Keywords: nutrition policy, models, economic, fat tax

Background/Objective: Previous studies have analysed impacts on average intakes. Agueably however intakes that are of real concern are those which are some distance from the recommendations. Fiscal measures may have a limited impact on such diets and, as a result, on health. We measure the impact of a scal intervention on the the risks of diet related disease accounting for the full range of diets.

Subjects/Methods: Demand equations are estimated with data from 6760 households in the UK Expenditure and Food Survey. The model is used to simulate the impacts of a policy in which a tax based on saturated fat content is combined with a subsidy on fruit and vegetables. Changes in consumption are used to compute the eects on the risks of a range of diet related disease using measures of relative risk. In contrast with other studies we simulate the impacts of the scal regime at the level of the individual households in the sample.

Results: The subsidy brings mean levels of fruit and vegetable consumption in line with dietary recommendations but the tax is insucient to achieve this goal for fat intakes. Once the changes in diet are converted into changes in the risks of disease the impacts of the policy are negligible. A substantial part of the population continues to consume an unhealthy diet.

Conclusion: Fiscally based interventions should be considered amongst a suite

of policy interventions which also include policies aimed at improving the poorest of diets.

Introduction

Public interest in the use of scal measures to regulate unhealthy diets has recently been reawakened by the proposal debated and rejected at the conference of the Scottish Local Medical Committee that a tax on chocolate might contribute to a reduction in obesity. A similar motion was debated in 2003 at the BMA annual representative meeting where a motion to impose a tax on saturated fat was defeated [START_REF] Beecham | Doctors vote against a tax on fatty food[END_REF][START_REF] Beecham | Doctors vote against a tax on fatty food[END_REF]. Interest extends to the US where a recent paper [START_REF] Brownell | Ounces of prevention -the public policy case for taxes on sugared beverages[END_REF]2009) has advocated the introduction of a tax on sugared drinks, an option which is amongst proposals that are currently under consideration by the United States Senate Committee on Finance as a means of raising revenue for health care reform. Evidence regarding the impacts of a scal policy on diet tends to focus on changes in the aggregate levels of food consumption of unhealthy products in the population as a whole. For example [START_REF] Marshall | Exploring a scal food policy: The case of diet and ischaemic heart disease[END_REF] and [START_REF] Mytton | Could targeted food taxes improve health?[END_REF] extend VAT in the UK to products regarded as the main sources of saturated fats. [START_REF] Mytton | Could targeted food taxes improve health?[END_REF] estimates that the ensuing variations in ischemic heart disease would lead to the avoidance of between 900 and 1,000 premature deaths every year. Whilst many studies (eg [START_REF] Chouinard | The eects of a fat tax on dairy products, CUDARE Working Paper[END_REF]2005;[START_REF] Kuchler | Taxing snack foods: Manipulating diet quality or nancing information programs?[END_REF]2005) nd that the impacts of a scal intervention on consumption are likely to be modest, these authors stress that a fat tax would be a useful tool to generate a revenue that could be allocated to prevention or information campaigns. In this vein and using Danish data, [START_REF] Jensen | Cost-eective design of economic instruments in nutrition policy[END_REF] investigate the eects of nutrient-and food-based taxes, coupled with subsidies in order to produce revenue-neutral scenarios. In line with other studies, they nd that dietary eects would be minimal, but, as far as nutrient intake is concerned, better results are obtained by focusing the tax on nutrient content rather than on specic food items (e.g., saturated fats vs. red meat).

A number of messages are emerging in this rapidly evolving literature. The results suggest that the impacts of a tax on diet may not be as large as might be hoped but by targeting taxes at specic nutrients more promising results may be obtained. Additionally, by coupling the negative impacts of taxes on unhealthy food with measures designed to promote healthy consumption, the eectiveness of the policy might be further enhanced. We therefore analyse the impacts of a tax based specically on the saturated fat content of foods coupled with a subsidy on fruit and vegetables. We also argue that it is important to to consider the distribution of nutrient consumption in the population and to measure not only the impacts on average intake but also on health across the population. In doing so we show that an undierentiated scal policy for all individuals has a limited impact on the overall health of the population.

Method

We report the results of a study which measures the impacts of a tax on saturated fats, coupled to a subsidy on fruit and vegetables, designed so as to create a revenue-neutral scheme. We explore the impacts of such a policy in achieving Department of Health guidelines and on the risk of being aected by diet related diseases. We estimate a system of demand equations as the basis for the model which we use to simulate the eects of the price changes. Our model employs the widely used Almost Ideal Demand System [START_REF] Deaton | An almost ideal demand system[END_REF][START_REF] Deaton | An almost ideal demand system[END_REF] which is represented as follows:

s it = α i + m+1 j=1 γ ij ln p jt + β i ln e t P t + ψ i h t + u it (1) i = 1, . . . , m + 1 and t = 1, ......T (2) (u 1t , . . . u mt ) ∼ N (0, Σ) (3)
where s it is the share of total expenditure (e t ) accounted for by expenditure on the i th good in the t th household, p jt is the price of the j th good to the t th household, P t = j p sjt jt is Stone's price index and h t is a vector of variables that describes the t th household's socio-demographic characteristics 1 . The use of a system of demand equations ensures that the changes in demand for the dierent food groups are consistent with one another fully taking into account cross price eects as suggested by [START_REF] Kennedy | Alternative nutrition outcome using a scal food policy. commentary tomarshall[END_REF], and [START_REF] Mytton | Could targeted food taxes improve health?[END_REF]. These cross price eects are important because, for example, a reduction in saturated fat consumption resulting from lower levels of butter demand could be partially oset as consumers substitute butter with lower fat spreads. Furthermore our model fully recognises that the consumer makes choices within a xed budget constraint. Thus the decision regarding how much less to spend on a taxed product will be partially inuenced by the savings that can be made elsewhere in the consumer's budget. We estimate the demand models using household data from the 2005-6 UK Expenditure and Food Survey (EFS). The data are collected through the completion a two-week diary for each individual over seven years of age which is supplemented with the use of till receipts. Each participating household does so voluntarily. An important consideration when estimating demand models is the treatment of censored observations where the level of consumption of a particular good in a household is zero during the survey period. In order to address this we employ a version of the Infrequency of Purchase Model (IPM) introduced by [START_REF] Blundell | Bivariate alternatives to the tobit-model[END_REF]. 2[Table 1 about here.] It is not possible to estimate a single model comprising all the food items required in our analysis. Instead, models comprising only a few food groups at a time must be estimated and these food groups are detailed in table 1. We use a hierarchical approach in which a top level model is used to explain the allocation of expenditure between the broad categories in the rst column of table 1. Six further models are estimated to explain the partition of expenditure amongst the narrower categories set out in the third column. The price elasticities obtained from each of these models assume that expenditure on the group of foods within the model remains constant as the price change takes place. For example the own price elasticity for fresh fruit and vegetables that is obtained from the fruit and vegetables system assumes that the total expenditure on the ve types of fruit and vegetables in the model remains constant. This assumption it likely to be unrealistic: a reduction in the price of fresh fruit and vegetables is likely to induce consumers to spend more on all fruit and vegetables. To address this problem, estimates from the two levels of the hierarchy are combined using the approach suggested by [START_REF] Edgerton | Weak seperability and the estimation of elasticities in multistage demandsystems[END_REF] to give overall elasticities which do not assume that expenditure within the 5 groupings remains constant.

The choice of the categories outlined in table 1 is a balance between nutritional rationale and specifying a model which can be estimated econometrically. From a nutritional perspective the ideal would be to estimate at a level that is suciently disaggregated to allow for all of the substitution possibilities that are likely to result from a tax that is imposed dierentially according to the saturated fat content of the food. Thus we include skimmed, semi skimmed and whole milk, and crisps, chips and fresh potatoes separately in the model.

In some cases however the aggregations are less than ideal from a nutritional perspective. For example we had to combine butter and margarine in a single aggregate because the levels of censoring across these two categories made it impossible to obtain meaningful estimates for a more disaggregated model. It should be noted however that the tax rate that is computed for a group such as this reects the observed composition of the aggregate within the sample and therefore, whilst substitution between butter and margarine might not be captured meaningfully, substitution away from the category as a whole will be picked up in the simulations.

We use our model to simulate the impact of a fat tax on intakes and on health.

We use Department of Health dietary reference values as a bench mark to assess the impacts of the policy. Dietary reference values recognise that nutrient requirements dier between individuals and are designed to ensure that almost the entire population's nutritional needs are met. Implicit in these guidelines therefore is a recognition that a distribution of nutrition intakes across the population in which a substantial number of people consume below the guidelines does not necessarily mean that a large number of people face a signicant health risk.

There is however evidence to suggest that some socio-economic groups have diets which are particularly poor (Food Standards Agency;2007) and it is unlikely that this variation is all attributable to dierences in nutrient requirements. We therefore base our analysis on the diets of the individual households within the sample so that we can assess the impacts of the policy on the whole distribution of nutrient intakes. Using our model of consumer demand, we estimate changes in consumption for every household in the sample after implementation of a tax policy.

To summarise the impact of the tax on the distribution of nutrient intakes on each of the individual households in the sample we convert the pattern of nutrient intakes to a single metric which gives an indication of the unhealthiness of particular distribution nutrient intakes. We measure dietary health using the odds O i of being aected by a particular diet related condition and, in common with much of the literature, assume that these are related to the level of exposure x i with a logistic function:

ln (O i ) = ln p i 1 -p i = α + βx i (4)
In order to measure the risk of being aected by a diet related condition across a range of intake levels we use estimates of β from the literature to compute the relative risk RR compared to the intake recommended in DoH guidelines (DoH; 1991):

RR i = O i O 0 = e β(xi-x0) (5)
where x 0 is the recommended intake level. The population risk associated with a given distribution of nutrient intakes is then computed as the weighted average:

P R = w i RR i (6
) where w i is the proportion of the population consuming x i . Our estimates of population risk therefore measure the average odds of the population being aected by a condition relative to the case where the whole population conforms to dietary guidelines for the food or nutrient in question. We compare the population risk before and after the policy to measure its eectiveness. The construction of this measure implies that diets which are far away from the reference value are given proportionately greater weight than those that are close to the value. Whilst we recognise that it is not universally true that an individual who consumes below the reference value inherently has a greater risk of disease than another individual who is close to the reference value, we argue that our measure is representative of general societal concern for those individuals who consume particularly poor diets.

A variety of alternative taxation regimes have been examined in the literature. [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] consider a range of scenarios in which VAT at 17.5% is charged either on a subset of foods (milk, butter, cheese, biscuits, cakes and pastries) which are deemed to be high in saturated fats or those which are deemed to be unhealthy because they have a WXYfm score over 4. They combine this with a subsidy on healthy food which is either at the same rate as the VAT or at a higher rate which is designed to make the policy revenue neutral. [START_REF] Jensen | Cost-eective design of economic instruments in nutrition policy[END_REF] also consider a variety of combinations of taxation and subsidy. In their case the fat tax is based on the amount of fat that is present in a particular food type and their subsidy is a either based on the amount of bre in a product or a blanket reduction in VAT on fruit and vegetables. 3Our approach combines aspects of both of these approaches. Like [START_REF] Jensen | Cost-eective design of economic instruments in nutrition policy[END_REF], we vary the fat tax rate according to the fat content to assess whether this more targeted approach is more eective than that of [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] which has the merit of administrative simplicity. We increase the price of fatty foods by 1% for every percent of saturated fats they contain; for instance, milk which contains 1.72% of saturated fats will see its price increasing by 1.72%. 4 We put a ceiling on the price increase of 15%. To oset this tax burden and to encourage consumption of fruit and vegetables, a subsidy on fruit and vegetables is introduced, so as to exactly cancel the costs of the fat tax paid by consumers. Table 2 [Table 2 about here.] presents the tax rates applied to the dierent food groups in our models, based on nutrient conversion tables available from the EFS data set. It can be seen that there is considerable variation in the rates across dierent foods but in most cases the magnitude of the tax is in an area which, whilst not being popular, is likely to be acceptable.

Results 5

Figures 1 and2 show the impacts of the policy on nutrient intakes together with the maximum and minimum recommended daily amounts based on the sample of households that is used to estimate the model. In gure 1 it can be seen that current consumption levels of all fats and the components of total fats except for PUFAs all currently exceed the maximum daily amounts measured as a percentage of energy intake and whilst the tax reduces intakes it does not bring them in line with the recommendation. By contrast intakes of PUFAs are below the recommended amounts and since the tax reduces consumption marginally it is ineective in helping to meet this target. Sugar consumption levels are also above the recommended amounts. The policy leads to a marginal increase in sugar consumption and there is therefore no impact in terms of this target either. In gure 2 the units dier between nutrients and are indicated with each column label. Figure 2 shows that the policy is eective in bringing fruit and vegetable consumption in line with the recommendations but it does not achieve this for sodium and bre intakes.

[Table 3

about here.]

Table 3 shows the impacts on health that result from the dietary changes that are induced by the policy. As noted above, the gures reported in the before and after tax columns of the table measure the average odds of being aected by a condition across the population relative to a situation in which the whole population conforms to dietary guidelines. As an example, the gures show that average odds of being aected by CHD as a result of elevated intakes of saturated fatty acids are 78% higher than they would be if everyone ate in accordance with dietary guidelines. Following the introduction of our proposed scal intervention, the average odds fall but only to the point where they are 72% higher than if everyone satised the guidelines. This pattern is reproduced throughout the table, in general the policy is only marginally eective in reducing the average odds of being aected by a condition. Even in the case of fruit and vegetables, where we have seen that the average levels of consumption in the population are moved into the `ve-a-day' region, the average odds of being aected by conditions are only changed slightly by the policy. In particular, after the imposition of the tax, the average odds of being aected by the two cancers considered are still 26% and 12% higher than they would be if everybody ate `ve-a-day'. These relatively small changes in health status are understandable when, for example, it is recognised that the eect of the subsidy on fruit and vegetable consumption is to only reduce the proportion of the population that consumes less than 100g of fruit and vegetables per day from 13% to 12%. Thus, whilst mean levels of consumption are moved into line with the recommendations, a substantial part of the the population remains at a level of consumption which is a considerable distance from the guidelines.

Conclusion

The results show that a policy based on a tax on saturated fats coupled with a subsidy will be eective in moving diets in the UK in a direction consistent with improvements in diet related health. In particular a subsidy approaching 15% of the price of fruit and vegetables has been shown to be eective in moving the average intakes to within the recommended `ve-a-day' region. By contrast the fat tax does not result in intakes of fat moving to the recommended amounts. Importantly however, we have shown that although mean levels of consumption move favourably in comparison with guidelines a large proportion of the population will remain some considerable distance from the recommended levels of intake. As a result the average level of diet related disease risk does not change substantially in the population. Whilst we acknowledge that, because of the large numbers of people involved, even small changes in the level of risk may have a large impact on disease rates, the fact that considerable numbers of people will continue to consume particularly bad diets is likely to be of concern.

Our study contributes to the growing literature on the impacts of a fat tax on dietary choice and consequent eects on health. Our study adds an analysis which is based on a rigorous theoretically consistent econometric model of consumer demand and in which the analysis is conducted at the level of the individual household. It complements that of [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] who use elasticities from the National Food Survey (NFS; 2000) to simulate the impacts of a number of policy scenarios on food choice. Our study extends the number of foods that are explicitly modeled in the simulation and in particular allows for a disaggregation of milk and cream into categories which have diering levels of saturated fat. The importance of doing this is noted by [START_REF] Andreyeva | The impact of food prices on consumption: A systematic review of research on the price elasticity of demand fo food[END_REF][START_REF] Andreyeva | The impact of food prices on consumption: A systematic review of research on the price elasticity of demand fo food[END_REF] given the importance of milk as a source of saturated fat and the potential for reducing intakes by encouraging substitution between milk of diering fat contents. In a similar vein we also estimate separate elasticities for fresh potatoes, crisps and chips because of the implications of consuming each of these potato based foods for health. As a consequence of this greater degree of disaggregation, our policy scenario is diers from those examined by [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF]. Where they place VAT at 17.5% on either a subset of foods (milk, butter, cheese, biscuits, cakes and pastries) which are deemed to be high in saturated fats or those which are deemed to be unhealthy because they have a WXYfm score over 4, we charge a variable tax according to the fat content of the product. The result is that we have lower but diering rates of tax on milk (2.6%, 1.14% and 0.13% respectively on whole, semi skimmed and skimmed milk respectively). We therefore see a much greater decline in consumption of whole milk (2.20%) than of skimmed milk (0.13%). It is not clear from the [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] paper how crisps are treated, however in our scenario the extremely high saturated fat content leads to a tax of 13.77% on these products and a resultant decline in consumption of 14.24%. Whilst it is not possible to perform a direct comparison between the impacts of the policies on saturated fat intakes between the two approaches,6 the results we report suggest that the reduction of fat that results from our more targeted but less burdensome policy is broadly similar to that reported by [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF]. A nal key dierence in our analysis is that we do not have the anomalous situation where the elasticities between dairy products and fruit and vegetables indicate complementarity. This results in the [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] tax on dairy products having a negative impact on the consumption of fruit and vegetables, although this is eect is countered by the introduction of a fruit and vegetable subsidy. In our study, as a result of ensuring that the theoretical properties of the economic model of demand are satised, we have these products as substitutes. As a consequence the tax and subsidy act in the same direction to increase consumption of fruit and vegetables. We conclude that the benecial health eects of policy induced increased fruit and vegetable consumption are likely to be understated by [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF].

Our analysis is restricted in the sense that it focuses only on reducing saturated fat intakes and increasing fruit and vegetable consumption. Arguably, other aspects of nutrition are as important to target, in particular a policy may be based additionally on calorie, sugar and salt contents of foods. Our broad conclusion that a targeted policy is likely to bring signicantly higher health benets is likely to be equally applicable when considering a broader range of nutrients. We have argued that the model that is used to estimate the elasticities that form the basis of our simulations is consistent with an economic model of choice and that it therefore avoids the inconsistencies that in particular may have aected the cross-price elasticities that have been used in previous studies. A caveat to our model is that the data we employ does not have information on prices, we therefore use expenditure and quantity purchased to compute the unit value of the product that is purchased. The unit value also includes an element reecting qualitative dierences in the product and may also be endogenous to model. As a result dierences in unit value may overstate the price dierence that explains why diering quantitative decisions are taken and our elasticities may be underestimates. Furthermore, because the data that we use is cross-sectional, the ability of our model to capture temporal aspects of the response to taxes. In particular there is likely to be an element of persistence in peoples choices which is not reected in our estimates. The adjustments which we simulate may therefore take some time to be realised.

A further important consideration is the dierential impact that a fat tax will have across society. Other studies point out the regressive distributional impacts of the type of policy analysed here: because food represents a greater proportion of total household expenditure in poorer households, the burden of a tax on food will be fall disproportionately on the poor. Whilst combining the tax with a subsidy on fruit and vegetables serves to neutralise the impact of a tax in aggregate it is possible that it may add to the regressivity of the policy if fruit and vegetables a consumed in proportionately larger quantities in rich households. As [START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] point out the regressivity might be justied if the health benets accrue proportionately more to the poor. Low income households tend to consume poor diets, therefore the fact that the tax has a proportionately larger economic impact on these households is positive in targeting the most needy in terms of their dietary health. In spite of this however our analysis of the dierential impacts of the policy across dierent demographic groups suggest that the eect of the policy on the risks of dietary disease are broadly similar across society [START_REF] Arnoult | Models of nutrient demand, tax policy and public health impact[END_REF]2008).

Our conclusion is that a fat tax should be seen as a component of a suite of instruments in tackling poor diets. Policies which target a number of dierent levels within society (eg. community, school, family, individual) referred to as social ecological approaches [START_REF] Stokols | The social ecology of health promotion: Implications for research and practice[END_REF]1996) are increasingly advocated as being more eective in delivering benecial changes in diets [START_REF] Gentile | Evaluation of a multiple ecological level child obesity prevention program: Switch what you do, view and chew[END_REF]2009). Experience in reducing the prevalence of smoking also points to an approach which does not rely entirely on price increases. [START_REF] Levy | The role of public policies in reducing smoking prevalence in california: Results from the california tobacco policy simulation model[END_REF]2007) nd that 59% of the reduction in the prevalence of smoking in California is attributable to increases in price whilst 28% is attributable to media policies. Whilst fat taxes have the potential to eect marginal changes in the diets of large numbers of people, and thus have a signicant impact on the incidence of disease across the population, more complex polices which target dierent levels in society are likely to be more eective in addressing the severe chronic dietary disease which aects some groups.

Conicts of interest

We declare that we have no conicts of interest

Table 1

 1

	: Food Categories Obs Component Foods 6315 Whole Milk Semi-skimmed Milk Skimmed Milk Other Dairy, Eggs and Fats 6687 Cheese and Cream Grouping Milk Other Dairy Eggs Fats Meat and Fish 6588 Beef Lamb Pork Poultry Fish Other Potatoes, Rice and Pasta 6262 Fresh potatoes Crisps Chips Rice and Pasta Cereals 6784 Bread Breakfast Cereals Other Cereals Confectionery Fruit and Vegetables 6588 Fresh Frozen Tinned Prepared Ready Meals Drinks 6414 Tea and Coee Soft Drinks Alcohol

Table 2 :

 2 Tax and subsidy rates used in the simulation The gures in parenthesis indicate the source of the relative risk estimates as follows: [1] Riboli and Norat (2003); [2] Hung et al. (2004); [3] Joshipura et al. (2001); [4][START_REF] Esrey | Relationships between dietary intake and coronary heart disease mortaility[END_REF].

	Food Full Fat Milk Semi Skimmed Milk Skimmed Milk Cheese and Cream Other dairy Eggs Fats Beef Lamb Pork Poultry Fish Other Fresh Potatoes Chips Crisps Rice and Pasta Bread Breakfast cereals Other cereals Confectionery Fresh Fruit and Vegetables Frozen Fruit and Vegetables Tinned Fruit and Vegetables Prepared Fruit and Vegetables Fruit and Vegetables based Ready Meals 2.27% Tax 2.60% 1.14% 0.13% 15.00% 2.83% 3.20% 15.00% 5.98% 6.38% 4.55% 1.93% 1.58% 5.40% 1.16% 1.55% 13.77% 0.31% 0.58% 0.88% 4.75% 5.94% -14.78% -14.78% -14.78% -14.78% Tea and Coee 0.55% Soft drinks 0.00% Alcohol 0.01%

We used an extensive list of demographic variable to describe: household composition; social class; region; age; sex and ethnicity.

Full details of our adaptation of the AIDS are available in a working paper[START_REF] Tin | Bayesian estimation of the infrequency of purchase model with an application to food demand in the uk[END_REF] 2008).

VAT is not charged on fruit and vegetables in the UK at present.

The saturated fat contents were obtained from data supplied in the Family Food module of the Expenditure and Food Survey. The majority of the data is from the Food Standards Agency's nutrient analysis programme, supplemented by values from manufacturers and retailers.

Full results for the econometric estimation of the models are available from the authors on request. Given their complexity, the models are robust with

67% of the estimated coecients signicant at the 95% level.

Our results are in terms of the proportion of energy derived from saturated fat, which declines by 2.00%, whilst[START_REF] Nnoaham | Modelling income group dierences in the health and economic impacts of targeted food taxes and subsidies[END_REF] express theirs in the quantity of saturated fat which declines by 3.11%.


Ysbrand J Schreuder

Barbara A Hutten

Manon Van Eijsden

Eugene H Jansen

Maud N Vissers

Marcel Th Twickler

Tanja G M Vrijkotte
email: t.vrijkotte@amc.uva.nl

Tgm Vrijkotte

Ethnic differences in maternal lipids

Keywords: Lipid profile, pregnancy, ethnicity, behavioural factors, ABCD study, prepregnancy BMI. Ethnic differences in maternal lipids

Background/Objectives: Lipid disturbances during pregnancy may lead to early onset of metabolic diseases in the offspring. However, there is little knowledge on ethnic differences in lipid levels during pregnancy. We evaluated ethnic differences in non-fasting total cholesterol (TC) and triglyceride (TG) levels during early gestation and the role of demographics, behavioural factors and clinical characteristics. Subjects/Methods: Non-diabetic pregnant women (N=3025) from the Amsterdam Born Children and their Development (ABCD) study. The studied ethnic groups were: Dutch, Surinam-Hindustani, African-Caribbean, Turkish, Moroccan and Ghanaian. A multilingual questionnaire was used to gather information on maternal demographics, behavioural factors and clinical characteristics. Non-fasting TC, TG, % saturated-fatty-acid (%SFA) and % linoleic acid status (%LA) were assessed in blood samples collected at the first antenatal visit.

Results: Ghanaian (-0.51 mmol/l), African-Caribbean (-0.19 mmol/l), and Moroccan (-0.15 mmol/l) women had significant lower TC levels compared to Dutch women. TG levels were lower in Ghanaian (log-transformed -0.12 mmol/l) but significantly higher in Surinam-Hindustani (0.10 mmol/l) and Turkish women (0.07 mmol/l). Age, physical activity, prepregnancy BMI, smoking, %SFA and %LA were independently related to TC and/or TG.

However, only pre-pregnancy BMI could partly explain observed disparities. Furthermore, pre-pregnancy BMI had a relatively large effect on TG levels in Surinam-Hindustani and Turkish women.

Conclusion:

TC and TG levels differed between ethnic groups during early gestation. Only pre-pregnancy BMI partly explained the ethnic differences to a relevant degree. Reduction in BMI before pregnancy may improve lipid profile, especially in Surinam-Hindustani and Turkish women.

Ethnic differences in maternal lipids 4

Introduction

Availability of maternal cholesterol and triglycerides (TG) is an essential factor in early pregnancy for an optimal development of both foetus and placenta. [START_REF] Herrera | Lipid metabolism in pregnancy and its consequences in the fetus and newborn[END_REF][START_REF] Woollett | Maternal cholesterol in fetal development: transport of cholesterol from the maternal to the fetal circulation[END_REF] When pregnancy progresses, specific changes in total cholesterol (TC), fractionated cholesterol and TG occur which leads to physiologically increased lipid levels towards the end of pregnancy. [START_REF] Sattar | Lipoprotein subfraction changes in normal pregnancy: Threshold effect of plasma triglyceride on appearance of small, dense low density lipoprotein[END_REF][START_REF] Brizzi | Lipoprotein metabolism during normal pregnancy[END_REF] It is thought that progressive increase in oestrogen levels during gestation induces most of these alterations in lipoprotein metabolism [START_REF] Desoye | Correlation of hormones with lipid and lipoprotein levels during normal pregnancy and postpartum[END_REF] which suggests the necessity of these metabolic changes for pregnancy maintenance and foetal growth. Indeed, reduced maternal TC levels are associated with intrauterine growth restriction and preterm delivery. [START_REF] Sattar | Lipid and lipoprotein concentrations in pregnancies complicated by intrauterine growth restriction[END_REF][START_REF] Edison | Adverse birth outcome among mothers with low serum cholesterol[END_REF] Conversely, marked elevated maternal TC and TG levels are associated with an increased risk on preeclampsia and other pregnancy related complications. [START_REF] Hj | Serum lipids in early pregnancy and risk of pre-eclampsia[END_REF][START_REF] Enquobahrie | Maternal plasma lipid concentrations in early pregnancy and risk of preeclampsia[END_REF] In addition, increased cholesterol deposition in foetal aortas could give rise to exaggerated lipid-rich lesions in both aortic arch and abdominal aorta in children. [START_REF] Napoli | Fatty streak formation occurs in human fetal aortas and is greatly enhanced by maternal hypercholesterolemia -Intimal accumulation of low density lipoprotein and its oxidation precede monocyte recruitment into early atherosclerotic lesions[END_REF][START_REF] Napoli | Influence of maternal hypercholesterolaemia during pregnancy on progression of early atherosclerotic lesions in childhood: Fate of Early Lesions in Children (FELIC) study[END_REF] These observations correspond to the 'foetal origins hypothesis' which states that an unfavourable intra-uterine metabolic environment predisposes for premature cardiovascular disease. [START_REF] Koklu | Increased aortic intima-media thickness is related to lipid profile in newborns with intrauterine growth restriction[END_REF][START_REF] Skilton | Intrauterine risk factors for precocious atherosclerosis[END_REF] Ethnic differences in lipid profile are well studied with in general a more favourable profile in the African populations, and a less favourable profile in the Indian-populations, compared to Western-populations in terms of cardiovascular risk. [START_REF] Bhalodkar | Comparison of highdensity and low-density lipoprotein cholesterol subclasses and sizes in Asian Indian women with Caucasian women from the Framingham Offspring Study[END_REF][START_REF] Cappuccio | Ethnicity and cardiovascular risk: variations in people of African ancestry and South Asian origin[END_REF][START_REF] Godsland | Mechanisms of Disease: lessons from ethnicity in the role of triglyceride metabolism in ischemic heart disease[END_REF] However, ethnic differences in lipid-profile during pregnancy is less frequently studied and results are inconclusive. [START_REF] Koukkou | Ethnic-Differences in Lipid and Lipoprotein Metabolism in Pregnant-Women of African and Caucasian Origin[END_REF]))

The underlying mechanisms that may explain possible ethnic differences in lipid profile are unknown. Genetic factors or structural variances in oestrogen levels could play a role in addition to behavioural factors. Behavioural factors related to lipid profile include smoking [START_REF] Willett | Effects of Cigarette-Smoking on Fasting Triglyceride, Total Cholesterol, and Hdl-Cholesterol in Women[END_REF], low physical activity [START_REF] Kelley | Aerobic exercise and lipids and lipoproteins in women: A meta-analysis of randomized controlled trials[END_REF], high fat diet [START_REF] Millen | Diet and plasma lipids in women .1. Macronutrients and plasma total and low-density lipoprotein Ethnic differences in maternal lipids 18 cholesterol in women: The Framingham nutrition studies[END_REF][START_REF] Sonnenberg | Diet and plasma lipids in women .2. Macronutrients and plasma triglycerides, high-density lipoprotein, and the ratio of total to high density lipoprotein cholesterol in women: The Framingham nutrition studies[END_REF] and alcohol use. [START_REF] Razay | Alcohol-Consumption and Its Relation to Cardiovascular Risk-Factors in British Women[END_REF] Large ethnic Ethnic differences in maternal lipids 6 differences in these behavioural factors have also been noted [START_REF] Zoratti | A review on ethnic differences in plasma triglycerides and high-density-lipoprotein cholesterol: Is the lipid pattern the key factor for the low coronary heart disease rate in people of African origin?[END_REF]Daryani et al., 2005b;[START_REF] Daryani | Reported macronutrient intake and metabolic risk factors: immigrant women from Iran and Turkey compared with native Swedish women[END_REF]Gentles et al., 2007) but up till now, it is unclear to what extent these factors contribute to ethnic differences in lipid profile during pregnancy. Knowledge about these ethnic differences in TC and TG and the explanatory role of behavioural factors is essential for initiating appropriate intervention strategies to prevent adverse pregnancy outcome and early predisposition of cardiovascular and related metabolic diseases.

We aimed to evaluate ethnic differences in TC and TG levels during pregnancy, and to explore to what extent maternal demographics, behavioural factors and clinical characteristics could explain possible differences.

Methods

This study is part of the Amsterdam Born Children and their Development study (www.abcd-study.nl). The ABCD study is a prospective community-based cohort study, which examines the relationship between maternal lifestyle and psychosocial conditions during pregnancy and the child's health at birth and later in life, with specific interest in ethnic differences. [START_REF] Van Eijsden | Maternal n-3, n-6, and trans fatty acid profile early in pregnancy and term birth weight: a prospective cohort study[END_REF]Goedhart et al., 2008a)

Study population

Between January 2003 and March 2004, all pregnant women living in Amsterdam (n =12.373) were invited to enrol in the ABCD study at their first prenatal visit to an obstetric caregiver (median: 12.9 weeks, interquartile range 11.9 -14.3 weeks). From these women, 8266 (response 67%) filled out an extensive pregnancy questionnaire including questions about socio-demographic characteristics, lifestyle, dietary habits and ethnic background. The questionnaire was in Dutch, accompanied by an English, Turkish or Arabic copy depending on the women's country of birth. These women were also asked to participate in the ABCD biomarker study. For this study, additional blood sampling took place after routine blood collection for screening purposes. The response rate for the ABCD biomarker study was 53% (n = 4389). TC and TG analyses were available for 95% (n = 4185). The study protocol was approved by the medical ethics committees of all Amsterdam hospitals and the Registration Committee of Amsterdam, and participants gave their written informed consent.

In this study, data of the six largest ethnic groups living in Amsterdam (Dutch, Surinam-Hindustani, African-Caribbean, Turkish, Moroccan and Ghanaian women) were used (N = 3073). Our analyses were based on data from the first and second trimester only.

Women using lipid altering medication (N = 39) and women with self reported pre-pregnancy diabetes (N = 9) were excluded due to influence on TC and TG levels, leaving 3025 women in the study population.

Laboratory Methods

Blood sampling was performed non-fasting. One blood sample was taken in a 10-mL EDTA (K) Vacutainer (Beckton Dickinson BV, Alphen aan de Rijn, the Netherlands) for the preparation of plasma and one blood sample was taken in a 9-mL Vacuette (Greiner BV, Alphen aan de Rijn, the Netherlands) for the preparation of serum. Aliquots were prepared by centrifugation (1600 x g for 10 min at room temperature) and stored at -80°C until analysis. [START_REF] Van Eijsden | Maternal n-3, n-6, and trans fatty acid profile early in pregnancy and term birth weight: a prospective cohort study[END_REF] TC and TG levels in the serum samples were determined at the Laboratory for Health Protection Research of the Dutch National Institute for Public Health and the Environment (RIVM). TC assay was performed using the CHOD-PAP method on a Hitachi 912 analyzer (Roche Diagnostics, Mannheim, Germany). TG was assayed with the GPO-PAP method (Roche Diagnostics, Mannheim, Germany). The interassay coefficient of variation (CV) was 1.0% for cholesterol and 2.3% for triglycerides.

The plasma samples were sent to the Analytical Biochemical Laboratory (ABL, Assen, the Netherlands) to perform fatty acid analysis using high performance capillary gas chromatography. [START_REF] Van Eijsden | Maternal n-3, n-6, and trans fatty acid profile early in pregnancy and term birth weight: a prospective cohort study[END_REF] The absolute amounts of SFA and LA (18:2-n6) (in mg/L plasma) were quantified on the basis of recovery from an internal standard and calculated in a relative value (percentage of total fatty acids).

Ethnic differences in maternal lipids 8 Measurements Ethnicity was based on the country of birth of the pregnant woman and her mother to include first and second generation women. [START_REF] Stronks | The utility of 'country of birth' for the classification of ethnic groups in health research: the Dutch experience[END_REF] We distinguished three groups of covariables: 1) demographic variables: maternal age, parity (0, 1, ≥2), cohabitant status (living together with partner vs. single) and maternal education (years of education after primary school: ≤5, 6-10, >10 year), 2) behavioural factors: self-reported physical activity (no exercise, low, moderate, vigorous), total %SFA (in quintiles), total %LA (in quintiles), smoking (no, yes and quit smoking because of pregnancy) and alcohol consumption (no vs. yes) and 3) clinical characteristics: maternal pre-pregnancy body mass index (BMI, based on self-reported height and weight, kg/m 2 ), pre-existent hypertension (no vs. yes) and depressive symptoms (CES-D scale [START_REF] Radloff | The CES-D scale: a self-report depression scale for research in the general population[END_REF]: lower vs. higher than the 90 th percentile). Physical activity during the previous week was scored by calculating a Metabolic equivalent (MET) score for the various reported activities using the compendium of physical activities. [START_REF] Ainsworth | Compendium of Physical Activities: an update of activity codes and MET intensities[END_REF] Percentage SFA was used as an indicator for saturated fatty acid consumption and %LA as an indicator for poly-unsaturated fatty acid consumption.

Statistical analysis

Differences in demographic variables, behavioural factors and clinical characteristics between the ethnic groups were tested with the Chi-square test for categorical variables and ANOVA for continuous variables. General linear models were built to study the relationship between ethnicity and TC or TG in more detail. First, univariate analyses were performed to explore the individual associations of ethnicity, maternal demographics, behavioural factors and clinical characteristics with TC and TG levels. As our results showed that TC and TG levels increased with gestational age (data not shown), we corrected for gestational age in further analyses.

We constructed three different extended models for multivariate analysis in a hierarchical approach. We used backward stepwise selection of variables to identify independent predictors of TC and TG. In model 1, demographics were added to ethnicity, model 2 subsequently added all behavioural factors to the model and in model 3, all clinical characteristics were added.

All potential first order interactions with ethnicity were tested. TG levels were log transformed to improve the normality of the residuals in the various models. P < 0.05 was considered significant. All statistical analyses were performed with the SPSS package version 15.0 (SPSS Inc, Chicago, USA).

Results

Study population

Compared to the non-response group (women of the same ethnicity as study population, but with no available blood samples, N = 2720), the study population (N = 3025) was significantly older (P = .004), more often nulliparous (P < .001), more often from Dutch origin P < .001), were higher educated (P < .001) and had a lower pre-pregnancy BMI (P < .001).

Table 1 presents the characteristics of the 3025 study participants according to ethnicity. Of the pregnant women, 74.8% were Dutch, 2.0% were Surinam-Hindustani, 7.8% African-Caribbean, 5.6% Turkish, 8.1% Moroccan, and 1.8% Ghanaian. Compared to the ethnic minority women, Dutch women were older, higher educated, more physically active, less often depressed, had a lower pre-pregnancy BMI and were more often nulliparous.

There were large differences in cohabitant status, the mean %SFA and mean %LA. Smoking during pregnancy was most common in Turkish women, while Dutch women were more likely to consume alcohol. Pre-pregnancy hypertension was most common among African-Caribbean women. The mean time at blood withdrawal did not differ between the ethnic groups (P = 0.61).

Total cholesterol

Mean levels of TC were highest in Surinam-Hindustani women (5.21 mmol/L) and lowest in Ghanaian women (4.65 mmol/L) (Table 1). Univariate analysis showed significantly Ethnic differences in maternal lipids 10 lower TC levels for African-Caribbean (β: -0.19 ± 0.06 (SE)), Moroccan (-0.15 ± 0.06) and Ghanaian (-0.51 ± 0.12) women compared to the Dutch women (Table 2).

Univariate analyses showed that maternal age was positively and parity was negatively associated with TC (Table 2). The relation between behavioural factors, clinical characteristics and TC was more pronounced. %SFA, smoking and pre-pregnancy BMI were univariately significantly associated with higher TC levels, while physical activity and %LA were negatively associated with TC (Table 2). Multivariate analysis adjusted for demographics (Table 2, model 1) showed that ethnic differences mostly remained the same.

Further adjustment for behavioural factors (Table 2, model 2) and clinical characteristics (Table 2, model 3) resulted in loss of significance in TC levels for Moroccan women compared to Dutch women (P = 0.077). In addition to ethnicity, maternal age, parity, physical activity, %SFA, %LA and pre-pregnancy BMI were all independently associated with TC in the final model.

Triglycerides

Median TG levels were highest in Surinam-Hindustani (1.56 mmol/L) women and lowest in Ghanaian women (1.11 mmol/L) (Table 1). Univariate analysis showed significantly higher TG levels (log transformed) for Surinam-Hindustani (β: 0.1 ± 0.03 (SE)) and Turkish (0.07 ± 0.02) women compared to Dutch women, while Ghanaian women had significant lower TG levels (-0.12 ± 0.03) (Table 3).

Further univariate analysis showed that maternal age, parity and maternal education were significantly associated with TG levels (Table 3). Behavioural factors %SFA and smoking were positively associated with TG levels, whereas physical activity, %LA and alcohol consumption were negatively associated. Pre-pregnancy BMI was also positively associated with TG levels, however, to a different degree within the ethnic groups (see below). Multivariate analysis adjusted for demographics (Table 3, model 1) showed that ethnic differences mostly remained the same. Further adjustment for behavioural factors (Table 3, model 2) slightly reduced the differences for Surinam-Hindustani, African-Caribbean and Turkish women compared to Dutch women, yet the differences remained significant. After adjustment for clinical characteristics and the interaction between ethnicity and pre-pregnancy BMI (Table 3, model 3), the difference in TG levels for Turkish women compared to Dutch women was slightly reduced. The African-Caribbean and Ghanaian women had significant lower TG levels compared to Dutch women in this final model. Furthermore, maternal age, education, physical activity, %SFA, %LA, smoking, alcohol consumption and pre-pregnancy BMI were all independently associated with TG levels.

Interaction pre-pregnancy BMI and ethnicity

The interaction between pre-pregnancy BMI and ethnicity was not significant for TC (P=0.09; Fig 1a), which means that the association between pre-pregnancy BMI and TC was the same for all ethnic groups. However, Figure 1b shows that the effect of increasing pre-pregnancy BMI has a larger effect on TG levels in Surinam-Hindustani and Turkish women compared to Dutch, African-Caribbean and Moroccan women. In Ghanaian women, pre-pregnancy BMI was not related to TG levels (P for interaction = 0.03).

Discussion

In this study, we evaluated ethnic differences in TC and TG levels during early pregnancy and the explanatory role of demographics, behavioural factors and clinical characteristics.

Compared to Dutch women, African-Caribbean, Moroccan and Ghanaian women displayed significant lower TC levels. Furthermore, Surinam-Hindustani and Turkish women had significantly higher TG levels, whereas Ghanaian women had significantly lower TG levels. Demographics and behavioural factors, except for cohabitant status, all contribute to gestational lipid profile independently. However, pre-pregnancy BMI was the only factor that could -partly -explain the ethnic differences in gestational lipid profile. The ethnic differences found in both TC and TG levels confirm results from previous studies in both non-pregnant [START_REF] Zoratti | A review on ethnic differences in plasma triglycerides and high-density-lipoprotein cholesterol: Is the lipid pattern the key factor for the low coronary heart disease rate in people of African origin?[END_REF][START_REF] Despres | Race, visceral adipose tissue, plasma lipids, and lipoprotein lipase activity in men and women -The health, risk factors, exercise training, and genetics (HERITAGE) family study[END_REF][START_REF] Lamon-Fava | Differences in serum sex hormone and plasma lipid levels in Caucasian and African-American premenopausal women[END_REF] and pregnant women [START_REF] Koukkou | Ethnic-Differences in Lipid and Lipoprotein Metabolism in Pregnant-Women of African and Caucasian Origin[END_REF], reporting more favourable TC and TG levels in African/Afro-Caribbean women as compared to Caucasian women. Taylor et al. (1987) found a significant difference in TG levels between postpartum African-Caribbean and Indian-Caribbean women which is similar to our observations for African-Caribbean and Surinam-Hindustani women. In contrast to their study, however, we also observed a significant difference in TC levels. The results of our comparison between Turkish and Dutch pregnant women, i.e. no difference in TC levels but significant higher TG levels, is in line with another study that compared Turkish to Swedish non-pregnant women. (Daryani et al., 2005b) However, no studies evaluated TC and TG levels as a result from early pregnancy between various ethnic populations in one living area.

Interestingly, previous results derived from the ABCD cohort showed that Ghanaian women have the highest risk for adverse birth outcomes, also after adjustments. (Goedhart et al., 2008a;Goedhart et al., 2008b) Since the Ghanaian women had the lowest TC and TG levels, this observation may confirm that reduced lipid levels during gestation may contribute to a reduced pregnancy outcome.

In our study, maternal behavioural factors and clinical characteristics were closely related to changes in TC or TG levels in early pregnancy. In men and non-pregnant women, %SFA, BMI and smoking have been demonstrated to be positively related with TC and/or TG levels whereas physical activity, %LA and alcohol intake were inversely related. [START_REF] Millen | Diet and plasma lipids in women .1. Macronutrients and plasma total and low-density lipoprotein Ethnic differences in maternal lipids 18 cholesterol in women: The Framingham nutrition studies[END_REF][START_REF] Sonnenberg | Diet and plasma lipids in women .2. Macronutrients and plasma triglycerides, high-density lipoprotein, and the ratio of total to high density lipoprotein cholesterol in women: The Framingham nutrition studies[END_REF][START_REF] Howard | Obesity and dyslipidemia[END_REF][START_REF] Willett | Effects of Cigarette-Smoking on Fasting Triglyceride, Total Cholesterol, and Hdl-Cholesterol in Women[END_REF][START_REF] Kelley | Aerobic exercise and lipids and lipoproteins in women: A meta-analysis of randomized controlled trials[END_REF]Daryani et al., 2005a;[START_REF] Razay | Alcohol-Consumption and Its Relation to Cardiovascular Risk-Factors in British Women[END_REF] Despite marked ethnic differences in various behavioural factors and clinical characteristics, only pre-pregnancy BMI explained ethnic disparities in gestational lipid profile. Indeed, pre-pregnancy BMI was significantly related to TG levels, especially in Surinam-Hindustani and Turkish women, and, to some extent, TC levels (Figure 1). In Ethnic differences in maternal lipids 13 contrast to waist circumference, BMI does not specify the fat location. Numerous studies have shown that the accumulation of central fat is more associated with abnormal lipid/lipoprotein profile compared to a general fat distribution. (Snijder et al., 2004b;[START_REF] Seidell | Waist and hip circumferences have independent and opposite effects on cardiovascular disease risk factors: the Quebec Family Study[END_REF][START_REF] Canoy | Serum lipid concentration in relation to anthropometric indices of central and peripheral fat distribution in 20,021 British men and women: Results from the EPIC-Norfolk population-based cohort study[END_REF] The ethnic differences might be explained by the variability in fat deposition, i.e. visceral vs. subcutaneous accumulation. [START_REF] Despres | Race, visceral adipose tissue, plasma lipids, and lipoprotein lipase activity in men and women -The health, risk factors, exercise training, and genetics (HERITAGE) family study[END_REF]Snijder et al., 2004b;Snijder et al., 2004a) For instance, Ghanaian women have more subcutaneous fat deposits compared with Caucasian women and also the lowest TC and TG levels (Table 2 and3). This indicates that there could be modulators of lipoprotein metabolism involved that are related to visceral fat, which may result in a different expression of lipid phenotype. Indeed, Després et al. [START_REF] Despres | Race, visceral adipose tissue, plasma lipids, and lipoprotein lipase activity in men and women -The health, risk factors, exercise training, and genetics (HERITAGE) family study[END_REF] found higher lipoprotein lipase and lower hepatic lipase activities in African-American than in white women and Bower et al. [START_REF] Bower | Ethnic differences in in vitro glyceride synthesis in subcutaneous and omental adipose tissue[END_REF] showed that African-American women have an increased capacity to synthesize locally TG in omental adipose tissue compared to Caucasian women. In African Americans, the relationship between BMI and waist circumference/visceral fat with assessment of CT scanning (L4 protocol) is less than in white American women. [START_REF] Carroll | Visceral fat, waist circumference, and BMI: Impact of race/ethnicity[END_REF] In addition, a different metabolic activity of abdominal fat and other environmental factors are closely related to differences in insulin resistance comparing African women with Caucasian women with subsequent effects on lipid levels. (Reimann et al., 2007) These intrinsic metabolic properties of local fat tissue, dependent on membership of an ethnic group, may explain the observed lipid differences despite a similar pre-pregnancy BMI among ethnic groups. Some limitations and strengths of our study should be acknowledged. Our study population consisted of a large, unselected cohort of pregnant women, who represented the main ethnic groups in the region of Amsterdam City, the Netherlands. Despite the large sample size, there was selective participation and our results apply to a relatively healthy sample. This could have made our estimates too conservative. Furthermore, blood sampling was non-fasting and performed just on one occasion, but this is expected to only dilute our results. We did not have data of other lipid fractions, such as high density lipoprotein cholesterol (HDL-c) or low density lipoprotein cholesterol (LDL-C). However, the aim of our analysis was not to perform a cardiovascular risk profile but to assess TG and TC levels.

Finally, although we adjusted for several factors, we cannot exclude some residual confounding, either resulting from unmeasured or inaccurately measured covariates. We did not have diet questionnaires although it is known that there are differences in dietary patterns between ethnic groups. (Daryani et al., 2005a;[START_REF] Daryani | Reported macronutrient intake and metabolic risk factors: immigrant women from Iran and Turkey compared with native Swedish women[END_REF][START_REF] Diaz | Race and diet in the overweight: association with cardiovascular risk in a nationally representative sample[END_REF].

However we did include %SFA and %LA in our analysis to reflect in average dietary composition. Behavioural and clinical characteristics were self-reported, which may have introduced some bias for measures, such as pre-pregnancy BMI, as women could misreport height and weight. However, in large epidemiological studies self report found to accurately measure BMI for the purpose of associational analysis. [START_REF] Mcadams | Comparison of self-reported and measured BMI as correlates of disease markers in US adults[END_REF] Whereas too low TC levels have been associated with adverse outcome at short term, too high levels may be predictive for atherosclerosis in the newborn at long term.

Facing the epidemic of overweight, lipid levels will rather be too high than too low at the population level. When the foetus is exposed to an atherogenic environment in utero, the vulnerability for enhanced risk for atherosclerosis-related disease may be higher already in early life. Based on the current data, this vulnerability may be more prominent in specific ethnic groups. However, the minimally required level of lipids that protect against adverse outcomes is also unknown. Altogether, more data is needed to define the optimal lipid ranges for both mother and child.

To decrease ethnic differences in cardiovascular health, prevention should start in the preconception phase and focus on weight reduction which can be achieved by dietary advises and physical activity programs. Although physical activity, %SFA and %LA did not explain the ethnic disparities in TC and TG levels, they remain important means to reduce pre-pregnancy BMI. In addition, smoking also contributed to adverse TG levels. This is particularly important for the Turkish women as 23.8% continued to smoke during pregnancy.

In conclusion, early gestational TC and TG levels differ between ethnic groups. Among all ethnic groups, this change in TG and TC levels is mostly explained by prepregnancy BMI. Another distribution of fat or intrinsic metabolic properties of both fat tissue and glucose/insulin homeostasis are all factors that characterize each ethnic group. In this background, preventive measures (eg. weight reduction) to lower TG and TC levels during pregnancy should be offered to especially Surinam-Hindustani and Turkish women already in the preconception phase. Before such preconception prevention strategy should be considered, more results are needed to acknowledge that an adverse gestational lipid profile is associated with pregnancy outcome and the occurrence of late onset disease in the newborn. *Model 2: adjusted for maternal age, parity, physical activity and %SFA and gestational age at blood sampling ‡ Model 2: adjusted for: maternal age, education, physical activity, %SFA, smoking, alcohol consumption and gestational age at blood sampling.

Figure 1

 1 Figure 1 Interaction between ethnicity and pre-pregnancy BMI on total cholesterol levels (a)* and triglyceride levels (b) ‡ .

  01 (0.01) Pre-existent hypertension 0.03 (0.02) -Depressed 0.02 (0.01) -*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 1 Univariate analysis: adjusted for gestational age at blood sampling.

Table 1

 1 Characteristics of study participants according to ethnicity.

	N	Dutch	Surinam -	African -	Turkish	Moroccan	Ghanaian	P value
			Hindustani	Caribbean

Table 2

 2 Univariate and multivariate associations between total cholesterol and ethnicity.

		Univariate analysis 1	Multivariate analysis Model 1 2	Multivariate analysis Model 2 3	Multivariate analysis Model 3 4
		β (SE)	β (SE)	β (SE)	Β (SE)
	Ethnicity
	Dutch	Reference	Reference	Reference	Reference
	Surinam-Hindustani	0.06 (0.11)	0.08 (0.11)	0.08 (0.11)	0.07 (0.11)
	African-Caribbean	-0.19 (0.06)***	-0.15 (0.06)*	-0.18 (0.06)**	-0.21 (0.06)***
	Turkish	-0.03 (0.07)	0.04 (0.08)	0.10 (0.08)	0.07 (0.08)
	Moroccan	-0.15 (0.06)**	-0.14 (0.07)*	-0.04 (0.06)	-0.09 (0.06)
	Ghanaian	-0.51 (0.12)***	-0.47 (0.12)***	-0.52 (0.12)***	-0.59 (0.12)***
	Demographics
	Maternal age (years)	0.01 (0.003)***	0.02 (0.004)***	0.02 (0.004)***	0.02 (0.004)***
	Parity
	0	Reference	Reference	Reference	Reference
	1	-0.05 (0.04)	-0.09 (0.04)*	-0.09 (0.04)**	-0.11 (0.04)**
	≥2	-0.11 (0.05)*	-0.17 (0.06)**	-0.14 (0.06)*	-0.17 (0.06)**
	Cohabitant status
	Living together	Reference	-	-	-
	Living apart	-0.06 (0.05)
	Education (years)
	0 -5	-0.02 (0.05)	0.14 (0.05)**	-	-
	6 -10	0.05 (0.03)	0.12 (0.04)***
	> 10	Reference	Reference
	Behavioural factors
	Physical activity
	No exercise	Reference		Reference	Reference
	Low	-0.08 (0.05)		-0.12 (0.05)*	-0.11 (0.05)*
	Moderate	-0.05 (0.05)		-0.15 (0.05)**	-0.14 (0.05)**
	Vigorous %SFA 5	-0.16 (0.07)*		-0.25 (0.07)***	-0.23 (0.07)***
	Q1	Reference		Reference	Reference
	Q2	0.12 (0.05)*		0.11 (0.05)*	0.11 (0.05)*
	Q3	0.20 (0.05)***		0.14 (0.05)**	0.14 (0.05)**
	Q4	0.23 (0.05)***		0.16 (0.05)***	0.16 (0.05)***
	Q5 % LA 6	0.43 (0.05)***		0.32 (0.05)***	0.32 (0.05)***
	Q1	0.40 (0.05)***		0.29 (0.05)***	0.26 (0.05)***
	Q2	0.28 (0.05)***		0.20 (0.05)***	0.19 (0.05)***
	Q3	0.27 (0.05)***		0.21 (0.05)***	0.19 (0.05)***
	Q4	0.15 (0.05)**		0.12 (0.05)*	0.11 (0.05)*
	Q5	Reference		Reference	Reference
	Smoking while pregnant
	No	Reference		-	-
	Quit smoking	0.04 (0.04)
	Yes	0.10 (0.05)*
	Alcohol while pregnant	-0.02 (0.04)		-	-
	Clinical characteristics BMI (kg/m 2 )	0.02 (0.004)***			0.02 (0.004)***
	Pre-existent hypertension 0.01 (0.09)			-
	Depressed	-0.02 (0.05)			-
	*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 1 Univariate analysis: adjusted for gestational age at blood sampling. 2 Model 1, adjusted for: maternal age, parity, maternal education and gestational age at blood sampling.

3

Model 2, adjusted for: maternal age, parity, physical activity, %SFA, %LA and gestational age at blood sampling.

4

Model 3, adjusted for: maternal age, parity, physical activity, %SFA, %LA, BMI and gestational age at blood sampling. 5 Saturated fatty acids: quintile distribution. 6 Linoleic acid: quintile distribution.

Table 3

 3 Univariate and multivariate associations between triglycerides (log transformed) and ethnicity.

	Univariate analysis 1	Multivariate analysis Model 1 2	Multivariate analysis Model 2 3	Multivariate analysis Model 3 4
	β (SE)	β (SE)	β (SE)	β (SE)	5
	Ethnicity
	Dutch
	Surinam-Hindustani
	African Caribbean
	Turkish
	Moroccan
	Ghanaian

Acknowledgements

We are grateful to all participating hospitals, obstetric clinics and GPs for their assistance in the implementation of the ABCD study. We like to thank all the participated pregnant women for their co-operation. We thank D. Bijlenga for her statistical advice, J.S.L.

de Munter for his help with calculating MET scores and Prof. K. Stronks for her critical comments on the manuscript.

Details of Ethics Approval

Approval for the ABCD study was obtained from the Central Committee on Research involving Human Subjects (CCMO; number P02.0335L, 2002), the Medical Ethical Committees of participating hospitals and the Registration Committee of Amsterdam.

Financial support for the Amsterdam Born Children and their Development study was granted by the Netherlands Organisation for Health Research and Development (ZonMw), The Hague. Dr ThB Twickler is financially supported by the Netherlands association of medical science (NWO-ZonMW).

Conflicts of interest

None declared.


Steve Bloom
email: s.bloom@imperial.ac.uk

Heather Ford

PhD Veronique Peters

PhD Niamh M Martin

Michelle Sleeth

PhD Mohammad A Ghatei

Heather E Ford Bsc

Michelle L Sleeth Bsc

PhD Gary S Frost

MD Steve R Bloom

Stephen R Bloom

Effects of oral ingestion of sucralose on gut hormone response and appetite in healthy normal-weight subjects

Keywords: obesity, sucralose, sweetener, gut hormone, appetite

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Introduction

Recently, there have been significant advances in our understanding of how hormonal signals released from the gastrointestinal (GI) tract interact with circuits within the central nervous system to control appetite and energy intake [START_REF] Murphy | Gut hormones and the regulation of energy homeostasis[END_REF]. The gut hormones peptide YY (PYY) and glucagon-like peptide (GLP)-1 are co-secreted from intestinal enteroendocrine L-cells and released post-prandially in proportion to the amount of energy ingested [START_REF] Adrian | Human distribution and release of a putative new gut hormone, peptide YY[END_REF][START_REF] Ghatei | Molecular forms of human enteroglucagon in tissue and plasma: plasma responses to nutrient stimuli in health and in disorders of the upper gastrointestinal tract[END_REF][START_REF] Le Roux | Attenuated peptide YY release in obese subjects is associated with reduced satiety[END_REF]. PYY and GLP-1 have both been shown to be satiety factors, reducing food intake when administered to rodents [START_REF] Batterham | Gut hormone PYY(3-36) physiologically inhibits food intake[END_REF][START_REF] Challis | Acute effects of PYY3-36 on food intake and hypothalamic neuropeptide expression in the mouse[END_REF]Chelikani et al., 2005a;Chelikani et al., 2005b;[START_REF] Chelikani | Daily, intermittent intravenous infusion of peptide YY(3-36) reduces daily food intake and adiposity in rats[END_REF][START_REF] Halatchev | Peptide YY3-36 inhibits food intake in mice through a melanocortin-4 receptor-independent mechanism[END_REF][START_REF] Talsania | Peripheral exendin-4 and peptide YY(3-36) synergistically reduce food intake through different mechanisms in mice[END_REF] and to humans [START_REF] Batterham | Gut hormone PYY(3-36) physiologically inhibits food intake[END_REF][START_REF] Degen | Effect of peptide YY3-36 on food intake in humans[END_REF][START_REF] Flint | Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans[END_REF][START_REF] Gutzwiller | Glucagon-like peptide-1: a potent regulator of food intake in humans[END_REF][START_REF] Le Roux | Attenuated peptide YY release in obese subjects is associated with reduced satiety[END_REF]. The incretin effect of GLP-1, augmentation of insulin secretion in response to an oral glucose load, has been well characterised [START_REF] Elrick | Plasma insulin response to oral and intravenous glucose administration[END_REF]. Secretion of PYY and GLP-1 is regulated by a complex neuro-humoral system in addition to direct nutrient contact with specific receptors expressed by intestinal L-cells. However, the mechanisms by which luminal nutrients stimulate the release of GLP-1 and PYY from L-cells remain poorly understood.

The two proteins T1r2 and T1r3 form a heterodimer and function together as a general sweettaste receptor [START_REF] Li | Human receptors for sweet and umami taste[END_REF][START_REF] Nelson | Mammalian sweet taste receptors[END_REF]. T1r2+T1r3 is coupled to the G-protein gustducin, which mediates transduction of sweet taste signals [START_REF] Wong | Transduction of bitter and sweet taste by gustducin[END_REF]. T1r, and the alpha subunit of gustducin (α-gust), are co-localised with GLP-1 and PYY in enteroendocrine L-cells of the intestinal brush border membranes [START_REF] Jang | Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1[END_REF][START_REF] Rozengurt | Colocalization of the alpha-subunit of gustducin with PYY and GLP-1 in L cells of human colon[END_REF][START_REF] Sutherland | Phenotypic characterization of taste cells of the mouse small intestine[END_REF]. Recently, a key role for α-gust and a functioning sweet-taste receptor in glucose stimulated GLP-1 secretion from the L-cell has been demonstrated [START_REF] Jang | Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1[END_REF]. Application of sucralose (a non-calorific, nonmetabolisable sweetener) to human L-cells in vitro stimulated GLP-1 secretion and this effect was inhibited with co-administration of a T1r3 inhibitor. This evidence supports a new signaling mechanism which regulates gut hormone secretion via the sweet-taste receptor T1r2+T1r3 in the GI tract.

One proposed factor in the increasing prevalence of obesity and type 2 diabetes is an increased consumption of processed foods containing high levels of sucrose and fructose [START_REF] Bray | Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity[END_REF][START_REF] Elliott | Fructose, weight gain, and the insulin resistance syndrome[END_REF][START_REF] Raben | Sucrose compared with artificial sweeteners: different effects on ad libitum food intake and body weight after 10 wk of supplementation in overweight subjects[END_REF]. To offset this, the food industry has attempted to replace sugars with artificial sweeteners. The ability of non-calorific sweeteners to enhance endogenous gut hormone release would represent a potentially exciting opportunity for their addition to foods as agents to control glucose homeostasis and appetite regulation in populations at risk of type 2 diabetes and obesity.

The aim of this study is to investigate whether oral ingestion of sucralose, at a dose that would be consumed in a normal diet, increases circulating GLP-1 or PYY concentrations in man.

Subjects and Methods

Subjects

Eight normal-weight, healthy volunteers were locally recruited. All were non-smokers, aged 22-27y (seven females and one male) with a stable body weight and a body mass index ranging from 18.8 to 23.9 kg/m 2 . Persons who disliked the study food, who had food allergies or food restrictions, who were taking medication that was likely to affect taste, smell or appetite or who reported recent weight loss or weight cycling were excluded. Subjects were screened using the standard Dutch Eating Behaviour questionnaire [START_REF] Van Strien | Life events, emotional eating and change in body mass index[END_REF] and SCOFF questionnaire [START_REF] Morgan | The SCOFF questionnaire: a new screening tool for eating disorders[END_REF] and were excluded if they demonstrated abnormal eating behaviour. Female volunteers attended all study days within the follicular stage of the menstrual cycle.

The study was conducted with local ethical approval (project registration number: 07/Q0406/62). Written informed consent was obtained from all volunteers and the study was performed in accordance with the Declaration of Helsinki.

Study design

The study had a randomised, single-blinded, cross-over design. Subjects were randomly assigned to receive one of four solutions on four separate study sessions. Study sessions lasted from 0830h until 1230h with at least three days between sessions. Subjects were asked to refrain from drinking alcohol and to keep evening meals and activity levels as similar as possible the day before each test session and to fast from 2100h, consuming only water.

On arrival at the study centre subjects were asked to be seated and to relax for 30 minutes following placement of the intravenous cannula. After two baseline blood samples subjects completed one of four experimental manipulations. Subjects ingested, in a single swallow, 50mL of either water (W), sucralose (S; 0.083% w/v, 2mmol/L Splenda®, Tate and Lyle PLC, Southampton, UK) or the positive control maltodextrin (MD) which was matched for sweetness with sucralose (50% w/v Polycose®, Abbott Laboratories Ltd, US, plus 0.083% sucralose). Each was followed by a one minute period of modified-sham-feeding (MSF) protocol of the same solution that was swallowed. The fourth experimental manipulation was designed to ascertain the involvement of stimulation of the sweet-taste receptors within the oral cavity independently of the sweet-taste receptors throughout the GI tract. In this instance subjects consumed 50mL water followed by the one-minute MSF of the sucralose solution (WS; 0.083% w/v sucralose). The test solutions used are described in Table 1. The MSF protocol involved drawing the solution up into the mouth through a straw, moving it around in the mouth and then spitting it out, doing so repeatedly until the entire volume of 200mL was finished and the one minute time limit was up. In order to investigate the cephalic effects of sucralose, the MSF was performed after ingestion of the test solution to ensure that no residual sucralose would be swallowed with the subsequent ingestion of water in the WS manipulation.

The dose of sucralose was chosen to represent a normal dietary load and the total volume ingested was kept to a minimum, as it is known that ingestion of large volumes of water alone can induce a gut hormone response [START_REF] Christofides | Release of gastrointestinal hormones following an oral water load[END_REF]. Maltodextrin (a five polymer chain of glucose) was used to assess the effect of glucose on gut hormone release without the potential confounder of high concentrations of glucose effecting gastric emptying.

The expectorate from the MSF was weighed to ensure compliance with the protocol. Blood samples and visual analogue scores (VAS) pertaining to subjective feelings of appetite, were taken for a further two hours. After the last blood sample at 120 minutes, a test meal of known energy content was given in excess and subjects were asked to eat until comfortably full. Energy intake was calculated from the weight of food eaten.

Blood samples

Two baseline blood samples were taken at -15 and 0 minutes before consumption of test solutions, then further samples were taken at 15, 30, 45, 60, 90 and 120 minutes after consumption of test solutions. For analysis of the cephalic phase insulin release (CPIR) and the cephalic phase GLP-1 response, blood samples were also taken at 2, 4, 6, 8 and 10 minutes for insulin and GLP-1 analysis only. Blood was collected in lithium heparin tubes containing 5000 kallikrein units of aprotinin (200µL; Trasylol; Bayer) and immediately centrifuged at 4ºC. Plasma was separated and stored at -20ºC until analysis.

Appetite ratings

Subjective feelings of appetite were assessed at -15, 0, 15, 30, 45, 60, 90 and 120 minutes using VAS [START_REF] Flint | Reproducibility, power and validity of visual analogue scales in assessment of appetite sensations in single test meal studies[END_REF] with questions pertaining to desire to eat, hunger and prospective food consumption. Subjects were also asked to score the palatability and sweetness of the test solutions. Subjects marked their answers to the questions on scales of 100mm in length anchored at either end with the most positive and the most negative response. The distance along the scale that the subjects placed their mark was measured from one end and the reading in millimeters was recorded.

Biochemical analyses

All samples were assayed in duplicate and in a single assay to eliminate inter assay variation.

Plasma PYY and GLP-1 were assessed using an established in-house radioimmunoassay (RIA) described previously [START_REF] Adrian | Human distribution and release of a putative new gut hormone, peptide YY[END_REF][START_REF] Kreymann | Glucagon-like peptide-1 7-36: a physiological incretin in man[END_REF]. The detection limit of the PYY and GLP-1 assays was 2.5pmol/L and 7.5pmol/L with an intra-assay coefficient variation (CV) of 5.8% and 5.4% respectively. Insulin was measured using Axsym analyser (Abbott Diagnostics, Maidenhead, UK). Sensitivity was 7 pmol/L with an intra-assay CV of 2.6%. Plasma glucose was measured using an Abbott Architect ci8200 analyser (Abbott Diagnostics, Maidenhead, UK). Sensitivity was 0.3mmol/L and intra-assay CV was 1%.

Statistics

All data are represented as mean values ± SEM. Plasma hormone and glucose concentrations were adjusted from baseline and represented as time course from change from baseline.

Incremental area under the curve (iAUC) was calculated over baseline by the trapezoidal rule.

Data for energy intake and iAUC were tested for normality and analysed using repeated measures one-way ANOVA with Bonferroni's test for post hoc comparisons (GraphPad Prism 4.03 Software, San Diego, USA). In all cases P<0.05 was considered to be statistically significant.

Results

Validation of MSF

After all test solutions, the expectorate weight was greater than the weight of the MSF solutions sipped due to the addition of saliva indicating a successful MSF with minimum swallowing of test solutions.

The water solution was significantly less sweet than the remaining three test solutions (70.9 ± 3 mm [WS], P<0.001; 65.9 ± 10.8 mm [S], P<0.01; 73.1 ± 8 mm [MD], P<0.001 vs. 5.9 ± 2 mm [W]; n=8). The WS, S and MD solutions were rated as having the same sweetness and palatability (42.8 ± 8.9 mm [WS]; 36.8 ± 10 mm [S]; 38.1 ± 8.5 mm [MD], 26.1 ± 6 mm [W]; n=8).

Appetite and food intake

For the two hour period following administration of the test solutions, there was no significant difference in the iAUC (0-120min) of subjective feelings of appetite (Table 2). Two hours after consumption of test solutions, there was no significant difference in energy intake or water intake at the buffet meal (Table 2).

Hormones and glucose

Plasma insulin and GLP-1 did not show any significant change during the first 10 min after the MSF of any solution (Table 2). iAUC (0-120min) for plasma GLP-1 and PYY concentrations were similar in all four groups. The MD group had a significantly higher iAUC (0-120min) of insulin and glucose concentrations compared with water, but there was no difference between any other solution tested (Figure 1 and Table 2).

Discussion

In the present study, we show that oral ingestion of a common dietary dose of the noncalorific, artificial sweetener sucralose does not increase plasma GLP-1 or PYY concentrations nor does it affect subjective feelings of appetite or energy intake at the next meal in healthy volunteers. This study mimics the physiological intake of a sweetened solution. Our data are in accord with recently published human data [START_REF] Ma | Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects[END_REF] and in vivo rat data [START_REF] Fujita | Incretin release from gut is acutely enhanced by sugar but not by sweeteners in vivo[END_REF] in which sucralose ingestion failed to stimulate a rise in two circulating incretin hormones, GLP-1 and the K cell derived glucose-dependent insulinotropic polypeptide (GIP). Ma et al. administered sucralose nasogastrically to healthy, normal weight volunteers and observed no effect on plasma GLP-1 or GIP concentrations [START_REF] Ma | Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects[END_REF]. Similarly, Fujita et al. demonstrated in rats that in contrast to sucrose gavage, oral gavage of sucralose did not induce a rise in plasma GLP-1 [START_REF] Fujita | Incretin release from gut is acutely enhanced by sugar but not by sweeteners in vivo[END_REF].

The only other published study to investigate the acute effect of oral sweetener ingestion on gut hormone release in humans used the sweetener aspartame. Although, ingestion of encapsulated aspartame was associated with a reduction in subsequent food intake, this effect did not seem to be mediated by GLP-1 release [START_REF] Hall | Physiological mechanisms mediating aspartame-induced satiety[END_REF]. We chose not to encapsulate sucralose in our study, as it remains intact throughout the GI tract and very little is absorbed [START_REF] Grice | Sucralose--an overview of the toxicity data[END_REF]. Therefore, sucralose may stimulate receptors on more distal L cells throughout the GI tract.

We did not observe a plasma GLP-1 response following ingestion of maltodextrin plus sucralose. GLP-1 response to glucose seems to be dependent on glucose been present in the distal duodenum where L-cells are present [START_REF] Parker | Molecular mechanisms underlying nutrient-stimulated incretin secretion[END_REF]. In this experiment we used relatively low amount of a glucose polymer which is cleared efficiently in the proximal duodenum before it can elicit a gut hormone response.

Oral ingestion of two non-calorific sweeteners, sucralose plus acesulfame K, followed by an oral glucose tolerance test, produces higher plasma peak GLP-1 concentrations compared to ingestion of water followed by an oral glucose tolerance test in healthy normal weight subjects [START_REF] Brown | Ingestion of Diet Soda before a Glucose Load Augments GLP-1 Secretion[END_REF]. Consistent with this sucralose plus glucose has an additive stimulatory effect on GLP-1 secretion from murine primary L-cells compared to sucralose or glucose alone [START_REF] Reimann | Glucose sensing in L cells: a primary cell study[END_REF]. Taken together these studies suggest that non-calorific sweeteners and sugars may act synergistically to stimulate GLP-1 release from L-cells. In the current study, the maltodextrin solution was matched for sweetness by addition of sucralose.

Therefore we cannot exclude a synergistic effect of maltodextrin and sucralose on plasma gut hormone concentrations, if a gut hormone response had occurred. It would be interesting to compare the effects of maltodextrin alone, without any match for sweetness, to the combination of maltodextrin plus sucralose to assess any additive effect of sucralose and maltodextrin on GLP-1 release.

Furthermore, we show that sucralose ingestion does not affect plasma glucose and insulin. This is consistent with previous human studies in which no effect on plasma glucose and insulin was observed following ingestion of encapsulated sucralose in diabetic patients [START_REF] Grotz | Lack of effect of sucralose on glucose homeostasis in subjects with type 2 diabetes[END_REF] or following intragastric infusion of sucralose in healthy subjects [START_REF] Ma | Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects[END_REF]. Similarly, oral gavage of sucralose in rats did not improve glucose homeostasis following an intraperitoneal glucose tolerance test [START_REF] Fujita | Incretin release from gut is acutely enhanced by sugar but not by sweeteners in vivo[END_REF], suggesting that there was no incretin effect mediated by the sucralose gavage.

Our study is the first to investigate the cephalic phase GLP-1 and insulin responses to sucralose. We demonstrate that stimulation of the oral cavity with a sucralose-sweetened solution does not lead to an early (0-10 min) increase in plasma GLP-1 and does not affect subsequent food intake. This is in keeping with previous studies which have not demonstrated a cephalic phase GLP-1 response to either ingestion of a mixed meal [START_REF] Ahren | The cephalic insulin response to meal ingestion in humans is dependent on both cholinergic and noncholinergic mechanisms and is important for postprandial glycemia[END_REF] or to a sham fed meal [START_REF] Luscombe-Marsh | The addition of monosodium glutamate and inosine monophosphate-5 to high-protein meals: effects on satiety, and energy and macronutrient intakes[END_REF]. Furthermore, we show that sucralose does not elicit a preabsorptive insulin response. This is consistent with previous studies using a similar MSF protocol to assess the ability of non-calorific sweeteners such as aspartame and saccharin to induce a CPIR in humans [START_REF] Abdallah | Cephalic phase responses to sweet taste[END_REF][START_REF] Teff | Sweet taste: effect on cephalic phase insulin release in men[END_REF].

The concentration of sucralose used in the present study (2mmol/L) was chosen to be both palatable and within the dose range (1-5mmol/L) previously shown to trigger GLP-1 release from intestinal L-cells in vitro [START_REF] Jang | Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1[END_REF]. However, with ensuing dilution in the gut lumen post-ingestion, it is possible that the concentration of sucralose reaching the small intestine was below 2mmol/l, which may have been insufficient to stimulate GLP-1 secretion. An alternative explanation for our findings is that sucralose does not stimulate GLP-1 release from intestinal enteroendocrine cells. In support of this, recent in vitro studies failed to demonstrate an effect of sucralose on GLP-1 [START_REF] Reimann | Glucose sensing in L cells: a primary cell study[END_REF] from enteroendocrine cells using a similar sucralose concentration to that used in the current study. Furthermore, a recent in vivo study has shown that intragastric infusion of up to 40mmol/L sucralose does not induce GLP-1 secretion in humans [START_REF] Ma | Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects[END_REF]. Together with our data, these studies suggest that there is no measurable acute enhancement of GLP-1 or PYY release in vivo following oral ingestion of sucralose. The reason for the apparent disparity of the effect of sucralose on gut hormone release in vitro and in vivo is not clear and requires further investigation.

In summary, we have shown that oral ingestion of sucralose does not elicit a cephalic phase GLP-1 or insulin response nor increase post-ingestive plasma GLP-1 or PYY concentrations, and therefore does not subsequently affect appetite. Our findings, using a dietary dose of sucralose, do not support the proposal that stimulation of the sweet taste receptor in the GI tract can stimulate release of GLP-1 and PYY from enteroendocrine L-cells.

Figure 1 .

 1 Figure 1. Change in plasma A) GLP-1, B) PYY, C) insulin and D) glucose from baseline following administration of test solutions (n=8). •=water, ○=cephalic sucralose, ■=sucralose, □=maltodextrin+sucralose. Data are represented as mean ± SEM.

Table 1 .

 1 Description of the test solutions used on the four study days.

	Study day	Test solution ingested	Test solution used for the
		(Volume=50ml)	one-minute MSF
			(Volume=200ml)
	water (W)	water	water
	cephalic sucralose (WS)	water	sucralose dissolved in water
	sucralose (S)	sucralose dissolved in water sucralose dissolved in water
	maltodextrin+sucralose (MD) maltodextrin plus sucralose	maltodextrin plus sucralose
		dissolved in water	dissolved in water

Table 2 .

 2 Incremental AUC data for plasma hormones, glucose and appetite scores measured between 0 and 120 minutes (unless specified) and energy and water intake at the buffet meal.W=water, WS= cephalic sucralose, S=sucralose, MD=maltodextrin+sucralose. Data are represented as mean ± SEM. a = p<0.05 compared to MD, b = p<0.01 compared to MD, c = p<0.001 compared to MD. n=8.

		W		WS		S		MD
	Insulin (0-10min) (pmol.min/L)	171 ± 60		235 ± 83		70 ± 25		68 ± 24
	GLP-1 (0-10min) (pmol.min/L)	882 ± 113		948 ± 171	922 ± 132	658 ± 78
	Glucose (mmol.min/L)	33.5 ± 6.9	a	51 ± 12.5 b	25.3 ± 20.4	c 122.3 ± 17.5
	Insulin (pmol.min/L)	287 ± 331 c	-471 ± 132	c	-459 ± 352	c	5669 ± 519
	GLP-1 (pmol.min/L)	-675 ± 1610	-248 ± 784	-359 ± 401	415 ± 610
	PYY (pmol.min/L)	-179 ± 119	-128 ± 119	-56 ± 192	283 ± 185
	Hunger (mm.min)	1724 ± 322	1641 ± 336	1993 ± 199	2017 ± 472
	Desire to eat (mm.min)	1376 ± 216	1128 ± 275	1330 ± 458	1441 ± 461
	Prospective food consumption (mm.min)	1318 ± 305	1623 ± 266	2002 ± 247	1676 ± 441
	Energy intake (kJ)	2355±227		2417±222	2597±277	2460±167
	Water intake (mL)	267.0±69.0	250.7±45.1	291.8±49.8	305.0±49.6

et al: Effects of oral ingestion of sucralose on gut hormone response and appetite in healthy normal- weight subjects.

Acknowledgements

We thank Mandy Donaldson and John Meek for glucose and insulin assays, the Sir John

McMichael research centre for Clinical Investigation and Research, Hammersmith Hospital and the volunteers. V.P. is funded through a European Union framework 6 Marie Curie fellowship (NuSISCO). N.M.M. is funded by a HEFCE Clinical Senior Lecturer Award.

Contributions of the authors:

H.E.F. and V.P. designed the experiment, collected and analysed data and wrote the manuscript. N.M.M. helped with the writing of the manuscript. M.S. contributed to the data analysis. M.A.G, G.S.F and S.R.B. provided significant advice.

This research is funded by program grants from the MRC (G7811974) and Wellcome Trust (072643/Z/03/Z) and by an EU FP6 Integrated Project Grant LSHM-CT-2003-503041. We are also grateful for support from the NIHR Biomedical Research Centre funding scheme.

We thank Tate and Lyle for the provision of sucralose. (GLP)-1 with co-administration of a T1r2+T1r3 inhibitor.

Conflict of interest:

The authors do not declare any conflict of interest.