Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(1),1^7 & 2001 Harcourt Publishers Ltd doi:10.1054/plef.2001.0281, available online at http://www.idealibrary.com on
Plasma fatty acid levels in autistic children S.Vancassel,1 G. Durand,1 C. Barthe¤le¤my,2 B. Lejeune,3 J. Martineau,2 D. Guilloteau,3 C. Andre's,4 S. Chalon3 1
Laboratoire de Nutrition et Se¤curite¤ Alimentaire, INRA, domaine de Vilvert, Jouy-en-Josas; 2INSERM U316, Service de Pe¤dopsychiatrie, CHU Bretonneau,Tours; INSERM U316, Laboratoire de Biophysique Me¤dicale et Pharmaceutique, Faculte¤ de Pharmacie,Tours; 4Laboratoire de Biochimie et Biologie Mole¤culaire,Tours; Faculte¤ de Me¤decine,Tours; France 3
Summary Phospholipid fatty acids are major structural components of neuronal cell membranes, which modulate membrane fluidityand hence function.Evidence from clinical and biochemical sourceshave indicated changesin the metabolism of fattyacids in several psychiatric disorders.We examined the phospholipid fatty acids in the plasma of a population of autistic subjects compared to mentally retarded controls. Our results showed a marked reduction in the levels of 22 : 6n-3 (23%) in the autistic subjects, resultingin significantly lower levels oftotal (n-3) polyunsaturated fattyacids (PUFA) (20%), without significant reduction in the (n-6) PUFA series, and consequently a significant increase in the (n-6)/(n-3) ratio (25%).These variations are discussed in terms of potential differences in PUFA dietary intake, metabolism, or incorporation into cellular membranes between the two groups of subjects.These results open up interesting perspectives for the investigation of new biological indices in autism. Moreover, this might have new therapeutic implications in terms of child nutrition. & 2001Harcourt Publishers Ltd
INTRODUCTION The human brain is the second only to adipose tissue in containing the highest concentration of lipids. Cerebral lipids are almost all structural and contribute to the composition of neuronal membranous phospholipids (PL). In the central nervous system (CNS), PL are exceptionally rich in highly unsaturated fatty acids (FA). Thus, about 20% of the dry weight of the brain consists of polyunsaturated fatty acids (PUFA) containing at least 20 atoms of carbon, such as arachidonic acid (20 : 4n-6, AA) and docosahexaenoic acid (22 : 6n-3, DHA). The mammalian body does not posses the enzymatic system required to synthesize these PUFA and they are directly provided by diet or derived from essential dietary precursors, linoleic acid (18 : 2n-6) and a-linolenic acid (18 : 3n-3), respectively.
Received 26 February 2001 Accepted 1June 2001 Correspondence to: S.Vancassel, Laboratoire de Nutrition et Se¤curite¤ Alimentaire, INRA, domaine de Vilvert, 78352 Jouy-en-Josas cedex; Phone: 01 34 65 28 36; Fax: 0134 65 23 11; E-mail: [email protected]
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A growing body of studies has demonstrated the involvement of PUFA in the regulation of many biochemical events such as neurotransmitter release and uptake, receptor function in the CNS1 and enzymatic processes.2 Moreover, many studies have reported abnormalities in lipid metabolism in several psychiatric disorders.3 In particular, evidence has been accumulated that PL metabolism in different biological tissues may be disturbed in schizophrenia. Several authors have reported significantly reduced levels of linoleic acid, AA and DHA in red blood cell membranes from different populations of schizophrenic patients, compared to healthy control subjects.4–6 PUFA studies have been since performed in plasma, thrombocytes, cultured fibroblasts and brains from patients suffering from schizophrenia in different countries and the results have recently been reviewed by Fenton et al.7 An impairment of PUFA metabolism has also been postulated to occur in children suffering from attention-deficit hyperactivity disorder8 (ADHD). This term is used to describe children, particularly boys, who are inattentive, impulsive and hyperactive.9 Mitchell et al.10,11 showed that the proportions of DHA and AA were significantly lower in the plasma of ADHD children. More recently, Stevens et al.12 and Burgess et al.13 Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(1), 1^7
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reported reduced concentrations of AA, DHA and 20 : 5n3 (EPA) in plasma polar lipids and also in red blood cell total lipids in a large cohort of subjects with ADHD as compared to controls. Approximately 40% of subjects with ADHD had a greater frequency of symptoms indicative of essential FA deficiency (increased thirst, frequent urination, high fluid consumption, dry hair), compared to 9% of subjects without ADHD.14 Furthermore, it has been reported that changes in the dietary intake of (n-3) and/or (n-6) PUFA, which modify amounts and composition of membrane FA, can have significant effects upon some symptoms of schizophrenia and ADHD.7,15–18 Thus, a potential therapeutic benefit of PUFA dietary supplementation has been postulated in these psychiatric disorders. A recent study has shown that abnormalities in plasma PUFA levels may provide an insight into the development of Rett syndrome.19 This syndrome is a serious neurological disorder that occurs only in females, and is characterized by the overall deceleration of psychomotor development, gradual loss of locomotion ability and disordered communication with the environment.20 The results of the study showed that very long chain saturated FA levels (22 : 0, 24 : 0, 26 : 0) were lower in the serum of girls with Rett syndrome than the reference range for healthy children. The impairment of communication abilities that is observed in children suffering from Rett syndrome is one of the major behavioral traits described in another prevalent infantile psychiatric disease, autism. Autism was first described by Kanner in 1943 but it has only recently reached official diagnostic status in the DSM classification system (DSM III-R21). Autism is an early and pervasive developmental disorder affecting communicative and also social, cognitive and imaginative development. It is a behaviorally defined syndrome with a complex etiology, and little is known about its pathological basis. Genetic vulnerability, environmental factors and neurochemical factors have been considered (see22 for review). The aim of this study was to measure the PL FA profile in plasma of autistic children compared to a population with mental retardation, but without autistic disorder.
METHODS AND MATERIALS
Patients All patients were attending the Child Psychiatry Day Care Unit of the Centre Hospitalier Re´gional Universitaire of Tours, France. The study population consisted of 15 children with autism (4 girls, 11 boys) aged between 3 and 17 years (mean age 8 years 4 months), and 18 mentally retarded children (5 girls, 13 boys) aged between 1 and 19 years (mean age 8 years 8 months). The Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(1), 1^7
diagnosis of autism and mental retardation was based on the DSM III-R and DSM-IV criteria.9,21 The patients of both groups received a complete diagnostic workup including medical, neurological, psychiatric and psychological evaluations, according to processes already described.23 The study was performed in accordance with the requirements of the Ethics Committee of the institution. The parents of all subjects gave informed written consent to the participation of their children for measurement of plasma biological parameters. All children were in excellent physical health, and none had a history of endocrine or systemic disease. Children are day-care patients; they all received an identical meal at 12.00 hours in the hospital.
Blood sampling Blood samples were collected on the second day (no fasting state), between 10.00 and 12.00 hours by venipuncture and transferred into Vacutainers tubes containing EDTA and mixed by gentle inversion. The blood was centrifuged and the plasma separated and frozen at 7808C until use.
Analysis of fatty acids Total lipids were extracted from plasma with a mixture of chloroform/methanol/water (1 : 2 : 0.8) according to the method of Bligh and Dyer24 and 0.02% of butyl hydroxytoluene (BHT) was added to prevent oxidation of the unsaturated FA. The extract was filtered through sodium sulfate, evaporated to dryness, weighed and taken up in 0.5 ml of a mixture of chloroform/methanol (50 : 50). The total lipid fractions were then separated by thin-layer chromatography on silica gel plates in a saturated environment of hexane/ether/formic acid (80 : 20 : 1) plus 0.02% BHT. After drying, plots were revealed by rhodamine pulverization. The PL fraction was scraped off and methylated using 10% BF3 in methanol.25 The resulting methyl esters of FA were separated and measured using a gas chromatograph ( JC 8000; ThermoQuest, Les Ulis, France) equipped with an on-column injector and capillary column (50 m; diameter, 0.32 mm; CP wax 52 CB; Chrompack, Les Ulis, France). Components were identified by their equivalent chain lengths in comparison with standards, the peaks being integrated by Nelson Software (SRA Instruments, Marcy l’Etoile, France).
Statistical analysis Because a number of measured variables did not come from normal distributions, they were analyzed using a non-parametric test. Results between mentally retarded and autistic subjects were compared using the Mann–Whitney test. & 2001Harcourt Publishers Ltd
Plasma fatty acid levels in autistic children 3
RESULTS The basic height and weight data of autistic and mentally retarded subjects are presented in Figures 1A & 1B, respectively. The means of height for autistic and mentally retarded subjects were 122.6 cm and 122.4 cm, respectively; the means of weight were 28.3 Kg and 26.8 Kg, respectively. Statistical analysis showed no difference between the two populations studied. The FA levels of total plasma PL measured in 15 autistic subjects and 18 mentally retarded subjects are shown in Table 1. The results showed that total saturated FA levels were 6% higher in autistic than in mentally retarded
Table 1 Fatty acid composition of plasma total phospholipids of15 autistic and18 mentally retarded subjects Fatty Acids
SFA MUFA 18 : 2n-6 20 : 4n-6 S (n-6) PUFA 18 : 3n-3 22 : 6n-3 S (n-3) PUFA S (n-6)þ(n-3) (n-6)/(n-3)
54.0+4.2 18.0+2.4 19.1+3.5 4.5+1.0 25.8+4.1 0.3+0.1 1.1+0.2 2.0+0.4 27.8+3.9 13.6+4.4
Mentally retarded subjects P = 0.086 P = 0.051 40.1 40.1 40.1 40.1 P = 0.080 P = 0.032 40.1 P = 0.039
51.1+3.1 19.9+3.1 18.4+4.4 5.5+2.1 26.1+4.9 0.3+0.1 1.4+0.7 2.5+0.8 28.6+5.3 11.0+3.1
Values are expressed as mg of fatty acids per100 g of fatty acids in total phospholipids (mean+SD). Statistical differences between the two groups of subjects were tested using the non-parametric Mann^Whitney test. Abreviations used: SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.
subjects. A significant 10% reduction in total monounsaturated FA levels was observed (19.9+3.1% FA in retarded subjects vs 17.9+2.4% FA in autistic subjects). The levels of plasma linoleic acid (18 : 2n-6), which is the precursor of the (n-6) series, were identical for the total PL of both groups of subjects. The results in autistic children showed a non-significant tendency to reduction (18%) in the levels of arachidonic acid (20 : 4n-6), which represents a major intermediate component in the long-chain derivatives of the (n-6) series. However, no statistical difference in the total (n-6) PUFA levels was observed between the autistic and the mentally retarded groups. The major variations observed in PUFA levels between the two groups of subjects involved the (n-3) PUFA series. The results showed that a-linolenic acid levels (18 : 3n-3) were identical in both groups of subjects. However, DHA levels (22 : 6n-3) were reduced by 23% in autistic children (1.1+0.2% FA), compared to mentally retarded subjects (1.4+0.7% FA). This reduction resulted in strong and significantly lower levels of total (n-3) PUFA (20%) in autistic children. Despite the significant decrease in total (n-3) PUFA levels and the moderate reduction in AA levels in autistic children, the total (n-6 þ n-3) PUFA levels were not statistically different between autistic and mentally retarded subjects. However, the differences in PUFA levels resulted in a significant increase in the (n-6)/(n-3) ratio values in autistic subjects (11.0+3.1 in mentally retarded subjects vs 13.6+4.4 in autistic subjects, 25%). Fig. 1 Basic height (A) and Weight data (B) according to age of autistic subjects (*; n = 15), and mentally retarded subjects (&; n = 18).Values are expressed in centimeters (cm) for height, in kilograms (Kg) for weight and in years for age. Statistical differences were tested using the non-parametric Mann^Whitney test.
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DISCUSSION Autism is an highly prevalent syndrome with a complex etiology. Extensive research has led to the hypothesis that Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(1), 1^7
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autism is a relating deficiency due to a developmental disorder of the central nervous system (see26 for review of recent advances). Findings from studies of major neurotransmitter systems (serotonin, catecholamines) strongly suggest that neurochemical factors could play a major role in autism.23,27,28 In particular, the serotonin transporter gene seems to be a primary candidate to mediate the genetic susceptibility to this disorder.29,30 Genetic and early environmental insults to brain development are also etiological determinants of the disorder. Thus, on the basis of family studies, many cases of autism are associated with fragility of chromosome X.31,32 Other studies have shown an association between autism and the locus containing the HRAS gene, which is involved in brain development.33,34 However, this list of parameters believed to be involved in the etiology of autism is not exhaustive and many other areas of research are in progress to understand this heterogeneous disorder. Modifications of lipid metabolism have been described in other psychiatric illnesses, in particular in schizophrenia7,17,35 and in ADHD.12–14 These syndromes and autism shared some common behavioral traits, in particular impairment of communication abilities.36 Moreover, polydipsia, which is considered as one of symptoms of essential FA deficiency,37 is described in ADHD14 and in autism.38 Evidence supporting the possibility that (n-3) PUFA may be of etiological importance in depression has recently been reviewed and therapeutic trials of (n-3) PUFA supplementation are in progress. A significant decrease in total (n-3) PUFA levels, and particularly DHA, has been reported in serum PL and cholesteryl esters, and in erythrocyte membrane PL from depressive patients.39–41 Furthermore, a significant negative correlation was found between dietary (n-3) PUFA intake and the severity of depression.42 Finally, an important role of (n-3) PUFA is now supported by a substantial body of direct and indirect evidence in the pathology and treatment of bipolar disorder (manic depressive illness), which is a frequent psychiatric disease.43 In the light of findings showing some impairment in lipid metabolism in patients suffering from psychiatric disorders reviewed above, we report the plasma FA profile of autistic subjects compared to a mentally retarded group. The latter was considered as the control group since subjects included failed to satisfy the diagnosis of autism, according to the DSM III-R.21 Thus, autism is the statistical independent variable which differed between the two groups of subjects. Our results showed that PUFA levels were different in the plasma of autistic children, compared to the mentally retarded group. More particularly, AA (20 : 4n-6) but especially DHA (22 : 6n-3) and total (n-3) PUFA levels were reduced by about 20% in the total PL of autistic children. A tendency to reduced levels of 20 : 5n-3 (EPA) Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(1), 1^7
was also observed. However, the levels measured in plasma were very low and the variations that we observed must be considered with caution (data not shown). A reduction in AA and DHA levels has been observed in the serum of children with ADHD12,13 and in the membranes of red blood cells from schizophrenic patients.4,5 Similar abnormalities have been observed in postmortem studies of membrane PL from the frontal cortex of drug-treated patients suffering of schizophrenia.35 The reasons for the lower concentrations of (n-6) and (n-3) PUFA are not well understood. Many hypotheses have been proposed: firstly, a primary deficiency may occur because of insufficient intake of PUFA or precursors. We did not investigate dietary habits in the subjects studied. However, values of weight and height showed no statistical difference, according to age and group of subjects. This suggests that eating habits did not induce gross difference between the two groups. Moreover, in our study, the plasma precursor levels of both PUFA series, i.e. 18 : n-6 and 18 : 3n-3, were not different between autistic and mentally retarded subjects. There was no indication in the plasma of autistic children of the presence of Mead acid (20 : 3n-9), which is elevated in essential FA deficiency (P = 0.19, data not shown). However, plasma fatty acid levels are sensitive to short-term fluctuations in diet and it would be very useful to correlate the variations in the PUFA levels observed between the two groups to dietary habits of the patients. Secondly, a poorer ability to convert 18-carbon FA to their longer and more highly unsaturated derivatives could be considered. Possible sites of this inefficiency include desaturase steps. A site linked to autism has been located on chromosome 11q22-23, in the viciny of the gene for delta-6 desaturase, which is the enzyme first involved in the production of PUFA-long chain derivatives of both (n-3) and (n-6) series.23,44 Because of their proximity on the chromosome, a concomitant impairment expression of these genes, or of the product of these genes, could then represent a causative factor in the lipid differences observed in autism. However, not all of the results were consistent with this reasoning since 22 : 5n-3 levels were not different in both groups. Another possible cause for the lower concentrations of 20 : 4n-6, 20 : 5n-3 and 22 : 6n-3 in the total plasma of autistic children might involve enhanced catabolism of the PUFA inserted in PL. This phenomenon is currently being proposed in schizophrenia and it has led to the concept of the ‘membrane hypothesis of schizophrenia’.45,46 This hypothesis is based on the fact that in schizophrenic patients, the activity of phospholipase A2 (PLA2) has been reported to be increased in plasma, serum and platelet membranes.47–49 Abnormalities in the & 2001Harcourt Publishers Ltd
Plasma fatty acid levels in autistic children 5
gene for PLA2 on chromosome 1 have also been described.50,51 PLA2 is an enzyme which acts at the sn-2 position of the PL and redeems PUFA, principally AA and DHA. Increased PLA2 activity may therefore be at the origin of an accelerated breakdown of PUFA and thus a lower PUFA content inserted in membranous PL. We could hypothesize that this phenomenon also occurs in autism. Moreover, genetic sites linked to autism on chromosome 8q22 are in the proximity of the gene for secretory soluble PLA252 (8q24). A related genetic modification in the expression and/or in the activity of PLA2 in plasma could therefore be considered in autistic children. Exploration of the activity and gene expression of PLA2 would thus be of great value. Finally, an abnormality in the incorporation of PUFA into membrane PL should be considered, as a lower incorporation of AA into platelets of schizophrenic patients has been shown.53 Such an abnormality could be the result of a variety of biochemical events controlling the incorporation of FA into membranes, such as enzyme reactions. This could provide another explanation for the reduced levels of (n-6) and (n-3) PUFA observed in the total PL plasma of autistic subjects. However, all these hypotheses are not exclusive and each should be considered as a possible mechanism for the variations that we report in the present study. All are worth investigating. Moreover, the examination of lipid profiles in erythrocyte membranes should more closely reflect the FA composition in neuronal brain membranes than measurements in plasma. However, the necessity of caution needs to be emphasized in view of the lack of controlled data, and it seems imperative to replicate this study under totally controlled conditions, including data from the dietary habits of the tested subjects. Indeed, it must be kept in mind that the mentally retarded subjects do not represent an absolute control group since they may be deviant with regard to their fatty acid levels. Then, Decsi and Kotetzko described in 199454 the fatty acid composition of plasma lipid classes in healthy subjects from birth to young adulthood. The authors suggested that their data may be used as reference values for healthy children consuming a diet typical for central Europe. In comparison to this study, we found variations in the fatty acid levels measured in total PL of mental retarded and autistic patients. Particularly, total (n-3) PUFA levels were 50% lower whereas total (n-6) PUFA levels were two-fold higher in the subjects we studied than in the corresponding age-class children studied by Decsi and Koletzko. Moilanen55 and Stevens et al.12 reported the fatty acid levels in serum phospholipids of control subjects aged 6 to 12 years, from Finland and North Indiana, respectively. These values are quite close to the means reported by Decsi and Koletzko54 in the same range of age. In our work, it seems, in the light of & 2001Harcourt Publishers Ltd
these data, that the mentally retarded subjects presented fatty acid levels slightly lower than the values of the literature, especially for 22 : 6n-3 and total (n-3) PUFA. However, despite these differences, total (n-3) levels remained significantly lower in the autistic subjects than in those suffering from mental retardation. Moreover, these studies dealt with patients from several countries and with different dietary habits as compared to the French subjects involved in our work. This represents another critical point which could explain in the differences between the data. Difference in the fasting state of the patients when blood was collected, in the storage of the plasma samples (7208C or 7808C in our study), or to difference between the methods used for lipid analysis could also be involved. In conclusion, this study showed for the first time that the levels of (n-3) PUFA and to a lesser extent AA were reduced in total PL, measured in the plasma of a population of autistic patients compared to mentally retarded ones. Similar variations have been described in other psychiatric diseases, such as schizophrenia, ADHD, major depression and bipolar disorder as compared to control subjects. Different hypotheses could be proposed to explain these changes. These results open up very interesting perspectives for the investigation of a new biological index in autism. Moreover, this could represent an additional approach to the treatment of autism, through dietary supplementation associated with the pharmacological strategies already being used.
ACKNOWLEDGMENTS We thank Jean-Paul Macaire and Alain Linard for their technical assistance, and Doreen Raine for editorial assistance. This work was supported by INRA, INSERM U316, INSERM Network 4R002B, and Fundation France Te´le´com.
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