Physiological network approach to prognosis in cirrhosis: A shifting paradigm.

Decompensated liver disease is complicated by multi-organ failure and poor prognosis. The prognosis of patients with liver failure often dictates clinical management. Current prognostic models have focused on biomarkers considered as individual isolated units. Network physiology assesses the interactions among multiple physiological systems in health and disease irrespective of anatomical connectivity and defines the influence or dependence of one organ system on another. Indeed, recent applications of network mapping methods to patient data have shown improved prediction of response to therapy or prognosis in cirrhosis. Initially, different physical markers have been used to assess physiological coupling in cirrhosis including heart rate variability, heart rate turbulence, and skin temperature variability measures. Further, the parenclitic network analysis was recently applied showing that organ systems connectivity is impaired in patients with decompensated cirrhosis and can predict mortality in cirrhosis independent of current prognostic models while also providing valuable insights into the associated pathological pathways. Moreover, network mapping also predicts response to intravenous albumin in patients hospitalized with decompensated cirrhosis. Thus, this review highlights the importance of evaluating decompensated cirrhosis through the network physiologic prism. It emphasizes the limitations of current prognostic models and the values of network physiologic techniques in cirrhosis.

Parenclitic Network Mapping Identifies Response to Targeted Albumin Therapy in Patients Hospitalized With Decompensated Cirrhosis.

INTRODUCTION: The efficacy of targeted albumin therapy in the management of decompensatory events in cirrhosis is unclear, with different reports showing conflicting results. It is possible that only certain subgroups of patients may benefit from targeted albumin administration. However, extensive conventional subgroup analyses have not yet identified these subgroups. Albumin is an important regulator of physiological networks and may interact with homeostatic mechanism differently in patients according to the integrity of their physiological network. In this study, we aimed to assess the value of network mapping in predicting response to targeted albumin therapy in patients with cirrhosis. METHODS: This is a substudy of the ATTIRE trial, a multicenter randomized trial conducted to assess the effect of targeted albumin therapy in cirrhosis. Baseline serum bilirubin, albumin, sodium, creatinine, CRP, white cell count (WCC), international normalized ratio, heart rate, and blood pressure of 777 patients followed up for 6 months were used for network mapping using parenclitic analysis. Parenclitic network analysis involves measuring the deviation of each patient from the existing network of physiological interactions in a reference population. RESULTS: Overall network connectivity and deviations along the WCC-CRP axis predicted 6-month survival independent of age and model for end-stage liver disease in the standard care arm. Patients with lower deviation along the WCC-CRP axis showed lower survival in response to targeted albumin administration over a 6-month follow-up period. Likewise, patients with higher overall physiological connectivity survived significantly less than the standard care group after targeted albumin infusion. DISCUSSION: The parenclitic network mapping can predict the survival of patients with cirrhosis and identify patient subgroups that do not benefit from targeted albumin therapy.

Prognosis and Survival Modelling in Cirrhosis Using Parenclitic Networks.

Background: Liver cirrhosis involves multiple organ systems and has a high mortality. A network approach to complex diseases often reveals the collective system behaviours and intrinsic interactions between organ systems. However, mapping the functional connectivity for each individual patient has been challenging due to the lack of suitable analytical methods for assessment of physiological networks. In the present study we applied a parenclitic approach to assess the physiological network of each individual patient from routine clinical/laboratory data available. We aimed to assess the value of the parenclitic networks to predict survival in patients with cirrhosis. Methods: Parenclitic approach creates a network from the perspective of an individual subject in a population. In this study such an approach was used to measure the deviation of each individual patient from the existing network of physiological interactions in a reference population of patients with cirrhosis. 106 patients with cirrhosis were retrospectively enrolled and followed up for 12 months. Network construction and analysis were performed using data from seven clinical/laboratory variables (serum albumin, bilirubin, creatinine, ammonia, sodium, prothrombin time and hepatic encephalopathy) for calculation of parenclitic deviations. Cox regression was used for survival analysis. Result: Initial network analysis indicated that correlation between five clinical/laboratory variables can distinguish between survivors and non-survivors in this cohort. Parenclitic deviations along albumin-bilirubin (Hazard ratio = 1.063, p < 0.05) and albumin-prothrombin time (Hazard ratio = 1.138, p < 0.05) predicted 12-month survival independent of model for end-stage liver disease (MELD). Combination of MELD with the parenclitic measures could predict survival better than MELD alone. Conclusion: The parenclitic network approach can predict survival of patients with cirrhosis and provides pathophysiologic insight on network disruption in chronic liver disease.

Short-term abstinence from alcohol and changes in cardiovascular risk factors, liver function tests and cancer-related growth factors: a prospective observational study.

OBJECTIVE: To assess changes in metabolic risk factors and cancer-related growth factors associated with short-term abstinence from alcohol. DESIGN: Prospective, observational study. SETTING: Single tertiary centre. PARTICIPANTS: Healthy subjects were recruited based on intention to: (1) abstain from alcohol for 1 month (abstinence group), or (2) continue to drink alcohol (control group). Inclusion criteria were baseline alcohol consumption >64 g/week (men) or >48 g/week (women). Exclusion criteria were known liver disease or alcohol dependence. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was change in insulin resistance (homeostatic model assessment (HOMA) score). Secondary outcomes were changes in weight, blood pressure (BP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and liver function tests. Primary and secondary outcomes were adjusted for changes in diet, exercise and cigarette smoking. RESULTS: The abstinence group comprised 94 participants (mean age 45.5 years, SD ±1.2) and the control group 47 participants (mean age 48.7 years, SD ±1.8). Baseline alcohol consumption in the abstinence group was 258.2 g/week, SD ±9.4, and in the control group 233.8 g, SD ±19.0. Significant reductions from baseline in the abstinence group (all p<0.001) were found in: HOMA score (-25.9%, IQR -48.6% to +0.3%), systolic BP (-6.6%, IQR -11.8% to 0.0%), diastolic BP (-6.3%, IQR -14.1% to +1.3%), weight (-1.5%, IQR -2.9% to -0.4%), VEGF (-41.8%, IQR -64.9% to -17.9%) and EGF (-73.9%, IQR -86.1% to -36.4%). None of these changes were associated with changes in diet, exercise or cigarette smoking. No significant changes from baseline in primary or secondary outcomes were noted in the control group. CONCLUSION: These findings demonstrate that abstinence from alcohol in moderate-heavy drinkers improves insulin resistance, weight, BP and cancer-related growth factors. These data support an independent association of alcohol consumption with cancer risk, and suggest an increased risk of metabolic diseases such as type 2 diabetes and fatty liver disease.

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: Chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models.

BACKGROUND: Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility. METHODS AND FINDINGS: In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced. CONCLUSIONS: The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury.

The metabolism and de-bromination of bromotyrosine in vivo.

During inflammation, leukocyte-derived eosinophil peroxidase catalyses the formation of hypobromous acid, which can brominate tyrosine residues in proteins to form bromotyrosine. Since eosinophils are involved in the pathogenesis of allergic reactions, such as asthma, urinary bromotyrosine level has been used for the assessment of children with asthma. However, little is known about the metabolism and disposition of bromotyrosine in vivo. The aim of this study was to identify the major urinary metabolites formed during bromotyrosine metabolism and to develop mass spectrometric methods for their quantitation. Deuterium-labeled bromotyrosine was synthesized by deuterium exchange. [D3]bromotyrosine (500 nmole) was injected intraperitoneally into Sprague-Dawley rats and urine was collected for 24h in a metabolic cage. (13)C-labeled derivatives of bromotyrosine and its major urinary metabolite were synthesized and used as internal standards for quantitation. Following solid phase extraction, urine samples were derivatized to the pentafluorobenzyl ester, and analyzed using isotope dilution gas chromatography and negative-ion chemical ionization mass spectrometry. A novel brominated metabolite, 3-bromo-4-hydroxyphenylacetic acid (bromo-HPA), was identified as the major brominated metabolite of bromotyrosine. Bromo-HPA only accounted for 0.43 ± 0.04% of infused [D3]bromotyrosine and 0.12 ± 0.02% of infused [D3]bromotyrosine was excreted in the urine unchanged. However, ~1.3% (6.66 ± 1.33 nmole) of infused [D3]bromotyrosine was excreted in the urine as the de-brominated metabolite, [D3]4-hydroxyphenylacetic acid, which is also a urinary metabolite of tyrosine in mammals. We also tested whether or not iodotyrosine dehalogenase can catalyse de-bromination of bromotyrosine and showed that iodotyrosine dehalogenase is able to de-brominate free bromotyrosine in vitro. We identified bromo-HPA as the main brominated urinary metabolite of bromotyrosine in rats. However, de-halogenation of bromotyrosine is the major metabolic pathway to eliminate free brominated tyrosine in vivo.

Nitric oxide-dependent bradycardia in mutant analbuminemic rats.

Nagase analbuminemic rats (NAR) are natural mutant Sprague-Dawley rats which do not express albumin due to a single splice mutation in the albumin gene. We accidentally discovered that NAR have a significant bradycardia compared with wild type Sprague-Dawley rats, and the present study was carried out to investigate the mechanism of bradycardia in analbuminemic rats. In vitro studies showed that the basal spontaneous beating rate of isolated atria is similar in NAR compared with wild type animals. However, the chronotropic responsiveness of isolated atria to cholinergic stimulation was markedly increased in NAR, an effect which was prevented by incubation with a nitric oxide synthase (NOS) or guanylyl cyclase inhibitor. NAR had a significant increase in plasma nitrite/nitrate concentrations. Administration of a NOS inhibitor for 5 days normalized heart rate in NAR. The level of NOS isoforms, caveolin-1 and caveolin-3 expression in the atria was assessed by real time PCR. There was no significant difference in the expression of NOS isoforms or caveolin-3 in NAR compared with wild type controls. However, NAR exhibited a significant decrease in caveolin-1 expression in the atria. Since caveolin-1 is known to inhibit endothelial NOS activity in cardiomyocytes, we suggest that decreased caveolin-1 levels may have a role in increased nitric oxide production in NAR. Our data suggest that a NOS/cGMP-dependent mechanism might be involved in increased responsiveness to vagal stimulation and bradycardia in analbuminemic condition.

Role of endogenous hydrogen sulfide in neurogenic relaxation of rat corpus cavernosum.

Relaxation of corpus cavernosum during penile erection is mediated by a non-adrenergic non-cholinergic (NANC) neurotransmission and by the endothelium via the release of nitric oxide. Hydrogen sulfide (H(2)S) is an endogenous gaseous mediator which is a potent vasodilator and a neurotransmitter. This study was initiated to characterize the role of H(2)S in NANC neurogenic transmission in rat corpus cavernosum. The expression of H(2)S producing enzymes was assessed using RT-PCR as well as Western blotting and showed the expression of cystathionine γ-lyase (CSE) in rat corporal tissue. Homogenates from rat corpus cavernosum convert l-cysteine to H(2)S and this was partially inhibited by a CSE inhibitor, propargylglycine. Electrical stimulation of corporal tissue strips caused NANC relaxation. This neurogenic relaxation was significantly enhanced by inhibition of CSE by propargylglycine indicating that endogenously produced H(2)S may have a negative regulatory role in neurogenic relaxation of rat corpus cavernosum. To investigate this further we used physiologically relevant concentrations of exogenous NaHS, and showed that nanomolar concentrations could inhibit corporal relaxation induced by a nitroxyl (HNO) donor (Angeli's salt) but not with nitrosonium (NO(+)) or NO donors. This suggests that an interaction between endogenously produced H(2)S and nitroxyl (HNO) might be involved in erectile function.

Endotoxemia is associated with partial uncoupling of cardiac pacemaker from cholinergic neural control in rats.

Cardiac cycle is regulated by a complex interplay between autonomic nervous system and cardiac pacemaker cells. Decreased heart rate variability (HRV) and increased cardiac rhythm regularity are associated with poor prognosis in patients with systemic inflammation (e.g., sepsis). However, the underlying mechanism of decreased HRV in systemic inflammation is not understood. It is known that greater regularity in a complex system could indicate uncoupling of the system's components. The present study aimed to test the hypothesis that impaired responsiveness of cardiac pacemaker to autonomic nervous system may lead to uncoupling of the cardiovascular regulatory mechanisms during systemic inflammation. Systemic inflammation was induced by intraperitoneal injection of endotoxin (lipopolysaccharide, 1 mg/kg) in rats. Cardiovascular signals were recorded in conscious animals using a telemetric system. Heart rate dynamics was analyzed using Poincaré plot, and cardiac cycle regularity was assessed by sample entropy analysis. Spontaneously beating atria were isolated, and chronotropic responsiveness to adrenergic and cholinergic stimulation was assessed using standard organ bath. Sample entropy decreased significantly 4 h after endotoxin injection in conscious rats. Vagal modulation of cardiac cycle (as assessed by Poincaré plot) also exhibited a significant reduction in endotoxemic rats. Acute endotoxin challenge was associated with a significant hyporesponsiveness of isolated spontaneously beating atria to cholinergic stimulation. The chronotropic responsiveness to adrenergic stimulation was identical in controls and endotoxin-treated rats. These data propose that systemic inflammation is linked to reduced cardiac responsiveness to cholinergic stimulation. This may lead to partial uncoupling of cardiac pacemaker cells from autonomic neural control and can explain decreased HRV during systemic inflammation.

d-Serine modulates neurogenic relaxation in rat corpus cavernosum.

d-Serine, an endogenous co-agonist for the N-methyl-d-aspartate (NMDA) receptor in mammals, is synthesized from l-serine by serine racemase. Although much attention has been focused on the role of d-serine within the central nervous system, the physiological role of d-serine in peripheral nerves such as corpus cavernosal nerves has not been investigated. The present study was aimed to study the expression, cellular localization and function of serine racemase/d-serine system in isolated rat corpus cavernosum. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis showed the expression of serine racemase in rat corpus cavernosum. Immunogold electron microscopy demonstrated the cellular localization of serine racemase in the cavernosal nerves' membrane of the tissue. The organ bath studies on isolated rat corpus cavernosum showed that d-serine increases the non-adrenergic non-cholinergic neurogenic relaxation of isolated rat corpus cavernosum in vitro. This effect of d-serine was inhibited by a variety of NMDA receptor antagonists (ketamine, MK 801 and ifenprodil), suggesting that NMDA receptors are involved in the effects of d-serine on the neurogenic relaxation of corporal tissue strips. These observations provide the first evidence for the role of d-serine in modulating the neurogenic relaxation of rat corpus cavernosum, and may open new therapeutic avenues for the treatment of impotence.

Acetaminophen inhibits hemoprotein-catalyzed lipid peroxidation and attenuates rhabdomyolysis-induced renal failure.

Hemoproteins, hemoglobin and myoglobin, once released from cells can cause severe oxidative damage as a consequence of heme redox cycling between ferric and ferryl states that generates radical species that induce lipid peroxidation. We demonstrate in vitro that acetaminophen inhibits hemoprotein-induced lipid peroxidation by reducing ferryl heme to its ferric state and quenching globin radicals. Severe muscle injury (rhabdomyolysis) is accompanied by the release of myoglobin that becomes deposited in the kidney, causing renal injury. We previously showed in a rat model of rhabdomyolysis that redox cycling between ferric and ferryl myoglobin yields radical species that cause severe oxidative damage to the kidney. In this model, acetaminophen at therapeutic plasma concentrations significantly decreased oxidant injury in the kidney, improved renal function, and reduced renal damage. These findings also provide a hypothesis for potential therapeutic applications for acetaminophen in diseases involving hemoprotein-mediated oxidative injury.

Making and working with hydrogen sulfide: The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: a review.

Hydrogen sulfide is rapidly emerging as an important vasoactive mediator formed in health and disease. Its biological action is centered on its reactivity with heme-proteins and its ability to activate K(ATP) channels. Hydrogen sulfide is a signalling molecule of the inflammatory and nervous systems, and in particular the cardiovascular system where it regulates vascular tone, cardiac work, and exerts cardioprotection. This has led to an explosion of papers in which the role of hydrogen sulfide generated in vitro has been used to stimulate biological responses, and where a variety of methods have been used to measure the concentration of this compound in biological fluids. Understanding the chemistry and the inherent problems in the analytical techniques used to measure hydrogen sulfide concentrations is critical to our expanding knowledge on the biology of hydrogen sulfide. In this brief review we will cover the chemistry of hydrogen sulfide, including sources of hydrogen sulfide, its speciation at physiological pH, the susceptibility of sulfide to aerobic oxidation, and the methods used to measure hydrogen sulfide concentrations in solution, including biological fluids. We also give a brief overview of knockout animals and inhibition of the enzymes involved in the formation of hydrogen sulfide in vivo.

Myocardial stunning is associated with impaired calcium uptake by sarcoplasmic reticulum.

Myocardial stunning (temporary post-ischaemic contractile dysfunction) may be caused by oxidative stress and/or impaired myocyte calcium homeostasis. Regional myocardial stunning was induced in open-chest pigs (segment shortening reduced to 68.3+/-4.7% of baseline) by repetitive brief circumflex coronary occlusion (I/R). Reduced glutathione was depleted in stunned myocardium (1.34+/-0.06 vs. 1.77+/-0.11 nmol/mg, p=0.02 vs. remote myocardium) indicating regional oxidant stress, but no regional differences were observed in protein-bound 3-nitrotyrosine or S-nitrosothiol content. Repetitive I/R did not affect myocardial quantities of the sarcolemmal sodium-calcium exchanger, L-type channel, SR calcium ATPase and phospholamban, or the kinetics of ligand binding to L-type channels and SR calcium release channels. However, initial rates of oxalate-supported (45)Ca uptake by SR were impaired in stunned myocardium (41.3+/-13.5 vs. 73.0+/-15.6 nmol/min/mg protein, p=0.03). The ability of SR calcium ATPase to sequester cytosolic calcium is impaired in stunned myocardium. This is a potential mechanism underlying contractile dysfunction.

Decreased heart rate variability in patients with cirrhosis relates to the presence and degree of hepatic encephalopathy.

Heart rate variability (HRV) is reduced in several clinical settings associated with either systemic inflammation or neuropsychiatric impairment. The possibility that the changes in HRV observed in patients with neuropsychiatric impairment might relate to the overproduction of inflammatory cytokines does not seem to have been considered in the studies undertaken to date. HRV is decreased in patients with liver cirrhosis but its relationship to the impairment of neuropsychiatric performance, commonly observed in these patients, is unknown. The aim of this study was to investigate the relationship between HRV, hepatic encephalopathy, and production of inflammatory cytokines in patients with cirrhosis. Eighty patients with cirrhosis [53 men, 27 women; mean (+/-1SD) age 54 +/- 10 yr], classified as neuropsychiatrically unimpaired or as having minimal or overt hepatic encephalopathy, and 11 healthy subjects were studied. HRV was assessed by applying Poincaré plot analysis to the R-R interval series on a 5-min ECG. Inflammatory cytokines (TNF-alpha, IL-6, IL-10, and IL-12) were measured in a subgroup of patients. Long-term R-R variability was significantly decreased in the patients with cirrhosis, in parallel with the degree of neuropsychiatric impairment (P < 0.01) and independently of the degree of hepatic dysfunction (P = 0.011). The relative risk of death increased by 7.7% for every 1-ms drop in this variable. Plasma levels of IL-6 significantly correlated with indexes of both HRV and neuropsychiatric performance. The changes observed in HRV and in neuropsychiatric status in patients with cirrhosis are significantly correlated, most likely reflecting a common pathogenic mechanism mediated by inflammatory cytokines.

Urinary excretion of the nitrotyrosine metabolite 3-nitro-4-hydroxyphenylacetic acid in preterm and term infants.

BACKGROUND: Newborn infants are exposed to various sources of oxidative and/or nitrative stress, which refers to either oxidation and/or nitration of endogenous proteins including loss of their original function. Nitrative stress is predominantly caused following synthesis of peroxynitrite. Particularly preterm infants with immature defense mechanisms against free radical injury appear at risk. OBJECTIVE: To test the feasibility of quantifying the degradation products of the peroxynitrite marker nitrotyrosine [3-nitro-4-hydroxyphenylacetic acid (NHPA) and para-hydroxyphenylacetic acid (PHPA)] in neonatal urine samples. METHODS: NHPA and PHPA were determined by gas chromatography/mass spectroscopy in urinary samples of preterm and term infants (mean gestational age = 28.4 and 39.6 weeks, respectively). RESULTS: The urinary NHPA levels were lower in preterm infants in comparison with term infants. When the NHPA levels were adjusted to the urinary PHPA levels, no differences were found between the two groups. CONCLUSIONS: Nitrotyrosine can be quantified in urinary samples of even the most immature infants. Nitration of endogenous PHPA in the gastrointestinal tract of term infants may have masked potentially higher levels of NHPA in preterm infants.

Diet-induced endogenous formation of nitroso compounds in the GI tract.

Red or processed meat, but not white meat or fish, is associated with colorectal cancer. The endogenous formation of nitroso compounds is a possible explanation, as red or processed meat--but not white meat or fish--causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC) and the formation of nitroso-compound-specific DNA adducts. Red meat is particularly rich in heme and heme has also been found to promote the formation of ATNC. To investigate the underlying mechanism of ATNC formation, fecal and ileal samples of volunteers fed a high red meat or a vegetarian diet were analyzed for nitrosyl iron, nitrosothiols, and heme. To simulate the processes in the stomach, food homogenates and hemoglobin were incubated under simulated gastric conditions. Nitrosyl iron and nitrosothiols were significantly (p < 0.0001) increased in ileal and fecal samples after a high red meat diet compared with a vegetarian diet; significantly more nitrosyl iron than nitrosothiols was detectable in ileal (p < 0.0001) and fecal (p < 0.001) samples. The strong correlation between fecal nitrosyl iron and heme (0.776; p < 0.0001) suggested that nitrosyl heme is the main source of nitrosyl iron, and ESR confirmed the presence of nitrosyl heme in fecal samples after a high red meat diet. Under simulated gastric conditions, mainly nitrosothiols were formed, suggesting that acid-catalyzed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl heme and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.

The metabolism and dechlorination of chlorotyrosine in vivo.

During inflammation, neutrophil- and monocyte-derived myeloperoxidase catalyzes the formation of hypochlorous acid, which can chlorinate tyrosine residues in proteins to form chlorotyrosine. However, little is known of the metabolism and disposition of chlorotyrosine in vivo. Following infusion of deuterium-labeled [D(4)]chlorotyrosine into Sprague-Dawley rats, the major urinary metabolites were identified by mass spectrometry. 3-Chloro-4-hydroxyphenylacetic acid was identified as the major chlorinated metabolite of chlorotyrosine and accounted for 3.6 +/- 0.3% of infused [D(4)]chlorotyrosine. The striking observation was that approximately 40% (39 +/- 1%) of infused [D(4)]chlorotyrosine was dechlorinated and excreted in the urine as deuterated 4-hydroxyphenylacetic acid, a major metabolite of tyrosine. 1.1 +/- 0.1% of infused [D(4)]chlorotyrosine was excreted as [D(4)]tyrosine. To determine whether protein-bound chlorotyrosine could undergo dechlorination, chlorinated albumin was incubated with liver homogenate from mutant rats, which did not synthesize albumin. There was approximately 20% decrease in the chlorotyrosine content over 1 h. This study is the first to describe the dechlorination of chlorotyrosine as the major metabolic pathway to eliminate this modified amino acid in vivo.

Endotoxemia produces coma and brain swelling in bile duct ligated rats.

UNLABELLED: This study explores the hypothesis that the inflammatory response induced by administration of lipopolysaccharide (LPS) exacerbates brain edema in cirrhotic rats; and if so whether this is associated with altered brain metabolism of ammonia or anatomical disturbance of the blood-brain barrier. Adult Sprague-Dawley rats 4 weeks after bile duct ligation (BDL)/Sham-operation, or naïve rats fed a hyperammonemic diet (HD), were injected with LPS (0.5 mg/kg, intraperitoneally) or saline, and killed 3 hours later. LPS administration increased brain water in HD, BDL, and sham-operated groups significantly (P < 0.05), but this was associated with progression to pre-coma stages only in BDL rats. LPS induced cytotoxic brain swelling and maintained anatomical integrity of the blood-brain barrier. Plasma/brain ammonia levels were higher in HD and BDL rats than in sham-operated controls and did not change with LPS administration. Brain glutamine/myoinositol ratio was increased in the HD group but reduced in the BDL animals. There was a background pro-inflammatory cytokine response in the brains of cirrhotic rats, and plasma/brain tumor necrosis factor alpha (TNF-alpha) and IL-6 significantly increased in LPS-treated animals. Plasma nitrite/nitrate levels increased significantly in LPS groups compared with non-LPS controls; however, frontal cortex nitrotyrosine levels only increased in the BDL + LPS rats (P < 0.005 versus BDL controls). CONCLUSION: Injection of LPS into cirrhotic rats induces pre-coma and exacerbates cytotoxic edema because of the synergistic effect of hyperammonemia and the induced inflammatory response. Although the exact mechanism of how hyperammonemia and LPS facilitate cytotoxic edema and pre-coma in cirrhosis is not clear, our data support an important role for the nitrosation of brain proteins.

pH-dependent nitration of para-hydroxyphenylacetic acid in the stomach.

The major urinary metabolite of nitrotyrosine is 3-nitro-4-hydroxyphenylacetic acid (3-Nitro-HPA). However, recent animal studies have shown that the majority of urinary 3-Nitro-HPA is derived from nitration of endogenous para-hydroxyphenylacetic acid (HPA), a metabolite of tyrosine. One potential site for the formation of 3-Nitro-HPA is the stomach, where nitrous acid is formed by the reaction of nitrite in saliva with gastric acid. The aim of this study was to determine whether there is pH-dependent nitration of salivary para-hydroxyphenylacetic acid or tyrosine, and the effects of dietary nitrate. Healthy volunteers (n = 18) ingested either a low or high nitrate diet, with and without the administration of omeprazole, a proton pump inhibitor. Urinary 3-Nitro-HPA excretion increased from 197 +/- 52 to 319 +/- 88 microg/day on switching from a low to a high nitrate diet (P < 0.05), and decreased (166 +/- 53 mug/day, P < 0.05) when gastric pH was increased by omeprazole. To determine whether 3-Nitro-HPA can be formed by nitration of para-hydroxyphenylacetic acid in the stomach, 500 microg of deuterated para-hydroxyphenylacetic acid was ingested with a high nitrate meal. This led to the excretion of both deuterated HPA and 3-Nitro-HPA in the urine, confirming that para-hydroxyphenylacetic acid is absorbed, and nitrated. Since omeprazole decreases the formation of 3-Nitro-HPA, presumably by decreasing the nitration of endogenous para-hydroxyphenylacetic acid present in saliva, and the observation that ingested deuterated para-hydroxyphenylacetic acid is nitrated and excreted, we conclude that endogenous para-hydroxyphenylacetic acid is nitrated in the stomach, absorbed, and excreted as 3-Nitro-HPA.