Diclofenac Prolongs Repolarization in Ventricular Muscle with Impaired Repolarization Reserve

Background

The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti-inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle.

Methods

Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model.

Results

Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 µM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl₂ application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 µg/kg) significantly lengthened the QT_c interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT_c. Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 µM) decreased the amplitude of rapid (I_Kr) and slow (I_Ks) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I_Ca) was slightly diminished, but the transient outward (I_to) and inward rectifier (I_K1) potassium currents were not influenced.

Conclusions

Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve.     

Low essential fatty acid and B-vitamin status in a subgroup of patients with schizophrenia and its response to dietary supplementation

We assessed essential fatty acid (EFA) and B-vitamin status, together with their determinants, in 61 patients with schizophrenia and established whether those with poor status responded biochemically to the appropriate dietary supplements. As a group, the patients had high erythrocyte saturated fatty acids (FAs), monounsaturated FA and low polyunsaturated FA of the omega3 and omega6 series. Patients reporting not to take vitamin supplements had low vitamin B12 and high homocysteine. Homocysteine variance proved best explained by folate in both the total group and male patients, and by vitamins B12 and B6 in females. Alcohol consumption and duration of illness are risk factors for low polyunsaturated FA status (< P2.5 of reference range), while male gender and absence of fish consumption predict hyperhomocysteinemia (> P97.5 of reference range). Two patients exhibited biochemical EFA deficiency and seven showed biochemical signs of omega3/docosahexaenoic acid (DHA) marginality. Four patients exhibited moderate hyperhomocysteinemia with plasma values ranging from 57.5 to 74.8 micromol/L. None of the five patients with either moderate hyperhomocysteinemia, biochemical EFA deficiency, or both, was predicted by their clinicians to have poor diets. That diet was nevertheless at the basis of these abnormalities became confirmed after supplementing 4 of them with B vitamins and with soybean and fish oils. We conclude that a subgroup of patients with schizophrenia has biochemical EFA deficiency, omega3/DHA marginality, moderate hyperhomocysteinemia, or combinations. Correction seems indicated in view of the possible relation of poor EFA and B-vitamin status with some of their psychiatric symptoms, but notably to reduce their high risk of cardiovascular disease.     

Impact of Hypokalemia on Electromechanical Window,Excitation Wavelength and Repolarization Gradients in Guinea-Pig and Rabbit Hearts

Normal hearts exhibit a positive time difference between the end of ventricular contraction and the end of QT interval, which is referred to as the electromechanical (EM) window. Drug-induced prolongation of repolarization may lead to the negative EM window, which was proposed to be a novel proarrhythmic marker. This study examined whether abnormal changes in the EM window may account for arrhythmogenic effects produced by hypokalemia. Left ventricular pressure, electrocardiogram, and epicardial monophasic action potentials were recorded in perfused hearts from guinea-pig and rabbit. Hypokalemia (2.5 mM K⁺) was found to prolong repolarization, reduce the EM window, and promote tachyarrhythmia. Nevertheless, during both regular pacing and extrasystolic excitation, the increased QT interval invariably remained shorter than the duration of mechanical systole, thus yielding positive EM window values. Hypokalemia-induced arrhythmogenicity was associated with slowed ventricular conduction, and shortened effective refractory periods, which translated to a reduced excitation wavelength index. Hypokalemia also evoked non-uniform prolongation of action potential duration in distinct epicardial regions, which resulted in increased spatial variability in the repolarization time. These findings suggest that arrhythmogenic effects of hypokalemia are not accounted for by the negative EM window, and are rather attributed to abnormal changes in ventricular conduction times, refractoriness, excitation wavelength, and spatial repolarization gradients.     

Short-term supplementation of low-dose gamma-linolenic acid (GLA), alpha-linolenic acid (ALA), or GLA plus ALA does not augment LCP omega 3 status of Dutch vegans to an appreciable extent

Vegans do not consume meat and fish and have therefore low intakes of long chain polyunsaturated fatty acids (LCP). They may consequently have little negative feedback inhibition from dietary LCP on conversion of alpha -linolenic acid (ALA) to the LCP omega 3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. We investigated whether supplementation of nine apparently healthy vegans with 2.01 g ALA (4 ml linseed oil), 1.17 g gamma-linolenic acid (GLA) (6 ml borage oil) or their combination increases the LCP omega 3 contents of erythrocytes (RBC) and platelets (PLT), and of plasma phospholipids (PL), cholesterol esters (CE) and triglycerides (TG). The supplements changed the dietary LA/ALA ratio (in g/g) from about 13.7 (baseline) to 6.8 (linseed oil), 14.3 (borage oil) and 6.4 (linseed + borage oil), respectively. ALA or GLA given as single supplements did not increase LCP omega 3 status, but their combination augmented LCP omega 3 (in CE) and EPA (in fasting TG) to a statistically significant, but nevertheless negligible, extent. We conclude that negative feedback inhibition by dietary LCP, if any, does not play an important role in the inability to augment notably DHA status by dietary ALA. The reach of a DHA plateau already at low dietary ALA intakes suggests that dietary DHA causes a non-functional DHA surplus, or is, alternatively, important for maintaining DHA status at a functionally relevant level.     

Effect of phospholipids containing omega-3 fatty acids on structural changes of microsomal lipids in cell membranes of functionally different cells

As a result of the experimental researches conducted it has been shown that administration of some normal animal marine phospholipids (PL) including in their structure omega-3 polyunsaturated fatty acids (PUFA) provides for quantitative changes of individual PL, fatty acids (FA) content and quantity in general and individual PL of liver, heart, brain and gonads microsomes. While estimating general microsomal PL fraction FA content under the action of PL omega-3 PUFA FA concentration change, unsaturation index (omega 6/omega 3) and relation of arachidonic acid to docosahexenic (AA/DHA) decrease have been identified. The decrease of AA/DHA relationship occurs due to AA and DHA quantitative changes. In the case of AA increase in some tissues there is observed the decrease of docosapentaenic acid and increase of DHA and eucosapentaenic (EPA) acidds. As a result of studying FA content in the individual PL composition it has been identified that certain PL classes characteristic for some tissues respond by changes of some certain FA. The relationship omega 6/omega 3 has been shown as decreasing in phosphatidilcholine (PC) all tissues microsomes (liver, gonads, heart, brain), in phosphatidilethanolamine (PEA) of liver and cardiac microsomes, in phosphatidilserine (PS) this relationship relationship decreases in the liver, brain and heart, for phosphatidilinositole (PI) the changes take place in liver, gonads, brain. Simultaneously, the decrease of AA/DHA relationship in the individual PL decrease of AA and increase of EPA and DHA depend on the tested tissues. The marine phospholipids might be supposed to render their effect on AA metabolism resulting in AA/DHA relationship in PEA and PS relationship displays itself as specific and depends on the tissues functions. The preference of PEA and PS use by certain tissues microsomes could be explained by their membrane protective capability.     

Effects of altered maternal folic acid, vitamin B12 and docosahexaenoic acid on placental global DNA methylation patterns in Wistar rats

Potential adverse effects of excess maternal folic acid supplementation on a vegetarian population deficient in vitamin B(12) are poorly understood. We have previously shown in a rat model that maternal folic acid supplementation at marginal protein levels reduces brain omega-3 fatty acid levels in the adult offspring. We have also reported that reduced docosahexaenoic acid (DHA) levels may result in diversion of methyl groups towards DNA in the one carbon metabolic pathway ultimately resulting in DNA methylation. This study was designed to examine the effect of normal and excess folic acid in the absence and presence of vitamin B(12) deficiency on global methylation patterns in the placenta. Further, the effect of maternal omega 3 fatty acid supplementation on the above vitamin B(12) deficient diets was also examined. Our results suggest maternal folic acid supplementation in the absence of vitamin B(12) lowers plasma and placental DHA levels (p<0.05) and reduces global DNA methylation levels (p<0.05). When this group was supplemented with omega 3 fatty acids there was an increase in placental DHA levels and subsequently DNA methylation levels revert back to the levels of the control group. Our results suggest for the first time that DHA plays an important role in one carbon metabolism thereby influencing global DNA methylation in the placenta.     

Voluntary Running in Young Adult Mice Reduces Anxiety-Like Behavior and Increases the Accumulation of Bioactive Lipids in the Cerebral Cortex

Combinatorial therapies using voluntary exercise and diet supplementation with polyunsaturated fatty acids have synergistic effects benefiting brain function and behavior. Here, we assessed the effects of voluntary exercise on anxiety-like behavior and on total FA accumulation within three brain regions: cortex, hippocampus, and cerebellum of running versus sedentary young adult male C57/BL6J mice. The running group was subjected to one month of voluntary exercise in their home cages, while the sedentary group was kept in their home cages without access to a running wheel. Elevated plus maze (EPM), several behavioral postures and two risk assessment behaviors (RABs) were then measured in both animal groups followed immediately by blood samplings for assessment of corticosterone levels. Brains were then dissected for non-targeted lipidomic analysis of selected brain regions using gas chromatography coupled to mass spectrometry (GC/MS). Results showed that mice in the running group, when examined in the EPM, displayed significantly lower anxiety-like behavior, higher exploratory and risky behaviors, compared to sedentary mice. Notably, we found no differences in blood corticosterone levels between the two groups, suggesting that the different EPM and RAB behaviors were not related to reduced physiological stress in the running mice. Lipidomics analysis revealed a region-specific cortical decrease of the saturated FA: palmitate (C16:0) and a concomitant increase of polyunsaturated FA, arachidonic acid (AA, omega 6-C20: 4) and docosahexaenoic acid (DHA, omega 3-C22: 6), in running mice compared to sedentary controls. Finally, we found that running mice, as opposed to sedentary animals, showed significantly enhanced cortical expression of phospholipase A2 (PLA₂) protein, a signaling molecule required in the production of both AA and DHA. In summary, our data support the anxiolytic effects of exercise and provide insights into the molecular processes modulated by exercise that may lead to its beneficial effects on mood.     

Omega-3 fatty acid supplementation accelerates chylomicron triglyceride clearance

Omega-3 fatty acids (FAs) reduce postprandial triacylglycerol (TG) concentrations. This study was undertaken to determine whether this effect was due to reduced production or increased clearance of chylomicrons. Healthy subjects (n = 33) began with a 4-week, olive oil placebo (4 g/d) run-in period. After a 4-week wash-out period, subjects were randomized to supplementation with 4 g/d of ethyl esters of either safflower oil (SAF), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) for 4 weeks. Results for EPA and DHA were similar, and therefore the data were combined into one omega-3 FA group. Omega-3 FA supplementation reduced the postprandial TG and apolipoprotein B (apo B)-48 and apoB-100 concentrations by 16% (P = 0.08), 28% (P < 0.001), and 24% (P < 0.01), respectively. Chylomicron TG half-lives in the fed state were reduced after omega-3 FA treatment (6.0 +/- 0.5 vs. 5.1 +/- 0.4 min; P < 0.05), but not after SAF (6.9 +/- 0.7 vs. 7.1 +/- 0.7 min). Omega-3 FA supplementation decreased chylomicron particle sizes (mean diameter; 293 +/- 44 vs. 175 +/- 25 nm; P < 0.01) and increased preheparin lipoprotein lipase (LPL; 0.6 +/- 0.1 vs. 0.9 +/- 0.1 micromol/h/ml; P < 0.05) activity during the fed state, but had no effect on postheparin LPL or hepatic lipase activities. The results suggest that omega-3 FA supplementation accelerates chylomicron TG clearance by increasing LPL activity, and that EPA and DHA are equally effective.     

Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenoic acid on breastmilk fatty acid composition

We investigated whether supplementation with arachidonic acid (20:4 omega 6; AA), or a combination of AA and docosahexaenoic acid (22:6 omega 3; DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition. Ten women were daily supplemented with 300 mg AA, eight with 300 mg AA, 110 mg eicosapentaenoic acid (20:5 omega 3; EPA) and 400 mg DHA, for one week and eight women served as unsupplemented controls. Milk samples were collected on days 0, 1 and 7. The fatty acid composition of the milk was analyzed by capillary gas chromatography with flame ionisation detection. Supplementation with AA alone had no effect on breastmilk AA, but tended to reduce EPA and DHA levels. Administration of a combination of AA, EPA and DHA tended to increase both milk AA and long chain PUFA (LCPUFA)omega 3 content. A larger simultaneous increase of milk AA, DHA and EPA than observed in the present study can probably be accomplished by the use of a combination of a lower LCPUFA omega 6/LCPUFA omega 3 ratio and higher AA, EPA and DHA dosages.     

The effect of n-3 long-chain polyunsaturated fatty acids and lipoic acid on the heart in the ovariectomized rat model of menopause

Menopause occurs as consequence of ovarian senescence that leads to a drop of oestrogen hormone. The decreased oestrogen levels combined with the impairment of the redox system may contribute to the increased risk of postmenopausal cardiovascular disease. Supplementation with antioxidants may be an alternative to reduce cardiovascular risk. The study evaluated the effect of dietary supplementation with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and α-lipoic acid (LA) for a period of 16 weeks on oxidative stress biomarkers in the hearts of ovariectomized 3-month-old rats. Ovariectomy did not increase the level of the damage markers malondialdehyde and carbonyl, and both were decreased by LA supplementation. Ovariectomy increased the levels of the endogenous antioxidants glutathione, vitamin C and H₂O₂ consumption, after restoration by DHA, EPA, and LA supplementation. Vitamin E, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase are not altered by ovariectomy. Lipid and protein damage are not increased after ovariectomy and a portion of the endogenous antioxidants concomitantly increased, suggesting that hearts may be protected by these antioxidants. DHA, EPA, and LA restored these endogenous antioxidants, showing that all evaluated supplements are effective in modulating the antioxidant redox system in the heart. LA showed additional effect on redox markers, decreasing lipid and protein damage markers.     

Essential fatty acid supplementation of DHA and ARA and effects on neurodevelopment across animal species: a review of the literature

Docosahexanoic acid (DHA) and arachidonic acid (ARA) are long chain essential fatty acids used as supplements in commercial infant formula. DHA/ARA deficient states are associated with adverse neurological outcomes in animals and humans. Preterm infants are at risk for DHA/ARA deficiency. A few clinical reports on the effects of fatty acid supplementation have shown benefit in preterm, low birth weight, and normal infants in the first year of life, whereas others did not. Studies in animals have reported shortened gestation, fetal growth retardation, reduced infant body mass, and increased fetal mortality with consumption of fatty acids during pregnancy. To understand the data that support fatty acid supplementation in infant formula, a review of the animal model literature was undertaken, to examine the effects of DHA/ARA on neurodevelopment, including the effects on visual acuity. Several points emerged from this review. (1) Animal studies indicate that requirements for DHA/ARA vary depending on developmental age. Alterations of the ratio of DHA/ARA can impact developmental outcome. (2) The available studies suggest that while supplementation of DHA/ARA in an appropriate ratio can increase tissue levels of these fatty acids in the brain and retina, tissues sensitive to depletion of fatty acids, the benefit of routine supplementation remains unclear. Few studies measure functional outcome relative to changes in physiologic pools of DHA/ARA after supplementation. (3) Animal literature does not support a clear long-term benefit of replenishing DHA/ARA tissue levels and administration of these fatty acids at concentrations above those in human milk suggests adverse effects on growth, survival, and neurodevelopment.     

Dietary Omega-3 Fatty Acid Supplementation Reduces Inflammation in Obese Pregnant Women: A Randomized Double-Blind Controlled Clinical Trial

Objective

Long-chain omega 3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) exert potent anti-inflammatory properties in humans. This study characterized the effects of omega-3 ω-3 fatty acids supplements (ω-3 FA) on the inflammatory status in the placenta and adipose tissue of overweight/obese pregnant women.

Study Design

A randomized, double-masked controlled trial was conducted in overweight/obese pregnant women that were randomly assigned to receive DHA plus EPA (2g/day) or the equivalent of a placebo twice a day from week 10–16 to term. Inflammatory pathways were characterized in: 1) adipose tissue and placenta of treated vs. untreated women; and 2) adipose and trophoblast cells cultured with long chain FAs.

Results

The sum of plasma DHA and EPA increased by 5.8 fold and ω-3 FA/ ω-6 FA ratio was 1.5 in treated vs. untreated women (p< 0.005). Plasma CRP concentrations were reduced (p<0.001). The adipose tissue and placenta of treated women exhibited a significant decrease in TLR4 adipose and placental expression as well as IL6, IL8, and TNFα In vitro, EPA and DHA suppressed the activation of TLR4, IL6, IL8 induced by palmitate in culture of adipose and trophoblast cells.

Conclusion

Supplementation of overweight/obese pregnant women with dietary ω-3 FAs for >25 weeks reduced inflammation in maternal adipose and the placental tissue. TLR4 appears as a central target of the anti-inflammatory effects at the cellular level.

Trial Registration

ClinicalTrials.gov NCT00957476     

Arrhythmogenic substrate and its modification by nicorandil in a murine model of long QT type 3 syndrome

The gain-of-function Scn5a+/ΔKPQ mutation in the cardiac Na⁺ channel causes human long QT type 3 syndrome (LQT3) associated with ventricular arrhythmogenesis. The K_ATP channel-opener nicorandil (20 μM) significantly reduced arrhythmic incidence in Langendorff-perfused Scn5a+/Δ hearts during programmed electrical stimulation; wild-types (WTs) showed a total absence of arrhythmogenicity. These observations precisely correlated with alterations in recently established criteria for re-entrant excitation reflected in: (1) shortened left-ventricular epicardial but not endocardial monophasic action potential durations at 90% repolarization (APD₉₀) that (2) restored transmural repolarization gradients, ΔAPD₉₀. Scn5a+/Δ hearts showed longer epicardial but not endocardial APD₉₀s, giving shorter ΔAPD₉₀s than WT hearts. Nicorandil reduced epicardial APD₉₀ in both Scn5a+/Δ and WT hearts thereby increasing ΔAPD₉₀. (3) Reduced epicardial critical intervals for re-excitation; Scn5a+/Δ hearts showed greater differences between APD₉₀ and ventricular effective refractory period than WT hearts that were reduced by nicorandil. (4) Reduced APD₉₀ alternans. Scn5a+/Δ hearts showed greater epicardial and endocardial alternans than WTs, which increased with pacing rate. Nicorandil reduced these in Scn5a+/Δ hearts to levels indistinguishable from untreated WTs. (5) Flattened restitution curves. Scn5a+/Δ hearts showed larger epicardial and endocardial critical diastolic intervals than WT hearts. Nicorandil decreased these in Scn5a+/Δ and WT hearts. The presence or absence of arrhythmogenesis in Scn5a+/Δ and WT hearts thus agreed with previously established criteria for re-entrant excitation, and alterations in these precisely correlated with the corresponding antiarrhythmic effects of nicorandil. Together these findings implicate spatial and temporal re-entrant mechanisms in arrhythmogenesis in LQT3 and their reversal by nicorandil.     

Climate warming is predicted to reduce omega‐3, long‐chain,polyunsaturated fatty acid production in phytoplankton

Phytoplankton are the main source of energy and omega‐3 (n‐3) long‐chain essential fatty acids (EFA) in aquatic ecosystems. Their growth and biochemical composition are affected by surrounding environmental conditions, including temperature, which continues to increase as a result of climate warming. Increasing water temperatures may negatively impact the production of EFA by phytoplankton through the process of homeoviscous adaptation. To investigate this, we conducted an exploratory data synthesis with 952 fatty acid (FA) profiles from six major groups of marine and freshwater phytoplankton. Temperature was strongly correlated with a decrease in the proportion of n‐3 long‐chain polyunsaturated FA (LC‐PUFA) and an increase in omega‐6 FA and saturated FA. Based on linear regression models, we predict that global n‐3 LC‐PUFA production will be reduced by 8.2% for eicosapentaenoic acid (EPA) and 27.8% for docosahexaenoic acid (DHA) with an increase in water temperature of 2.5 °C. Using a previously published estimate of the global production of EPA by diatoms, which contribute to most of the world's supply of EPA, we predict a loss of 14.2 Mt of EPA annually as a result of ocean warming. The n‐3 LC‐PUFA are vitally important for an array of key physiological functions in aquatic and terrestrial organisms, and these FA are mainly produced by phytoplankton. Therefore, reduced production of these EFA, as a consequence of climate warming, is predicted to negatively affect species that depend on these compounds for optimum physiological function. Such profound changes in the biochemical composition of phytoplankton cell membranes can lead to cascading effects throughout the world's ecosystems.     

Oscillation in cycle length induces transient discordant and steady-state concordant alternans in the heart

Alternans is a beat-to-beat alternation of the cardiac action potential duration (APD) or intracellular calcium (Ca(i)) transient. In cardiac tissue, alternans can be spatially concordant or discordant, of which the latter has been shown to increase dispersion of repolarization and promote a substrate for initiation of ventricular fibrillation. Alternans has been studied almost exclusively under constant cycle length pacing conditions. However, heart rate varies greatly on a beat-by-beat basis in normal and pathological conditions. The purpose of this study was to determine if applying a repetitive but non-constant pacing pattern, specifically cycle length oscillation (CLO), promotes or suppresses a proarrhythmic substrate. We performed computational simulations and optical mapping experiments to investigate the potential consequences of CLO. In a single cell computational model, CLO induced APD and Ca(i) alternans, which became "phase-matched" with the applied oscillation. As a consequence of the phase-matching, in one-dimensional cable simulations, neonatal rat ventricular myocyte monolayers, and isolated adult guinea pig hearts CLO could transiently induce spatial and electromechanical discordant alternans followed by a steady-state of concordance. Our results demonstrated that under certain conditions, CLO can initiate ventricular fibrillation in the isolated hearts. On the other hand, CLO can also exert an antiarrhythmic effect by converting an existing state of discordant alternans to concordant alternans.     

Determinants of DHA levels in early infancy: differential effects of breast milk and direct fish oil supplementation

IntroductionAlthough omega (n)-3 long-chain polyunsaturated fatty acids (LCPUFA), particularly docosahexaenoic acid (DHA), intakes are important during infancy, the optimal method of increasing infant status remains unclear. We hypothesized that high-dose infant fish oil supplementation would have greater relative effects upon n-3 LCPUFA status at six months of age than breast milk fatty acids.Patients and methodsInfants (n=420) were supplemented daily from birth to six months with fish oil or placebo. In a subset of infants, LCPUFA levels were measured in cord blood, breast milk and in infant blood at 6 months.ResultsDHA levels increased in the fish oil group relative to placebo (p<05). Breast milk DHA was the strongest predictor of infant erythrocyte DHA levels (p=<001). This remained significant after adjustment for cord blood DHA, supplementation group and adherence.ConclusionIn this cohort, breast milk DHA was a greater determinant of infant erythrocyte n?3 LCPUFA status, than direct supplementation with fish oil.     

Double enrichment of chicken eggs with conjugated linoleic acid and n-3 fatty acids through dietary fat supplementation

Yolk fat fatty acid (FA) concentrations, sensory quality and firmness of eggs and laying hen performance were evaluated with respect to the combined inclusion in the diet of conjugated linoleic acid (CLA), high n-3 oil sources and high-oleic sunflower oil (HOSO). Nine diets were arranged factorially, with three levels of n-3 FA supplementation (2.9, 3.7 and 4.5 g/kg) from three different sources (two fish oils highly concentrated in eicosapentanoic (EPA) or docosahexanoic acid (DHA) and one algae oil with a very high-DHA content) in diets added with fixed amounts of CLA (2.5 g/kg) and HOSO (30 g/kg). A commercial feed with no CLA, n-3 or HOSO added, and another one containing 4.5 g/kg of high-DHA fish oil but not CLA or HOSO were also formulated. An increase in n-3 FA supplementation had little effect on proportions of CLA, monounsaturated FA, saturated FA or total polyunsaturated FA in yolk fat, but increased (P<0.005) long-chain n-3 FA and decreased (P<0.001) long-chain n-6 FA. An increment of dietary n-3 FA also impaired linearly (P<0.001) egg acceptability by consumers. An increment in the proportion of DHA with respect to total n-3 FA from 0.28 to 0.96 increased yolk concentrations of DHA (P<0.001) and total n-3 FA (P<0.01), but decreased (P<0.001) concentrations of EPA and docosapentanoic acid FA. Current data indicate that addition of HOSO to diets supplemented with moderate amounts of CLA and n-3 FA allows the production of double enriched eggs while maintaining sensory quality for consumers at acceptable levels.     

Effects of the new antiarrhythmic peptide ZP123 on epicardial activation and repolarization pattern

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH(2)) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH(2)). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H(2)O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 +/- 6% and 146 +/- 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCalpha inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivo studies of antiarrhythmic peptides.     

Plasma and Red Blood Cell Fatty Acids in Peroxisomal Disorders

The demonstration of abnormal levels of fatty acids or plasmalogens in plasma or red blood cells is key to the diagnosis of peroxisomal disorders. We report the levels of 62 fatty acids and plasmalogens in patients with X-linked adrenoleukodystrophy (X-ALD), Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), both at baseline and after dietary interventions. Lorenzo's Oil therapy in X-ALD normalizes the levels of saturated very long chain fatty acids in plasma, but leads to reduced levels of omega 6 and other omega 3 fatty acids, and requires monitoring and appropriate dietary supplements. Patients with ZS, NALD and IRD have reduced levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) which can be normalized by the oral administration of microencapsulated DHA and AA.     

CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.