Serum Metabolomic Profiling in Acute Alcoholic Hepatitis Identifies Multiple Dysregulated Pathways

Background and Objectives

While animal studies have implicated derangements of global energy homeostasis in the pathogenesis of acute alcoholic hepatitis (AAH), the relevance of these findings to the development of human AAH remains unclear. Using global, unbiased serum metabolomics analysis, we sought to characterize alterations in metabolic pathways associated with severe AAH and identify potential biomarkers for disease prognosis.

Methods

This prospective, case-control study design included 25 patients with severe AAH and 25 ambulatory patients with alcoholic cirrhosis. Serum samples were collected within 24 hours of the index clinical encounter. Global, unbiased metabolomics profiling was performed. Patients were followed for 180 days after enrollment to determine survival.

Results

Levels of 234 biochemicals were altered in subjects with severe AAH. Random-forest analysis, principal component analysis, and integrated hierarchical clustering methods demonstrated that metabolomics profiles separated the two cohorts with 100% accuracy. Severe AAH was associated with enhanced triglyceride lipolysis, impaired mitochondrial fatty acid beta oxidation, and upregulated omega oxidation. Low levels of multiple lysolipids and related metabolites suggested decreased plasma membrane remodeling in severe AAH. While most measured bile acids were increased in severe AAH, low deoxycholate and glycodeoxycholate levels indicated intestinal dysbiosis. Several changes in substrate utilization for energy homeostasis were identified in severe AAH, including increased glucose consumption by the pentose phosphate pathway, altered tricarboxylic acid (TCA) cycle activity, and enhanced peptide catabolism. Finally, altered levels of small molecules related to glutathione metabolism and antioxidant vitamin depletion were observed in patients with severe AAH. Univariable logistic regression revealed 15 metabolites associated with 180-day survival in severe AAH.

Conclusion

Severe AAH is characterized by a distinct metabolic phenotype spanning multiple pathways. Metabolomics profiling revealed a panel of biomarkers for disease prognosis, and future studies are planned to validate these findings in larger cohorts of patients with severe AAH.     

Inflammation and Resolution Are Associated with Upregulation of Fatty Acid β-Oxidation in Zymosan-Induced Peritonitis

Inflammation is a fundamental defensive response to harmful stimuli. However, it can cause damage if it does not subside. To avoid such damage, organisms have developed a mechanism called resolution of inflammation. Here we applied an untargeted metabolomics approach to a sterile and self-resolving animal model of acute inflammation, namely zymosan-induced peritonitis in mice, to examine the effect of inflammation and resolution on the metabolomic profiles. Significant and time-dependent changes in metabolite profiles after zymosan administration were observed in both peritoneal wash fluid (PWF) and plasma. These metabolomic changes correlated well with inflammatory chemokine or cytokine production. In PWF, most of metabolites that could detected increased in zymosan-treated mice, which is suggestive of inflammation, oxidative stress and increased energy demands. In plasma, most metabolites in the central metabolic pathway (glycolysis and TCA cycle) were significantly downregulated after zymosan administration. The concentration of the ketone body 3-hydroxybutyric acid (3-HB) in plasma and PWF increased in zymosan-injected animals indicating upregulation of fatty acid β-oxidation. Increased 3-HB level was observed in the cells that infiltrated into the peritoneal cavity and these infiltrated cells might contribute, at least in part, to the production of 3-HB in the peritoneal cavity.     

Effect of gamma irradiation on liver metallothionein synthesis and lipid peroxidation in rats.

Several studies have recently shown that metallothionein (MT), a protein characterized by a high thiol content and that binds Zn2+ and Cu+, might be involved in the protection against oxidative stress and can act as a free radical scavenger. Oxidative stresses, such as irradiation, increase lipid peroxidation (LP) and subsequent tissue damage through free radical production. The induction of hepatic MT synthesis by gamma-irradiation (20 Gy) at 8, 24, 30 and 48 hrs. post-irradiation in two different age groups of Sprague-Dawley rats (39-40 and 48-49 days old) was studied. LP measured by the thiobarbituric acid reactive substances (TBARS) assay and Cu and Zn levels in liver have also been determined. In the younger group, the gamma-irradiation induced hepatic MT synthesis and increased LP that peaked 24 hrs. after irradiation. During the first 30 hrs. post-irradiation, a positive and statistically significant correlation between hepatic MT content and LP level in liver was found. In the older group, liver MT synthesis was only increased 1.7-fold and LP levels were not altered at 24 hrs. post-irradiation compared with sham-irradiated rats.Therefore it appears that LP is not necessary for induction of MT synthesis by gamma-irradiation.     

Repetitive Transcranial Magnetic Stimulation Ameliorates Anxiety-Like Behavior and Impaired Sensorimotor Gating in a Rat Model of Post-Traumatic Stress Disorder

BackgroundRepetitive transcranial magnetic stimulation (rTMS) has been employed for decades as a non-pharmacologic treatment for post-traumatic stress disorder (PTSD). Although a link has been suggested between PTSD and impaired sensorimotor gating (SG), studies assessing the effects of rTMS against PTSD or PTSD with impaired SG are scarce.AimTo assess the benefit of rTMS in a rat model of PTSD.MethodsUsing a modified single prolonged stress (SPS&S) rat model of PTSD, behavioral parameters were acquired using open field test (OFT), elevated plus maze test (EPMT), and prepulse inhibition trial (PPI), with or without 7 days of high frequency (10Hz) rTMS treatment of SPS&S rats.ResultsAnxiety-like behavior, impaired SG and increased plasma level of cortisol were observed in SPS&S animals after stress for a prolonged time. Interestingly, rTMS administered immediately after stress prevented those impairment.ConclusionStress-induced anxiety-like behavior, increased plasma level of cortisol and impaired PPI occur after stress and high-frequency rTMS has the potential to ameliorate this behavior, suggesting that high frequency rTMS should be further evaluated for its use as a method for preventing PTSD.     

Untargeted Plasma Metabolomics Identifies Endogenous Metabolite with Drug-like Properties in Chronic Animal Model of Multiple Sclerosis

We performed untargeted metabolomics in plasma of B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation, and membrane stability were observed to be significantly altered between EAE and control (p < 0.05, false discovery rate <0.10). Bioinformatics analysis revealed six metabolic pathways being impacted and altered in EAE, including α-linolenic acid and linoleic acid metabolism (PUFA). The metabolites of PUFAs, including ω-3 and ω-6 fatty acids, are commonly decreased in mouse models of multiple sclerosis (MS) and in patients with MS. Daily oral administration of resolvin D1, a downstream metabolite of ω-3, decreased disease progression by suppressing autoreactive T cells and inducing an M2 phenotype of monocytes/macrophages and resident brain microglial cells. This study provides a proof of principle for the application of metabolomics to identify an endogenous metabolite(s) possessing drug-like properties, which is assessed for therapy in preclinical mouse models of MS.     

Inflammation and Oxidative Stress Are Elevated in the Brain,Blood, and Adrenal Glands during the Progression of Post-Traumatic Stress Disorder in a Predator Exposure Animal Model

This study sought to analyze specific pathophysiological mechanisms involved in the progression of post-traumatic stress disorder (PTSD) by utilizing an animal model. To examine PTSD pathophysiology, we measured damaging reactive oxygen species and inflammatory cytokines to determine if oxidative stress and inflammation in the brain, adrenal glands, and systemic circulation were upregulated in response to constant stress. Pre-clinical PTSD was induced in naïve, male Sprague-Dawley rats via a predator exposure/psychosocial stress regimen. PTSD group rats were secured in Plexiglas cylinders and placed in a cage with a cat for one hour on days 1 and 11 of a 31-day stress regimen. In addition, PTSD group rats were subjected to psychosocial stress whereby their cage cohort was changed daily. This model has been shown to cause heightened anxiety, exaggerated startle response, impaired cognition, and increased cardiovascular reactivity, all of which are common symptoms seen in humans with PTSD. At the conclusion of the predator exposure/psychosocial stress regimen, the rats were euthanized and their brains were dissected to remove the hippocampus, amygdala, and pre-frontal cortex (PFC), the three areas commonly associated with PTSD development. The adrenal glands and whole blood were also collected to assess systemic oxidative stress. Analysis of the whole blood, adrenal glands, and brain regions revealed oxidative stress increased during PTSD progression. In addition, examination of pro-inflammatory cytokine (PIC) mRNA and protein demonstrated neurological inflammatory molecules were significantly upregulated in the PTSD group vs. controls. These results indicate oxidative stress and inflammation in the brain, adrenal glands, and systemic circulation may play a critical role in the development and further exacerbation of PTSD. Thus, PTSD may not be solely a neurological pathology but may progress as a systemic condition involving multiple organ systems.     

Posttraumatic Stress Disorder Disturbs Coronary Tone and Its Regulatory Mechanisms

Posttraumatic stress disorder (PTSD) is associated with myocardial injury, but changes in coronary regulatory mechanisms in PTSD have not been investigated. This study evaluated the effect of PTSD-inducing stress on coronary tone and its regulation by nitric oxide (NO) and voltage-gated K⁺ channels. PTSD was induced by exposing rats to predator stress, 15 min daily for 10 days, followed by 14 stress-free days. Presence of PTSD was confirmed by the elevated plus-maze test. Coronary tone was evaluated from changes in coronary perfusion pressure of Langendorff isolated hearts. Predator stress induced significant decreases in coronary tone of isolated hearts and in blood pressure of intact rats. L-NAME, a non-selective NO synthase (NOS) inhibitor, but not S-MT, a selective iNOS inhibitor, and increased coronary tone of control rats. In PTSD rats, both L-NAME and S-MT increased coronary tone. Therefore, the stress-induced coronary vasodilation resulted from NO overproduction by both iNOS and eNOS. NOS induction was apparently due to systemic inflammation as evidenced by increased serum interleukin-1β and C-reactive protein in PTSD rats. Decreased corticosterone in PTSD rats may have contributed to inflammation and its effect on coronary tone. PTSD was also associated with voltage-gated K⁺ channel dysfunction, which would have also reduced coronary tone.     

Plasma metabolomics and proteomics profiling after a postprandial challenge reveal subtle diet effects on human metabolic status

We introduce the metabolomics and proteomics based Postprandial Challenge Test (PCT) to quantify the postprandial response of multiple metabolic processes in humans in a standardized manner. The PCT comprised consumption of a standardized 500?ml dairy shake containing respectively 59, 30 and 12 energy percent lipids, carbohydrates and protein. During a 6?h time course after PCT 145 plasma metabolites, 79 proteins and 7 clinical chemistry parameters were quantified. Multiple processes related to metabolism, oxidation and inflammation reacted to the PCT, as demonstrated by changes of 106 metabolites, 31 proteins and 5 clinical chemistry parameters. The PCT was applied in a dietary intervention study to evaluate if the PCT would reveal additional metabolic changes compared to non-perturbed conditions. The study consisted of a 5-week intervention with a supplement mix of anti-inflammatory compounds in a crossover design with 36 overweight subjects. Of the 231 quantified parameters, 31 had different responses over time between treated and control groups, revealing differences in amino acid metabolism, oxidative stress, inflammation and endocrine metabolism. The results showed that the acute, short term metabolic responses to the PCT were different in subjects on the supplement mix compared to the controls. The PCT provided additional metabolic changes related to the dietary intervention not observed in non-perturbed conditions. Thus, a metabolomics based quantification of a standardized perturbation of metabolic homeostasis is more informative on metabolic status and subtle health effects induced by (dietary) interventions than quantification of the homeostatic situation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0320-5) contains supplementary material, which is available to authorized users.     

A combination of untargeted and targeted metabolomics approaches unveils changes in the kynurenine pathway following cardiopulmonary resuscitation

The mechanisms responsible for post-resuscitation myocardial and cerebral dysfunction are not well understood, especially in the early post-resuscitation phases. In this investigation, we first adopted unbiased mass spectrometry-based metabolomic profiling to identify perturbations in circulating metabolites in a rat model of cardiac arrest and cardiopulmonary resuscitation. Our findings strongly indicated early alterations in a major route of the tryptophan catabolism, namely the kynurenines pathway, after resuscitation. Specific metabolites involved in the tryptophan catabolism were quantified absolutely using liquid chromatography-multiple reaction monitoring-mass spectrometry. Tryptophan plasma concentration fell significantly very early in the post-resuscitation phase, while its metabolites, l-kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and 5-hydroxyindoleacetic acid, rose significantly. Changes in their concentration reflected changes in rat post-resuscitation myocardial dysfunction. Elevated plasma level of kynurenic acid, 3-hydroxyanthranilic acid were associated with significant decrease in ejection fraction and stroke volume. It is well known that kynurenines pathway is involved in the pathogenesis of numerous central nervous system disorders. By implication, altered levels of tryptophan metabolites in the early post resuscitation phase might contribute to the degree of cognitive recovery. Our results suggest that kynurenine pathway is activated early following resuscitation from cardiac arrest and might account for the severity of post-resuscitation syndrome. Our explorative investigation indicate that metabolomics can help to clarify unexplored biochemical pathways in cardiopulmonary resuscitation.     

The effect of repeated immobilization on the level of plasma corticosterone and on the activity of several liver enzymes in rats.

The effect of repeated stress on the level of plasma corticosterone and on the activity of several target enzymes for this hormone in the liver was studied. In adult male rats immobilized for 2.5 hrs daily, on day 7 the response of both plasma corticosterone and hepatic tyrosine aminotransferase is modified: After similar increases immediately after immobilization as in aminals stressed for the first time, in the conditioned rats precocious decreases to initial values take place. Moreover, on day 4, 24 hrs after a third immobilization, there are increases arise partly at least as a consequence of diminished food intake, as shown by comparing them with data from pair-fed rats. Partial fasting leading also to slight increase of hepatic glucose-6-phosphatase activity constitutes an important part of repeated stress with substantial impacts on metabolic processes.     

The potential role of hypocortisolism in the pathophysiology of PTSD and psoriasis

Different physical, chemical and psychological stressors can provoke a unique but different endocrine response involving activation of the hypothalamo-pituitary-adrenal (HPA) axis. Inability of adequate compensatory reaction can lead to many disorders. The aim of our study was comparison of cortisol values in diseases provoked by various stressors. Our investigation included 34 posttraumatic stress disorder (PTSD) patients, as an example of disorder caused by extremely strong, acute stressful stimulus, 19 psoriatic patients, as an example of chronic stress stimulus and 17 healthy volunteers. In each patient we determined 24-hour urinary cortisol, serum cortisol at 8 a.m. and 5 p.m., and cortisol in dexamethasone suppression test by the standard radioimmunoassay (RIA) method. PTSD patients showed lower urinary 24-hour cortisol values, (361 +/- 28 nmol/24 h), "stronger" circadian rhythm of serum cortisol (595 +/- 57 nmol/l at 8 a.m. and 242 +/- 23 nmol/l at 5 p.m.) and attenuated suppression of cortisol in dexamethasone suppression test (197 +/- 45 nmol/l) in comparison to healthy volunteers (590 +/- 87 nmol/24 h urine, 590 +/- 32 nmol/l at 8 a.m., 402 +/- 31 nmol/l, and < 86 nmol/l in dexa test). Psoriatic patients showed markedly lower 24-hour cortisol values (150 +/- 98 nmol/24 h), even in comparison to PTSD patients, then serum cortisol values (404 +/- 138 nmol/l at 8 a.m., 187 +/- 80 nmol/l at 5 p.m.) and enhanced suppression of cortisol (23 +/- 5 nmol/l). The model of attenuated feedback inhibition in PTSD patients shows that they are unusually reactive to stress and represents an alternative model of acute stress reaction to extremely strong stressful stimulus. Unusually low cortisol values in psoriatic patients correlate to our hypothesis that in chronic stress-related disease, as psoriasis is, exists, by still undefined mechanism, altered HPA axis function, which is obviously incompetent to realise its immunoregulatory function, so consequentially, clinical signs of psoriasis persist.     

Post-traumatic Stress Disorder in children and adolescents: from Sigmund Freud's "trauma" to psychopathology and the (Dys)metabolic syndrome.

Post-traumatic Stress Disorder (PTSD) is an anxiety syndrome that develops after exposure to traumatic life events. Symptoms include re-experience of the initial trauma, avoidance of stimuli associated with the trauma and symptoms of excessive arousal. Neuroendocrine studies in adults with PTSD have demonstrated that basal cerebrospinal fluid (CSF) CRH levels are elevated and urinary cortisol levels are variable--low in the majority of cases--whereas other studies demonstrate no differences in urinary and plasma cortisol concentrations. Urinary catecholamine excretion is higher in PTSD patients than those of control subjects and other psychiatric disorders. Children may differ from adults in their psychologic and physiologic responses to severe stressors. Also, exposure to stress during critical periods of development may have irreversible effects on behavioral maturation and may affect specific vulnerable brain areas, altering CNS development. Similar to findings in adult studies, PTSD in children is characterized by increased sympathetic nervous system (SNS) activity, as indicated by elevated norepinephrine levels in the periphery. High cortisol levels in urine or saliva have been reported in most studies of childhood PTSD, while prospective longitudinal studies concerning the natural history of neuroendocrine changes in pediatric PTSD after an acute stressor are limited. The identification of neurobiologic changes in response to early adverse experiences is of major importance for the prognosis, prevention, management, and treatment of children and adolescents at risk for or suffering from PTSD.     

创伤后应激障碍机制的研究进展

创伤后应激障碍(Post-traumatic stress disorder;PTSD)是一种由严重强烈的伤害事件造成的精神障碍,随着近年来社会应激事件的增多和自然灾害的发生,创伤应激障碍的发病率逐渐增高。同时为了研究对应的治疗方法,人们对创伤应激障碍的机制进行了更深入的探索,也有了新的进展。本文着重从激素、神经营养因子、免疫系统等方面来总结创伤后应激障碍发生的生物学机制。激素方面,PTSD主要与交感肾上腺髓质系统(Sympatho-adrenomedullarysystem,SAS)和下丘脑-垂体-肾上腺轴(Hypothalamicpituitary-adrenal axis,HPA)的功能异常有关;神经营养因子方面,其产生与分泌的异常增加或减少可能是PTSD产生的重要机制;免疫系统方面,PTSD可能与免疫系统相关的蛋白质、细胞的数量和功能变化有关。整合神经生物学与分子生物学、表观遗传学、蛋白质组学及分子影像学的成果将对PTSD的研究产生推动作用。     

Increase of sialyltransferase activity in the small intestine following thermal injury in rats

The acute phase protein response following inflammation is associated with an increased total protein-bound carbohydrate content in plasma in the form of glycoproteins. Glycosyltransferases in liver may serve as a regulator of this increased glycosylation activity in the plasma and may also serve as a marker for the acute phase response. Sialyltransferase is an example of a glycosyltransferases in which sialic acid is transferred to oligosaccharides of glycopeptides in the Golgi prior to glycopeptide secretion. In this study, sialyltransferase activities were determined in plasma, liver, and intestinal mucosa following a standardized 25% body surface area thermal injury in the rat. A statistically significant increase in sialyltransferase activity was found in liver and small intestine which were maximal at 24 hours after the injury. These increased sialyltransferase activities were accompanied by a statistically significant 2 to 4 fold elevation in plasma sialyltransferase activity at 24 hours. The plasma and liver elevations in these activities were similar to elevations seen in other models of acute inflammation using turpentine injections and bacterial infections. The increased sialyltransferase activity within the rat intestine was comparable to increases in intestinal sialyltransferase activity following colchicine treatment and may represent a similar mechanism(s).     

Green tea extract supplement reduces D-galactosamine-induced acute liver injury by inhibition of apoptotic and proinflammatory signaling

Oxidative stress and inflammation contributed to the propagation of acute liver injury (ALI). The present study was undertaken to determine whether D-galactosamine (D-GalN) induces ALI via the mitochondrial apoptosis- and proinflammatory cytokine-signaling pathways, and possible mechanism(s) by which green tea (GT) extract modulates the apoptotic and proinflammatory signaling in rat. D-GalN induced hepatic hypoxia/hypoperfusion and triggered reactive oxygen species (ROS) production from affected hepatocytes, infiltrated leukocytes, and activated Kupffer cells. D-GalN evoked cytosolic Bax and mitochondrial cytochrome C translocation and activated proinflammatory nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) translocation, contributing to the increase of intercellular adhesion molecule-1 expression, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)-positive hepatocytes, multiple plasma cytokines and chemokines release, and alanine aminotransferase (ALT) activity. An altered biliary secretion profile of several acute phase proteins directly indicates oxidative stress affecting intracellular trafficking in the hepatocyte. GT pretreatment attenuated ROS production, mitochondrial apoptosis- and proinflammatory cytokine-signaling pathway, plasma ALT and cytokines levels, biliary acute phase proteins secretion and hepatic pathology by the enhancement of anti-apoptotic mechanisms. In conclusion, D-GalN induced ALI via hypoxia/hypoperfusion-enhanced mitochondrial apoptosis- and proinflammatory cytokine-signaling pathway, contributing to oxidative stress and inflammation in the liver. GT can counteract the D-GalN-induced ALI via the attenuation of apoptotic and proinflammatory signaling by the upregulation of anti-apoptotic mechanism.