Cardiovascular, Metabolic and Mental Health Benefits of Vasoactive Nutrients Professor Peter Howe Nutritional Physiology Research Centre University of South Australia Newcastle, 11th February, 2011

Goal: attain and maintain optimal health Functional capacity (physical & mental fitness) Early Life Growth and development Adult Life Maintaining highest level of function Older Age Maintaining health and independence Range of function in individuals genes diet lifestyle Disability threshold Age

Preventable Causes of Death in the US in 2005 Danaei G et al. PLoS Med 2009;6(4): e1000058

Obese Australians

Obesity Inflammation Metabolic Syndrome Heart disease Diabetes high blood glucose, insulin high blood pressure high blood fats Heart disease Diabetes $1.4B p.a. $1.2B p.a. Physical disability Cancer $0.9B p.a. Mental disorders obese individuals are 74% more likely to have dementia than normal weight individuals AIHW, 2008

Is metabolic syndrome a circulatory disorder? Endothelial cells inhibit cell adhesion/aggregation regulate vasomotor tone maintain barrier function (selective permeability) maintain vascular integrity (inhibit smooth muscle cell migration, proliferation)

% change in diameter of artery Flow mediated dilatation G Born & C Schwartz Vascular Endothelium smoking high BP high blood sugar high blood fat obesity NO % change in diameter of artery Flow mediated dilatation

Impaired Endothelial Function in Obesity Obese Lean P Davison K, Bircher S, Hill A, Coates A, Howe P, Buckley J. J Obes (accepted 29 Dec 2010)

reduced fat, glucose metabolism Circulatory effects of endothelial dysfunction Cognitive decline depression neuro-degenerative disorders endothelium Endothelial dysfunction Obesity High blood pressure High blood sugar High blood fats (incl cholesterol) Smoking high BP coronary disease angina stroke impaired blood flow Can be improved/restored by regular aerobic exercise and supplementation with specific bioactive nutrients physical incapacity reduced fat, glucose metabolism

Bioactive nutrients from plants Erdman J et al. J Nutr 2007;137:718S Cocoa flavanols in dark chocolate

Effect of Cocoa Intake on Blood Pressure: meta-analysis Taubert D et al Arch Intern Med 2007;167:626-634 Cocoa flavanols attenuate Davison K, Berry N, Misan G, Coates AM, Buckley JD, Howe PRC 24hr Ambulatory BP Dose-related effects of flavanol-rich cocoa on blood pressure. J Hum Hypertens 2010

Flavanol-rich cocoa attenuates BP responses to exercise Responses to exercise were tested in borderline hypertensives 2 hours after cocoa by continuously monitoring of beat to beat BP for 5 min at rest (Finapres) then for 10 min whilst cycling at 75% of age-predicted max HR. (Berry NM et al, Br J Nutr 2010) BP increase (mmHg) SBP MAP ( AUC) Time (sec) DBP  LF 30mg flavanols  HF 600mg flavanols

FMD responses to flavanol-rich cocoa supplementation Acute response 2hrs after single dose * 1 2 3 4 5 6 7 8 LF HF FMD (%) Before After Chronic response measured > 10hrs after previous dose * 1 2 3 4 5 6 7 8 LF HF FMD (%) Week 0 Week 6 Week 12 Davison K, Coates AM, Buckley JD, Howe PRC. Int J Obesity 2008;32:1289-96

Endothelial functions are dependent on NO production Cocoa flavanols reduce LDL oxidation inhibit platelet aggregation modulate inflammatory eicosanoids and cytokines Cocoa flavanol supplementation can lower BP, attenuate BP responsiveness to stress and counteract hypertension not due to antioxidant activity attributable to increased production of endothelial NO and enhanced vasodilatation probably mediated by epicatechin and related flavanols Endothelial functions are dependent on NO production l-arginine l-arginase NO synthase free radical oxidation urea nitric oxide (NO) NOx

Meta-analysis: effects of flavonoids on FMD Hooper, L. et al. Am J Clin Nutr 2008;88:38-50 Acute Chronic

Effects of Soy on Arterial Function A Thorp et al. J Hypertens 2009;27(Suppl 4):S153 Soy/Dairy + Iso Soy + Iso Dairy Large Artery Elasticity Index n=91) Flow mediated vasodilatation (n=55) * * Normal dilatation % ▲ in dilatation from baseline diameter ( ml/mmHg x100) * Sig difference in dilatation compared to Dairy (P<0.05) Small Artery Elasticity Index (n=91) Nitroglycerine vasodilatation (n=55) The second outcome measure we assessed when on the different diets was arterial compliance. As you can see there was no difference in either large or small arterial elasticity for all out subjects irrespective of what diet they were on And again equol production appeared to had no effect % ▲ in dilatation from baseline diameter ( ml/mmHg x100)

Flavonoid Intake and Cognitive Decline in men aged 65–70 years intake quartiles Letenneur L et al. Am J Epidemiol 2007;165:1364–1371

Cognition is linked to endothelial function in older adults with cardiovascular disease 88 independent, community-dwelling older adults (average 70 years) with mild to severe CVD but without neurological disease and dementia significant correlation with FMD Forman DE et al. Artery Research 2008;2:35-43

Can soy isoflavones improve cognitive function? ERβ are localised in brain regions associated with learning and memory -- Resnick et al 2004 Estrogen acts on ERβ to increase cerebral blood flow - Duckles & Krause 2007 cerebellum hippocampus basal forebrain prefrontal cortex ER β ER α amygdala pituitary hypothalamus ER α & ER β Limbic System cerebral cortex Important in visuospatial tasks & memory recall Important in spatial working memory Relative binding affinities ERα ERβ Estradiol 100 Genistein 0.7 13 Daidzein 0.01 0.04 The sex hormone estrogen has since been show to play an integral role in the organisation and maintenance of these cognitive sex differences by acting on estrogen receptors in the brain There are two types of estrogen receptors in the brain – alpha and beta And they are shown to be both selectively expressed and co localised in areas such as the limbic system and cerebral cortex which are associated with memory and learning. Localisation of estrogen beta receptors specifically in the hippocampus and prefrontal cortex which control cogntive functions known to differ between women and men is of particular importance as it suggest estrogen acts on these receptors to induce genomic and non genomic pathways that upregulate neuronal activity We recently found that isoflavone supplementation enhances spatial working memory in men. (Thorp A, Sinn N, Buckley J, Coates A, Howe PR. Br J Nutr 2009)

Cerebral blood flow Local regulation of cerebral blood flow critical for brain function Ongoing supply required for delivery of glucose, oxygen & nutrients in response to requirements Evidence of reduced cerebral blood flow in psychopathologies mild cognitive impairment (Sun et al 2007) dementias including Alzheimer’s Disease (Crawford 1996, 1998; Warkentin et al 2004) ADHD (Bradley & Golden 2001) Depression (Yazici et al 1992) Schizophrenia (Mori et al 1999) High overlap between coronary heart disease and psychopathology Relatively little attention has been given to the possibility that omega-3s may also act on cerebral blood flow. We know that local regulation of blood flow in the brain is critical for ongoing brain function. In fact, although the brain only accounts for 2-2.7% of body weight, it needs 25% of the body’s glucose supply and 19% of the blood supply at rest, and these increase by 50% in response to cerebral activity (Haller 2005). Glucose is the brain’s primary source of energy for metabolic activities and the brain has very limited capacity for storing glucose, hence the importance of a continuous reliable supply of blood. The brain also has high requirements for nutrients, which must also be supplied by the blood and cross the blood-brain barrier. There is ample evidence of reduced cerebral blood flow in psychiatric illness. There is also a high incidence of coronary heart disease in psychopathologies such as depression and dementia. These point to a possible common underlying vascular problem. healthguide.howstuffworks.com/stroke-in-depth.htm 20

The Blood-Brain Barrier Neurovascular Unit in Health and Disease Hawkins & Davis, Pharmacol Rev 57:173–185, 2005

Impaired Blood Brain Barrier in Alzheimer's Disease? A generalized mechanism for AD pathogenesis that includes breakdown of the blood-brain barrier, the leak of plasma components including soluble amyloid peptide (Aβ42) and autoantibodies into the brain tissue, the binding of both of these components to the surfaces of neurons, and their internalization and accumulation in neurons. Accumulation of Aβ42 causes loss of dendrites and synapses, impairment of neuronal function and eventual cell death. Nagele RG, New Jersey Institute for Successful Aging, http://www.umdnj.edu/research/publications/fall06/4.htm

Cocoa flavanols and brain perfusion Transcranial Doppler ultrasound Vasodilator response to breathing 5% CO2 18% increase in flow velocity in the MCA after eating flavanol-rich cocoa for 1 week Fisher N et al. J Cardiovasc Pharmacol 2006;47:S210

Resveratrol & cardiovascular health Acute dose-related improvement of FMD *P<0.05 relative to placebo Mean ± SEM. N=18 Trans-resveratrol (3,4,5’-trihydroxystilbene) (Wong RHX, Howe PRC, Buckley JD, Coates AM, Kunz I, Berry NM. Nutr Metab Cardiovasc Dis 2010)

Red wine polyphenol extract (600mg)1 Resveratrol & cardiovascular health Acute dose-related improvement of FMD +3.7 +2.5 +2.4 Red wine polyphenol extract (600mg)1 Cocoa flavanols (902mg)2 Tea (450ml)3 EGCG (300mg)4 1Lekakis et al. 2005;Eur J Cardiov Prev Rehab:12:596-600, 2Davison et al. 2008;Int J Obes: 32:1289-1296, 3Widlansky et al. 2005; Free Rad Bio Med: 38:499-506, 4Widlansky et al. 2007; J Am Coll Nutr: 26:95-102

Omega-3

Health Benefits of Omega-3 PUFA Infant development & growth Promoting fitness (physical, mental, reproductive) Counteracting chronic disease (prevention, treatment) Health benefits of 3 PUFA Inflammatory disorders psoriasis/dermatitis rheumatoid arthritis inflammatory bowel disease immune renal disease periodontal disease osteoporosis? asthma? Behavioural disorders depression, bipolar disorder cognitive impairment, ADHD schizophrenia, autism? Cancer? Cardiovascular disease lipids (TG, HDL-C) blood pressure arterial compliance endothelial dilatation platelet aggregation heart rate / variability arrhythmia cardiac hypertrophy heart failure kidney damage stroke Diabetes insulin resistance abdominal adiposity

a-Linolenic acid C18:3 LNA Physiological effects of PUFAs Omega 6 Omega 3 plants safflower sunflower corn, soy Plants linseed, canola NUTS Linoleic acid C18:2 LA a-Linolenic acid C18:3 LNA Arachidonic acid C20:4 AA Eicosapentaenoic acid C20:5 EPA fish, fish oil animals Docosahexaenoic acid C22:6 DHA Docosapentaenoic acid C22:5 DPA fish fish, fish oil EICOSANOIDS 2-series prostaglandins 3-series 4-series leukotrienes 5-series excess may promote inhibit Thrombosis vasoconstriction inflammation inhibits thromboxane synthase Resolvins,Protectins Nucleus PPAR DNA cardiovascular and inflammatory disorders Modulates gene expression in regulatory pathways, e.g. metabolism, inflammation

Greatest cardiovascular protection Omega-3 Index - a new marker of health status Harris and von Schacky, Preventive Medicine 2004 Least Protection 8.1% Greatest cardiovascular protection GISSI-P2: 9-10% CHS3: 8.8% DART4:  8-9% SCIMO5: 8.3% 5 epi. studies:  8% PHS6: 7.3% Seattle7: 6.5% 4% 8% PHS6: 3.9% Seattle7: 3.3% 6% 10% 1Nilsen. AJCN. 74:50, 2001; 2Marchioli. Circulation. 105:1897, 2002; 3Mozaffarian. Circulation.107:1372, 2003; 4Burr. Lancet. 2:757, 1989; 5von Schacky Ann Intern Med 130:554, 1999; 6Albert. NEJM. 346:1113, 2002; 7Siscovick. JAMA. 274:1363, 1995 SCIMO5: 3.4% EPA + DHA % total fatty acids in red blood cells Current levels in Australians ~5%

Effects of Omega-3 and Exercise on CV risk factors Hill A, Murphy K, Buckley J, Howe P, Am J Clin Nutr 2007;85:1267 Change in TG (mmol/L) * p < 0.05 Change in HDL (mmol/L) * p < 0.05 * FMD Change in artery diam. (mm) p = 0.05 0.000 0.004 0.008 0.012 0.016 FO FOX SO SOX *

Effects on Energy intake, Weight & Body Composition Hill A, Murphy K, Buckley J, Howe P, Am J Clin Nutr 2007;85:1267 Change in body weight kg p = 0.06 Change in fat mass kg Change in lean mass * * P < 0.05 for oil x time and FO FOX SO SOX Change in energy intake kJ When we investigated changes in body composition as measured by DEXA, we could see that changes in body weight was primarily associated with a reduction in fat mass, shown exclusively in the FOX group, and not changes in lean tissue. exercise x time interactions Nutritional Physiology Research Group

Relationships between erythrocyte LC n-3 PUFA and body composition Cross sectional analysis BMI, waist circumference, DEXA (% body fat) GC analysis of erythrocyte fatty acids n = 291 BMI WC % Body Fat EPA -0.08 -0.16* -0.17** DPA -0.05 -0.13 -0.14* DHA -0.28** -0.35** -0.33** Omega -3 Index -0.24** -0.32** -0.31** n = 185 BMI WC % Body Fat EPA 0.09 -0.06 -0.15 DPA -0.02 -0.20* -0.23** DHA -0.08 -0.14 -0.19* Omega -3 Index -0.04 -0.13 Data for this study was collected at baseline from volunteers who participated in 5 intervention trials conducted between 2005-2009 in South Australia. As you can see there were mixed inclusion criteria but for the majority were overweight or obese and there was no restriction of how much fish they ate. A range of different techniques were used to assess body composition depending on the study with the first 2 studies only using BMI and the latter 3 also including waist circumference and DEXA to determine % body fat. The fatty acid profiles of erythrocytes membrane were determined using GC

Waist Circumference vs Omega-3 Index women men r= -0.36 P<0.0001 r= -0.18 P> 0.05

Omega-3 and mental health Omega-3 deficiency is associated with depression, schizophrenia, aggression, Alzheimer’s Disease, bipolar disorder and developmental disorders. Animal studies: omega-3 supplementation linked to improved learning and behaviour in rats. Humans trials: increasing evidence of beneficial impact (particularly EPA) on mental health outcomes, especially mood and cognition. So not surprisingly, researchers have started investigating omega-3 fatty acids in the brain and have found deficiencies in people with a range of mental illnesses like depression, schizophrenia, Alzheimer’s Disease, bipolar disorder and developmental disorders like dyslexia, learning disorders, autism and ADHD. Some animal studies have found improved learning and behaviour in rats after omega-3 supplementation and a small number of clinical trials with humans have started to find evidence that omega-3s can improve symptoms in some of these conditions.

ADHD Intervention Trial ADHD is most commonly diagnosed childhood disorder Characterised by hyperactivity; poor impulse control; difficulty sustaining attention Randomised, placebo-controlled, double-blind 30 wk trial 132 children aged 7-12, 2/3rds boys, unmedicated and on upper end of Conners’ ADHD Index Evaluated effects of omega-3 supplementation on inattention hyperactivity impulsivity cognition Outline intervention trial. Sinn N & Bryan J. J Dev Behav Pediatr 2007

Reductions in inattention Reductions in hyperactivity-impulsivity 10 11 12 13 14 15 16 17 18 Baseline 15 weeks 30 weeks Parent ratings of Hyperactive-Impulsive PUFA Placebo 12 13 14 15 16 17 18 19 20 21 Baseline 15 weeks 30 weeks Parent ratings of DSM Inattention PUFA Placebo F = 11.24, p < .01 (Effect size .61) Improvements in both fish oil groups compared to placebo on core symptoms; no additional improvements with micronutrients (therefore have been grouped together). Placebo group represented by orange line - explain results after one-way crossover. F = 7.68, p < .01 (Effect size .20)

Reductions in cognitive problems Reductions in overall ADHD ratings 16 17 18 19 20 21 22 23 24 25 26 Baseline 15 weeks 30 weeks Parent ratings of Cognitive Problems PUFA Placebo 18 19 20 21 22 23 24 25 26 27 28 Baseline 15 weeks 30 weeks Parent ratings of ADHD Index PUFA Placebo F = 10.06, p < .01 (Effect size .52) F = 9.09, p < .01 (Effect size .59)

Current studies on omega-3 in mental health collaborative studies at UniSA and QUT Comparative effects of EPA and DHA in children with ADHD 7-12 year old children with ADHD and learning difficulties 12-month study: 3 x 4 mth crossover EPA-rich oil, DHA-rich oil, placebo Baseline red blood cell PUFA results:- lower n-3 PUFA = poorer literacy higher DHA = better word reading Omega-3 and MCI Emerging evidence that n-3 supplementation slows the rate of cognitive decline in adults with MCI or early AD (Panza et al 2007, Kotani et al 2006, Freund-Levi et al 2006) Comparative effects of EPA and DHA in adults with MCI 6-month parallel comparison Some children with ADHD-related problems with attention and behaviour may benefit from n-3 PUFA: 30-40% improved notably after 15 weeks; 40-50% after continued supplementation over 30 weeks This has very important implications for the treatment of learning and behaviour problems in childhood. Further research required to determine which children will benefit, longer term outcomes and biological mechanisms via blood analyses and other neuropsychological tests. Further research is also needed to investigate the effect of other nutrients and nutrients in combination with each other. In my study, the levels might not have been high enough E.g. children with ADHD are often deficient in zinc – one study found cognitive improvements in children after supplementation with 20mg of zinc per day; which also assists in omega-3 metabolism – MVM supplement in this study only contained less than 2mg zinc per day… 39

ADHD Study: baseline RBC n-3 PUFA status CM Milte, N Sinn, AM Coates, J Buckley, PRC Howe. ISSFAL June, 2010 % of total fatty acids N = 74 1 2 3 4 5 6 7 8 EPA DPA DHA EPA+DHA n - With learning difficulties Without learning difficulties * N-3 index is low, but compares to what seen in typical adult population in studies in Adelaide before. LD may represent a subgroup which may benefit from PUFA supplementation. *p < .03 Controlling for age

Change in RBC PUFA status after 4 months % of total fas

DHA predicts improvements in child cognition Word reading Switching attention p .005 Beta .421 p .005 Beta .415 Increase in cognitive measure indicates an improvement

DHA predicts improvements in child cognition Learning Difficulties Subgroup (N = 17) Word reading Switching attention p .010 Beta .630 p .018 Beta .611 Divided attention p .013 Beta .682

DHA predicts improvements in parent ratings Learning Difficulties Subgroup (N = 17) Hyperactivity Restless/impulsive P .005 Beta .658 P .003 Beta .676

Memory and brain function Erythrocyte polyunsaturated fatty acid status, memory and cognition in older adults with mild cognitive impairment and healthy controls CM Milte, N Sinn, AM Coates, J Buckley, PRC Howe ISSFAL June, 2010 Study measures Memory and brain function cognitive tests: RAVLT, Stroop, Boston naming task, letter fluency, digits forward/backward, trail making, letter number sequencing delayed and immediate recall memory, executive function, attention, information processing speed, mental flexibility, naming Memory Functioning Questionnaire (MFQ) Quality of life/mood Satisfaction with life scale (SWLS) Geriatric Depression Scale (GDS) Health Survey (SF-36) Blood samples RBCs: PUFA content Memory and brain function were assessed using various cognitive tests which measured delayed and immediate recall memory, executive function, attention, information processing speed, mental flexibility and naming. Self-reported memory was also measured using the memory functioning questionnaire. Self-reported quality of life and mood were measured using questionnaires including the geriatric depression scale. We also took blood samples to assess PUFA content of erythrocytes.

PUFAs in MCI compared with healthy controls Higher omega-6 Lower omega-3 * We found those with MCI had higher n-6 status than healthy controls including DPA n-6, and lower n-3 status including EPA after controlling for age. This led to those with MCI also having an elevated AA/EPA and n-6 to n-3 ratio compared with out healthy controls. Omega-6 Omega-3 *P < .05, **P < .01

n-6 PUFA and impaired cognition ↓ verbal paired associates performance ↑ DPA n-6 ↓ excluded letter fluency ↑ DPAn-6 ↓ RAVLT delayed recall ↑ LCn-6 PUFA ↑ LCn-6 PUFA (20 min) ↓ digits backwards span performance ↑ AA MCI Healthy Beta= -.272 P < .05 R² = 0.1206 We did regression analysis for the whole cohort controlling for age, gender and education. Higher levels of DPAn-6 predicted poorer performance on verbal paired associates, n=73 Age + gender + education

n-6 PUFA and impaired cognition ↓ verbal paired associates performance ↑ DPA n-6 ↓ excluded letter fluency ↑ DPAn-6 ↓ RAVLT delayed recall ↑ LCn-6 PUFA ↑ LCn-6 PUFA (20 min) ↓ digits backwards span performance ↑ AA MCI Healthy Beta = -.276 P < .01 R² = 0.0662 And excluded letter fluency n=73 Age + gender + education

n-6 PUFA and impaired cognition ↓ verbal paired associates performance ↑ DPA n-6 ↓ excluded letter fluency ↑ DPAn-6 ↓ RAVLT delayed recall ↑ LCn-6 PUFA ↑ LCn-6 PUFA (20 min) ↓ digits backwards span performance ↑ AA MCI Healthy Beta = -.239 P < .05 R² = 0.0715 Higher levels of LCn-6 PUFA predicted poorer performance on the RAVLT delayed memory recall task. n=73 Age + gender + education

n-6 PUFA and impaired cognition ↓ verbal paired associates performance ↑ DPA n-6 ↓ excluded letter fluency ↑ DPAn-6 ↓ RAVLT delayed recall ↑ LCn-6 PUFA ↑ LCn-6 PUFA (20 min) ↓ digits backwards span performance ↑ AA MCI Healthy Beta = -.295 P < .05 R² = 0.1281 And higher levels of n-6 AA were associated with poorer performance on the digits backwards. n=73 Age + gender + education

Depressive scores ↑ in MCI 7 * GDS 6 8 MCI volunteers & no controls in possible depression range 5 4 * P < 0.01 3 2 1 MCI Healthy Controls And when we compared depressive scores we found those with MCI had worse scores on the geriatric depression scale than healthy controls. And 8 MCI volunteers in the range for a possible diagnosis of depression, whereas no of the healthy controls were. This is expected when we consider we found worse scores on the GDS predicted poorer cognition and self-reported memory functioning. Our data support previous work relating PUFA intake to dementia: high n-6 PUFA and low n-3 PUFA in erythrocytes may predict memory problems relationship of n-3 PUFA status to self-reported health may be due to depression

Summary of key findings Effects of EPA and DHA vs LA on depressive symptoms (N=38; mixed model analysis N=50) Mixed model analysis EPA (p=.03) and DHA (p=.01) vs LA significant after controlling for basal EPA+DHA Correlated with change in ↑EPA+DHA r=.039* ↑AA r=-.031, ↓AA/EPA r=-0.34* Summary of key findings Both EPA and DHA improved depressive symptoms in elderly with MCI Improvements correlated with increases in erythrocyte EPA+DHA DHA was associated with improved health-related quality of life, and to a small degree this explained improved mood Improved mood in elderly with MCI may reduce risk of dementia This pilot study was one of the first clinical trials with omega-3 and mental illness. 30 inpatients with unstable bipolar disorder were given over 9g omega-3 per day in addition to their existing medication (none were started on any new medication or psychotherapy) – 4 were unmedicated in each group. There were 10 drop-outs in the placebo group compared with 2 in the treatment group; most due to worsening of their depressed state. The omega-3 group performed better than placebo on nearly all outcomes, and in those who showed improvements, this was clinically significant. Sinn, Milte, Street, Buckley, Coates, Howe (in preparation)

Conclusions Increased intakes of bioactive nutrients such as omega-3 PUFA, flavanols and other polyphenols offer multiple physical and mental health benefits These benefits may be partly attributable to improvements of circulatory function in chronic cardiometabolic and inflammatory disorders Functional foods enriched with bioactive nutrients have the potential to optimise health status, especially when combined with regular exercise They may also deliver therapeutic benefits, when used alone or as adjuncts to medication

Investigating nutrition and exercise strategies to improve human health and performance