Francesco Sardanelli
email: f.sardanelli@grupposandonato.itfrancesco.sardanelli@unimi.it

Prof Francesco Sardanelli

Additional Findings at Preoperative MRI. A Simple Golden Rule for a Complex Problem?

Additional Findings at Preoperative MRI. A Simple Golden Rule for a Complex Problem?

Preoperative magnetic resonance imaging (MRI) is a hot topic, a complex problem which probably will remain unresolved for several years.

The starting point is the great body of evidence for the higher sensitivity of MRI compared with mammography and ultrasonography (US) in detecting malignant lesions in the breast harboring the index cancer [START_REF] Houssami | Accuracy and surgical impact of magnetic resonance imaging in breast cancer staging: systematic review and meta-analysis in detection of multifocal and multicentric cancer[END_REF] and in the contralateral breast [START_REF] Brennan | Magnetic resonance imaging screening of the contralateral breast in women with newly diagnosed breast cancer: systematic review and meta-analysis of incremental cancer detection and impact on surgical management[END_REF]. However, the discussion about advantages [START_REF] Sardanelli | Overview of the role of pre-operative breast MRI in the absence of evidence on patient outcomes[END_REF] and disadvantages [START_REF] Solin | Counterview: Pre-operative breast MRI (magnetic resonance imaging) is not recommended for all patients with newly diagnosed breast cancer[END_REF] is challenging [START_REF] Sardanelli | Magnetic resonance imaging of the breast: recommendations from the EUSOMA working group[END_REF].

Potential advantages of preoperative MRI can be summarized as follows:

1. better short-term patient outcome (reduced re-excision rate; reduced amount of removed tissue due to a tailored surgical strategy); 2. better mid-term patient outcome (reduced rate of ipsilateral local recurrence and contralateral cancer); 3. better long term patient outcome (longer disease free and overall survival); 4. better psychological patient status due to the use of the most sensitive tool for local staging.

However, none of these potential advantages have been clearly demonstrated by high-level studies, i.e. by a large multi-institutional randomized controlled trial (RCT). On the other side, drawbacks of preoperative MRI can be outlined as follows:

1. overtreatment due to false positive MRI findings; 2. overdiagnosis and overtreatment of malignant additional lesions which could have been cured by radiotherapy and/or systemic adjuvant chemotherapy or hormone therapy; 3. delay in definition of surgical strategy due to difficult managing of MRI additional findings, especially those visible only at MRI, hence requiring an MR-guided needle biopsy and/or localization; 4. increase in patient anxiety due to treatment delay and/or uncertainties related to the interpretation and management of additional findings.

Obviously, advantages and disadvantages can be mixed in variable combinations. The problem is hard because we have in our hands a technique surely being the best option for evaluating ipsilateral disease extent and possible contralateral cancers but we are not sure that, using this technique, we have a better treatment for our patients. We could have a worse (i.e., an avoidable more aggressive) treatment. Moreover, patients know that MRI is highly sensitive and self-referred presentation is possible.

Looking at the secondary evidence, meta-analyses found a rate of more aggressive surgery for true positive MRI findings of 11.1% for the ipsilateral breast [START_REF] Houssami | Accuracy and surgical impact of magnetic resonance imaging in breast cancer staging: systematic review and meta-analysis in detection of multifocal and multicentric cancer[END_REF] and 4.1% for the contralateral breast [START_REF] Brennan | Magnetic resonance imaging screening of the contralateral breast in women with newly diagnosed breast cancer: systematic review and meta-analysis of incremental cancer detection and impact on surgical management[END_REF]. The rate of MRI-induced ipsilateral wider surgical treatment should be compared with the rate of positive margins after breast conserving treatment (BCT), reported to be from 20% to 40% or more, and that of local recurrences after BCT, usually considered from 5% to 10% at ten years and reported about 9% at 20 years. The rate of MRI-induced contralateral surgery for synchronous cancer should be compared with 0.5-1% annual risk of contralateral breast cancer in women with a previous history of breast cancer [START_REF] Sardanelli | Overview of the role of pre-operative breast MRI in the absence of evidence on patient outcomes[END_REF]. Notably, only ipsilateral recurrences or contralateral cancers which would have appeared in the first years after a conservative treatment might be avoided by pre-operative MRI. Thus, we obtain a relatively balanced result for the contralateral breast: 3-4% of MRI-detected contralateral cancers versus 2-3% cumulative rate of expected contralateral cancers. Conversely, we have an 11% of MRI-induced wider ipsilateral surgery versus and only 2-3% of cumulative rate of expected ipsilateral recurrences [START_REF] Sardanelli | Overview of the role of pre-operative breast MRI in the absence of evidence on patient outcomes[END_REF].

As a consequence, the risk of overdiagnosis and overtreatment by preoperative MRI is probable at least for the ipsilateral breast. However, we must consider that: a) the rate of MRI-detected ipsilateral and contralateral cancers is probably overestimated due to the fact that preoperative MRI has been performed in non-consecutive (selected) patients with a probable higher likelihood of these lesions; b) a publication bias can be hypothesized; c) ipsilateral and/or contralateral overtreatment could be compensated by the reduction of additional surgical interventions needed to achieve free margins; d) considering the role of radiation and systemic therapy, a patient-based perspective should evaluate the combined effect on both breasts. This last point means that MRI could determine an unnecessary ipsilateral excision but also anticipate the diagnosis of a contralateral canceror vice versaavoiding the future second cancer event. For an individual patient, preoperative MRI may determine a spectrum of possibilities from a bilateral advantage to no change of treatment planning to a bilateral overtreatment [START_REF] Sardanelli | Overview of the role of pre-operative breast MRI in the absence of evidence on patient outcomes[END_REF]. Finally, if preoperative MRI would be demonstrated to increase free disease or overall survival, overdiagnosis and overtreatment would change in simple diagnosis and treatment. But this is beyond the current strategic horizon.

A shared consensus was recently reached by an interdisciplinary working group promoted by the European Society of Breast Cancer Specialists (EUSOMA) in considering the following four indications for preoperative MRI [START_REF] Sardanelli | Magnetic resonance imaging of the breast: recommendations from the EUSOMA working group[END_REF]:

1. patients newly diagnosed with an invasive lobular cancer;

2. patients at high-risk for breast cancer;

3. patients under 60 years of age with discrepancy in size>1 cm between mammography and US with expected impact on treatment decision; 4. patients eligible for partial breast irradiation on the basis of clinical breast examination and conventional imaging.

My personal view is that other two groups of women could be considered for preoperative MRI [START_REF] Sardanelli | Overview of the role of pre-operative breast MRI in the absence of evidence on patient outcomes[END_REF]: 5. women with mammographically heterogeneously or extremely dense breast; and 6. women with a multifocal or multicentric or bilateral cancer already diagnosed at conventional imaging.

The indication concerning invasive lobular cancer is in agreement with the study recently published by Mann et al in this journal [START_REF] Mann | The impact of preoperative breast MRI on the re-excision rate in invasive lobular carcinoma of the breast[END_REF]. With the limitation of a retrospective design, they demonstrated a significant reduction in the re-excision rate in the women who did undergo preoperative MRI (9%) versus those who did not (27%) without any increase either of mastectomy rate (48% versus 59%) or mean time to final pathology (40 versus 44 days, respectively) [START_REF] Mann | The impact of preoperative breast MRI on the re-excision rate in invasive lobular carcinoma of the breast[END_REF]. Thus, the paradigm is inverted showing the possibility to use MRI preoperatively with the aim to reduce the aggressiveness of surgical treatment.

However, all nonrandomized studies on this matter have intrinsic bias and limitations. This is true also for other retrospective studies which reported results in favor [START_REF] Fischer | The influence of preoperative MRI of the breasts on recurrence rate in patients with breast cancer[END_REF] or partially in favor [START_REF] Pengel | The impact of preoperative MRI on breast-conserving surgery of invasive cancer: a comparative cohort study[END_REF] of preoperative MRI as well as for retrospective studies which reported results against [START_REF] Solin | Relationship of breast magnetic resonance imaging to outcome after breast-conservation treatment with radiation for women with early-stage invasive breast carcinoma or ductal carcinoma in situ[END_REF][START_REF] Hwang | Magnetic resonance imaging in the planning of initial lumpectomy for invasive breast carcinoma: its effect on ipsilateral breast tumor recurrence after breastconservation therapy[END_REF][START_REF] Bleicher | Association of routine pretreatment magnetic resonance imaging with time to surgery, mastectomy rate, and margin status[END_REF] preoperative MRI. Two interesting small studies from a Japanese group showed important advantages of a surgical approach precisely tailored on the basis of preoperative MRI performed in supine surgical position. In one non randomized study [START_REF] Nakamura | Breast-conserving surgery using supine magnetic resonance imaging in breast cancer patients receiving neoadjuvant chemotherapy[END_REF], they demonstrated significantly reduced excision area and additional excision rate compared to US-guided dye application in the surgical treatment of invasive ductal carcinomas after neoadjuvant chemotherapy. In a second study [START_REF] Sakakibara | Breast-conserving surgery using projection and reproduction techniques of surgical-position breast MRI in patients with ductal carcinoma in situ of the breast[END_REF], they randomized 52 patients with localized ductal carcinoma in situ: 24 of them received breast conserving surgery (BCS) using projection and reproduction techniques of surgical-position breast MRI; 28 of them underwent conventional prone-position breast MRI and BCS using mammography-guided hookwire. Average volume of the pathologic specimens in the new technique group was significantly smaller than that in the conventional BCS group (27.5 cm 3 versus 57.6 cm 3 ) while the positive margin rate was significantly lower (12.5% versus 39.3%, respectively). These experiences indicate that a precise MRI-based tailored BCS is the way for the future, as is shown also by a recent research on US navigation of breast MRI volumes acquired in supine position [START_REF] Alderliesten | On the feasibility of MRI-guided navigation to demarcate breast cancer for breast-conserving surgery[END_REF]. The key point is the translation of a three-dimensional (3D) information from the MR room to the operating theatre.

Preoperative MRI is a typical matter which should be solved by means of large RCTs. This is due to the fact that this use of breast MRI is not for a simple diagnosis (we are already aware of the index cancer). It may seem a paradox, but preoperative MRI is a kind of screening looking for other cancers than the index one. If the results of preoperative MRI impact on treatment, this diagnostic tool should be regarded as a therapy.

Few months ago the results of the COMICE trial came to publication [START_REF] Turnbull | Comparative effectiveness of MRI in breast cancer (COMICE) trial: a randomised controlled trial[END_REF]. The investigators enrolled more than 1,800 women with a a newly diagnosed breast cancer randomized into two groups, with (n=816) and without (n=807) preoperative MRI. The reoperation rate (primary end-point) was 19% in both groups; the total mastectomy rate was 13% versus 9%, respectively. These results are important. They show the possible consequences of a suboptimal use of preoperative MRI. Several limitations of the study have been already discussed [START_REF] Morris | Should we dispense with preoperative breast MRI?[END_REF]. Importantly, the investigators faced a very slow enrollment by a large number of centers, with many of them enrolling less than 4-5 patients/year, only 2-3 randomized to MRI. The results show no systematic use of needle biopsy and localization (including MR-guided procedures), with many cases of changed treatment due to unverified false positive findings. Regarding the initial surgical treatment in the MRI arm, the investigators say: "Of the 58 patients who underwent a mastectomy, 32 had an additional biopsy, 11 did not have a biopsy, and data were unavailable for the remaining 15 patients. Of the 16 patients who underwent an avoidable mastectomy, three did and six did not have a biopsy, and data are missing for the remaining seven patients" [START_REF] Turnbull | Comparative effectiveness of MRI in breast cancer (COMICE) trial: a randomised controlled trial[END_REF].

In this large RCT, the management of MRI additional findings found has been a key point. In this perspective, an expert use of second look targeted US is essential. This requires not only to be familiar with (supine) breast US but also with (prone) breast MRI as well as with the different lesion location at the two imaging techniques. Six recent studies reported percentages of US correlate for initially MR-detected findings variable from 46% to 82% [START_REF] Linda | Outcome of initially only magnetic resonance mammography-detected findings with and without correlate at second-look sonography: distribution according to patient history of breast cancer and lesion size[END_REF][START_REF] Demartini | Utility of targeted sonography for breast lesions that were suspicious on MRI[END_REF][START_REF] Destounis | Community-based practice experience of unsuspected breast magnetic resonance imaging abnormalities evaluated with second-look sonography[END_REF][START_REF] Meissnitzer | Targeted ultrasound of the breast in women with abnormal MRI findings for whom biopsy has been recommended[END_REF][START_REF] Abe | MR-directed ("Second-Look") ultrasound examination for breast lesions detected initially on MRI: MR and sonographic findings[END_REF][START_REF] Carbognin | Utility of second-look ultrasound in the management of incidental enhancing lesions detected by breast MR imaging[END_REF]. Pooling their results, we have a total of 1,208 findings, 759 of them with a US correlate (63%). While the mean malignancy rate is 27% (range, 22%-29%), that of lesions with a US-correlate is 32%, (range, 27%-36%), and that of lesion without a US correlate is 18% (range, 10%-28%), showing a variability of results probably depending also on the specific experience and expertise for targeted US examination. Thus, MR-guided procedures are necessary only in a minority of cases, those where targeted US fails to find the MRI finding. However, the rate of malignancy of MRI findings without US correlate does not go below 10% also when the rate of US detection reaches 82% [START_REF] Linda | Outcome of initially only magnetic resonance mammography-detected findings with and without correlate at second-look sonography: distribution according to patient history of breast cancer and lesion size[END_REF] In this context, in the current issue of this journal, Lotte E. Elshof et al [START_REF] Elshof | A practical approach to manage additional lesions at preoperative breast MRI in patients eligible for breast conserving therapy: results[END_REF] report their experience on 690 consecutive patients with 698 pathology-proven index cancers planned for BCT based on clinical examination and conventional imaging who underwent preoperative breast MRI. MRI additional findings were prospectively managed without using MR-guided procedures. They defined additional findings located within a 3-cm 3D space including the index tumor as "multifocal", those located outside that space as "multicentric", those in the other breast as contralateral. Multifocal findings were not sent to targeted US and these findings typically led to BCS with larger excision to include the additional findings. Multicentric and contralateral findings were sent to targeted US. If they were found, needle biopsy was performed. If malignant disease was confirmed over a region too large to allow cosmetically acceptable conserving treatment, mastectomy was planned. The key rule of this approach is as follows: If pathology proof could not be obtained (i.e., if targeted US failed to find the lesion), the therapy plan was not changed and follow-up was advised.

At preoperative MRI, 141 additional findings were detected in 121/690 patients (18%). MRI additional findings without pathology proofnamed unidentified bright objects (UBOs) by the authorswere found in 81/690 patients (12%). Importantly, of 141 additional findings, 44 multicentric and contralateral findings (31%) were followed up at least with conventional imaging (40 of them being MRI BI-RADS 3).

The median follow up was 55 months (range, 22-103 months). In none of the patients these findings turned out to be detected as suspicious and confirmed to be malignant (only four additional findings were lost to follow-up). Multifocal lesions were significantly more often malignant than multicentric and contralateral lesions.

This approach resulted in a rate of change of surgical planning inferior to 10%, composed of mastectomy for 24 breasts in 23/690 patients (3.3%), wider ipsilateral excisions in 40/690 patients (5.8%), and contralateral excision in 3/690 (0.4%). Of 40 wider ipsilateral excisions, 32 (80%) were pathologically confirmed after surgery.

This experience contains a golden rule and a possible misleading message, the latter already acknowledged by the authors. The golden rule is: Do not convert a BCS to mastectomy on the basis of MRI additional finding(s) not pathologically verified to be malignant. The possible misleading message is: The use of MRguidance is not necessary in the preoperative setting.

To perform mastectomy or contralateral surgery for MRI additional findings without needle biopsy verification of malignancy should be regarded as malpractice. Moreover, MR-guided biopsy should be integrated in clinical practice as it was for needle biopsy under stereotactic guidance. A large experience in MR-guided biopsy is reported in the literature, including a large prospective multi-institutional study of 538 lesions [START_REF] Perlet | Magnetic resonance-guided, vacuum-assisted breast biopsy: results from a European multicenter study of 538 lesions[END_REF], many recent single-center experiences [START_REF] Tozaki | MR-guided vacuum-assisted breast biopsy: is it an essential technique?[END_REF][START_REF] Han | Outcome of MRI-guided breast biopsy[END_REF][START_REF] Khouli | MRI-guided vacuum-assisted breast biopsy: a phantom and patient evaluation of targeting accuracy[END_REF][START_REF] Li | MRI follow-up after concordant, histologically benign diagnosis of breast lesions sampled by MRI-guided biopsy[END_REF][START_REF] Friedman | Magnetic resonance imaging-guided vacuum-assisted breast biopsy: an initial experience in a community hospital[END_REF][START_REF] Malhaire | Vacuum-assisted biopsies under MR guidance: results of 72 procedures[END_REF][START_REF] Schrading | MRI-guided breast biopsy: influence of choice of vacuum biopsy system on the mode of biopsy of MRI-only suspicious breast lesions[END_REF], also evaluating problems and limitations [START_REF] Meeuwis | Targeting difficult accessible breast lesions: MRI-guided needle localization using a freehand technique in a 3.0 T closed bore magnet[END_REF][START_REF] Liberman | Underestimation of atypical ductal hyperplasia at MRI-guided 9gauge vacuum-assisted breast biopsy[END_REF][START_REF] Lee | Underestimation of DCIS at MRI-guided vacuum-assisted breast biopsy[END_REF][START_REF] Lee | Imaging histologic discordance at MRI-guided 9-gauge vacuumassisted breast biopsy[END_REF], as well as the results of a multidisciplinary consensus meeting on the use of vacuum-assisted MR-guided biopsy [START_REF] Heywang-Köbrunner | Consensus Group (2009) Interdisciplinary consensus on the uses and technique of MR-guided vacuum-assisted breast biopsy (VAB): results of a European consensus meeting[END_REF]. The mean time needed for an MR-guided procedure is not different than that of a stereotactic biopsy in the film-screen era: about 45 minutes [START_REF] Fischer | Effort, accuracy and histology of MRguided vacuum biopsy of suspicious breast lesions--retrospective evaluation after 389 interventions[END_REF]. Using MR-guidance, the rate of unnecessary ipsilateral wider excisions reported by Elshof et al [START_REF] Elshof | A practical approach to manage additional lesions at preoperative breast MRI in patients eligible for breast conserving therapy: results[END_REF] -8/40 (20%)could have been drastically reduced. Also MRguidance has limitations and some findings can be difficult or impossible to be reached. In these cases, the golden rule by Elshof et al should be applied. Notably, the application of this rule demonstrated that multicentric and contralateral findings without US correlate were not detected as malignant at follow up. However, we do not know how many of them were benign and how many of them were malignant but cured by radiation or systemic therapy.

The general recommendations of the EUSOMA interdisciplinary working group still appear to be valid:

1. irrespective of whether the clinical team routinely uses preoperative MRI or not, women newly diagnosed with breast cancer should always be informed of the potential risks and benefits of preoperative MRI if this is under consideration prior to therapy; 2. results of preoperative MRI should be interpreted taking into account clinical breast examination as well as mammography and US (whenever mammography and US are indicated); 3. MRI findings with impact on patient treatment should be verified by percutaneous biopsy whenever possible; 4. lesions visible on MRI alone require MR-guidance for needle biopsy with pathological assessment and, if needed, presurgical localization, implying the availability of specialized equipment and personnel; 5. the total treatment delay due to preoperative MRI and possible workup should be no longer than 1 month; 6. possible changes in therapeutic planning resulting from the findings of preoperative MRI should be decided by a multidisciplinary team [START_REF] Sardanelli | Magnetic resonance imaging of the breast: recommendations from the EUSOMA working group[END_REF].

At any rate, the use of preoperative MRI remains an open issue and high-quality research using the patient's outcome as primary end-point is still warranted.


PhD Rebecca Golley

PhD Ed Baines

MSc Paul Bassett

PhD Lesley Wood

MSc Jo Pearce

PhD Michael Nelson
email: michael.nelson@sft.gsi.gov.uk

S1 4pq

School lunch and learning behaviour in primary schools: an intervention study

Keywords: School lunch, behaviour, learning, primary school, UK, intervention study

Background/Objectives: In addition to the nutritional benefits of healthier school food, anecdotes describe improvements in children's behaviour and educational outcomes when school food or the school dining room environment is improved. This study hypothesised that a school food and dining room intervention would improve pupils' learning-related classroom behaviour.

Subjects/Methods: A controlled intervention trial involving six primary schools matched in triplets and randomly assigned to a 12 week intervention (promotion of healthier school food at lunchtime and changes in the school dining environment) or 12 week waitlisted control group. Study outcome was learning-related behaviours measured in a random sample of 146 pupils in years 3-5.

Results: On-task and off-task behaviours were observed and used as proxy measures for concentration and disengagement (disruption) respectively. Teacher-pupil on-task engagement was 3.4 times more likely in the intervention schools compared with the control schools (adjusted model OR=3.40 (95% CI: 1.56, 7.36), p=0.009). However, on-task pupilpupil behaviour was less likely in the intervention group (adjusted model OR=0.45 (95% CI: 0.28, 0.70), p<0.001). Similarly, off-task pupil-pupil behaviour was more likely in the intervention group than in the control group in both the unadjusted model (OR=2.18 (95% CI:

1.52, 3.13), p<0.001) and the adjusted model (OR=2.28 (95% CI: 1.25, 4.17), p=0.007).

Conclusions:

This study offers some support for the hypothesis that a school food and dining room intervention can have a positive impact on pupils' alertness. However, if raised alertness is not channelled and supervised, it may result in increased off-task behaviour when pupils are working together.

Introduction

Food provided by schools at lunch and other times of the day has the potential to support children's nutrition, growth and development (School Meals Review Panel, 2005). The reintroduction of standards (School meals review panel 2005, Statutory Instrument 2000No. 1777, Statutory Instrument 2006No. 2381, Statutory Instrument 2007No. 2359) and Government investment in school food in England aims to facilitate food consumption and nutrient intake in keeping with Government healthy eating guidelines.

Anecdotes from teachers and parents describe improvements in children's concentration, behaviour, learning and academic performance when healthier school food is introduced.

Additionally, best practice guidance promotes improvements in the school dining environment to support children's behaviour, well being and learning (School Meals Review Panel, 2005). These wider benefits of improved school food are not well supported by existing evidence.

Studies exploring the benefits of school food generally lack clear and objective behavioural and educational endpoints; have been carried out in populations not generalisable to the wider school community; or have focused on the role of single foods, nutrient or meals in mediating educational improvements, but not addressed the way in which nutrition interventions themselves alter the environment in which children's behaviour is being evaluated, nor the potential interactions between nutritional and environmental interventions (Ells et al, 2008).

Only two studies cited in the University of Teeside review (Ells et al, 2008) explored school lunches and educational outcomes (Dunifon & Kowaleski-Jones, 2002, Gietzen & Vermeersh 1980). Neither explored the impact of better eating at lunchtime on classroom learning behaviour. A recent study (Belot & James, 2009) comparing the impact of a campaign to improve school lunches in the London Borough of Greenwich against seven similar 'control' boroughs (i.e. no campaign) showed better average academic achievement scores at age 11 in English and Science and reduced authorised absenteeism. There were no direct measures of learning behaviour.

In the present study, it was hypothesised that providing and promoting healthier school food at lunch time and improving the school dining room environment (e.g. physical setting, lunchtime routine or lunchtime behaviour management) would result in improvements in pupils' learning-related behaviour in the classroom after lunch.

Methods

Study design

A 12-week controlled trial involving six primary schools in Sheffield, England, matched in triplets (see Figure 1) with school pairs randomly assigned to one of three treatment groups: Nutrition first (NF): introduction and promotion of healthier school food at lunchtime over six weeks followed by changes in the school dining environment Environment first (EF): changes in the school dining environment over six weeks followed by the introduction and promotion of healthier school food at lunchtime Control: wait-listed schools in which there was no intervention for 12 weeks. Control schools were then provided with intervention support. The primary study outcome was the learning-related behaviour of a randomly selected group of pupils in the post-lunch period. This was assessed by trained observers in the classroom setting.

Treatment order (nutrition first versus environment first) was randomly allocated within the design to assess their independent effects. In practice, however, the two intervention components were undertaken simultaneously (not sequentially), so analysis of their independent effects was not possible. The results have therefore been analysed as a single (multi-component) intervention at the end of 12 weeks rather than by phase of intervention delivery.

The study was approved by the University of Sheffield Research and Ethics Committee.

Study schools

Sheffield primary schools within a 10 mile radius of the School Food Trust office willing to undertaking a dining room intervention in Spring term 2007 were invited to participate.

Interested schools were screened (Figure 1), with six schools selected by researchers in order to achieve three pairs of schools best matched for; baseline food provision (i.e. toward the interim food-based standards, Statutory Instrument 2006 No. 2381) and the dining environment indicating room for improvement, type of catering provider, school roll, free school meal eligibility, Healthy School status and school meal take up. The three pairs of schools were then randomly assigned to study group.

Study intervention

The intervention comprised both nutrition and dining room components which together facilitated improvements in the provision, promotion and consumption of school food and to the dining room environment. The intervention was designed to be representative of the dining room recommendations promoted in current guidelines (North Yorkshire Business and Education Partnership, 2004) and was based on a checklist designed to assess the quality of the dining room environment. To achieve a standardised intervention, a framework was used to enable schools to implement a tailored and flexible intervention underpinned by a consistent process, content themes and timeline. Food provision was assessed against Government standards and the take up of school meals was also recorded. Intervention components were categorised in five themes under two headings; 'nutritional characteristics' (food provision and food promotion) and 'environmental characteristics' (dining space, lunchtime management and good relations) (Figure 1).

Pupil sample

Schools nominated one year four class (in one school the nominated class was a combined year 3/4 class, with two study pupils in year three) and one year five class to be observed.

Parents and pupils in the study classes were sent a study information sheet and asked to participate. Within each school, 24 pupils were randomly selected via random number generation (stratified for year level, gender, attainment level and whether they were registered as taking a school meal or packed lunch; and excluding those declining participation (2%) or children with acute or chronic illness based on school records). Packed lunch pupils were included because they were potentially influenced by the interventions.

Data collection

School and pupil characteristics Demographic information was collected at baseline (attainment level, gender, birth date, ethnicity, English as an additional language, usual type of lunch eaten (i.e. school lunch or packed lunch), free school meal registration and special needs status

Food and dining room environment

A 91-item checklist was developed for the study by reviewing the literature to identify factors that shape the school lunch dining service. The checklist had four sections; an interview conducted with the head teacher and head caterer, a menu checker (for compliance with standards), a pre-service observation checklist and a during-service observation checklist. Nutritional characteristics covered; food provision (compliance with the food based standards for school lunch 2006 and 2008, cooking practices and variety), menu promotion (price, menu visibility/accessibility, dining room rules/contingencies, taster sessions, parent/pupil involvement, links with packed lunches and incentives). Environmental characteristics included aspects of the dining space (appearance, layout, blockage points), lunchtime management (management of space and pupil numbers, noise levels and behaviour management) and good relations (staff training, presence, involvement and value as well as pupil ownership of the lunch service). Questions relating to either school lunch provision or school dining room were summed for nutritional and environmental sub-scores. These were totalled for the final dining room checklist score. Responses were scored with a higher score indicative of a positive dining room environment.

Systematic classroom observation

Systematic observation (SO) (Blatchford et al, 2006) provided a direct measure of pupil learning-related behaviours in the classroom without reliance on pupil or teacher reported behaviour. SO builds up data by recording, in real time, the incidence of clearly defined low inference behaviours.

Observation categories

The observation schedule was modified from Blatchford et al, 2006. It comprised categories that provided a description of time spent in different work settings (Individual, Pupil-pupil interaction, Adult led group, Whole Class, Pupil Plenary, Other), teaching topic, and social modes (interacting with their teachers, interacting with other children, not interacting). Within each of these three social modes were mutually exclusive categories that covered engagement in work, procedural, social and off-task activity. Observation categories addressed learning behaviours of interest to the research question, looking at pupil concentration (being 'ontask'), disengagement/ disruption ('off-task') and pupil mood.

Organisation of Observations

Observations of each 'target' child were collected over five minutes in ten 30-second blocks: 10 seconds for observers to 'tune in' to the target's behaviour, 10 seconds to observe behaviour, and 10 seconds to record what took place during the observation period. After each block of ten observations, observer attention switched to the next pupil. Each observer collected observations on 12 selected 'target' pupils over approximately one hour in the same classroom on five consecutive days. The order in which pupils were observed differed each day.

Fieldworkers and training

Six observers were recruited from the National Centre for Social Research (NatCen), an, independent UK social research institute. Over two days of training, observers focused on how to use the behavioural codes, the sampling framework and schedule. Observers practised coding (with periodic checks of accuracy and category understanding) using classroom video tape footage, followed by in-class observations. Weekly support sessions were held to discuss field visits. A retraining session was held prior to follow up visits. Observers were blinded to school study group allocation.

Fieldworker reliability checks

Initial training, inter-coder reliability coefficients for the main sets of mutually exclusive categories were good to high with Kappa coefficients ranging from 0.65 -0.98. Reliability coefficients for the recalibration session were fair to good ranging from 0.48 -0.87. For the ratings of observed mood, however, Kappa was low (0.23). Observed mood was not therefore included in the analytical model.

Data analysis

Data were entered in to a spreadsheet by two experienced personnel using pre-specified ranges. Five percent of cases were checked for errors and found to be within acceptable limits (less than 5% errors) and a range of validation and logical checking techniques were used to detect and remove any errors that remained. Data was imported into SPSS (SPSS Inc, Chicago, 2006) and MLWin (Goldstein, 1998) for analysis.

Multilevel statistical analysis of the systematic observation data, was undertaken appropriate for a cluster randomised trial, (Goldstein, 1995;Paterson & Goldstein, 1991). The 10-second observation interval was the unit of analysis with observation outcomes at each time point recorded as binary variables (present or absent). As a result, multilevel logistic regression was used for all analyses. Two-level models were used with individual observations nested within pupils. Observations in intervention (NF and EF combined) and control schools were compared at baseline and 12 weeks.

The model included terms for treatment group (Intervention vs. Control), visit (baseline vs.

follow up), and a two-way interaction (treatment group by visit). The interaction term was of primary interest, showing whether differences in behaviours between visits varied significantly between intervention and control groups. An examination of a three-way interaction between visit, intervention and lunch type showed that results did not vary by lunch type (school lunch versus packed lunch on the day of observation), so analyses are reported only for the two way interaction effects. Two analytical models were assessed.

Model 1 examined the effect of the intervention on behaviours, accounting only for differences between schools (unadjusted model). Model 2 included potentially confounding class and pupil characteristics in the analysis: class size, presence of additional adults in the classroom (yes/no), English as an additional language (EAL), free school meal eligibility (FSM), gender, special educational need (SEN) status, ethnicity (White British or 'other') and lunch type (school lunch or packed lunch) (adjusted model). These multi level logistic regression models were fitted using MLwiN software (Goldstein et al, 1998).

Statistics are reported as odds ratios (OR), confidence interval, and p value. An OR above 1.0 shows that the outcome is more likely in the intervention pupils than the control pupils; OR less than 1.0 shows that the outcome is less likely in the intervention pupils than the control pupils. 1. There were 96 year 3-5 pupils in the intervention schools (47 nutrition-first, 49 environment-first) and 50 year 4 and 5 pupils in the control schools; 72 girls and 74 boys; and 67 low attainment, 53 medium attainment and 9 high attainment pupils (data for the remaining 17 pupils were not available).

Results

Sample characteristics at school and pupil level are shown in Table

Gender and attainment levels were balanced across intervention and control schools. Other characteristics were similar between schools, except for FSM eligibility which was lower in the control schools (7.3%) than in the intervention schools (23.6% and 27%, NF and EF schools, respectively).

Class characteristics

Classes were of similar size (NF,25.6;EF,25.8;Control,24.9)

Learning behaviours

On-task and off-task behaviours were observed and used as proxy measures for concentration and disengagement (disruption), respectively (Table 2). Total levels of on-task behaviour were high at around 80% in both intervention and control schools at baseline and postintervention. Conversely, total levels of off-task were low, between 9.7% and 15.4%. The changes in on-task and off-task behaviours between baseline and 12 weeks, by intervention group and social-mode, are shown in Figures 2 and3, respectively. The odds ratios for the three social modes ('individual', 'pupil-pupil' 'teacher-pupil') as well as for the total on-task and off-task behaviours observed are shown in Table 3.

In the unadjusted model (Model 1), there were no statistically significant time-treatment interactions for total on-task or off-task behaviours (Table 3). Thus, there was no statistically significant effect of the intervention on pupils' behaviour that was consistent across all socialmodes. There were, however, significant interactions between treatment and time for specific social-modes in both the unadjusted model and the adjusted models. Consistent with the hypothesis, teacher-pupil on-task engagement in the hour after lunch was 3.4 times more likely in the intervention schools compared with the control schools in the adjusted model (OR=3.40 (95% CI: 1.56, 7.36), p=0.009), but not in the unadjusted model. Contrary to expectations, however, on-task pupil-pupil behaviour was less likely in the intervention group compared with the control group in both the unadjusted model (OR=0.72 (95% CI: 0.54, 0.95), p=0.02) and the adjusted model (OR=0.45 (95% CI: 0.28, 0.70), p<0.001). Similarly, off-task pupil-pupil behaviour was more likely in the intervention group than in the control group in both the unadjusted model (OR=2.18 (95% CI: 1.52, 3.13), p<0.001) and the adjusted model (OR=2.28 (95% CI: 1.25, 4.17), p=0.007).

Food and environment

A 2.2% increase in the percentage take-up of school meals was observed at the end of the study, although this change occurred in both control and intervention schools. Intervention schools met, on average, 18 of 19 Government food-based standards for school lunch at the start of the study which was maintained throughout along with improvements to food presentation, choice, quality and promotion. Control schools met on average 17 standards at the start of the study and 16.5 at the end. Environmental changes were implemented by schools according to their tailored intervention plan. Over the study intervention school's implemented the following changes; newsletters to parents about school meals, behaviour management training for dining room supervisors, sticker schemes for pupils tasting new foods or eating fruit or vegetables, dining room displays and school assemblies on healthy eating, introduction of dining room rules, tasting sessions, promotion of menus at parents evening and via mail outs. Some schools also staggered lunchtime to reduce queuing, introduced music in to the dining room, changed the layout of chairs and tables, introduced 'grab and go' school lunches and involved pupils in the dining service. Planned changes to the dining room were reviewed during the intervention period and modified depending on their ongoing success. The precision of the instrument for assessing the dining room environment was valuable in deciding on which interventions were appropriate but was not adequate to show how the dining room environment changed over time when administered pre and post intervention. The impact of changes to the food and environment on children's nutritional intakes are reported elsewhere.

Discussion

The key finding suggested by these results is that a school food and dining room intervention was followed by an increase in levels of alertness (concentration/engagement) among pupils in the hour after lunch, but that the expression of this raised alertness was different in the individual, pupil-pupil, and teacher-pupil social modes. Although the odds ratios were statistically significant only for the teacher-pupil on-task and the pupil-pupil on-task and offtask analyses, the general trends observed in Figures 2 and3 are consistent with an overall pattern of raised alertness in the intervention schools.

A high proportion of the OR for the pupil-teacher on-task findings is likely to be explained by the decrease in on-task pupil-teacher behaviour observed in the control schools compared with the intervention schools. This is reflected in part in the wide confidence intervals for the OR.

The lower levels of concentration (and higher levels of disengagement) evident during the pupil-pupil social mode may be explained in a number of ways:

• Poorly defined peer interactive activities. Pupil-pupil interaction is the least easily controlled by an adult (Baines et al., 2003, Bennett et al., 1984, Galton et al., 1999, Galton et al., 1980) (teacher-pupil is the most controlled). If a reduction in levels of ontask behaviour was to be expected, it is in this context where it would most likely be observed.

• Disruption due to the intervention implementation. Interventions often take time to have a positive impact and may cause a disruption before a positive effect becomes evident (Blatchford et al., 2005). Classroom observations were made directly following the final stages of the intervention implementation, and the results could therefore reflect pupils' immediate response rather than the final impact of change.

• Changes in alertness. Lower levels of on-task behaviour in the pupil-pupil social-mode may be attributable to greater levels of alertness post-intervention with a greater need to interact. Teachers may not have catered sufficiently to these changes (Baines et al., 2003, Hastings & Schweiso, 1995). This greater need to interact could also be expected in the individual (working alone) mode, but this was not observed here. Rather, differences between baseline and post intervention in the individual setting, although small and not statistically significant (and potentially lacking in power), are consistent with the suggestion that pupils in the intervention group are more alert and more likely to be on-task and less likely to be off-task compared with the pupils in the control group.

The present study examined the impact of changes in lunchtime school food provision on education-related behavioural outcomes, and its strength is the direct measures of learningrelated behaviours in the classroom setting rather than indirect evidence such as attendance, exclusions, etc.

The study has limitations. It focused on 146 randomly selected pupils in six primary schools in one city, with schools selected by researchers based on their willingness and capacity to implement the study intervention. There is no reason to suspect, however, that the schools or pupils are very different from other primary schools and pupils in England. The matching of schools between study groups, pupil selection stratified for a number of characteristics, relatively large number of observations (over 17,000), the careful training and monitoring of the fieldworkers, and the 12 week intervention all support the study's generalisability to other motivated schools. While free school meal eligibility at the school level was matched, lack of pupil level data for sample selection stratified for free school meal eligibility resulted in higher eligibility in the intervention than control schools.

Any study of this kind is subject to the Hawthorne effect (short-term changes in behaviour resulting from an intervention per se rather than specific intervention). A second control group with some form of intervention exposure, not related to school, and a longer-term follow up would be necessary to determine a sustainable impact. The Greenwich study (Belot & James, 2009) found changes persisting after one year, supporting the interpretation of a real effect. While attempts were made to measure the impact the food and dining room intervention, this was limited due to a lack of relevant and valid assessment tools. Although the dining room check list tool was useful to assess the initial quality of the dining room environment and inform intervention development, the tool was not sensitive to be able to show any differences in environment at follow up, despite numerous changes in each school. This tool could be refined to more effectively monitor change. Some observers may have held an understanding of the behavioural categories not consistent with training. Data were, therefore, compared to observations collected on pupils in Years 4-6 as part of the SPRinG and class size projects (Blatchford et al., 2005, Baines et al., 2008). * There is the possibility of the co-occurrence of Teacher-Pupil and Pupil-Pupil contexts (i.e. these 2 contexts are not mutually exclusive). This situation is on the whole a relatively rare occurrence but may happen, for example, when a teacher is leading a reading group, when the teacher is talking to the class while a student is simultaneously talking to another student and so on. The total number of off task and not-off task in the two sections of the table are not the same because 'not on task' includes various 'off task' variables PLUS other variables (in this study procedure, social and other). * Unadjusted: differences between intervention and control groups ** Adjusted: differences between intervention and control groups, adjusted for class size (number of pupils), presence of additional adults in the classroom (yes/no), EAL, FSM, Gender, SEN status, Ethnicity (White British or other ethnic group) and Lunch type (school lunch or packed lunch).

  , and of mixed ability. Two thirds of classes had a female teacher, 16% had another teacher, and 16% SEN teacher/support staff. The number of additional adults varied across the treatment conditions (means across all observations were: NF = 0.16, EF = 0.68, Control = 0.75), adjusted for in analytical Model 2. Observations A total of 17,306 observations were collected across the two time points and allowed for an analysis of reasonable power. At baseline, 5702, 10-second observations were collected in the intervention schools and 2941 in the control schools. At 12 weeks, 5811 and 2852 observations were collected, respectively. On average, 63.6 observations were collected for each 'target' pupil at each time point. Allowing for missing values, data for 132 pupils with observations at both baseline and week 12 have been included in Model 2.

•

  Hastings, N. & Schweiso, J. (1995) Tables and tasks: the effects of seating arrangements on task engagement in primary classrooms. Educational Research, 37 (3), 279-291. • North Yorkshire Business & Education Partnership, Business & Education South Yorkshire. 2004). The Dining Room Environment Project. Food in Schools Programme (DfES, DH). • Paterson, L., & Goldstein, H. (1991). New statistical methods for analyzing social structures: An introduction to multilevel models. British Educational Research Journal, 17, 387-393. • School Meals Review Panel (2005). Turning the Tables -Transforming School Food: The Development and implementation of nutritional standards for school lunches. http://www.schoolfoodtrust.org.uk/UploadDocs/Library/Documents/SMRP_Report_FIN AL.pdf • SPSS for Windows, version 15.0. (2006). SPSS Inc. Chicago. • Statutory Instrument 2000 No. 1777. Education (nutritional standards for School Lunches) (England) Regulations 2000. London: TSO. • Statutory Instrument 2006 No. 2381.The Education (Nutritional Standards for School Lunches) (England) Regulations 2006. London: TSO. • Statutory Instrument 2007 No. 2359. The Education (Nutritional Standards and Requirements for School Food) (England) Regulations 2007. London: TSO.

Figure 1 Figure 2

 12 Figure 1 Study flow of school recruitment, intervention content and data collection timing

Figure 3

 3 Figure 3 Percentage of off-task behaviour at baseline and 12 weeks, by intervention group and setting.

Figure 1 Figure

 1 Figure 1

Table Legends Table 1 .

 Legends1

	1 Special education needs
	2 Free school meals
	*based on 151 pupils
	** from information reported by school

Table 2 .

 2

Table 3 .

 3 Occurrence of On-task and Off-task behaviour overall and for each social mode separately for the Nutrition first and Environment first intervention groups combined relative to the Control group.

Table 2 .

 2 Number of observations * and percentage of time spent in specified activities, by intervention group, at baseline and post-intervention (week 12).

	Baseline	Post-intervention
	Control	Intervention	Control	Intervention

Table 3 .

 3 Occurrence of On-task and Off-task behaviour overall and for each social mode separately for the Nutrition first and Environment first intervention groups combined relative to the Control group.

	Group (Intervention : Control)	P
	Odds Ratio	95% CI

Acknowledgements

The authors would like to thank the staff and pupils of the Sheffield primary schools who participated in the study and all the organisations and individuals who contributed to its design and implementation: the Sheffield Healthy Schools Team, Sheffield PCT, Chartwells, Chaucer Catering, Sheffield Children and Young People's Directorate, Sheffield local authority and the National Centre for Social Research (NatCen). In addition, the authors would like to thank Claire Storey and Pauline Ashfield-Watt, both of the School Food Trust, for their help in revising the manuscript.

Conflict of interest

The authors disclose no conflict of interest.

Comparisons can only be considered indicative since the present study included a limited numbers of classes observed by a small number of observers. Differences were seen in relation to setting: teacher-pupil and pupil-pupil interactions. The SPRinG/Class size data on the pupil-pupil variables showed higher levels of on-task (74% on-task in present data set compared with 56%) and lower levels of off-task. Observations in the present study were collected by a single set of observers who each worked in at least three schools (including at least one intervention and one control school), therefore, it could reasonably be assumed that any biases in observation were balanced across all schools.

Finally, because of the relatively short timescale and the inability to separate the nutrition and environment phases, it was not possible to resolve whether a nutrition-only or a nutrition-first intervention would differ from an environment-only or an environment-first intervention.

On balance, the present study offers some support for the hypothesis that a dining room intervention that changes both food provision and environment can have a positive impact on pupils' alertness, particularly during interactions between teacher and pupil which represent the majority of classroom experiences in primary schools. The findings also suggest that if that raised alertness is not appropriately channelled and adequately supervised, it may result in increased off-task behaviour when pupils are working together. • Goldstein, H., Rasbash, J., Plewis, I., Draper, D., Browne, W., Yang, M., Woodhouse, G., & Healy, M. (1998). A User's Guide to MlwiN. Multilevel Models Project, Institute of Education. University of London.


PhD Lena Ohlsson
email: lena.ohlsson@med.lu.se.

PhD Hans Burling

Prof Rui-Dong Duan

MD Åke Nilsson

Prof Affiliation

Effects of

Keywords: sphingomyelin, postprandial, triacylglycerol, milk polar lipids, cholesterol

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Dietary polar lipids may have beneficial health effects. Glycerophospholipids as soy lecithin lower LDL and increase HDL, although the LDL lowering effect is hard to differentiate from the effect of the high content of linoleic acid in this formulation (Knuiman et al 1989, O'Brien and[START_REF] O'brien | Influence of dietary egg and soybean phospholipids and triacylglycerols on human serum lipoproteins[END_REF]. Sphingolipids (SL) are a small component in the normal diet [START_REF] Vesper | Sphingolipids in food and the emerging importance of sphingolipids to nutrition[END_REF], and studies in which SL enriched formulations are given to humans have earlier been hard to conduct. A dairy formulation, enriched in butter milk polar lipids is now available. This formulation can be administered in a milk drink like formulation containing 2-3 times the normal daily intake of dietary SL per serving.

We showed earlier that the digestion of SL is extended and catalyzed by mucosal enzymes [START_REF] Nilsson | Absorption and lipoprotein transport of sphingomyelin[END_REF]. When cholesterol was given together with sphingomyelin (SM) an inhibitory effect on cholesterol absorption could be demonstrated in intact [START_REF] Nyberg | A mutual inhibitory effect on absorption of sphingomyelin and cholesterol[END_REF] as well as lymphatic duct cannulated rats [START_REF] Noh | Milk sphingomyelin is more effective than egg sphingomyelin in inhibiting intestinal absorption of cholesterol and fat in rats[END_REF]) and mice, and in CaCo2 cell cultures [START_REF] Eckhardt | Dietary sphingomyelin suppresses intestinal cholesterol absorption by decreasing thermodynamic activity of cholesterol monomers[END_REF]. In apoE Leyden mice, a genetically modified animal model that reacts to dietary changes more like humans than wild type mice sphingolipids were shown to decrease plasma cholesterol and triglyceride levels when the animals were on a high fat and high cholesterol diet. The effect was shown to be linked to an inhibitory effect on cholesterol-and lipid absorption. In this experimental model the sphingolipids also decreased the accumulation of fat in the liver [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF]. In vitro addition of sphingolipids inhibit the action of pancreatic lipases [START_REF] Patton | Inhibition of human pancreatic lipase-colipase activity by mixed bile salt-phospholipid micelles[END_REF]. In this study we raised the question, whether a recently developed tasty milk drink formulation, enriched in milk fat globule membrane polar lipids, influenced the postprandial lipid and lipoprotein profile. These parameters are well known indicators of cardiovascular risk and can be influenced by numerous dietary factors [START_REF] Lopez-Miranda | Dietary, physiological, genetic and pathological influences on postprandial lipid metabolism[END_REF].

Methods

The test drink A used in this study was based on processed and enriched butter milk and contains 4,0 % Lacprodan PL-20 powder from Arla Foods Amba. This powder contains 20 % (w/w) milk phospholipids originating from the serum phase of butter oil production. The test drink (A) contains 700 mg SM, 180 mg glucoceramides, and 95 mg gangliosides (mainly GD3). Total amount of SL was 975 mg in one portion of 340 ml. A placebo drink (B) was made by 5,82 % skim milk powder containing 37% protein in which the phospholipids have been replaced by 2/3 of egg PL isolate by Fresenius GmbH and 1/3 butter oil. The placebo drink contains 119 mg of SL per portion. The cholesterol content was adjusted to be the same in both drinks, i.e. 11 mg per 100 mL or 38 mg per serving. Based on dry weight both formulations contained 58,5 % milk protein and 6,6 % lactose as major constituents. The drinks has been subjected to heat treatment at 143 o C for 6 s and poured into Tetra Brick containers under aseptic conditions. The sterility of the drink has been verified by a certified bacteriological control laboratory. Test and placebo drink was manufactured at Trenums Industries, Tingsryd, Sweden, under supervision of Arla-experts. Trenums is an approved facility for manufacturing of food items. The drinks were stored in refrigerator and it is recommended to shake it a little before consumption.

Twenty male healthy volunteers in the age of 21-65 years, mean 39 years of age and mean BMI 24.3 were recruited through announcements on local intranet and advertisement in different work places in Lund. Exclusion criteria were abnormal fasting blood glucose levels, and hyperlipidemia in addition to signs of liver or kidney disease or inflammatory activity as indicated by raised C-reactive protein, liver tests (bilirubin, alkaline phosphatase, gammaglutamyltranspeptidase and aminotransferases), and creatinine levels. All participants gave their written informed consent prior to the study. The study was registered and approved by the Regional Human Ethics Committee of Lund-Malmö, Sweden.

The subjects were fed a standard breakfast containing the milk drink or the control formulation in a randomized crossover design and the wash out period between trial days was 10-14 days. The breakfast consisted of one 275 g portion of semolina pudding (8,0 g fat, 1029 KJ), 28 mL cream (10,1 g fat, 392 KJ), 20 g butter (16,2 g fat, 596 KJ), one slice of bread (0,8 g fat, 298 KJ), 17 g liver paste or 15 g cheese (4,0 g fat, 202-214 KJ) and 16 g blackberry jam (128 KJ) (table 1). Coffee or tea was served, without sugar, and the cream could be taken with these drinks or with the semolina pudding and blackberry jam. The breakfast except the test drinks thus contained 2645-2657 KJ (637-640 Kcal) and 39,1 g fat of which about two thirds was milk fat. The total cholesterol intake from the breakfast is 130 mg without the milk drink.

Blood samples were taken immediately before the breakfast, when the subjects were fasting since 10 o´clock the night before, and after 1, 3, 5 and 7 hours. The samples were analyzed for triglycerides, cholesterol, blood glucose, insulin, HDL and LDL cholesterol, apoAI and apoB, liver tests, C-reactive protein, hemoglobin, white blood cell count and platelet count. Baseline concentrations of plasma lipids before each trial are shown in table 2. All analyses were done at the Department of Clinical Chemistry, University Hospital of Lund, Sweden using the accredited routine methods developed by Roche Diagnostics (MSDS -COBAS Integra ® -General Chemistry). Two mL EDTA-plasma were frozen for other analyses if motivated.

Participants conducted food registrations in the form of diaries over four days before experiments were performed and the intake was analyzed by the use of Dietist XP software with the Swedish National Food Administration database (20080306) (Kost och Näringsdata, Sweden). Dietary intakes were related to recommended Swedish dietary guidelines [START_REF] Snf A | Swedish Nutrition recommendations[END_REF].

Statistics

Significance of differences between the results obtained with the A and the B formulation were examined using paired ANOVA multiple measures. Bonferroni post hoc test was carried out when significant differences between diets were found. The calculation of incremental area under curve (iAUC) and AUC were done using the trapezoid rule. The Students paired ttest was used to calculate if there were any difference between cholesterol in TG-rich lipoproteins after formulation A and B after one hour since this was the time for the highest lipoprotein output. All statistical analyses and calculations of iAUC were performed with GrahpPad Prism (version 5.00, GraphPad software, San Diego, Ca USA).

Results

Characteristics of the plasma lipid levels from18 of the 20 participants in fasting state are shown in table 2. One did not fulfill the study and values from one participant were excluded due to protocol deviation. The fasting values for this subject was 7,5 mmo/l TG and 13 mIU/L insulin probably due to heavy eating and drinking later than 10 pm. The study was participant blinded and randomized with regard to the order in which formulation A and B were taken. We received registration of food intake before each trial from nine individuals but only eight participants are included in table 3. We excluded the registration from the person who deviated from the protocol and presented elevated fasting TG levels mentioned above.

The effects on postprandial parameters of the two lipid formulations are shown in Figure 1.

As shown in Figure 1a the postprandial triglyceride responses were almost identical after formulations A and B. The concentrations were highest after 1 hour and then declined. After 7 hours they were slightly lower than the fasting values. In case of total cholesterol the postprandial concentrations were slightly lower after formulation A, but the differences were small and not statistically significant (Figure 1b). There were no statistically significant difference between the A and the B formulation in insulin and glucose levels (Figure 1c andd). The postprandial decreases in HDL-and LDL cholesterol 1 and 3 hours after the B formulation exceeded those obtained with formulation A (Figure 1 e andf). At the later time intervals, HDL cholesterol was higher after the A than after the B formulation but the difference was, however, not significant with paired ANOVA multiple measures. To estimate the postprandial concentrations of cholesterol in TG-rich lipoproteins we subtracted both LDL and HDL-cholesterol from total cholesterol (Figure 2a). We observed a 30 % lower iAUC with formulation A but the difference was not statistically significant. At one hour postprandially, the time point at which the intestinal output of TG-rich lipoproteins is culminating, there was a trend (p = 0,063) that cholesterol concentrations in TG-rich lipoproteins was lower after formulation A. Since the intestine is a source of apolipoprotein A1 and B48 we measure the postprandial concentrations of the individual proteins as well as the ratio. The apo B/ApoA1 ratio reflects to some extent the concentration of small dense LDL (figure 2b). In this study the Apo B and apo A1 (individual concentrations not shown), or the apoB/apoA1 ratio did not differ postprandially between the formulations.

The ingestion of the meal took about 20 minutes and the measurements were performed one hour after the end of the meal. The iAUC for the different parameters were not significantly different between formulation A and B (Table 4).

Discussion

This paper shows that a dairy formulation containing 975 mg sphingolipids, of which 700 mg is sphingomyelin, and polar dairy glycerophospholipids as well, did not influence the postprandial triglyceride increase from a high fat breakfast. There was a trend that cholesterol concentrations in TG-rich lipoproteins (total cholesterol excluding HDL and LDL) was lower after formulation A. It is possible that a larger study could have presented a true difference but with present data we do not consider this as proof of a SL-mediated inhibition of cholesterol absorption.

Earlier animal experiments demonstrated both an inhibition of cholesterol absorption with sphingomyelin [START_REF] Eckhardt | Dietary sphingomyelin suppresses intestinal cholesterol absorption by decreasing thermodynamic activity of cholesterol monomers[END_REF][START_REF] Noh | Milk sphingomyelin is more effective than egg sphingomyelin in inhibiting intestinal absorption of cholesterol and fat in rats[END_REF][START_REF] Nyberg | A mutual inhibitory effect on absorption of sphingomyelin and cholesterol[END_REF] and a cholesterol-and triglyceride lowering effect of several sphingolipids in apoE Leyden mice fed a cholesterol-and lipid rich diet which were fed 0,2-1 % of the respective sphingolipid in the diet for four weeks [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF]. Recently, milk polar lipids were shown to reduce hepatic steatosis and hyperlipidemia in C57BL/6 mice [START_REF] Tandy | Dietary phospholipid-rich dairy milk extract reduces hepatomegaly, hepatic steatosis and hyperlipidemia in mice fed a high-fat diet[END_REF]. Since the reason behind the effects in rodents has not been clarified, any explanation for the lack of effect on postprandial lipids in humans must be speculative. Indirect measurements of triglyceride fatty acid absorption in the ApoE Leyden mice suggested that 1% phytosphingosine decreased fatty acid absorption after a fat bolus dose [START_REF] Duivenvoorden | Dietary sphingolipids lower plasma cholesterol and triacylglycerol and prevent liver steatosis in APOE*3Leiden mice[END_REF]. Such an effect might be exerted at the level of intestinal lipolysis [START_REF] Patton | Inhibition of human pancreatic lipase-colipase activity by mixed bile salt-phospholipid micelles[END_REF], via an effect of the sphingoid bases in the mucosal cells [START_REF] Garmy | Interaction of cholesterol with sphingosine: physicochemical characterization and impact on intestinal absorption[END_REF] or on the postprandial fatty acid metabolism. Whatever the mechanism, it may obviously be influenced by dose of sphingolipids as wells as the amount of fat and cholesterol given, and other dietary conditions. The dose of sphingolipids given in this study i.e. 975 mg should be in a range within which effects might be realistically expected, but which may still be insufficient.

Furthermore the effects seen in the apoE Leyden mice may require the exposure to sphingolipids over longer time periods, whereas this study examines acute effects only.

As a general feature HDL-and LDL-cholesterol decreases initially after dietary lipid intake by the action of cholesterol ester transfer protein (CETP) which transfer cholesterol ester from both HDL and LDL to TG-rich lipoproteins during the postprandial phase. In this study HDL decreased less with formulation A, and it is therefore difficult to conclude, whether dietary SL cause inhibition of cholesterol absorption or influence the CETP mediated transfer of cholesterol ester from other lipoprotein classes. We also do not know at present if this difference is due to the presence of sphingolipids in A or is caused by other components of milk fat globule membranes. For instance the presence of milk polar lipids in the diet might increase the formation of polar lipid surface coat material which is transferred to HDL as the triglyceride rich lipoproteins are metabolized by lipoprotein lipase. An earlier study showed that feeding soy lecithin with a triglyceride rich meal decreased the average size of postprandial lipoproteins and thus the amount of polar surface component per mass of triglyceride [START_REF] Beil | Studies on plasma lipoproteins during absorption of exogenous lecithin in man[END_REF]. Soy phosphatidylcholine was also shown to increase the levels of HDL 2 levels (O'Brien and Andrews 1993), i.e. the most phospholipid-rich HDL sub fraction. Further studies including the size spectrum of triglyceride rich lipoproteins, activity of CETP and lipid transfers after ingestion of polar milk lipids are necessary to explain the cholesterol findings in this study.

The relation between glucose-, insulin action and ceramide in diabetes and obesity is important [START_REF] Summers | Ceramides in insulin resistance and lipotoxicity[END_REF]. We have, however, neither found any differences in insulin or glucose levels in this study, and so far, nor been able to demonstrate any change in plasma sphingolipids after feeding the milk sphingolipids (unpublished).

Diet registrations revealed no significant difference between the intakes before the two occasions, indicating that no false negative findings were caused by change in diet.

In summary, we found no immediate significant effect of milk polar lipids on postprandial plasma lipid levels. Thus, the result from this relatively small study on healthy individuals does not support our hypothesis that milk polar lipids affect our plasma lipids and lipoprotein profile. However, the formulation enriched in milk polar lipids was well tolerated and suitable for further human studies e.g. in diabetic and obese subjects with high fasting and postprandial triglyceride levels and low HDL, in which beneficial effects are plausible.

Legend to figures

Figure 1 .

 1 Figure 1. Normalized postprandial plasma lipids, lipoproteins, insulin and glucose

Figure 2

 2 Figure 2. a) Cholesterol in TG-rich lipoproteins in mmol/L calculated by subtracting values

Table 1 .

 1 Macro-and micro nutrient composition of the test meal.Fasting concentrations in all participants (n=18) before ingestion of formulations A and B.Postprandial values are presented in Figure1a-h as differences from the fasting values.

		Breakfast meal: 275 g semolina	Test drink/ placebo
		pudding, 28 mL cream, 20 g	(in 350 ml)
		butter, 1 slice of bread, 17g
		liver paté, 16 g blackberry jam
	KJ/Kcal	2683/643	807/193
	Protein (g)	16.9	8,19
	Fat (g)	39.4	3.85
	Carbohydrates (g)	54.3	31.2
	Calcium (mg)	315	130/271
	Fiber (g)	3.4	0
	Cholesterol (mg)	99.3

Table 3 .

 3 Amounts of nutrient intake of participants (n=8) before each test day. Values are mean of 8 subjects and four days intake ± SD.

		Before intake of formula A	Before intake of formula B
	Energy intake
	(kJ)	10828 ± 2846	9731 ± 2666
	Fat intake
	(g)	102.7 ± 35.3	91.5 ± 38.7
	Saturated fatty acids
	(g)	40.1 ± 17.1	37.6 ± 16.7
	Monoenoic fatty acids
	(g)	38.5 ± 15.5	32.2 ± 15.5
	Polyenoic fatty acids
	(g)	14.7 ± 4.6	11.2 ± 4.8
	Cholesterol intake
	(mg)	429.1 ± 194.7	394.8 ± 200
	Fiber
	(g)	19.7 ± 6.4	19.9 ± 7.8
	Calcium
	(mg)	928 ± 424	1092 ± 607
	Fat intake the day before
	(g)	112.3 ± 65.9 1	107.1 ± 73.6 1
	Energy intake the day
	before (KJ)	11591 ± 116.6 1	10792 ± 108.7 1

1 Intake the day prior to trial. Mean of 8 subjects.

Table 4 .

 4 The iAUC for normalized values from 0h to 7 hrs for formula A and B and different plasma components. The values are mean and 95% CI, n = 18

		Formula A	Formula B	Statistic analysis
		Total Peak area (95 %	Total peak area (95%	(Students paired t-
		CI)	CI)	test p>0.05 = n.s)
	TG	2.41 (1.71-3.12)	2.69 (1.87-3.51)	0.35
	Insulin	18.11 (12.63-23.60)	17.17 (11.93-22.42)	0.74
	Glucose	0.71 (-0.09-1.50)	0.57 (0.04-1.11)	0.71
	Cholesterol	0.39 (0.17-0.60)	0.52 (0.12-0.93)	0.52
	HDL	0.15 (0.05-0.25)	0.12 (0.02-0.21)	0.66
	LDL	0.15 (0.06-0.24)	0.19 (0.03-0.35)	0.69
	Cholesterol in TG-	1.61 (0.87-2.36)	2.10 (1.42-2.77)	0.27
	rich lipoprotein
	ApoA1	0.12 (0.05-0.18)	0.13 (0.05-0.22)	0.82
	ApoB/ApoA1	0.21 (-0.17-0.56)	0.01 (0.00-0.22)	0.30
	ApoB	0.04 (0.02-0.07)	0.04 (-0.00-0.09)	0.99

Acknowledgement

This study was in collaboration with ARLA-foods which include financial support and testdrinks. Other financial supports are grants from the Albert Påhlsson Foundation, Research Funds of the Lund University Hospital, the Swedish Research Council, and the Swedish Research Council Formas.

Financial supports are grants from the Albert Påhlsson Foundation, Research Funds of the Lund University Hospital, the Swedish Research Council, and the Swedish Research Council Formas. This study was in collaboration with ARLA-foods which include financial support and test-drinks. The authors have no conflict of interest.