Alleviation of liver cirrhosis and associated portal-hypertension by Astragalus species in relation to their UPLC-MS/MS metabolic profiles: a mechanistic study.

Liver cirrhosis is a late-stage liver disease characterized by excessive fibrous deposition triggering portal-hypertension (PH); the prime restrainer for cirrhosis-related complications. Remedies that can dually oppose hepatic fibrosis and lower PH, may prevent progression into decompensated-cirrhosis. Different Astragalus-species members have shown antifibrotic and diuretic actions with possible subsequent PH reduction. However, A.spinosus and A.trigonus were poorly tested for eliciting these actions. Herein, A.spinosus and A.trigonus roots and aerial parts extracts were subjected to comprehensive metabolic-fingerprinting using UHPLC-MS/MS resulting in 56 identified phytoconstituents, followed by chemometric untargeted analysis that revealed variable metabolic profiles exemplified by different species and organ types. Consequently, tested extracts were in-vivo evaluated for potential antifibrotic/anticirrhotic activity by assessing specific markers. The mechanistic prospective to induce diuresis was investigated by analyzing plasma aldosterone and renal-transporters gene-expression. Serum apelin and dimethylarginine-dimethylaminohydrolase-1 were measured to indicate the overall effect on PH. All extracts amended cirrhosis and PH to varying extents and induced diuresis via different mechanisms. Further, An OPLS model was built to generate a comprehensive metabolic-profiling of A.spinosus and A.trigonus secondary-metabolites providing a chemical-based evidence for their efficacious consistency. In conclusion, A.spinosus and A.trigonus organs comprised myriad pharmacologically-active constituents that act synergistically to ameliorate cirrhosis and associated PH.

PM20D1 is a circulating biomarker closely associated with obesity, insulin resistance and metabolic syndrome.

OBJECTIVE: Peptidase M20 domain containing 1 (PM20D1), a secreted enzyme catalysing condensation of fatty acids and amino acids into the bioactive lipids N-acyl amino acids (NAAA), induces uncoupling protein 1 (UCP1)-independent adaptive thermogenesis in brown/beige adipocytes in mice. This study aimed to explore the associations of the circulating levels of PM20D1 and major NAAA with obesity-related metabolic complications in humans. DESIGN AND METHODS: Serum concentrations of PM20D1 and NAAA (C18:1-Leu and C18:1-Phe) in 256 Chinese subjects, including 78 lean and 178 overweight/obese individuals with or without diabetes, were measured with immunoassays and liquid chromatography-mass spectrometry, respectively. The impact of sulfonylurea and rosiglitazone on their circulating levels was examined in 62 patients with type 2 diabetes. RESULTS: Serum PM20D1 level was significantly elevated in overweight/obese individuals and was closely associated with circulating levels of C18:1-Leu and C18:1-Phe. Furthermore, serum PM20D1, C18:1-Leu and C18:1-Phe concentrations correlated positively with several parameters of adiposity as well as fasting and 2 h postprandial glucose, HbA1c, fasting insulin and HOMA-IR independent of BMI and age. Moreover, a significant elevation in PM20D1, C18:1-Leu and C18:1-Phe concentrations corresponding with increases in the number of components of the metabolic syndrome (MetS) was observed. Treatment with sulfonylurea significantly decreased circulating PM20D1, C18:1-Leu and C18:1-Phe in patients with type 2 diabetes. CONCLUSIONS: Increased serum levels of PM20D1 and its catalytic products NAAA are closely associated with obesity-related glucose dysregulation, insulin resistance and MetS and can be potentially used as clinical biomarkers for diagnosing and monitoring these disorders.

Longitudinal data in peripheral blood confirm that PM20D1 is a quantitative trait locus (QTL) for Alzheimer's disease and implicate its dynamic role in disease progression.

BACKGROUND: While Alzheimer's disease (AD) remains one of the most challenging diseases to tackle, genome-wide genetic/epigenetic studies reveal many disease-associated risk loci, which sheds new light onto disease heritability, provides novel insights to understand its underlying mechanism and potentially offers easily measurable biomarkers for early diagnosis and intervention. METHODS: We analyzed whole-genome DNA methylation data collected from peripheral blood in a cohort (n = 649) from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and compared the DNA methylation level at baseline among participants diagnosed with AD (n = 87), mild cognitive impairment (MCI, n = 175) and normal controls (n = 162), to identify differentially methylated regions (DMRs). We also leveraged up to 4 years of longitudinal DNA methylation data, sampled at approximately 1 year intervals to model alterations in methylation levels at DMRs to delineate methylation changes associated with aging and disease progression, by linear mixed-effects (LME) modeling for the unchanged diagnosis groups (AD, MCI and control, respectively) and U-shape testing for those with changed diagnosis (converters). RESULTS: When compared with controls, patients with MCI consistently displayed promoter hypomethylation at methylation QTL (mQTL) gene locus PM20D1. This promoter hypomethylation was even more prominent in patients with mild to moderate AD. This is in stark contrast with previously reported hypermethylation in hippocampal and frontal cortex brain tissues in patients with advanced-stage AD at this locus. From longitudinal data, we show that initial promoter hypomethylation of PM20D1 during MCI and early stage AD is reversed to eventual promoter hypermethylation in late stage AD, which helps to complete a fuller picture of methylation dynamics. We also confirm this observation in an independent cohort from the Religious Orders Study and Memory and Aging Project (ROSMAP) Study using DNA methylation and gene expression data from brain tissues as neuropathological staging (Braak score) advances. CONCLUSIONS: Our results confirm that PM20D1 is an mQTL in AD and demonstrate that it plays a dynamic role at different stages of the disease. Further in-depth study is thus warranted to fully decipher its role in the evolution of AD and potentially explore its utility as a blood-based biomarker for AD.

Investigation of Safety and Tolerability of ASP3652 Based on Clinical Studies of Cerebrospinal Fluid Transfer After Multiple Doses and Exposure After Single Doses at High Dose Levels.

INTRODUCTION: The studies described here were conducted to investigate the central nervous system (CNS) transfer of ASP3652, a peripherally acting inhibitor of fatty acid amide hydrolase, after multiple doses at around the anticipated therapeutic dose and the safety, tolerability, and pharmacokinetics after single doses at corresponding supratherapeutic doses in healthy subjects. METHODS: Study 1 was an open-label multiple dose study in which ASP3652 (300 mg bid) or matching placebo was administered in multiple doses to healthy subjects. Study 2 was a placebo-controlled, randomized 4 × 4 crossover study in which ASP3652 was given as three single ascending doses of ASP3652 (600-1800 mg) or matching placebo to healthy subjects. Levels of ASP3652 and endocannabinoids (eCBs) in plasma, cerebrospinal fluid (CSF) (study 1 only), and safety were evaluated. RESULTS: In study 1, ASP3652 was readily absorbed to reach Cmax at 1 h after dosing. AUCtau and Cmax of ASP3652 in CSF were approximately 0.2% and 0.06% of the AUCtau and Cmax in plasma after multiple doses of ASP3652 300 mg bid. At steady state the area under the response-time curve (AURC) from 0 to 12 h and the maximum response for anandamide in plasma were approximately 550-fold and 230-fold higher than those in CSF. In study 2, the Cmax and AUC of ASP3652 increased higher than dose proportionally in subjects receiving 600-1800 mg ASP3652. For eCBs, although the AURC increased less than dose proportionally, maximum plasma levels were comparable across all treatment groups. The incidence of adverse events (AEs) was similar across all treatment groups including the placebo group. There was no evidence of CNS-related side effects. CONCLUSIONS: ASP3652 showed low CNS penetration at the anticipated therapeutic dose and was well tolerable without any CNS-related AEs at supratherapeutic doses, supporting that the drug can be safely tested at the anticipated therapeutic dose. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02034734 for study 1, NCT01815684 for study 2.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ASP3652, a Reversible Fatty Acid Amide Hydrolase Inhibitor, in Healthy, Nonelderly, Japanese Men and Elderly, Japanese Men and Women: A Randomized, Double-blind, Placebo-controlled, Single and Multiple Oral Dose, Phase I Study.

PURPOSE: This study aimed to evaluate the safety, tolerability, and pharmacokinetic and pharmacodynamic properties of ASP3652, a peripherally acting inhibitor of peripheral fatty acid amide hydrolase (FAAH) after 30-, 100-, 300-, 600-, and 900-mg single and 100- and 300-mg BID multiple oral dose in Japanese patients. METHODS: This was a randomized, double-blind, placebo-controlled, single and multiple oral dose Phase I study in healthy, nonelderly men and elderly men and women. The study consisted of 2 parts: in the single oral dose part, 40 healthy, nonelderly men were randomized to receive placebo or ASP3652; in the multiple oral dose part, 48 enrolled nonelderly men and elderly men and women were randomized to receive placebo or ASP3652. In both parts, the investigator judged whether the individuals were healthy based on the results of physical examinations and screening. The safety profile was assessed by examining adverse events, defined as any untoward medical occurrence in an individual administered the study drug and that did not necessarily have a causal relationship with the study treatment; clinical laboratory evaluations; vital signs; the Profile of Mood States scale; and standard 12-lead ECGs and 12-lead ECGs for QT assessment. Pharmacokinetic parameters were estimated using unchanged ASP3652 concentrations in plasma and urine. Pharmacodynamic parameters were estimated using FAAH activity and plasma anandamide, oleoylethanolamide, and palmitoylethanolamide concentrations. Safety and tolerability profiles were compared with the placebo group. FINDINGS: ASP3652 was rapidly absorbed to reach Cmax in a single dose and near steady-state at approximately 3 days after the start of multiple dosing. The Cmax and AUC of ASP3652 were slightly higher than dose proportional after a single dose of ASP3652 at 30-900 mg. There was no apparent accumulation based on Cmax and AUC0-12 after multiple doses. Although no differences were found in Cmax or AUC0-12 by age in men, Cmax and AUC0-12 were slightly higher in elderly women than elderly men. FAAH activity was inhibited in a dose-dependent manner, and plasma levels of anandamide, oleoylethanolamide, and palmitoylethanolamide increased in all dose groups after single and multiple doses of ASP3652. The incidence of adverse events after multiple doses, which ranged from 44.4% to 66.7%, was similar across all treatment groups, including the placebo group. IMPLICATIONS: Single and multiple doses of ASP3652 were well tolerated and increased endogenous cannabinoids.

FAAH levels and its genetic polymorphism association with susceptibility to methamphetamine dependence.

The fatty acid amide hydrolase (FAAH) gene was involved in the modulation of reward and addiction pathophysiology of illicit drugs abuse, and its polymorphisms might be associated with risk of methamphetamine (METH) dependence. This study aimed to investigate the FAAH mRNA levels in peripheral blood mononuclear cells and plasma protein levels and to analyze the 385C/A polymorphism (rs324420) between METH-dependent patients and controls. The levels of FAAH mRNA in METH dependence were significantly lower than in controls (P < 0.001), however, its plasma protein underwent a significant ∼2-fold increase (P < 0.001). The A allele of the 385C/A polymorphism significantly increased the METH dependence risk (P < 0.001, odds ratio [OR] = 1.646, 95% confidence interval [CI] = 1.332-2.034). The carried A genotypes (AA, AC, and AA/AC) of 385C/A polymorphism also increased METH-dependence risks under a different genetic model (AA vs. CC: P = 0.017, OR = 2.454, 95%CI = 1.171-2.143; AC vs. CC: P < 0.001, OR = 1.818, 95%CI = 1.404-2.353; AC/AA vs. CC: P < 0.001, OR = 1.858, 95%CI = 1.444-2.319). The similar results were obtained after adjusting for age and sex. Unfortunately, we failed to find that any genotype of 385C/A polymorphism affected the mRNA or plasma protein levels in controls, respectively (P > 0.05). These data indicate that the FAAH may play an important role in the pathophysiological process of METH dependence, and the 385C/A polymorphism may be associated with METH dependence susceptibility in a Chinese Han population.

Inhibition of Dimethylarginine Dimethylaminohydrolase 1 Improves the Outcome of Sepsis in Pregnant Mice.

Sepsis is one of the most important causes of maternal mortality. In our previous work, we established a polymicrobial sepsis (cecal ligation and puncture [CLP]) model in murine pregnancy and found that pregnant mice had a greater susceptibility to septic shock. In this model, mortality seemed to be associated with the development of early hemodynamic dysfunction and although circulating cytokine levels were similar, "off target" lung inflammatory cell numbers were greater in pregnant mice.Here, we have used the same CLP model to test the hypothesis that inhibiting the metabolism of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine would improve the outcome of sepsis in pregnancy. We used a dimethylarginine dimethylaminohydrolase 1-selective inhibitor (L-257), which reduces vascular nitric oxide synthesis without impairing immune cell function, in combination with a broad-spectrum antibiotic (Imipenem) and studied the outcome of septic shock in pregnant mice. Treatments were administered 3 h after CLP and samples were taken 3 h later. Both Imipenem and L-257 treatment alone slightly improved mortality rates from 13% (NaCl) to 20% (Imipenem) and 33% (L-257), whereas the combination of Imipenem and L-257 significantly improved survival to 50%. Imipenem and L-257 together prevented cardiovascular collapse and improved both organ function and bacterial killing, but did not reduce lung inflammatory cell numbers and actually increased lung cytokine levels.These data suggest that conventional management in combination with selective inhibition of DDAH1 may have therapeutic potential in the management of sepsis in pregnancy.

Prediction of preterm labour from a single blood test: The role of the endocannabinoid system in predicting preterm birth in high-risk women.

OBJECTIVE: To determine if plasma concentrations of the N-acylethanolamines (NAEs) N-arachidonoylethanolamine (AEA), N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA) increase in women at high risk for preterm birth (PTB) and whether these could be used to predict preterm delivery and if so, how they compare with current methods. DESIGN: Prospective cohort study. SETTING: A large UK teaching hospital. POPULATION: 217 pregnant women were recruited between 24 and 34 gestational weeks at 'high-risk' for PTB, recruited from a prematurity prevention clinic or antenatal wards. METHODS: Plasma AEA, OEA, and PEA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry whilst FAAH enzyme activity was measured by fluorometric radiometric assay and CL by ultrasound scan. The clinical usefulness of these measurements were determined by ROC and multivariate analyses. RESULTS: AEA and PEA concentrations were significantly higher in women who delivered prematurely. An AEA concentration >1.095 nM predicted PTB, the gestational age at delivery and the recruitment to delivery interval (RTDI). A PEA concentration >17.50 nM only predicted PTB; FAAH enzyme activity was not related to these changes. Multivariate analysis (all variables) generated an equation to accurately predict the RTDI. CONCLUSIONS: A single plasma AEA or PEA measurement can predict PTB. A single AEA measurement predicts the gestational age of delivery and the remaining period of pregnancy with reasonable accuracy and better than existing conventional tests thus offering a better window for primary prevention of PTB.

Characterization of Parkinson's disease using blood-based biomarkers: A multicohort proteomic analysis.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disease affecting about 5 million people worldwide with no disease-modifying therapies. We sought blood-based biomarkers in order to provide molecular characterization of individuals with PD for diagnostic confirmation and prediction of progression. METHODS AND FINDINGS: In 141 plasma samples (96 PD, 45 neurologically normal control [NC] individuals; 45.4% female, mean age 70.0 years) from a longitudinally followed Discovery Cohort based at the University of Pennsylvania (UPenn), we measured levels of 1,129 proteins using an aptamer-based platform. We modeled protein plasma concentration (log10 of relative fluorescence units [RFUs]) as the effect of treatment group (PD versus NC), age at plasma collection, sex, and the levodopa equivalent daily dose (LEDD), deriving first-pass candidate protein biomarkers based on p-value for PD versus NC. These candidate proteins were then ranked by Stability Selection. We confirmed findings from our Discovery Cohort in a Replication Cohort of 317 individuals (215 PD, 102 NC; 47.9% female, mean age 66.7 years) from the multisite, longitudinally followed National Institute of Neurological Disorders and Stroke Parkinson's Disease Biomarker Program (PDBP) Cohort. Analytical approach in the Replication Cohort mirrored the approach in the Discovery Cohort: each protein plasma concentration (log10 of RFU) was modeled as the effect of group (PD versus NC), age at plasma collection, sex, clinical site, and batch. Of the top 10 proteins from the Discovery Cohort ranked by Stability Selection, four associations were replicated in the Replication Cohort. These blood-based biomarkers were bone sialoprotein (BSP, Discovery false discovery rate [FDR]-corrected p = 2.82 × 10-2, Replication FDR-corrected p = 1.03 × 10-4), osteomodulin (OMD, Discovery FDR-corrected p = 2.14 × 10-2, Replication FDR-corrected p = 9.14 × 10-5), aminoacylase-1 (ACY1, Discovery FDR-corrected p = 1.86 × 10-3, Replication FDR-corrected p = 2.18 × 10-2), and growth hormone receptor (GHR, Discovery FDR-corrected p = 3.49 × 10-4, Replication FDR-corrected p = 2.97 × 10-3). Measures of these proteins were not significantly affected by differences in sample handling, and they did not change comparing plasma samples from 10 PD participants sampled both on versus off dopaminergic medication. Plasma measures of OMD, ACY1, and GHR differed in PD versus NC but did not differ between individuals with amyotrophic lateral sclerosis (ALS, n = 59) versus NC. In the Discovery Cohort, individuals with baseline levels of GHR and ACY1 in the lowest tertile were more likely to progress to mild cognitive impairment (MCI) or dementia in Cox proportional hazards analyses adjusting for age, sex, and disease duration (hazard ratio [HR] 2.27 [95% CI 1.04-5.0, p = 0.04] for GHR, and HR 3.0 [95% CI 1.24-7.0, p = 0.014] for ACY1). GHR's association with cognitive decline was confirmed in the Replication Cohort (HR 3.6 [95% CI 1.20-11.1, p = 0.02]). The main limitations of this study were its reliance on the aptamer-based platform for protein measurement and limited follow-up time available for some cohorts. CONCLUSIONS: In this study, we found that the blood-based biomarkers BSP, OMD, ACY1, and GHR robustly associated with PD across multiple clinical sites. Our findings suggest that biomarkers based on a peripheral blood sample may be developed for both disease characterization and prediction of future disease progression in PD.

Microdeletion in a FAAH pseudogene identified in a patient with high anandamide concentrations and pain insensitivity.

The study of rare families with inherited pain insensitivity can identify new human-validated analgesic drug targets. Here, a 66-yr-old female presented with nil requirement for postoperative analgesia after a normally painful orthopaedic hand surgery (trapeziectomy). Further investigations revealed a lifelong history of painless injuries, such as frequent cuts and burns, which were observed to heal quickly. We report the causative mutations for this new pain insensitivity disorder: the co-inheritance of (i) a microdeletion in dorsal root ganglia and brain-expressed pseudogene, FAAH-OUT, which we cloned from the fatty-acid amide hydrolase (FAAH) chromosomal region; and (ii) a common functional single-nucleotide polymorphism in FAAH conferring reduced expression and activity. Circulating concentrations of anandamide and related fatty-acid amides (palmitoylethanolamide and oleoylethanolamine) that are all normally degraded by FAAH were significantly elevated in peripheral blood compared with normal control carriers of the hypomorphic single-nucleotide polymorphism. The genetic findings and elevated circulating fatty-acid amides are consistent with a phenotype resulting from enhanced endocannabinoid signalling and a loss of function of FAAH. Our results highlight previously unknown complexity at the FAAH genomic locus involving the expression of FAAH-OUT, a novel pseudogene and long non-coding RNA. These data suggest new routes to develop FAAH-based analgesia by targeting of FAAH-OUT, which could significantly improve the treatment of postoperative pain and potentially chronic pain and anxiety disorders.

The iridoid loganic acid and anthocyanins from the cornelian cherry (Cornus mas L.) fruit increase the plasma l-arginine/ADMA ratio and decrease levels of ADMA in rabbits fed a high-cholesterol diet.

BACKGROUND: Although fruit and vegetable-rich diets have beneficial effects on cardiovascular diseases, we have little knowledge of the impact of fruits and their constituents, iridoids and anthocyanins, on the l-arginine-ADMA-DDAH pathway. Our previous study demonstrated the modulation of those factors by the oral administration of the cornelian cherry fruit. HYPOTHESIS/PURPOSE: We have assessed the effects of the oral administration of two main constituents isolated from the cornelian cherry fruit, iridoid loganic acid and anthocyanins, on l-arginine, its derivatives (ADMA, SDMA), metabolites (DMA, l-citrulline), and the hepatic DDAH activity and its isoform expression in rabbits fed a high-cholesterol diet. We have also analyzed eNOS expression in the thoracic aorta as well as the redox status in blood. STUDY DESIGN: In the present study, we used an animal model of diet induced atherosclerosis. For 60 days, white New Zealand rabbits were fed a standard diet, a 1% cholesterol enriched diet, or concomitantly with the investigated substances. l-arginine, ADMA, SDMA, DMA, and l-citrulline were assessed using the LC-MS/MS method. DDAH activity and redox parameters were analyzed spectrophotometrically. DDAH1 and DDAH2 isoform expressions were assessed by western blotting, mRNA expression of eNOS was quantified by real-time PCR. RESULTS: We demonstrated that the administration of loganic acid (20 mg/kg b.w.), and to a lesser extent of anthocyanins (10 mg/kg b.w.), caused an increase in the l-arginine level and the l-arginine/ADMA ratio. Also, both substances decreased ADMA, DMA, and l-citrulline, but not SDMA levels. Anthocyanins, but not loganic acid, enhanced the activity of DDAH in the liver. Anthocyanins also significantly enhanced both DDAH1 and DDAH2 expression, while loganic acid to a lesser extent enhanced DDAH1 but not DDAH2 expression. Both loganic acid and anthocyanins pronouncedly increased mRNA expression of eNOS in thoracic aortas. Both loganic acid and anthocyanins reversed the blood glutathione level depleted by dietary cholesterol. Cholesterol feeding decreased the blood GPx level, and the change was not reversed by anthocyanins or loganic acid. We did not observe any significant differences in the blood levels of MDA or SOD among the groups. CONCLUSION: Iridoids and anthocyanins may modulate the l-arginine-ADMA pathway in subjects fed a high-cholesterol diet.

Oleoylethanolamide restores alcohol-induced inhibition of neuronal proliferation and microglial activity in striatum.

Previous findings demonstrate a homeostatic role for oleoylethanolamide (OEA) signaling in the ethanol-related neuroinflammation and behavior. However, extensive research is still required in order to unveil the effects of OEA on a number of neurobiological functions such as adult neurogenesis, cell survival and resident neuroimmunity that become notably altered by alcohol. Daily consumption of ethanol (10%) for 2 weeks (6.3 ± 1.1 g/kg/day during last 5 days) caused hypolocomotor activity in rats. This effect appears to rely on central signaling mechanisms given that alcohol increased the OEA levels, the gene expression of OEA-synthesizing enzyme Nape-pld and the number of PPARα-immunoreactive neurons in the striatum. Ethanol-related neurobiological alterations such as a reduction in the number of microglial cells expressing iNOS (a cytokine-inducible immune defense) and in adult neural stem/progenitor cell (NSPC) proliferation (phospho-H3 and BrdU) and maturation (BrdU/ß3-tubulin), as well as an increase in damage cell activity (FosB) and apoptosis (cleaved caspase 3) were also observed in the rat striatum. Pharmacological administration of OEA (10 mg/kg) for 5 days during ethanol exposure exacerbated ethanol-induced hypolocomotion and cell apoptosis in the striatum. Interestingly, OEA abrogated the impaired effects of ethanol on PPARα-positive cell population and NSPC proliferation and maturation. OEA also decreased astrocyte-related vimentin immunoreactivity and increased microglial cell population (Iba-1, iNOS) in the striatum. These results suggest that OEA-PPARα signaling modulates glial activation, cell apoptosis and NSPC proliferation and maturation in response to striatal-specific neurobiological alterations induced by prolonged ethanol intake in rats.

Evidence for a protective role for the rs805305 single nucleotide polymorphism of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in septic shock through the regulation of DDAH activity.

BACKGROUND: Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates the synthesis of nitric oxide (NO) through the metabolism of the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA). Pilot studies have associated the rs805305 SNP of DDAH2 with ADMA concentrations in sepsis. This study explored the impact of the rs805305 polymorphism on DDAH activity and outcome in septic shock. METHODS: We undertook a secondary analysis of data and samples collected during the Vasopressin versus noradrenaline as initial therapy in septic shock (VANISH) trial. Plasma and DNA samples isolated from 286 patients recruited into the VANISH trial were analysed. Concentrations of L-Arginine and the methylarginines ADMA and symmetric dimethylarginine (SDMA) were determined from plasma samples. Whole blood and buffy-coat samples were genotyped for polymorphisms of DDAH2. Clinical data collected during the study were used to explore the relationship between circulating methylarginines, genotype and outcome. RESULTS: Peak ADMA concentration over the study period was associated with a hazard ratio for death at 28 days of 3.3 (95% CI 2.0-5.4), p < 0.001. Reduced DDAH activity measured by an elevated ADMA:SDMA ratio was associated with a reduced risk of death in septic shock (p = 0.03). The rs805305 polymorphism of DDAH2 was associated with reduced DDAH activity (p = 0.004) and 28-day mortality (p = 0.02). Mean SOFA score and shock duration were also reduced in the less common G:G genotype compared to heterozygotes and C:C genotype patients (p = 0.04 and p = 0.02, respectively). CONCLUSIONS: Plasma ADMA is a biomarker of outcome in septic shock, and reduced DDAH activity is associated with a protective effect. The polymorphism rs805305 SNP is associated with reduced mortality, which is potentially mediated by reduced DDAH2 activity. TRIAL REGISTRATION: ISRCTN Registry, ISRCTN20769191 . Registered on 20 September 2012.

Electrochemical Molecular Switch for the Selective Profiling of Cysteine in Live Cells and Whole Blood and for the Quantification of Aminoacylase-1.

A first-of-a-kind latent electrochemical redox probe, ferrocene carbamate phenyl acrylate (FCPA), was developed for the selective detection of cysteine (Cys) and aminoacylase (ACY-1). The electrochemical signal generated by this probe was shown to be highly specific to Cys and insensitive to other amino acids and biological redox reactants. The FCPA-incorporated electrochemical sensor exhibited a broad dynamic range of 0.25-100 µM toward Cys. This probe also proficiently monitored the ACY-1-catalyzed biochemical transformation of N-acetylcysteine (NAC) into Cys, and this proficiency was used to develop an electrochemical assay for quantifying active ACY-1, which it did so in a dynamic range of 10-200 pM (0.1-2 mU/cm3) with a detection limit of 1 pM (0.01 mU/cm3). Furthermore, the probe was utilized in real-time tracking and quantification of cellular Cys production, specifically in Escherichia coli W3110, along with a whole blood assay to determine levels of Cys and spiked ACY-1 in blood with a reliable analytical performance.

Ratiometric Fluorescent Probe for Imaging of Pantetheinase in Living Cells.

Pantetheinase, which catalyzes the cleavage of pantetheine to pantothenic acid (vitamin B5) and cysteamine, is involved in the regulation of oxidative stress, pantothenate recycling and cell migration. However, further elucidating the cellular function of this enzyme is largely limited by the lack of a suitable fluorescence imaging probe. By conjugating pantothenic acid with cresyl violet, herein we develop a new fluorescence probe CV-PA for the assay of pantetheinase. The probe not only possesses long analytical wavelengths but also displays linear ratiometric (I628/582 nm) fluorescence response to pantetheinase in the range of 5-400 ng/mL with a detection limit of 4.7 ng/mL. This probe has been used to evaluate the efficiency of different inhibitors and quantitatively detect pantetheinase in serum samples, revealing that pantetheinase in fetal bovine serum and new born calf serum is much higher than that in normal human serum. Notably, with the probe the ratiometric imaging and in situ quantitative comparison of pantetheinase in different living cells (LO2 and HK-2) have been achieved for the first time. It is found that the level of pantetheinase in LO2 cells is much larger than that in HK-2 cells, as further validated by Western blot analysis. The proposed probe may be useful to better understand the specific function of pantetheinase in the pantetheinase-related pathophysiological processes.

Markers of nitric oxide are associated with sepsis severity: an observational study.

BACKGROUND: Nitric oxide (NO) regulates processes involved in sepsis progression, including vascular function and pathogen defense. Direct NO measurement in patients is unfeasible because of its short half-life. Surrogate markers for NO bioavailability are substrates of NO generating synthase (NOS): L-arginine (lArg) and homoarginine (hArg) together with the inhibitory competitive substrate asymmetric dimethylarginine (ADMA). In immune cells ADMA is cleaved by dimethylarginine-dimethylaminohydrolase-2 (DDAH2). The aim of this study was to investigate whether concentrations of surrogate markers for NO bioavailability are associated with sepsis severity. METHOD: This single-center, prospective study involved 25 controls and 100 patients with surgical trauma (n = 20), sepsis (n = 63), or septic shock (n = 17) according to the Sepsis-3 definition. Plasma lArg, hArg, and ADMA concentrations were measured by mass spectrometry and peripheral blood mononuclear cells (PBMCs) were analyzed for DDAH2 expression. RESULTS: lArg concentrations did not differ between groups. Median (IQR) hArg concentrations were significantly lower in patient groups than controls, being 1.89 (1.30-2.29) µmol/L (P < 0.01), with the greatest difference in the septic shock group, being 0.74 (0.36-1.44) µmol/L. In contrast median ADMA concentrations were significantly higher in patient groups compared to controls, being 0.57 (0.46-0.65) µmol/L (P < 0.01), with the highest levels in the septic shock group, being 0.89 (0.56-1.39) µmol/L. The ratio of hArg:ADMA was inversely correlated with disease severity as determined by the Sequential Organ Failure Assessment (SOFA) score. Receiver-operating characteristic analysis for the presence or absence of septic shock revealed equally high sensitivity and specificity for the hArg:ADMA ratio compared to the SOFA score. DDAH2 expression was lower in patients than controls and lowest in the subgroup of patients with increasing SOFA. CONCLUSIONS: In patients with sepsis, plasma hArg concentrations are decreased and ADMA concentrations are increased. Both metabolites affect NO metabolism and our findings suggest reduced NO bioavailability in sepsis. In addition, reduced expression of DDAH2 in immune cells was observed and may not only contribute to blunted NO signaling but also to subsequent impaired pathogen defense.

Hereditary C1 inhibitor deficiency is associated with high spontaneous amidase activity.

BACKGROUND: Angioedema diagnosis classically targets the complement system (via C1 inhibitor (C1Inh) function and antigenic C4 level) and contact phase activation (via amidase activity). Bradykinin is responsible for angioedema attacks and is produced from contact phase activation secondary to failed C1Inh control. OBJECTIVE: We aimed to compare the diagnostic performances of spontaneous amidase activity and antigenic C4 level in C1Inh hereditary angioedema (C1Inh-HAE) patients. METHODS: Samples from 185 C1Inh-HAE patients (81 men, 104 women; confirmed by SERPING1 gene mutations) and from 99 blood donors (50 men, 49 women) were tested for C1Inh function, antigenic C4 level and spontaneous amidase activity. RESULTS: In the C1Inh-HAE group, antigenic C4 level was decreased (n=135) and amidase activity was increased (n=181). Receiver operating characteristic analyses showed higher diagnostic performance values for the spontaneous amidase assay compared to those of antigenic C4. CONCLUSION: The spontaneous amidase activity assay should replace antigenic C4 level testing and should be tested alongside the C1Inh function for both AE screening and follow up of HAE patients.

Fatty acid amide hydrolase-morphine interaction influences ventilatory response to hypercapnia and postoperative opioid outcomes in children.

AIM: Fatty acid amide hydrolase (FAAH) degrades anandamide, an endogenous cannabinoid. We hypothesized that FAAH variants will predict risk of morphine-related adverse outcomes due to opioid-endocannabinoid interactions. PATIENTS & METHODS: In 101 postsurgical adolescents receiving morphine analgesia, we prospectively studied ventilatory response to 5% CO2 (HCVR), respiratory depression (RD) and vomiting. Blood was collected for genotyping and morphine pharmacokinetics. RESULTS: We found significant FAAH-morphine interaction for missense (rs324420) and several regulatory variants, with HCVR (p < 0.0001) and vomiting (p = 0.0339). HCVR was more depressed in patients who developed RD compared with those who did not (p = 0.0034), thus FAAH-HCVR association predicts risk of impending RD from morphine use. CONCLUSION: FAAH genotypes predict risk for morphine-related adverse outcomes.

Identification and clinical significance of an elevated level of serum aminoacylase-1 autoantibody in patients with hepatitis B virus-related liver cirrhosis.

Chronic hepatitis B (CHB) is prevalent worldwide and can develop into liver cirrhosis and liver carcinoma. Early discrimination of liver cirrhosis from chronic hepatitis is critical for effective treatment and optimal prognosis. The aim of the present study was to assess the diagnostic value of a panel of cellular proteins that can be recognized by autoantibodies in patient serum for hepatitis B virus (HBV)­related liver cirrhosis. Twenty­two candidate autoantigens screened using a serum proteomics assay in our previous study were assessed retrospectively in 443 participants, comprising 89 patients with HBV­related liver cirrhosis, 89 patients with CHB, and 265 healthy controls. The levels of autoantibodies against the candidate autoantigens were measured by protein microarrays containing the candidate antigen proteins. Receiver operating characteristic (ROC) curves were used to calculate the diagnostic accuracy. The present study determined that seven of the 22 candidate autoantibodies differed significantly in serum level between HBV­related liver cirrhosis and CHB (P<0.0001), with area under curve (AUC) values >0.7. The seven autoantibodies recognized aminoacylase­1 (ACY1), histidine triad nucleotide­binding protein 1, insulin­like growth factor 2 mRNA­binding protein 2, heat shock 70 kDa protein 6, peroxiredoxin 3, apoptosis­inducing factor and regucalcin. Among these, the ACY1 autoantibody had the highest value for discriminating HBV­related liver cirrhosis from CHB, with an AUC value of 0.872 (95% confidence interval: 0.810­0.934, P<0.0001), sensitivity of 77.3% and specificity of 85.0%. In conclusion, with the elevated level in the disease progression of CHB, ACY1 autoantibody may be a valuable serum biomarker for discriminating HBV­related liver cirrhosis from CHB.