Metabolomics and a Breath Sensor Identify Acetone as a Biomarker for Heart Failure.

BACKGROUND: Multi-omics delivers more biological insight than targeted investigations. We applied multi-omics to patients with heart failure with reduced ejection fraction (HFrEF). METHODS: 46 patients with HFrEF and 20 controls underwent metabolomic profiling, including liquid/gas chromatography mass spectrometry (LC-MS/GC-MS) and solid-phase microextraction (SPME) volatilomics in plasma and urine. HFrEF was defined using left ventricular global longitudinal strain, ejection fraction and NTproBNP. A consumer breath acetone (BrACE) sensor validated results in n = 73. RESULTS: 28 metabolites were identified by GCMS, 35 by LCMS and 4 volatiles by SPME in plasma and urine. Alanine, aspartate and glutamate, citric acid cycle, arginine biosynthesis, glyoxylate and dicarboxylate metabolism were altered in HFrEF. Plasma acetone correlated with NT-proBNP (r = 0.59, 95% CI 0.4 to 0.7), 2-oxovaleric and cis-aconitic acid, involved with ketone metabolism and mitochondrial energetics. BrACE > 1.5 ppm discriminated HF from other cardiac pathology (AUC 0.8, 95% CI 0.61 to 0.92, p < 0.0001). CONCLUSION: Breath acetone discriminated HFrEF from other cardiac pathology using a consumer sensor, but was not cardiac specific.

Multiomics, virtual reality and artificial intelligence in heart failure.

Aim: Multiomics delivers more biological insight than targeted investigations. We applied multiomics to patients with heart failure (HF) and reduced ejection fraction (HFrEF), with machine learning applied to advanced ECG (AECG) and echocardiography artificial intelligence (Echo AI). Patients & methods: In total, 46 patients with HFrEF and 20 controls underwent metabolomic profiling, including liquid/gas chromatography-mass spectrometry and solid-phase microextraction volatilomics in plasma and urine. HFrEF was defined using left ventricular (LV) global longitudinal strain, EF and N-terminal pro hormone BNP. AECG and Echo AI were performed over 5 min, with a subset of patients undergoing a virtual reality mental stress test. Results: A-ECG had similar diagnostic accuracy as N-terminal pro hormone BNP for HFrEF (area under the curve = 0.95, 95% CI: 0.85-0.99), and correlated with global longitudinal strain (r = -0.77, p < 0.0001), while Echo AI-generated measurements correlated well with manually measured LV end diastolic volume r = 0.77, LV end systolic volume r = 0.8, LVEF r = 0.71, indexed left atrium volume r = 0.71 and indexed LV mass r = 0.6, p < 0.005. AI-LVEF and other HFrEF biomarkers had a similar discrimination for HFrEF (area under the curve AI-LVEF = 0.88; 95% CI: -0.03 to 0.15; p = 0.19). Virtual reality mental stress test elicited arrhythmic biomarkers on AECG and indicated blunted autonomic responsiveness (alpha 2 of RR interval variability, p = 1 × 10-4) in HFrEF. Conclusion: Multiomics-related machine learning shows promise for the assessment of HF.

Lay abstract Multiomics is the integration of multiple sources of health information, for example, genomic, metabolite, etc. This delivers more insight than targeted single investigations and provides an ability to perceive subtle individual differences between people. In this study we applied multiomics to patients with heart failure (HF) using DNA sequencing, metabolomics and machine learning applied to ECG echocardiography. We demonstrated significant differences between subsets of patients with HF using these methods. We also showed that machine learning has significant diagnostic potential in identifying HF patients more efficiently than manual or conventional techniques.

Protein Levels and Microstructural Changes in Localized Regions of Early Cartilage Degeneration Compared with Adjacent Intact Cartilage.

OBJECTIVE: It was hypothesized that the respective protein profiles of bovine cartilage from sites of localized mild to moderate (GI to GII) degeneration versus adjacent sites of intact tissue would vary in accordance with the tissue microstructural changes associated with a pre-osteoarthritic state. METHODS: A total of 15 bovine patellae were obtained for this study. Paired samples of tissue were collected from the lateral region of each patella. If the patella contained a site of degeneration, a paired tissue set involved taking one sample each from the degenerated site and the intact tissue adjacent to it. Sufficient tissue was collected to facilitate 2 arms of investigation: microstructural imaging and proteome analysis. The microstructural analysis used a bespoke tissue preparation technique imaged with differential interference contrast optical microscopy to assess fibrillar scale destructuring and underlying bone spicule formation. An iTRAQ-based proteome analysis was performed using liquid chromatography-tandem mass spectrometry to identify the differential levels of proteins across the intact and degenerated cartilage and further, the results were validated with multiple reaction monitoring assay. RESULTS: In the healthy cartilage pairs, there was no significant variation in protein profiles between 2 adjacent sample sites. In pairs of tissue that contained a sample of GI/GII tissue, there were both significant microstructural changes as well as the difference in abundance levels of 24 proteins. CONCLUSIONS: From the known functions of the 24 proteins, found to be strongly aligned with the specific microstructural changes observed, a unique "proteins ensemble" involved in the initiation and progression of early cartilage degeneration is proposed.

Differentially Regulated Host Proteins Associated with Chronic Rhinosinusitis Are Correlated with the Sinonasal Microbiome.

The chronic inflammatory nature of chronic rhinosinusitis (CRS) makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling) and microbiome (bacterial 16S rRNA gene sequencing) analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS (n = 10) and healthy controls (n = 9). Of the total 606 proteins identified in this study, seven were significantly (p < 0.05) more abundant and 104 were significantly lower in the CRS cohort compared with healthy controls. The majority of detected proteins (82% of proteins identified) were not significantly correlated with disease status. Elevated levels of blood and immune cell proteins in the CRS cohort, together with significantly higher numbers of B-cells and macrophages in the underlying tissue, confirmed the inflammatory status of CRS individuals. Protein PRRC2C and Ras-related protein (RAB14) (two of the seven elevated proteins) showed the biggest fold difference between the healthy and CRS groups. Validation of the elevated levels of these two proteins in CRS samples was provided by immunohistochemistry. Members of the bacterial community in the two study cohorts were not associated with PRRC2C, however members of the genus Moraxella did correlate with RAB14 (p < 0.0001, rho = -0.95), which is a protein involved in the development of basement membrane. In addition, significant correlations between certain members of the CRS bacterial community and 33 lower abundant proteins in the CRS cohort were identified. Members of the genera Streptococcus, Haemophilus and Veillonella were strongly correlated with CRS and were significantly associated with a number of proteins with varying functions. The results from this study reveal a strong association between the host and microbes in the sinonasal cavity. Proteins identified as associated with CRS could be new targets for drug therapies and biomarkers for assessment of treatment efficacy.

Increased collagen within the transverse tubules in human heart failure.

AIMS: In heart failure transverse-tubule (t-tubule) remodelling disrupts calcium release, and contraction. T-tubules in human failing hearts exhibit increased labelling by wheat germ agglutinin (WGA), a lectin that binds to the dystrophin-associated glycoprotein complex. We hypothesized changes in this complex may explain the increased WGA labelling and contribute to t-tubule remodelling in the failing human heart. In this study we sought to identify the molecules responsible for this increased WGA labelling. METHODS AND RESULTS: Confocal and super-resolution fluorescence microscopy and proteomic analyses were used to quantify left ventricle samples from healthy donors and patients with idiopathic dilated cardiomyopathy (IDCM). Confocal microscopy demonstrated both WGA and dystrophin were located at t-tubules. Super-resolution microscopy revealed that WGA labelling of t-tubules is largely located within the lumen while dystrophin was restricted to near the sarcolemma. Western blots probed with WGA reveal a 5.7-fold increase in a 140 kDa band in IDCM. Mass spectrometry identified this band as type VI collagen (Col-VI) comprised of α1(VI), α2(VI), and α3(VI) chains. Pertinently, mutations in Col-VI cause muscular dystrophy. Western blotting identified a 2.4-fold increased expression and 3.2-fold increased WGA binding of Col-VI in IDCM. Confocal images showed that Col-VI is located in the t-tubules and that their diameter increased in the IDCM samples. Super-resolution imaging revealed Col-VI was restricted to the t-tubule lumen where increases were associated with displacement in the sarcolemma as identified from dystrophin labelling. Samples were also labelled for type I, III, and IV collagen. Both confocal and super-resolution imaging identified that these collagens were also present within t-tubule lumen. CONCLUSION: Increased expression and labelling of collagen in IDCM samples indicates fibrosis may contribute to t-tubule remodelling in human heart failure.

Randomized clinical trial of expressive writing on wound healing following bariatric surgery.

OBJECTIVE: Writing emotionally about upsetting life events (expressive writing) has been shown to speed healing of punch-biopsy wounds compared to writing objectively about daily activities. We aimed to investigate whether a presurgical expressive writing intervention could improve surgical wound healing. METHOD: Seventy-six patients undergoing elective laparoscopic bariatric surgery were randomized either to write emotionally about traumatic life events (expressive writing) or to write objectively about how they spent their time (daily activities writing) for 20 min a day for 3 consecutive days beginning 2 weeks prior to surgery. A wound drain was inserted into a laparoscopic port site and wound fluid analyzed for proinflammatory cytokines collected over 24 hr postoperatively. Expanded polytetrafluoroethylene tubes were inserted into separate laparoscopic port sites during surgery and removed after 14 days. Tubes were analyzed for hydroxyproline deposition (the primary outcome), a major component of collagen and marker of healing. Fifty-four patients completed the study. RESULTS: Patients who wrote about daily activities had significantly more hydroxyproline than did expressive writing patients, t(34) = -2.43, p = .020, 95% confidence interval [-4.61, -0.41], and higher tumor necrosis factor-alpha, t(29) = -2.42, p = .022, 95% confidence interval [-0.42, -0.04]. Perceived stress significantly reduced in both groups after surgery. CONCLUSIONS: Expressive writing prior to bariatric surgery was not effective at increasing hydroxyproline at the wound site 14 days after surgery. However, writing about daily activities did predict such an increase. Future research needs to replicate these findings and investigate generalizability to other surgical groups. (PsycINFO Database Record

Differential Changes in Gut Microbiota After Gastric Bypass and Sleeve Gastrectomy Bariatric Surgery Vary According to Diabetes Remission.

BACKGROUND: It is unclear whether specific gut microbiota is associated with remission of type 2 diabetes (T2D) after distinct types of bariatric surgery. AIMS: The aim of this study is to examine gut microbiota changes after laparoscopic Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) surgery in obese patients with T2D. METHODS: Whole-metagenome shotgun sequencing of DNA fragments using Illumina HiSeq2000 was obtained from stool samples collected from 14 obese T2D patients pre-operatively (while on very low calorie diet) and 1 year after randomisation to laparoscopic SG (n = 7) or RYGB (n = 7). Resulting shotgun reads were annotated with Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: Body weight reduction and dietary change was similar 1 year after both surgery types. Identical proportions (n = 5/7) achieved diabetes remission (HbA1c < 48 mmol/mol without medications) 1 year after RYGB and SG. RYGB resulted in increased Firmicutes and Actinobacteria phyla but decreased Bacteroidetes phyla. SG resulted in increased Bacteroidetes phyla. Only an increase in Roseburia species was observed among those achieving diabetes remission, common to both surgery types. KEGG Orthology and pathway analysis predicted contrasting and greater gut microbiota metabolism changes after diabetes remission following RYGB than after SG. Those with persistent diabetes post-operatively had higher Desulfovibrio species pre-operatively. CONCLUSIONS: Overall, RYGB produces greater and more predicted favourable changes in gut microbiota functional capacity than SG. An increase in Roseburia species was the only compositional change common to both types of surgery among those achieving diabetes remission.

Graded perturbations of metabolism in multiple regions of human brain in Alzheimer's disease: Snapshot of a pervasive metabolic disorder.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder that displays pathological characteristics including senile plaques and neurofibrillary tangles. Metabolic defects are also present in AD-brain: for example, signs of deficient cerebral glucose uptake may occur decades before onset of cognitive dysfunction and tissue damage. There have been few systematic studies of the metabolite content of AD human brain, possibly due to scarcity of high-quality brain tissue and/or lack of reliable experimental methodologies. Here we sought to: 1) elucidate the molecular basis of metabolic defects in human AD-brain; and 2) identify endogenous metabolites that might guide new approaches for therapeutic intervention, diagnosis or monitoring of AD. Brains were obtained from nine cases with confirmed clinical/neuropathological AD and nine controls matched for age, sex and post-mortem delay. Metabolite levels were measured in post-mortem tissue from seven regions: three that undergo severe neuronal damage (hippocampus, entorhinal cortex and middle-temporal gyrus); three less severely affected (cingulate gyrus, sensory cortex and motor cortex); and one (cerebellum) that is relatively spared. We report a total of 55 metabolites that were altered in at least one AD-brain region, with different regions showing alterations in between 16 and 33 metabolites. Overall, we detected prominent global alterations in metabolites from several pathways involved in glucose clearance/utilization, the urea cycle, and amino-acid metabolism. The finding that potentially toxigenic molecular perturbations are widespread throughout all brain regions including the cerebellum is consistent with a global brain disease process rather than a localized effect of AD on regional brain metabolism.

Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of cholesterol-fed rabbits.

The cholesterol-fed rabbit is commonly used as a model to study the vascular effects of hypercholesterolemia and resulting atherosclerotic lesions. Here we undertook a proteomic case-control investigation of ascending aortas from male New Zealand White rabbits after 10 weeks on a high-cholesterol (2% w/w) diet (HCD, n = 5) or control diet (n = 5), in order to determine the changes in response to the HCD. Histology confirmed intimal thickening in the HCD group consistent with atherosclerosis, and LC-MS/MS analysis of individually-obtained ascending aortic extracts labelled with isobaric (iTRAQ) tags enabled the identification and quantitation of 453 unique proteins above the 1% false discovery rate threshold. Of 67 proteins showing significant differences in relative abundance (p < 0.05), 62 were elevated and five decreased in ascending aortas from HCD-fed rabbits compared to controls. Six proteins were selected for validation using Multiple Reaction Monitoring, which confirmed the iTRAQ results. Many of the observed protein changes are consistent with known molecular perturbations in the ascending aorta that occur in response to hypercholesterolemia, e.g. elevation of tissue levels of apolipoproteins, extracellular matrix adhesion proteins, glycolytic enzymes, heat shock proteins and proteins involved in immune defense. We also made a number of novel observations, including a 15-fold elevation of glycoprotein (trans-membrane) nmb-like (Gpnmb) in response to HCD. Gpnmb has previously been linked to angiogenesis but not to atherosclerosis. This and additional novel observations merit further investigation as these perturbations may play important and as yet undiscovered roles in the pathogenesis of atherosclerosis in rabbits as well as humans.

Lower fetuin-A, retinol binding protein 4 and several metabolites after gastric bypass compared to sleeve gastrectomy in patients with type 2 diabetes.

BACKGROUND: Bypass of foregut secreted factors promoting insulin resistance is hypothesized to be one of the mechanisms by which resolution of type 2 diabetes (T2D) follows roux-en-y gastric bypass (GBP) surgery. AIM: To identify insulin resistance-associated proteins and metabolites which decrease more after GBP than after sleeve gastrectomy (SG) prior to diabetes remission. METHODS: Fasting plasma from 15 subjects with T2D undergoing GBP or SG was analyzed by proteomic and metabolomic methods 3 days before and 3 days after surgery. Subjects were matched for age, BMI, metformin therapy and glycemic control. Insulin resistance was calculated using homeostasis model assessment (HOMA-IR). For proteomics, samples were depleted of abundant plasma proteins, digested with trypsin and labeled with iTRAQ isobaric tags prior to liquid chromatography-tandem mass spectrometry analysis. Metabolomic analysis was performed using gas chromatography-mass spectrometry. The effect of the respective bariatric surgery on identified proteins and metabolites was evaluated using two-way analysis of variance and appropriate post-hoc tests. RESULTS: HOMA-IR improved, albeit not significantly, in both groups after surgery. Proteomic analysis yielded seven proteins which decreased significantly after GBP only, including Fetuin-A and Retinol binding protein 4, both previously linked to insulin resistance. Significant decrease in Fetuin-A and Retinol binding protein 4 after GBP was confirmed using ELISA and immunoassay. Metabolomic analysis identified significant decrease of citrate, proline, histidine and decanoic acid specifically after GBP. CONCLUSION: Greater early decrease was seen for Fetuin-A, Retinol binding protein 4, and several metabolites after GBP compared to SG, preceding significant weight loss. This may contribute to enhanced T2D remission observed following foregut bypass procedures.

Bioengineering silicon quantum dot theranostics using a network analysis of metabolomic and proteomic data in cardiac ischemia.

Metabolomic profiling is ideally suited for the analysis of cardiac metabolism in healthy and diseased states. Here, we show that systematic discovery of biomarkers of ischemic preconditioning using metabolomics can be translated to potential nanotheranostics. Thirty-three patients underwent percutaneous coronary intervention (PCI) after myocardial infarction. Blood was sampled from catheters in the coronary sinus, aorta and femoral vein before coronary occlusion and 20 minutes after one minute of coronary occlusion. Plasma was analysed using GC-MS metabolomics and iTRAQ LC-MS/MS proteomics. Proteins and metabolites were mapped into the Metacore network database (GeneGo, MI, USA) to establish functional relevance. Expression of 13 proteins was significantly different (p<0.05) as a result of PCI. Included amongst these was CD44, a cell surface marker of reperfusion injury. Thirty-eight metabolites were identified using a targeted approach. Using PCA, 42% of their variance was accounted for by 21 metabolites. Multiple metabolic pathways and potential biomarkers of cardiac ischemia, reperfusion and preconditioning were identified. CD44, a marker of reperfusion injury, and myristic acid, a potential preconditioning agent, were incorporated into a nanotheranostic that may be useful for cardiovascular applications. Integrating biomarker discovery techniques into rationally designed nanoconstructs may lead to improvements in disease-specific diagnosis and treatment.

Birds and longevity: does flight driven aerobicity provide an oxidative sink?

Birds generally age slower and live longer than similar sized mammals. For birds this occurs despite elevated blood glucose levels that for mammals would in part define them as diabetic. However these data were acquired in respiration states that have little resemblance to conditions in healthy tissues and mitochondrial RS production is probably minimal in healthy animals. Indeed mitochondria probably act as net consumers rather than producers of RS. Here we propose that (1) if mitochondria are antioxidant systems, the greater mitochondrial mass in athletic species, such as birds, is advantageous as it should provide a substantial sink for RS. (2) The intense drive for aerobic performance and decreased body density to facilitate flight may explain the relative insensitivity of birds to insulin, as well as depressed insulin levels and apparent sensitization to glucagon. Glucagon also associates with the sirtuin protein family, most of which are associated with caloric restriction regulated pathways, mitochondrial biogenesis and life span extension. (3) We note that telomeres, which appear to be unusually long in birds, bind Sirtuins 2 and 4 and therefore may stabilize and protect nuclear DNA. Ultimately these flight driven responses may suppress somatic growth and protect DNA from oxidative damage that would otherwise lead to ageing and non-viral cancers.

A simple and rapid method for identifying and semi-quantifying peptide hormones in isolated pancreatic islets by direct-tissue matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

We describe a new, simple, robust and efficient method based on direct-tissue matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that enables consistent semi-quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet-endocrine cells: (α-cells) glucagon, glicentin-related polypeptide/GRPP; (ß-cells) insulin I, insulin II, C-peptide I, C-peptide II, amylin; (δ-cells) somatostatin-14; and (PP-cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non-ß-cell-hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non-ß-cells. It was also effective in characterizing hormonal phenotype in hemizygous human-amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide-hormone discovery by identifying a prominent new Gcg-gene-derived peptide (theoretical monoisotopic molecular weight 3263.5 Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide-ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies.

A unique case of neural amyloidoma diagnosed by mass spectrometry of formalin-fixed tissue using a novel preparative technique.

We report here a unique amyloidoma of the radial nerve which could not be subtyped by available techniques, including immunohistochemistry and standard clinical and laboratory evaluation. In order to identify the amyloid monomer, we developed a novel preparative procedure designed to optimize conditions for liquid chromatography tandem mass spectrometry analysis of formalin-fixed/paraffin-embedded (FFPE) tissue. Subsequent mass spectrometric analysis clearly identified kappa light chain as the monomer, with no evidence of lambda light chain. Manual interpretation of the matched spectra revealed no evidence of polyclonality. This study also enabled detailed characterisation of twelve likely amyloid matrix components. Finally, our analysis revealed extensive hydroxylation of collagen type I but, unexpectedly, an almost complete lack of hydroxylated residues in the normally heavily-hydroxylated collagen type VI chains, pointing to structural/functional alterations of collagen VI in this matrix that could have contributed to the pathogenesis of this very unusual tumour. Given the high quality of the data here acquired using a standard quadrupole-time of flight tandem mass spectrometer of modest performance, the robust and straightforward preparative method described constitutes a competitive alternative to more involved approaches using state-of-the-art equipment.

Illuminating the molecular basis of diabetic arteriopathy: a proteomic comparison of aortic tissue from diabetic and healthy rats.

Arterial disease is a major diabetic complication, yet the component molecular mechanisms of diabetic arteriopathy remain poorly understood. In order to identify major proteins/pathways implicated in diabetic arteriopathy, we studied the effect of 16-wk untreated streptozotocin-induced diabetes on the rat aortic proteome. Specific protein levels in isolated aortas were compared in six discrete, pair-wise (streptozotocin-diabetic and non-diabetic age-matched controls) experiments in which individual proteins were identified and quantified by iTRAQ combined with LC-MS/MS. A total of 398 unique non-redundant proteins were identified in at least one experiment and 208 were detected in three or more. Between-group comparisons revealed significant changes or trends towards changes in relative abundance of 51 proteins (25 increased, 26 decreased). Differences in levels of selected proteins were supported by Western blotting and/or enzyme assays. The most prominent diabetes-associated changes were in groups of proteins linked to oxidative stress responses and the structure/function of myofibrils and microfilaments. Indexes of mitochondrial content were measurably lower in aortic tissue from diabetic animals. Functional cluster analysis also showed decreased levels of glycolytic enzymes and mitochondrial electron transport system-complex components. These findings newly implicate several proteins/functional pathways in the pathogenesis of arteriosclerosis/diabetic arteriopathy.

Changes in the mesenteric lymph proteome induced by hemorrhagic shock.

Biologically active factors produced by the intestine and transported by the aqueous and protein fraction of mesenteric lymph are now thought to contribute significantly to the development of distant organ failure in hemorrhagic shock. Despite the likely relevance of the protein composition of mesenteric lymph conditioned by hemorrhagic shock, there is no detailed description of its proteome. The aim of this study was to provide the first comprehensive description of the proteome of hemorrhagic shock-conditioned mesenteric lymph. Mesenteric lymph was collected from 16 male Wistar rats randomized to group 1 (n = 8) sham control and group 2 (n = 8) with hemorrhagic shock. The lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry. Sixty of the 245 proteins had a significant increase in their relative abundance in the hemorrhagic shock group. A bioinformatics approach highlighted the importance of the key gene ontology pathways relating to response to injury and metabolic responses as changing most significantly in shock. Using an interactome, we identified several highly connected proteins: 14-3-3 Zeta, 14-3-3 epsilon, actin, aldolase A, calmodulin, cofilin 1, cystatin C, fatty acid-binding protein 4, profilin 1, prolyl 4-hydrolase, peptidylprolyl isomerase, and transgelin. This study provides the first detailed description of protein changes in hemorrhagic shock-conditioned mesenteric lymph, and using a bioinformatics approach, we identified several targets for possible further research.

The proteome of rodent mesenteric lymph.

Mesenteric lymph contributes to normal homeostasis and has an emerging role in the pathogenesis of multiple organ dysfunction syndrome. The aim of this study was to define the proteome of normal rodent mesenteric lymph in the fasted and fed states. Eight male Wistar rats fed a standard rodent diet were randomized to two groups. Group 1 (fasted, n = 4) were fasted for 24 h before anesthetized collection of mesenteric lymph. Group 2 (fed, n = 4) were allowed ad libitum access to food before lymph collection. Mesenteric lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry (LC-MS/MS). One hundred fifty proteins, including 26 hypothetical proteins, were identified in this study. All proteins were identified in lymph from both the fasted and fed states. The relative distribution profiles of protein functional classes in the mesenteric lymph differed significantly from that reported for plasma. The most abundant classes identified in lymph were protease inhibitors (16%) and proteins related to innate immunity (12%). In conclusion, this study provides the first detailed description of the normal mesenteric lymph proteome in the fed and fasted states using iTRAQ and LC-MS/MS.

Is the failing heart out of fuel or a worn engine running rich? A study of mitochondria in old spontaneously hypertensive rats.

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause-and-effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20-month-old SHR and Wistar-Kyoto controls by iTRAQ-labelling combined with multidimensional LC/MS/MS. Of 137 high-scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.

Reversal of diabetes-evoked changes in mitochondrial protein expression of cardiac left ventricle by treatment with a copper(II)-selective chelator.

Cardiac disease is the commonest cause of death amongst diabetic patients. Diabetic cardiomyopathy, which has a poor prognosis, is characterized by left ventricular hypertrophy and impaired cardiac function and mitochondrial damage is said to contribute to its development. We recently showed that treatment with the Cu(II) -selective chelator, triethylenetetramine (TETA), improved cardiac structure, and function in diabetic subjects without modifying hyperglycemia. Thus, TETA has potential utility for the treatment of heart disease. To further understand the molecular mechanism by which it causes these effects, we have conducted the first study of the effect of oral TETA on protein abundance in the cardiac left ventricle of rats with severe streptozotocin-induced diabetes. Proteomic methods showed that of 211 proteins changed in diabetes, 33 recovered after treatment. Through MS, 16 proteins were identified which may constitute major targets of drug action. Remarkably, most of these were mitochondrial proteins with roles in energy metabolism. In addition to components of the mitochondrial respiratory chain and enzymes involved in fatty acid oxidation, TETA treatment normalized both myocardial expression and enzymatic activity of carnitine palmitoyltransferase 2. These findings indicate that mitochondria constitute major targets in the mechanism by which TETA restores cardiac structure and function in diabetes.

Characterization of proteomic changes in cardiac mitochondria in streptozotocin-diabetic rats using iTRAQ™ isobaric tags.

Diabetes now affects more than 5% of the world's population and heart failure is the most common cause of death amongst diabetic patients. Accumulating evidence supports a view that myocardial mitochondrial structural and functional changes are central to the onset of diabetic heart failure, but the exact nature of these changes at the proteomic level remains unclear.Here we report on proteomic changes in diabetic rat heart mitochondria following 120 days of streptozotocin-diabetes using the recently developed iTRAQ™ labeling method, which permits quantification of proteins directly from complex mixtures, bypassing the limitations associated with gel-based methods such as 2-DE. Of 252 unique proteins identified, 144 were represented in at least three of six individual paired experiments. Relative amounts of 65 proteins differed significantly between the groups, confirming that the cardiac mitochondrial proteome is indeed impacted by diabetes. The most significant changes were increased protein levels of enzymes involved in mitochondrial oxidation of long-chain fatty acids, which was also confirmed by enzyme assays, and decreased levels of multiple enzymes involved in oxidative phosphorylation and catabolism of short-chain fatty acids and branched-chain amino acids. We also found significant changes in levels of several enzymes linked to oxidative stress.