FAST-IT: Find A Simple Test - In TIA (transient ischaemic attack): a prospective cohort study to develop a multivariable prediction model for diagnosis of TIA through proteomic discovery and candidate lipid mass spectrometry, neuroimaging and machine learning-study protocol.

INTRODUCTION: Transient ischaemic attack (TIA) may be a warning sign of stroke and difficult to differentiate from minor stroke and TIA-mimics. Urgent evaluation and diagnosis is important as treating TIA early can prevent subsequent strokes. Recent improvements in mass spectrometer technology allow quantification of hundreds of plasma proteins and lipids, yielding large datasets that would benefit from different approaches including machine learning. Using plasma protein, lipid and radiological biomarkers, our study will develop predictive algorithms to distinguish TIA from minor stroke (positive control) and TIA-mimics (negative control). Analysis including machine learning employs more sophisticated modelling, allowing non-linear interactions, adapting to datasets and enabling development of multiple specialised test-panels for identification and differentiation. METHODS AND ANALYSIS: Patients attending the Emergency Department, Stroke Ward or TIA Clinic at the Royal Adelaide Hospital with TIA, minor stroke or TIA-like symptoms will be recruited consecutively by staff-alert for this prospective cohort study. Advanced neuroimaging will be performed for each participant, with images assessed independently by up to three expert neurologists. Venous blood samples will be collected within 48 hours of symptom onset. Plasma proteomic and lipid analysis will use advanced mass spectrometry (MS) techniques. Principal component analysis and hierarchical cluster analysis will be performed using MS software. Output files will be analysed for relative biomarker quantitative differences between the three groups. Differences will be assessed by linear regression, one-way analysis of variance, Kruskal-Wallis H-test, χ2 test or Fisher's exact test. Machine learning methods will also be applied including deep learning using neural networks. ETHICS AND DISSEMINATION: Patients will provide written informed consent to participate in this grant-funded study. The Central Adelaide Local Health Network Human Research Ethics Committee approved this study (HREC/18/CALHN/384; R20180618). Findings will be disseminated through peer-reviewed publication and conferences; data will be managed according to our Data Management Plan (DMP2020-00062).

A rapid method for the screening of fatty acids in lipids in plasma or serum without prior extraction.

Serum or plasma are the commonly used blood fractions to determine the relationship between dietary and circulating fatty acids in health and disease. Most methods available for the measurement of fatty acids in serum or plasma (referred to as serum henceforth) require prior extraction with organic solvents. We have determined that it is possible to directly convert the lipids in aqueous biological samples to fatty acid methyl esters (FAME) without prior extraction, providing that the ratio of serum to transmethylation solvent does not exceed 10%. Our in-vial transmethylation system uses 50uL serum pipetted into 2 mL screw top GC vials containing 1 mL of 1% H2SO4 in methanol at 50 °C and subsequent FAME extracted in the same vial into 300uL heptane. The system yields both compositional and quantitative analysis of the fatty acids of serum identical to conventional standard methods. Evaluation of our new serum assay confirms significant correlations between the fatty acid measures and those obtained from conventional standard assay for all fatty acids (r > 0.99, P<0.0001), including the n-6 (r = 0.998, P<0.0001) and n-3 long chain polyunsaturated fatty acids (r = 0.993, P<0.0001). There were high levels of agreement between methods on Bland -Altman analysis, indicating the interchangeability of the methods. These results establish our new method as reliable for the assessment of fatty acid composition of small volumes of serum useful for high throughput situations that limits the volume of organic solvents and technical input.

Increased inspired oxygen concentration does not adversely affect oxidative stress and the resolution of inflammation during reperfusion in patients undergoing knee replacement surgery.

The level of inspired oxygen during surgery may modify free radical release, and reperfusion injury. This controlled trial examined the effect of inspired oxygen on F2-isoprostanes (F2-IsoPs), isofurans (IsoFs), and specialized mediators of inflammation resolution (SPM) during knee replacement surgery. Patients received either 30% O2 (control n = 21), 50% O2 (n = 20), or 80% O2 (n = 19) O2, in a parallel design. Hemoglobin (Hb) was measured throughout the surgery and F2-IsoPs, IsoFs and SPM were analyzed by mass spectrometry. The effect of O2 on F2-IsoPs and IsoFs was examined during tourniquet inflation and after tourniquet release. SPM were measured at baseline and the end of surgery. There was a significant interaction between O2 and Hb concentrations with plasma IsoFs during tourniquet inflation. An increase in plasma IsoFs over time was attenuated in the 80% O2 group (p=.012) compared with the 30% O2 group after adjusting for Hb concentration. After tourniquet release, plasma F2-IsoPs were significantly lower in the 50% and 80% O2 groups (p=.009 and p=.001, respectively) compared with the 30% O2 group after adjustment for Hb concentration. The SPM RvD2 and RvE2 were increased with 50% and 80% O2 (RvD2, p=.014 and p=.002, respectively; RvE2, p=.032 with 50% O2) compared with the 30% O2 group, in analyses that corrected for Hb concentration. We have shown for the first time that higher O2 levels may be beneficial in reducing oxidative stress and increasing resolution of inflammation during surgery that involves reperfusion after application of a tourniquet.

Effects of antiemetic doses of dexamethasone on plasma mediators of inflammation resolution and pain after surgery in women.

BACKGROUND: Dexamethasone is commonly given as an antiemetic during surgical procedures. It has immunosuppressive effects and can affect key enzymes involved in the synthesis of specialised lipid mediators of inflammation resolution (SPM) that direct inflammation resolution and have anti-nociceptive actions. This study examined the effect of dexamethasone on plasma SPM, and the relationship between SPM and perceived pain in women undergoing surgery. METHODS: Plasma SPM were measured in samples obtained from two double-blind controlled interventions. The first, included 51 women mean age 53 ± 1.5 years, undergoing breast surgery allocated to either intravenous saline, or dexamethasone (4 mg or 8 mg) after induction of anaesthesia. The second study included 31 women of mean age 44 ± 0.5 years undergoing laparoscopic gynecological surgery that were allocated to either saline, or dexamethasone (4 mg). SPM (18-HEPE, 17-HDHA, RvE2, RvD1 17R-RvD1 and RvD2) were measured in plasma collected prior to induction of anaesthesia and at 24 h, and 6 weeks post-surgery. Pain was assessed using a verbal analogue scale at discharge from the post-anaesthesia recovery unit. The data from each study was combined to examine the effect of dexamethasone on plasma SPM. The relationship between pain score and SPM was examined using ordinal logistic regression. RESULTS: The SPM 18-HEPE, 17-HDHA, RvE2, RvD1 17R-RvD1 and RvD2 were detectable in all plasma samples. There was no significant difference in any SPM due to dexamethasone over the duration of the study. There was a fall in 17-HDHA between baseline and 24 h in both the dexamethasone and saline groups (P = 0.003) but no change in the downstream SPM (RvD1, 17R-RvD1 and RvD2) or 18-HEPE and RvE2. Pain score was negatively related to levels of RvE2 measured prior to induction of anaesthesia (rho = -0.2991, P = 0.006) and positively related to BMI (rho = 0.279, P = 0.011). In ordinal logistic regression the odds ratio for RvE2 was 0.931 (CI 0.880, 0.986; P = 0.014); after adjusting for the effect of BMI indicating that an increase in RvE2 of 1 pg/ml would result in a 6.9 % fall in pain score. Allocation to a dexamethasone group did not influence the pain score or the relationship between RvE2 and pain score. CONCLUSION: Dexamethasone administered as an anti-emetic does not affect plasma SPM levels. An elevated RvE2 level prior to surgery is predictive of a lower perceived pain score post-anaesthesia.

Potency test to discriminate between differentially over-inactivated rabies vaccines: Agreement between the NIH assay and a G-protein based ELISA.

The NIH assay is used to assess the potency of rabies vaccine and is currently a key measure required for vaccine release. As this test involves immunization of mice and subsequent viral challenge, efforts are being made to develop alternative analytical methods that do not rely on animal testing. Sanofi Pasteur has reported the development of a G-protein specific ELISA assay that has shown agreement with the NIH test. In this study we have generated several non-conform vaccine lots by an excessive inactivation with ß-propiolactone (BPL) and assessed the capacity of both tests to detect the corresponding consequences. Excessive BPL inactivation causes G-protein unfolding, altering in turn viral morphology and the continuity of the G-protein layer in the viral particle. Both the NIH and the ELISA tests were able to monitor the consequences of excessive inactivation in a similar manner. Of note, the experimental error of the ELISA test was well below that of the NIH test. These results increase the prospect that the ELISA test could be considered a suitable candidate for the replacement of the NIH test.

Antiemetic doses of dexamethasone and their effects on immune cell populations and plasma mediators of inflammation resolution in healthy volunteers.

INTRODUCTION: The synthetic glucocorticoid dexamethasone is a commonly administered antiemetic. It has immunosuppressive effects and may alter postoperative blood glucose concentrations. Dexamethasone can effect key enzymes involved in inflammation resolution that is an active process driven by specialised lipid mediators of inflammation resolution (SPM). The purpose of this study in healthy volunteers was to examine whether dexamethasone effects cell populations and synthesis of SPM that are critical for the resolution of inflammation. METHODS: Thirty-two healthy volunteers were randomly allocated to receive saline (Control) or dexamethasone 2 mg, 4 mg or 8 mg intravenously. Venous blood samples were collected at baseline before administration of treatment, and at 4 h, 24 h and one-week post-treatment. At each time point, measurements included blood glucose and macrophage migration inhibition factor (MMIF), full blood count including lymphocyte subsets, monocytes, neutrophils, eosinophils and basophils by flow cytometry, and plasma SPM using liquid chromatography tandem mass spectrometry. The effect of dexamethasone dose and time on all measures was analysed using linear mixed models. RESULTS: There was a dose-dependent increase in neutrophil count after dexamethasone that persisted for 24 h. In contrast, there was a dose-dependent reduction in counts of monocytes, lymphocytes, basophils and eosinophils 4 h after dexamethasone, followed by a rebound increase in cell counts at 24 h. Seven days after administration of dexamethasone, all cell counts were similar to baseline levels. MMIF concentration, glucose and natural killer cell counts were not significantly affected by dexamethasone. There was a significant gender effect on plasma SPM such that levels of 17-HDHA, RvD1, 17R-RvD1 and RvE2 in females were on average 14%-50% lower than males. In a linear mixed model that adjusted for neutrophil count, there was a significant interaction between the dose of dexamethasone and time, on plasma 17R-RvD1 such that plasma 17R-RvD1 fell in a dose-dependent manner until 4 h after administration of dexamethasone. There were no significant effects of dexamethasone on the other plasma SPM (18-HEPE, RvE2, 17-HDHA, RvD1, RvD2 and 14-HDHA) measured. DISCUSSION: This is the first study in healthy volunteers to demonstrate that commonly employed antiemetic doses of dexamethasone affect immune cell populations and plasma levels of 17R-RvD1 an SPM with anti-nociceptive properties. If similar changes occur in surgical patients, then this may have implications for acute infection risk in the post-operative period.

The effects of alcohol on plasma lipid mediators of inflammation resolution in patients with Type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus is characterized by peripheral insulin resistance and low-grade systemic inflammation. Inflammation resolution is recognised as an important process driven by specialised pro-resolving mediators of inflammation (SPMs) and has the potential to moderate chronic inflammation. Alcohol has the potential to affect synthesis of SPMs by altering key enzymes involved in SPM synthesis and may influence ongoing inflammation associated with Type 2 diabetes mellitus. AIMS: (i) To examine the effects of alcohol consumed as red wine on plasma SPM in men and women with Type 2 diabetes in a randomised controlled trial and (ii) compare baseline plasma SPM levels in the same patients with those of healthy volunteers. METHODS: Twenty-four patients with Type 2 diabetes mellitus were randomized to a three-period crossover study with men drinking red wine 300 ml/day (∼31 g alcohol/day) and women drinking red wine 230 ml/day (∼24 g alcohol/day), or equivalent volumes of dealcoholized red wine (DRW) or water, each for 4 weeks. The SPM 18-hydroxyeicosapentaenoic acid (18-HEPE), E-series resolvins (Rv) (RvE1-RvE3), 17-hydroxydocosahexaenoic acid (17-HDHA), and D-series resolvins (RvD1, 17R-RvD1, RvD2, RvD5), 14-hydroxydocosahexaenoic acid (14-HDHA) and Maresin 1 were measured at the end of each period. A baseline comparison of plasma SPM, hs CRP, lipids and glucose was made with healthy volunteers. RESULTS: Red wine did not differentially affect any of the SPM measured when compared with DRW or water. Baseline levels of the hs-CRP and the SPM 18-HEPE, 17-HDHA, RvD1 and 17R-RvD1 in patients with Type 2 diabetes mellitus were all significantly elevated compared with healthy controls and remained so after adjusting for age and gender. CONCLUSION: Moderate alcohol consumption as red wine does not alter plasma SPM in patients with Type 2 diabetes mellitus. The elevation of SPM levels compared with healthy volunteers may be a homeostatic response to counter ongoing inflammation.

Hyperbaric oxygen therapy is not associated with oxidative stress assessed using plasma F2-isoprostanes and isofurans.

BACKGROUND AND AIMS: Hyperbaric oxygen (HBO) therapy is increasingly used in medical practice as a means of enhancing the formation of collagen matrix and angiogenesis, thus promoting healing in wounds and necrotic tissue. However, there are concerns that oxygen can also associate with increased production of oxygen free radicals and oxidative stress. F2-Isoprostanes (F2-IsoPs) formed by non-enzymatic oxidation of arachidonic acid (AA) are reliable measures for assessing oxidative stress in vivo. In addition, under conditions of high oxygen tension isofurans (IsoFs) are preferentially formed from AA and are considered to better reflect oxidative stress in the setting of high oxygen tension. This study aimed to measure plasma IsoFs and F2-IsoP in patients receiving HBO therapy to treat osteonecrosis secondary to radiation therapy. Our hypothesis was that IsoFs would continue to rise with increasing oxygen pressures in contrast to F2-IsoPs whose synthesis would be reduced. METHODS: Twelve patients receiving hyperbaric therapy to treat osteonecrosis secondary to radiation therapy were studied during hyperbaric treatment. Blood samples were collected prior to, during and after cessation of HBO therapy that lasted for 119min. Seven serial blood samples were collected for measurement of plasma F2-IsoPs and IsoFs, blood gases and haemoglobin. RESULTS: Oxygen saturation and venous oxygen partial pressure (PvO2) rose significantly during hyperbaric therapy. However, there were no significant changes in plasma IsoFs or F2-IsoPs during the hyperbaric therapy session. CONCLUSION: In this study of patients with osteonecrosis, HBO therapy at a maximum pressure of 2.4atm with up to 100% oxygen did not worsen oxidative stress assessed using plasma F2- IsoFs and IsoPs.

Effects of prenatal n-3 fatty acid supplementation on offspring resolvins at birth and 12 years of age: a double-blind, randomised controlled clinical trial.

Resolution of inflammation is an active process involving specialised pro-resolving mediators (SPM) generated from the n-3 fatty acids EPA and DHA. n-3 Fatty acid supplementation during pregnancy may provide an intervention strategy to modify these novel SPM. This study aimed to assess the effect of n-3 fatty acid supplementation in pregnancy on offspring SPM at birth and 12 years of age (12 years). In all, ninety-eight atopic pregnant women were randomised to 3·7 g daily n-3 fatty acids or a control (olive oil), from 20 weeks gestation until delivery. Blood was collected from the offspring at birth and at 12 years. Plasma SPM consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), E-series resolvins, 17-hydroxydocosahexaenoic acid (17-HDHA), D-series resolvins, 14-hydroxydocosahexaenoic acid (14-HDHA), 10 S,17S-dihydroxydocosahexaenoic acid, maresins and protectin 1, were measured by liquid chromatography-tandem MS. We identified the resolvins RvE1, RvE2, RvE3, RvD1, 17R-RvD1 and RvD2 for the first time in human cord blood. n-3 Fatty acids increased cord blood 18-HEPE (P<0·001) derived from EPA relative to the control group. DHA-derived 17-HDHA at birth was significantly increased in the n-3 fatty acid group relative to the controls (P=0·001), but other SPM were not different between the groups. n-3 Fatty acid supplementation during pregnancy was associated with an increase in SPM precursors in the offspring at birth but the effects were not sustained at 12 years. The presence of these SPM, particularly at birth, may have functions relevant in the newborn that remain to be established, which may be useful for future investigations.

A Randomized Trial of Effects of Alcohol on Cytochrome P450 Eicosanoids, Mediators of Inflammation Resolution, and Blood Pressure in Men.

BACKGROUND: Cardiovascular effects of alcohol consumption may be influenced by both pro- and anti-inflammatory mechanisms. We previously showed that chronic alcohol consumption increased blood pressure (BP), oxidative stress, and 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoconstrictor and pro-inflammatory eicosanoid synthesized by cytochrome P450 (CYP450) enzymes from arachidonic acid. This study in men examined the effect of consuming red wine (RW) on BP in relation to changes in 20-HETE, oxidative stress (F2 -isoprostanes), markers of inflammation, anti-inflammatory CYP450 epoxyeicosatrienoic acids (EETs), and specialized pro-resolving mediators of inflammation (SPMs) derived from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). METHODS: Normotensive men (n = 22) were randomly allocated to drink RW (375 ml/d) or the equivalent volume of dealcoholized red wine (DRW) or water for 4 weeks in a 12-week, 3-period crossover trial. BP, heart rate, 20-HETE, F2 -isoprostanes, and SPM were measured at baseline, 4, 8, and 12 weeks. RESULTS: Drinking RW increased BP (p < 0.05), plasma and urinary 20-HETE (p < 0.05), plasma F2 -isoprostanes (p < 0.0001), and the SPMs 18-hydroxyeicosapentaenoic acid (18-HEPE) from EPA, and resolvin D1 (RvD1) and 17R-resolvin D1 (17R-RvD1) from DHA (all p < 0.05) compared with DRW and water. EETs and high-sensitivity C-reactive protein were unaffected by RW. Plasma 18-HEPE was positively related to urinary 20-HETE (p < 0.008) only after RW. CONCLUSIONS: This study has shown that men consuming moderate-to-high alcohol as RW for 4 weeks had increased BP, 20-HETE, and oxidative stress, as well as specific SPM that resolve inflammation. These paradoxical findings require further studies to determine whether alcohol stimulates different CYP450 enzymes and whether the findings can be replicated in females.

Does Furosemide Increase Oxidative Stress in Acute Kidney Injury?

Furosemide, a loop diuretic, is used to increase urine output in patients with acute kidney injury (AKI). It remains uncertain whether the benefits of furosemide in AKI outweigh its potential harms. We investigated if furosemide influenced oxidative stress in 30 critically ill patients with AKI by measuring changes in F2-isoprostanes (F2-IsoPs), markers of in vivo oxidative stress, in plasma and urine following intravenous furosemide. Urine F2-IsoPs were higher in sepsis (p = 0.001) and increased in proportion to urine furosemide (p = 0.001). The furosemide-induced increase in urine F2-IsoPs differed depending on AKI severity (p < 0.001) and was greatest in those with the most severe AKI. Furosemide had no effect on plasma F2-IsoPs. We demonstrate for the first time that furosemide increases renal oxidative stress in AKI and find that patients with the most severe AKI-to whom the largest doses are likely to be administered-showed the greatest increase in oxidative stress. These findings lead to the hypothesis that the common practice of administering high-dose furosemide to convert oliguric to nonoliguric AKI may induce harmful oxidative stress in the kidneys, and an adequately powered, randomized controlled trial is required to determine if clinical benefits of this dosing strategy justify its potential harms. Antioxid. Redox Signal. 26, 221-226.

Controlled moderate hypovolaemia in healthy volunteers is not associated with the development of oxidative stress assessed by plasma F2-isoprostanes and isofurans.

Hypovolaemia can be associated with substantial morbidity, particularly when it occurs in the setting of trauma and in patients with comorbid diseases. Hypovolaemia and inflammation such as occur in the setting of trauma and surgery, are associated with systemic oxidative stress and free-radical injury. Free-radical injury that results from hypovolaemia-induced organ reperfusion may further augment inflammatory processes. It is unknown exactly what proportion of free-radical injury is associated with isolated hypovolaemia as opposed to the contribution from inflammation from surgery or trauma. In the first human study of its kind, we exposed 8 adult male volunteers to venesection-induced hypovolaemia in progressive aliquots of 5% of total blood volume until 20% had been removed. This blood was subsequently reinfused. Plasma F2-isoprostanes and isofurans, markers of in vivo lipid oxidation, were measured by gas chromatography-mass spectrometry at each 5% aliquot venesected and at each 5% reinfused. Between baseline and maximal blood loss there was a minor fall in haemoglobin concentration from 143.9g/l to 138.8g/l (p=0.004, 95% CI 2.2, 8.0g/L). No significant change from baseline occurred in the concentrations of either plasma F2-isoprostanes or isofurans during venesection (p=0.116 and p=0.152, respectively) or blood reinfusion (p=0.553 and p=0.736, respectively). We can conclude that in healthy adult volunteers, isolated hypovolaemia to 20% total blood volume loss is not associated with detectable systemic oxidative stress. The free-radical injury identified in surgical and trauma patients may represent the effects of tissue damage and inflammation, with an uncertain contribution from tissue ischemia as may occur with hypovolaemia.

Determinants of Urinary Output Response to IV Furosemide in Acute Kidney Injury: A Pharmacokinetic/Pharmacodynamic Study.

OBJECTIVES: This study assessed the determinants of urinary output response to furosemide in acute kidney injury; specifically, whether the response is related to altered pharmacokinetics or pharmacodynamics. DESIGN: Prospective cohort. SETTING: Tertiary ICU. PATIENTS: Thirty critically ill patients with acute kidney injury without preexisting renal impairment or recent diuretic exposure. INTERVENTION: A single dose of IV furosemide. MEASUREMENTS AND MAIN RESULTS: Baseline markers of intravascular volume status were obtained prior to administering furosemide. Six-hour creatinine clearance, hourly plasma/urinary furosemide concentrations, and hourly urinary output were used to assess furosemide pharmacokinetics/pharmacodynamics parameters. Of 30 patients enrolled, 11 had stage-1 (37%), nine had stage-2 (30%), and 10 had stage-3 (33%) Acute Kidney Injury Network acute kidney injury. Seventy-three percent were septic, 47% required norepinephrine, and 53% were mechanically ventilated. Urinary output doubled in 20 patients (67%) following IV furosemide. Measured creatinine clearance was strongly associated with the amount of urinary furosemide excreted and was the only reliable predictor of the urinary output after furosemide (area under the receiver-operating-characteristic curve, 0.75; 95% CI, 0.57-0.93). In addition to an altered pharmacokinetics (p < 0.01), a reduced pharmacodynamics response to furosemide also became important when creatinine clearance was reduced to less than 40 mL/min/1.73 m (p = 0.01). Acute kidney injury staging and markers of intravascular volume, including central venous pressure, brain-natriuretic-peptide concentration, and fractional urinary sodium excretion were not predictive of urinary output response to furosemide. CONCLUSIONS: The severity of acute kidney injury, as reflected by the measured creatinine clearance, alters both pharmacokinetics and pharmacodynamics of furosemide in acute kidney injury, and was the only reliable predictor of the urinary output response to furosemide in acute kidney injury.

Acute effects of quercetin-3-O-glucoside on endothelial function and blood pressure: a randomized dose-response study.

BACKGROUND: Epidemiologic studies have suggested that a flavonoid-rich diet can reduce the risk of developing cardiovascular disease. Certain flavonoids, in particular quercetin, have been shown to ameliorate endothelial dysfunction and reduce blood pressure (BP), possibly by increasing the bioavailability of the potent vasodilator nitric oxide (NO). Several studies have indicated that improvements in measures of cardiovascular health do not occur linearly, but rather, plateau or decrease with an increasing dose of flavonoids. OBJECTIVES: We determined whether the acute administration of increasing doses of a common quercetin glycoside (quercetin-3-O-glucoside) improves endothelial function and reduces BP in a dose-dependent manner. We also explored whether any effects were correlated with changes in plasma NO production. DESIGN: A randomized, controlled, crossover study was performed in 15 healthy volunteers who each completed 5 visits with a minimum washout period of 1 wk between testing days. Participants received each of the following 5 interventions in a random order: 1) 0, 2) 50, 3) 100, 4) 200, or 5) 400 mg quercetin-3-O-glucoside. Endothelial function and BP were assessed before and 60 min after intervention. A blood sample was taken before and 90 min after intervention for the analysis of plasma nitrate and nitrite as markers of NO production as well as of plasma quercetin metabolites. RESULTS: Although we observed a significant correlation between the dose of quercetin-3-O-glucoside and plasma concentrations of total quercetin (R(2) = 0.52, P < 0.001) and isorhamnetin (R(2) = 0.12, P = 0.005), we showed no improvements in endothelial function or BP and no changes in NO production after any dose. CONCLUSION: From these results, we conclude that there are no acute changes in BP or the NO-mediated endothelium-dependent relaxation of the brachial artery with doses of quercetin ranging from 50 to 400 mg in healthy men and women. This trial was registered at www.anzctr.org.au as ACTRN12615001338550.

Specialised pro-resolving mediators of inflammation in inflammatory arthritis.

INTRODUCTION: Specialised pro-resolving mediators (SPM) are derived from n-3 long chain polyunsaturated fatty acids (n-3FA). They promote resolution of inflammation and may contribute to the beneficial effects of n-3FA in patients with arthritis. This study compared SPM in knee effusions and plasma of patients with arthritis taking n-3FA, and plasma of healthy volunteers taking n-3FA. METHODS: Thirty six patients taking n-3FA undergoing arthrocentesis for an inflammatory knee effusion and 36 healthy volunteers who had taken n-3FA (2.4g/day) for 4 weeks were studied. SPM in synovial fluid and plasma were measured by liquid chromatography-tandem mass spectrometry included 18-hydroxyeicosapentaenoic acid (18-HEPE), the precursor of the E-series SPM (RvE1, RvE2, RvE3, 18R-RvE3), and 17-hydroxydocosahexaenoic acid (17-HDHA), the precursor of the D-series SPM (RvD1, 17R-RvD1, RvD2). Other SPM included protectin D1 (PD1), 10S,17S-dihydroxydocosahexaenoic acid (10,17S-DHDHA), maresin-1 (MaR-1) and 14-hydroxydocosahexaenoic acid (14-HDHA) derived from docosahexaenoic acid (DHA). RESULTS: E- and D-series SPM and the precursors 18-HEPE and 17-HDHA were present in synovial fluid and plasma of the patients with inflammatory arthritis. Plasma SPM were negatively related to erythrocyte sedimentation rate in arthritis patients (P<0.01) and synovial fluid RvE2 was negatively associated with pain score (P=0.02). Conversion from 18-HEPE and 17-HDHA to E- and D-series SPM was greater in synovial fluid (P<0.01). Most plasma SPM in arthritis patients were elevated (P<0.05) compared with healthy volunteers, and conversion to E- and D-series SPM was greater (P<0.01). CONCLUSIONS: SPM are present in chronic knee effusions and although the levels are lower than in plasma, the association between synovial fluid RvE2 and reduced pain scores suggests that synthesis of SPM at the site of inflammation is a relevant mechanism by which n-3FA alleviate the symptoms of arthritis.

Chlorogenic acid improves ex vivo vessel function and protects endothelial cells against HOCl-induced oxidative damage, via increased production of nitric oxide and induction of Hmox-1.

Dietary polyphenols are potential contributors toward improved cardiovascular health. Coffee is one of the richest sources of dietary polyphenols in a coffee-drinking population, the most abundant form being chlorogenic acid (CGA). Endothelial dysfunction is an early and major risk factor for cardiovascular disease. Nitric oxide (NO) is a key factor in regulation of endothelial function. Heme oxygenase-1 (Hmox-1), an inducible isoform of heme oxygenase that is produced in response to stressors such as oxidative stress, may also play a role in vascular protection. The aim of this study was to investigate the effect of CGA on endothelial function with oxidant-induced damage in isolated aortic rings from C57BL mice. We further examine the mechanism by investigating cell viability, activation of eNOS and induction of Hmox-1 in human aortic endothelial cells (HAECs). We found that pretreatment of isolated aortic rings with 10-µM CGA-protected vessels against HOCl-induced endothelial dysfunction (P<0.05). Pretreatment of cultured HAECs with 10-µM CGA increased endothelial cell viability following exposure to HOCl (P<0.05). Moreover, CGA increased NO production in HAECs in a dose-dependent manner, peaking at 6 h (P<0.05). CGA at 5 µM and 10 µM increased eNOS dimerization at 6 h and induced Hmox-1 protein expression at 6 h and 24 h in HAECs. These results are consistent with the cardiovascular protective effects of coffee polyphenols and demonstrate that CGA can protect vessels and cultured endothelial cells against oxidant-induced damage. The mechanism behind the beneficial effect of CGA appears to be in part via increased production of NO and induction of Hmox-1.

n-3 Fatty acid supplementation and proresolving mediators of inflammation.

PURPOSE OF REVIEW: The review presents recent developments in the identification of specialized proresolving mediators (SPMs) of inflammation following supplementation with n-3 fatty acids in humans. RECENT FINDINGS: A number of reports have measured SPMs in human plasma after n-3 fatty acid supplementation. Although studies have shown some variability in plasma SPM levels, there is strong evidence that a number of resolvins are increased after n-3 fatty acids to concentrations that have been shown to have biological activity. SPM concentrations at the inflammatory site would be expected to be higher than that in blood. SPMs derived from docosapentaenoic acid require further investigation. SUMMARY: Resolution of inflammation is an active process with SPM playing a vital role in maintaining homeostasis. Studies in humans are providing evidence to suggest that this may be a relevant mechanism that can be stimulated by n-3 fatty acid supplementation. Further research is now required to determine SPM profiles in patients with different chronic conditions and to examine whether supplementation with n-3 fatty acids affects SPMs in relation to their clinical outcome.

A randomized controlled trial of the effects of n-3 fatty acids on resolvins in chronic kidney disease.

BACKGROUND AND OBJECTIVE: The high incidence of cardiovascular disease (CVD) in chronic kidney disease (CKD) is related partially to chronic inflammation. n-3 Fatty acids have been shown to have anti-inflammatory effects and to reduce the risk of CVD. Specialized Proresolving Lipid Mediators (SPMs) derived from the n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) actively promote the resolution of inflammation. This study evaluates the effects of n-3 fatty acid supplementation on plasma SPMs in patients with CKD. METHODS: In a double-blind, placebo-controlled intervention of factorial design, 85 patients were randomized to either n-3 fatty acids (4 g), Coenzyme Q10 (CoQ) (200 mg), both supplements, or control (4 g olive oil), daily for 8 weeks. The SPMs 18-HEPE, 17-HDHA, RvD1, 17R-RvD1, and RvD2, were measured in plasma by liquid chromatography-tandem mass spectrometry before and after intervention. RESULTS: Seventy four patients completed the 8 weeks intervention. n-3 Fatty acids but not CoQ significantly increased (P < 0.0001) plasma levels of 18-HEPE and 17-HDHA, the upstream precursors to the E- and D-series resolvins, respectively. RvD1 was significantly increased (P = 0.036) after n-3 fatty acids, but no change was seen in other SPMs. In regression analysis the increase in 18-HEPE and 17-HDHA after n-3 fatty acids was significantly predicted by the change in platelet EPA and DHA, respectively. CONCLUSION: SPMs are increased after 8 weeks n-3 fatty acid supplementation in patients with CKD. This may have important implications for limiting ongoing low grade inflammation in CKD.

Specialized proresolving lipid mediators in humans with the metabolic syndrome after n-3 fatty acids and aspirin.

BACKGROUND: The metabolic syndrome (MetS) is associated with a chronic low-grade inflammatory state and may be affected by the ability to resolve inflammation, which is an active process that involves specialized proresolving lipid mediators (SPMs) derived from n-3 (ω-3) fatty acids. OBJECTIVE: We compared plasma concentrations of SPMs in men and women with features of the MetS and in healthy matched control subjects in response to intakes of n-3 fatty acids and aspirin. DESIGN: MetS volunteers (n = 22) and healthy, matched controls (n = 21) were studied in parallel for 4 wk. Both groups took n-3 fatty acids (2.4 g/d) for 4 wk with the addition of aspirin (300 mg/d) during the last 7 d. Blood was collected at baseline and at 3 and 4 wk. Plasma SPMs were measured with the use of liquid chromatography-tandem mass spectrometry and included 18-hydroxyeicosapentaenoic acid (18-HEPE), E-series resolvins, 17-hydroxydocosahexaenoic acid (17-HDHA), D-series resolvins, 14-hydroxydocosahexaenoic acid (14-HDHA), and maresin-1. RESULTS: Baseline SPMs did not differ between groups. There was an increase in the SPM precursors 18-HEPE, 17-HDHA, and 14-HDHA after n-3 fatty acid supplementation that was significantly attenuated in the MetS (P < 0.05). However, the E-series resolvins increased to a similar extent in the groups after n-3 fatty acid supplementation, and the D-series resolvins were not different from those at baseline. The addition of aspirin to n-3 fatty acids did not alter any SPMs in either group. CONCLUSIONS: Volunteers with MetS had reduced plasma concentrations of the precursors of the E- and D- series resolvins as well as of 14-HDHA in response to n-3 fatty acid supplementation. However, plasma E-series resolvins were increased to a similar extent after n-3 fatty acid supplementation in both groups, and the addition of aspirin to n-3 fatty acid supplementation did not alter any of the plasma SPMs in MetS and control subjects. Additional studies in the MetS are required to determine whether SPMs affect the ability to mount an appropriate response to infection. This trial was registered at the Australian New Zealand Clinical Trials Registry as ACTRN12610000708055.

n-3 fatty acids reduce plasma 20-hydroxyeicosatetraenoic acid and blood pressure in patients with chronic kidney disease.

BACKGROUND: Metabolism of arachidonic acid by cytochrome P450 ω-hydroxylase leads to the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) that regulates vascular function, sodium homeostasis and blood pressure (BP). Supplementation with n-3 fatty acids is known to alter arachidonic acid metabolism and reduce the formation of the lipid peroxidation products F2-isoprostanes, but the effect of n-3 fatty acids on 20-HETE has not been studied. METHOD: We previously reported a significant effect of n-3 fatty acids but not coenzyme Q10 (CoQ) to reduce BP in a double-blind, placebo-controlled intervention, wherein patients with chronic kidney disease (CKD) were randomized to n-3 fatty acids (4 g), CoQ (200 mg), both supplements or control (4 g olive oil), daily for 8 weeks. This study examined the effect of n-3 fatty acids on plasma and urinary 20-HETE in the same study, as well as plasma and urinary F2-isoprostanes, and relate these to changes in BP. RESULTS: Seventy-four patients completed the 8-week intervention. n-3 fatty acids but not CoQ significantly reduced plasma 20-HETE (P = 0.001) and F2-isoprostanes (P < 0.001). In regression models adjusted for BP at baseline, postintervention plasma 20-HETE was a significant predictor of the fall in SBP (P < 0.0001) and DBP (P < 0.0001) after n-3 fatty acids. CONCLUSION: This is the first report that n-3 fatty acid supplementation reduces plasma 20-HETE in humans and that this associates with reduced BP. These results provide a plausible mechanism for the reduction in BP observed in patients with CKD following n-3 fatty acid supplementation.