Trace Elements and Cardiovascular Diseases Evidences From the Comparative Postmortem Analysis of Tissues

i Dissertation of the 2nd Cycle of Studies Leading to Master's Degree in Analytical, Clinical and Forensic Toxicology TRACE ELEMENTS AND CARDIOVASCULAR DISEASES Evidences from the comparative postmortem analysis of tissues Anne Sophie Pereira Alves Supervised by: Prof. Doutor Agostinho Almeida (Faculdade de Farmácia da Universidade do Porto) Prof. Doutor Agostinho Santos (Faculdade de Medicina da Universidade do Porto; Instituto Nacional de Medicina Legal e Ciências Forenses) September 2014 ii AUTHOR’S DECLARATION The integral reproduction of this dissertation is only authorized for research purposes when provided a written declaration for permission of use. iii ACKNOWLEDGMENTS I would like to express my gratitude to all the people involved in this work and who helped me to make this work possible. I would like to thank in particular: To my supervisor, Prof. Agostinho Almeida, for his guidance and support during the course of this work. To my co-supervisor, Prof. Agostinho Santos, for his support. To all forensic pathologist and technicians involved in sample collection at Instituto Nacional de Medicina Legal. I would like to refer a special thanks to Ricardo, Débora and Maria. To Patricia Ramos for all the support, patience and sympathy demonstrated since the first day. To Mariana, thank you for the support and understanding. To all my friend, that even from distance supported me. Catarina, Marisa, Lis thank you for everything. And last but not least to my parents without them none of this would be possible. Thank you for support and love iv ABSTRACT Cardiovascular diseases (CVD) are a major public health problem. Disruption of trace element homeostasis may lead to oxidative stress, and consequent DNA damage, lipid peroxidation, protein modification and other biochemical effects, causing CVD. The main goal of this work was to directly study the disease-related changes on some trace elements levels (Cu, Cd, Pb, Se and Zn) in different tissues: kidney (medulla and cortex) and aorta (with and without atheroma plaque). Comparing the two kidney regions, higher levels were found in cortex, but the difference reached statistical significance only for Zn (166.1±100.2 vs 115.7±86.5 µg/g; p≤0.01) and Cd (72.57±45.43 vs 41.37±38.72 µg/g; p≤0.0001). For kidney medulla, a gender-related difference was found for Pb, with higher levels in men (0.32±0.23 µg/g) than women (0.16±0.10 µg/g; p≤0.01). It was also observed that smoking habits have an influence in some trace elements levels in this kidney structure. Higher levels of Zn (16671±112.1 vs 91.46±63.47 µg/g; p≤0.01), Cd (75.97±61.04 vs 27.78±20.24 µg/g; p≤0,001) and Pb (0.39±0.19 vs 0.21±0.20 µg/g; p≤0.05) were found in smokers. In kidney cortex no significant difference was found between genders for any of the five elements studied. Regarding smoking habits, only Cd showed a significant difference between smokers (110.1±37.74 µg/g) and non-smokers (51.06±27.62 µg/g; p≤0.0001). In both kidney regions, a slight tendency for an age-related increase was observed for the five elements. However, the Cd levels in renal cortex only increased up to 50 years old; then a subsequent decrease was observed. In aorta without plaque no significant genderor smoking habits-related differences were found for any of five elements. In aorta with plaque, smokers showed significantly decreased Zn levels: 78.44±32.25 µg/g vs 126.45±66.60 µg/g in non-smokers (p≤0.05). When comparing both tissues (aorta with and without plaque), increased levels of Zn and decreased levels of Se were found in aorta with plaque (110.9±58.42 vs 89.74±28.45 µg/g; p≤0,05 and 3.85±2.50 vs 4.71±2.33 µg/g; p≤0,01, respectively). In aorta without plaque only Pb showed to be related (increased) with age. In aorta with plaque, an age-related tendency for decreased levels was observed for Cu, Cd and Se. On the contrary, Zn and Pb showed a tendency to increase with age. Individuals with CVD showed significantly lower levels of Cu and Se in both tissues – Cu kidney: 5.61±3.82 vs 7.96±3.71 µg/g; Cu aorta: 3.18±1.32 vs 4.48±2.69 µg/g; Se kidney: 3.90±2.80 vs 5.48±5.35 µg/g; Se aorta 3.59±2.45 vs 4.46±2.14 µg/g). Interestingly, decreased levels of Cd and Pb were also observed. Keywords: Trace elements; cardiovascular diseases; ICP-MS; postmortem analysis; kidney; aorta. v RESUMO As doenças cardiovasculares (DCVs) são um sério problema de saúde pública. A perturbação da homeostase dos elementos vestigiais pode levar a stress oxidativo e consequente dano no ADN, peroxidação lipídica e modificação de proteínas, entre outros efeitos bioquímicos, causando DCV. O principal objetivo deste trabalho foi estudar diretamente as alterações, relacionadas com as DCVs, nos níveis de alguns elementos vestigiais (Cu, Cd, Pb, Se e Zn) em diferentes tecidos: rim (medula e córtex) e aorta (com e sem placa de ateroma). Comparando as duas regiões do rim, foram encontrados níveis mais elevados no córtex, mas a diferença só foi estatisticamente significativa para Zn (166,1±100,2 vs 115,7±86,5 µg/g; p≤0,01) e Cd (72,57±45,43 vs 41,37±38,72 µg/g; p≤0,0001). Em relação à medula renal, foi observada uma diferença entre sexos para o Pb, com níveis mais elevados nos homens (0,32±0,23 µg/g, vs 0,16±0,10 µg/g nas mulheres; p≤0,01). Observou-se também que o tabagismo influencia os níveis de alguns elementos nesta região do rim. Nos fumadores foram encontrados níveis mais elevados de Zn (166.73±112.1 vs 91.74±63.47 µg/g; p≤0.01), Cd (75.97±61.04 vs 27.78±20.24 µg/g; p≤0,001) e Pb (0,39±0,19 vs 0,21±0,205 µg/g; p≤0.05). No córtex renal não foram encontradas diferenças significativas entre sexos para qualquer dos cinco elementos estudados. Em relação ao tabagismo, somente o Cd mostrou uma diferença significativa entre fumadores (110,1±37,74 µg/g) e não fumadores (51,06±27,62 µg/g; p≤0.0001). Em ambas as regiões do rim, foi observada uma ligeira tendência para um aumento relacionado com a idade nos níveis dos cinco elementos. No entanto, os níveis de Cd no córtex renal aumentaram somente até aos 50 anos de idade, diminuindo depois. Na aorta sem placa não foram encontradas diferenças significativas entre sexos ou relacionadas com os hábitos tabágicos para qualquer dos cinco elementos. Na aorta com placa, os fumadores apresentaram uma diminuição significativa dos níveis de Zn: 78,44±32,25 vs 126,45±66,60 µg/g em não-fumadores (p≤0,05). Na comparação entre os tecidos (aorta com e sem placa), foi observado um aumento significativo dos níveis de Zn e uma diminuição dos níveis de Se na aorta com placa (110.9±58.42 vs 89.74±28.45 µg/g; p≤0.05 e 3.85±2.50 vs 4.71±2,34 µg/g; p≤0.01, respetivamente). Na aorta sem placa só o Pb mostrou estar relacionado (aumentado) com a idade. Na aorta com placa observou-se uma tendência para a diminuição com a idade dos níveis de Cu, Cd e Se. Pelo contrário, o Zn e o Pb mostraram uma tendência para aumentarem com a idade. Os indivíduos com DCV apresentaram níveis significativamente mais baixos de Cu e Se em ambos os tecidos – Cu renal: 5.61±3.82 vs 7.96±3.71 µg/g; Cu aorta: 3.18±1.32 vs 4.48 ± 2.69 µg/g; Se renal: 3.90±2.80 vs 5.48±5.35 µg/g; Se aorta 3.19±1,60 vs 4.00±1.53 µg/g). Curiosamente, também foi observada uma diminuição dos níveis de Cd e Pb. Palavras-chave: Elementos vestigiais; doenças cardiovasculares; ICP-MS; análise postmortem; rim; aorta. vi TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................... iii ABSTRACT ................................................................................................................. iv RESUMO ...................................................................................................................... v INDEX OF FIGURES .................................................................................................. viii INDEX OF TABLES ...................................................................................................... x ABBREVIATIONS LIST ............................................................................................... xii 1.1 introductory note .................................................................................................... 2 1.2 Cardiovascular diseases ........................................................................................ 4 1.2.1 Arteriosclerosis ................................................................................................. 5 1.2.2 Atherosclerosis ................................................................................................. 5 1.3 Risk factors ............................................................................................................ 8 1.3.1 Age ................................................................................................................... 8 1.3.2 Hypertension .................................................................................................... 9 1.3.3 Tobacco ......................................................................................................... 10 1.3.4 Dietary habits ................................................................................................. 10 1.3.5 Obesity ........................................................................................................... 11 1.3.6 Diabetes ......................................................................................................... 12 1.3.7 Hyperlipidaemia .............................................................................................. 12 1.3.8 Oral contraceptives......................................................................................... 13 1.4 Trace elements .................................................................................................... 14 1.4.1 Cadmium ........................................................................................................ 15 1.4.2 Copper ........................................................................................................... 16 1.4.3 Lead ............................................................................................................... 18 1.4.4 Selenium ........................................................................................................ 20 1.4.5 Zinc ................................................................................................................ 21 1.5 Trace element analysis ........................................................................................ 24 2 Objectives ............................................................................................................... 29 3.1 Laboratory ware ................................................................................................... 31 3.2 Subjects ............................................................................................................... 31 3.3 Sample collection ................................................................................................. 32 3.4 Sample preparation .............................................................................................. 32 3.5 Sample analysis ................................................................................................... 33 3.6 Analytical quality control ....................................................................................... 34 3.7 Statistical analysis ................................................................................................ 34 vii 4.1 Kidney .................................................................................................................. 37 4.1.1 Medulla ........................................................................................................... 37 a) All the samples ............................................................................................. 37 b) Men vs Women ............................................................................................ 37 c) Smokers vs Non-smokers ............................................................................ 39 d) Differences by area of residence .................................................................. 41 4.1.2 Cortex............................................................................................................. 43 a) All the samples ............................................................................................. 43 b) Men vs Women ............................................................................................ 43 c) Smokers vs Non-smokers ............................................................................ 44 d) Differences by area of residence .................................................................. 46 4.1.3 Cortex vs Medulla ........................................................................................... 47 4.2 AORTA TISSUE................................................................................................... 49 4.2.1 Aorta tissue without plaque of atheroma ........................................................ 49 a) All samples................................................................................................... 49 b) Men vs Women ............................................................................................ 49 c) Smokers vs Non-smokers ............................................................................ 50 4.2.2 Aorta tissue with plaque of atheroma .......................................................... 51 a) All samples................................................................................................... 51 b) Men vs Women ............................................................................................ 51 c) Smokers vs non smokers ............................................................................. 52 d) Differences by area of residence .................................................................. 53 4.2.3 Aorta tissue with and without plaque of atheroma ....................................... 54 4.3 AGE-RELATED CHANGES ................................................................................. 57 4.3.1 Kidney ......................................................................................................... 57 4.3.2 Aorta ........................................................................................................... 60 4.4 HEALTHY INDIVIDUALS vs INDIVIDUALS WITH CVD ........................................ 63 CONCLUSIONS AND FUTURE RESEARCH ............................................................. 68 REFERENCES ........................................................................................................... 71 ATTACHMENT ........................................................................................................... 76 viii INDEX OF FIGURES Figure 1 Normal artery (left) and an artery affected by atherosclerosis (right). .................... 7 Figure 2 Fenton reaction. .................................................................................................... 17 Figure 3 Chemical background for the flexibility of zinc ion in changing the redox environment (redox zinc switch). Zinc is coordinated in a reduced sulphur-containing protein domain and is released under oxidation. ................................................................. 22 Figure 4 Role of zinc in cardiovascular diseases ................................................................ 23 Figure 5 Diagram of ICP-MS instruments. .......................................................................... 25 Figure 6 (a) Schematic representation of kidney anatomy; (b) Nephron structure ............ 32 Figure 7 Lead levels (g/g) in kidney medulla in men (n=35) and women (n=18) ............. 38 Figure 8 Cadmium and zinc levels (µg/g) in kidney medulla of smokers (n=13) and non-smokers (n=28) ............................................................................................................. 40 Figure 9 Lead levels (g/g) in kidney medulla of smokers (n=13) and non-smokers (n=28) ...................................................................................................... 41 Figure 10 Average concentration (µg/g) of Cu, Se and Pb in renal medulla according to individuals’ area of residence. .............................................................................................. 42 Figure 11 Average concentration (µg/g) of Zn and Cd in renal medulla according to individuals’ area of residence. .............................................................................................. 42 Figure 12 Cadmium levels (g/g) in the kidney cortex of smokers (n=14) and non smokers (n=29) ...................................................................................................... 46 Figure 13 Average concentration (µg/g) of Cu, Se and Pb in renal cortex according to individual’s area of residence. .............................................................................................. 46 Figure 14 Average concentration (µg/g) of Zn and Cd in cortex according to individual’s area of residence. ................................................................................................................. 47 Figure 15 Cadmium and zinc levels (g/g) in kidney cortex (n=56) and medulla (n=57) ... 47 Figure 16 Zinc levels (g/g) in aorta tissue with plaque of atheroma of smokers (n=10) and non-smokers (n=20); ..................................................................................................... 53 Figure 17 Average concentration (µg/g) of Cu, Cd, Pb and Se in aorta according to individuals’ area of residence. .............................................................................................. 53 ix Figure 18 Average concentration (µg/g) of Zn in aorta according to individuals’ area of residence. ............................................................................................................................. 54 Figure 19 Zinc levels (g/g) in aorta tissue without plaque of atheroma (n=52) and aorta tissue with plaque of atheroma (n= 41) ................................................................................ 54 Figure 20 Selenium levels (g/g) in aorta tissue without plaque of atheroma (n=45) and in aorta tissue with plaque of atheroma (n=36)........................................................................ 55 Figure 21 Relationship between Cd levels (µg/g) in kidney and age (years). .................... 57 Figure 22 Relationship between Cu levels (µg/g) in kidney and age (years). .................... 58 Figure 23 Relationship between Zn levels (µg/g) in kidney and age (years). .................... 58 Figure 24 Relationship between Pb levels (µg/g) in kidney and age (years). .................... 59 Figure 25 Relationship between Se levels (µg/g) in kidney and age (years). .................... 59 Figure 26 Relationship between Cu levels (µg/g) in aorta and age (years). ...................... 60 Figure 27 Relationship between Zn levels (µg/g) in aorta and age (years). ....................... 61 Figure 28 Relationship between Cd levels (µg/g) in aorta and age (years). ...................... 61 Figure 29 Relationship between Pb levels (µg/g) in aorta and age (years)........................ 62 Figure 30 Relationship between Se levels (µg/g) in aorta and age (years)........................ 62 Figure 31 Copper levels (g/g) in kidney and aorta tissue in healthy individuals (n=19) and individuals with CVD (n=23) .......................................................................................... 64 Figure 32 Cadmium levels (g/g) in aorta tissue of healthy individuals (n=19) and individuals with CVD (n=23) ................................................................................................. 65 Figure 33 Lead levels (g/g) in kidney in healthy individuals (n=19) and individuals with CVD (n=23) ........................................................................................................................... 65 Figure 34 Selenium levels (g/g) in kidney and aorta tissue from healthy individuals (n=19) and individuals with CVD (n=23) ............................................................ 66 4 1.2 CARDIOVASCULAR DISEASES CVD are the leading cause of morbidity and mortality in developed countries, accounting for over a third of all deaths (9). According to WHO (10), CVD are a group of diseases of the heart and blood vessels comprising: 1. CVD due to atherosclerosis:  Ischaemic heart disease or coronary artery disease (e.g. heart attack)  Cerebrovascular disease (e.g. stroke)  Diseases of the aorta and arteries, including hypertension and peripheral vascular disease 2. Other CVD  Congenital heart disease  Rheumatic heart disease  Cardiomyopathies  Cardiac arrhythmias Myocardial infarctions (“heart attack”) and stroke are usually acute events and are mainly caused by a blockage that stops blood from flowing to the heart or the brain. The most common cause is the formation of fatty deposits on the inner walls of blood vessels. Strokes may be caused by bleeding from a blood vessel in the brain (hemorrhagic stroke) or by a vessel blockade due to a blood clot (ischemic stroke). Rheumatic heart disease is caused by damage to the heart muscle and heart valves from rheumatic fever. Malformations of heart structures present at birth are known as congenital heart defects. They may be caused by: (i) a close blood relation between parents (consanguinity); (ii) maternal infections (e.g. rubella); (iii) maternal abuse of alcohol and drugs (e.g. warfarin); and (iv) poor maternal nutrition (e.g. deficiency of folic acid). In some cases the cause remains unknown. Examples of congenital heart disease include holes in the septum of the heart, abnormal valves and abnormalities in heart chambers (10). Other CVD, such as disorders of the electrical conduction system of the heart (e.g. cardiac arrhythmias), disorders of the heart muscle (e.g. cardiomyopathy) and heart valve diseases are less common than heart attacks and strokes. The costs of CVD are one of the higher, in the different group of diagnosis. Thus, prevention and minimization of the various risk factors, may be the most effective means of preventing clinical events during the life (11). 5 The American Heart Association (AHA) has established a new concept, the "ideal cardiovascular health”, which emphasizes seven positive behaviours and the factors that increase the likelihood of living free of CVD, and stroke in particular. This concept consists in: 1. Simultaneous presence of four healthy behaviours: a) abstinence from smoking in the last year; b) ideal body mass index (BMI); c) physical activity; d) a dietary pattern that promotes cardiovascular health; 2. Simultaneous presence of three factors: a) total cholesterol less than 200 mg/dL; b) arterial blood pressure lower than 120/80 mmHg; c) absence of diabetes mellitus; 3. No clinically established CVD (e.g., coronary artery disease, stroke). 1.2.1 Arteriosclerosis Arteriosclerosis is a general term for the thickening and hardening of arteries. Due to the decreased elasticity of the arterial wall, systolic blood pressure increases and the diastolic blood pressure decreases. Arteriosclerosis is almost universally present in the elderly and it is more prevalent in males (12). 1.2.2 Atherosclerosis Atherosclerosis is a type of arteriosclerosis. It is an inflammatory disease (13). Atherosclerotic lesions are often found in the aorta and major aortic branches. They are also common in the coronary arteries, where the condition is called “coronary artery disease” (also called coronary heart disease or ischemic heart disease). The underlying pathology is characterized by a chronic inflammatory process of the arterial wall which disturbed the blood laminar flow, particularly at the branch points (14). On initiation of an atherogenic diet, rich in cholesterol and saturated fat, one of the first ultra-structural alterations is the accumulation of small lipoprotein particles in the intima. The binding of lipoproteins to proteoglycan in the intima captures and retains these particles, accounting for their prolonged residence time. Lipoprotein particles bound to proteoglycan seems to exhibit increased susceptibility to oxidative or other chemical modifications, considered by many to be an important component of the pathogenesis of early atherosclerosis (2). The second morphologically definable event in the initiation of atheroma (a mass or plaque of degenerated thickened arterial intima, occurring in atherosclerosis) is leukocyte recruitment and accumulation. Under normal conditions, endothelial cells inhibit leukocyte adhesion to the vascular surface. However, early after initiation of hypercholesterolemia, 6 leukocytes adhere to the vascular surface and begin to accumulate lipids and transform into “foam cells”. Via secretion of cytokines, leukocytes penetrate the endothelial cell and enter the arterial wall (2). Whereas the early events in atheroma initiation involve primarily altered endothelial function and recruitment and accumulation of leukocytes, the subsequent evolution of atheroma into more complex plaques involves migration of circulating monocytes, T cells and smooth muscle cells from the media, leading to an accumulation of these cells within the vascular intima. In addition, smooth muscle cell death may also participate in complication of the atherosclerotic plaque (2). Plaques often develop areas of calcification as they evolve. Some subpopulations of smooth muscle cells may foster calcification by enhanced secretion of cytokines such as bone morphogenetic proteins, homologues of TGF-beta (2). These lesions (atheromatous plaques) enlarge as cells and lipids accumulate and begin to bulge into the vessel lumen (Figure 1). When the process continues, there is thinning of the fibrous cap accompanied by fissuring of the endothelial surface of the plaque, which may rupture. With the rupture of the plaque, lipid fragments and cellular debris are released into the vessel lumen. These are exposed to thrombogenic agents on the endothelial surface, resulting in the formation of a thrombus. If the thrombus is large enough, and a coronary blood vessel or a cerebral blood vessel is blocked, this results in a heart attack or a stroke (10). Atherosclerosis is clinically manifested in 10% of the population over 50 years, its development is slow and progressive, and significant arterial obstruction is needed, about 75% of the caliber of an artery, to lead to early ischemic symptoms (12). 7 Figure 1 Normal artery (left) and an artery affected by atherosclerosis (right). Certain drugs can reduce the risk associated with atherosclerosis. These include statins, which reduce the level of cholesterol and other fats in the blood, as well as anticoagulant drugs such as aspirin that prevent the formation of blood clots. In large arteries such as the aorta or carotid, the sections blocked by atheroma plaques may be surgically removed and replaced by synthetic materials. They can also be removed by atherectomy, in which fatty deposits are removed carefully using a blade inserted into the vessel through a catheter. In the case of completely obstructed coronary arteries, the lives of patients have been saved through coronary artery bypass surgery, in which sections of blood vessels from other parts of the body are used to divert blood flow around the blockages. Some occlusions can be opened by balloon angioplasty, in which a catheter is inserted into the site of the obstruction and a balloon is inflated in order to dilate the artery and flatten the deposits (atherosclerotic plaques) (15). 8 1.3 RISK FACTORS Risk factor is a particular condition which is associated with increased probability of an individual to suffer or die from a disease. The knowledge of risk factors allows then to predict, determine the aetiology, diagnose or prevent the disease (16). The rate of progression of atherosclerosis is influenced by cardiovascular risk factors. A classic classification of cardiovascular risk factors (CRF) is based on the possibility of the patient to modify them or not. The modifiable risk factors are those that can be changed through the adoption of new behaviours or lifestyles: hypertension (HT), diabetes mellitus (DM), hypercholesterolemia, dyslipidaemia, obesity, smoking, sedentary lifestyle, dietary habits, alcoholism, stress and others. The non-modifiable risk factors are those that do not depend on the will of the individual, so they cannot be changed, such as gender, age, previous personal history and family history. According to WHO, 75% of CVD can be attributed to modifiable risk factors (16). Table 1 Modifiable and non-modifiable risk factors for cardiovascular disease. Modifiable risk factors Non-modifiable risk factors Hypertension Gender Diabetes mellitus Age Hypercholesterolemia Previous history Dyslipidaemia Family history Obesity Smoking Sedentary Eating habits Alcoholism Stress 1.3.1 Age Of all the risk factors, age showed to have the strongest and most consistent association with CVD (17). Studies indicate that the incidence of stroke increases exponentially from 50-60 years and more than 80% of CVD mortality occurs in individuals older than 65 years (18, 19). As age advances, there is an increase in intimal thickening of arteries by diffuse accumulation of smooth muscle cells, connective tissue, decreased elastin and gradual increase in the stiffness of the vessels. The weakness of the arterial wall is recognized as a phenomenon that increases with age, especially after age 60 (17, 18). 9 1.3.2 Hypertension Worldwide, approximately 62% of strokes and 49% of cases of coronary heart disease (CHD) are attributed to a higher blood pressure (> 115 mmHg systolic), a factor that accounts for more than 7 million deaths per year (2). According to the study by Lawes et al. (20), it is estimated that about 14% of deaths and 6% of disability-adjusted life years (DALY) 1 are caused by a non-optimal blood pressure. When blood pressure rises, the resistance to the expulsion of the blood increases and the heart, to move the same volume of blood, is subject to a larger effort. To address this situation the heart has adaptation mechanisms, which is the cardiac hypertrophy. The heart is able to gradually increase the thickness of its walls, thereby increasing the force with which it contracts. However, there are limits, and permanently increased arterial resistance causes the heart to fail after a few years, exceeding its resilience and adaptability. Then, heart begins to dilate and stops working as efficiently, moving into a period of incapacity to promote adequate blood flow to the body's needs, thus resulting in heart failure. This mechanism is just one of the dangers of hypertension. The other one is the fact that hypertension functions as risk factors for atherosclerosis. Thus, excessive pressure in the arteries favors the deposition of fat in the walls and the consequent development of atherosclerosis. And the greater the values of blood pressure, especially the "minimum" (diastolic), the higher is the probability of appearance of lesions and their complications, the most common being the stroke and heart attacks, as mentioned above. If hypertension is associated with other factors (smoking habits, increased blood fats, diabetes, etc.) the risk increases much more (21). Blood pressure levels have been shown to be positively and progressively related to the risk of stroke and coronary heart disease. In some age groups, the risk of CVD doubles for each increase of 20/10 mmHg on blood pressure, starting as low as 115/75 mmHg. In addition to coronary heart disease and cerebrovascular disease, uncontrolled blood pressure causes heart failure, renal impairment, peripheral vascular disease, damage to retinal blood vessels and visual impairment (10). The stroke and heart attack risk of people with high cardiovascular risk and/or raised blood pressure can be reduced through non-pharmacological (e.g. low salt diet, physical activity) and pharmacological measures. These measures are particularly important for people with diabetes, as they are also particularly vulnerable to heart attacks and strokes 1 In practice, the sum of years lost due to premature mortality and years lived with disability, adjusted for the severity of the disease. 10 (10). Policies to reduce salt consumption can shift the population distribution of blood pressure so that there is a reduction in cardiovascular risk. 1.3.3 Tobacco Smoking is estimated to cause nearly 10% of CVD. There is a large body of evidence from prospective cohort studies regarding the beneficial effect of smoking cessation on coronary heart disease mortality. A 50-year follow-up of British doctors demonstrated that, among ex-smokers, the age of quitting has a major impact on survival prospects: those who quit between 35 and 44 years of age had the same survival rates as those who had never smoked (10). Tobacco use is the leading preventable cause of death. Almost 6 million people die from tobacco use and exposure each year, accounting for 6% of all female and 12% of all male deaths in the world. “Second hand smoke” is also well established as a cause of coronary heart disease (2). Tobacco is certainly incriminated as a causative factor for atherosclerosis. The combined action of nicotine and carbon monoxide results in the onset of atherosclerosis, especially coronary atherosclerosis. Nicotine causes increased heart work and carbon monoxide decreases the oxygen available, which leads to the possibility of arrhythmias and sudden cardiac death (21). 1.3.4 Dietary habits There is a considerable body of evidence regarding the nutritional background of atherosclerosis in general and coronary heart disease in particular. High dietary intakes of saturated fat, cholesterol and salt, and low intake of fruits, vegetables and fish are linked to cardiovascular risk (10). Worldwide, high levels of cholesterol cause about 56% of ischemic heart disease and 18% of stroke, a total of 4.4 million deaths annually. Changes in lifestyle that accompany urbanization clearly play an important role, since plasma cholesterol levels tend to be higher in the urban than in the rural population. These changes are largely due to the increased dietary consumption of fat, especially animal products and processed vegetable oils, and decreased physical activity (2). The amount of dietary salt consumed is an important determinant of blood pressure levels and overall cardiovascular risk. In order to help the prevention of CVD, WHO recommends 11 a population salt intake of less than 5 grams/person/day. Adequate consumption of fruit and vegetables reduces the risk of CVD (10). High consumption of saturated fats and trans-fatty acids is linked to heart disease; elimination of trans-fat and replacement of saturated with polyunsaturated vegetable oils lowers coronary heart disease risk (10). The increase of cholesterol and lipid levels in blood contributes to the development of atherosclerosis and thus coronary disease. It is the increase in blood cholesterol that leads to the penetration and deposition in the inner layers of the arterial wall, this phenomenon being greater the higher is the concentration of cholesterol in blood (21). Thus, the number of people who fall ill and die from coronary disease correlates perfectly with cholesterol values. Individuals with a cholesterol level higher than 300 mg/dL have a risk of developing CVD four times higher than individuals with cholesterol concentrations below 200 mg/dL (21). A healthy diet can contribute to a healthy body weight, a desirable lipid profile and a desirable blood pressure (10). 1.3.5 Obesity Obesity is a cardiovascular risk factor closely linked to diet and physical inactivity. Obesity results when there is an imbalance between energy intake in the diet and energy expenditure. Regular physical activity can prevent obesity by increasing the expended energy (10). Worldwide, at least 2.8 million people die each year as a result of being overweight or obese, and an estimated 35.8 million (2.3%) of global DALYs are caused by overweight or obesity (10). Obesity is a major risk factor for CVD in adults (4). Overweight and obesity are associated with numerous co-morbidities, such as CVD, type 2 diabetes and certain cancers. The rising prevalence of obesity represents a global public health issue, with an estimated 30% of CHD and ischaemic stroke and almost 60% of hypertensive disease in developed countries being attributable to increased BMI (4). CHD is more frequent in obese people, and the risk increases with the degree of overweight. Obesity is often associated with high blood pressure, diabetes and increased levels of fats and uric acid in the blood. The very marked obesity also causes other cardiac problems. In particular, it requires a permanent increase of heart work that leads to hypertrophy, dilatation and onset of heart failure (21). 12 To achieve optimal health, the median BMI for adult populations should be in the range of 21-23 kg/m2, while the goal for individuals should be to maintain a BMI in the range 18.524.9 kg/m2. 1.3.6 Diabetes Diabetes is a major risk factor for CVD. Diabetes is defined as having a fasting plasma glucose value ≥7.0 mmol/L (126 mg/dL). Impaired glucose tolerance and impaired fasting glycaemia are an important risk for future development of CVD (10). DM affects approximately 180 million people worldwide, and it is estimated that this number could double by 2030. Most cases are in the age group of 45 to 64 years in developing countries, while in developed countries the prevalence is higher in individuals over 65 years (2). Increased levels of glucose are also a factor responsible for atherosclerosis. Atherosclerosis is not only very common in diabetics as they have earlier and more extensive lesions than non-diabetics. Atherosclerotic disease in diabetics is often widespread, premature and severe, leading to stroke, disturbed circulation in the legs and changes in vision. It is believed that sugar acts directly or by favouring the formation of fat in the body and its penetration into the arterial wall (21). Cardiovascular risk increases with raised glucose values. Furthermore, abnormal glucose regulation tends to occur together with other known cardiovascular risk factors such as central obesity, elevated blood pressure, low high density lipoprotein (HDL) cholesterol and high triglyceride levels (10). 1.3.7 Hyperlipidaemia The lipoprotein profile includes: (i) low density lipoprotein (LDL) cholesterol, also called “bad” cholesterol); (ii) HDL, also called “good” cholesterol; and (iii) very low density lipoprotein cholesterol. LDL cholesterol is deposited in the walls of arteries and causes atherosclerosis. In general, low LDL cholesterol levels are better for vascular health. HDL cholesterol protects against vascular disease by removing the LDL cholesterol out of the wall of arteries. Excess calories are converted into triglycerides and stored into fat cells throughout the body. High triglycerides also increase the risk of atherosclerotic CVD (10). 13 1.3.8 Oral contraceptives Oral contraceptives increase the risk of venous thrombosis, stroke and myocardial infarction. Although controversial, it has been accepted that the use of oral contraceptives is really another factor that increases the risk of atherosclerosis (21). 20 with high occupational exposure have reported an association between Pb exposure and markers of oxidative stress (51). Epidemiological studies have reported that low-level Pb exposure has an association with several diseases such as hypertension and peripheral arterial disease (52, 53). 1.4.4 Selenium The trace element Se is a non-metallic essential nutrient of fundamental importance to human biology. The biological role of Se in mammals, including man, is mainly attributed to the presence of selenocysteine at each of the four catalytic sites of the enzyme glutathione peroxidase (GPx). This enzyme uses glutathione to reduce peroxides in the cell and, this way, protects the lipid membranes and possibly proteins and nucleic acids from damage caused by free radicals and other oxidative agents. The need for Se is related to the degree of oxidative activity and the supply of nutrients such as Zn, Cu, Mn, Fe and vitamin E (26). The most extensively documented deficiency of Se in humans is Keshan disease, an endemic cardiomyopathy first discovered in Keshan County, People's Republic of China in 1935. The disease is clinically characterized by varying degrees of cardiomegaly and cardiac decompensation. The histopathologic exam of the myocardium shows degeneration and necrosis of myocardial fibers and their replacement by fibrosis and areas of healing. Se deficiency is also one of the factors that influence the risk of hypertension and CVD. Furthermore, myocardial ischemia can also be worsened in case of Se deficiency (54). Experimental studies suggest that Se may reduce CVD risk via several mechanisms (Table 2). For example, antioxidant defences may reduce vascular and tissue injury resulting from formation of ROS due to shear stress, hypoxia, hypertension, hyperlipidemia or diabetes. Systems related to Se may also decrease the oxidation of lipids and protect the vascular endothelium from damage due to oxidized LDL particles. In animal studies, Se consumption has shown to increase cardiomyocyte GPx activity, to improve cardiac recovery from ischemia-reperfusion and to reduce the size of myocardial infarction (55). 21 Table 2 Experimentally-observed effects of selenium that may reduce CVD risk. (55) Systemic effects Direct cardiovascular effects Antioxidant defence against free radicals and reactive oxygen species Increased myocardial antioxidant glutathione peroxidase activity Decreased lipid peroxidation Improved cardiac recovery from ischemiareperfusion injury Protection against vascular damage from oxidized LDL particles Limitation of ischemia-induced and diabetesinduced ultrastructural damage Antithrombotic effects from decreased plasma thromboxane A2 Reduction in myocardial infarct size Restoration of altered myocyte ion currents Reduced incidence of ischemia-induced ventricular arrhythmias 1.4.5 Zinc Zn is an essential trace element that is vital in maintaining normal physiology and cellular functions. Normal Zn levels in plasma are in the range of 70 to 120 mg/dL. Low levels of Zn (“deficiency”) are usually defined as Zn plasma levels lower than 60 mg/dL (56). The absorption of Zn from the gastrointestinal tract is homeostatically regulated. About 2030% of ingested Zn is absorbed into systemic blood circulation. Zn uptake from the intestinal lumen involves passive diffusion and a carrier-mediated process through specific Zn transporters, such as ZnT-1. Intestinal absorption of Zn can be reduced by dietary fiber, phytates, Ca and phosphorus, while amino acids, picolinic acid and prostaglandin E2 can enhance Zn absorption. Once absorbed, Zn is widely distributed throughout the body. The total content of Zn in the human body is in the range of 1.5-3 g. Most of this is found in muscle (60%), bone (30%), skin/hair (8%), liver (5%) and pancreas (3%). The highest concentrations of Zn are found in prostate, pancreas, liver and kidney. In plasma, Zn is mostly bound to albumin (60-80%), which represents the metabolically active pool of the element. The remainder is bound to α2-macroglobulin and transferrin. Zn is excreted both by urine and feces. The concentration of Zn in plasma is not a sensitive indicator of Zn status and does not reflect Zn levels in the tissues and its effects at the various target sites. Zn ions are involved as interand intracellular messengers and the homeostasis of Zn has to be tightly controlled (26). Zn is an effective inducer of Mt synthesis and, when 22 Mt become saturated in intestinal cells, Zn absorption is decreased. Mt are also an important intracellular Zn storage (26). Zn is found in more than 200 metalloenzymes, including acid phosphatase, alkaline phosphatase, alcohol dehydrogenase, carbonic anhydrase, superoxide dismutase and DNA and RNA polymerases. Zn contributes to gene expression and chelates with either cysteine or histidine in a tetrahedral configuration, forming looped structures know as Zn fingers, which bind to specific DNA regions. Other functions of Zn include membrane stabilization, vitamin A metabolism and the development and maintenance of the nervous system. Zn and Cu concentration generally have an inverse relationship in the serum, with elevated Zn concentrations resulting in decreased Cu concentrations (33). It is important to note that, although Zn ions per se are redox inert, they have profound effects on redox state and, conversely, redox state has a profound effect on Zn metabolism. Increased oxidative stress can release Zn from its binding sites, where it performs coordination function, as reported in a number of proteins (Fig. 3). Zn ions released from proteins may potentially act on signal transduction pathways, modify mitochondrial metabolism, and can affect the redox status of the cell. Zn at various concentrations can increase the cell’s antioxidant capacity or the release of toxic ROS (56). Figure 3 Chemical background for the flexibility of zinc ion in changing the redox environment (redox zinc switch). Zinc is coordinated in a reduced sulphur-containing protein domain and is released under oxidation. Numerous studies have explored the association of Zn with cardiomyopathies, arrhythmias and coronary diseases. Several investigators have shown decreased blood Zn levels in patients with ischemia/myocardial infarction, congestive heart failure, conduction abnormalities and heart transplant, resulting in poor outcomes (56). 23 The 'response to injury' hypothesis of atherosclerosis by Ross and Glomset (60) states that atherosclerosis begins with endothelial cell injury. It is now known that endothelial cells undergo apoptosis, possibly as a result of increased oxidative stress from oxidized LDL. Evidence suggests that Zn may be protective by maintaining the integrity of endothelial cells and thus reducing vessel susceptibility to atherosclerosis. Therefore, deficiency of Zn may corollary promote endothelial cell injury. A mediator of inflammatory responses in many cells is nuclear factor-kB (NF-kB), a transcription factor that regulates the gene expression associated with apoptosis and inflammation. The NF-kB binding to DNA is dependent on Zn and, consequently, the NF-kB transcriptional activity is regulated by Zn. In endothelial cells the Zn ionophore pyrithione inhibits the regulatory activity of NFkB, thereby inhibiting the inflammatory process responsible for the atherosclerotic process (56). Figure 4 Role of zinc in cardiovascular diseases. (56) Zn has not only an influence in the atherosclerotic process but also in other CVD like arrhythmias and myocardial infarction. In addition, Zn is involved in insulin signalling and the pathogenesis of diabetes, further indicating the potential role in diabetic cardiomyopathy. Zn supplementation also protects cardiomyocytes from acute redox stress and prevents inflammatory processes that are triggered during myocardial damage. Zn is also a wound-healing agent, and it is believe that it may support survival of cardiac stem cells that are essential components of cardiac healing. Some studies seem to corroborate the hypothesis that Zn may have an antiatherosclerotic role in both animals (57-59) and humans (60). 24 1.5 TRACE ELEMENT ANALYSIS As previously discussed, there is considerable evidence supporting the hypothesis that atherosclerosis involves chronic inflammation and oxidative stress, although the origin of oxidation is still not fully established. Various substances, including transition metal ions have been suggested as sources of oxidizing species in atherosclerotic lesions (5). Heavy metals ions are shown to be present at high levels in some animal models and also in human atherosclerotic plaques, which are in agreement with the hypothesis that metal ions contribute to the formation of plaque as well as for their destabilization (8, 61, 62). Many chemical elements occur in biological matrices at so low levels that they could not be detected at the beginning of the development of instrumental analysis (63). However, the extraordinary development of analytical techniques observed in last decades made possible a reliable quantification of these chemical species (“trace elements”), and contributed to the current understanding of the importance of these elements in human health and disease. One of the first important contributions to metal analysis in clinical chemistry was the introduction of the flame photometer, based on atomic emission spectrometry, which significantly improved Na and K determination (64). Later, the development of atomic absorption spectrometry (AAS) with flame atomization allowed faster determinations of several important metals (e.g., Ca, Mg, Fe, Cu, Zn), with great accuracy and precision, in small sample volumes. However, the range of analyzable metals was low due to its poor sensitivity (high detection limits). This limitation was overtaken in the 1970s with the introduction of the graphite furnace as atomization system, which greatly improves the efficiency of the atomization process (65). The development of atomic absorption spectrophotometry with graphite furnace (or electrothermal) atomization allowed to decrease by about 10 to 100 times the limits of detection for most metallic elements, making possible its quantification at the parts per billion (ppb) level (66). In practice, all spectroscopic elemental techniques, with different requirements regarding sample preparation and with an applicability dependent on the sensitivity and selectivity needed, can be used for the determination of trace elements in biological samples. Highlight for AAS, in its different atomization modes (flame atomization, electrothermal atomization and hydride generation), inductively coupled plasma-atomic emission spectrophotometry (ICP-AES), X-ray fluorescence and inductively coupled plasma-mass spectrometry (ICP-MS) (67). 25 In particular, ICP-MS is currently the “golden standard” for trace element analysis. The potential of ICP-MS in the determination of trace elements in various fluids (whole blood, plasma, serum, urine) and biological tissues is extensively illustrated in the literature (68). The combination of excellent metrological characteristics (very low limits of detection, typically in the order of 10-4-10-5 g/L, wide dynamic range, of nine orders of magnitude, and high precision) with very rapid multi-elemental analysis capability is the basis of the high potential of this analytical technique. This last feature (the ability to fast multielemental analysis), in particular, makes ICP-MS a very important tool in the evaluation of the interactions and reciprocal effects of trace elements in the human body (67). A mass spectrometer comprises three main components: (i) an ion source (the plasma, in ICP-MS), which converts sample components in ions (mainly mono-elemental and positive, monovalent) in the gas phase; (ii) one (or more) mass analyzer, which separates the ions (ionized analytes) based on their mass-to-charge ratio (m/z); and (iii) a detector that counts the number of ions of each m/z value, producing a mass spectrum. The use of a high temperature plasma (6000-10000 ºC) as ion source for elemental mass spectrometry has been exploited over the last decades of the twentieth century, and the more successful combination was ICP-MS. After its commercial introduction in the early 1980s (69), ICP-MS has become the leading technique for trace element analysis, allowing the fast determination of more than 70% of the elements of the periodic table. This stems from the fact that, due to the high temperature of the plasma, ionization and subsequent atomization is highly effective, i.e., a complete fragmentation of molecules in its constituent atoms and subsequent ionization occurs in a very large extent (70). Figure 5 Diagram of ICP-MS instruments (71). 26 One of the major problems in the analysis of biological fluids and tissues is the possible contamination of the samples at all stages of the analytical procedure (from sampling and preservation to sample pre-treatment and final instrumental analysis). It is well known that even slight hemolysis can increase the concentration of Fe, Cu, Zn, Pb, Rb, Mn and Mg in serum from several tens to several hundred percent. With the use of needles for sampling, the blood may be contaminated with Al, Cr, Ni and Zn. Glassware is another source of contamination, and is preferable to use plastic materials. The use of anticoagulants itself (heparin, EDTA) can contaminate the samples at the stage of sampling (67). The complex matrix of biological samples in most cases requires a step of sample preparation. Depending on the circumstances, sample preparation can range from a simple dilution of the sample to a thermal mineralization or an acid digestion in a microwave oven (67). The ideal process for solubilization of samples should be able to solubilize the sample completely, leaving no residue, should be reasonably fast and reagents used should not interfere with the subsequent determination or should be easily removable. It must also be taken into account that the reagents used should be available in high purity grade (to not contaminate the sample), and losses of analyte(s) must be insignificant. Together with these criteria, it must be taken into account that the reactants/reaction products should not attack the containers where solubilisation procedure is carried-out, and this should be a safe procedure. There are essentially two methods of solubilisation of samples: "dry ashing" (incineration, ashing; dry mineralization) and "wet ashing" (digestion; wet mineralization). The first is the classical method used to mineralize large amounts of samples with high organic content (e.g. biological samples, solid waste, sediments). The samples are heated at elevated temperatures (in a furnace) to destroy organic matter. The ashes are then treated as if they were a simple inorganic sample. This method has several limitations, such as the possibility of losing some of the more volatile elements, some ashes can be difficult to solubilize completely and there is a great potential for sample contamination. In the second case (wet digestion) the solubilisation is performed by using concentrated strong acids (e.g., HClO4, HNO3, HCl, H3PO4, H2SO4, HF) and/or other reagents such as H2O2, which lead to the decomposition of organic matter. This procedure can be done in two different ways: a) In open vessels – by simply bringing the mixture [acid(s) + sample] to boiling in a beaker covered with a watch glass; or b) in closed vessels – in a microwave oven, where microwave radiation provides the energy to heat the mixture. Although it can only be used for small sample masses (up to 1 g), the last one is a very efficient (because 27 it occurs at high pressures/temperatures) and safe solubilization process (it minimizes losses and contamination). The efficiency of wet (acid) digestion in a microwave oven depends on the nature of the sample, the adjustment of heating time, pressure and temperature, the mass of the sample and the type of acids used. Among these parameters, the choice of acid(s) is very important. Several concentrated acids such as nitric acid, hydrochloric acid, sulfuric acid or a mixture of these acids have been widely used to solubilize samples (68). The microwave heating is very efficient as heat is developed within the mixtures [sample + reagent (s)] and is not "driven" through the vessel walls as it happens when the sample is put into a vessel over an electric plate. Microwave heating also produces a very uniform heating. The acid digestion in closed vessels in a microwave oven has become the most important procedure for organic samples solubilization. As already highlighted, this procedure minimizes the amount of reagents required and, as a result, a reduced possibility of contamination (and the “sample blank” value). There is also a significant decrease in time spent on this task – the decomposition of the samples occurs at higher temperatures, which greatly increases the reaction rate. Importantly, the use of closed vessels makes it possible to avoid uncontrollable losses of trace elements, which inevitably occur when solubilisation is made in open vessels. This is a very important issue, especially when determining trace elements that form hydrides and/or volatile halides, such as As, B, Cr, Hg, Sb, Sn and Se (67). 28 CHAPTER 2 OBJECTIVES 29 2 OBJECTIVES The link between trace element imbalances and CVD is not fully understood. Most of the existing studies on this subject are simply based on the analysis of body fluids (blood, serum, plasma, urine), since they can be easily obtained. From a theoretical point of view, a potentially more conclusive approach could be the direct study of trace elements changes in the target organs and tissues, and this was the rational for the study performed. The study focused on five trace elements: Zn, Cu, Se – three elements with a wellrecognized “essentiality”, needed for very important physiological functions; Cd and Pb – two important "toxic" trace elements, whose role in atherosclerosis and CVD is long suspected. Basically, we aimed to directly look, in the tissues themselves, for evidences of significant changes in trace elements levels that could be related to CVD. For a better understanding of these changes, the age-related changes in “normal” (nondiseased individuals), the gender-related differences and the effect of smoking habits were also assessed. 36 CHAPTER 4 RESULTS AND DISCUSSION 37 4.1 KIDNEY 4.1.1 Medulla a) All the samples Table 4 Concentration (µg/g) of elements in kidney medulla. Cu Zn Se Cd Pb N 57 57 53 57 57 Mean 6.72 115.7 3.97 41.37 0.26 SD 3.98 86.48 2.18 38.72 0.21 Median 6.29 89.74 3.86 31.36 0.19 Minimum 1.28 16.93 0.81 4.47 0.04 Maximum 15.82 437.4 9.05 210.7 1.01 b) Men vs Women In medulla, no significant difference was found between genders for Cd, Cu, Se and Zn (Tables 5 and 6). Most of the available literature is focused on the metal content of kidney cortex. Results for renal medulla are very scarce. Lower Zn and Se levels have been found in women blood serum (74), but apparently this is not reflected in the content of renal medulla tissue. Table 5 Concentration (µg/g) of elements in kidney medulla in men. Men Cu Zn Se Cd Pb N 35 35 34 32 35 Mean 6.69 121.8 4.60 36.95 0.32 SD 4.19 97.15 5.78 23.79 0.23 Median 6.29 84.42 3.66 34.74 0.27 Minimum 1.28 16.93 0.81 4.47 0.05 Maximum 15.82 437.4 35.32 83.90 1.01 38 Table 6 Concentration (µg/g) of elements in kidney medulla in women. Women Cu Zn Se Cd Pb N 18 18 17 17 18 Mean 7.02 109.3 4.80 24.72 0.16 SD 3.82 68.30 2.88 14.10 0.10 Median 6.56 106.8 4.03 26.53 0.14 Minimum 1.47 21.31 1.55 5.57 0.04 Maximum 13.97 301.0 11.90 54.96 0.46 For Pb, a higher concentration was found in men (0.32±0.23 versus 0.16±0.10 in women; p≤0.01) (Figure 7). In general, men have higher blood Pb levels than women. Pb in blood is bound to hemoglobin, inside the erythrocytes. Therefore, since men have higher hematocrit values (and higher haemoglobin values), it is expected that higher Pb levels are also found in men (75). Our findings may also be due to a higher percentage of smokers in the men sub-group (35,5% vs 13,6% in the women sub-group), and smoking is an important source of human exposure to Pb (29). M e n W om e n 0 .0 0 .2 0 .4 0 .6 [P b ] (g /g ) * * Figure 7 Lead levels (g/g) in kidney medulla in men (n=35) and women (n=18), **p ≤0.01 39 c) Smokers vs Non-smokers Results for Cu, Zn, Se, Cd and Pb in the renal medulla tissue of smokers and nonsmokers are shown in next tables. Table 7 Concentration (µg/g) of elements in kidney medulla of smokers. Smokers Cu Zn Se Cd Pb N 13 13 11 13 13 Mean 7.46 166.7 3.26 75.97 0.39 SD 4.71 112.1 1.77 61.04 0.19 Median 6.66 146.7 2.66 52.00 0.33 Minimum 1.28 24.53 0.81 7.03 0.14 Maximum 15.82 437.4 6.05 210.7 0.70 Table 8 Concentration (µg/g) of elements in kidney medulla of non-smokers. Non-smokers Cu Zn Se Cd Pb N 28 28 28 28 28 Mean 6.16 91.46 4.06 27.78 0.21 SD 3.60 63.47 2.51 20.24 0.20 Median 5.83 76.15 3.61 20.87 0.16 Minimum 1.44 16.93 1.16 4.47 0.05 Maximum 13.97 301.0 11.90 80.29 1.01 Plants have evolved physiological mechanisms to gain access to essential elements from the soil. On the other hand, plants can accumulate toxic metals, which is an ability used by modern biotechnology to remove metals from contaminated soils. However, regarding Nicotiana tabacum farming for cigarette production, this ability of tobacco plant becomes a health problem. Toxic metals such as Cd, Pb, Hg and Ni are found in tobacco, and consequently in cigarette smoke (29). Recent data suggests that smoking interferes with the metal homeostasis of the human body and plays a crucial role in the pathogenesis of a number of diseases. It was demonstrated that metals present in cigarette smoke are essential in the process that led to the injury of the vascular endothelium (76). And as already pointed out, the dysfunction 40 and disruption of vascular endothelium is the primary event in the genesis of atherosclerosis (29). We found a significant difference between smokers and non-smokers regarding the kidney medulla levels of Cd, Zn (Figure 8) and Pb (Figure 9). The average content of Zn in cigarettes has been estimated in 24 µg/g and about 70% is transferred to the smoke. Studies regarding the relationship between plasma Zn concentration and smoking habits found no differences in Zn levels between smokers and non-smokers (74, 77). In our study, however, a significantly higher Zn content in the renal medulla of smokers (166.7±112.1 µg/g versus 91.46±63.47 µg/g in non-smokers; p≤0.01) was found (Figure 7). It seems that excretion by the kidney tends to increase to remove the excess of Zn inhaled via smoke. Smokers also showed significantly higher levels of Cd (75.97±61.04 µg/g vs 27.78±20.24 µg/g in non-smokers) (Figure 8). This is in accordance with Scott et al. (78), who also found increased levels of Cd in smokers kidney tissue. Cd Zn 0 100 200 300 (g /g ) S m okers N on S m okers *** * * Figure 8 Cadmium and zinc levels (µg/g) in kidney medulla of smokers (n=13) and non-smokers (n=28); **p≤0.01, ***p≤0.001 The Pb content of a cigarette has been estimated around 1.2 µg, and about 6% of this total amount is thought to pass over to mainstream smoke, which is further inhaled by smokers (29). Navas-Acien et al. (53) and Mortada et al. (79) reported higher Pb levels in smokers blood. In our study, Pb was significantly higher in the smokers’ renal medulla: 0.39±0.19 µg/g versus 0.21±0.20 µg/g in non-smokers (Figure 9). 41 S m ok ers Non S m ok ers 0 .0 0 .2 0 .4 0 .6 0 .8 [P b ] (g /g ) * Figure 9 Lead levels (g/g) in kidney medulla of smokers (n=13) and non-smokers (n=28); *p≤0.05 d) Differences by area of residence Because of the large inter-individual variability, different number of smokers and unequal distribution of subjects across the different geographical areas, it is not possible to draw significant conclusions about the influence of the area of residence in the trace element levels in kidney medulla. However, individuals from Gondomar showed lower Cu levels in kidney medulla (without reaching statistical significance) (Figure 10) and Zn (p≤0.05) (Figure 11). Individuals from Santo Tirso and Vila do Conde showed lower concentrations of Cd (Figure 11). 42 Figure 10 Average concentration (µg/g) of Cu, Se and Pb in renal medulla according to individuals’ area of residence. Figure 11 Average concentration (µg/g) of Zn and Cd in renal medulla according to individuals’ area of residence. 43 4.1.2 Cortex a) All the samples Table 9 Concentration (µg/g) of the elements in kidney cortex. Cu Zn Se Cd Pb N 56 56 51 56 56 Mean 7.52 166.1 4.83 72.57 0.34 SD 4.10 100.2 2.54 45.43 0.39 Median 7.61 144.9 4.84 67.97 0.26 Minimum 1.68 31.54 1.47 0.03 0.02 Maximum 16.63 409.5 12.30 198.5 2.56 b) Men vs Women No significant difference was found between genders for any of the five analysed elements in kidney cortex (Tables 10 and 11). These results are in close agreement with those found in other studies where no significant gender-related differences were also found regarding Pb, Cd, Cu, Se and Zn levels in the kidney (80-82). Table 10 Concentration (µg/g) of elements in kidney cortex of men. Men Cu Zn Se Cd Pb N 33 33 29 33 32 Mean 7.20 168.05 4.34 78.54 0.35 SD 4.28 105.46 2.14 47.35 0.30 Median 5.68 139.54 4.06 75.42 0.28 Minimum 1.68 31.54 1.47 18.87 0.02 Maximum 16.63 409.48 9.88 198.49 1.20 44 Table 11 Concentration (µg/L) of elements in kidney cortex of women. Women Cu Zn Se Cd Pb N 20 20 19 20 20 Mean 8.25 171.7 5.69 68.92 0.25 SD 3.87 94.88 2.99 42.97 0.16 Median 8.79 154.0 4.90 60.83 0.25 Minimum 2.34 48.97 1.68 0.03 0.04 Maximum 12.90 403.0 12.30 179.5 0.63 c) Smokers vs Non-smokers No significant differences were found between smokers and non-smokers regarding Cu, Zn, Se and Zn levels in kidney cortex (Tables 12 and 13). Studies on the concentration of Cu in serum (77, 83) showed higher levels of this element in smokers. Since free Cu2+ ions are potent catalysts in Fenton reaction (Figure 2), these observations might indicate and increased risk of oxidative stress. No clear evidence exists about the influence of smoking in Zn levels. Garcia et al. (80) didn’t found a significant difference in Zn levels between smokers and non-smokers, but Blanusa et al. (84) found higher levels in smokers. Table 12 Concentration (µg/g) of elements in kidney cortex of smokers. Smokers Cu Zn Se Cd Pb N 14 14 12 14 14 Mean 8.31 210.0 4.28 110.1 0.43 SD 4.65 103.9 1.87 37.74 0.40 Median 9.39 195.1 4.72 110.6 0.280 Minimum 1.98 66.59 1.87 48.09 0.02 Maximum 16.63 409.5 8.09 179.5 1.20 45 Table 13 Concentration (µg/g) of elements in kidney cortex of non-smokers. Non Smokers Cu Zn Se Cd Pb N 29 29 26 29 29 Mean 7.49 159.9 5.91 51.06 0.34 SD 3.85 103.6 2.98 27.62 0.46 Median 7.67 124.8 6.20 44.74 0.27 Minimum 2.15 31.54 1.68 0.03 0.04 Maximum 15.94 403.0 12.30 110.7 2.59 Cd showed significant higher levels in smokers (110.1±37.74 µg/g versus 51.06±27.62 µg/g in non-smokers) (Figure 12). This is in close agreement with other studies on the influence of smoking habits in the Cd content of kidney cortex (81, 82, 84). Smoking is assumed to be the main source of human exposure to Cd. Although the amounts of Cd varied, the average content of this element has been found between 0.5 and 1.5 µg per cigarette (29). When the cigarette is smoked, Cd is transformed into CdO, which is then inhaled. It is estimated that approximately 10% of the Cd inhaled is deposited in the lungs, and 20-50% of this amount is transferred to the blood circulation. Cd not only increases in the circulation, but also accumulates in kidney, mainly in the kidney cortex, where Mt chelate and immobilize it. As already stressed, studies have shown that Cd accumulation in kidney causes tubular dysfunction and renal failure, but there is also some evidence that the amount of Cd released with the smoke is not enough to cause kidney failure (29). 52 c) Smokers vs non smokers Table 22 Concentration (µg/g) of the elements in aorta with plaque of atheroma of smokers. Smokers Cu Zn Se Cd Pb N 10 10 9 10 10 Mean 3.47 78.44 3.93 0.91 1.84 SD 1.03 32.25 1.62 1.63 1.69 Median 3.49 70.09 3.77 0.16 1.29 Minimum 1.75 39.67 1.81 0.01 0.09 Maximum 5.09 147.2 7.19 5.23 5.83 Table 23 Concentration (µg/g) of the elements in aorta with plaque of atheroma of nonsmokers. Non-Smokers Cu Zn Se Cd Pb N 20 20 20 17 20 Mean 3.36 126.4 3.33 0.47 3.51 SD 2.94 66.60 1.56 1.29 2.67 Median 2.50 106.8 3.65 0.07 3.06 Minimum 1.10 40.88 0.52 0.01 0.10 Maximum 14.69 350.2 7.10 23.28 9.45 A significant difference was found for Zn levels in aorta with plaque. Smokers showed a lower Zn concentration: 78.44±32.25 µg/g vs 126.5±66.60 in non-smokers (p≤0.05) (Figure 16). The mechanism through which cigarette smoke reduces Zn levels is the aorta with plaque of atheroma is thought to be the result of an increased blood Cd concentration, which induces the expression of Mt. Mt complexes not only bind Cd but also Zn, and later this complexes accumulate in the renal cortex (29). 53 S m ok e rs Non S m ok e rs 0 5 0 100 150 200 250 [Z n ] (g /g ) * Figure 16 Zinc levels (g/g) in aorta tissue with plaque of atheroma of smokers (n=10) and non-smokers (n=20); *p≤0.05 d) Differences by area of residence As also observed in renal medulla, individuals from Santo Tirso and Vila do Conde showed lower concentrations of Cd in aorta tissue (with and without plaque) (Figure 17). Figure 17 Average concentration (µg/g) of Cu, Cd, Pb and Se in aorta according to individuals’ area of residence. 54 Figure 18 Average concentration (µg/g) of Zn in aorta according to individuals’ area of residence. 4.2.3 Aorta tissue with and without plaque of atheroma Significant differences were found between both aorta tissues (i.e., with and without plaque of atheroma) for Zn and Se. For Zn, significantly higher concentrations were found in aorta tissue with plaque of atheroma: 111.0±58.42 µg/g vs 89.74±28.45 µg/g in aorta without plaque (Figure 19). Aorta w ithout pla que Aorta w ith pla que 0 5 0 100 150 200 [Z n] (g /g ) * Figure 19 Zinc levels (g/g) in aorta tissue without plaque of atheroma (n=52) and aorta tissue with plaque of atheroma (n= 41); *p≤0.05 55 Our results are in agreement with those obtained by Tohno et al. (86), who studied the human thoracic aorta and found a lack of correlation between Zn and some independent markers of protein oxidation (tyrosine nitration and thiol oxidation), suggesting that Zn is unlikely to protect against transition metals induced lipoprotein oxidation. Moreover, a highly significant correlation between Zn and Ca in all lesions was observed. It seems that high Zn levels may merely be an indicator of Ca accumulation and fibrosis. Regarding Se, we found a significantly higher concentration of this element in the aorta tissue without plaque of atheroma (4.71±2.33 µg/g) when compared with the aorta tissue with plaque of atheroma (3.85±2.50 µg/g) (Figure 20). Aorta w ithout pla que Aorta w ith pla que 0 2 4 6 8 [S e ] (g /g ) * * Figure 20 Selenium levels (g/g) in aorta tissue without plaque of atheroma (n=45) and in aorta tissue with plaque of atheroma (n=36); **p≤0.01 The higher Se concentration in aorta without plaque seems to confirm that Se is a cardioprotector element (87). In a study by Lubos et al. (88), the plasma Se concentration of individuals with acute coronary syndrome was inversely associated with cardiovascular mortality, independently of classical risk factors. The exact mechanism through which low plasma Se levels act in CVD is not yet known. Some authors have pointed out that low Se concentrations in atherosclerosis may facilitate the formation of lipid hydroperoxides, which could attack vascular endothelium. Other authors have argued that Se modifies prostaglandin synthesis, improving 56 thromboxane levels in platelets and diminishing prostacyclin concentration in vascular endothelium. In these circumstances, Se, as an antioxidant agent, is closely related to prostaglandin metabolism, since it acts as a GPx cofactor. Another mechanism would be the protective action of Se against the toxicity of heavy metals (88). 57 4.3 AGE-RELATED CHANGES 4.3.1 Kidney For Cd levels in renal cortex, an age-related increase up to 50 years old with a subsequent decrease was found (Figure 21). This behaviour has already been reported from previous studies (80). The decline in Cd levels after 50 years of age may be due to an age-related degeneration or Cd-induced damage of the kidney tissue. In medulla, no significant age-related changes in Cd levels were found. Figure 21 Relationship between Cd levels (µg/g) in kidney and age (years). For Cu, Zn, Pb and Se linear regression analysis of metal content versus age produced positive slopes, suggesting a tendency for an increase in the elements levels with age (Figures 22-25). For Zn, this is in disagreement with Garcia et al. (79), who reported no age-related changes in kidney tissue levels. This is also in disagreement with the same authors (79) for Pb, as they reported a decrease in kidney Pb levels with age. y = -0,0263x2 + 2,8879x - 5,6781 R² = 0,08 y = 0,1203x + 28,639 R² = 0,0069 0 20 40 60 80 100 120 140 020 40 60 80 100 [Cd] µg/g Age (years) Cortex Medulla 58 Figure 22 Relationship between Cu levels (µg/g) in kidney and age (years). Figure 23 Relationship between Zn levels (µg/g) in kidney and age (years). y = 0,101x + 0,4766 R² = 0,5272 y = 0,0877x + 0,5047 R² = 0,463 0 2 4 6 8 10 12 14 16 18 020 40 60 80 100 [Cu] µg/g Age (years) Cortex Medulla y = 2,4099x + 9,5951 R² = 0,5312 y = 1,5117x + 7,7823 R² = 0,4291 0 50 100 150 200 250 300 350 400 450 020 40 60 80 100 [Zn] µg/g Age (years) Cortex Medulla 59 Figure 24 Relationship between Pb levels (µg/g) in kidney and age (years). Figure 25 Relationship between Se levels (µg/g) in kidney and age (years). y = 0,0045x + 0,0144 R² = 0,4146 y = 0,0038x + 0,0171 R² = 0,4173 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 020 40 60 80 100 [Pb] µg/g Age (years) Cortex Medulla y = 0,0646x + 0,2277 R² = 0,5279 y = 0,0577x + 0,1744 R² = 0,547 0 2 4 6 8 10 12 14 020 40 60 80 100 [Se] µg/g Age (years) Cortex Medulla 60 4.3.2 Aorta Except for Pb, which showed to increase with age (Figure 29), Cu, Zn, Cd and Se in aorta tissue without plaque of atheroma showed quite constant levels with the age increasing (Figures 26, 27, 28, 30). For aorta tissue with plaque of atheroma linear, Cu, Cd and Se showed a tendency for a decrease with age (Figure 26, 28 and 30), while Zn and Pb showed a tendency to increase (Figures 27 and 29). Figure 26 Relationship between Cu levels (µg/g) in aorta and age (years). y = -0,0005x + 4,2342 R² = 6E-05 y = -0,0315x + 5,175 R² = 0,141 0 1 2 3 4 5 6 7 020 40 60 80 100 [Cu] µg/g Age (years) Aorta sem placa Aorta com placa Aorta without plaque Aorta with plaque 61 Figure 27 Relationship between Zn levels (µg/g) in aorta and age (years). Figure 28 Relationship between Cd levels (µg/g) in aorta and age (years). y = 0,3015x + 70,041 R² = 0,0415 y = 0,6586x + 67,978 R² = 0,0361 0 50 100 150 200 250 300 350 400 020 40 60 80 100 [Zn] µg/g Age (years) Aorta sem placa Aorta com placa y = -0,0006x + 0,1631 R² = 0,0029 y = -0,0015x + 0,1905 R² = 0,0335 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 10 30 50 70 90 [Cd] µg/g Age (years) Aorta sem placa Aorta com Placa Aorta without plaque Aorta with plaque Aorta without plaque Aorta with plaque 68 CONCLUSIONS AND FUTURE RESEARCH This work aimed to study the link between trace element imbalances and CVD. A direct approach (i.e., the analysis of the “target” tissues themselves) was used, instead of the usually performed analysis of body fluids (blood, urine), which is a particular and distinctive feature of this work. It is widely assumed that this may be a potentially more conclusive approach. The direct study of the changes of three important trace elements (Zn, Cu and Se) and two important “heavy metals” (Cd and Pb) in kidney tissue (cortex and medulla) and aorta wall (both with and without atheroma plaque) was performed. Data obtained clearly show that:  The two kidney regions (cortex and medulla) present significantly different levels of Zn and Cd (higher in cortex).  In kidney medulla a gender-related difference was observed for Pb (higher values in men). Smoking habits leads to increased levels of Zn, Cd and Pb.  In kidney cortex smoking habits showed to induce significantly increases levels of Cd.  In both kidney regions a slight tendency for an age-related increase in trace elements levels was observed. However, Cd in kidney cortex showed an interesting behaviour: after a increase up to 50 years old, a subsequent decreased was observed.  For aorta without plaque no gender or smoking habits-related differences were found for the five elements.  In aorta with plaque, smokers showed significantly decreased Zn levels. Aorta with plaque showed higher levels of Zn and lower levels of Se than aorta without plaque. The comparison between results from “healthy” individuals and individuals with CVD showed:  Significantly lower levels of Se in both kidney and aorta tissues, indicating a potential cardio-protector role for Se.  However Cu (in both kidney and aorta tissues), Cd (in aorta) and Pb (in the kidney) were also significantly lower in CVD, which is may be considered as unexpected findings, requiring further investigation. The data presented should be considered as preliminary results. However, they seem to corroborate the hypothesis that trace elements play an important role in CVD and 69 important trace element imbalances may occur in tissues. A main question is whether these changes are a cause or a consequence of CVDs. Further investigation of trace elements imbalances in body tissues and its correlation with CVD must be expanded to include more individuals, more elements and more tissues. 70 CHAPTER 6 REFERENCES 71 REFERENCES 1. World Health Organization. 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No Vila do Conde Traumatic Brain Injury M 43 Yes No Alfandega da Fé Left ventricular hypertrophy M 43 No No Vila do Conde Alcohol intoxication associated with aspiration of vomit M 44 Yes No Gondomar Traumatic Brain Injury M 44 Yes No Felgueiras ? M 46 ? Yes Santo Tirso Left hypertrophic cardiomyopathy F 47 Yes ? Vila do Conde Drug intoxication F 47 No No Porto Acute Myocardial Infarction M 48 Yes Yes Gondomar 50-59 Bronchopneumonia M 50 No Yes ? Acute Myocardial Infarction M 50 Yes Yes Porto Asphyxia from hanging M 51 ? ? Porto Asphyxiation due to aspiration of foreign body M 52 No ? Gaia Asphyxia from hanging M 53 ? No Santo Tirso Acute Myocardial Infarction M 54 Yes Yes Gaia Hepatorrenal syndrome M 55 Yes Yes Gondomar Pneumonia F 56 Yes No Gaia Traumatic Brain Injury F 57 ? No Porto Acute Myocardial Infarction M 59 Yes Yes Matosinhos 60-69 Traumatic Brain Injury M 64 No Yes Mirandela Acute Myocardial Infarction M 65 Yes Yes Gondomar Asphyxia from hanging M 67 ? Yes Gaia 77 ? F 67 No Yes Matosinhos Acute Myocardial Infarction M 68 No Yes Maia Pulmonary embolism F 69 No No Gondomar 70-79 Traumatic Brain Injury M 70 Former smoker ? Matosinhos ? M 71 ? ? Gaia ? F 71 No No Gaia Pneumonia M 73 No No Barcelos Sepsis F 74 No Yes Porto Asphyxia from hanging M 74 ? No Gondomar Pulmonary embolism F 74 No Yes Porto Asphyxia from hanging F 76 ? ? Gaia Acute Myocardial Infarction F 79 Yes No Vila do Conde ? M 79 Former smoker ? Vila do Conde Traumatic injuries M 79 No Yes Porto 80-89 Traumatic injuries M 82 No Yes Gondomar Multiorgan failure due to pneumonia F 83 No Yes Marco de Canaveses Stroke F 84 No Yes Gaia Abdominal infection F 85 No ? Vila do Conde Traumatic Brain Injury M 86 No Yes Chaves Traumatic Brain Injury F 87 No Yes Porto ≥90 ? F 91 No Yes Porto Pulmonary embolism F 93 No Yes Porto