Identifying young adults at high risk of cardiometabolic disease using cluster analysis and the Framingham 30-yr risk score.

BACKGROUND AND AIMS: Current strategies to reduce cardiovascular disease (CVD) risk in young adults are largely limited to those at extremes of risk. In cohort studies we have shown cluster analysis identified a large sub-group of adolescents with multiple risk factors. This study examined if individuals classified at 'high-risk' by cluster analysis could also be identified by their Framingham risk scores. METHODS AND RESULTS: Raine Study data at 17- (n = 1048) and 20-years (n = 1120) identified high- and low-risk groups by cluster analysis using continuous measures of systolic BP, BMI, triglycerides and insulin resistance. We assessed:- CVD risk at 20-years using the Framingham 30 yr-risk-score in the high- and low-risk clusters, and cluster stability from adolescence to adulthood. Cluster analysis at 17- and 20-years identified a high-risk group comprising, 17.9% and 21.3%, respectively of the cohort. In contrast, only 1.2% and 3.4%, respectively, met the metabolic syndrome criteria, all of whom were within the high-risk cluster. Compared with the low-risk cluster, Framingham scores of the high-risk cluster were elevated in males (9.4%; 99%CI 8.3, 10.6 vs 6.0%; 99%CI 5.7, 6.2) and females (4.9%; 99%CI 4.4, 5.4 vs 3.2%; 99%CI 3.0, 3.3) (both P < 0.0001). A score >8 for males and >4 for females identified those at high CVD risk with 99% confidence. CONCLUSION: Cluster analysis using multiple risk factors identified ∼20% of young adults at high CVD risk. Application of our Framingham 30 yr-risk cut-offs to individuals allows identification of more young people with multiple risk factors for CVD than conventional metabolic syndrome criteria.

Alcohol and Hypertension-New Insights and Lingering Controversies.

PURPOSE OF REVIEW: To examine outstanding issues in the relationship of alcohol to hypertension. These include whether the increase in BP with alcohol is causally related, the nature of the relationship in women, the contribution of alcohol-related increases in BP to cardiovascular disease and the aetiology of alcohol-related hypertension. RECENT FINDINGS: Intervention studies and Mendelian randomisation analyses confirm the alcohol-BP relationship is causal. The concept that low-level alcohol intake reduces BP in women is increasingly unsustainable. Alcohol-related hypertension is in the causal pathway between alcohol use and increased risk for several cardiovascular outcomes. The aetiology of alcohol-related hypertension is multifactorial with recent data highlighting the effects of alcohol on the vasoconstrictor 20-HETE and oxidative stress. The high prevalence of both alcohol use and hypertension mandates a careful alcohol history in every patient with elevated BP. Early intervention for excessive alcohol use offers the promise of lower levels of BP and reduced risk of adverse cardiovascular outcomes.

The effect of n-3 fatty acids and coenzyme Q10 supplementation on neutrophil leukotrienes, mediators of inflammation resolution and myeloperoxidase in chronic kidney disease.

BACKGROUND: Neutrophils release leukotriene (LT)B4 and myeloperoxidase (MPO) that may be important mediators of chronic inflammation in chronic kidney disease (CKD). The n-3 fatty acids (n-3 FA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have the potential to attenuate inflammation through production of LTB5 and the Specialized Proresolving Lipid Mediators (SPM) that promote the resolution of inflammation. In animal models, coenzyme Q10 (CoQ) also attenuates inflammation by reducing MPO and LTB4. OBJECTIVE: This study evaluated the independent and combined effects of n-3 FA and CoQ supplementation on neutrophil leukotrienes, the pro-inflammatory eicosanoid 5-hydroxyeicosatetraenoic acid (5-HETE), SPM, and plasma MPO, in patients with CKD. DESIGN: In a double-blind, placebo-controlled intervention of factorial design, 85 patients with CKD were randomized to either n-3 FA (4 g), CoQ (200 mg), both supplements, or control (4 g olive oil), daily for 8 weeks. Plasma MPO and calcium ionophore-stimulated neutrophil release of LTs, 5-HETE and SPM were measured at baseline and after 8 weeks. RESULTS: Seventy four patients completed the intervention. n-3 FA, but not CoQ, significantly increased neutrophil LTB5 (P < 0.0001) and the SPM 18-hydroxyeicosapentaenoic acid (18-HEPE), resolvin E1 (RvE1), resolvin E2 (RvE2) and resolvin E3 (RvE3) that derive from EPA, as well as 17-hydroxydocosahexaenoic acid (17-HDHA) and resolvin D5 (RvD5) that derive from DHA (all P < 0.01). Neutrophil LTB4 and its metabolites, and 5-HETE were not significantly altered by n-3 FA or CoQ. Plasma MPO was significantly reduced with n-3 FA alone (P = 0.013) but not when given in combination with CoQ. CONCLUSION: n-3 FA supplementation in patients with CKD leads to increased neutrophil release of LTB5 and several SPM, as well as a reduction in plasma MPO that may have important implications for limiting chronic inflammation.

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.

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.

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.

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.

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.

Effects of maternal n-3 fatty acid supplementation on placental cytokines, pro-resolving lipid mediators and their precursors.

The aim of this study was to determine whether supplementation with fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy modifies placental PUFA composition, the accumulation of specialised pro-resolving lipid mediators (SPMs, specifically resolvins (Rv), protectins (PD) and upstream precursors) and inflammatory gene expression. Placentas were collected from women (n=51) enrolled in a randomised, placebo controlled trial of n-3 PUFA supplementation from 20-week gestation. Lipids were extracted for fatty acid analysis and SPMs were quantitated by mass spectrometry. Gene expression was determined by qRT-PCR. Using multiple regression analysis, data were correlated for placental n-3 PUFA and SPM levels with PUFA levels in maternal and cord blood erythrocytes. Supplementation with n-3 PUFAs increased placental docosahexaenoic acid (DHA) levels, but not eicosapentaenoic acid (EPA) levels (P<0.05), and increased the levels of the SPM precursors 18-hydroxyeicosapentaenoic acid and 17-hydroxydocosahexaenoic acid (17-HDHA) by two- to threefold (P<0.0005). RvD1, 17R-RvD1, RvD2 and PD1 were detectable in all placentas, but concentrations were not significantly increased by n-3 PUFA supplementation. Placental DHA levels were positively associated with maternal and cord DHA levels (P<0.005), and with placental 17-HDHA concentrations (P<0.0001). Placental mRNA expression of PTGS2, IL1ß, IL6 and IL10 was unaffected by n-3 PUFA supplementation, but TNFα expression was increased by 14-fold (P<0.05). We conclude that n-3 PUFA supplementation in pregnancy i) enhances placental accumulation of DHA and SPM precursors, ii) does not alter placental EPA levels, and iii) has no stimulatory effects on inflammatory gene expression. Further studies are required to ascertain the biological significance of SPMs in the placenta and the potential immunomodulatory effects of elevating placental SPM levels.

Mediators of inflammation resolution and vasoactive eicosanoids in gestational diabetes and preeclampsia.

OBJECTIVE: Women with gestational diabetes (GDM) have an increased risk of preeclampsia and postpartum diabetes. Inflammation associates with both GDM and preeclampsia. This study examined specialized proresolving mediators (SPM) that direct inflammation resolution and eicosanoids that are involved in inflammation, in relation to the development of preeclampsia and ongoing postpartum glucose intolerance in GDM. METHODS: Participants were selected from a prospective study examining the development of preeclampsia in women with GDM. Four groups of age-matched women were studied: GDM ( n  = 20), GDM who developed preeclampsia (GDM+PE, n  = 21), GDM who remained glucose-intolerant postpartum (GDM+PPIGT, n  = 20), or pregnancies with glucose tolerance within the normal range (NGT, n  = 21). Measurement of SPM (E-series resolvins and D-series resolvins), SPM pathway intermediates (14-HDHA, 18-HEPE and 17-HDHA), 20-hydroxyeicosatetraenoic acid (20-HETE), and the urinary metabolite of the vasodilator prostacyclin 2,3-dinor-6-Keto-PGF 1α , were made at 28, 32 and 36 weeks gestation and at 6 months postpartum. RESULTS: Compared with GDM, GDM+PE had elevated levels of 20-HETE and the SPM pathway intermediates 14-HDHA, 18-HEPE, 17-HDHA, at 32 weeks, and the SPM RvE1 at 32 and 36 weeks gestation. Compared with NGT and regardless of whether they developed preeclampsia or PPIGT, GDM had lower levels of 2,3-dinor-6-Keto-PGF 1α during pregnancy. CONCLUSION: Reduced levels of the prostacyclin metabolite 2,3-dinor-6-Keto-PGF 1α may contribute to the increased risk of preeclampsia in women with GDM. The increase in 20-HETE, a vasoconstrictor and mediator of inflammation, and SPM that contribute to inflammation resolution, prior to the onset of preeclampsia require further investigation to clarify their clinical significance.

Lupin and soya reduce glycaemia acutely in type 2 diabetes.

Addition of fibre or protein to carbohydrate-rich foods can reduce the glycaemic response to those foods. This may assist with glycaemic management in individuals with type 2 diabetes. Lupin is a legume rich in fibre and protein. We assessed the acute effects of lupin- and soya-based beverages on glucose and insulin responses in type 2 diabetic individuals. We hypothesised that the lupin and soya beverages would lower the acute glycaemic response compared with a control beverage containing no protein or fibre, and that lupin would reduce the postprandial glucose more than soya. In a randomised, controlled, cross-over trial, twenty-four diabetic adults (nineteen men and five women) attended three testing sessions, each 1 week apart. At each session, participants consumed a beverage containing 50 g glucose (control), 50 g glucose plus lupin kernel flour with 12·5 g fibre and 22 g protein (lupin), or 50 g glucose plus 12·5 g fibre and 22 g protein from soya isolates (soya). Serum glucose, insulin and C-peptide were measured periodically for 4 h following beverage consumption. Compared with the control beverage, the 4 h post-beverage glucose response was lower (P < 0·001), and the 4 h post-beverage insulin and C-peptide responses were higher (P < 0·001) for lupin and soya. Glucose (P = 0·25) and C-peptide (P = 0·07) responses did not differ significantly between lupin and soya, but lupin resulted in a lower insulin response compared with soya (P = 0·013). Adding lupin or soya to a carbohydrate-rich beverage reduces glycaemia acutely in type 2 diabetic individuals. This may have a beneficial role in glycaemic management.

A significant proportion of F2-isoprostanes in human urine are excreted as glucuronide conjugates.

The measurement of urinary F(2)-isoprostanes is noninvasive and widely used to assess in vivo oxidative stress in humans. Most studies measure urinary F(2)-isoprostanes in the free form; however, many eicosanoids are excreted as urine glucuronide conjugates. Using gas chromatography-mass spectrometry, we examined the extent of glucuronide conjugation of F(2)-isoprostanes in urine collected from healthy men (n = 20) and women (n = 15). Incubation of urine with exogenous glucuronidase led to F(2)-isoprostane concentrations that were approximately 40% higher than untreated samples (P < 0.001). We conclude that a significant proportion of F(2)-isoprostanes in urine are conjugated as glucuronides.

Flaxseed oil supplementation increases plasma F1-phytoprostanes in healthy men.

Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been reported to reduce lipid peroxidation products formed from arachidonic acid (F(2)-isoprostanes) in healthy humans, as well as in those under oxidative stress. alpha-Linolenic acid (ALA) is a precursor to EPA and DHA; however, its conversion in humans is thought to be inefficient. ALA can also undergo free radical oxidation, forming compounds known as F(1)-phytoprostanes, which are found in all plants and are in high concentrations in plant pollens. In this study, we examined the effect of ALA supplementation on plasma and urine F(1)-phytoprostane and F(2)-isoprostane concentrations in men. Thirty-six nonsmoking men, aged 20-65 y, were recruited from the general population and randomly allocated to consume 9 g/d of either flaxseed oil (62% ALA, 5.4 g/d) or olive oil (placebo) for 4 wk in a parallel design. At baseline and after 4 wk of supplementation, blood samples and a 24-h urine sample were collected for measurement of plasma and urinary F(1)-phytoprostanes and F(2)-isoprostanes and plasma fatty acids. Compared with the olive oil group, plasma phospholipid ALA was greater (P < 0.0001), as were F(1)-phytoprostanes in plasma (P = 0.049) and urine (P = 0.06) in the flaxseed oil group after 4 wk supplementation. Flaxseed oil did not affect plasma or urinary F(2)-isoprostanes. The greater plasma F(1)-phytoprostane concentration in the flaxseed oil group most likely resulted from the increased plasma concentration of the ALA substrate and/or the F(1)-phytoprostane content of the flaxseed oil. Future studies are needed to determine the physiological importance of increased plasma and urine F(1)-phytoprostanes and their relevance to heart disease prevention.

Maternal fish oil supplementation in pregnancy modifies neonatal leukotriene production by cord-blood-derived neutrophils.

Fish oil supplementation has been shown to reduce neutrophil production of inflammatory LTB4 (leukotriene B4) in adults. The present study is the first to examine the effects on neonatal neutrophil function following supplementation in pregnancy. Pregnant women with allergic disease (n=98) were randomized to receive either fish oil [3.7 g of n-3 long-chain PUFAs (polyunsaturated fatty acids)/day] or a placebo supplement for the final 20 weeks of pregnancy. Leukotriene production by neonatal neutrophils was measured after stimulation with the calcium ionophore A23187. This was examined in relation to supplementation, cell membrane fatty acid levels and mononuclear cytokine production. Neutrophil LTB4 production was significantly reduced in neonates whose mothers had received fish oil in pregnancy. This was most evident for isomer 2 of LTB4 (P=0.031), although this was also observed for total LTB4 (P=0.051) and isomer 1 (P=0.088). There was also a trend for lower production of other PUFA metabolites, namely 5-HETE (5-hydroxyeicosatetraenoic acid; P=0.054) in the fish oil group. Accordingly, LTB4 levels were inversely related to membrane n-3 PUFA levels. Less inflammatory products (LTB5) were only produced at very low levels, although there was a trend for higher levels of this metabolite in the fish oil group. Consistent with this, LTB5 levels were positively correlated with n-3 PUFA membrane levels, particularly EPA (eicosapentanoic acid) and negatively correlated with n-6 PUFAs. Neonates with lower neutrophil LTB4 production also had lower production of pro-inflammatory IL (interleukin)-6 responses (r=0.35, P=0.005) and regulatory IL-10 responses (r=0.37, P=0.003) by LPS (lipopolysaccharide)-stimulated neonatal mononuclear cells. In conclusion, maternal dietary changes can modify neonatal neutrophil function. This has implications for the early immune programming, which can be influenced by the inflammatory milieu of local tissues during initial antigen encounter. It also provides evidence of another pathway through which long-chain PUFAs status can influence early immune development.

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.

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.

F2-Isoprostanes in HDL are bound to neutral lipids and phospholipids.

Low HDL cholesterol (HDL-C) is a risk factor for coronary artery disease (CAD). However, interventions that raise HDL-C have failed to reduce cardiovascular events. We previously reported that HDL is the main carrier of plasma F2-isoprostanes (F2-IsoPs) that are markers of oxidative stress formed upon oxidation of arachidonic acid. F2-IsoPs are predominantly associated with phospholipids. However, there is evidence that F2-IsoPs in the liver of rats treated with carbon tetrachloride associate with the neutral lipids. To date it is not known whether F2-IsoPs are found in the neutral lipids in HDL in humans. Possible candidate neutral lipids include cholesteryl esters, triglycerides, diglycerides, and monoglycerides. This study aimed to identify the lipid classes within native and oxidized HDL that contain F2-IsoPs. We showed that F2-IsoPs in HDL are bound to neutral lipids as well as phospholipids. HDL-3 contained the highest concentration of F2-IsoPs in all lipid classes before and after in vitro oxidation. Using targeted LC/MS and high resolution MS, we were unable to provide conclusive evidence for the presence of the synthesized standards 15(R)-15-F2t-isoP cholesterol and 1-ent-15(RS)-15-F2t-isoprostanoyl-sn-glycerol in the neutral lipids of HDL. Our findings show that oxidized lipids such as F2-IsoPs are found in the core and surface of HDL. However, the exact molecular species remain to be definitively characterized. Future studies are required to determine whether the presence of F2-IsoPs in neutral lipids alters HDL function.

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.

Fish oil supplementation in pregnancy lowers F2-isoprostanes in neonates at high risk of atopy.

The anti-inflammatory properties of n-3 polyunsaturated fatty acids (n-3 PUFA) have suggested a potential role of these nutrients in dietary modification for prevention of allergic disease in early life. As oxidative stress is known to modify antigen presenting cell (APC) signalling and resulting immune responses, we examined the effects of maternal n-3 PUFA supplementation in pregnancy on markers of oxidative stress and APC function in neonates at high risk of allergy. Eighty-three pregnant atopic women were randomised to receive 4 g daily of either fish oil (n = 40) or olive oil (n = 43) capsules in a controlled trial from 20 weeks gestation until delivery. Plasma (cord blood) and urinary F2-isoprostanes were measured as markers of lipid peroxidation. Cord erythrocyte fatty acids and markers of APC function (HLA-DR expression and cytokine responses) were measured and related to levels of plasma F2-isoprostanes. Maternal fish oil supplementation lowered plasma (p < 0.0001) and urinary (p = 0.06) F2-isoprostanes. HLA-DR expression on APC was not different between the groups. In multiple regression analysis, 28.8% of the variance in plasma F2-isoprostanes was explained by positive relationships with erythrocyte arachidonic acid (AA) and monocyte HLA-DR expression and a negative relationship with erythrocyte eicosapentaenoic acid (EPA). This study shows that maternal supplementation with fish oil can attenuate neonatal lipid peroxidation. Clinical follow-up of these infants will help to determine if there are sustained effects on postnatal oxidative stress and expression of allergic disease.