ARCLI: AntioxidantsTAMARA SIMPSON & EMMA BARVICH.

Antioxidants• Molecules which prevent the oxidation of other molecules• Oxidation reactions can lead to free radical damage• Free radical damage can lead to cell death• Some also act as free radical scavengers

• Broadly classed as either: 1) Hydrophillic 2) Lipophillic• Hydrophillic antioxidants react with oxidants in cellular cytosol as well as blood plasma• Lipophillic antioxidants protect cell membranes from lipid peroxidation

Antioxidants can also become pro-oxidant following reductionof oxidizing molecules (e.g. Vitamin C)

Oxidative stress• Oxidative stress is a condition whereby cellular

antioxidant defences are insufficient to keep the level of reactive oxygen species (ROS) below a toxic threshold.

• It can be thought of as a balancing act between antioxidants and free radicals/ROS

• This is because both free radicals and ROS have important roles to play.

• ROS: cellular redox signalling, particularly H2O2• Free radicals: thought to play a role in inducing

an endogenous response to protect against exogenous radicals (i.e. radiation, smoking, etc)

• Lipid peroxidation, the oxidation of cellular lipids, is a central feature of oxidative stress.

• Lipid peroxidation is implicated as playing a causative role in the pathophysiology of a number of diseases.

Pycnogenol What is it?

◦ French Maritime Tree Bark. ◦ Pro-cyanidins (flavanols) make up 65 – 75 % of the extract

What does it do?◦ Pro-cyanidins in the bark show high anti-oxidant; free radical scavenging activity◦ Anti-inflammatory

• Going back to the broad classes of antioxidants, pro-cyanidins are moderately water soluble - Pycnogenol’s antioxidant component is likely to react with oxidants in the blood plasma and cytosol of the cell

Pycnogenol research findings

Note: PYC = Pycnogenol, RCT = Randomized Controlled Trial, CDR = Cognitive Drug Research computerized assessment system, F2I = F2 Isoprostanes, BP = Blood Pressure, GSH = Glutathione, GSSG = glutathione oxidised disulfide

Author/Year Intervention/dosage

Design summary Result

Ryan et al. 2008 PYC – 150 mg daily 3 arm – 3 months – RCT- sample 60 – 85 Yrs (n = 101)

PYC sign. Improved working memory (CDR) – decr. F2I

Enseleit et al. 2011 PYC – 200 mg daily 2 arm – 2 months – RCT – clinical sample (n = 23)

F2I decr. – No sign. Improvements for BP

Dvorakova et al. 2006

PYC – 1 mg/kg body weight daily

2 arm – 1 month – RCT – Sample ADHD 6 – 14 yrs (n = 43)

PYC improved GSH levels as well as GSH:GSSG ratio

Bacopa monnieriWhat is it?• a traditional Ayurvedic herb• used for memory decline, inflammation, pain, fever, epilepsy and as a sedative• Steroidal sapponins, Bacosides A and B are the active constituents believed to be

responsible for improving both learning and memory

Antioxidant effect:• may be due to the phytochemicals (alkaloids, flavanoids, steroids) in the plant• may act at the initiation or termination level or as a chain breaker in free radical

reaction• it is believed the metal chelating effect on transition metals (ferrous iron) act to

inhibit the formation of free radicals• high concentrations of Bacopa have showed a marked enhancement in the rate of

oxidation (GSH), thus there may be a balancing act between dosage and duration

Bacopa randomised controlled trial examples

Author/Year Intervention/dosage

Design summary Result

Stough et al.(2001)

BM/300mg daily

2 arm, 3 months, parallel group RCT. Sample aged 18-60y (n=46)

BM significantly improved speed of visual information processing (IT), learning rate and memory (AVLT) & state anxiety

Roodenrys et al.(2002)

BM/300mg daily

2 arm, 3 months, parallel group RCT. Sample aged 40-65y (n=76)

BM significantly improved retention of new information in delayed recall of word pairs.

Stough et al. (2008)

BM/300mg daily

2 arm, 3 months, parallel group RCT. Sample aged 18-60y (n=62)

BM significantly improved working memory (CDR) and RVIP

Calabrese et al.(2008)

BM/300mg daily

2 arm, 3months, BM significantly improved learning rate and memory (AVLT) & state anxiety

Peth-Nui et al.(2012)

BM/300mg or 600mg daily

3 arm, 3 months, parallel group RCT. Sample mean age 62.62y, SD 6.46 (n=60)

BM improved WM decrease in N100 &P300 latencies. Reduced plasma AChE occurred.

Benson et al.(2013)

BM/320mg or 640mg

2 arm, acute, cross over RCT.Sample aged 18-44y (n=17)

BM improved processing speed/selective attention (Stroop/Letter Search) 1hr & 2hrs post consumption and positive mood effects and reduction in cortisol.

Note: PYC = Pycnogenol, BM = Bacopa, RCT = Randomized Controlled Trial, AVLT= Auditory Verbal Learning Test, IT = Inspection Time, RVIP = Rapid Visual Information Processing, CDR = Cognitive Drug Research computerized assessment system

Active Ingredient Dose Role Action

Folic acid 400µg Essential for lots of bodily functions • Synthesise and repair DNA• regulate homocysteine levels• reduce neural tube defects• required in methylation

Vitamin B12 500µg AKA cobalamin, plays a key role in the normal functioning of the brain and nervous system

• amino acid, fatty acid metabolism• DNA synthesis and regulation• identified to reduce brain atrophy associated with AD and

impaired cognitive function• required in methylation reactions and regulate homocysteine

levels

Vitamin B 6 25mg Coenzyme involved in metabolic processes • amino acid, glucose and lipid metabolism• neurotransmitter, histamine synthesis• haemoglobin synthesis and function• Gene expression

Phosphatidylserine 50mg Phospholipid for brain cells • responsible for cell cycle signaling, specifically in apoptosis

Lipoic acid 300mg AKA alpha lipoic acid, essential for aerobic metabolism, antioxidant from red meat, broccoli, spinach, potatoes, carrots, water and fat soluble

• involved in the Krebs cycle – converting carbs into energy• In a ‘free’ state, excessive amounts act as an antioxidant

removing heavy metals • may also assist in regenerating antioxidants Vitamins C and E

Vitamin E 30IU ~20mg

Fat soluble antioxidant • stops the production of ROS when fat undergoes oxidation• Involved in cell signaling, gene expression (connective tissue

growth factor)• plays a role in cognition

CoQ10 50mg Found in the mitrochondria, part of electron transport and aerobic cellular respiration, powerful antioxidant

• key role in producing energy (ATP) for mitochondria• believed to increase energy production in heart muscle• may increase dopamine which is low in PD patients• may assist with heart related conditions due to improving cell

energy production and preventing blood clot formation• may assist in lowering glycemic index of diabetic patients

Blackmores® MultivitaminProprietary blend of micronutrients and vitamins, not yet commercially available

Multivitamin and B vitamin RCT examples

Author/Year Intervention/dosage

Design summary Result

Cognitionde Jager et al. (2011) 0.8mg FA, 0.5 mg

B12, 20mg B6 (VITACOG-MCI)

2 arm, 2yrs, parallel group RCT. Sample aged ≥ 70y (n=266)

BV improved global cognition (MMSE), episodic memory (p=0.001), semantic memory (p=0.037)

Pipingas et al.(2014)

Swisse Men’s or Women’s Ultivite

2 arm, 4 month, parallel group RCT. Sample aged 20-50y (n=138)

Strong trend for males’ (p=0.01) improvement in selective attention/response inhibition(Stroop incongruent). No cognitive benefits for women. Multivitamins increased blood levels of Vit B6, B12 and folate for both genders, decreased homocysteine in men.

Kennedy et al.(2011)

Berocca®, 1 tablet per ay

2 arm, 1 month, parallel group RCT. Male sample aged 30-55y (n=198)

No cognitive enhancing effects. Subjective ratings of having greater ‘physical stamina’ across assessments and weeks. Subjective ratings of greater ‘concentration’ and mental stamina’ during the wo

AtrophyDouaud et al. (2013) 0.8mg FA, 0.5 mg

B12, 20mg B6 (VITACOG-MCI)

2 arm, 2yrs, parallel group RCT. Sample aged ≥ 70y (n=156)

Whole brain shrinkage slowed as well as, bilateral hippocampus, parahippocampal gyrus, retrosplenial precuneus, lingual and fusiform gyrus cerebellum (p < 0.001 FWE corrected for multiple comparisons)

Antioxidant biomarkers recruited in ARCLI

Glutathione (GSH) in vivo(hopefully GSH assay)

and

F2 isoprostanes

Glutathione• Most abundant antioxidant in the human body

• Protects cells against reactive oxygen species - from free radical damage and peroxides and maintains exogenous antioxidants (Vits C & E) in their active form

• It is represented as a reduced form (GSH) and as an oxidised disulphide (GSSG)

• GSH is the major tissue antioxidant. More than 90% of the total glutathione pool is in this reduced form in healthy cells

• Glutathione peroxidase (GPx) reduces lipid hydroperoxide to their corresponding alcohols and hydrogen peroxide to water. Low levels have been correlated with free radical related disorders

• GPx catalyzes, generating oxidised glutathione (GSSG)

• Glutathione reductase (GR) is coupled to GPx and recycles GSSG to GSH

cont…• Plays an essential role in the synthesis and degradation of proteins and DNA

synthesis and repair

• Conjugates with foreign proteins (drugs) and with compounds formed in metabolism ( eg. estrogens, prostaglandins) participating in their metabolism

• A depletion of GSH is indicative of mitochondrial dysfunction and decreased NAA

• Higher concentrations of GSH has been identified in:• astrocytes vs neurons, • grey matter vs white matter and • females vs males

• GSH levels can be raised by mild stress, possibly as a measure of support to handle increases in stress levels

• With age, GSH levels are diminished

Rae, Neurochem Res (2014)39; 1-36

• GSH is made from: glutamate (Glu), cysteine (Cys) and glycine (Gly) in a two step pathway requiring ATP

• GSH converted to GSSG via GPx redox (oxidation-reduction) reactions

• GSSG can leave the cell via a multidrug resistance pump as: GSH, GSSG or as a mixed bonded disulphide

• In astrocytes, the major source of cysteine for GSH synthesis is from cystine

• Cystine enters astrocytes via the cystine/glutamate exchanger

• Cysteine is also made through transsulfuration using methionine via

S-adenosylmethionine, S-adenosylhomocysteine, homocysteine and cystathione

Glutathione in neurons & astrocytes

Measuring GSH endogenously

•blood assay measuring total glutathione (GSH: GSSG) ratio• Kits incorporate glutathione reductase to enable GSH & GSSG to be

measured, reflecting total glutathione

•blood assay measuring GSH peroxidase (GPx)• kits incorporate cumene hydroperoxide to measure GPx activity• GPX reduces cumene hdroperoxide while oxidising GSH to GSSG. GSSG is reduced to GSH with the

consumption of NADPH by GR. The decrease in NADPH (measured at A340 monitored as a function of time) is proportional to GPx activity

GSSG (oxidised state) accumulates when cells are exposed to increased levels of oxidative stress, thus the ratio of GSSG to GSH increases

An increased ratio of GSSG to GSH is indicative of oxidative stress

The monitoring of reduced and oxidised GSH in biological samples is essential for evaluating the redox and detoxification status of the cells and tissues against oxidative and free radicals mediated cell injury

Research including blood assays isolating GSH and the relationship to atherosclerosis

114 healthy adults underwent blood assays (GSH:GSSG, high-sensitivity C-reactive protein, HDL, LDL, triglycerides). BMI, Age, Framingham risk score and carotid intimamedia thickness (IMT) measured using ultrasound, were also collected.

GSH:GSSG was identified to be an independent predictor for the presence of atherosclerosis (narrowing of arteries due to plaques) in an otherwise healthy cohort.

Measuring GSH in vivoMagnetic Resonance Spectroscopy (MRS)

• a technique that complements standard MRI to examine small molecules

• The signal is obtained from the abundance of hydrogen protons (1H) to determine metabolite peak areas that reflect concentrations of metabolites in brain tissue

• Each metabolite is associated with a peak that occurs at a known frequency

Brennan et al., Biological Psychiatry,(2013)73:24-31.

Research applying the technique of MRS isolating GSH in response to supplements

Ethyl-eicosapentaenoic Acid (E-EPA- 2g), an omega-3 fatty acid, was administered for 12 weeks with 24 first episode psychosis patients. GSH increased bilaterally and glutamate/glutamine increased in the left hemisphere post E-EPA administration. Improvement in negative symptoms correlated with increased GSH (r=-0.57).

Research applying the technique of MRS isolating GSH in response to intravenous application of N-acetylcysteine

Infusion of 150mg/kg NAC with 3 PD, 3GD and 3 healthy controls increased blood GSH redox ratios. This was followed by an increase in GSH neurometabolite concentrations across the participants identified using MRS.

F2 - isoprostanes•Prostaglandin-like compounds formed from free radical induced peroxidation of arachadonic acid (ubiquitous omega-6 polyunsaturated acid from food eg. egg, red meat)

•Lipid peroxidation can be very damaging, it leads to alterations in the biophysical properties of the cell membrane, impairing normal cellular function

•F2 isoprostanes are a significant and accurate marker of oxidative stress in vivo in humans and animals

•They are the most studied prostaglandin due to their stability which enables the most accurate measure of oxidative stress

•Elevated levels have been linked to a myriad of human disorders eg. smoking, diabetes, Huntington’s disease, AD, Chron’s diesease, atherosclerosis

•Clinically, F2 isoprostanes are useful for monitoring disease and response to therapy

•F2 isprostanes in ARCLI are measured through blood assays

Biological effects of F2 - isoprostanes

Isoprostanes:• produce inflammation and

atherogenesis (formation of plaques in arteries) via Mitogen-Activated Protein kinases

• Promote platelet activation and induces mitogenesis (triggering of mitosis) in vascular smooth muscle cells, stimulates responses in fibroblasts, alters endothelial cell biology (COX activation-produces inflammation, PGF2a-induces labour)

• Asprin and ibuprofen inhibit COX activation

Kaviarasan et al. J Clin Biochem Nutr (2009)45(1):1-8.

Diabetes:• Associated with high F2

levels• Deficiencies in: ascorbate,

GSH, superoxide dismutase.

• Low levels of GSH found in diabetic neutrophils (white blood cell, hallmark of acute inflam) and monocytes (white blood cell, elicit immune response)

• Low levels of ascorbate found in both diabetic plasma and mononuclear cells (lymphocytes, monocytes, dendritic cells)

Examples of F2 isoprostane research

Weight loss in obese women was associated with a significant reduction in isoprostane formation.

Based on 3,000 participants in the Framingham Heart study, elevated F2 isoprostane levels in both males and females was strongly associated with increasing BMI.

Possible directions using baseline data

Baseline correlations of F2 isoprostane levels, BMI, weight and diet (FFQ)

Baseline correlations of F2 isoprostane levels and GSH concentrations in vivo to elucidate markers of oxidative stress

Baseline correlations of F2 isoprostane levels, GSH:GSSG bloods, different age ranges to determine the trajectory of normal age related cognition

GSH:GSSG ratio and the relationship to F2 isoprostane levels

Baseline assessment of brain atrophy between different age groups and correlation to cognition or WASI

Possible directions usinglongitudinal data

Pycnogenol

Cognition: Based on Ryan et al. (2008) – improvements expected for PYC group on CDR

F2 Isoprostanes: Expected decrease in PYC group compared to control (Ryan et al., 2008 & Enseleit et al., 2011)

GSH levels: Based on quite tentative findings, we could see an increase in PYC group (Dvorakova et al., 2006)

Bacopa

Cognition: Based on the body of CHP work and others, improvement is expected at 3mths and more importantly 6mths and 12mths. Any permutation of the cognitive variables in ARCLI with MRS, F2’s and/ or cardio measures to investigate improvement over time.

GSH levels: If assayed will be interesting to investigate any changes that occur, as low doses for a long duration has been suggested to possibly be more effective (Tripathi et al., 1996).

Multivitamin

Cognition: Correlation to forestalling of brain atrophy over time (Duoud et al. 2013).

F2 Isoprostanes: A reduction in levels may be mediated through B Vits and antioxidants in the blend - speculative

Imaging

Cognition: correlation to MRS (NAA, Cho, Cre) and supplements, correlation to GSH in vivo and supplements, correlation to MRS, GSH in vivo and blood flow (ASL), supplements and PWV, correlation to resting state (DMN) and supplements

Thank you

Dr Doug MitchellSwinburne Alumni Benefactor

Acknowledgements: