omega-3 polyunsaturated fatty acids ( -3 pufas ) have received an increasing attention by the scientific community and the society as well due to their status of natural molecules with a number of claimed positive effects on a large variety of conditions , in particular in the prevention of cardiovascular disease . most common -3 pufas are eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) found in fish oil and -linolenic acid c18:3 n-3 , derived from plants . the hypotriglyceridemic effect is the best defined metabolic action of -3 pufas , with a mechanism likely to be related to activation of peroxisome proliferator - activated receptors . a host of other potential beneficial effects of -3 pufas has been suggested . besides the demonstrated or claimed beneficial cardiovascular effects ( reduction of susceptibility to ventricular arrhythmia ; antithrombogenic and antioxidant effect ; retardation of the atherosclerotic plaque growth by reduced expression of adhesion molecules and platelet - derived growth factor ; promotion of endothelial relaxation by induction of nitric - oxide production ; and mild hypotensive effect ) , a more general anti - inflammatory effect , either direct or indirect has been reported [ 68 ] . however , the molecular mechanisms underlying the -3 pufas effects in the regulation of the inflammatory process are still poorly understood and an emerging major field of research [ 911 ] . several factors may indeed contribute to limit our understanding in this specific area of the lipid metabolism , including the type of pufas , formulation , dose , duration , age and underlying condition of the target subjects , and however , a relatively recent report in adult smoker subjects   demonstrates that -3 pufas are able to affect plasma protein expression by regulating acute phase response ( apr ) . rett syndrome ( rtt ) is a devastating neurodevelopmental disorder mainly caused by sporadic mutations in the gene encoding the methyl - cpg binding protein 2 ( mecp2 ) protein . rtt affects almost exclusively females with an average frequency of 1  :  10,000 female live births and is considered to be the second commonest cause of severe cognitive impairment in this gender . rtt has been formerly included in the autism spectrum disorders ( asds ) , although evident differences with autism exist   and is no longer classified in the asds group . nevertheless , transient autistic features are always present in the rtt natural history . therefore , this relatively rare disease actually offers a good opportunity to test the effects of -3 pufas in an objective way in an autistic condition , given that ( 1 ) a persistent redox imbalance has been demonstrated ; ( 2 ) -3 pufas have been suggested to reduce phenotypical severity and improve the redox balance in supplemented patients at several stages of their clinical history ; ( 3 ) an impaired cholesterol metabolism has been very recently demonstrated in a mecp2-null mouse model of rtt , with statin treatment leading to improvement of motor symptoms and conferring increased longevity ; and ( 4 ) an unexplained hypercholesterolemia has been reported by our group in rtt patients . since only one study has examined the plasma proteome of rtt , here we hypothesized that -3 pufas may have an impact on the rtt plasma proteome by modulating the apr . the study included a total of 24 female patients with a clinical diagnosis of typical rtt ( mean age : 14.4  8.0 years , range 433 years ) with demonstrated mecp2 gene mutations [ i.e. , t158  m ( n = 5 ) , c - terminal deletions ( n = 4 ) , r255x ( n = 4 ) , r270x ( n = 3 ) , r133c ( n = 2 ) , early truncating mutations ( n = 1 ) , large deletions ( n = 1 ) other mutations ( n = 4 ) ] . clinical stages distribution was stage i ( n = 4 ) , stage ii ( n = 6 ) , stage iii ( n = 7 ) , and stage iv ( n = 7 ) . rtt diagnosis and inclusion / exclusion criteria were based on the recently revised rtt nomenclature consensus [ 19 , 20 ] . all the patients were admitted to the rett syndrome national reference centre of the university hospital of the azienda ospedaliera universitaria senese ( head : professor j. hayek ) . blood samplings in the patients ' group were performed during the routine follow - up study at hospital admission , while the samples from the control group were carried out during routine health checks , sports , or blood donations , obtained during the periodic clinical checks . the healthy control subjects ( n = 24 ) were gender ( given that over 98% of rtt patients are females , we selected a female control group ) and age matched ( mean age : 14.4  8.2 years , range 4.133 years ) . the study was conducted with the approval by the institutional review board and all informed consents were obtained from either the parents or the legal tutors of the enrolled patients . the aim of the present study was to assess the effects on the plasma proteome of a supplementation already tested to be effective in the clinical setting [ 2123 ] . therefore , as a consequence we did not include a placebo arm for ethical reasons and limited the study to three subjects population , that is , healthy controls ,  untreated  rett syndrome , and -3 pufas supplemented rett syndrome . administered -3 pufas were in the form of fish oil ( norwegian fish oil as , trondheim , norway , product number ho320 - 6 ; italian importer : transforma as italia , forl , italy ; italian ministry registration code : 10 43863-y ) at a dose corresponding to dha , 71.9  13.9  mg / kg  b.w./day and epa , 115.5  22.4  mg / kg  b.w./day , with a total -3 pufas of 242.4  47.1  mg / kg  b.w./day . the dose used in this specific rtt girls cohort is likely to be 5 to 6 times higher than the standard one , which is typically 2  g per day in adult subjects . the rationale for it is contained in a prior paper , in which we proposed a very high dose in rett syndrome . after several attempts , the final dose per kg / day was found empirically in the clinical setting . all samplings from rtt patients and healthy controls were carried out around 8 am after overnight fasting . blood was collected in heparinized tubes and all manipulations were carried out within 2  h after sample collection . the blood samples were centrifuged at 2400  g for 15  min at 4c ; the platelet poor plasma was saved ; and the buffy coat was removed by aspiration . 2-de was performed according to grg et al . , and samples containing 60  g of protein as determined by bradford   were denatured with a solution containing 10% of sodium dodecyl sulfate ( sds ) and 2.3% of dithiothreitol ( dtt ) heated to 95c for 5  min . the sample was then combined solubilizing buffer composed by 8  m urea , 2% of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate ( chaps ) , 0.3% dtt , and 2% of immobilized ph gradient ( ipg ) buffer and loaded into 18  cm ipg strips 310 non linear on an ettan ipgphor ( ge healthcare ) apparatus system and rehydrated for 7  h. isoelectric focusing ( ief ) was carried out for a total of 32  kv  h. the strips were first equilibrated with a buffer containing 50  mm tris - hcl , ph 8.8 , 6  m urea , 2%  w / v sds , 30%  v / v glycerol , and 1%  w / v dtt for 15  min ; then they were equilibrated again with the same buffer described above , except it contained 4%  w / v iodoacetamide instead of dtt . the second dimension was performed on an ettan daltsix electrophoresis system ( ge healthcare ) . ipg strips were embedded at the top of a 1.5  mm thick vertical polyacrylamide gradient gel ( 816%  t ) using 0.5%  w / v agarose and run at a constant current of 40  ma / gel at 20c . images of gels were analyzed using imagemaster 2d platinum v7.0 software ( ge healthcare ) . the reference gel for each group ( i.e. , healthy controls , untreated rtt , and rtt after -3 supplementation ) was defined and used for the comparative analyses . the background was subtracted from all gels using the average - on - boundary method . spot volume was expressed as a ratio of the total protein percentage volume ( % v ) detected from the entire gel to minimize differences between gels ( gel normalization ) , for pooling them . only spots appearing in all gels of the same group were matched with those of the reference gel . after mass spectrometry compatible silver staining , a spot - picking list was generated and exported to ettan spot picker ( ge healthcare ) . the spots were excised and delivered into 96-well microplates where they were destained and dehydrated with acetonitrile ( acn ) for subsequent rehydration with trypsin solution . each protein spot digest ( 0.75  ml ) was spotted into the maldi instrument target and allowed to dry . then , 0.75  ml of the instrument matrix solution ( saturated solution of -cyano-4-hydroxycinnamic acid in 50% acn and 0.5%  v / v trifluoroacetic acid ) was applied to dried samples and dried again . mass spectra were obtained , as described , using an ultraflextreme maldi - tof / tof ( bruker corporation , billerica , ma , usa ) . after tryptic peptide mass acquisition , mass fingerprint searching was carried out in swiss - prot / trembl and ncbinr databases using mascot ( matrix science , london , uk , http://www.matrixscience.com/ ) . all variables were tested for normal distribution ( d'agostino - pearson test ) and data were presented as median and interquartile range unless otherwise . statistical analysis for protein expressed differently in the groups was carried out using student 's t - test and one - way anova test . unmatched spots or spots with significantly different % v were considered  differently expressed  in the groups . comparisons between differently expressed proteins of untreated rtt and rtt after -3 supplementation were evaluated using either mann - whitney rank sum test or kruskal - wallis test . the effects of small population sizes on possible type i ( )/type ii (  ) errors in the data interpretation were examined using a sampling size algorithm . a two - sided p < 0.05 was considered to indicate statistical significance , the medcalc version 12.1.4 statistical software package ( medcalc software , mariakerke , belgium ) was used . the expression of 16 proteins , mainly related to the apr , was found to be changed at the baseline level in the untreated rtt patients . as compared to healthy controls , the whole rtt group showed significant increase in 10 protein spots [ i.e. , complement factor b ( cfab ) , fibrinogen alpha chain ( fiba ) , serum albumin ( albu , spots number 3 , number 7 c terminal fragment and number 14 n terminal fragment ) alpha-1-antitrypsin ( a1at , spots number 4 and number 5 ) , haptoglobin ( hpt , spots number 9 and number 15 ) , and transthyretin ( tthy spot number 11 ) ] and decrease in 6 protein spots [ i.e. , vitamin d - binding protein ( vtdb ) , apolipoprotein a4 ( apoa4 ) , clusterin ( clus ) , apolipoprotein a1 ( apoa1 ) , retinol - binding protein 4 ( ret4 ) , and transthyretin ( tthy spot number 16 ) ] ( table 1 and figures 1(a ) , 1(b ) , and 2(a ) ) . a full list of the known biological functions for the identified plasma proteins is shown in table 2 . following -3 pufas supplementation , the expression of 10 out of 16 ( 62% ) proteins was found to be comparable to that of control subjects ( figure 2(c ) ) . in particular , after -3 pufas , plasma proteins expression levels were comparable to those of the control population , with the exception of persistent overexpression of a1at ( spot number 4 ) , vtdb ( spot number 6 ) , albu c terminal fragment ( spot number 7 ) , and hpt ( spot number 15 ) and the persistent underexpression of fiba ( spot number 2 ) and albu ( spot number 3 ) . by comparing the plasma protein profile of treated rtt with the one before treatment , significant decreases in 9 protein spots previously overexpressed before treatment , including cfab , fiba , albu ( spots number 3 and number 14 ) , a1at , hpt , and tthy ( spot number 11 ) were reported , whereas significant increases were observed for 5 protein spots that were underexpressed before the -3 pufas supplementation and included vtdb , apoa4 , clus , apoa1 , and ret4 . after -3 pufas supplementation , the levels albu ( spot number 7 ) and tthy ( spot number 16 ) remained unchanged compared to untreated rtt ( table 1 and figures 1(a ) , 1(c ) , and 2(b ) ) . the mechanism of action of -3 pufas is a major area of research , which has led in the last two decades to the discovery of protectins , resolvins and maresins , and all lipid mediators involved in the active resolution of the inflammatory process . our results indicate that -3 pufas are able to modulate plasma protein expression in rtt , having a major impact on the modulation of the apr , with a partial ( approximately 62% ) rescue of the protein changes observed at baseline . these findings well fit with the known anti - inflammatory properties of this fatty acids family . the apr is a highly conserved adaptive mechanism   and is a core part of the innate immune response . profound changes occur in the plasma proteome as the consequence of apr , reflecting a highly regulated process as part of a more generalized reprogramming of signaling events under the influence of cytokines . the involved proteins ( i.e. , apr proteins ) are known to be predominantly synthesized in the liver , and the signaling events result in either an upregulation or downregulation of apr proteins . more than 200 plasma proteins are known to vary in the apr , some of which may control tissue damage and participate in tissue repair , although their role still remains speculative . omega-3 pufas have multiple health benefits mediated at least in part by their anti - inflammatory actions . a recent paper   demonstrated that epa and dha are competitors for arachidonic acid ( aa ) in binding to the 5-lipooxygenase enzyme , since -3 pufas displace aa in membrane phospholipids , reducing the production of aa - derived eicosanoids ( i.e. , prostaglandin e2 ) while increasing those generated from -3 pufas activation . as reported in several trials , -3 pufas supplementation is able to reduce plasma and urine levels of eicosanoids such as leukotriene e4 [ 3235 ] . besides the anti - inflammatory effects based on the interruption of the aa cascade , -3 pufas impart their anti - inflammatory effects via a decreased activation of nuclear factor - kappa b ( nf - kb ) , a potent inducer of the production of proinflammatory cytokines , including interleukin-6 and tumor necrosis factor-. overall , enrichment of cellular membranes with -3 pufas disrupts dimerization and recruitment of toll - like receptor-4 , which might contribute to anti - inflammatory effects by downregulation of nf - kb activation . further evidence demonstrates that -3 pufas can repress lipogenesis and increase generation of resolvins and protectins , ultimately leading to reduced inflammation . finally , the effects of epa and dha include their ability to increase secretion of adiponectin , an anti - inflammatory adipokine . when considering the effects of -3 pufas on the cellular function , their direct modulation of g - protein - coupled receptor is noteworthy and might contribute to the anti - inflammatory properties . at the same time , -3 pufas show a direct regulation on gene expression vianuclear receptors and transcription factors , which are in turn modulated by cytoplasmic lipid binding proteins transporting these fatty acids into the nucleus . regulation of -3 pufas on gene expression could explain the altered protein expression that we reported on the rtt plasma proteome and is in line with previous findings in adult smokers following a short course ( i.e. , 5 weeks ) of -3 pufas enriched diet . in this latter study , proteins related to the antioxidant , anti - inflammatory , and antiatherosclerotic properties of hdl were upregulated , in contrast with down - regulation of complement activation and apr proteins . in particular , in our study major changes for proteins involved in apr ( cfab , fiba , albu ( spot number 3 c terminal fragment and spot number 14 n terminal fragment ) , a1at ( spots number 4 and number 5 ) and hpt ( spots number 9 and number 15 ) ) , immunity , unfolded protein response ( clus ) , blood coagulation ( fiba ) , transport pathways ( tthy ( spots number 11 and number 16 ) , ret4 and vtdb ) , and lipid metabolism ( apoa4 and apoa 1 ) were evidenced at the proteome analysis of plasma samples from a population of patients with typical rtt at different stages and harboring a variety of mecp2 gene mutations . overexpressed plasma proteins in the unsupplemented rtt patients were mainly related to apr and underexpressed spots corresponded to negative apr proteins , unfolded protein response , and proteins involved in the lipid metabolism . our data indicated that -3 pufas almost completely rescued the apr detected at the baseline . the molecular mechanisms of the -3 pufas action remain only partially understood and include changes in membrane structures and gene expression , direct interactions with ion channels , and alterations in eicosanoid biosynthesis . epa and dha , key -3 pufas , have been reported to compete with aa for the conversion by cytochrome p450 enzymes , thus resulting in the formation of alternative , physiologically active , metabolites , given that cytochrome p450 enzymes are known to efficiently convert epa and dha to epoxy and hydroxy metabolites ( 17,18-epoxyeicosatetraenoic , and 19,20-epoxydocosapentaenoic acid , resp . ) , which could likely mediate some of their beneficial effects . the present study strongly suggests that the main beneficial action of -3 pufas ( or their secondary metabolites ) in rtt is the modulation of an unrecognized subclinical inflammatory status and fits well with the known anti - inflammatory properties of this fatty acids family and suggesting that the super - family of the multiple actions attributed to -3 pufas could be attributable to the apr modulation . a subclinical inflammatory state has been previously reported in autistic subjects with significant changes in inflammation - related proteins . overall , our findings of a subclinical apr in rtt that can be modulated by a dietary supplementation of -3 pufas provide new insights into the role of inflammation in autistic disorders and support the role of -3 pufas as key nutraceuticals .
the mechanism of action of omega-3 polyunsaturated fatty acids ( -3 pufas ) is only partially known . 
 prior reports suggest a partial rescue of clinical symptoms and oxidative stress ( os ) alterations following -3 pufas supplementation in patients with rett syndrome ( rtt ) , a devastating neurodevelopmental disorder with transient autistic features , affecting almost exclusively females and mainly caused by sporadic mutations in the gene encoding the methyl cpg binding protein 2 ( mecp2 ) protein . here , we tested the hypothesis that -3 pufas may modify the plasma proteome profile in typical rtt patients with mecp2 mutations and classic phenotype . 
 a total of 24 rtt girls at different clinical stages were supplemented with -3 pufas as fish oil for 12 months and compared to matched healthy controls . 
 the expression of 16 proteins , mainly related to acute phase response ( apr ) , was changed at the baseline in the untreated patients . following -3 pufas supplementation , 
 the detected apr was partially rescued , with the expression of 10 out of 16 ( 62% ) proteins being normalized . 
 -3 pufas have a major impact on the modulation of the apr in rtt , thus providing new insights into the role of inflammation in autistic disorders and paving the way for novel therapeutic strategies .