. Student's two-tailed paired t test and one-way ANOVA with Scheffé corrected Post hoc test was used to determine significant methylation and expression differences. Two-tailed Pearson test was used to determine correlations between methylation level, gene expression, and DNA strand break amount. RESULTS: The intervention resulted in significantly higher CpG methylation in two particular MLH1 promoter regions and the MGMT promoter. DNA strand breaks and methylation levels correlated significantly. The expression of MLH1, DNMT1, and the promoter methylation of MSH2 remained stable. CpG methylation levels and gene expression did not correlate. CONCLUSION: This vitamin and antioxidant rich diet affected the CpG methylation of MLH1. The higher methylation might be a result of the ROS scavenging antioxidant rich diet, leading to lower activity of DNA demethylating enzymes. Our results suggest the hypothesis of CpG demethylation via DNA repair enzymes under these circumstances. NIDDM2 and IFG patients benefit from this simple dietary intervention involving epigenetic and DNA repair mechanisms.