Lack of benefit of linoleic and α-linolenic polyunsaturated fatty acids on seizure latency, duration, severity or incidence in rats

Results There were no significant effects of SR-3 on seizure latency, duration or severity ( P > 0.05). There were also no significant differences in the incidence of myoclonic jerks, forelimb and hindlimb clonus, forelimb and hindlimb tonus or running fits in rats that received SR-3, as compared to control rats ( P > 0.05). Conclusion Linoleic and α-linolenic polyunsaturated fatty acids have no beneficial effects on seizure latency, duration, average severity or incidence. Keywords Linoleic acid α-Linolenic acid Polyunsaturated fatty acids SR-3 Seizures Pentylenetetrazol 1 Introduction Epilepsy is a neurological disorder characterized by spontaneous, recurrent seizures ( Burnham, 1998 ). Although 60–70% of patients respond to conventional anticonvulsant drug treatment, 30–40% of patients continue to experience seizures despite the best anticonvulsant therapy ( Vining, 1999 ). New therapies are required to help these patients with drug-resistant seizures. The high-fat ketogenic diet is a commonly used treatment for drug-resistant epilepsy ( Stafstrom, 2004 ). The classic ketogenic diet contains 80% fat, mainly in the form of saturated fatty acids, derived from butter. Despite the diet's efficacy ( Vining, 1999 ), there is some concern regarding its unfavorable, atherogenic effect on plasma lipid profiles. It elevates triglycerides, LDL-cholesterol and total cholesterol ( Kwiterovitch et al., 2003; Fuehrelein et al., 2005 ). Polyunsaturated fatty acids (PUFAs), which have anti-atherogenic properties, have been considered as a potential alternate therapy for drug-resistant seizures ( Schlanger et al., 2002; Fuehrelein et al., 2005 ). PUFAs, such as docosahexaenoic acid and arachidonic acid, are essential for normal brain function, due to their role as structural components of membranes and their involvement in neurotransmission, cell signaling and gene regulation ( Rapoport, 2003; Kitajka et al., 2004 ). They are synthesized in the liver from dietary linoleic and α-linolenic acids, or obtained directly from the diet ( Sprecher, 2000 ). Studies have reported that dietary or infused PUFAs, such as linoleic, α-linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acids, confer seizure protection in cell cultures ( Fraser et al., 1993; Vreugdenhil et al., 1996; Keros and McBain, 1997; Lauritzen et al., 2000; Young et al., 2000 ), animal models ( Yehuda et al., 1994; Voskuyl et al., 1998; Blondeau et al., 2002; Rabinovitz et al., 2004 ), and, most recently, human cases of drug-resistant epilepsy ( Schlanger et al., 2002 ). In particular, Yehuda et al. (1994) have reported that a mixture of linoleic and α-linolenic acids in a 4:1 ratio (i.e. the “SR-3 compound”) reduces latency and severity in young rats in the maximal pentylenetetrazol (PTZ) seizure model. This observation has recently been replicated by Rabinovitz et al. (2004) . Although these studies are promising, they are somewhat flawed, in that they compared the anticonvulsant properties of the SR-3 PUFA mixture in experimental rats, to control rats that were injected with saline as a vehicle, rather than the mineral oil which was used to dissolve the PUFA mixture. Thus, it is not clear whether the reported anticonvulsant effects of the SR-3 compound in the PTZ seizure model were due to its PUFA content (i.e. linoleic and α-linolenic acids), or to an anticonvulsant effect of the mineral oil. The present study was, therefore, conducted to determine whether a PUFA-based mixture, containing linoleic and α-linolenic acids in a 4:1 ratio (SR-3), confers protection against PTZ induced seizures in rats, as compared to controls treated with mineral oil. 2 Materials and methods 2.1 SR-3 preparation The SR-3 mixture was prepared as described by Rabinovitz et al. (2004) . Briefly, 0.05 ml of non-esterified α-linolenic acid (0.90 g/ml) and 0.2 ml of non-esterified linoleic acid (0.92 mg/ml; Sigma–Aldrich, St. Louis, Missouri, USA) were dissolved in 0.73 ml of mineral oil (Sigma–Aldrich, St. Louis, Missouri, USA), containing 0.02 ml of α-tocopherol. The SR-3 mixture was stored at −20 °C until use. PTZ (Sigma–Aldrich) was dissolved in 0.9% saline on the day of seizure testing. 2.2 Subjects and treatments The following experiments were conducted according to the guidelines of the Canadian Council of Animal Care, and approved by the Animal Care Committee of the Faculty of Medicine of the University of Toronto. One-month-old male Long-Evans hooded rats (Charles River, La Prairie, Que., Canada), weighing on average 116 g, served as subjects. Subjects were individually housed in plastic cages with corn-cob bedding and maintained in a 12-h light:12-h dark cycle (lights on at 7 a.m.) at 21 °C. Water and Purina ® rat chow were available ad libitum to both control and experimental groups. The Purina rat chow contained (g/kg diet) 234.0 protein, 45.0 fat, 623.5 carbohydrates, 58.0 fiber, 0.3 vitamins and 39.2 minerals. After 7 days in the facility, subjects were randomly divided into experimental ( n = 12) and control ( n = 8) groups. The experimental subjects received daily intraperitoneal injections with 40 mg/kg SR-3 in mineral oil. The control subjects received intraperitoneal injections with an equal volume of vehicle (mineral oil). Subjects were injected daily for 21 consecutive days as previously done by Rabinovitz et al. (2004) , and were weighed each day prior to receiving the injections. 2.3 Seizure testing On experimental day 22, subjects were weighed and then seizure tested using the PTZ procedure ( Krall et al., 1978 ). Eighty milligram per kilogram of PTZ were injected intra-peritoneally. The subjects were then placed in an open field and videotaped for 30 min. Videotapes were subsequently scored by two independent “blinded” observers. Latency (seconds) was measured between PTZ injection and the onset of: (1) myoclonic jerks, (2) forelimb and hindlimb clonus, (3) forelimb and hindlimb tonus and (4) running fits. “Seizure duration” was also measured. It was defined as the time from seizure onset (myoclonic jerks, clonus, tonus or running fits) until the cessation of convulsions, unless the rat exhibited severe running fits, in which case the rat was immediately sacrificed by an intra-peritoneal injection of sodium pentobarbital (MCT Pharmaceuticals, Cambridge, Ont.) and excluded from the seizure duration analysis. “Seizure severity” was scored according to the following scale: stage 1, myoclonic jerks; stage 2, forelimb or hindlimb clonus; stage 3, forelimb or hindlimb tonus; stage 4, running fits. Scores were averaged in order to yield a measure of “seizure severity” (out of 4). “Seizure incidence”, defined as the percentage of rats experiencing stages 1, 2, 3, 4 seizures, was also determined. It was calculated by dividing the number of rats experiencing convulsions at a certain seizure stage by the total number of rats, and multiplying by 100%. 2.4 Fatty acid analysis The fatty acid composition of each component of the SR-3 mixture (i.e. linoleic acid, α-linolenic acid and mineral oil) was verified by gas chromatography as previously described ( Taha et al., 2005 ). Briefly, total fatty acids from each compound were extracted from 4–5 samples, and derivitized according to the method of Folch et al. (1957) . The resulting fatty acid methyl esters were quantified on a HP6890 gas chromatograph (Agilent Technologies, Mississauga, Ont.), equipped with a flame ionization detector, and separated on a fused silica capillary SP2560 100 m column (Supelco, Bellefonte, PA) with 0.2 μm film thickness and 0.25 mm internal diameter. One microlitre of fatty acid methyl esters from each sample was injected into the column in splitless mode, using helium gas as a carrier at a constant flow rate of 1.3 ml/min. A 5-stage temperature program was used to acquire the fatty acid methyl ester profile. The initial temperature was 60 °C. This was followed by a ramp up at 10 °C/min to 170 °C and a 5 min hold, a 5 °C/min ramp up to 175 °C, a 2 °C/min ramp up to 185 °C, a 1 °C/min ramp up to 190 °C, and a final 10 °C/min ramp up to 240 °C and an 18 min hold (total run time = 50 min). Fatty acid peaks were identified by comparing the retention time of each peak against the retention times of a fatty acid standard of known composition (GLC463, NuCheck Prep., Ont., Canada). 2.5 Statistical analysis The data are presented as means ± S.E. Data analysis was performed on Statistical Analysis Software (version 8.02, SAS Institute, Cary, NC) and Sigma Stat v.3.2 (Jandel Corporation). A 2-way analysis of variance was used to determine the effects of treatment and time on body weight gain. Seizure threshold and duration were analyzed using an unpaired t -test after verifying the normality of the data. The data for seizure duration and severity did not have a normal distribution, and, therefore, the Mann–Whitney U -test was used. Outliers falling more than 2 standard deviations from the mean were excluded from the statistical analyses. Fisher's exact test was used to compare the incidence of seizures at each stage in the control and SR-3 groups. Statistical significance was accepted at P < 0.05. 3 Results 3.1 Body weights Body weights of control and experimental subjects are presented in Fig. 1 . All subjects gained weight over time ( P < 0.05). There was no significant difference in body weights, however, between control and experimental subjects at any time point ( P > 0.05). 3.2 Fatty acid profile of SR-3 constituents The purity of the SR-3 constituents was verified by gas-chromatography. The purities of linoleic and α-linolenic acids, on a percent composition basis, were 96.2 ± 1.6 and 91.3 ± 1.4, respectively. As expected, mineral oil, being a petroleum hydrocarbon chain, contained no fatty acids. 3.3 Seizure latency All animals in the control and experimental groups exhibited seizure activity after PTZ administration. The data related to seizure latency are presented in Fig. 2 . As indicated by Fig. 2 , latencies in most subjects were in the range of 60–70 s. Outliers that were excluded from the analysis included one rat from the control group which seized at 15 min post PTZ injection, and two rats from the SR-3 group which, respectively, seized at 10 and 25 min after PTZ administration. With the outliers excluded, mean seizure latency did not differ significantly between the control and the SR-3 groups (69.7 ± 2.8 s in controls versus 67.9 ± 2.5 s in SR-3 group; P > 0.05). 3.4 Seizure duration The data for seizure duration are presented in Fig. 3 . As indicated by Fig. 3 , seizure duration in most subjects fell in the range of 80–100 s. One out of the eight control rats and two out of the 12 experimental rats were excluded from the seizure duration analysis because they had severe running fits, and were therefore euthanized immediately. As a result, their seizure duration was not determined. The data for seizure duration was not normally distributed, and therefore, the Mann–Whitney U -test was used to detect significance in ranking between the two groups. The results showed that seizure duration did not differ significantly between the control and SR-3 groups (81.9 ± 19.0 s in controls versus 96.5 ± 15.8 s in SR-3 group, P > 0.05). 3.5 Seizure severity The data for seizure severity are presented in Fig. 4 . As indicated, most subjects had clonic seizures, that were ranked between 2 and 2.5. Because the data for seizure severity were not normally distributed, the Mann–Whitney U -test was used to analyze the data. The results showed that there were no significant differences in seizure severity between the control and SR-3 groups ( P > 0.05). 3.6 Seizure incidence within each seizure score category The incidence of seizures within each seizure score category is shown in Table 1 . There were no significant differences between the percentage of rats experiencing myoclonic jerks (stage 1), forelimb and hindlimb clonus (stage 2), forelimb and hindlimb tonus, running fits (stage 4), or forelimb and hindlimb tonus and running fits combined (stage 3 + 4). 4 Discussion The primary objective of the present study was to determine the potential anticonvulsant properties of a PUFA-based mixture containing linoleic and α-linolenic acids. This has been termed the “SR-3 mixture” ( Yehuda et al., 1994 ). Our results indicate that the SR-3 PUFA mixture did not alter seizure latency, duration or severity, as compared to controls that received a mineral oil vehicle. It also did not alter the incidence of myoclonic jerks, forelimb and hindlimb clonus, forelimb and hindlimb tonus or running fits in SR-3 treated subjects, as compared to control subjects. The lack of effect of the SR-3 mixture on seizure latency, duration, severity or incidence contrasts with the reports of Yehuda et al. (1994) and Rabinovitz et al. (2004) . A similar experimental design and the same seizure model were used in this study, so differences in design or seizure model cannot explain the differences in the results. The differing results, however, may relate to the fact that we injected our control subjects with mineral oil (the SR-3 vehicle) instead of saline. This raises the possibility that the mineral oil may possess anticonvulsant properties. Mineral oil is a petroleum hydrocarbon containing n -alkanes and cyclic paraffin ( Christensen et al., 2005 ). Previous research has demonstrated that after 5 h of oral administration of H 3 labeled mineral oil to rats, 80% of the label appeared in faeces, 1–5% was absorbed and stored in liver, kidney and adipose tissue, whereas 15% was detected in brain, liver and other tissues as an unidentified H 3 labeled mineral oil metabolite ( Ebert et al., 1966 ). It is possible that a metabolite of the mineral oil accumulated in brain after 21 days of daily administration, and increased seizure latency in the control group, thereby masking any potential anticonvulsant properties of the SR-3 compound. It is also possible that SR-3 did not raise brain PUFA composition to the necessary threshold for detecting seizure protection following 3 weeks of SR-3 administration. SR-3 contains linoleic and α-linolenic acids, which are converted primarily in the liver to their elongation/desaturation products, arachidonic and docosahexaenoic acids, respectively ( Sprecher, 2000 ). Arachidonic and docosahexaenoic acids are considered to be the bioactive products of SR-3 in the brain, because (1) they constitute more than 50% of brain total lipids (versus <2% for linoleic and α-linolenic acids), and (2) they have been reported to confer seizure protection in animal models and humans ( Voskuyl et al., 1998; Schlanger et al., 2002 ). Brain PUFAs were not measured in the present study due to the possibility that brain fatty acid composition would be altered after seizure induction ( Kulagina et al., 2000 ). SR-3, however, has been previously shown to significantly raise brain arachidonate and docosahexaenoate composition ( Yehuda et al., 1996 ). New evidence suggests that the seizure protective effects of the ketogenic diet may in part be attributed to its ability to increase brain PUFA composition, particularly arachidonic acid and docosahexaenoic acid by at least 15% each ( Fraser et al., 2003; Taha et al., 2005 ). Thus, the possibility remains that the duration of the trial was too short to achieve the desired threshold concentrations of arachidonic and docosahexaenoic acids (>15%) in brain, that may lead to seizure protection. We have suggested in previous publications that the elevation of ketones, particularly acetone, may contribute to the anticonvulsant effects of the ketogenic diet in humans ( Likhodii and Burnham, 2002 ). It is not clear, however, that the ketogenic diet produces significant elevations of ketone bodies in rats ( Nylen et al., 2005; Taha et al., 2005 ). Thus, it is tempting to speculate that the previously reported alterations in brain PUFA composition observed in rats fed a ketogenic diet ( Taha et al., 2005 ) may potentially be partially or fully responsible for the diet's ability to ameliorate seizure severity ( Cunnane, 2004; Taha et al., 2005 ). We conclude that SR-3 did not alter seizure latency, duration, severity or incidence in young rats. The lack of benefit of SR-3 on seizures may be due to the possibility that PUFAs have no measurable influence on these outcomes at the dose used in this study. Alternatively, a mineral oil metabolite may have raised seizure threshold and reduced duration and severity in the control group to the extent of masking any potential benefits of SR-3. Finally, it is possible that the duration of the trial was not long enough to produce an effect on these parameters. Further studies assessing the potential anticonvulsant effects of PUFAs at higher doses and prolonged periods of intake are warranted. Acknowledgements The authors would like to acknowledge Mr. Jerome Cheng for his help in scoring the seizures. NSERC provided financial support for this study. References Blondeau et al., 2002 N. Blondeau M. Widmann M. Lazdunski C. Heurteaux Polyunsaturated fatty acids induce ischemic and epileptic tolerance Neuroscience 109 2 2002 231 241 Burnham, 1998 W.M. Burnham Principles of Medical Pharmacology sixth ed. 1998 Oxford University Press pp. 250–256 Cunnane, 2004 S.C. Cunnane Metabolism of polyunsaturated fatty acids and ketogenesis: an emerging connection Prostagalndins Leukot Essent Fatty Acids 70 3 2004 237 241 Christensen et al., 2005 J.H. Christensen A.B. Hansen U. Karlson J. Mortensen O. Anderson Multivariate statistical methods for evaluating biodegradation of mineral oil J. Chromatogr. A 1090 1–2 2005 133 145 Ebert et al., 1966 A.G. Ebert C.R. Schleifer S.M. Hess Absorption, disposition, and excretion of 3H-mineral oil in rats J. Pharm. Sci. 55 9 1966 923 929 Folch et al., 1957 Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226 (1), 497–509. Fraser et al., 1993 D.D. Fraser K. Hoehn S. Weiss B.A. MacVicar Arachidonic acid inhibits sodium currents and synaptic transmission in cultured striatal neurons Neuron 11 4 1993 633 644 Fraser et al., 2003 D.D. Fraser S. Whiting R.D. Andrew E.A. MacDonald K. Musa-Veloso S.C. Cunnane Elevated polyunsaturated fatty acids in blood serum of obtained from children on the ketogenic diet Neurology 60 2003 1026 1029 Fuehrelein et al., 2005 B.S. Fuehrelein M.S. Rutenberg J.N. Silver M.W. Warren D.W. Theriaque G.E. Duncan P.W. Stacpoole M.L. Brantly Differential metabolic effects of saturated versus polyunsaturated fats in ketogenic diets Clin. Endocrinol. Metab. 89 4 2005 1641 1645 Keros and McBain, 1997 S. Keros C.J. McBain Arachidnoic acid inhibits transient potassium currents and broadens potentials during electrographic seizures in hippocampal pyramidal and inhibitory neurons J. Neurosci. 17 10 1997 3476 3487 Kitajka et al., 2004 K. Kitajka A.J. Sinclair R.S. Weisinger H.S. Weisinger M. Mathai A.P. Jayasooriya J.E. Halver L.G. Pukas Effects of dietary omega-3 polyunsaturated fatty acids on brain gene expression Proc. Natl. Acad. Sci. U.S.A. 101 30 2004 10931 10936 Krall et al., 1978 R.L. Krall J.K. Penry B.G. White H.J. Kupferberg E.A. Swinyard Antiepileptic drug development. II. Anticonvulsant drug screening Epilepsia 19 4 1978 409 428 Kulagina et al., 2000 T.P. Kulagina I.K. Kolomiitseva V.I. Arkhipoc Effect of picrotoxin-induced seizures on lipid composition of cortical tissue homogenate and its nuclear fraction in rats Bull. Ex. Biol. Med. 130 9 2000 864 866 Kwiterovitch et al., 2003 P.O. Kwiterovitch Jr. E.P. Vining P. Pyzik R. Skolasky Jr. J.M. Freeman Effect of a high-fat ketogenic diet on plasma levels of lipids, lipoproteins, and apolipoproteins in children JAMA 290 7 2003 912 920 Lauritzen et al., 2000 I. Lauritzen N. Blondeau C. Heurteaux C. Widmann G. Romey M. Lazdunski Polyunsaturated fatty acids are potent neuroprotectors EMBO J. 19 8 2000 1784 1793 Likhodii and Burnham, 2002 S.S. Likhodii W.M. Burnham Ketogenic diet: does acetone stop seizures? Med. Sci. Monit. 8 8 2002 19 24 Nylen et al., 2005 K. Nylen S. Likhodii P.A. Abdelmalik J. Clarke W.M. Burnham A comparison of the ability of a 4:1 ketogenic diet and a 6:3:1 ketogenic diet to elevate seizure thresholds in adult and young rats Epilepsia 46 8 2005 1198 1204 Rabinovitz et al., 2004 S. Rabinovitz D.I. Mostofsky S. Yehuda Anticonvulsant efficiency, behavioral performance and cortisol levels: a comparison of carbamazepine (CBZ) and a fatty acid compound (SR-3) Psychoneuroendocrinology 29 2004 113 124 Rapoport, 2003 S.I. Rapoport In vivo approaches to quantifying and imaging brain arachidonic and docosahexaenoic acid metabolism J. Pediatr. 143 4 2003 S26 S34 Schlanger et al., 2002 S. Schlanger M. Shinitzky D. Yam Diet enriched in omega-3 fatty acids alleviates convulsion symptoms in epilepsy patients Epilepsia 43 1 2002 103 104 Sprecher, 2000 H. Sprecher Metabolism of highly unsaturated n-3 and n-6 fatty acids Biochim. Biophys. Acta 1486 2–3 2000 219 231 Stafstrom, 2004 C.E. Stafstrom Dietary approaches to epilepsy treatment: old and new options on the menu Epilepsy Curr. 4 6 2004 215 222 Taha et al., 2005 A.Y. Taha M.A. Ryan S.C. Cunnane Despite transient ketosis, the classic high-fat ketogenic diet induces marked changes in fatty acid metabolism in rats Metabolism 54 9 2005 1127 1132 Vining, 1999 E.P.G. Vining Clinical efficacy of the ketogenic diet Epilepsy Res. 37 1999 181 190 Voskuyl et al., 1998 R.A. Voskuyl M. Vreugdenhil J.X. Xang A. Leaf Anticonvulsant effects of polyunsaturated fatty acids in rats, using the cortical stimulation model Eur. J. Pharmacol. 341 1998 145 152 Vreugdenhil et al., 1996 M. Vreugdenhil C. Bruehl R.A. Voskuyl J.X. Xang A. Leaf Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons PNAS 93 1996 12559 12563 Yehuda et al., 1994 S. Yehuda R.L. Carasso D.I. Mostofsky Essential fatty acid preparation (SR-3) raises seizure threshold in rats Eur. J. Pharmacol. 254 1994 193 198 Yehuda et al., 1996 S. Yehuda Y. Brandys A. Blumenfeld D.I. Mostofsky Essential fatty acid preparation reduces cholesterol and fatty acids in rat cortex Int. J. Neurosci. 86 3–4 1996 249 256 Young et al., 2000 C. Young P.W. Gean L.C. Chiou Y.Z. Shen Docosahexanoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus Synapse 37 2000 90 94