Increases in nervonic acid following RYGB.

<p>A significant increase in serum abundance of nervonic acid occurred following RYGB in both OB and OB/D subjects, as assessed using a mixed-effects ANOVA with group:time interaction (** <i>p</i><0.01 versus T0). Overall, nervonic acid levels were significantly different at each time point between OB and OB/D subgroups (p<0.01, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007905#pone-0007905-t002" target="_blank"><b>TABLE 2</b></a>). No group:time interaction was identified. Mean metabolite abundance±SEM is indicated in red.</p

Estimates for HOMA-IR before and after RYGB.

<p>HOMA-IR was estimated for 12 subjects, 9 OB and 3 OB/D subjects. The OB/D subjects were treated with metformin and not with insulin. A significant reduction (T0 → T3, p = 0.014; T0 → T6, p = 0.001; T3 → T6, p = 0.123) in HOMA-IR occurred following RYGB, as assessed using a Friedman test. Prior to RYGB, significant variability in HOMA-IR estimations was observed between subjects (because OB and OB/D subjects are combined); however, post RYGB, all subjects demonstrated a major improvement in insulin sensitivity (illustrated by smaller error bars). Box plots indicate no outlying data (i.e. above or below the whiskers), and the band in the middle of the box indicates the median. ** <i>p</i><0.01.</p

Increases in 1,5-anhydrosorbitol and decreases in ascorbic acid following RYGB.

<p>The top two metabolites distinguishing OB and OB/D subjects were identified with the mixed-effects ANOVA allowing for group:time-interactions, where * indicates <i>p</i><0.05 (versus T0). White circles and black circles correspond to OB/D or OB subjects, respectively. Furthermore, symbols and numbers are consistently used for the same subject in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007905#pone-0007905-g004" target="_blank">Figures 4</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007905#pone-0007905-g005" target="_blank">5</a>. Mean metabolite abundance±SEM is indicated in red. A) A significant increase in 1,5-anhydrosorbitol (1,5-AG) occurred at T6 vs. T0 in OB/D subjects, while a non-significant increase was seen in OB subjects (group:time interaction was allowed for by the linear model). B) For ascorbic acid, the ANOVA model did not detect group-specific changes over time (no group:time interaction), but indicated significant decreases from T0 to T6 in both groups. Overall, 1,5-anhydrosorbitol and ascorbic acid were significantly different at each time point between OB and OB/D subgroups (p<0.01, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007905#pone-0007905-t002" target="_blank"><b>TABLE 2</b></a>).</p

Clinical data in subjects at all 3 time points (T0, T3, and T6) examined.

<p>All 14 subjects are included in the analysis of parameters related to body weight and lipids. Decreases after RYGB were observed for BMI, weight, fat mass, fat free mass, resting energy expenditure and triglycerides. Leptin was also decreased significantly. While HDL-cholesterol decreased from T0 to T3, HDL-Cholesterol levels recovered by T6 and are confirmed by the lack of change in Apo-A1 levels. Total caloric intake decreased after RYGB; however, the relative proportion of lipid, carbohydrate, and protein consumed remained stable. When considering the 12 subjects not treated with insulin, glucose and insulin levels decreased post RYGB. Estimates for HOMA-IR and HOMA%B decreased while HOMA%S increased after surgery. Data presented as mean±standard error. * represents p<0.1 and ** represents p<0.05, assessed by a Friedman test.</p

Various profiles for metabolites that change significantly at some point following RYGB.

<p>A profile is characterized by 3 dots, which represent T0 (prior to RYGB), T3 and T6 (post surgery). An angled slope between two time points indicates a significant change (p<0.05) and a flat slope between two time points indicates non-significant changes. Based on data derived from the mixed-effects ANOVA using all 14 subjects together. (*1): Structure annotation is based on strong analytical evidence (combinations of chromatography, mass spectrometry, chemical reactions, deuterium-labeling, database and literature search, as well as comparisons to similar/homologue/isomeric reference compounds). (*2): Metabolite exhibits identical qualitative analytical characteristics (chromatography and mass spectrometry) compared to status (*1). Further structural and analytical investigations of this metabolite - also in comparison to structurally identified or status (*1) metabolites - are still pending.</p

Metabolite lists differentiating OB from OB/D subjects at each time point.

<p>Metabolites were identified using a mixed-effects ANOVA: 33 metabolites at T0, 32 metabolites at T3, and 28 metabolites at T6 (p<0.05), with indication of whether serum levels are higher in obese (OB) or obese/diabetic (OB/D) subjects. Those metabolites present in all three lists are indicated in bold font. The profiles for metabolites whose abundance changed post surgery are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007905#pone-0007905-g001" target="_blank"><b>FIGURE 1</b></a>. (*1): Structure annotation is based on strong analytical evidence (combinations of chromatography, mass spectrometry, chemical reactions, deuterium-labeling, database and literature search, as well as comparisons to similar/homologue/isomeric reference compounds). (*2): Metabolite exhibits identical qualitative analytical characteristics (chromatography and mass spectrometry) compared to status (*1). Further structural and analytical investigations of this metabolite - also in comparison to structurally identified or status (*1) metabolites - are still pending.</p

A New Metabolomic Signature in Type-2 Diabetes Mellitus and Its Pathophysiology

<div><p>Objective</p><p>The objective of the current study was to find a metabolic signature associated with the early manifestations of type-2 diabetes mellitus.</p><p>Research Design and Method</p><p>Modern metabolic profiling technology (MxP™ Broad Profiling) was applied to find early alterations in the plasma metabolome of type-2 diabetic patients. The results were validated in an independent study. Eicosanoid and single inon monitoring analysis (MxP™ Eicosanoid and MxP™ SIM analysis) were performed in subsets of samples.</p><p>Results</p><p>A metabolic signature including significantly increased levels of glyoxylate as a potential novel marker for early detection of type-2 diabetes mellitus was identified in an initial study (Study1). The signature was significantly altered in fasted diabetic and pre-diabetic subjects and in non-fasted subjects up to three years prior to the diagnosis of type-2 diabetes; most alterations were also consistently found in an independent patient group (Study 2). In Study 2 diabetic and most control subjects suffered from heart failure. In Study 1 a subgroup of diabetic subjects, with a history of use of anti-hypertensive medication further showed a more pronounced increase of glyoxylate levels, compared to a non-diabetic control group when tested in a hyperglycemic state. In the context of a prior history of anti-hypertensive medication, alterations in hexosamine and eicosanoid levels were also found.</p><p>Conclusion</p><p>A metabolic signature including glyoxylate was associated with type-2 diabetes mellitus, independent of the fasting status and of occurrence of another major disease. The same signature was also found to be associated with pre-diabetic subjects. Glyoxylate levels further showed a specifically strong increase in a subgroup of diabetic subjects. It could represent a new marker for the detection of medical subgroups of diabetic subjects.</p></div

Other than glucose, glyoxylate levels are strongly increased in a defined subgroup of diabetic patients.

<p>Scatter plots of glucose and glyoxylate levels show an increase of glyoxlate during hyperglycemic stress (OGTT_t = 120) which was observed to be stronger in a specific subgroup of diabetic patients with a history of anti-hypertensive medication intake. For glucose, no such specific increase in diabetic patients with a history of taking anti-hypertensive medication was seen. Subjects with anti-hypertension medication (med) are represented by circles; subjects with no history of anti-hypertensive medication (no med) are represented by crosses. Study participants were categorized as type-2 diabetics (n = 47) or control (n = 51) based on FPG and/or OGTT_t = 120 levels. P-values for the difference between diabetic and control subjects in subjects with vs. subjects without a history of anti-hypertensive medication are 0.02 for glyoxylate and 0.77 for glucose.</p

Metabolites showing a difference in diabetic and healthy subjects at OGTT time-point 0 vs. 120.

<p>Branched-chain amino acids and metabolites of the hexosamine pathway similar to glyoxylate and glucose were all significant for a difference in diabetic and control patients at OGTT time point 0 vs. 120. P-value cutoff was p = 0.05.</p

Metabolite levels differ in diabetic subjects having a history an intake of anti-hypertensive medication.

<p>Subjects with anti-hypertension medication (med) are dark red or blue; subjects with no history of anti-hypertensive medication (no med) are light red or blue. OGTT_t = 120 samples were analyzed with the SIM method. The study participants were categorized as type-2 diabetic (n = 47) or control (n = 51) based on FPG and/or OGTT_t = 120 levels. Levels of glyoxylate, fructosamine and dihomo-gamma linolenic acid are depicted. Furthermore, OGTT_t = 0 samples were analyzed by MxP™ Eicosanoid analysis. The study participants here were categorized as type-2 diabetic (n = 58) or control (n = 177) based on FPG and/or OGTT_t = 120 levels. Levels of the eicosanoids metabolites thromboxane B2, prostaglandin E2, and 15-hydroxyeicosatetranoic are depicted. When comparing diabetic and control subjects with and without a history of anti-hypertensive medication, metabolite levels differ: hexosamines are more strongly increased in diabetic subjects with a history of anti-hypertensive medication, while eicosanoid and eicosanoid precursor levels are regulated in different directions in diabetic patients with or without a history of anti-hypertensive medication compared to their corresponding non-diabetic controls. P-values for the difference between diabetic and healthy subjects in subjects with vs. subjects without a history of anti-hypertensive medication are 0.02 for glyoxylate, 0.01 for dihomo-gamma linolenic acid, 0.04 for fructosamine, 0.02 for 5-Hydroxyeicosatetraenoic 15-Hydroxyeicosatetraenoic acid, 0.01 for thromboxane B2 and 4.58E-04 for prostaglandine E2.</p