A dose response study of the effects of increased fruit and vegetable intake on vascular function. Final Report to the Food Standards Agency

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1 A dose response study of the effects of increased fruit and vegetable intake on vascular function Final Report to the Food Standards Agency Project N2030 Project Leader: Professor TAB Sanders Nutritional Sciences Research Division King s College London Franklin-Wilkins Building 150 Stamford Street London SE1 9NH Modified 14 November2007 1

2 CONTENTS 1. Executive Summary Background Rationale and Objectives Approach Outcome/key findings What it means and why it is important Technical Report Background Aims and objectives of the investigation Pilot study Main study; experimental procedure Study design Recruitment Dietary intervention Outcome variables Compliance to dietary intervention Subject contact Quality assurance of data entry Statistical analysis Results Screening and Recruitment Reported fruit and vegetable intake & urinary excretion of potassium and sodium Body weight Blood pressure Arterial stiffness and endothelial function measures Serum C-reactive protein and soluble ICAM-1 concentrations Urinary vitamin K and plasma vitamin C, tocopherol and carotenoid concentrations Discussion References...51 Modified 14 November2007 2

3 1. Executive Summary 1.1. Background The project was undertaken in response to the research requirement RRD10/N02/A To characterise the dose-response relationship between the dietary intake of specific plant foods and vascular function. Cross-sectional as well as prospective cohort epidemiological studies show the consumption of fruit and vegetables to be associated with a substantially lower risk of stroke and a slightly lower risk of coronary heart disease. The effect appears to be stronger for fruit than vegetables. As raised blood pressure is the major predictor of risk of stroke and also contributes to causing heart disease, increasing the intake of fruit and vegetables may help lower blood pressure. It has been proposed the additional potassium provided by fruit and vegetables helps counteract some of the adverse effects of salt on blood pressure. It has also been suggested that fruit and vegetables contain non-nutritive components that may have beneficial effects on blood vessel health. Desirable blood pressure is less than 120/80 mm Hg but a high proportion of the UK adults have raised blood pressure and this becomes more prevalent with increasing age; over the half of the population over the age of 55 years have raised blood pressure 140/90 mm Hg. A reduction in average blood pressure by 5 mm Hg would reduce the incidence of stroke by 22% and coronary heart disease by 16% and would prevent some 75,000 deaths a year in the UK as well as much disability. High blood pressure is a self-amplifying condition and prevention of hypertension (elevated blood pressure) is most effective in the earlier stages of its development. In this context diet is believed to be very important. The current UK average intake of fruit and vegetables in the UK is about 3 portions a day with lower intakes in low income groups. Current UK government advice is to consume five portions of fruit and vegetables daily. Information is required to show whether such an increase in consumption will lower blood pressure or whether even high intakes of fruit and vegetables are needed as has been suggested Rationale and Objectives This project was designed to test whether increasing the intake of potassium-rich fruit and vegetables from the UK average of 3 portions a day to the recommended level (5 portions a day) or higher (approximately 10 portions a day) lowers blood pressure among subjects with high normal blood pressure or elevated blood pressure. The study also compared the effects of an increased intake of potassium provided as a supplement. In order to investigate the potential mechanisms by which fruit and vegetable intake have been proposed to have a beneficial effect on blood vessel health, a series of physiological measurements of vessel function including measures of large artery stiffness and response of the artery in the arm to changes in blood flow were also made. The Modified 14 November2007 3

4 subjects were asked to keep a record of their fruit and vegetable consumption during the study and their body weights were monitored to ensure their weight remained stable. In order to verify that subjects were complying with the dietary advice, measurements of biomarkers of dietary intake were made in blood and urine collected during the study Approach A randomized controlled trial was designed to evaluate the impact of the increased intake of potassium from fruit and vegetables on indices of vascular function with blood pressure being the primary outcome. The aim was to recruit 48 non-smoking subjects (equal numbers of men and women) aged years with high normal or raised blood pressure. The trial had a crossover design of 4 treatments; this means each subject received every treatment. The treatments involved the manipulation of fruit and vegetable intake to supply three levels of potassium intake supplying an additional 0, 20 or 40 mmol potassium daily, on top of a background diet providing a similar amount of fruit and vegetables and potassium to the average UK intake. These intakes correspond roughly to intakes of 3, 5 and 10 portions of fruit and vegetables per day. In order to test whether an increased intake of potassium alone would be effective, capsules providing 40 mmol potassium citrate per day or matching placebo capsules were administered on the lowest level of fruit and vegetable intake. The subjects were not told which was the active or placebo capsules (i.e. they were blinded to treatment allocation). Prior to the main dietary intervention, the subjects followed a run-in period on a low level of fruit and vegetables for 3 weeks. At the end of this period, measurements were made. The subjects were then randomly allocated to different treatment sequences so that every subject received each treatment in random order for 6 weeks. Further measurements were made at the end of each treatment period. A break of at least 3 weeks before the subject started the next treatment was planned but in practice the break was longer (mean ± SD 60.5±12.5 days; range 36 to 129 days). The following measures were assessed at the end of the run in period and after 6 weeks on each treatment: self-reported intake of fruit and vegetables daily urinary excretion of potassium and sodium to assess potassium intake and salt intake systolic and diastolic blood pressure using 24 hour ambulatory blood pressure monitoring large artery stiffness and supine blood pressure measured in a blood pressure clinic measures of endothelium dependent and independent response of the brachial artery urinary vitamin K excretion Plasma vitamin C, vitamin E and carotenoid concentrations Modified 14 November2007 4

5 Markers of inflammation and endothelial function: C-reactive protein, solube I- CAM Outcome/key findings A total 55 subjects were recruited to the trial and 48 subjects completed the study. Of these subjects. 25 subjects (52%) met the definition of Stage I hypertension (day-time ambulatory BP>135/85), 12 (25%) met the definition for high normal BP (day-time ambulatory BP >125/80 mm Hg) and the remaining 11 subjects (23%) had normal blood pressure (day-ambulatory BP>=120/80 mm Hg). Self-reported fruit and vegetable intakes and capsule counts indicated good compliance to the dietary advice. This was corroborated by the predicted increase in urinary potassium excretion. Plasma vitamin C concentrations were slightly greater on the highest levels of fruit and vegetable intakes but urinary excretion of vitamin K metabolites, plasma carotenoids and vitamin E concentrations were unaffected by increased intakes perhaps not surprisingly as these are fat-soluble vitamins. Plasma concentrations of β-cryptoxanthin tended to increase with the higher intakes of fruit and vegetables. Body weights were stable throughout the study. There were no changes in blood pressure whether measured in the clinic or by ambulatory blood pressure monitoring, arterial stiffness, endothelial function (as determined by flow mediate dilatation of the brachial artery) or serum CRP concentration. Plasma concentrations of s-icam-1 fell in the women on the highest level of fruit and vegetable intake but did not change in the men What it means and why it is important The findings are in contrast to the fall in blood pressure predicted from the epidemiological studies. Discordance between the findings of intervention studies and predictions from epidemiological are not uncommon, e.g. as with dietary antioxidant supplements. This study was not designed to study the effects of lower intakes of fruit and vegetables (less than 3 portions per day), it may well be that the benefits of increased fruit and vegetable consumption in terms of decreased cardiovascular risk may already be maximal at a level of intake of 3 portions per day. It may well be that the blood pressure lowering effect is only seen in subjects with very low intakes of potassium (about 40 mmol/d). It is to be noted that the intake of potassium on the lowest level of intake in this study was similar to the average intake in the UK population, which is higher than the intake in the USA. The conclusions from this study are that dietary advice to increase fruit and vegetable consumption to 5 portions a day or more or potassium supplementation is unlikely to be of value in the prevention or management of high blood pressure. The results of the present supplementation study are in agreement with a recent Cochrane Systematic Review which found no statistically significant effect of potassium supplementation on blood pressure. The majority (88%) of the subjects in this study Modified 14 November2007 5

6 were overweight/obese and 68% had salt intakes above the recommended 6g/d. These factors, which contribute to high blood pressure, were not influenced by the intervention. Some of the benefits associated with an increased intake of fruit and vegetables in epidemiological studies may be a consequence of individuals consuming fewer less healthy foods (crisps, cakes, biscuits and confectionery) that contribute towards obesity. The present study did not attempt to decrease the consumption of these foods. On the basis of these findings, dietary advice for the prevention of high blood pressure should focus on weight control, moderating alcohol intake and restricting the intake of salt with less emphasis on promoting high intakes of fruit and vegetables and more on using an increased intake of fruit and vegetables to substitute foods high in food energy.. Modified 14 November2007 6

7 2. Technical Report 2.1. Background The project was proposed in response to the research requirement RRD10/N02/A To characterise the dose-response relationship between the dietary intake of specific plant foods and vascular function. Cross-sectional as well as prospective cohort studies show the consumption of fruit and vegetables to be associated with a substantially lower risk of stroke and a slightly lower risk of coronary heart disease (1,2). The effect appears to be stronger for fruit than vegetables and pickled or salted vegetables which have even been associated with increased risk of stroke. Dietary advice to increase the consumption of fruit and vegetables does not decrease body weight or alter plasma lipoprotein concentrations (3) neither does it affect folate status (4). However, increased fruit and vegetable intake has been reported to result in a significant fall in blood pressure (3,5) and there are indications of favourable effects on vascular function (6,7). A blood pressure lowering effect of fruit and vegetables might explain why fruit and vegetable intake is more strongly associated with risk of stroke than CHD because elevated blood pressure is a more powerful risk factor for stroke than CHD (8). Blood pressure rises with age particularly in economically developed societies and is positively associated with body mass index, alcohol and salt intake and negatively with potassium intake as assessed by urinary excretion (9). There is evidence to show that an increased intake of vitamin C, beta-carotene and tocopherol does not result in changes in blood pressure or effect stroke or CHD incidence (10). Consequently, the most plausible mechanism for the fall in blood pressure with increased fruit and vegetables intake is because of an increased intake of potassium which is the major intracellular cation. Randomized placebo controlled trials of potassium supplements (11) at the time of commissioning the study indicated that an average a 3mm reduction in systolic BP occurred in normotensive subjects and even larger reduction ~7mm in moderately hypertensive subjects. However, potassium supplements are problematic as they result in gastric irritation. Consequently, an increased intake of potassium in its natural food matrix is safer and fruit and vegetables also contain other minerals such as magnesium which is present in chlorophyll and may contribute to the blood pressure lowering effect. The DASH study (12) demonstrated a substantial reduction (7mm) in blood pressure in mildly hypertensive subjects with diet rich in fruit and vegetables that remained stable after two weeks on the diet. However, this diet provided 10 portions of fruit and vegetables/day (twice the current Modified 14 November2007 7

8 recommendation) supplying an additional ~45mmol potassium/d. This blood pressure lowering effect of increased fruit and vegetable consumption is claimed to occur even in the presence of a high sodium intake (13) but a concomitant reduction in sodium intake led to a greater fall in BP in the DASH-2 study. More recently, a community based intervention (3) reported a 4mm Hg reduction in systolic BP with a self reported increase from 3 portions of fruit and vegetables to 5 portions/d. However, the study was not designed to test blood pressure as a primary outcome and this important observation requires confirmation. Furthermore, the difference was also partly attributable to a slight rise in BP in the control group. Blood pressure is probably the single most important modifiable risk factor for cardiovascular disease and relatively small reductions in population blood pressure are likely to have a major impact on cardiovascular mortality. For example, it has been argued that a reduction in average blood pressure by 5 mm Hg would reduce the incidence of stroke by 22% and CHD by 16% and may prevent some 75,000 deaths a year in the UK as well as much disability (14). Usual dietary intake of potassium in the UK is about 80 mmol/d in men and 60mmol/d in women and the COMA Report on Diet and Cardiovascular Disease recommended an intake 90mmol/d (1). Most studies with potassium supplements have supplied an additional 60mmol K/d. In these studies, the increment in urinary K excretion is only 20mmol/d (15) which would suggest that at these high levels of intake a significant proportion is excreted in faeces. There is a need for better information on the use of potassium as biomarker for fruit and vegetable intake. The epidemiological evidence would suggest that intakes of fruit and vegetables supplying an additional mmol K/d is associated with decreased risk of cardiovascular disease (16,17). In the Nurses Health Study, supplementation with an additional 40mmol potassium/d resulted in a 3mm Hg reduction in systolic BP in predominantly normotensive subjects (18). There is a lack of information on lower doses. An open-label study found that a dietary supplement of 24 mmol slow release potassium chloride/d lowered systolic BP by ~5mm Hg (19). The mechanism by which the intake of potassium results in a reduction in BP is uncertain as potassium concentrations are under tight homeostatic regulation but it may involve changes in vessel wall tone and elasticity (compliance). Fruit and vegetables contain a variety of bioactive compounds that are vasoactive e.g. caffeine. There is currently much interest in the vasoactive effects of flavonoids because these compounds are potent antioxidants and also share structural homology with steroid hormones. High intakes of soya result in decreased blood pressure (20) and some isoflavones have direct actions on the vascular endothelium (21) and result in changes in arterial compliance (22). There is also some evidence to show that cocoa and tea influence endothelial function (23). However, there is Modified 14 November2007 8

9 considerable variability in the bioavailability of flavonoids (24). The consumption of a Mediterranean style diet rich in fruit and vegetables has been claimed in some studies to result in improvements in endothelial function measured by flow mediated dilatation (FMD) of the brachial artery (6). FMD reflects the bioactivity of endothelium-derived NO which plays an important role in blood pressure regulation. It has been claimed that there are beneficial effects of a Mediterranean diet (7) on both by endothelium and endothelium responses assessed by brachial arterial plethysmography studies but which is not attributable to vitamin C, which is consistent with other studies that conclude that high intakes of vitamin C do not influence endothelial function (25). However, it is not possible to attribute this effect to fruit and vegetables as the type of fat was also changed in the diet. To summarise, at the time of undertaking the study the evidence suggested that fruit and vegetables that are rich in potassium but low in sodium are likely to have a beneficial effect on blood pressure and vascular function. It appears that intakes of fruit and vegetable providing an additional 20-40mmol K/d may result in a reduction of blood pressure which would have important public health implications for the prevention of stroke and heart disease. There is no clear conclusion concerning the effect of increased fruit and vegetable consumption of aortic compliance and endothelial function. It is important to address the latter as it is believed to initiate arterial disease (26). Advice to eat five portions of fruit and vegetables a day is currently recommended by the DoH Five-a-Day campaign but the lack of dose response studies means that there is no sound evidence base for this recommendation. Although, there is a need to identify constituents responsible for the blood pressure lowering effects and the level of intake required, there is a more pressing need to establish whether the current recommended intakes have effects on blood pressure and vascular function. Especially, as it is necessary to study relatively large numbers of subjects to demonstrate small but clinically relevant changes in blood pressure and vascular function. Furthermore, there has been much confusion surrounding the definition of a portion of fruit and vegetables particularly with regard to processed foods which may be high in fat (coleslaw), salt (pickled cucumbers, baked beans in tomato sauce) and sugar (fruit in syrup). Clarification of what constitutes a portion of fruit and vegetables as proposed by the Department of Health has been helpful. Furthermore, there are concerns that high intakes of certain cruciferous vegetables such as spinach and lettuce would result in the upper tolerable intakes for nitrate/nitrite being exceeded. Hence there is a need to demonstrate a beneficial risk/benefit ratio of moderately increased intakes of fruit and vegetables. The focus of the study was to investigate the dose response relationship of an increased consumption of fruit and Modified 14 November2007 9

10 vegetables (low in added fat, sugar and salt) without exceeding tolerable intakes for nitrate/nitrite. Consideration was given to a parallel design but this would have less statistical power to discriminate dose response effects and would require a far greater number of subjects and would increase costs greatly. Previous studies have shown that it is possible to use a smaller number of subjects to demonstrate the blood pressure lowering effects of potassium if they have moderate (140/90mm Hg) or borderline (130/80 mmhg) hypertension. There is ample justification for studying such subjects as they constitute about 1/5 of the adult population and half of UK adults over 55 years have blood pressures in excess of 140/90 mm Hg. Consequently, in order to address the dose response relationships a randomized crossover trial is proposed in subjects with moderately elevated blood pressure Aims and objectives of the investigation The aim of this study was to compare the effects of three levels of fruit and vegetable intake providing 0, 20 and 40 mmol extra K/d versus 40 mmol extra K/d as a supplement (potassium citrate) on blood pressure, arterial compliance and endothelial function using a crossover design in subjects with moderately elevated BP. The study aimed to address the hypothesis that: 1. An increase in K intake from fruit and vegetables by 20 to 40mmol/d will lower BP. 2. An increase in fruit and vegetable intake will have beneficial effects on arterial compliance and endothelial function. 3. That the effect is attributable to an increase in K intake. The specific objectives as outlined in the Scope of Work were as follows: 01 Protocol refinement and ethical approval 02 To recruit 9 subjects into a pilot study to quantify urinary potassium excretion at intakes of fruit and vegetables providing 40, 60 and 90 mmol potassium/d. In order to establish a benchmark for fruit and vegetable consumption 03 To recruit a total of 48 subjects (24 males and 24 females) into a randomized crossover study. Subjects will be selected on the basis of their seated blood pressure with a diastolic BP of >80mm Hg and a systolic BP <160mm Hg. Subjects will be recruited as two cohorts (n=24). State of the Art: Given that the proportion of older persons is increasing in the population, factors that lower blood pressure will have a major impact on stroke, which is a major cause of death and disability with a high social cost. Modified 14 November

11 Ascertaining the effect of moderate increments of fruit and vegetables on blood pressure is urgently required. 04 To conduct a dietary intervention study involving the manipulation of fruit and vegetable intake to supply three levels of potassium intake supplying an additional 0, 20 or 40 mmolk /d using a single blind crossover Latin square design; in order to discriminate between an effect of potassium and additional treatment of 40 mmol K/d will be tested by using a slow release K preparation versus matching placebo. Forty eight subjects (24 male, 24 female) will undergo a run in period on a diet containing diets with a similar fruit and vegetable content to the average UK diet (80mmol/d in men and 60 mmol/d in women) and will then be randomized to one of four orthogonal treatment sequences so that a similar number of subjects are on each treatment during each phase of the study. The treatment phases will last for 6 weeks with a three week washout period between treatments. 05 To evaluate the impact of the increased intake of potassium from fruit and vegetables on indices of vascular function with blood pressure being the primary outcome. The following outcome measures will be assessed at the end of the run in period and after 6 weeks on each treatment: systolic and diastolic blood pressure using ambulatory blood pressure monitoring habitual dietary intake of fruit and vegetables will be determined using a food frequency questionnaire potassium intake will be estimated from urinary excretion of potassium large artery compliance by determining pulse wave velocity endothelial function by measurement of endothelium dependent and independent dilatation of the brachial artery as determined by B-wave ultrasound To determine biochemical indices of endothelial function CRP, s- ICAM-1 and biomarkers of fruit and vegetable intake. State of the Art: Decreased arterial compliance has emerged as a powerful predictor of risk of cardiovascular events. Impaired endothelial function as determined by flow mediated dilatation of the brachial artery is regarded as critical event in the atherogenesis but also increases risk of thrombosis. Modified 14 November

12 2.3. Pilot study The pilot study was conducted to assess the validity of urinary K excretion as a biomarker of fruit and vegetable intake, and to characterise the dose-response relationship compared with a dietary supplement of K citrate. A copy of the full report submitted previously to the FSA is in Appendix 25. Using an orthogonal crossover study design, 12 volunteers were provided with different intakes of fruit and vegetable equivalent to additional K intakes of 0, 20 and 40 mmol/d, and a dietary K citrate supplement equivalent to 40 mmolk/d (manufactured for the study to the specifications shown in Appendix 14), for four consecutive 1-week periods. 24h urine collections were obtained at the end of each intervention and daily K excretion (mmol and mmol:mmol creatinine) was calculated. A highly significant linear trend was found between increasing fruit and vegetable intakes and K (P=0.0006) and K:creatinine (P=0.0019) excretion (Figure 1 and Figure 2 respectively), suggesting that both measures can be used as reliable biomarkers of fruit and vegetable intake. An increased intake of 40mmol K resulted in a 30 mmol K excretion or an increase in 3mmol K/mmol creatinine. This suggested that about 75% of the additional potassium is excreted in the urine. Modified 14 November

13 Figure 1. Daily potassium excretion during each treatment period (mean with SE) in 12 subjects. Potassium excretion Potassium mmol/d run-in Low Low + K Medium High Parameter Bonferroni's Multiple Comparison Test run-in vs Low Low vs Low + K Low vs Medium Low vs High Low + K vs High Medium vs High Value Mean Diff Low t Low + K P value P < P < Medium 95% CI of diff to to to to to to Modified 14 November

14 Figure 2. K:creatinine ratio during each treatment period (mean with SE) in 12 subjects. Urinary potassium/creatinine Urinary potassium/creatinine run-in Low Low + K Medium High Parameter Bonferroni's Multiple Comparison Test run-in vs Low Low vs Low + K Low vs Medium Low vs High Low + K vs High Medium vs High Value Mean Diff Low t Low + K P value P < 0.01 P < Medium 95% CI of diff to to to to to to Main study; experimental procedure Study design A randomized crossover design was used to test the effects of 3 dose levels of fruit and vegetable intake on BP, aortic compliance and endothelial function. A cross-over design was selected because changes in BP occur rapidly (2 weeks) and because within subject comparisons are more sensitive in assessing dose response relationships. Each treatment was separated by a washout period of a minimum of 3 weeks and a 6 week treatment period was chosen so as to avoid any possible carryover effects. In order to increase the sensitivity of the study, subjects with mild to moderately increased blood pressure were used. Previous studies (12) have shown larger reductions in the order of 7mm Hg in systolic blood pressure in such subjects compared with 3mm in normotensive subjects. A high proportion of the adults in the UK have raised blood pressure. Cut-offs for defining hypertension have been revised downwards over the last few years and the latest British Hypertension Society Guideline define optimal BP <120 / <80, normal BP <130 / <85, high-normal BP / 85-89, Modified 14 November

15 grade 1 hypertension (mild) / grade 2 hypertension (moderate) / mm Hg. The aim of this study was to recruit subjects with high normal blood or hypertension but only including subjects who do not qualify for drug therapy of hypertension according to the British Hypertension Society guideline (27). Measurement of blood pressure is subject to large day-day variation and observer bias but the use of standardized protocols and repeat measures enables more reliable characterization of true blood pressure. In the present study subjects were required to have evidence of elevated diastolic BP (>80 to 100 mm Hg) on at least two visits to be eligible for inclusion in the study. In order to familiarize subjects with the dietary intervention and methods, and to standardize intakes prior to the test interventions subjects completed a 3-week run in on the control diet (men 80mmol K/d and women 60mmol K/d) which are potassium intakes typical of the UK diet (approximately 3 portions of fruit and vegetables daily). Measurements were made in the last week of intervention. Following the completion of the run-in period subjects were randomized to orthogonal treatment sequences so that the same number of subjects was on each treatment at the same time in order to neutralize any possible seasonal effects or time trends. Each treatment period lasted 6 weeks and measurements were made in the last week of each treatment. Each treatment was separated by a wash-out period of at least 3 weeks between treatment periods. Thus the total study time for each subject was 36 weeks. The study design is outlined in Figure 1. The study was run in 2 cohorts; the first cohort (cohort 1a) (n=20) commenced in Jan 2005, additional subjects to add to cohort 1 (cohort 1b) (n=12) commenced in March 2005 (due to greater than anticipated drop-outs) and the second (cohort 2) (n=24) commenced in Jan Sequence orders for each cohort were as follows: Cohort 1a: 1) ABCD, 2) BADC, 3) CDAB, 4) DCBA. Cohort 1b: 1) ADBC, 2) BCAD, 3) CBDA, 4) DACB. Cohort 2: 1) ACDB, 2) BDCA, 3) CABD, 4) DBAC. Where A = placebo capsules, B = 40 mmol K citrate capsules, C = additional 20 mmol K/d from fruit and vegetables, D = additional 40 mmol K/d from fruit and vegetables. Modified 14 November

16 Figure 3. Study design Last 2 weeks of each intervention; 24hr ABP measurement, 24hr urine collection Run-in Intervention 1 Wash out Intervention 2 Wash out Intervention 3 Wash out Intervention 4 Week Clinic visit: last week of each intervention, following overnight fast, measurement of FMD, PWV, PWA, DVP, weight and waist circumference and collection of fasting blood sample (for determination of ICAM, CRP, Vit C) PWV Pulse wave velocity, DPV Digital pulse volume, FMD - Flow mediated dilatation. Run-in = control diet; run-in on average UK fruit and vegetable intake to equal approximately; men 80 mmol K/d and women 60 mmol K/d approximately 3 portions of fruit and vegetables per day. Four different interventions were; placebo capsules (A), 40 mmol K citrate capsules (B), additional 20 mmol K/d (C) or 40 mmol K/d from fruit and vegetables (D) Recruitment Sample size considerations Power calculations were based on 80% power to detect differences between treatment at P=0.01 (in order to account for multiple comparisons among the treatment levels). The initial power calculations were based on a sample size of 32 completing subjects. The project aimed to recruit 48 subjects to allow for drop-outs. However, recruitment exceeded this target, in total 55 subjects were recruited to the study and 48 subjects completed the study. Sample size was estimated on the following outcome variables: Outcome variable 1: Blood pressure A sample size of 32 subjects/group will have 80% power to detect a difference in means of 4 mm change in systolic BP. This estimate is based on a within subject SD of 5mm Hg as determined by ABP monitoring. For a sample size of 48 the study had power to detect changes of 3.5mm Hg. Outcome variable 2: Aortic compliance Modified 14 November

17 A sample size of 32 subjects/group has 80% power to detect a difference in means of 1m/sec in pulse wave velocity based on a mean value of 7.6 and within subject SD of 1.1m/sec. For a sample of 48 subjects the power was 0.77 m/sec. Outcome variable 3: Endothelial function (FMD) The estimated variability in measurement of FMD in our laboratory was 0.46% units and for measurements on different days 1.1% units. The mean value of FMD (all measurements on all subjects) was 6.2% units. We estimated that the current study would have sufficient power to detect a 1% unit difference between treatments for 32 subjects completing each treatment (α=0.01, β=0.80). For a sample size of 48 subjects, the power was 0.8% units Inclusion criteria A principal aim was to identify and recruit men and women (aged years) with moderate elevation of blood pressure. Eligible subjects had seated diastolic BP >80mm Hg and <100mm Hg and a systolic BP <160 mm. As blood pressure tends to regress towards the mean value, measurement of blood pressure were made on two occasions at least one week a part Exclusion criteria Current smokers were excluded due to the confounding influences of smoking on measurement of endothelial function. A reported history of myocardial infarction or cancer. Diabetes mellitus (fasting plasma glucose greater than 7 mmol/l). Recent use of oral lipid lowering therapy, systemic corticosteroids, androgens, phenytoin, erythromycin or thyroid hormones. Current use of antihypertensive medication. Those receiving drugs for regulating haemostasis but excluding aspirin, or who have been exposed to any investigational agent within 30 days of the study. Chronic coronary, renal or bowel disease or history of cholestatic liver disease or pancreatitis. Presence of gastrointestinal disorder or use of a drug, which is likely to alter gastrointestinal motility or nutrient absorption. History of substance abuse or alcoholism. Currently pregnant, planning pregnancy or having had a baby in the last 12 months. Allergy or intolerance to intervention foods. Unwilling to follow the protocol and/or give informed consent. Modified 14 November

18 Unwilling to refrain from use of dietary supplements. Weight loss at >3kg in the preceding 2 months. Alcohol intake exceeding a moderate intake (>24-36 g alcohol/day). Body Mass Index <20 and >35 kg/m 2. Subjects with a BP and other risk factors that make them eligible for drug treatment of raised BP according to the UK guidelines of the British Hypertension Society (BHS) (as calculated by BHS risk calculator) Recruitment methodology Subjects were recruited through a variety of methods including; recruitment from staff and students of KCL, parents and teachers from various schools in the London area and pupils from an adult education centre. For an outline of the recruitment methodology see Figure 4a and Figure 4b. For subjects recruited from within KCL, volunteers were initially interviewed via a telephone questionnaire to assess whether the study was suitable for them (Appendix 1). Volunteers, who meet the inclusion criteria, were then invited to attend a screening session during which BP, height and weight were measured. For subjects recruited from educational establishments, newsletters (example in Appendix 2) were initially sent out to all the parents, teachers and in the case of adult education centres, to all the pupils, inviting them to attend a BP check afternoon when collecting their children from school. The BP check afternoons were held within the educational establishment and volunteers had their BP checked and were asked to complete a questionnaire (Appendix 1). All subjects were provided with a copy of the Patient Information Sheet (Appendix 3) and a detailed study outline booklet (Appendix 4). If BP met the screening criteria, the subjects were then asked to attend for a second BP measurement to confirm their eligibility and to provide a fasting blood sample taken in order to determine normal liver function and blood counts at KCL. Eligible subjects were asked to sign a consent form (Appendix 5) complete a food frequency and health questionnaire (FFQ) similar to that used in the European Prospective Investigation into Cancer study to evaluate habitual dietary intake and use of medicines and supplements (Appendix 6). Subjects were excluded at this stage if their BP was not within the prescribed limits for the trial and if there were clinically abnormal findings from haematology or liver function tests. Modified 14 November

19 Figure 4a. Recruitment time-line for KCL Newsletter BP check afternoon scheduled School BP check: Questionnaire 1 st BP check Eligible subjects Screening visit at KCL: 2 nd BP check at KCL Body composition Consent form Fasting blood sample FFQ Figure 4b. Recruitment time-line for educational establishments Telephone questionnaire Eligible subjects; invited for BP check & provided with detailed study outline 1 st BP check at KCL Body composition Eligible subjects Screening visit at KCL: 2nd BP check at KCL Consent form Fasting blood sample FFQ For full details on methods for the following screening measurements; height, weight, body composition, seated BP, serum lipids, liver function tests, plasma glucose and full blood counts, refer to Appendices 16, 17 and Dietary intervention The dietary intervention compared 3 levels of fruit and vegetable intakes designed to supply additional K intakes of 0, 20 and 40 mmol K/d, versus an additional 40 mmol K/d provided as a supplement. All subjects were given verbal and written dietary advice for each intervention (Appendices 9-13). Written general dietary advice (Appendix 7) and details on portion sizes (Appendix 8) and K contents of fruit and vegetables (Appendix 11) were also provided Run-in period During the 3-week run-in period subjects were instructed to consume a daily intake of fruit and vegetables of 3 portions, equivalent to approximately 15 mmol K/d (Appendix 9). This intake was intended to represent the mean daily contribution of fruit and vegetables to K intake for the UK population as measured in the NDNS 2002 survey. The NDNS data shows that of the mean daily K intake of 76 mmol for men (calculated from 7 day diet diaries), 9% (7.7 mmol) came from Modified 14 November

20 vegetables and 6% (5.4 mmol) from fruit and nuts, a total of 13.1mmol K/d. For women the mean daily K intake was calculated as 68 mmol, with 11% (7.5 mmol) from vegetables and 9% (6.1 mmol) from fruit and nuts, a total of 13.6 mmol per day Capsules Slow release K citrate capsules and matching placebos were manufactured for the study by Penn Pharmaceuticals to the specifications shown in Appendix 14. Subjects received active/placebo capsules in a plastic pill bottle and were given verbal instructions to take 2 capsules, 4 times a day (Appendix 10). They were advised to take the capsules with food to reduce any likelihood of gastric irritation, and instructions were also written on the label on the pill bottle. Subjects were requested to return any unused capsules at the end of the treatment period. Subjects were provided with empty small pocket sized pill bottles to carry their capsules around with them and a pill dispenser with the days of the week listed to aid their compliance Additional Fruit and Vegetables For two of the dietary interventions, subjects were requested to increase their fruit and vegetable intake to provide an additional 20 or 40 mmol K/d from fruit and vegetables. Subjects were provided with a simple guide of how much K is in each fruit and vegetable (Appendix 11), using a unit system, whereby 1 unit of K is equal to 5mmol K; such that subjects consuming an additional 20 mmol K/d from fruit and vegetables were requested to consume 4 units of K/d in addition to their background diet of 3 portions fruit and vegetables/d (Appendix 12), and subjects consuming an additional 40 mmol K/d from fruit and vegetables were requested to consume 8 units of K/d in addition to their background diet of 3 portions fruit and vegetables/d (Appendix 13). Fruit and vegetables were supplied to subjects via 2 processes: 1) Subjects were contacted weekly and asked for a fruit and vegetables order which was processed for home delivery by supermarket. 2) Subjects not wishing to receive a home delivery of fruit and vegetables bought their own fruit and vegetables and retained receipts for re-imbursement. Subjects were asked to fill in a record of their fruit and vegetable intake and were also contacted regularly by telephone Outcome variables At the end of the run in period and each subsequent treatment period, measurements of outcome variables were made. 24-h ambulatory blood pressuring monitoring was carried out between weeks Modified 14 November

21 5 and 6. In week 6, subjects made a 24 h urine collection (to assess compliance to the dietary advice; determination of urinary K, creatinine, sodium and vitamin K excretion) and attended St Thomas Hospital for a clinic visit. On the day before the clinic visit, subjects were asked to fast overnight and the following morning body weight and body composition were measured, 3 clinic seated BP measurements were made and a fasting blood sample collected. Blood samples were processed as outlined in Appendix 17. After 15 minutes rest, supine BP and femoral to carotid pulse wave velocity (PWV c-f ), augmentation index (AIx), stiffness index and reflection index was determined. Endothelial function was then measured using B-wave ultrasound in response to hyperaemic and glycerol trinitrate stimuli in order to determine endothelium and non-endothelium dependent responses. Subjects were given detailed guidelines on the last week of each intervention (Appendix 15). Clinic visits were scheduled at approximately the same time of day for each visit for the subjects Urine sample collection Participants were asked to collect a 24-hour urine sample following the instructions in Appendix 20. Subjects were provided with a plastic jug and one or two 2.5 litre plastic containers. Subjects were advised to make their collections on the day preceding their visit to St Thomas so that they could bring it with them to St Thomas which would be within a few hours of the last collection. Between collections they were advised to store it in a cool, dark place. Upon receipt, the urine volume was measured prior to separation into aliquots and storage (Appendix 21). Subjects were also asked to complete a Last day of intervention food record card (Appendix 24) on the day that they made the urine collection, this was for reference in case of any unusual results (ie if subjects consumed K rich foods such as potatoes during the collection time). Urine samples were analysed for K, sodium, creatinine and vitamin K according to the methods in Appendix 22. Urine samples were also set aside for assays not specified in the FSA contract (flavonoids and isoprostanes) Ambulatory BP monitoring The A&D TM-2430 ABP monitoring devices were used (graded A/A by the BHS;28). Measurements were made towards the end of each treatment period (week 5 or 6) but not on the day prior to a blood sample. Measurements were made in accordance with BHS guidelines (29). Subjects were provided with a 24 hour BP Monitoring Subject Information Sheet (Appendix 18) and the procedure was carefully explained to them. Subjects were asked to complete a diary card during the 24 hour measurement to illustrate what activity they were doing during each ABP Modified 14 November

22 measurement and record sleep time (Appendix 19) Pulse wave velocity and pulse wave analysis Pulse wave analysis (PWA) and carotid to femoral pulse wave velocity (PWV c-f ) were measured using a non-invasive Sphygmocor system (SphygmoCor VW apparatus with Sphygmocor analysis software; SphygmoCor version 7.01 AtCor Medical Pty, Australia). Prior to each measurement subjects rested in the supine position for 15 min and BP was recorded using an automated sphygmomanometer (Omron 70CP). The measurements were made by a single observer Karen McNeil who was blind to the treatment allocation. Pulse wave velocity: Aortic compliance was assessed by measuring carotid to femoral pulse wave velocity (PWV c-f.) was computed from the time delay between the upstroke of the arterial pressure wave at the carotid and femoral arteries and the anatomical carotid to femoral distance. The distance between the surface markings of the sternal notch and the femoral artery site was used to estimate the difference in path length between the arteries in order to calculate PWV c-f. A total of 3 measurements were made with the criteria that the coefficient of variation was less than 5% for 3 measurements for the results to be acceptable. Pulse wave analysis: AIx was derived from measurements of the carotid to radial pulse wave form using the Sphymocor analysis software in the pulse wave analysis mode. Left ventricular ejection generates a pressure wave that travels outward to the periphery of the circulation. This pressure wave is then reflected back from small arteries mainly in the lower body. The pressure waveform at any point in the vascular tree is thus a composite of a forward going wave and a reflected wave. Normally the reflected wave returns to the heart in diastole, increasing myocardial perfusion. With increased vascular tone and arterial stiffening the reflected wave arrives earlier in systole reducing myocardial perfusion during diastole and imposing a higher load on the heart during systole. Stiffness index and reflection index: For measurement of stiffness index (SI DVP ), a photoplethysmograph (Micro Medical, Gillingham, Kent, UK) was placed on the index finger of the right hand to obtain the digital pulse volume (DVP). BP and DVP waveforms were recorded over 10 second periods over 15 minutes with 5 minute intervals between measurements. The average (peak-to-peak time, PPT) at 5, 10 and 15 minutes was calculated using the PulseTrace Softwave (version 1.0, Micro Medical) and SI DVP index was calculated by dividing the PPT value into height in m and multiplying the result by 1000 to convert the result into m/sec. The reflection index (RI) was calculated as the height of the diastolic peak of the DVP relative to that of the systolic peak. Modified 14 November

23 Endothelial function measurements Endothelial function was assessed by measuring flow mediated dilation (FMD) of the brachial artery according to current guidelines (30). All measurements were made by the same observer Dr Benju Jiang who was blind to the allocation of treatment. A high resolution ultrasound (Siemens Accuson CV70) system with 7-10 MHz linear array transducer, positioned by a stereotactic manipulator, is used to scan the brachial artery in a longitudinal section 2 to 15 cm above the elbow. After optimal positioning of the transducer a baseline scan is recorded. Increased flow was then induced by inflation of a pneumatic tourniquet placed around the forearm (distal to the arterial segment being scanned) to a pressure of 250 mmhg for 5 min, followed by release. A second scan commenced 10 sec before release of the cuff and continued for 3 min after cuff deflation. After 10 min to allow vessel recovery, another resting scan was taken. Sublingual glycerol trinitrate (GTN, 25 µg) is then administered, and a final scan performed 3 to 4 min later. Images are coded and recorded on VHS videotape, then digitized for subsequent blinded analysis using automated edge detection software (Brachial Analyser, Medical Imaging Applications, LCC, Iowa, USA). FMD is expressed as the percentage increase in brachial artery diameter from baseline to maximal dilation which occurs 30 to 90 sec after release of the cuff. Dilation to GTN is expressed as the percentage increase in brachial artery diameter from baseline to maximal dilation after GTN and indicates nonendothelium dependent vasorelaxation Biochemical analysis Blood samples collected at the end of run-in and each intervention were analysed for high sensitivity C-reactive protein (scrp), soluble ICAM-1, vitamin C, carotenoids and vitamin E, according to the protocols outlined in Appendix 22. Additional plasma assays not specified in the FSA contract were also carried out for analysis of; plasma flavonoids, isoprostanes, insulin, glucose, total, HDL & LDL cholesterol, triacylglycerol, folate and homocysteine Compliance to dietary intervention The primary compliance marker was 24hr urinary K excretion. The following were also measured; urinary sodium, creatinine and Vit K; plasma vitamin C, carotenoids and vitamin E; self-reported fruit and vegetable intake and capsule return Urine and plasma measurements Urinary electrolytes were determined in the Department of Clinical Biochemistry, King s College Modified 14 November

24 Hospital under the supervision of Dr Roy Sherwood, and the absolute excretion of K, sodium and creatinine calculated according to the protocol in Appendix 22. Urinary vitamin K (which indicates the bioavailability of phylloquinones from fruit and vegetables) was determined by Dr Martin Shearer in the Haemophilia Centre, St Thomas Hospital, according to the method in Appendix 22. Plasma vitamin C was measured under the supervision of Professor Frank Kelly in the Lung Biology Centre, KCL according to the method in Appendix 22. Carotenoids (which indicate the intake of specific vegetables e.g. lycopene tomatoes, lutein dark green vegetables, beta-carotene carrots) and vitamin E were measured in the Department of Nutrition & Dietetics, KCL under the supervision of Professor Tom Sanders according to the method in Appendix Fruit and vegetable record card Subjects completed a 1 week Fruit and Vegetable record card (Appendix 23) during week 3 of each intervention, to monitor how many portions of fruit, vegetables and mmol of K they consumed per day. Information regarding the type of fruit and vegetables consumed was also collected from the record cards Capsule count An additional 20% of capsules were given to subjects. Subjects were not informed as to how many spare capsules they were provided with and they were asked to return any unused capsules. Upon return, the number of unused capsules was weighed and the percent capsules consumed was calculated. For subjects returning less that 20% capsules a maximum of 100% intake was used Subject contact Subjects were contacted on a regular basis throughout the study both by and telephone. During the intervention periods subjects were contacted via telephone on a weekly basis to arrange fruit and vegetable delivery orders and check that subjects were following the dietary instructions. All subjects were contacted via telephone on the day prior to their visit to St Thomas to confirm their appointment and the instructions for the day before. s were also sent out on a regular basis to remind subjects when to start the interventions and to arrange visits to St Thomas and collection of BP monitors. Any important information from subject correspondence and / or unusual circumstances during visits was recorded on subjects individual files. Modified 14 November