Anticonvulsant effects of linolenic acid are unrelated to brain phospholipid cell membrane compositions

Authors

  • Natacha Porta,

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
    2. Pediatric Neurology Department, Lille University Hospital, Lille, France
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  • Béatrice Bourgois,

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
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  • Claude Galabert,

    1. CERM, Hôpital Renée Sabran, Giens Hyères, CHU Lyon, France
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  • Cécile Lecointe,

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
    2. Pediatric Neurology Department, Lille University Hospital, Lille, France
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  • Pierre Cappy,

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
    2. Pediatric Neurology Department, Lille University Hospital, Lille, France
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  • Régis Bordet,

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
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  • Louis Vallée,

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
    2. Pediatric Neurology Department, Lille University Hospital, Lille, France
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  • Stéphane Auvin

    1. Department of Pharmacology, EA 1046, Institut de Médecine Prédictive et de Recherche Thérapeutique, Université de Lille-2 et Centre Hospitalier Universitaire, Lille, France
    2. Pediatric Neurology Department, Lille University Hospital, Lille, France
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Address correspondence to Stéphane Auvin, Service de Neurologie Pédiatrique, Hôpital Roger Salengro, Lille, France. E-mail: auvin@invivo.edu

Summary

Purpose: Recent studies have revealed that polyunsaturated fatty acids (PUFAs) have anticonvulsive properties. Clinical trials using PUFAs reported conflicting results. It was suggested that PUFAs have anticonvulsant effects via modifications of brain phospholipids. Moreover, some authors suggested that the effect of the ketogenic diet (KD) leads to a high PUFA content. The aim of the study was to evaluate the anticonvulsant properties of a mixture containing α-linolenic acid (ALA) and linolenic acid (LA).

Methods: Four-week-old male Wistar rats were fed one of the following diets for 30 days: KD, standard diet, and standard diet with daily LA/ALA oral supplementation. Pentylenetetrazol (PTZ) threshold was used to assess the anticonvulsive effects of the diets. Nutritional status was monitored by body composition evaluation. Fatty acids composition of both plasma and brain phospholipids were also assessed.

Results: Animals fed the KD and those who had the daily LA/ALA supplementation exhibited an increase in PTZ threshold. The animals did not show any modification of body composition or brain phospholipid composition. The plasma fatty acids composition was modified by KD and LA/ALA. A decrease in arachidonic acid (AA) concentrations was observed in both the KD and LA/ALA groups, while an increase in eicosapentanoic acid (EPA) and ALA concentrations was only observed in the LA/ALA group.

Conclusions: Our study shows that LA/ALA supplementation exerts anticonvulsive properties comparable to KD. Nutritional status can not explain the anticonvulsive effects of PUFAs supplementation. Brain phospholipids were not different within groups. The anticonvulsive effects of LA supplementation seem to be unrelated to brain phospholipid composition.

Approximately one-third of patients with epilepsy continues to experience seizures, despite the adequate use of available antiepileptic drugs (Vining, 1999). New therapies are required to help patients with drug-resistant seizures.

The high fat ketogenic diet (KD) has been used since the 1920s to treat intractable childhood epilepsy. Multicenter trials showed that 40%–50% of children on the KD exhibit a greater than 50% reduction in seizure frequency (Freeman et al., 1998; Vining et al., 1998). Similar anticonvulsant effects of the KD have been demonstrated in rats (Appleton & DeVivo, 1974; Hori et al., 1997; Bough & Eagles, 1999; Bough et al., 1999a, 1999b, 2000). However, the mechanisms underlying its anticonvulsant properties remain unknown.

Recent studies have revealed that polyunsaturated fatty acids (PUFAs) have anticonvulsant actions. Linoleic acid [LA; C18:2(n-6)], α-linolenic acid [ALA; C18:3(n-3)], arachidonic acid [AA; C20:4(n-6)], eicosapentanoic acid [EPA; C20:5(n-3)], and docosahexaenoic acid [DHA; C22:6(n-3)] confer seizure protection in both in vitro (Vreugdenhil et al., 1996; Keros & McBain, 1997; Xiao & Li, 1999; Lauritzen et al., 2000; Young et al., 2000) or in vivo models. PUFAs raise the seizure threshold using pentylenetetrazol (PTZ) (Yehuda et al., 1994; Rabinovitz et al., 2004; Taha et al., 2007), kaïnate (Blondeau et al., 2002), cortical irritation by iron chloride (Yehuda et al., 1994), electrical cortical stimulation (Voskuyl et al., 1998), or audiogenic-seizure (Yehuda et al., 1994) in rats.

Clinical trials reported conflicting results. In an open study, 21 patients with intractable epilepsy received 3.25 g n-3 PUFAs daily (46% DHA, 18% EPA, 1% ALA) for 6 months. Five patients completed the study with a reduction in both frequency and strength of the seizures (Schlanger et al., 2002). In a randomized study, the administration of a mixture EPA/DHA (10:7) showed a decrease in seizure frequency over the first 6-week treatment period, but the effect was not sustained (Yuen et al., 2005). Bromfield and coworkers (2008) did not succeed in obtaining seizure protection with an EPA/DHA (3:2) mixture given for 12 weeks.

The majority of the PUFAs of cell membranes are synthesized from LA and ALA, which act as precursors for the synthesis of those longer-chained PUFAs via a series of desaturation and elongation reactions (Qi et al., 2002). In the brain membrane, AA is a bilayer component, but the major of PUFA is usually DHA (Spector, 2001). PUFAs may exhibit anticonvulsant effects via fatty acid modifications of the brain membrane (Yehuda et al., 1996, 1998). Several studies have reported that membrane bilayer structure modifications induced functional changes (Ehringer et al., 1990; Stillwell et al., 1993; Yehuda et al., 1999; Haag, 2003). Pharmacology studies have shown that the extracellular application of EPA and DHA raises the stimulatory thresholds of CA1 neurons in hippocampal slices (Xiao & Li, 1999). PUFAs caused a significant shift in steady state inactivation in the hyperpolarizing direction for both Na+ and Ca2+ currents (Vreugdenhil et al., 1996; Voskuyl et al., 1998) and induced a stabilization of the neuronal membrane by suppressing voltage-gated Ca2+ currents and Na+ channels (Xiao et al., 1995, 1997).

The aim of the study was to evaluate the anticonvulsant properties of ALA (mixture LA/ALA 25%–70%) in rats using a PTZ threshold. Both brain and plasma fatty acid compositions of phospholipids (phosphatidylethanolamine and/or phosphatidylcholine) were also assessed to evaluate its role in the anticonvulsive effect.

Methods

Animals

Four-week-old male Wistar rats (Charles River, L'arbresle, France) were used in the present study. Rats were housed 6 to 8 per cage on an alternating 12:12 light/dark cycle with lights on at 07:00 a.m. All procedures involving animals and their care were performed in agreement with the local ethical committee for animal experimentation and in compliance with our institutional guidelines, which in turn comply with current national and international laws and recommendation.

Treatments

All animals were maintained on rodent chow and water ad libitum before diet onset. Rats were weighed and divided into three diet treatment groups: (1) A standard diet containing 2.46% PUFAs (2.31% LA and 0.15% ALA; control group, n = 23, Harlan Teklad Global 2016); (2) an isocaloric KD 4:1 ratio containing 17.5% PUFAs (16% LA and 1.5% ALA; KD group, n = 22, Ketocal® SHS); (3) standard diet with daily oral supplementation by a blend (Sigma-Aldrich, St. Louis, MO, U.S.A) of 70% ALA, 25% LA, and 5% oleic acid (LINO group, n = 19). The rats were kept on the diets for 30 days. Standard diet was served ad libitum while KD was given based on the caloric intake for laboratory rats: 0.3 kcal/g body weight/day (Rogers, 1979). The ALA/LA blend was administrated by mouth (p.o.) at 6 g/kg body weight/day. This amount was determined to have a similar quantity of LA plus ALA in both KD and LINO groups. All rats were weighed at least twice weekly.

PTZ threshold

A PTZ (Sigma-Aldrich, Saint-Quentin Fallavier, France) threshold was reached between 9:00 a.m. and 12:00 a.m., 30 days after each diet treatment. PTZ was dissolved in physiologic saline at 10 mg/ml and was infused with a syringe pump in the tail vein of the freely moving rat at constant rate (2 ml/h). Time to first forelegs myoclonus was used as the end point. The total amount of drug infused at this time was calculated from the duration of the infusion and expressed as a dose (mg/kg) representing PTZ threshold (control, n = 14; KD, n = 9; LINO, n = 6) (Pollack & Shen, 1985; Loscher et al., 1991; Auvin et al., 2006).

Body composition

Six days before diet onset (D-0) and on the last day (D-30) of the study (after PTZ threshold), the rats were anesthetized [300 mg/kg intraperitoneally (i.p.) chloral hydrate] and scanned using a Lunar PIXImus densitometer (GE Lunar, Madison, WI, U.S.A.) ( Fig. 1) for body composition measurement (control, n = 12; KD, n = 10; LINO, n = 7).

Figure 1.

 Percentage of body fat 6 days before diet onset (D-0) and the last day of diet (D-30) in control (n  = 12), KD (n  = 9), and LINO (n  = 7) groups measured with the PIXImus densitometer (upper left corner). Values are mean ± SEM. Upper left corner: PIXImus densitometer. Upper right corner: scan of a studied rat. Body fat composition (%) was comparable between groups at the start and the end of the study.

Lipids analysis

After the body composition measurement (D-30), rats were killed by an overdose of pentobarbital injected i.p (180 mg/kg). Blood samples were drawn by cardiac punctures after which the brains were removed. Blood samples were centrifuged for 20 min at 3,500 rpm and 4°C, and then plasma was separated and stored at −80°C. Excised brains were frozen in liquid nitrogen and stored at −80°C.

Lipid extraction from serum and from homogenized brains was performed with chloroform/methanol as described previously (Bligh & Dyer, 1959). About 300–600 mg brain tissue and 200 μl serum were used for each analysis. Organic phase obtained after extraction was concentrated, and individual lipid classes within extract were separated by one-dimensional (serum) or two-dimensional (brain) high-performance thin layer chromatography (HPTLC) as described by  Galabert et al. (1987). Briefly, HPTLC plate was developed with chloroform/methanol/16.5 N aqueous ammonia vol/vol/vol, 65:25:5 in the first dimension, followed by chloroform/acetone/methanol/acetic acid/water vol/vol/vol/vol/vol, 3:4:1:1:0.5 in the second dimension. Isolated phospholipids subclasses phosphatidylcholine and phosphatidylethanolamine were scraped off the HPTLC plate and transesterified in 14% boron trifluoride-methanol (code 18017; Alltech Biotechnologies, Carquefou, France) in a sealed vial in nitrogen atmosphere at 100°C for 45  min according to Morrison and Smith (1964). The resulting fatty acid methylesters (FAME) were extracted with pentane, dehydrated over sodium sulfate decahydrated (Na2SO4, 10 H2O), evaporated in a vacuum concentrator, and prepared for gas chromatography (GC) by sealing pentane extract in nitrogen. FAME were separated and quantified by capillary GC using a gas chromatograph (model 5890; Hewlett-Packard, Wilmington, DE, U.S.A.) equipped with a 60 m/0.32 mm DB-23 capillary column (J&W Scientific, Folsom, CA, U.S.A.) and flame-ionization detector. A time-programmed temperature from 170° to 230°C was used. Individual fatty acids were identified by comparison of retention time with known standard and quantified with an HP3396 integrator.

Statistical analysis

Data are expressed as the mean ± SEM. Statistical analysis was performed using a Kruskal-Wallis test and Dunn post hoc analysis using SPSS software (15.00 version; LEAD Technologies, Chicago, IL, U.S.A.). Statistical significance was considered to be p < 0.05.

Results

Weight and body composition were comparable between groups and tend to increase between D-0 and D-30

Animals which freely consumed the KD or the standard diet (control and LINO groups) did not show any signs of ill health and remained well groomed during the study. No difference was observed in mean fat component of the body between KD, LINO, and control groups 6 days before diet onset (D-0) and at day-30 (D-30), the last day of treatment (Fig. 1). Mean weights were also similar within groups.

PTZ thresholds were increased on rats fed KD and in LINO group

PTZ thresholds (PTZth) were significantly increased in KD and LINO groups in comparison to the control animal group. PTZth were: KD, 51.2 ± 2 mg/kg; LINO, 50.2 ± 3 mg/kg; versus control, 43.2 ± 2 mg/kg with p < 0.05 (Fig. 2).

Figure 2.

 Effects of 30-day preventive treatments on myoclonic PTZ thresholds (mg/kg) in control (n  = 14), KD (n  = 9), and LINO (n  = 6) groups. Values are mean ± SEM. *, p ≤ 0.05; **, p  < 0.005 in comparison with control. PTZ thresholds were increased in both KD and LINO group.

Fatty acid composition of the plasma was modified by the different diets

AA was decreased in both the KD and LINO groups compared to the control group. EPA and ALA were increased in the LINO group compared to the KD and control groups (p < 0.02). When phosphatidylcholine (PC) data was analyzed according to n-6:n-3 ratio, the ratio was statistically lower in the LINO and KD groups compared to the control group (p < 0.01). No difference was found between groups for DHA and LA (Fig. 3).

Figure 3.

 Plasma phosphatidylcholine fatty acid composition in KD (n  = 9), LINO (n  = 13), and control (n  = 9) groups. Results are expressed as percent of total lipids. The plasma fatty acid composition in AA, EPA, and ALA was modified in both KD and LINO groups. The n-6:n-3 ratio was lower in LINO and KD groups compare to the control group. LA, linoleic acid; ALA, α-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PC, phosphatidy-lcholine; *, p ≤ 0.02; **, p ≤ 0.01; ***, p ≤ 0.001.

Fatty acid composition of the brain was not modified by diets

No difference was found in the fatty acid composition of brain tissue. In both PC and phosphatidylethanolamine (PE) fractions, AA, DHA, EPA, LA, and ALA were not different between groups. Consistent with these data, PC and PE n-6:n-3 ratios were not modified under the three different diets (Figs. 4 and 5).

Figure 4.

 Fatty acids of PC fraction in brain after in KD (n  = 13), LINO (n  = 10), and control (n  = 6) groups. Results are expressed as percent of total fatty acids. The fatty acid of PC fraction in the brain was similar in both KD and LINO groups. LA, linoleic acid; ALA, α-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PC, phosphatidylcholine.

Figure 5.

 Fatty acids of PE fraction in brain in KD (n  = 10), LINO (n = 13), and control (n = 6) groups. Results are expressed as percent of total fatty acids. The fatty acid of PC fraction in the brain was similar in both KD and LINO groups. LA, linoleic acid; ALA, α-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; PE, phosphatidylethanolamine.

Discussion

Daily ALA/LA supplementation exhibits anticonvulsive properties comparable to the KD in rats. Using weight and body composition measurements, we were able to exclude the idea that the nutritional status may explain our results. Thirty days after the diet modifications, the fatty acid composition of the plasma phospholipids was modified, while no modification was observed in the brain phospholipids composition. The underlying mechanisms of the anticonvulsive effect seem to be unrelated to the central nervous system (CNS) cell membrane composition.

Clinical studies on PUFAs showed conflicting results. In a randomized trial, patients' alimentation was supplemented by 1 g EPA and 0.7 g DHA daily (10:7) or by mineral oils. Fifty-six patients with refractory epilepsy (29 PUFAs and 27 placebos) completed a 12-week double-bind phase. Seizure frequency was reduced by 50% over a 6-week period of treatment in the supplement group (5 of 29), but the effect was not sustained (Yuen et al., 2005). In a prospective randomized study, adults with uncontrolled epilepsy were randomized to either mineral oil or a PUFAs supplement (EPA/DHA, 2.2 mg/day, 3:2). Following a 4-week prospective baseline and 1-week titration, subjects entered a 12-week treatment period, followed by an optional 4-week open-label phase. Of 21 patients (12 PUFAs and 9 placebos), 0 on PUFAs versus 2 on placebo had at least a 50% decrease in seizure frequency from baseline. Overall, seizure frequency increased 6% on PUFAs and decreased 12% on placebo (Bromfield et al., 2008). In the open-label study done by Schlanger et al. (2002), a high number of patients discontinued the study (16 of 21). However, it is the only study using a mixture containing ALA. Five patients completed the study (6 months) and exerted a marked seizure reduction.

In animal models, it has been shown that ALA has anticonvulsant and neuroprotective properties (Lauritzen et al., 2000; Blondeau et al., 2002). Among all tested PUFAs (DHA, AA, and ALA), ALA represented the most efficacious and reproducible effect against a kainate-induced cell injury (Blondeau et al., 2002). Intracerebroventricular injection of ALA prior to kainate blocked the epileptiform activity and reduced cell loss in both CA1 and CA3 neurons (Lauritzen et al., 2000). ALA was also found to be neuroprotective when it was administrated intravenously (i.v.) either before or after the kainate injection (Lauritzen et al., 2000). Other studies using PTZ-induced seizures showed that rats treated for 21 days (i.p.) with the SR-3 mixture (LA and ALA mixture, 4:1) exerted a decrease in seizure severity, frequency, and duration (Yehuda et al., 1994; Rabinovitz et al., 2004). The SR-3 mixture was also effective against cortical irritation by iron chloride and audiogenic seizures (Yehuda et al., 1994). Using SR-3 mixture, a recent study with a 21-day treatment did not reduce seizure latency, frequency, or severity in rats (Taha et al., 2006).

Both human and animal data suggest that ALA can have anticonvulsive properties. However, underlying mechanisms remain unknown. The cell membrane phospholipids composition reflects the dietary fatty acids intake (Yehuda et al., 1999; Youdim et al., 2000). It has been reported that in the brain, PUFAs may induce changes in cell membrane fluidity (Zerouga et al., 1991; Yehuda et al., 1999). Free fatty acids, lipids metabolites, and phospholipids can also modify the function of membrane proteins including enzymes, receptors, membrane transporters, and ion channels (Ordway et al., 1991; Keros & McBain, 1997). In our study, we did not find any change in CNS phospholipids fatty acid composition. The AA, EPA, DHA, ALA, and LA compositions were similar between groups in both PC and PE fractions. PC is considered as a reflection of the extern cell membrane layer, while PE reflects the intern cell membrane layer (Janmey & Kinnunen, 2006). The anticonvulsive effects of ALA/LA in rats seem to be unrelated to the brain phospholipid cell membrane composition.

Several mechanisms may explain the anticonvulsive properties of ALA and/or LA. First of all, free fatty acids are well known to activate peroxysome proliferator-activated receptor-α (PPARα) (Kliewer et al., 1997). PPARα is likely to regulate expression of many genes encoding enzymes of amino acid and/or neurotransmitter metabolisms in the brain (Cullingford, 2004). Free fatty acids may also act by direct action on ion channels modeling cell membrane excitability (Keros & McBain, 1997; Xiao et al., 1997).

KD is a high-fat, low-carbohydrate, and low-protein diet with demonstrated anticonvulsive effects. It has been suggested that plasmatic and/or brain fatty acid composition can explain its efficacy. In rats treated by KD with different fatty acids supplementation, the concentration of total fatty acids in the brain was no different to the control group, while seizures induced by PTZ were most severe in the control group (Dell et al., 2001). When KD was supplemented by LA/ALA (21% and 48%, respectively), the proportion of AA in brain total lipids was decreased. No change was observed regarding brain level of DHA. The authors suggest the absence of a correlation between seizure protection and brain lipids composition. Other studies, using KD or SR-3 mixture supplementation, reported brain lipids modifications. However, the seizure protection was not evaluated in the animals studied (Yehuda et al., 1996; Taha et al., 2005). An increase in DHA brain level was correlated to an increase in seizure latency induced by PTZ using transgenic fat-1 mice. Fat-1 gene encodes an n-3 fatty acid desaturase that converts n-6 to n-3 fatty acids, which is usually absent in mammals. The fat-1 transgenic mice are capable of producing n-3 fatty acids from the n-6 type, leading to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in their organs and tissues (Taha et al., 2007).

In our study, rats treated with both the ALA/LA mixture and the KD did not exert brain phospholipid modifications. Rats exerted plasma fatty acid changes and PTZ-induced seizure protection. The plasma phospholipid fatty acids composition may explain the anticonvulsive effects observed in this study. An ALA i.v. administration was neuroprotective against kainite-induced seizures and cell death (Lauritzen et al., 2000). Moreover, several clinical studies have suggested that a change in plasmatic AA concentrations may be linked to treatment effects of KD or EPA/DHA supplementation (Fraser et al., 2003; Yuen et al., 2005). An inverse variation of AA was reported in these studies. A 6-week EPA/DHA supplementation produced a fall in AA and LA concentration and an increase in EPA and DHA in the 5 of 29 patients with seizure reduction (Yuen et al., 2005). A positive correlation between plasma AA increase and seizure reduction was reported in patients treated with the KD (Fraser et al., 2003). However, an explanation for the divergent results described may be the fatty acid composition of the diet used in all these studies. Finally, the conflicting results observed in clinical and experimental studies could lead to the different amount of fatty acids and/or duration of diet modifications. Negative results should be analyzed according to the design of the study.

Our study shows that supplementation by a mixture of LA/ALA exerts anticonvulsive properties comparable to those of the KD. It is the first study showing that nutritional status cannot explain the anticonvulsive effects of PUFA supplementation. In animals with antiepileptic effects induced by diets, we found a change in the fatty acids composition of plasma phospholipids, while brain phospholipids were not different among the studied groups. The underlying mechanisms of the anticonvulsive effect seem to be unrelated to brain cell membrane composition. The role of plasma fatty acids on the anticonvulsive effects remains unclear.

Acknowledgments

This work was supported by the AEAC association. N.P. was supported by the grant “Conseil Régional Nord-Pas-de-Calais et CHRU de Lille.”

Conflict of interest: We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The authors have no conflicts of interest to disclose.

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