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Università degli Studi di Palermo
DIPARTIMENTO SCIENZE AGRARIE E FORESTALI
BIO/10
DOTTORATO DI RICERCA INTERNAZIONALE IN FRUTTICOLTURA MEDITERRANEA - XXV CICLO
Oxidative stress, oxidative stress-based diseases and
Mediterranean diet.
Raman spectroscopy monitoring of skin carotenoids in healthy individuals and in breast cancer operated patients under controlled diets and in vitro assessment of platelet aggregability.
PhD student
Dr. Anna Perrone
Ciclo XXV
ANNO CONSEGUIMENTO TITOLO 2015
Oxidative stress, oxidative stress-based diseases and
Mediterranean diet.
Raman spectroscopy monitoring of skin carotenoids in healthy individuals and in breast cancer operated patients under controlled diets, and in vitro assessment of platelet aggregation.
Acknowledgements
I would like to thank my PhD supervisor, Prof. M.A Livrea for her supervision, guidance, support and being an invaluable source of knowledge, expertise and guidance. Besides, I express my sincere gratitude for giving me the opportunity to conduct such an enjoyable and successful project .
I would like to express my sincere gratitude to Dr. Anna Maria Pintaudi for her support, guidance, expertise and words of encouragement.
I am also very grateful to Prof. Luisa Tesoriere for her scientific advice and suggestions.
I would like to express my sincere gratitude to Dr. Carla Gentile who helped me to learn biochemical and cellular colture techniques.
I would like to extend my appreciation to Dr Mario Allegra for giving me the opportunity to go to William Harvey Research Institute of London and start my research on platelet aggregability, under supervision of Prof. Tim Warner.
I also have to thank the members of my phD committee, Professor M.A. Germanà, Dr. Farina, Dr. Lo Bianco for their suggestions in general.
I would also like to thank my present and ex colleagues.
I would especially like to thank Dr. G. Carruba, Dr. A. Traina, Dr. L. Blasi and all staff at the Department of Oncology ARNAS Ospedali Civico e Benfratelli G. Di Cristina e M. Ascoli, Palermo. They supported me when I collected data for my Ph.D. thesis.
I wish to thank all of the volunteers who were invited to participate in this study. Without them this project would been impossible.
I would like to extend my sincerest thanks to members of my thesis reading committee Dr. Antonio Cilla Tatay (PhD, University of Valencia) and Dr. Magali Pederzoli Ribeil (Lecturer in Immunology, University of Paris) for spending their time on careful reading of my thesis as well as for valuable comments.
I would like to express my gratitude and appreciation to Prof. Tim Warner for his supervision during period of visiting research. He gave me the opportunity to learn techniques about platelet aggregation.
I would like to extend my appreciation to Dr. Ivana Vojnovic for her words of encouragement.
I also want to thank Prof. Fulvio DAcquisto, Prof. Jane Mitchell, Dr. P. Armstrong, Dr. Bennet Shih, Dr. T. Hoefer and Dr. N. Kirkby, Dr. R.Knowles, Dr. Elias Petrou and Dr.Francesca Rauzi for all their advise and assistance.
Last but not least, special thanks go to my family. Words can not express the gratitude to my parents, my brother Giovanni and my boyfriend Eng. Gianfilippo Di Clemente for their continuing supports and understanding. Anna Perrone
Abstract
Evaluating the level of body antioxidants may reveal a more or less severe oxidative stress condition in either healthy people or subjects with pathologies, and can be suggestive for suitable dietary interventions to possibly affect it. In this work taking advantage from the Raman spectroscopic technology to measure the level of skin carotenoids in a simple and non-invasive manner, healthy people (age 15 to 70, n=155) and women who had surgery for breast cancer (BC) (age 38 to 76, n=71) have been screened to assess to what extent an increased intake of fruit and vegetables (healthy people and patients), associated with diets based on a classical Mediterranean style and that did not include sugared drinks, alcoholic beverages, animal fats and processed meat (patients), might affect the individual antioxidant status. Our data clearly showed that in healthy individuals an optimal body redox state, as expressed by the skin carotenoid score (SCS), positively correlated with the intake of fruit and vegetables, but was inversely correlated with body mass index (BMI), an acknowledged risk factor for cardiovascular disease and cancer. Daily supplements of fruit servings, fresh orange juice in particular, ameliorated significantly the SCS, i.e. decreased the individual oxidative stress. Findings from BC women, monitored for three years (2011-2014), showed that the patients had a significant increase of SCS as strictly adhered to the Mediterranean dietary regimen, again with a higher SCS associated with a lower BMI.
Controlling platelet aggregation may prevent thrombotic vascular events. Assessment of platelet aggregation under conditions mimicking a currently prescribed dual antiplatelet therapy, with the use of a novel aggregability assay, has been carried out as a preliminary study to eventually examine the potential modulatory activity of phytochemicals. Our data suggest that the utilized set-up can be an useful approach for further investigation on either whole extracts or purified compounds from Mediterranean vegetal species.
Table of contents
1. Introduction
1.1 Premises and aims of the study
.
...
.9
Carotenoids and antioxidant molecules
...
.13
1.3 Resonance Raman Spectroscopy for the detection of carotenoids in skin and influence of the nutrition on the antioxidative status.
..
.. ..17
1.4 Mediterranean Diet may improve obesity, chronic degenerative pathologies and reduce risk of cancer
..20
1.5 Platelet Aggregability. Search for a potential modulatory activity of redox active phytochemicals
.
.
.22
2. Subjects and Methods
2.1 Measurement of carotenoids in skin
. . 25
2.2 Subjects and experimental protocol (healthy population)
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...25
2.2.1 Effect of consumption of fresh orange juice on Skin Carotenoid Score (SCS) of healthy subjects
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.
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.26
2.3 Subjects and experimental protocol (DIANA study)
.. 26
2.4 Platelet aggregation and inhibition
...
....28
2.5 Statistical analysis
.28
3.Results
3.1 SCS of healthy subjects compared to individual features
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..30
3.1.1 SCS of healthy subjects before and after consumption of fresh orange juice
..36
3.2 SCS of women from DIANA-5
.. .37
3.3 Platelet aggregation in the presence of aspirin and prasugrel alone or in combination with nitric oxide
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. 48
4.Conclusions
.... .56
5. References
.
..58
6. Result diffusion
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.
67
Index of tables
Table 1.WCRF/AICR 2007 recommendations
27
Table 2. Age, BMI and skin carotenoid score of healthy subjects (n=155)
.. 31
Table 3: Baseline Characteristics of women from DIANA study (2011)
....38
Table 4. Anthropometric data and SCS of women from DIANA study in 2014
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...40
Table 5. BMI and WC of DIANA women from (2011 versus 2014)
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.....44
Index of figures
Figure 1. Chemical structure of major carotenoids present in human blood
.14
Figure 2. Vitamin A and provitamin A carotenoid chemical structures
.......14
Figure 3. Carotenoids with no vitamin A activity
..15
Figure 4. Energy level diagram representing Quantum Energy Transitions for Rayleigh Scattering and Raman Scattering
17
Figure 5. Biophotonic Scanner S2 (Pharmanex®, NuSkin, USA)
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..18
Figure 6. Representation of skin carotenoid score (SCS) index
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...19
Figure 7. Histogram showing the distribution of skin carotenoid score (SCS) in the healthy subjects analyzed
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..30
Figure 8. Skin carotenoid score (SCS) among the groups of different intake of fruit and vegetables.
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..31
Figure 9. Comparison of SCS (means + SD) among the groups of different BMI.
...
32
Figure 10. Correlation between skin carotenoid score (SCS) and BMI in healthy subjects.
. 32
Figure 11. Comparison of SCS (means + SD) among the groups of different BMI, reporting high intake of fruits and vegetables.
.
..
. 33
Figure 12. Effect of increasing fruit and vegetable intake on Skin carotenoid score (SCS) of healthy subjects
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34
Figure 13. Effect of mental stress on Skin carotenoid score (SCS) of healthy students.
35
Figure 14. Skin carotenoid score (SCS) in smokers volunteers as compared with no smokers volunteers.
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. 35
Figure 15. Correlation between skin carotenoid score (SCS) of smokers and number of cigarettes for each smoker.
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... 36
Figure 16. Skin carotenoid score (SCS) of each subject as compared with SCS 15 days after consumption of orange juice.
...
..
. 37
Figure 17. Comparison of baseline SCS (2011) among intervention (blue, n= 38), control (green, n=33) and healthy (grey, n= 37) women.
.
.39
Figure 18. Skin carotenoid score (SCS) of each subject (blue group, intervention group) compared with corresponding measurements 3 years later.
..
41
Figure 19. Skin carotenoid score (SCS) of each subject (green group, control group) compared with corresponding measurements 3 years later
.
... 41
Figure 20. Skin carotenoid score (SCS) of each subject (healthy women) compared with corresponding measurements 3 years later.
.42
Figure 21. SCS of patients of the blue group (n= 38) consuming daily high amounts of fruit and vegetables (> 3 serving fr/veg; n= 21) as compared with low daily intake of fruit and vegetables (< 3 serving fr/veg; n= 17).
..43
Figure 22. SCS of patients of the green group (n= 33) consuming daily high amounts of fruit and vegetables (> 3 serving fr/veg; n= 18) as compared with low daily intake of fruit and vegetables (< 3 serving fr/veg; n=15 ).
.
.
43
Figure 23. Correlation between SCS and BMI in patients of DIANA-5 study
... 44
Figure 24. Correlation between SCS and WC (waist circumference) in patients of DIANA-5 study.
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45
Figure 25. SCS values of 19 patients of the blue group from 2011 to 2014.
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..47
Figure 26. SCS values of 12 patients of the green group from 2011 to 2014.
...
. .47
Figure 27. The effect of ASA on thrombin-induced DEA-NONOate-dependent platelet aggregation
49
Figure 28. The effect of PAM on thrombin-induced DEA-NONOate-dependent platelet aggregation.
..
50
Figure 29 (a-c). Inhibition of platelet aggregation induced by variable thrombin concentrations at varied DEA-NONOate concentrations (0.001 to 0.1 mðM) in the presence of 6 mðM PAM. ..& 51
Figure 30 (a-c). Inhibition of platelet aggregation induced by variable thrombin concentrations at varied DEA-NONOate concentrations (1.0 to 100 mðM) in the presence of 6 mðM PAM. & & .. 52
Figure 31 (a-d). The effect of PAM on the DEA-NONOate-dependent platelet aggregation induced by 0.0625 to 0.5 mðM thrombin. & & & & & & & & & & & & & & ...& & & & & & & & & & .53
Figure 32. The effect of PAM, ASA, or their combination on thrombin -induced platelet aggregation & & & & & & & & & & & & & ............................................................................. 54
List of abbreviations
ANOVAAnalysis of varianceADPAdenosine diphosphateASAAcetylsalicylic acidATPAdenosine triphosphateBCBreast cancerBMIBody mass index CRPC-reactive proteinDEA/NONOateDiethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2 diolateDIANADiet And AndrogensDMSOdimethylsulfoxideDPBDiastolic pressure bloodEREstrogen receptorGPxGlutathione peroxidase H2O2Hydrogen peroxideHPLCHigh performance liquid chromatographyHO.Hydroxyl radical IGF-IInsuline-like growth factor -1MTHTyrodes HEPES bufferMSMetabolic Syndrome NADHNicotinamide adenine dinucleotideNADPHNicotinamide adenine dinucleotide phosphateNONitric oxideNOO.Peroxynitrite NSAIDNon-steroidal anti-inflammatory drugO2.-Superoxide anion radical (O2.-),PAMPrasugrel active metaboliteRONSReactive nitrogen speciesROSReactive oxygen speciesRRSRaman resonance spectroscopyPRPPlatelet Rich PlasmaSCSSkin Carotenoid ScoreSDStandard deviationSEMStandard error of meansGCSoluble guanylate cyclaseSODSuperoxide dismutaseSPBSystolic pressure bloodTXA2Tromboxane A2WCWaist circumferenceWCRFWorld Cancer Research Fund WPsWashed platelets
1. Introduction
1.1 Premises and aims of the study
Oxygen plays a vital role in human life and other living organisms. Aerobic organisms, need oxygen for breathing processes and their metabolism. Oxygen is used as an electron acceptor at the end of the mitochondrial respiratory chain to generate chemical energy. An electron (e-) and a proton (H+) are removed from reduced redox cofactors such as nicotinamide adenine dinucleotide (NADH), then the electron is transferred to the first component of the respiratory chain, and the proton is released into the surrounding fluid. During this reaction, NADH is oxidized to NAD+ and the component that accepts electrons is reduced. The NAD+ can be used again to accept new hydrogen atoms that are generated during oxidation of nutrients. Electrons moving through the respiratory chain are transferred from a component to another to generate sufficient energy to produce molecules of ATP. Molecular oxygen can accept a total of four electrons and the corresponding number of protons to generate two molecules of water. Though oxygen is normally completely reduced, moderate amount of different oxygen radicals are formed during this process.
Reactive oxygen species (ROS) are highly reactive molecules or atoms having one or more unpaired electrons (1,2) and formed in cells as a consequence of aerobic energy metabolism. In addition to catabolic reactions, reactive species are generated by the reaction of NADPH oxidase. In cells, free radicals like superoxide anion radical (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (HO.), peroxynitrite (NOO.) are derived from oxygen and/or nitrogen and are known as reactive oxygen/nitrogen species (ROS/RONS).
Living organisms produce low or moderate concentrations of these compounds, in physiological cell processes, like defence against infectious agents and in the activation of cellular signalling network as observed during induction of a mitogenic response (3). Generally, the formation of free radicals is not dangerous due to either enzyme or non enzyme resources, known as antioxidant defences, that remove these oxidant species. When there is an overproduction of ROS, an imbalance between oxidants and antioxidants occurs. Endogenous antioxidant enzymes such as superoxide dismutas, glutathione peroxidase, catalase, reducing molecules such as glutathione, and exogenous molecular antioxidants such as vitamin E, vitamin C, vitamin A cannot neutralize the excess oxidants resulting in oxidative stress (1,2). Various environmental factors, such as air pollutants, ionizing radiation, UV light, cigarette smoke or inflammation induce ROS production thus damaging cell lipids, proteins and DNA. This in turn can bring about cell death or remarkable alteration of their normal function (4-6), and also activation of several stress-induced transcription factors, with production of proinflammatory and anti-inflammatory cytokines (7-9). Oxidative stress contributes to outset and development of many pathologies, including various types of cancer, atherosclerosis, cardiovascular disease, chronic inflammation, neurological disorder (10-13).
Monitoring individual oxidative stress status in healthy as well as subjects with pathologies may be used to prevent or slow down onset and development of disease, improve our understanding of disease pathogenesis and allow development of new therapeutic strategies. Numerous epidemiological studies have shown that dietary consumption of fruit and vegetable plays a protective role on health and is associated with a lowered risk of cancer, heart disease and stroke (14-16). These properties have appeared to be linked to the presence of phytochemicals generally acting in maintaining the appropriate cell redox balance through diverse mechanisms including antioxidant activity.
Apart the antioxidant vitamins A, C and E, and carotenoids, a ever increasing number of redox-active phytochemicals including polyphenols, have been considered to have a positive effect on human health. Although they are not considered essential nutrients and are present in small amount, current research is showing that plant phytochemicals play important protective roles in many chronic and age-related diseases. In this context, a number of studies have shown that the traditional Mediterranean diet, including large amounts of plant foods and derivatives, such as fruits, vegetables, olive oil, red wine, legumes, has to be considered the preferred dietary program and plays an important role in the prevention of human disease and maintaining of good health; the bioactive phytochemicals indeed appear to work in concert to help and protect from diseases (17-20).
There are thousands phytochemicals in fruits and vegetables, and most of them are coloured compounds. Some well-known ones are:
Flavonoids are reddish pigments, found in red grape skins and citrus fruits.
Isoflavones can be found in peanuts, lentils, soy, and other legumes.
Carotenoids which include yellow, orange, and red pigments in fruits and vegetables
They can work in different ways to improve health. Though each of them may have a particular activity and mechanism of action, it is important to understand that when introduced in a dietary context they become part of a pool of molecules and act all together (21).
Due to the importance of reducing antioxidant activity of either endogenous or exogenous compounds in cell functioning and well being, measuring the total body antioxidant status has appeared as a tool to provide indication on the individual capacity to maintain a proper redox balance.
In clinical and research settings, carotenoids in blood or in tissues may be detected following dietary intake (22). In addition, a study demonstrated that the serum level of carotenoids is predictive of the serum level of other antioxidants (23, 24). Therefore, plasma carotenoids may be a suitable indicator of the total antioxidant status (25). However, measurement of carotenoids in plasma is an invasive method and needs a long time to proceed.
In these years, new methodologies have been developed using an in vivo novel and non invasive optical method, based on the Raman resonance spectroscopy (RRS) to measure carotenoids in the skin. RRS is a spectroscopic technique using visible light at low intensity radiation (471.3 473 nm) that interacts with carotenoids in the skin; thus the energy of the reflected light is shifted to higher wavelength, in the green region. Carotenoid molecules, with their conjugated double bonds, possess vibrational/rotational features and generate strong and unique Raman signals. These properties allow detection and quantification of carotenoids in skin (26), as well as in oral mucosa (27), and in macula lutea (23,24). Importantly, other studies have shown the validity of RRS measurement of carotenoids in skin and its significant correlation to total carotenoid content measured by high- performance liquid chromatography (HPLC), which however requires invasive collection of skin samples (28). Other authors have shown that skin carotenoid levels, measured by RRS are significantly correlated to levels of carotenoids in the diet and blood (26-29); for these reasons, RRS is now considered a non-invasive, rapid, accurate, and safe assessment of carotenoid levels in the skin (26-27) and appears to reflect the blood level of carotenoids. Therefore, Raman spectroscopy is a valid, simple and non invasive technique to be used in humans and can have applications for assessing the antioxidant status of healthy individuals as well as for diseases related to oxidative stress. This technique is easy to use and applicable to epidemiological studies. In addition, the assessment of antioxidant status may suggest dietetic interventions when necessary.
In this study a portable Raman spectroscope has been used to measure carotenoid levels in the skin of healthy volunteers and women operated for breast cancer (BC), from the DIANA (Diet And Androgens) cohort. Different epidemiological studies suggest a protective effect of vegetables and fruits on BC risk (30-32). In this context, the purpose of our study was to monitor the level of carotenoids in the skin of BC patients as a biomarker of the entire antioxidant status (27) and ascertain whether positive changes occurred after adoption of a dietary intervention based on Mediterranean Diet. The latter is adopted to lose weight, reduce body mass index (BMI) and waist circumference. Indeed, improvement of these anthropometric measurements are associated with a decrease in breast cancer risk (33). On these basis, our RRS measurements of skin carotenoids have first been correlated to fruit and vegetable consumption. In addition, relationship between skin carotenoids and risk factors contributing to chronic disease (waist circumference, overweigh, obesity) have been analyzed.
1.2 Carotenoids as biomarker of fruit and vegetable intake
Carotenoids are natural pigments with polyisoprenoid structure and conjugated chain of double bond that contribute to yellow, orange and red pigmentations of plants and fruits (34-35). They are important constituents of photosynthetic organelles present in all higher plants, mosses, ferns and algae, and in photosynthetic membranes of phototropic bacteria and cyanobacteria (36). In plants, they are involved in photosynthesis and in photoprotection. Carotenoids are not synthesized by humans and animals, however they occur in their blood and tissues. More than 700 carotenoids have been identified in nature (37). Among them, about 50 are present in a typical human diet (38), however only 20 carotenoids are been identified in human blood and tissues (39). The most important ones include beta-carotene, alpha-carotene, lycopene, lutein, zeaxanthin, beta-cryptoxantin, alpha-cryptoxantin, gamma carotene, phytoene, phytofluene, neurosporene, carotene (Figure 1), all present in human plasma (38-40). These antioxidants having characteristic of lipophilic molecules exist in many tissue, including the stratum corneum of skin, the sole of foot, forehead, and palm of hand (30).
Figure 1. Chemical structure of major carotenoids present in human blood.
Alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, and zeaxanthin are the most common dietary carotenoids. Alpha-carotene, beta-carotene and beta-cryptoxanthin are provitamin A carotenoids and therefore, they can be converted to retinol by the body (Figure 2). Lutein, lycopene, and zeaxanthin cannot be converted to retinol, so they havent vitamin A activity (Figure 3).
Figure 2. Vitamin A and provitamin A carotenoid chemical structures.
Figure 3. Carotenoids with no vitamin A activity.
Orange vegetables and fruits, including carrots, sweet potatoes, winter squash, pumpkin, papaya, mango, and cantaloupe, are rich sources of the carotenoid B-carotene. Tomatoes, watermelons, pink grapefruits, apricots, and pink guavas are the most common sources of lycopene. Dark green vegetable contain lutein .
Carotenoids have lipophilic characteristics and for this reason, they are very efficient physical and chemical quenchers of singlet molecular oxygen (O2) and peroxyl radicals generated in the process of lipid peroxidation (41-42). These antioxidant molecules are also known as scavengers of other reactive oxygen species (ROS), thus playing a protective role in a number of ROS-related chronic diseases. Furthermore, it is known that hydrophilic ascorbic acid (vitamin C), hydrophobic alpha-tocopherol (vitamin E) and beta carotene can act synergistically to give cell protection against different free radicals. Carotenoids are also involved to quench free radical species generated in the skin exposed to UVA and causing skin damage (43). Indeed several studies have shown the protective effects of carotenoids against premature skin photoaging (44).
Different epidemiological studies and clinical trials have clearly shown that an increased intake of fruit and vegetables rich of carotenoids is inversely related to the risk of chronic diseases such as certain types of cancer, cardiovascular disease, and metabolic syndrome (45-46). Accordingly, several in vitro and in vivo studies have reported that beta carotene and other carotenoids inhibit cancer cell growth (47). In recent years, extensive research has revealed important roles of lycopene, beta carotene and some other carotenoids in human health (25). Carotenoids are also involved in: (i) immune system stimulation; (ii) cell cycle and apoptosis regulation; (iii) growth factors modulation; (iv) cell differentiation; (v) modulation of various types of receptors or adhesion molecules (48).
Different factors such as genetic factor, nutritional status, infection, gender and aging influence bioavailability of carotenoids (49,50). Interactions with drugs (e.g., aspirin and antibiotics) are associated with a decreased b-carotene availability (51). Other studies show that the interaction of different types of carotenoids with other food components may influence absorption, metabolism and serum clearance of these molecules (51,52).
The level of carotenoids in blood and skin can be regarded as a biomarker of fruit and vegetable intake and used in nutritional studies. The gold standard procedure to measure carotenoids in skin or in blood is extraction followed by high performance liquid chromatography (HPLC) (26,53). Relatively high concentrations of carotenoids accumulate in human skin, where their level is quite stable. However, skin carotenoids HPLC measurement requires tissue biopsy and is then highly invasive. On the other hand, blood measurement of these antioxidants are indicative of short-term dietary intake of carotenoids and does not provide the state of tissue accumulation.
Recently, a novel non invasive optical technology based on Raman spectroscopy has been validated as a method to quantify carotenoids in the skin in vivo. These skin measurements are predictive of the antioxidant status, without the inconvenience of invasive methods (26,53), which makes this technology suitable for epidemiological, nutritional or clinical studies, with healthy as well as with patients and subjects that have risk to develop disease.
1.3. Resonance Raman spectroscopy for the detection of carotenoids in skin and influence of the nutrition on the antioxidative status.
A novel optical method, based on the Raman resonance spectroscopy (RRS), allows measurement of carotenoids in skin, using a small probe with a blue wavelength laser (lð =473nm). RRS is a type of laser spectroscopy that interacts with molecules having vibrational and rotational characteristics, such as carotenoids. When light irradiates molecules having conjugated double bonds, most of them are scattered elastically. The elastically scattered light has the same frequency as the incident light, and it is called Rayleigh Scattering. However, a small amount of light is scattered inelastically, and it is termed Raman Scattered light, that owns a frequency shift respect to the incident light. The frequency shift corresponds exactly to the vibrational and rotational energy transitions of these molecules.
Figure 4. Energy level diagram representing Quantum Energy Transitions for Rayleigh Scattering and Raman Scattering.
Carotenoids are capable of absorbing strongly in the blue wavelength region and emitting in the green region. The Raman spectrum gives a spectral fingerprint of the molecules, and the intensity of the Raman peak is directly related to the concentration of molecules. The use of RRS for the measurement of carotenoids in human tissues in vivo has been developed and validated in several reports. A study showed a highly significant correlation between carotenoid measurement in the skin by Raman spectroscopy and carotenoid serum level (54,55), thus validating the Raman spectroscopy as a method to assess these compounds in skin. Other studies demonstrated a significant correlation between skin carotenoid levels measured by Raman spectroscopy and dermal carotenoids quantified by HPLC method (26). All these studies have validated Raman technology as a method to assess carotenoids in the skin in vivo, and also predict the individual antioxidant status (55) without the inconvenience of blood collection.
To monitor the skin carotenoid levels the Biophotonic Scanner S2 (PharmanexR, NuSkin, USA) was used in the present work.
Figure 5. Biophotonic Scanner S2 (Pharmanex ®, NuSkin, USA)
This instrument measures carotenoid levels in the 0.1 mm stratum corneum of the hand skin. The scanner emitted a low intensity 473 nm radiation that interacts with the skin carotenoids. A unique fingerprint spectrum with a peak at 510 nm is generated by carotenoids and therefore this scattered light is captured by a highly sensitive light detector that converts the Raman intensity in counts [skin carotenoids score, SCS] from 0 to 80,000 reported on a computer screen, and represented on a colour scale from red (low SCS) to blue (high SCS). SCS can be converted to laboratory measurement using the equation Y = 12703 * X + 5891.7; where Y is SCS and X is the carotenoid expressed as micrograms/ml of serum. Thus, for example, a SCS value of 25,000 units on the SCS scale represents 1.5 mðg of carotenoid/ml of blood or serum (source: Nuskin/Pharmanex).
The normal concentration of blood beta-carotene is around 0.40 mðM.
The SCS can provide evidence of the individual redox balance status, which in turn may be related to consumption of suitable amounts of antioxidants, especially those of fruits and vegetables. Different factors including erratic lifestyle, intensive physical activity, physical and psychological stress, and exposure to sun, toxins and pollution are known to lower the content of antioxidants. Variations of SCS may also be influenced by the genetic ability to absorb carotenoids. On the other hand, antioxidant status can be significantly improved by increasing daily assumption of fruits and vegetables (5 servings per day, according to World Health Organization (WHO) criteria).
Figure 6: Representation of skin carotenoid score (SCS) index.
In conclusion, RRS is a valid and rapid technique for non invasive measurement of carotenoids in skin and for these reasons may be used as a simple method to monitor nutritional and antioxidant status in humans, and is suitable for epidemiological studies.
1.4 Mediterranean Diet may improve obesity, chronic degenerative pathologies and reduce risk of cancer.
Until the last decades of the 19th century, developed countries were still characterized by poverty and malnutrition, except for Greece, South Italy, Spain and other countries in the Mediterranean region that observed Mediterranean Diet, based on high consumption of olive oil, grains, fresh fruits and vegetables, nuts, legumes and fish, and low consumption of red meats and other fats. During the 20th century, the development of industrial productivity carried out an important reduction of malnutrition and a change of lifestyle, and obesity became a condition associated with high socioeconomic status and a public health problem of the developed world, primarily the USA and Europe.
Obesity is defined as an excess of body adiposity that may have a negative effects on health. People are considered obese according to their body mass index (BMI), a value obtained by dividing weight by the square of height; a value exceeding 30 HYPERLINK "http://en.wikipedia.org/wiki/Kilogram" \o "Kilogram"kg/HYPERLINK "http://en.wikipedia.org/wiki/Square_metre" \o "Square metre"m2 defines obesity, while in the range of 25-30 HYPERLINK "http://en.wikipedia.org/wiki/Kilogram" \o "Kilogram"kg/HYPERLINK "http://en.wikipedia.org/wiki/Square_metre" \o "Square metre"m2 is considered HYPERLINK "http://en.wikipedia.org/wiki/Overweight" \o "Overweight"overweight, for both men and women (56). Excess adiposity and weight are major risk factors for cardiovascular disease (CVD), hypertension, chronic inflammation, insulin resistance, type 2 diabetes and some type of cancer (57,58). In particular, epidemiological studies show that obesity is directly related to mortality from colon cancer in men, and from breast cancer predominantly in post-menopausal women. In addition, in women, evidence has been provided that a high waist circumference (WC) is associated with an increased risk for CVD, type 2 diabetes, dyslipidemia, and hypertension (59,60).
The term Metabolic Syndrome (MS) is used to define a cluster of dysmetabolic traits, such as abdominal obesity (WC > 88 cm), high tryglicerides (>150 mg/ 100 ml serum), low HDL cholesterol (< 50 mg/100 ml serum), hypertension (systolic blood pressure, SPB > 130 and diastolic blood pressure, DPB > 85 mm Hg) and hyperglycemia (>110 mg/100 ml serum) (61). MS can also include hyperinsulinemia, insulin resistance, chronic inflammation, non-alcoholic fatty liver disease and high androgen levels (62). In different epidemiological studies, MS has been associated with BC risk (63). Generally, women with MS have three or more traits and have a significantly higher risk of BC than women without MS, and in this context, obesity represents a higher risk factor for both MS and BC after menopause (64,65). The risk of metastases also appeared significantly higher in patients with MS than in patients without dysmetabolic traits (66), because each trait was associated with an increased risk; for example large waist circumference predicts worse prognosis (67,68). Obesity, and particularly abdominal obesity, is associated with higher serum levels of sex hormones, insulin and somatomedin (Insulin-like Factor 1, IGF-I). Abdominal obesity is also associated with a chronic inflammatory status, and several studies showed an association of high levels of C-reactive protein (CRP) with poor prognosis (69-71). MS is a reversible condition associated with sedentary lifestyle, and a diet characterized by high glycaemia or fat index food, energy drinks, salt and alcohol. In this context, the Mediterranean Diet, characterized by consumption of olive oil, whole grain, fruits, vegetables, nuts, cereals and legumes and low in animal fat, can reduce MS parameters (67,72). Diet may influence prognosis for BC diagnosis (73) and reduce cardiovascular risk (20). However, only a few epidemiological studies have investigated the role of diet in BC and mortality for this disease. Cohort studies showed a significantly reduction of BC associated with higher plasma concentration of carotenoid or dietary intake of beta-carotene, vitamin C and vegetables (74, 75).
In our three years study, we used a Raman spectroscope to measure carotenoid levels in the skin in healthy volunteers and women operated for breast cancer belonging to the DIANA-5 study (Diet and Androgens). Different epidemiological studies suggest a protective effect of vegetables and fruits on BC risk (25,32). DIANA study is a randomized controlled trial based on traditional Mediterranean and macrobiotic recipes and moderate physical activity. This behaviour allows a significant decrease of the serum level of sex hormones, insulin and IGF-I, weight loss, and reduction of waist circumference, which may reduce additional breast cancer events and the risk of metastasis (31). The DIANA multicenter study involved 2,092 patients recruited between 2008 and 2012 in eleven Italian centres 0-5 years after surgical treatment, who were followed up to the end of 2013 for BC related events, including BC-specific mortality, distant metastasis, local recurrences and controlateral BC.
In this context, in the present study the level of skin carotenoids as biomarker of the global antioxidant status (76) was monitored in 71 women among the 391 DIANA study subjects recruited at the Department of Oncology ARNAS Ospedali Civico e Benfratelli G. Di Cristina and M. Ascoli, Palermo, Italy. We wanted to investigate whether the adoption of dietary intervention based on Mediterranean Diet may improve redox status, and anthropometric measurements associated with breast cancer risk (77). On this basis, RRS measurements were first correlated with fruit and vegetable consumption. In addition, relationships between skin carotenoids and risk factors contributing to chronic disease (WC, overweigh, obesity) have been analyzed.
Since the Raman spectroscopy measurements of antioxidant status are not invasive, the compliance was optimal and measurements were usually carried out. Moreover, after the first measurement, any eventual improvement worked as a stimulus for each subject to increase servings of fruits and vegetables.
1.5 Platelet Aggregability. Potential modulatory activity of redox-active phytochemicals.
Platelets play a key role in haemostasis and thrombosis. They may also contribute to several physiologic and pathologic processes, such as inflammation, anti-microbial defence, and tumor growth and metastasis (78). Haemostasis is a physiological process that arrests blood loss from damaged blood vessels, thus being essential to life. Under certain circumstances, however, this process can result in adverse clinical events such as thrombotic occlusion of a vessel. Thrombosis is the pathological formation of haemostatic plug in the absence of bleeding. Under normal and healthy conditions, circulating platelets keep the integrity of vascular system (79) and contribute to maintain blood fluidity. In pathological conditions associated with cardiovascular risk, such as diabetes mellitus, hypercholesterolemia, smoking and hypertension, an increase in platelet activity, aggregability and adhesiveness has been observed (80-82). Under these circumstances, platelets may finally become activated at the level of injured areas of endothelial cells, where early formation and rupture of atherosclerotic plaque may occur, starting the cascade of events culminating in a rapid thrombus formation. This can result in a number of severe consequences from angina to peripheral artery disease and stroke. In this scenery, maintaining a healthy status of platelets, thus preventing their activation and aggregability, has been deemed beneficial to prevent arterial thrombosis (83-85).
Overproduction of reactive oxygen species may have an important role in thrombotic events because of either direct or indirect platelet activation. Indeed oxidative stress is an important mediator of both abnormal platelet function and dysfunctional endothelial-dependent vasodilation in setting of cardiovascular disease. Excess superoxide production drive further platelet activation and recruitment leading to greater thrombus formation (86). All this body of knowledge suggests that platelet protection by redox active phytochemicals may play a role in the widely acknowledged beneficial effects of green food in preventing and delay development of atherogenesis and cardiovascular diseases.
A number of studies focussed on the importance of antioxidants in regulating platelet function. Antioxidants may indirectly inhibit aggregation of platelets through scavenging activity of reactive oxygen species (87,88). A decrease of antioxidant content in human platelets is associated with enhanced platelet activation responses (88). Vitamin E may provide cardiovascular protection. One study shows that platelets incorporate vitamin E both in vitro and in vivo which causes a significant dose-dependent inhibition of platelet aggregation in response to agonists such as arachidonic acid (89). Other studies have proved that flavonoids prevent platelet aggregation (antithrombotic effects), modify eicosanoid biosynthesis (anti-inflammatory responses) and protect low-density lipoprotein from oxidation for prevention of atherosclerotic plaque formation (90). A dual antiplatelet therapy is often prescribed against thrombotic vascular events. Thus it may be interesting to study interaction of phytochemicals and antiplatelets agents in the control of platelet aggregation.
The aggregation of platelets can be assessed in plates of 96 wells, using a modified light transmission method, first described by Warner et al. (91,92). Application of this innovative technique might help to investigate the effects of active components of fruits on human platelet responses and potential mechanisms of protective effects. Fruit extracts or purified components may be tested on isolated platelets in appropriate conditions and other ex vivo research may be carried out on aggregability of platelets before and after a period of controlled dietary regimen in accordance with the classical Mediterranean Diet.
2. Subjects and Methods
2.1 Measurement of carotenoids in skin
A portable Raman spectroscopy, Pharmanex® Biophotonic Scanner S2 (NuSkin, USA) measures carotenoids in the 0.1 cm stratum corneum of the skin of the hand and provided information on the redox state in healthy subjects and patients.
The scanner emitted a low intensity, 473 nm radiation that interacts with the skin carotenoids. The scattered light is detected at 510 nm by the scanner, that converts the Raman intensity in counts [skin carotenoids score, SCS]. A computer then analyze the scanner signal and converts in a coloured scale going from red (< 19,000 low SCS) to blue ( > 50,000 high SCS).
2.2 Subjects and experimental protocol (healthy population).
Healthy volunteers, 62 male and 93 female, aged 15 to 70 (32.73 + 12.57) were enrolled in the present study, after informed consent. A baseline questionnaire including demographic data (age, sex, weight, height, body mass index) and usual amount of fruit and vegetable intake was filled in. The food frequency questionnaires included information about daily or weekly servings of fruits and vegetables. Fruit and vegetable intakes were grouped in 3 categories as follows: low intake ( 1 serving per day). In accordance with the definition of the United States Department of Agriculture (USDA) a serving size for vegetables or fruit has to be equal to about one-half cup, except for greens like spinach and HYPERLINK "http://nutrition.about.com/od/askyournutritionist/f/lettuce_info.htm"lettuce, which have a serving size equal to one full cup. Other focal points of interview were the possible state of mental stress, alcohol consumption, smoking, inflammation state, correctly noted. Subjects did not take any supplement or drugs.
During the day of the SCS measurements, the volunteers cant use dermatological or cosmetic products containing antioxidants during the measurements. All measurements were performed in duplicate.
2.2.1 Effect of consumption of fresh orange juice on SCS of healthy subjects.
Fresh oranges (Cultivar Valencia) were used to extract juice. Healthy volunteers from the faculty of Agriculture of the University of Palermo were invited to participate in this study.
Healthy subjects recruited, 19 male and 17 female, aged 15 to 66 (38.75 + 11.12) received information about the study design and a baseline measurement was carried out for each subject. A baseline questionnaire including demographic data (age, sex, weight, height, BMI) and amount of fruit and vegetable intake were recorded, and state of stress, alcohol consumption, smoking, inflammatory state, were correctly noted. Exclusion criteria used were the use of vitamin supplements, medications, and vegetarian or other restrictive dietary requirements. Each Volunteer consumed 500 ml of fresh orange juice every day for two weeks. Participants were required not to change lifestyle and diet for the period of study, after which a second measurement was carried out. All measurements were performed in duplicate.
2.3 Subjects and experimental protocol (DIANA study).
391 Patients surgically treated for BC in the previous 5 years, who had not developed distant metastasis or second primary BC, in the last 5 years, were enrolled into DIANA-5 (Diet and Androgen-5) study at the Department of Oncology ARNAS Ospedali Civico e Benfratelli G. Di Cristina e M. Ascoli, Palermo, Italy. DIANA-5 study is a randomized dietary intervention in postmenopausal women with high risk factors for BC or recurrences, based on traditional Mediterranean Diet and moderate physical activity, aimed at reducing additional breast cancer events. Dietary intervention prescribed consumption of seasonal vegetables and fruits, unrefined grains, legumes, olive oil, whereas sugared drinks, alcoholic beverages and processed meat were forbidden. For these reasons, before randomization, all participants received the WCRF guidelines for the prevention of cancer through a correct diet and physical activity.
Table 1.WCRF/AICR 2007 recommendations
Be as lean as possible within the normal range of body weight
Be physically active as part of everyday life
Limit consumption of energy-dense food & avoid sugary drinks
Eat mostly food of plant origin, with a variety of non-starchy vegetables
and fruit every day and unprocessed cereals and/or pulses
within every meal
Limit intake of red meat & avoid processed meat
Limit alcoholic drinks
Limit consumption of salt & avoid mouldy cereals or pulses
Aim to meet nutritional needs through diet alone
Mothers to breastfeed; children to be breastfed
Cancer survivors: follow the recommendations for cancer prevention
WCRF, World Cancer Research Fund; AICR, American Institute of Cancer Research.
The intervention diet was expected to reduce level of sex hormones, insulin and IGF-I, reduce weight and waist circumference, both risk factors associated with breast prognosis and may reduce additional breast cancer events and the risk of metastasis (32). All patients with one or more of high risk traits (metabolic syndrome, Estrogen Receptor, ER negative tumor, high serum testosterone or insulin level) were randomly assigned to A) control (green) group or B) an active intervention (blue) group (33). Green group received only WCRF/AICR recommendations, whereas the blue group, received WCRF/AICR recommendations, and also participated in kitchen courses and physical exercise sessions.
In 2011, 38 women from the intervention group, aged 38 to 76, and 33 women from control group, aged 39 to 73 were invited for measurement of skin carotenoid score (SCS), and at the same time anthropometric parameters (height, weight, waist circumference) were collected. As a reference, a group of 37 healthy women, aged 34 to 66 years who usually consumed high amount of fruit and vegetables have been considered. All participants filled in a questionnaire including demographic data (age, sex, weight, height, body mass index), and the amount of fruit and vegetable intake were recorded. A number of blue (n=19) and of green (n=12) women had additional measurements in 2012, and 2013 before the end of observation in 2014, when all patients were called back and new measurements of SCS were carried out.
2.4 Platelet aggregation and inhibition.
Blood was collected by venipuncture of healthy volunteers who abstained from non-steroidal anti-inflammatory drug (NSAID) consumption for the preceding 14 days. Collected blood contained tri-sodium citrate (3.2% w/v) and was centrifuged at 1100 g for 15 minutes at 25oC to obtain platelet rich plasma (PRP). PRP was transferred in separate tubes and centrifuged at 2300 g for 10 min in the presence of PGI2 (2 mðg/ml ) and apyrase (1 mðg/ml). The platelet pellet obtained was washed twice by resuspension in MTH buffer (134 mM NaCl; 2.9 mM KCl; 0.34 mM Na2HPO4; 12 mM NaHCO3; 20 mM Hepes; 1 mM MgCl2; glucose (0.1%) and apyrase (0.02 U/ml), pH adjusted to 7.4). The washed platelets (WPs) were counted using a counter machine and their concentration was adjusted to 3 x 106 platelets/ml. Aggregation of WPs was determined using 96-well plates (98). WPs were pre-incubated at 25°C for 30 min prior the addition of Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2 diolate [DEA/NONOate ] (0.001 mðM 100 mðM) or vehicle (0.01M NaOH) and the pharmacological effects of aspirin and prasugrel alone or combined were evaluated. Platelet activation was induced by the addition of 10 mðl of thrombin (0.03- 0.5 U/ml) and mixed (2300 g) for 5 min at 38oC. Absorbance was determined at 595 nm using a microplate reader (Sunrise microplate reader, Tecan, Switzerland).
2.5 Statistical analysis
Age, BMI, SCS, WC in each group considered, are expressed as means and standard deviation (SD).
Differences in skin carotenoid score among groups of fruit and vegetable intakes and BMI were tested by analysis of variance (ANOVA) and Bonferroni post hoc tests, with P < 0.05 being taken as significant.
Pearsons correlation were used to determine the relationship between SCS and BMI or waist circumference .
All statistical analyses were done using GRAPHPAD PRISM v5 (GraphPad Software, San Diego, California USA), SYSTAT version 10.0 (SPSS, Chicago, IL, USA) and Microsoft Excel.
Data of platelet aggregation were analyzed by GRAPHPAD PRISM v5 (GraphPad Software, San Diego, California USA). Agonist concentration response curves were plotted and analyzed according to the four parameter logistic equation:
Y = Bottom + (Top-Bottom) / ( 1+ 10 (LogEC50 X) x HillSlope)
Concentration-response curves were compared using two way ANOVA and Bonferronis post-hoc test.
3. Results
3.1 SCS of healthy subjects compared to individual features.
Among 155 healthy volunteers, 93 of them were females and 130 were non cigarette smokers. The mean age of the volunteers was 32.66 + 12.58 years and their BMI was 23.02 + 3.8 kg/m2.
Figure 7 shows the distribution of SCS values (one duplicate measurement) for each participant. The mean of SCS value was 34.32 with a standard deviation of 10.88. The data were approximately normally distributed (Anderson-Darling test for normality, P*OJQJmH sH
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ÿÿÿÿsH 69;*B*OJQJmH phÿsH jh±Lh±LOJQJU h±Lh±LOJQJmH sH h&+hOJQJmH sH ARLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Johnson%20LK%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Johnson LK, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Mayne%20ST%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Mayne ST, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Cartmel%20B%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Cartmel B, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Picklo%20MJ%20Sr%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Picklo MJ Sr, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Ermakov%20IV%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Ermakov IV, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Gellermann%20W%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Gellermann W, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Whigham%20LD%5BAuthor%5D&cauthor=true&cauthor_uid=25008856" Whigham LD (2014) Skin and plasma carotenoid response to a provided intervention diet high in vegetables and fruit: uptake and depletion kinetics. HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/25008856" \o "The American journal of clinical nutrition." Am J Clin Nutr 100(3):930-7. doi: 10.3945/ajcn.114.086900.
107. HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Scarmo%20S%5Bauth%5D" Scarmo S, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Henebery%20K%5Bauth%5D" Henebery K, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Peracchio%20H%5Bauth%5D" Peracchio H, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Cartmel%20B%5Bauth%5D" Cartmel B, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Lin%20H%5Bauth%5D" Lin HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Ermakov%20I%5Bauth%5D" IV, Ermakov H, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Gellermann%20W%5Bauth%5D" Gellermann W, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Bernstein%20P%5Bauth%5D" Bernstein PS, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Duffy%20V%5Bauth%5D" Duffy WB, HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/?term=Mayne%20S%5Bauth%5D" Mayne ST (2012) Skin carotenoid status measured by resonance Raman spectroscopy as a biomarker of fruit and vegetable intake in preschool children. HYPERLINK "http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=22434053" \t "pmc_ext" Eur J Clin Nutr 66(5): 555560. doi: 10.1038/ejcn.2012.31.
6. Result diffusion
1) Perrone A, Tesoriere L, Pintaudi AM, Attanzio A, Rigano P, Maggio A, et al. (2014) Raman spectroscopy technology to monitor the carotenoids in skin of thalassemia patients: a novel non-invasive tool relating oxidative stress with iron burden. thalassemia reports, 4(s1), 38-42.
2) Gentile C, Perrone A, Attanzio A, Tesoriere L, Livrea MA (2014) Sicilian pistachio (Pistacia vera L.) nut inhibits expression and release of inflammatory mediators and reverts the increase of paracellular permeability in IL-1b-exposed human intestinal epithelial cells. European Journal of Nutrition, 00.
3) Farina V, Corona O, Todaro A, Moreno S, Barone F, Gentile C, Perrone A, Mazzaglia A. et al. (2014) Pomological Traits, Sensory Characteristics, and Antioxidant Activity in Fruits of Nine Loquat Cultivars Grown in Sicily. [ submit]
PAGE
PAGE 16
PhD supervisor
Prof. M.A. Livrea
PhD coordinator
Prof. M. A. Germanà
Low SCS Moderate SCS High SCS
n=75
a
b
n=28
n=52
b
a
n=124
a,b
b
n=9
n=22
Daily intake of fruits and vegetables
(> 1 serving per day)
a
n=58
b
n=4
b
n=13
*
b
a,b
a
DIANA STUDY (intervention group, 2014 )
*
n=17
n=21
DIANA STUDY (control group, 2014)
*
n=18
n=15
B
A
DIANA STUDY
Control group
WC
DIANA STUDY
Intervention group
B
***
Vehicle
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*** *** ***
**
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*
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** **
**
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HEALTHY WOMEN
FIRST MEASUREMENT SECOND MEASUREMENT
FIRST MEASUREMENT SECOND MEASUREMENT
HIGH INTAKE OF FRUIT AND VEGETABLE
INCLUDEPICTURE "../../AppData/Local/Temp/Res_SempRoot/syo5484/emf5014.png" \* MERGEFORMAT
FIRST MEASUREMENT SECOND MEASUREMENT
Figure 16. Skin carotenoid score (SCS) of each subject compared with SCS 15 days after consumption of orange juice. (n=36; P=0.019, Students paired t-test).
2011 2014
DIANA STUDY INTERVENTION GROUP
2011 2014
DIANA STUDY CONTROL GROUP
INCLUDEPICTURE "../../AppData/Local/Temp/Res_SYSTATTempRoot/syo3392/emf5004.png" \* MERGEFORMAT
2011 2014
2011 2014
DIANA STUDY 2014
INTERVENTION GROUP
Figure 22. SCS of patients of the green group (n=33 ) consuming daily high amounts of fruit and vegetables (> 3 serving fr/veg; n= 18) as compared with low daily intake of fruit and vegetables (< 3 serving fr/veg; n=15). Values were significant with *P=0.0016 (Students t-test).
DIANA STUDY 2014
CONTROL GROUP
High intake fr/veg
(>1 serving/day)
Moderate intake fr/veg
Low intake fr/veg
BMI
BMI
DIANA STUDY 2014
INTERVENTION GROUP
DIANA STUDY 2014
INTERVENTION GROUP
DIANA STUDY 2014
INTERVENTION GROUP
WC
DIANA STUDY 2014
CONTROL GROUP
A
***
***
[Thrombin] (0.03 U/ml)
[Thrombin] (0.03 U/ml)
[DEA-NONOate]0.001 mðM
Vehicle
Vehicle
6 mðM PAM
[DEA-NONOate]0.01 mðM
Vehicle
6 mðM PAM
[DEA-NONOate]0.1 mðM
Vehicle
6 mðM PAM
DEA/NONOate 1¼M
DEA/NONOate 10¼M
DEA/NONOate 100¼M
Vehicle
6 mðM PAM
Vehicle
6 mðM PAM
Vehicle
6 mðM PAM
[Thrombin] (0.0625 U/ml)
[Thrombin] (0.125 U/ml)
[Thrombin] (0.25 U/ml)
[Thrombin] (0.5 U/ml)
Vehicle
6mðM PAM
Vehicle
6mðM PAM
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