Pablo Perez-Martinez

Jose Maria Garcia-Quintana

Elena M Yubero-Serrano

Inmaculada Tasset-Cuevas

Isaac Tunez

Antonio Garcia-Rios

Javier Delgado-Lista

Carmen Marin

Francisco Perez-Jimenez

Helen M Roche

Jose Lopez-Miranda

Reina

Postprandial oxidative stress is modified by dietary fat: evidences from a human intervention study

Keywords: oxidative stress, metabolic syndrome, dietary fat, postprandial lipemia, LIPGENE study

Previous evidences support the concept that increased oxidative stress may play an important role in the metabolic syndrome (MetS)-related manifestations. Dietary fat quality has been proposed to be critical in oxidative stress and the pathogenesis of the MetS. We investigated whether oxidative stress parameters are affected by diets with different fat quantity and quality during the postprandial state in subjects with the MetS. Patients were randomly assigned to one of four isoenergetic diets distinct in fat quantity and quality: high-saturated fatty acid (HSFA); high-monounsaturated fatty acid (HMUFA) and two low-fat, high complex carbohydrate (diets, supplemented with 1.24 g/day of long chain n-3 polyunsaturated fatty acid (LFHCC n-3) or with placebo (LFHCC) for 12 weeks. HMUFA diet enhanced postprandial reduced glutathione levels and the glutathione/oxidized glutathione ratio, compared to the other three diets. In addition, after the HMUFA diet postprandial lipid peroxides levels, protein carbonyls concentration, superoxide dismutase activity and plasma hydrogen peroxide plasma levels were lower compared to subjects adhering to the HSFA diet. Both LFHCC diets had an intermediate effect relative to the HMUFA and HSFA diets. Our data support the notion that the HMUFA diet improves postprandial oxidative stress in patients with the MetS. These findings suggest that the postprandial state is important for understanding possible cardio-protective effects associated with monounsaturated dietary fat particularly in subjects with the MetS (NCT00429195)

Introduction

The metabolic syndrome (MetS) refers to the aggregation of cardiometabolic risk factors, including insulin resistance, dyslipidemia, hyperglycemia, and hypertension [START_REF] Reaven | Metabolic syndrome: pathophysiology and implications for management of cardiovascular disease[END_REF]. It is often characterized by oxidative stress, a condition in which an imbalance results between the production and inactivation of reactive oxygen species (ROS) [START_REF] Roberts | Oxidative stress and metabolic syndrome[END_REF]. Previous studies support the concept that increased oxidative stress may play an important role in MetS related manifestations, including atherosclerosis, hypertension and type 2 diabetes mellitus (T2DM) [START_REF] Ceriello | Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited[END_REF].

Patients with MetS have elevated oxidative damage, as evidenced by decreased antioxidant protection, and increased lipid peroxidation, protein carbonyls, and hydrogen peroxide (H 2 O 2 ) plasma levels [START_REF] Armutcu | Oxidative stress markers, Creactive protein and heat shock protein 70 levels in subjects with metabolic syndrome[END_REF]. Nevertheless, although some of the constituent characteristics of the MetS are known to share common pathogenic mechanisms of damage, the impact of hereditary predisposition and the role of environment and dietary habit in determining inflammatory process-triggered oxidation are still unclear.

Recent dietary intervention studies have demonstrated that altering fat composition can significantly reduce oxidative stress in the MetS [START_REF] Roberts | Effect of diet and exercise intervention on blood pressure, insulin, oxidative stress, and nitric oxide availability[END_REF][START_REF] Roberts | Effect of a short term diet and exercise intervention on oxidative stress, inflammation, MMP-9 and monocyte chemotactic activity in men with metabolic syndrome factors[END_REF]. A number of clinical trials examined short-or intermediate-term effects of the Mediterranean diet on different circulating markers of oxidative stress [START_REF] Ambring | Effects of a Mediterranean-inspired diet on blood lipids, vascular function and oxidative stress in healthy subjects[END_REF][START_REF] Stachowska | Elements of Mediterranean diet improve oxidative status in blood of kidney graft recipients[END_REF][START_REF] Hagfors | Antioxidant intake, plasma antioxidants and oxidative stress in a randomized, controlled, parallel, Mediterranean dietary intervention study on patients with rheumatoid arthritis[END_REF][START_REF] Fito | Effect of a traditional Mediterranean diet on lipoprotein oxidation: a randomized controlled trial[END_REF]. Recently, Dai et al. reported a robust inverse association between adherence to the Mediterranean diet and oxidative stress as measured by reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, independent of a wide range of known cardiovascular disease risk factors [START_REF] Dai | Association between adherence to the Mediterranean diet and oxidative stress[END_REF]. Other trials, however, yielded conflicting results. Given the potential of altering dietary fat composition (i.e n-3 polyunsaturated fatty acids (PUFA) or low fat diets) in the prevention and therapy of a number of chronic diseases including MetS [START_REF] Perez-Martinez | n-3 PUFA and lipotoxicity[END_REF] and the inconsistencies found so far, this subject is in need of more in depth investigation.

Much of our knowledge of the relationship between lipids, lipoprotein metabolism and the development of atherosclerosis and cardiovascular disease is based on characterising fasting metabolic markers. However humans spend the majority of time in a nonfasting, postprandial state, with a continual fluctuation in the degree of lipemia throughout the day. In line with this notion, oxidative stress has received considerable attention over the past several years in the fasting state; however there is a paucity of data on postprandial oxidative stress. With regard to the postprandial state, several previous studies have demonstrated that a breakfast enriched in saturated fat resulted in an increase in biomarkers of oxidative stress [START_REF] Ursini | Postprandial plasma lipid hydroperoxides: a possible link between diet and atherosclerosis[END_REF][START_REF] Devaraj | High-fat, energy-dense, fast-food-style breakfast results in an increase in oxidative stress in metabolic syndrome[END_REF][START_REF] Cardona | Fat overload aggravates oxidative stress in patients with the metabolic syndrome[END_REF]. Based in these previous evidences, we investigated whether oxidative stress is affected by diets with different fat quantity and quality during the postprandial state in MetS patients from the LIPGENE study. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Material and Methods

Design

The current study was conducted within the framework of the LIPGENE study ("Diet, genomics and the metabolic syndrome: an integrated nutrition, agro-food, social and economic analysis"), a Framework 6 Integrated Projected funded by the European Union. A subgroup of patients with the MetS was randomly stratified to one of four dietary interventions. MetS was defined by published criteria [START_REF] Grundy | Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition[END_REF][START_REF] Grundy | Obesity, metabolic syndrome, and coronary atherosclerosis[END_REF], which conformed to the LIPGENE inclusion and exclusion criteria [START_REF] Perez-Martinez | Dietary fat differentially influences regulatory endotelial function during the postprandial state in patients with metabolic syndrome: From the LIPGENE study[END_REF]. Pre-and post-intervention was administered a fatty meal with a fat composition similar to that consumed in each of the diets (Figure 1). The intervention study design and the dietary strategy protocol have been previously described in detail by Shaw et al [START_REF] Shaw | LIPGENE food-exchange model for alteration of dietary fat quantity and quality in freeliving participants from eight European countries[END_REF].

Participant and recruitment

Randomization and intervention

Each volunteer was randomly stratified to one of four dietary interventions for 12 weeks (Figure 1). Randomisation was completed centrally, according to age, gender and fasting plasma glucose concentration using the MINIM (Minimisation Programme for Allocating patients to Clinical Trials, Dept of Clinical Epidemiology, The London Hospital Medical College, UK) randomisation programme. The composition of the four diets was as follow:

-High-fat (38% energy), SFA-rich diet (16% SFA, 12 % MUFA 6% PUFA) (HSFA).

-High-fat (38% energy), MUFA-rich diet (8% SFA, 20% MUFA, 6% PUFA) (HMUFA).

-Low-fat (28% energy), high-complex carbohydrate diet (8% SFA, 11%; MUFA; 6% PUFA), with 1g/d high oleic sunflower oil (placebo) (LFHCC) -Low-fat (28% energy), high-complex carbohydrate diet (8% SFA, 11% MUFA; 6% PUFA), with 1.24 g/d LC n-3 PUFA (LFHCC n-3).

Post-intervention (week 12), we performed a postprandial challenge with the same fat composition as consumed on the assigned dietary period (Figure 1). Patients presented at the clinical centre at 8:00 a.m following a 12 h fast, refrained from smoking during the fasting period and abstained from alcohol intake during the preceding 7 days, in the laboratory and, after canulation a fasting blood sample was taken before the test meal, which then was ingested under supervision within 20 min. The test meal reflected fatty acid composition of each subject chronic dietary intervention. Subsequent blood samples were drawn at 2 and 4 hours. Test meals provided an equal amount of fat (0.7 g/kg body weight), cholesterol (5 mg/kg of body weight) and vitamin A (60,000 IU/m 2 body surface area). The test meal provided 65% of energy as fat, 10% as protein and 25% as carbohydrates. During the postprandial assessment subjects rested, did not consume any other food for 9 hours, but were allowed to drink water. The composition of the breakfasts was as follow: HSFA breakfast (38% SFA, 21% MUFA 6% PUFA); HMUFA breakfast (12% SFA, 43% MUFA,10% PUFA); LFHCC breakfast with placebo capsules (21% SFA, 28% MUFA, 16% PUFA); LFHCC with LC n-3 PUFA (21% SFA, 28%; MUFA, 16% PUFA (1.24 g/d of LC n-3 PUFA).

Measurements

Blood was collected in tubes containing ethylene diaminetetraacetic acid (EDTA) to give a final concentration of 0.1% EDTA. Plasma was separated from red cells by centrifugation at 1500 × g for 15 min at 4 ºC. Biomarkers determined in frozen samples were realized by laboratory investigators who were blinded to the interventions. Lipid parameters were assessed with a DDPPII Hitachi modular analyzer (Roche, Basal, Switzerland), using specific reagents (Boehringer-Mannheim, Mannheim, Germany). Plasma triglycerides (TG) and cholesterol concentrations were assayed by enzymatic procedures [START_REF] Bucolo | Quantitative determination of serum triglycerides by the use of enzymes[END_REF][START_REF] Allain | Enzymatic determination of total serum cholesterol[END_REF]. Apolipoprotein (Apo) A-I and apo B were determined by turbidimetry [START_REF] Riepponen | Immunoturbidimetric determination of apolipoproteins A-1 and B in serum[END_REF]. HDL-C was measured by precipitation of a plasma aliquot with dextran sulphate-Mg2+, as described by Warnick et al [START_REF] Warnick | Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol[END_REF]. Low density lipoprotein-cholesterol (LDL-C) was calculated by using the following formula; plasma cholesterol -(high density lipoprotein-cholesterol (HDL-C) + large triacylglycerol rich lipoprotein (TRL)-C+ small TRL-C). Plasma glucose concentrations were measured using the Architect-CG16000 analyzer (Abbott Diagnostics, Tokio, Japan) by the exoquinase method and plasma insulin concentrations were measured by chemoluminescence with an Architect-I2000SR analyzer (Abbott Diagnostics, Tokio, Japan). Plasma fatty acid composition was determined by the enzymatic colorimetric assay for the determination of free fatty acids, Half-micro test (Roche Diagnostic GmbH).

Determination of oxidative stress biomarkers

Lipid peroxides (LPO) in plasma were estimated using the method of Eldermeier et al. [24]. This method uses a chromatogenic reagent which reacts with the lipid peroxidation products malondialdehyde and 4-hydroxyalkenals at 45 ± 1ºC, yielding a stable chromophore with maximum absorbance 586 nm. Protein carbonyls content was carried out in plasma samples using the method of Levine [START_REF] Levine | Determination of carbonyl content in oxidatively modified proteins[END_REF]. Samples were incubated with 500ml of a 10 mM solution of 2,4dinitrophenylhydrazine in 2N HCl for 60 min. Subsequently, the proteins were precipitated from the solutions using 500 ml of 20% trichloroacetic acid. The proteins were then washed three times with a solution of ethanol and ethylacetate (1:1 vol/vol) and dissolved in 1 ml of 6M guanidine (containing 20 mM phosphate buffer, pH 2,3, in trifluoroacetic acid) at 37º C. The carbonyls were evaluated in a spectrophotometer (UV-1603 Shimadzu, Kyoto, Japan) at wavelength 360 nm [START_REF] Luo | Methionine in proteins defends against oxidative stress[END_REF].

Antioxidant capacity in plasma was measured using BIOXYTECH® AOP-490 TM (OXIS International). This assay is based upon the reduction of Cu2 + to Cu + by the combined action of all antioxidants present in the sample. Thus a chromogenic ragent forms a complex with Cu + which has an absorbance at 490 nm [START_REF] Price | Application of manual assessment of oxygen radical capacity (ORAC) for use in high throughput assay of "total" antioxidant activity of drugs and natural products[END_REF]. Total glutathione (data non-shown), reduced glutathione (GSH) and GSH/oxidized glutathione (GSSG) ratio were carried out in plasma samples using BIXYTECH® GSH-420, GSH-400 and GSH/GSSG-412 kits, respectively. The determination of total glutathione levels is based on the formation of a chromophoric thione which has an absorbance at 420 nm. GSH concentration is based on reaction which leads to the formation of a chromophore with absorbance at 400 nm. The GSH-412 method uses the thiol-scavenging reagent and this technique is based on Tietze method [START_REF] Rahman | Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method[END_REF]. To determine GSSG levels we used the following equation: GSSG = A-B;

where A= total glutathione and B= GSH. Plasma hydrogen peroxide (H 2 O 2 ) concentration was measured using the BIOXYTECH ® H 2 O 2 -560 Assay (OXIS International). This kit allows for colorimetric quantitative determination of hydrogen peroxide (total hydroperoxides) in aqueous samples. This assay is based on method of ferric-xylenol orange hydroperoxide assay [START_REF] Gay | Hydroperoxydes assay with the ferric-xylenol oragen complex[END_REF][START_REF] Fabiani | Producction of hydrogen peroxide is responsable for the induction of apoptosis by hydroxytyrosol on HL60 cells[END_REF].

Antioxidant enzyme activity

Total superoxide dismutase (SOD; E.C: 1.15.1.1) activity was determined by colorimetric assay in plasma at wavelengths of 525 nm according to the laboratory method described by McCord and Nebot [START_REF] Mccord | Superoxide dismutase: the firs twenty years (1968-1988)[END_REF][START_REF] Nebot | Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol[END_REF]. Glutathione peroxidase (GPx; E.C.: 1.11.1.9) activity was evaluated in plasma using the Glutathione Peroxidase Assay Kit (Cayman Chemical). This assay is based on the oxidation of NADPH to NAD + , catalysed by a limiting concentration of glutathione reductase, with maximum absorbance at 340 nm. The GPx activity was measured based on the formation of GSSG from GPx-catalyzed oxidation of GSH by H 2 O 2 , coupled with NADPH consumption in the presence of exogenously added glutathione reductase, with maximum absorbance at 340 nm. This assay is on based in method of Flohé [START_REF] Jones | Redox state of glutathione in human plasma[END_REF].

Although GSH/GSSG ratio and antioxidant enzymes activities may be measured in erythrocyte, their plasma measurements provide an approach to the blood state, reflecting the similar change at the intracellular level [START_REF] Jones | Redox state of glutathione in human plasma[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Statistical Analyses

All data presented in the text and tables are expressed as mean values and standard deviations. SSPS 17 for Windows (SSPS INC., Chicago) was used for the statistical analysis.

The data were analyzed using repeated measures analysis of variance and Student t test for paired data analysis and analysis of variance for repeated measures. In this analysis we studied:

1. the postprandial time points; 2. the effect of the type of fat meal ingested, independent of time (represented by dietary fat); and 3. the interaction of both factors, indicative of the degree of the postprandial response in each group of subjects with each breakfast (represented by time*dietary fat interaction). Post hoc statistical analysis was completed by using the protected least-significant-difference test to identify significant differences between dietary treatments.

The contrast statistic used when the sphericity assumption was not satisfied was Huynh-Feldt. 1). There were no significant differences in dietary composition at baseline between the four diet groups. However, during the intervention period we observed significant differences between the different diets. The HSFA diet group consumed 17.9% of energy as saturated fat compared to the HMUFA group who consumed 21% as monounsaturated fat, while carbohydrate consumption fell to 38.2% and 40.7% respectively. The LFHCC diet group decreased their intake of total fat to 27% and the LFHCC n-3 diet to 26.5%, while carbohydrate consumption rose to 51.2% and 54% respectively. The LFHCC n-3 diet group attained their target intake of EPA and DHA at 1.8g/day. In addition, analysis of plasma fatty acids obtained after each dietary period showed good adherence in the different intervention stages. During the HSFA diet, we observed an increase in plasma C16:0 palmitic (11%), C18:0 stearic (20%) and C14:0 myristic acids (90%) compared with the baseline state. Consistently, the chronic intake of the LFHCC n-3 diet induced an increase in the C22:6n3 docosahexaenoic (96%) and C20:5n3 eicosapentaenoic acids (206%). Finally, in our typically Mediterranean population, subjects consumed a high intake of C18:1 oleic acid at baseline (21.6% energy from MUFA) and no significantly increase were observed at the end of the HMUFA period (21.1% energy from MUFA).

Results

Dietary compliance was good, with close attainment of the dietary intervention targets (Table

Table 2 shows the age, baseline BMI and lipid related risk factors of the 75 subjects with MetS randomized to each dietary intervention.

The effect of dietary fat quality and quantity on postprandial plasma TG, glucose, insulin, glutathione (GSH), glutathione/oxidized glutathione (GSH/GSSG) ratio, lipoperoxides (LPO), protein carbonyl, hydrogen peroxide (H 2 O 2 ), superoxide dismutase activity (SOD), total antioxidant capacity, and glutathione peroxidase (GPx) were assessed post-intervention.

Postprandial glucose and insulin levels after the intervention were not significantly different between diets (data not shown). We observed a significantly increment in the area under the curve (AUC) for postprandial TG during the HSFA (5231.87 mmol x min/L) diet compared with the HMUFA (2095.32 mmol x min/L) and LFHCC n-3 (2354.65 mmol x min/L) diets (HSFA vs HMUFA, P<0.013 and HSFA vs LFHCC n-3, P<0.018).

Figure 2A shows that postprandial plasma GSH levels were higher 2 h after the intake of the HMUFA diet (P<0.05), compared to the other three diets. Plasma GSH levels remained significantly higher 4 h after the HMUFA diet compared with the HSFA diet (P<0.05). In addition, at 2 h after the HSFA diet we observed lower GSH plasma levels (P<0.05) compared to the LFHCC and LFHCC n-3 diets. As an indicator of redox status we measured the GSH/GSSG ratio. Interestingly we showed a consistent postprandial increase in GSH/GSSG (P=0.002) ratio 2 h after the HMUFA diet (P<0.001), compared to the other three diets (Figure 2B). GSH/GSSG remained significantly higher 4 h after the HMUFA diet compared with the LFHCC and HSFA diets (P<0.003). Consistently, postprandial plasma GSSG levels were lower 2 h after the HMUFA diet compared with the other 3 diets (P<0.004) and remained significantly lower at 4 h compared with the LFHCC and HSFA diets (Figure 2C). Moreover, GSSG levels were significant higher 4 h after the HSFA diet compared with the LFHCC n-3 (P<0.05). We also measured lipid peroxidation given the important potential effect of fat composition. Interestingly, plasma LPO levels were significantly higher 2 h after the HSFA diet, compared to subjects adhering to the HMUFA and LFHCC n-3 diets (P<0.05), (Fig 3A).

Moreover, plasma LPO level remained lower later in the postprandial phase 4 h after the HMUFA diet, compared to all other diets (Figure 3A). In the case of postprandial protein carbonyls concentration, a significant time * diet interaction (P< 0.02) confirmed that levels were significantly higher 2 h after the HSFA diet but lower 2h following the HMUFA diet (Figure 3B). Consistent with this effect postprandial SOD was significantly lower 2 h and 4 h after the HMUFA diet, compared with the HSFA and LFHCC diets (P<0.001) (Figure 3C).

Again, an intermediate effect was found after the intake of the two LFHCC diets compared with the HSFA (Figure 3C). Postprandial plasma H 2 O 2 levels were significant higher 2 h after the HSFA diet compared with the other three diets (Figure 3D). Postprandial total antioxidant capacity (Figure 3E) and plasma GPx levels were not significantly different between diets (Figure 3F). On the other hand, with the aim of identifying the effects of long-term consumption of the four diets, we also explored the effect of dietary fat on GSH, GSH/GSSG ratio, GSSG, LPO, protein carbonyls, H 2 O 2 , SOD, total antioxidant capacity, and GPx at baseline (before the test meal) . In this context we found significantly differences related to the chronic dietary intervention for GSH, protein carbonyl and SOD plasma levels. Thus, the results of our study demonstrated that the long-term consumption of the HMUFA diet increased the GSH plasma levels compared with the HSFA, LFHCC and LFHCC n-3 diets (P<0.002) (Figure 2A).

Moreover GSH levels remained significantly lower after the HSFA diet compared with the LFHCC diet (P<0.05) (Figure 2A). Protein carbonyls concentrations were significantly lower after the HMUFA diet compared with the other 3 diets (P<0.001) (Figure 3B). Finally, SOD was significantly higher after the HSFA diet, compared with the HMUFA (P<0.002) and LFHCC n-3 diets (P<0.043) (Figure 3C).

Discussion

Oxidative stress has been associated with all the individual components and with the onset of cardiovascular complications in subjects with the MetS. Although some aspects of diet have been linked to individual features of the MetS [START_REF] Wirfalt | Food patterns and components of the metabolic syndrome in men and women: a cross-sectional study within the Malmo Diet and Cancer cohort[END_REF], the role of diet in the etiology of the syndrome is poorly understood. Our study supports the notion that the HMUFA diet improves postprandial oxidative stress parameters as measured by GSH and GSH/GSSG ratio. In addition, the HMUFA diet induced lower postprandial plasma levels of LPO, protein carbonyls concentration and SOD compared to subjects adhering to the other three diets. Furthermore, postprandial plasma H 2 O 2 levels were unfavourable increased during the HSFA diet compared to the other three diets.

Fasting is not the typical physiological state of the modern human being, which spends most the time in the postprandial state. Therefore, the assessment of the postprandial lipemic response may be more relevant to identify disturbances in metabolic pathways related to oxidative stress than measures taken in the fasting state. Although previous studies demonstrated that patients with the MetS have greater levels of oxidative stress after a fat overload [START_REF] Devaraj | High-fat, energy-dense, fast-food-style breakfast results in an increase in oxidative stress in metabolic syndrome[END_REF][START_REF] Cardona | Fat overload aggravates oxidative stress in patients with the metabolic syndrome[END_REF], the current well controlled intervention study is in our knowledge, the first to examine the effect of four isoenergetic diets distinct in fat quantity and quality during the postprandial state.

One biomarker used to evaluate oxidative stress in humans is the redox state GSH/GSSG and this quotient appears to correlate with the biological status of the cell [START_REF] Jones | Redefining oxidative stress[END_REF].

Biochemically, GSH/ GSSG redox decreases lipid hydroperoxides by reducing these peroxides into alcohols and suppressing their generation [START_REF] Girotti | Lipid hydroperoxide generation, turnover, and effector action in biological systems[END_REF]. Moreover, a lower GSH/GSSG ratio may result in protein glutathionylation and oxidatively altered GSH/GSSG redox signalling and associated gene expression and apoptosis, which may contribute to atherosclerosis [START_REF] Jones | Redox potential of GSH/GSSG couple: assay and biological significance[END_REF]. In our study, the HMUFA diet produced a postprandial increase in GSH plasma levels and GSH/GSSG ratio compared to the other three diets (HSFA and LFHCC diets). Thus, our data carried out in the postprandial state support previous evidences in the fasting state suggesting that dietary patterns similar to those of the Mediterranean-style diet exert positive effects on GSH/GSSG ratio [START_REF] Dai | Association between adherence to the Mediterranean diet and oxidative stress[END_REF]. Additionally, Fitó et al. found that olive oil caused increases in glutathione reductase plasma [START_REF] Fitó | Postprandial and short-term effects of dietary virgin olive oil on oxidant/antioxidant status[END_REF]. This data supports the idea that HMUFA may trigger the increase of GSH and GSH/GSSG ratio, together with a reduction of GSSG by mean the stimulation GR activity. The major levels of GSH may help to regenerate the most important antioxidant such as ascorbic acid, alpha-tocopherol and others [START_REF] Valko | Free radicals and antioxidants in normal physiological functions and human disease[END_REF]. This situation may explain the highest antioxidant effect of HMFA than LFHCC and LFHCC n-3 on oxidative stress characterized by a reduction in lipid peroxidation, carbonylated protein and hydrogen peroxide levels. On the other hand a possible mechanism to explain the increased plasma GSH/GSSG ratio observed after the intake of the HMUFA diet could be through decreased GSSG concentrations and enhanced GSH levels, like it is suggested by our data. Initially, GSH is oxidized into GSSG by the enzyme GPx; in this process, GSH quenches peroxides. GSSG reverts to GSH via gluthathione reductase with concomitant oxidation of NADPH [START_REF] Fico | Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis[END_REF]. Diverse nutrients and biofactors in foods characteristic of the Mediterranean diet may provide higher NADPH [START_REF] Le | Protection of myocytes against free radical-induced damage by accelerated turnover of the glutathione redox cycle[END_REF] and up-regulate gluthathione reductase activity [START_REF] Fang | Free radicals, antioxidants, and nutrition[END_REF], which may lead to a decrease in GSSG and a resulting in higher GSH/GSSG ratio. Interestingly the effects observed by the present study were the results of the consumption of diets over a period of 12 weeks. As a result, chronic ingestion results in a more faithful translation of the effects of the different dietary models, in that meal consumption is not an isolated phenomenon, and in the type of design that employs only acute ingestion of fats it is impossible to separate out the potential effects of the background diet on the oxidative stress status. In this line, we demonstrated that the long-term consumption of the HMUFA diet increased the GSH plasma levels and decreased protein carbonyls concentrations compared with the HSFA, LFHCC and LFHCC n-3 diets. These findings suggest that background dietary MUFA consumption may influence the nature and extent of postprandial oxidative stress. Our results are important, particularly in view of the lack of randomized controlled trials assessing the effects of diets rich in MUFA typical for the traditional Mediterranean diet on glutathione redox pathways in the general population and in patients with metabolic syndrome. Emerging evidence thus points to the notion that oxidative stress may contribute to insulin resistance, the key characteristic of MetS [START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF]. Mitochondria are generally considered a major source of ROS, which can lead to lipid peroxidation [START_REF] Adam-Vizi | Bioenergetics and the formation of mitochondrial reactive oxygen species[END_REF]. In particular, the mitochondrial matrix is sensitive to peroxide-induced oxidative damage and must be protected against the formation and accumulation of lipids and lipid peroxides, where free fatty acids (FFA) can modulate signaling functions of ROS [START_REF] Schönfeld | Fatty acids as modulators of the cellular production of reactive oxygen species[END_REF]. In the current study, the HMUFA diet induced lower postprandial plasma levels of LPO and protein carbonyl concentration compared to subjects adhering to the other three diets. Although not explored, and beyond the remit of our study, a probable mechanistic explanation for the observed effects lies in the high intakes of MUFA together with natural antioxidants present in the Mediterranean diet that could prevent against the accumulation of the lipid peroxides. According with our results a recent PREDIMED substudy demonstrated that adherence to the Mediterranean diet was associated with a reduced oxidative status, as shown by lower serum concentrations of oxidized LDL compared with the low-fat diet [START_REF] Fito | Effect of a traditional Mediterranean diet on lipoprotein oxidation: a randomized controlled trial[END_REF]. On the other hand, our data are in agreement with previous postprandial studies, suggesting that the acute intake of fat load enriched in saturated fat results in increased postprandial oxidative stress in patients with MetS [START_REF] Devaraj | High-fat, energy-dense, fast-food-style breakfast results in an increase in oxidative stress in metabolic syndrome[END_REF][START_REF] Cardona | Fat overload aggravates oxidative stress in patients with the metabolic syndrome[END_REF].

During the past few years, several studies have shown that n-3 PUFA have potentially cardioprotective effects, especially in high-risk patients with dyslipidemia, and might therefore be expected to be of benefit in T2DM. In our study, as compared with the HSFA diet the effect of the two LFHCC diets was favourable intermediate. In the same line, a previous study showed that daily supplementation of EPA and DHA for three months reduced levels of 8-iso-PGF 2α measured in plasma, but not 15-keto-dihydro-PGF 2α , compared to controls among healthy subjects in the KANWU study [START_REF] Nalsen | Dietary (n-3) fatty acids reduce plasma F2-isoprostanes but not prostaglandin F2alpha in healthy humans[END_REF]. An isoprostane reducing effect of n-3 PUFA has also been observed in treated-hypertensive type 2 diabetic patients [START_REF] Mori | Effect of eicosapentaenoic acid and docosahexaenoic acid on oxidative stress and inflammatory markers in treated-hypertensive type 2 diabetic subjects[END_REF]. Contrary to these previous evidences, PUFA has been suggested to be more prone to oxidation than SFA and MUFA [START_REF] Turpeinen | A high linoleic acid diet increases oxidative stress in vivo and affects nitric oxide metabolism in humans[END_REF][START_REF] Wagner | Free radical-mediated lipid peroxidation in cells: oxidizability is a function of cell lipid bis-allylic hydrogen content[END_REF]. Although only suggestive, these results need to be confirmed and extended.

In response to oxidative stress, cells attempt to fortify the antioxidant arsenal as the first line of defence. The natural antioxidant system consists of a series of antioxidant enzymes and numerous endogenous and dietary antioxidant compounds that react with and inactive ROS. Superoxide is converted to H 2 O 2 by a group of enzymes known as SOD. H 2 O 2 , in turn, is converted to water and molecular oxygen by either catalase or GPx. In addition, GPx can reduce lipid hydroperoxides and other organic hydroperoxides. Previous studies [START_REF] Mates | Antioxidant enzymes and human diseases[END_REF] show that the expression of SOD is upregulated by superoxide so the decrease in the SOD after consumption of the HMUFA could be explained to a lower generation of SOD in this diet. Moreover, it has been reported that several genes encoding antioxidant enzymes, such as SOD, contain antioxidant response elements sequences in its promoter that bind the transcription factor Nrf2 in response to increased ROS, promoting an increase in the expression of these enzymes [START_REF] Osburn | Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults[END_REF]. In our study, the long-term consumption of HSFA diet also increased the SOD compared with the HMUFA and LFHCC n-3 diets. Therefore, the higher SOD activity obtained after the chronic intake of the HSFA diet could be explained by increased activation of Nrf2 which we believe could be a direct consequence of a higher production of O 2-in this situation.

In our study, GPx plasma levels and total antioxidant capacity were not significantly different among the four diets during the postprandial period. One reason for this finding could be explained because some pathways but not others may be differentially influenced by dietary fat in the postprandial state. However it is also interesting to emphasize that although we did not find significantly differences for these paramenters a trend was observed between the HSFA vs HMUFA diet. Interestingly, our results also expand our recent finding in this same population, of an inverse association between HMUFA diet and inflammation [START_REF] Perez-Martinez | Dietary fat differentially influences regulatory endotelial function during the postprandial state in patients with metabolic syndrome: From the LIPGENE study[END_REF], since inflammation and oxidative stress are tightly inter-dependent. A synergistic relationship exists between oxidative damage and inflammation, and both of these are related to endothelial dysfunction [START_REF] Perez-Martinez | Dietary fat differentially influences regulatory endotelial function during the postprandial state in patients with metabolic syndrome: From the LIPGENE study[END_REF]. Hyperlipidemia and hyperglycemia have been associated with increased oxidative damage affecting lipoproteins and the antioxidant status [START_REF] Sies | Nutritional, dietary and postprandial oxidative stress[END_REF]. Previous evidences suggest that prolonged elevations in TRL, associated with decreased chylomicron clearance, result in a restriction in vitamin E transfer to LDL and HDL along with greater susceptibility to oxidation [START_REF] Sies | Nutritional, dietary and postprandial oxidative stress[END_REF]. In this context, we have demonstrated in a recent study that a HMUFA diet can lower postprandial lipoprotein abnormalities associated with the MetS [START_REF] Jimenez-Gomez | Beneficial long-term effects of a low-fat, high complex carbohydrate diet supplemented with long-chain n-3 PUFA on postprandial lipoprotein metabolism in patients with metabolic syndrome[END_REF]. Also the adverse TG Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

raising effects of the long-term LFHCC diets were avoided by concomitant LC n-3 PUFA supplementation. These findings suggest that the beneficial effects observed during the consumption of the HMUFA diet could be mediated at least in part by a more efficient clearance of the postprandial lipoproteins which is accompanied by the improvement of the postprandial oxidative stress observed in these patients. However, the detailed relations among hyperlipidemia, hyperglycemia, hyperinsulinemia, and oxidative stress are still under research.

Our study presents some limitations. Ensuring adherence to dietary instructions is difficult in a feeding trial. However, adherence to recommended dietary patterns was good, as judged by measurements. On the other hand, our design has the strength of reproducing real-life conditions with home-prepared foods, reflecting usual practice.

In summary, our results support the notion that the HMUFA diet improves postprandial oxidative stress parameters in patients with the MetS. Both LFHCC diets had an intermediate effect relative to the HMUFA and HSFA diets. These findings suggest that the postprandial state is important for understanding possible cardio-protective effects associated with the Mediterranean diet particularly in subject with the MetS. In addition, these findings support recommendations to consume a HMUFA diet as a useful tool to prevent cardiovascular diseases in MetS patients. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t

  75 patients were included. All participants gave written, informed consent and underwent a comprehensive medical history, physical examination and clinical chemistry analysis before enrolment. This study was carried out in the Lipid and Atherosclerosis Unit at the Reina Sofia University Hospital, from February 2005 to April 2006. The experimental protocol was approved by the local ethic committee, according to the Helsinki Declaration. The study was registered with The US National Library of Medicine Clinical Trials registry (NCT00429195).

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t

  P<0.05 was considered statistically significant. Clinical Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2010 Portland Press Limited

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2010 Portland Press Limited

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t List of Abbreviations Apo: apolipoprotein; EDTA: ethylene diaminetetraacetic acid; FFA: free fatty acids; GPx: glutathione peroxidise; GSH: reduced glutathione; GSH/GSSG: reduced glutathione/oxidized glutathione; GSSG: oxidized glutathione; HDL-C: high density lipoprotein-cholesterol; H 2 O 2 : hydrogen peroxide; HMUFA: high monounsaturated fatty acid rich-rich diet; HSFA: high saturated fatty acid-rich diet; LDL-C: low density lipoprotein-cholesterol; LFHCC: low fat, high-complex carbohydrate diet; LPO: lipid peroxides; MetS: metabolic syndrome; PUFA: polyunsaturated fatty acid; ROS: reactive oxygen species; SOD: superoxide dismutase activity; T2DM: type 2 diabetes mellitus; TRL: triacylglycerol rich lipoprotein. Clinical Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2010 Portland Press Limited

A c c e

  p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2010 Portland Press Limited

Figure 1 :

 1 Figure 1: Intervention study design. Each volunteer was randomly stratified to one of four dietary interventions for 12 weeks. Pre-and post-intervention was administered a fatty meal with a fat composition similar to that consumed in each of the diets.

Figure 2 :

 2 Figure 2: (A) Reduced glutathione (GSH), (B) reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and (C) oxidized glutathione (GSSG) in the postprandial state at the end of each dietary period. n=75. Results are expressed as mean ± SEM. Data were analysed using ANOVA for repeated measures. *p<0.05: HMUFA vs HSFA, LFHCC and LFHCC n-3; † p<0.05: HSFA vs HMUFA, LFHCC and LFHCC n-3; # p<0.05: HSFA vs LFHCC; ‡ p<0.05: HSFA vs LFHCC n-3; ¶ p<0.05: HMUFA vs HSFA and LFHCC. HSFA: high SFA-rich diet; HMUFA: high MUFA-rich diet; LFHCC: low fat, high-complex carbohydrate diet with 1g/d high oleic sunflower oil (placebo); LFHCC n-3: Low fat, highcomplex carbohydrate with 1.24 g/d LC n-3 PUFA diet.

Figure 3 :

 3 Figure 3: (A) Lipid peroxides, (B) protein carbonyls, (C) superoxide dismutase, (D) hydrogen peroxide, (E) total antioxidant capacity and (F) glutathione peroxidase in the postprandial state at the end of each dietary period. n=75. Results are expressed as mean ± SEM. Data were analysed using ANOVA for repeated measures. *p<0.05: HMUFA vs HSFA, LFHCC and LFHCC n-3; †p<0.05: HSFA vs HMUFA, LFHCC and LFHCC n-3; ‡ p<0.05: HSFA vs LFHCC n-3; §p<0.05: MUFA vs SFA; ¶ p<0.05: HMUFA vs HSFA and LFHCC. HSFA: high SFA-rich diet; HMUFA: high MUFA-rich diet; LFHCC: low fat, high-complex carbohydrate diet with 1g/d high oleic sunflower oil (placebo); LFHCC n-3: Low fat, highcomplex carbohydrate with 1.24 g/d LC n-3 PUFA diet.

Figure 1 .

 1 Figure 1.

  Figure 2

3

 3 Figure 3

TABLE 1 :

 1 Dietary intake at baseline and end of intervention period, alongside dietary targets.

			Clinical Science Immediate Publication. Published on 26 Apr 2010 as manuscript CS20100015
			HSFA	HMUFA	LFHCC	LFHCC n-3
		Baseline
		n	17	18	20	20
		Energy (MJ/d)	8.81 ± 0.5 a	8.12 ± 0.4 a	8.48± 0.4 a	8.84 ± 0.4 a
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100015	%E from fat %E from SFA %E from MUFA %E from PUFA %E from CHO %E from protein Total EPA and DHA (g/d) Target %E from fat %E from SFA %E from MUFA %E from PUFA Total EPA and DHA (g/d) End of intervention n Energy (MJ/d) %E from fat %E from SFA %E from MUFA %E from PUFA %E from CHO %E from protein Total EPA and DHA (g/d) Abbreviations: %E, percentage energy; HSFA, SFA-rich diet; HMUFA, MUFA-rich diet; 43.3 ± 1.3 a 42.8 ± 1.2 a 41.4 ±1.1 a 45.5 ± 1.4 a 11.6 ± 0.5 a 10.7 ± 0.5 a 10.2 ± 0.4 a 12.0 ± 0.4 a 20.9 ± 0.9 a 21.6 ± 0.7 a 20.4 ± 0.7 a 22.7 ± 0.9 a 4.45 ± 0.2 a 4.84 ± 0.2 a 4.39 ± 0.2 a 4.80 ± 0.2 a 37.5 ± 1.3 a 38.9 ± 1.0 a 40.9 ± 1.3 a 36.5 ± 1.7 a 17.2 ± 0.5 a 17.6 ± 0.7 a 16.6 ± 0.6 a 16.8 ± 0.5 a 0.39 ± 0.06 a 0.42 ± 0.1 a 0.36 ± 0.07 a 0.43 ± 0.06 a 38 38 28 28 16 8 8 8 12 20 11 11 6 6 6 6 1.24 17 18 20 20 8.2 ± 0.4 a 7.7 ± 0.4 a 7.7 ± 0.4 a 9.2 ± 0.5 a 40.3 ± 0.5 a 40.2 ± 0.7 a 27.1 ± 0.5 b 26.5 ± 0.5 b 17.9 ± 0.3 a 9.1 ± 0.4 b 6.6 ± 0.3 c 6.4 ± 0.3 c 12.8 ± 0.3 a 21.1 ± 0.4 b 11.5 ± 0.3 c 11.1 ± 0.3 c 6.1 ± 0.3 a 5.7 ± 0.2 a 5.3 ± 0.2 a 5.0 ± 0.2 a 38.3 ± 1.0 a 40.7 ± 1.0 a 51.2 ± 1.1 b 54.1 ± 0.9 b 19.2 ± 0.7 a 19.2 ± 0.7 a 21.2 ± 1 b 18.8 ± 0.6 a,b 0.42 ± 0.1 a 0.41 ± 0.1 a 0.47 ± 0.08 a 1.83 ± 0.1 b LFHCC, Low fat, high-complex carbohydrate diet with 1g/d high oleic sunflower oil (placebo); LFHCC n-3, Low fat, high-complex carbohydrate diet with 1.24 g/d LC n-3 PUFA diet. Differences (P<0.05) between diet groups were assessed by one-way ANOVA. a,b,c Mean values within a row with unlike superscript letters were significantly different. Values presented are the mean ± SEM of each diet group. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

TABLE 2 :

 2 Baseline characteristics of subjects with the MetS assigned to each diet. SFA-rich diet; HMUFA, MUFA-rich diet; LFHCC, Low fat, highcomplex carbohydrate diet with placebo; LFHCC n-3, Low fat, high-complex carbohydrate diet with 1.24 g/d LC n-3 PUFA diet; BMI, body mass index; TG, triglycerides; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol; apo, apolipoprotein.

	Characteristics	HSFA	HMUFA	LFHCC	LFHCC n-3 P value
	n	17	18	20	20
	Age, years	58.58 (1.9)	54.61 (1.8)	56.35 (1.8)	55.30 (1.4)	0.449
	BMI, Kg/m2	35.27 (0.8)	34.45 (0.8)	35.48 (0.6)	35.15 (0.7)	0.798
	Total Cholesterol, mg/dL 200.02 (9.8) 189.13 (6.9) 207.16 (10.3) 189.49 (8.0) 0.405
	Total TG, mg/dL	171.13 (13.3) 143.74 (13.4) 144.48 (13.0) 138.43 (14.3) 0.654
	LDL-C, mg/dL	136.06 (7.7) 131.64 (5.6) 147.58 (8.5) 131.47 (7.6) 0.374
	HDL-C, mg/dL	43.2 (2.5)	44.6 (2.3)	44.5 (2.3)	42.1 (1.9)	0.834
	ApoB, mg/dL	92.1 (4.1)	89.06 (3.8)	101 (5.6)	90.7 (5.05)	0.291
	ApoA-1, mg/dL	133.5 (4.02) 135.03 (5.7) 135.1 (5.3)	130.2 (4.3)	0.876
	Abbreviations: HSFA,

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgments

Dr Lopez-Miranda had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Funding

This study was supported in part by research grants from the European community (LIPGENE European Integrated Project-505944), from the Ministerio de Ciencia e Innovación (AGL 2004-07907, AGL2006-01979, AGL2009-12270, to JL-M ), Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P06-CTS-01425 to JL-M); Consejería de Salud, Junta de Andalucía (06/128, 07/43, PI-0193 to JL-M). and the Spanish Ministry of Health (CB06/03/0047 (CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII)).

HRM is a recipient by Science Foundation Ireland Principal Investigator Programme (06/IM.1/B105). All decisions regarding the design, conduct, collection, analysis, or interpretation of the data and the decision to submit the manuscript for publication were made independently by the authors. None of the authors has any conflict of interest that could affect the performance of the work or the interpretation of the data.


Craig Heath

Sarah Anne Cooper

Katy Murray

Andrea Lowman

Colm Henry

Margaret Macleod

Gillian E Stewart

Martin Zeidler

Jan Mackenzie

S G Knight

Heath Ca

Cooper Sa

Macleod Ma

Mckenzie Jm

Will Rg

Professor R G Will

Diagnosing variant Creutzfeldt-Jakob disease: A retrospective analysis of the first 150 cases in the UK

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Introduction

Establishing an early clinical diagnosis in variant Creutzfeldt-Jakob disease (vCJD) can be difficult, resulting in extended periods of uncertainty for many families and sometimes a view that patients have been subjected to unnecessary investigations. This issue is accentuated by the progressive nature of vCJD and by the difficulty in achieving a confident clinical diagnosis before an advanced stage of illness. Although diagnostic delay may be a result of the non-specific early clinical features, a systematic analysis of the process of diagnosis was undertaken, with the aim of trying to achieve earlier diagnosis of vCJD.

Methods

Retrospective case file analysis was undertaken of the first 150 definite and clinically probable cases of vCJD identified by the UK surveillance system.

Results

There is a significant interval between illness onset and presentation to a primary care physician, which is influenced by the nature of the initial clinical features.

Neurological review is invariably sought following the development of clinical signs and a diagnosis is then established relatively quickly. Despite the progressive clinical course, a confident clinical diagnosis is not usually achieved until a relatively advanced stage of illness (mean time to diagnosis 10.5 months) with a more rapid clinical progression accounting for those cases diagnosed earlier after symptom onset.

Introduction

A novel form of human prion disease, variant Creutzfeldt-Jakob disease (vCJD), was reported in 1996 [START_REF] Will | A new variant of Creutzfeldt-Jakob disease in the UK[END_REF] . vCJD has provided both public health authorities and physicians with many challenges and achieving accurate early diagnosis has proved difficult [START_REF] Will | Psychiatric features of new variant Creutzfeldt-Jakob disease[END_REF][START_REF] Myles | Variant Creutzfeldt-Jakob disease: costs borne by families[END_REF] . This has left some families feeling that their affected relatives have been misdiagnosed or subjected to unnecessary, sometimes invasive, investigations. This issue, among others, was addressed by the BSE Inquiry, which concluded that 'Early diagnosis of this disease has been and continues to be a major problem as the first symptoms are depression and other non-specific psychiatric features. [START_REF] Phillips | The BSE Inquiry[END_REF] Difficulty in diagnosing vCJD may be the result of subtle early clinical features which are often insidious in onset. Non-specific psychiatric features dominate the early clinical course with few cases exhibiting objective neurological impairment. Neurological features develop early in a minority of patients, but are usually subjective symptoms, for example painful sensory symptoms [START_REF] Zeidler | New variant Creutzfeldt-Jakob disease: neurological features and diagnostic tests[END_REF] , which are not associated with neurological signs on examination. An analysis of a small number of the initial cases identified through UK surveillance supported the proposition that delay in diagnosis reflects the early clinical characteristics 2 , but a detailed analysis of the diagnostic process in vCJD has now been undertaken. The aim is to provide a detailed description of the diagnostic process in a large cohort of patients and to identify the reasons for diagnostic delay. This may have significant implications for counselling, public health and treatment. Although there is no proven treatment for vCJD, a number of promising agents are being tested in animal models and early introduction of therapy may be critical to efficacy [START_REF] Trevitt | A systematic review of prion therapeutics in experimental models[END_REF] . This study aims to explore the diagnostic process of vCJD in detail and aims to determine whether earlier diagnosis is possible.

Methods

Prospective surveillance of CJD in the UK has been ongoing since 1990. The methodology of the National CJD Surveillance Unit has been described in previous publications [START_REF] Spencer | First hundred cases of variant Creutzfeldt-Jakob disease: retrospective case note review of early psychiatric and neurological features[END_REF][START_REF] Will | Diagnosis of new variant Creutzfeldt-Jakob disease[END_REF] .

Included in this study are the first 150 "definite" and "probable" cases of vCJD identified in the UK. Cases were classified according to current diagnostic criteria, Table 1 9 . The cases were referred to the UK National CJD Surveillance Unit (NCJDSU) between 1995 and 2005. Retrospective analysis of case data held at the NCJDSU was undertaken and included hospital records (n=149), GP records (n=147) and a structured questionnaire (n=147), which was completed during an interview with the next of kin. The symptoms and signs elicited during the record review were classified as psychiatric or neurological according to the system described in a previous publication on clinical features in vCJD [START_REF] Spencer | First hundred cases of variant Creutzfeldt-Jakob disease: retrospective case note review of early psychiatric and neurological features[END_REF] . (Table 2)

Results

Basic demographics

One hundred and fifty cases of vCJD were identified in the UK between 1995 and 2005. Autopsy was undertaken in 106 cases (definite cases) and 44 cases were classified as clinically probable. Of 150 cases, 84 were male and 66 were female (gender ratio=1.2; 1). The median age at onset was 26 years (mean 28 years, range 12-74 years) with a median age at death 28 years (mean 30.1 years; range 15-75 years). The median duration of illness was 14 months (mean 15.7 months; range 6.5-40 months). One case remains alive, 96 months after onset.

Diagnosing vCJD a) Seeking medical attention

Individuals with vCJD rarely sought medical attention early in the clinical course.

Overall, the median time from onset to the first point of medical contact was 2.5 months (mean 3.4 months; range: 0-12 months). The time to presentation to a medical practioner was dependent on the nature of the early clinical features.

Those presenting with neurological features (ie. cases with isolated neurological symptoms or a combination of neurological and psychiatric symptoms) presented earlier to a medical practioner in comparison to those with isolated psychiatric features (neurological onset: 2 months; psychiatric onset: 3.3 months; combined 1.7 months; Kruskall Wallis test-p=0.04). Once medical attention was sought a neurological aetiology was suspected in a minority of cases (27/150) and the proposed initial diagnosis in the majority of cases was of a psychiatric disorder (Table 3). vCJD was not considered a potential diagnosis in any case at the first point of medical contact.

b) Neurological referral

The mean time from onset to referral to a neurologist was 7.4 months (95% CI: 6.5 to 8). There was a trend towards those with neurological features being referred earlier, but this did not reach statistical significance (neurological onset = 6.8 months; psychiatric onset= 7.9 months; both features at onset: 6.8 months Kruskall Wallis=0.056). The mean time from neurological referral to review was only 16 days. Figure 1 illustrates the source of neurology referral. 48% of referrals came from general practitioners, 24% from psychiatrists and 19% from physicians. Nine referrals (6%) came from other sources including paediatrics, a chronic pain team clinician, orthopaedics, obstetrics, a maxillary facial surgeon and a doctor working in armed forces. In one case a family member insisted on a neurology review (by-passing the GP) and in 2 cases the information was not available c) Neurology review 149/150 a individuals were reviewed by a neurologist during the illness. 116 cases were initially reviewed either in an outpatient clinic or as a ward review and 33 were directly admitted via primary care. Overall, 138 cases were admitted as a In a single case investigated overseas it is unclear whether a neurological opinion was sought.

inpatients during the illness and 11 remained as outpatients. The proposed differential diagnosis following the initial neurological review is summarised in Table 4. The diagnosis of vCJD was suspected in 54/149 (36%) following neurological review.

d) Suspecting CJD b

The diagnosis of CJD was suspected in 147 cases with a mean time from onset to a suspect diagnosis of 8.9 months (95% CI: 8.3 -9.7 months; representing 60% of mean total illness duration). The time to suspect CJD was independent of clinical features at onset (psychiatric 9.1 months (95% CI: 8.3 -9.8); neurological 9.4 months (95% CI: 7.9 -110, both=7.9 months (95% CI 6.1 -9.7), Kruskall Wallis Test p=0.3). A neurologist most often suspected the diagnosis, but in about a third of cases the diagnosis was proposed by another speciality. It is of note that one in 9 cases of vCJD were suspected by a radiologist underlining the diagnostic value of brain MRI in diagnosing vCJD (Table 5).

Two cases were only identified following autopsy and in one case detailed information was not available. Of the two cases not identified in life, both were significantly older than the mean (74 years old and 68 years old at onset) and neither of these cases retrospectively fulfilled the diagnostic criteria for "probable" vCJD in life (Table 1).

It is of interest to assess whether time to diagnosis has changed during the period of the study. Figure 2 provides an overview of the time to suspect CJD by year of onset. This illustrates that the most significant decrease in time to diagnosis occurred during the first years following the identification of vCJD, namely 1994 and 1995, but there has been little subsequent change in this parameter. In addition, there is no statistically significant variation in time to suspect CJD by b The more ambiguous term of CJD is used. The reason for this was two fold. Firstly it allowed the first ten cases to be included prior to the initial description of vCJD. Prior to this report vCJD was not recognised as a clinical entity. Secondly, more often than not when vCJD was being considered at an early stage of the diagnostic process vague terms like CJD or prion disease were used.

UK region (Table 6) despite regional variation in vCJD incidence 10 . There is also no good evidence of variation in time to vCJD diagnosis by neurological centre (not shown). e) Providing an early diagnosis (< 6 months from onset)

The diagnosis of vCJD was suspected in 38 cases within 6 months of onset. The median age at onset in this cohort was 25 years (range: 13-62 years) and the median duration of illness was 10.5 months (range: 6.5-26 months). 17/38 cases presented with psychiatric features at onset, 11 with isolated neurological features and 10 with a combination of features. The symptoms at onset were not significantly different from those diagnosed after 6 months (58/109, 35/109, 16/109 respectively). Despite this, there was a trend for those diagnosed earlier coming to medical attention earlier with the median time from onset to first medical contact of 1.7 months (range 0-6.5 months) in comparison to 3.2 months (range 0-12 months) in the later diagnosed cohort (Mann-Whitney p=0.08). In addition, the time from onset to neurology referral was significantly shorter ; 3.5 months (range 1.4-6.3 months) versus 8.4 months (range 1-23 months: Mann-Whitney p<0.0001). This may be explained by the development of early neurological symptoms/signs. Figure 3 summarises the median time from onset to the development of specific neurological symptoms or signs and demonstrates that cases diagnosed earlier developed neurological features at an earlier time after the onset of illness. It is also of note that the median time taken for individuals to become dependent was also significantly shorter in this cohort of patients.

Independent mobility was lost at 5.2 months (range 4.5-25 months; compared to 10.2 months, range 7-28.5 months: Mann-Whitney p<0.0001) and the time until bed bound was 8.1 months (range 4.5-25 months; compared to 14.2 months, range 7-28.5 months; Mann-Whitney p<0.0001). Independent feeding was also lost at an earlier stage, as was continence. (6.2 months, range 2.6-8.8 months compared to 12.5 months, range: 2.9-27.6) months; 5.5 months, range 1.5-14 months compared to 12.8 months, range 2.9-27.6 months), respectively (Mann Whitney p<0.0001).

. f) Confirmation of the clinical diagnosis

The mean time for vCJD to be considered the most likely diagnosis in life (or in 10 cases pathologically proven following cortical biopsy) was 10.5 months (95% CI: 9.8-11.2 months). A local neurologist confirmed the diagnosis in almost 70% of cases; the NCJDSU or National Prion Clinic (NPC) in 25% of cases. By the time the diagnosis was judged most likely 72% of cases were classifiable as definite or probable vCJD and 9% were classifiable as possible according to the diagnostic criteria (Table 1). Despite vCJD being considered as the most likely diagnosis, 25 cases did not fulfil the criteria for either a probable or possible case at this time. In this cohort 3 had a duration of illness of less than 6 months and thus could not be classified as a probable case and the remainder lacked some of the "core" clinical features. 20/25 cases that did not fulfil the diagnostic criteria for definite or probable vCJD had a characteristic pulvinar sign on brain MRI.

Of the ten patients undergoing cortical biopsy 7 fulfilled diagnostic criteria for probable vCJD at the time of the procedure and 8 fulfilled these criteria at some stage during life. Of the remaining two cases, one could not be classified in view of complex co-morbidity.

Twenty nine patients underwent tonsil biopsy and 28 were positive (1 provided an equivocal result), but the time to reach a confident clinical diagnosis of vCJD in these cases was similar to patients that did not undergo tonsil biopsy (mean time to confident clinical diagnosis; 10.9 months vs 10.5 months). All cases undergoing tonsil biopsy fulfilled criteria for probable vCJD at some stage and 21/29 fulfilled the criteria for a probable case prior to tonsil biopsy.

The mean time from achieving a confident clinical diagnosis to death was only 5.2 months.

Of the 148 cases in whom the diagnosis of CJD was suspected in life, 147 were referred to the NCJDSU in life. In one case vCJD was proposed as a potential diagnosis but referral to the NCJDSU was not undertaken. The overall mean time to referral was 9.8 months (95% CI: 9.2-10.6 months, representing 65% of the mean total illness duration)

Discussion

This study highlights the difficulties in achieving an early diagnosis in vCJD and this was not possible in the great majority of cases. The explanation is multi-factorial but one component is the time taken to seek an initial medical opinion, with few patients attending a primary care physician within the first few months of illness (Figure 4). This is likely to reflect the nature of the early clinical features, which are often insidious in onset, subtle in character and common in the general population. This hypothesis is supported by sub-group analysis, which showed that the cohort of individuals exhibiting early neurological features sought medical attention earlier. At the initial medical contact a variety of diagnoses were proposed, but vCJD was not considered in a single case and a neurological aetiology was suspected in only a small number of cases. This is not surprising given the frequency of psychiatric features seen by primary care physicians [START_REF] Lepine | Depression in the community: The first paneuropean study[END_REF] . Primary care physicians refer to what they consider to be the most appropriate hospital specialist, but utilisation of these services can take time and may result in delay in diagnosis if, as in vCJD, the clinical features at referral are misleading as to the true cause of the illness. Almost half the cases of vCJD were reviewed by a psychiatrist prior to neurological referral.

A neurological aetiology was usually suspected promptly following the development of objective neurological features and resulted in neurological referral in all cases (it is likely that the UK case investigated in another country also saw a neurologist).

Neurological review was arranged promptly (a mean of 16 days from referral to neurology review) indicating that delay in waiting to see a neurologist contributed little to the overall time to achieve a diagnosis, which is of note if one considers that many individuals were seen promptly when waiting time for a neurology appointment in some areas was in excess of 6 months 12, during the period of study. This also suggests that, although the diagnosis of vCJD may not have been considered at this stage, the referral to neurology was judged to be urgent because of progressive neurological deficits occurring in individuals usually in younger age groups.

The single most important determinant of early diagnosis was the presence of objective neurological features. The cohort of individuals diagnosed within 6 months had a more rapidly progressive illness course, with earlier objective neurological features, and this is likely to account for the earlier diagnosis. The reason for the short duration of illness in some cases is unknown, but may reflect variation about a mean rather than a specific biological factor.

Once suspected, the diagnosis of vCJD was confirmed relatively quickly, obviating the need for further investigations such as brain biopsy and alleviating the stress of diagnostic uncertainty for the next of kin. The study also indicates that diagnostic delay is not due to an absence of helpful diagnostic investigations, indeed one in 9 cases were initially suspected following MRI brain scan. Only rarely did brain MRI fail to support the clinical diagnosis 7/150 cases) and the majority of negative scans did not include the most sensitive MRI sequence, namely FLAIR [START_REF] Collie | Diagnosing variant Creutzfeldt-Jakob disease with the pulvinar sign: MR imaging findings in 86 neuropathologically confirmed cases[END_REF] . Tonsil biopsy was used to support the diagnosis in the minority of cases 14 , but did not lead to a significantly earlier diagnosis. However, this procedure may be of particular value in cases in which the MRI brain scan is not diagnostic or in countries with limited experience of the clinical features of vCJD.

The difficulty in achieving an accurate early diagnosis in vCJD has an important influence on patient management. Once the diagnosis is confirmed most individuals have significant neurological impairment and usually require assistance to mobilise and, after a short period, assistance to feed. Once neurological signs develop there is rapid progression with accumulating deficits with implications for management at home or in the community [START_REF] Myles | Variant Creutzfeldt-Jakob disease: costs borne by families[END_REF][START_REF] Weller | An overview of the care issues for Creutzfeldt-Jakob disease[END_REF] . There may be a need for domiciliary nursing care, adaptations to accommodation and an appropriate care package. It is therefore crucial that families who wish to care for their affected relatives at home have support made available without delay. In view of the rapid neurological deterioration in vCJD, delay in accessing appropriate aids and services is clearly inappropriate and the Department of Health have put in place a dedicated National Care Package for patients with human prion disease 15.

An analysis of changes in the time to suspect vCJD was carried out to determine whether there has been a decrease in the time to diagnose vCJD as clinicians have gained experience of this new disease. Significant reductions in time to diagnosis occurred in the early years after the identification of vCJD, but have remained stable since. This is evident in both the time to suspect vCJD (Figure 2) and the time to confirm vCJD (not shown). These findings indicate that increasing awareness of the clinical phenotype of vCJD has not resulted in an incremental decrease in time to diagnosis with the implication that further measures to increase knowledge of vCJD may not necessarily lead to an earlier diagnosis. It may be that the clinical presentation, with non-specific early features, makes prompt diagnosis intrinsically difficult.

On the other hand all but two cases of vCJD in the UK were suspected in life. The two cases in which vCJD had not been considered were significantly older than the mean and this may have led to a failure to consider the diagnosis, although in both of these cases there were complicating factors: in one case an MRI scan was carried out but did not include a FLAIR or DWI sequence and in the other the illness occurred in the context of a severe general medical condition with encephalopathy. An important question is whether cases are being overlooked in an elderly population. Although vCJD may not be as readily recognised in the elderly, the clinical phenotype does not vary with age and there is a significant decrease in mortality rates in those aged 40-60 years and older, which includes patients in age groups in which intensive investigation is likely. There is no evidence of regional variations in time to diagnose vCJD and support for the NCJDSU from clinicians in the UK has been outstanding with cases promptly referred once suspected from all regions in the UK.

This study has significant implications for therapeutic development. Extrapolation from both in-vitro and in-vivo studies indicates that to achieve significant efficacy it is likely that any proposed therapy should be introduced at an early stage of illness [START_REF] Knight | Therapeutic possibilities in CJD: patents 1996-1999[END_REF] . This paper indicates that early identification of vCJD is likely to be very difficult prior to the development of objective neurological features and raises the question as to whether accurate early diagnosis and early introduction of therapy is an achievable goal in the majority of cases. In addition the issue of diagnostic delay may have public health implications. To date, there have been four instances of secondary transmission of vCJD by blood transfusion [START_REF] Llewelyn | Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion[END_REF][START_REF] Peden | Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient[END_REF][START_REF] Wroe | Clinical presentation and premortem diagnosis of variant Creutzfeldt-Jakob disease associated with blood transfusion: a case report[END_REF][START_REF]Fourth case of transfusion-associated variant-CJD infection[END_REF] and four affected individuals have donated blood in the early clinical phase. There is also concern about the potential for secondary transmission via contaminated surgical instruments [START_REF]Patient safety and reduction of risk of transmission of Creutzfeldt-Jakob disease (CJD) via interventional procedures[END_REF] and twenty surgical interventions were carried out after symptom onset and before diagnosis (Table 6).

suggest that achieving this in vCJD is difficult and the non-specific early clinical features may prohibit early diagnosis in the majority of cases.

Ackowledgements

The National CJD Surveillance Unit is funded by the Department of Health and the Scottish Government. The surveillance system for CJD in the UK has depended on a remarkable level of cooperation from neurologist and other clinicians since 1990 and would not have been possible without the help of patients and their families.

Licence for Publication:

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JNNP and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence.

Table 1

 1 the typical appearance of the EEG in sporadic CJD consists of generalised triphasic periodic complexes at approximately one per second. These may occasionally been seen in the late stages of variant CJD. d tonsil biopsy is not recommended routinely, nor in cases with EEG appearances typical of sporadic CJD, but may be useful in suspect cases in which the clinical features are compatible with vCJD and MRI does not show bilateral pulvinar high signal. e spongiform change and extensive PrP deposition with florid plaques throughout the cerebrum and cerebellum.

		Current diagnostic criteria for vCJD
	I	A Progressive neuropsychiatric disorder
		B Duration of illness > 6 months
		C Routine investigations do not suggest an alternative diagnosis
		D No history of potential iatrogenic exposure
		E No evidence of a familial form of TSE
	II A Early psychiatric features a
		B Persistent painful sensory symptoms b
		C Ataxia
		D Myoclonus or chorea or dystonia
		E Dementia
	III A EEG does not show the typical appearance of sporadic CJD c in the early stages of
		illness
		B Bilateral pulvinar high signal on MRI scan
	IV A Positive tonsil biopsy d

Definite: IA and neuropathological confirmation of vCJD e Probable: I and 4/5 of II and IIIA and IIIB or I and IVA Possible: I and 4/5 of II and IIIA a depression, anxiety, apathy, withdrawal, delusions. b this includes both frank pain and/or dysaesthesia. c

Table 3 Proposed diagnosis following initial presentation to a medical practitioner

 3

		Number of cases	Percentage(%)
	Depression	76	50.7
	"Neurological"	27	18
	Diagnosis	19	12.7
	"unclear"
	*Other	12	8
	Anxiety	7	4.6
	"Stress"	4	2.7
	Psychotic illness	2	1.3
	Functional illness	1	0.7
	No diagnosis	2	1.3
	suggested
	Total	150	100

*"other" includeorthopaedic problems (n=3), glandular fever (n=3), an endocrine abnormality, asthma, a dental problem, a viral illness, anorexia nervosa and a grief reaction.

Table 4 : Proposed differential diagnosis at first review by a neurologist (some cases had more than one possible diagnosis)

 4

	Differential Diagnosis	Frequency
	vCJD	54
	Infectious/post-infectious	41
	Vasculitis	38
	Demyelination (MS)	34
	Wilson's disease	34
	"Metabolic disease"	29
	Malignancy/paraneoplastic	25
	Non-organic	16
	Huntington's disease	11
	Neuroacanthocytosis	8
	Drugs (illicit drug use or adverse	7
	drug reactions)
	"Encephalopathy-unknown origin"	7
	"Inflammatory disorder"	7
	Porphyria	4
	Hereditary ataxia (SCA, FA)	4
	sCJD	4
	Whipple's disease	3
	Mitochondrial disorder	3
	Posterior fossa "lesion"	3
	Other*	34
	* Other includes: "sub-acute cerebellar syndrome", complex partial status, Alzheimer's, fronto-
	temporal dementia, Parkinson's disease, normal pressure hydrocephalus, alcohol related cerebellar
	degeneration, glioma, AV fistula, Leigh's syndrome, "leukodystrophy", sub-acute combined
	degeneration, "storage disorder", limbic encephalitis, Wernicke's Encephalopathy, toxins

Table 5 Who suggested the diagnosis of CJD?

 5 Including: psychology, genetics, NCJDSU, paediatrics, accident and emergency and next of kin. ** In two cases the diagnosis was not suspected and in a single case it is impossible to determine who made the diagnosis.

	Diagnosis by:	Frequency Percentage(%)
	Neurologist	100	66.7
	Radiologist	17	11.4
	Other*	11	7.3
	Psychiatrist	9	6.0
	GP	8	5.3
	Not suspected**	3	2.0
	Physician	2	1.3
	Total	150	100

*

Table 6 Regional variation in "time to suspect CJD"

 6

	Region	No of cases Mean time months (95% CI)
	South East England	38	8.6 (7.6 to 9.7)
	North West England	24	9.6 (8.1 to 11.1)
	Scotland	21	8.3 (6.7 to 9.9)
	North East England	18	8.3 (6 to 10.7)
	South West England	16	9.6 (7 to 12.2)
	"Midlands"	11	9.7 (4.8 to 14.7)
	"Yorkshire"	11	10.1 (7.5 to 12.7)
	Wales	5	8.6 (5.8 to 11.3)
	Northern Ireland	3	5.1 (0 to 12.7)
	Total	147	8.9 (8.3 to 9.7)

"The Midlands"= Birmingham, Nottingham, Leicester "Yorkshire"= Leeds, Bradford, Sheffield

Table 7 Surgical procedures* (n=20) after onset and before diagnosis in 18/150 cases.

 7

	Case	Procedure
	1	Sutures to eyelid following road traffic accident
	2	Caesarian section
	3	Colonoscopy
	4	Tooth extraction
	5	Fenton's procedure (repair of episiotomy)
	6	Skin removed surgically after burn to foot
	7	Caesarian section
	8	Wisdom teeth removed
	9	Removal of epididymal cyst left testicle
	10	Arthroscopy
		Elevation of left nasal bone
	11	Caesarian section
	12	Caesarian section
	13	Pins and plate inserted in ankle
	14	Hysteroscopy
	15	Endoscopy and small bowel biopsies
	16	MUA and percutaneous K wire second left metacarpal
		Hand infected -K wire removed
	17	Watson/Jones reconstruction of ankle
	18	Sutures to eyebrow

*procedures relating to work up of the current diagnosis are not included

Figure 4 Time course in variant CJD in relation to the diagnostic process (Mean time in months)

			1 st point of	Neuro referral Ne uro CJD su spected
			medical contact	review		CJD " confirmed "
	Onset						Death
	Time	0	3.4	7.4	8.9	10.5	15.7 months
	(


Theodoros N Sergentanis
email: tsergentanis@sni.gr

Konstantinos P Economopoulos

Validating the meta-analytical results on MDM2, CASP8, XRCC3 polymorphisms and breast cancer risk: examination of Hardy-Weinberg Equilibrium

Validating the meta-analytical results on MDM2, CASP8, XRCC3 polymorphisms and breast cancer risk: examination of Hardy-Weinberg Equilibrium

Dear Editor,

Following the valuable methodological remarks by Mao et al. [1] regarding our metaanalysis on glutathione-S-transferase polymorphisms and breast cancer risk [2], we decided to present elaborate sensitivity analyses on Hardy-Weinberg Equilibrium (HWE) in our three previous meta-analyses examining MDM2 SNP309 [3], CASP8 [4] and XRCC3 Thr241Met [START_REF] Economopoulos | XRCC3 Thr241Met polymorphism and breast cancer risk: a meta-analysis[END_REF] polymorphisms and breast cancer risk. Sensitivity analyses were performed excluding studies whose allele frequencies in controls exhibited significant deviation from HWE, given that the deviation may denote bias [START_REF] Thakkinstian | A method for meta-analysis of molecular association studies[END_REF]. For the assessment of the deviation from HWE, the appropriate goodness-of-fit chi-square test was performed [START_REF] Thakkinstian | A method for meta-analysis of molecular association studies[END_REF][START_REF] Rohlfs | Distributions of Hardy-Weinberg equilibrium test statistics[END_REF].Of note a deviation from HWE in a mixed control population was allowed as the underlying assumptions of HWE are not fulfilled [START_REF] Yu | systematic review of the relationship between polymorphic sites in the estrogen receptor-beta (ESR2) gene and breast cancer risk[END_REF]. For the evaluation of the goodness-of-fit chi-square test, statistical significance was defined as p<0.05.

Concerning MDM2 SNP309 polymorphism only one study [START_REF] Schmidt | Do MDM2 SNP309 and TP53 R72P interact in breast cancer susceptibility? A large pooled series from the breast cancer association consortium[END_REF] included controls deviating from HWE. Significant deviation from HWE was noted in the controls of the Chinese part in the study by Haiman et al. [START_REF] Haiman | A promoter polymorphism in the CASP8 gene is not associated with cancer risk[END_REF] regarding CASP8 -652 6N del polymorphism; no deviation was noted regarding studies on CASP8 D302H polymorphism. With respect to XRCC3 Thr241Met polymorphism, deviation was noted in three individual case-control studies [START_REF] Costa | DNA repair polymorphisms might contribute differentially on familial and sporadic breast cancer susceptibility: a study on a Portuguese population[END_REF][START_REF] Dufloth | DNA repair gene polymorphisms and susceptibility to familial breast cancer in a group of patients from Campinas, Brazil[END_REF][START_REF] Krupa | Polymorphism of the homologous recombination repair genes RAD51 and XRCC3 in breast cancer[END_REF], as well as the Madrid study of the article published by the Breast Cancer Association Consortium [START_REF]Commonly studied singlenucleotide polymorphisms and breast cancer: results from the Breast Cancer Association Consortium[END_REF].

We are happy to report that the sensitivity analysis replicated all the results pertaining to MDM2 SNP309 and XRCC3 Thr241Met. Concerning CASP8 -652 6N del polymorphism, it should be declared that pooling of Chinese studies was no more feasible at the sensitivity analysis, as only one study remained. Of note, no sensitivity analysis was needed regarding CASP8 D302H in the light of no deviation. Detailed results are presented in the Table .   Taken as a whole, this Letter supports the validity of our previously published results as they proved robust enough to persist at the sensitivity analysis presented herein.

Table:

Pooled Odds Ratios (ORs) as initially published in the meta-analyses regarding MDM2 SNP309, CASP8 -652 6N del, CASP8 D302H and XRCC3 Thr241Met polymorphisms, as well as pooled ORs after the exclusion of studies whose controls significantly deviated from Hardy-Weinberg Equilibrium. Bold cells denote statistically significant ORs. OR: Odds Ratios; CI: confidence interval; HWE: Hardy-Weinberg Equilibrium; R: Random effects; §: in non-mixed studies. We read with great interest the article by Sergentanis et al [1]. The authors performed sensitivity analyses on Hardy-Weinberg Equilibrium (HWE) in their three previous meta-analyses and concluded that the results of their previous studies were robust. We thank them for accepting our suggestion on sensitivity analyses by excluding the HWE-violating studies, but we are concerned about the following issues.

Models

First, the authors stated that deviation from HWE in a mixed control population was allowed as the underlying assumptions of HWE are not fulfilled. They cited the article by Yu et al as a reference [2]. However,this reference was not an original article for this issue. In addition, even if deviation from HWE in a mixed control population was allowed, it is more appropriate to consider the studies with a mixed control population as an independent group when performing subgroup analyses or sensitivity analyses. However,the author neglected this point when they performed subgroup analyses or sensitivity analyses. They just divided ethnicity into Chinese and non-Chinese in their two previous studies.

Second,for MDM2 SNP309 polymorphism, the OR with 95%CI for heterozygous changed from 1.056 (1.000-1.115) to 1.077 (1.002-1.158) when they performed sensitivity analyses by excluding HWE-violating studies. The results for this genetic model should be interpreted with caution.

Third, for CASP8 -652 6N del, CASP8 D302H and XRCC3 Thr241Met polymorphisms, the authors stated that significant heterogeneity was detected in some comparisons. As we know, heterogeneity is a potential problem that may affects the interpretation of the results. It is important to present the changes for heterogeneity when the authors performed sensitivity analyses by excluding HWE-violating studies. Maybe departure from HWE is the main factor contributing to substantial heterogeneity Fourth, the authors published six meta-analyses on the journal of Breast Cancer Research and Treatment. They only presented data for three studies. Even if the results of these three studies are robust, it does not guarantee the robust results of the remaining studies. Take the article for GSTP1 polymorphism as an example [3], when sensitivity analyses were performed by excluding one HWE-violating study, the results for Chinese population were materially changed [4].

Furthermore, considering the influence of sample-sizes, heterogeneity and publication bias on the results of meta-analysis, it may not be appropriate for the authors to conclude that their previously published results were robust as proved by sensitivity analyses.