Mitigating HIV/AIDS in Sub-Saharan Africa through Selenium in Food

Transcription

1 Mitigating HIV/AIDS in Sub-Saharan Africa through Selenium in Food Dr. Alida Melse-Boonstra Pleunie Hogenkamp Prof. Dr. Obed I. Lungu May 2007 \ Golden Valley Agricultural Research Trust P.O. Box 50834, Lusaka, Zambia, gart@zamnet.zm.

2 Contents Abbreviations... iii Preface...iv Executive Summary...vi 1. Background HIV/AIDS and nutritional status HIV/AIDS and the immune function Oxidative stress Why selenium? Evidence for a beneficial role of selenium in HIV/AIDS Selenoproteins Effects on the immune system Selenium Interaction between selenium and other nutrients Mechanisms of selenium deficiency on HIV-progression GSHPx activity Cytokine production Viral replication Viral mutations DNA damage Depletion of the Se pool Effect of selenium status on HIV-progression Observational studies Clinical trials Null and negative findings Ongoing studies Conclusion Selenium in the food chain in Sub-Saharan Africa Soil Plants Accumulators and non-accumulators Uptake, transport and distribution Selenium toxicity within plants Animals Chicken Dairy cows Humans Absorption and metabolism Markers of selenium status Recommendations for selenium intake Selenium toxicity in humans Foods Selenium content of foods Effect of processing on the selenium content of foods Bioavailability Factors affecting selenium bioavailability Bioavailability studies in animals Bioavailability studies in humans Selenium status of populations in Sub-Saharan Africa Selenium intake in Sub-Saharan Africa Blood concentrations of selenium in Sub-Saharan Africa Risk maps of selenium deficiency in South Africa Strategies to improve selenium status in sub-saharan Africa Supplementation vs. food-based strategies Dietary diversification Food fortification Agronomic fortification Se-enriched fertilizers Supplementation of livestock Bio-fortification... 40

3 4.5 Reaching the targets: Which strategy is the best? Selenium content of some soil and food crops from Zambia Introduction Materials and Methods Results Discussion Conclusions Conclusion and recommendations Glossary References Abbreviations AIDS GSHPx HIV IL Met RDA ROS Se SeCys SeMC SeMet Acquired Immunodeficiency Syndrome Glutathione peroxidase Human Immunodeficiency Virus Interleukin Methionine Recommended Daily Allowance Reactive Oxygen Species Selenium Selenocysteine Selenium-methyl selenocysteine Selenomethionine iii

4 Preface This report brings together tangible findings supporting the importance of selenium in food for strengthening people s resilience against HIV infection as well as developing AIDS if infected. It presents the outcomes of an extensive literature review on this subject and identifies gaps in the current knowledge and recommends areas of work for agricultural research and development. While much of the report is of a wider significance, specific reference is made to Sub-Saharan Africa. The authors recommend that in order to guarantee adequate and equitable levels of selenium intake by the people in Sub-Saharan Africa, a first critical step should be to assess the selenium contents of major soils and food crops and bring the results together into a selenium map. A second step would be to use this selenium map and work out best-bet strategies using available technologies to increase natural Se-levels in common people s diets. The preference should be organic forms of selenium. A prevailing shortage of selenium could actually be dealt with at different entry points in the food chain. Strategies could include fortification of fertilizers and animal feed and the promotion of the best selenium accumulators among the food crops. The authors emphasised the importance to involve all relevant actors in the process right from the beginning, including the agro-industry as well as researchers, policy makers and extension workers. The report also presents the preliminary results on the analysis of selenium in a series of soils and crops of Zambia. The study was commissioned by Golden Valley Agricultural Research Trust (GART) to Agrotechnology Consult Africa B.V. (ACA) under GART s iv

5 regional project on Strengthening HIV/AIDS and Food Security Mitigating Mechanisms amongst Smallholder Farmers in Botswana, Lesotho, Namibia and Zambia. GART implements this project together with its partners in the Government and NGO sector in these countries on behalf of the Regional HIV/AIDS Team in Lusaka with funding by Sida. The principal investigator, Dr. Alida Melse-Boonstra, is senior research fellow at the Wageningen University and Research Centre in The Netherlands. She also worked as post-doctoral fellow at the Department of Nutrition of North-West University in Potchefstroom, South Africa. Pleunie Hogenkamp did part of the research for her MSc-degree in Nutrition at Wageningen University, the Netherlands. Professor Obed I. Lungu, Senior Soil Scientist at the University of Zambia (UNZA) and who is also actively involved in the research programmes of GART, did the soil and crop analyses as presented and discussed in chapter 4. GART wishes to thank the authors and ACA for producing this valuable document. Both the literature review and the preliminary soil and crop analyses point to the need of more substantial research. Stephen W. Muliokela (PhD) Director GART v

6 Executive Summary In Sub-Saharan Africa, the prevalence of HIV and AIDS is still increasing. By the end of 2005, almost 26 million people were living with HIV/AIDS in these countries. AIDS is a wasting syndrome that affects the body in a similar way as malnutrition: it causes impaired growth and development and the immune system is weakened. Optimal nutrition has been shown to improve the quality of life of people living with HIV/AIDS and to slow down the progression of HIV to AIDS. Selenium is an antioxidant that plays an essential role in the immune system. Similar to other antioxidants, e.g. vitamins A, C, and E, zinc, and iron, selenium can inhibit or delay oxidative stress caused by cell entry of HIV and resulting in oxidation of DNA, membranes, lipids and proteins. Furthermore, selenium deficiency impairs macrophage activity that leads to decreased resistance to invading pathogens. In the human body, selenium forms part of selenoproteins. The most important selenoprotein in this respect is glutathione peroxidase (GSHPx). GSHPx controls oxidative stress in tissues and is thereby essential for maintaining immunity against infections. The activity of GSHPx enzymes diminishes rapidly at early stages of selenium deficiency. Selenium supplementation has also been shown to diminish cellular inflammatory responses, mediated by the production of cytokines. Low selenium concentrations stimulate viral replication due to increased oxidative stress. Mutation of viruses into more virulent strains due to selenium deficiency has been reported in animal models. Although the scientific literature is indicative of a beneficial effect of selenium in HIV/AIDS patients, at present no definite conclusions can be made. Selenium has been shown to be a strong predictor of HIVprogression, and it has been suggested that low plasma selenium concentrations form a greater risk for HIV-related mortality than a low concentration of any of the other antioxidants. In a large observational cohort study in HIV-infected pregnant women performed in Tanzania, it was found that low plasma Se concentrations correlated with increased risk of mortality, foetal death, HIV transmission and child death. Studies in which the effect of selenium supplementation on HIV is evaluated are small, scarce and mostly performed in HIV-patients in westernized countries such as the USA. Results of such studies indicate beneficial effects of selenium supplementation on GSHPx activity, increased CD4+ counts and lower hospital admission due to infections. A few vi

7 supplementation studies are currently ongoing in Tanzania, Botswana and South Africa; results are expected in 2008 or after. Soils in sub-saharan Africa are assumed to be selenium deficient in certain areas, but not universally. However, data are limited and this requires further study. The content of selenium in crops does not only depend on the content of soil selenium. Soil characteristics such as acidity and nonpermeability reduce the availability of selenium to plants. Furthermore, plants that are selenium accumulators take up selenium against a concentration gradient up to concentrations of 1,000 mg Se/kg. Examples of such accumulators are species of Brassica (broccoli, cabbage, Indian mustard) and Allium (garlic, leek, onion). Most plants that accumulate higher amounts of selenium, such as certain types of nuts, are toxic to animals and humans. Grasses, forage, grains and most vegetables and fruits belong largely to the category of non-accumulators with typical selenium concentrations below 1 mg Se/kg. Among the grains, wheat was found to be the best accumulator, followed by rice, maize, barley and oats. However, further research on grains typically grown in Africa is required. Supplementation of chicken with organic selenium (selenomethionine, selenocysteine) was shown to result in increased incorporation of selenium in muscle tissue as compared to inorganic forms of selenium (selenite, selenate). Sufficient selenium in the chicken diet had a positive effect on egg production and egg quality maintenance. In cattle, optimal selenium in the diet has been shown to prevent mastitis and to increase selenium concentrations in milk. Although inorganic forms of selenium are readily absorbed in the human duodenum, storage of such forms in tissues is low. SeMet is actively absorbed as methionine and accumulated in tissues such as blood, muscle and liver incorporated into haemoglobin or albumin. SeCys is the bioactive form of selenium that can be incorporated at the active site of selenoproteins. Plasma selenium concentrations reflect short-term changes in dietary selenium intake, whereas concentrations in whole blood, hair and toenail reflect long-term selenium status. Other markers of selenium status that can be used are urinary selenium concentrations, GSHPx activity or selenoprotein P. The recommended intake of selenium is 55 µg per day for adults. Average values of selenium in food products vary per region. Selenium concentrations in cereals usually range from 1-55 µg/ 100 g fresh weight, in dairy products from µg/ 100 g, and in fruits from µg/100 g. vii

8 Some legume varieties contain a reasonable amount of selenium. Processing procedures do not appear to affect selenium content of foods to a great extent, although losses have been reported after removal of bran by milling of grains and after dry heating of cereals. Bioavailability of selenium depends on the outcome measure that is used and on the ingested form. Organic selenium has been shown to be more bio-available than inorganic selenium when maximal GSHPx activity was taken as the outcome measure. Bioavailability of selenium from fish may be inhibited by interactions with heavy metals. No information is available on other factors, such as acidity that may inhibit selenium bioavailability. Studies from South Africa and Malawi showed that selenium intakes of women and children were mostly below the recommended intake levels. Inadequate plasma selenium concentrations were found in various African countries, such as Burundi, Zambia, Nigeria, Malawi and Zaire. The available data indicate large intra-country variability. Mapping of selenium deficient areas has so far been limited to South Africa. As part of the present study, a modest start was made in Zambia with the analysis of soil and food crop samples. The results are reported in this document. Clearly the data indicate generally low levels of selenium. It can be concluded that inadequate selenium supply is present in certain segments of populations living in sub-saharan Africa. Moreover, it is likely that adequate selenium status can improve the quality and duration of life of HIV/AIDS patients. Various strategies can be followed to increase the dietary supply of selenium in this region. Addition of selenium supplementation to routine medication could be an option. However, since medication is usually given rather late in the course of HIV, this would not provide any benefit in earlier stages of infection. Moreover, supplementation strategies are costly, can cause adverse effects, and often show low coverage and poor compliance. Therefore, food-based strategies provide a more sustainable solution. Dietary diversification could help to increase the intake of selenium. This requires better access to selenium-rich foods as well as behavioural changes in the food pattern. Addition of selenium to processed foods, i.e. food fortification could also be considered. Prerequisites for a successful fortification programme are that eligible processed foods are available and that the target population consumes such products regularly. Moreover, excessive selenium intake by non-target population groups should be avoided. Foods that could be considered for selenium fortification include viii

9 salt, wheat flour and maize meal. Compulsory fortification is only warranted, however, after surveys at national and regional levels have identified population groups at risk of selenium deficiency. Agronomic fortification, i.e. increasing the selenium content of agricultural products, provides a safe and sustainable strategy. Fertilizers enriched with selenium can be used to increase the selenium content of crops. This strategy has been applied successfully in Finland, China and USA. Environmental concerns with regard to contamination of ground water by leaching out of selenium can form an obstacle. Improvement of favourable soil conditions for selenium uptake by crops could also be a topic for further study. Supplementation of livestock can be applied to increase the selenium content of meat, milk and eggs. Selenium-enriched milk and eggs are already on the market in various countries. The potential effectiveness of selenium enrichment of animal products in sub-saharan Africa should be evaluated based on food consumption patterns of populations at risk of selenium deficiency. Bio-fortification, which comprises the selection and breeding of crop varieties that have the genetic potential to accumulate an increased amount of selenium, forms a promising self-sustaining strategy. Crops that could be investigated in this respect are wheat, maize, sorghum, legumes, nuts and species of the Brassica and Allium families. If the clinical trials that are currently ongoing provide further evidence that selenium can mitigate HIV/AIDS in sub-saharan Africa, strategies to alleviate selenium deficiency should be defined. As a first step, it is recommended to identify populations at risk of selenium deficiency in sub- Saharan Africa by mapping selenium concentrations in soils, plants, animals, foods and humans. After that, the best strategy or combination of strategies for each country or region should be defined. Food consumption surveys should identify foods that can serve as carriers for either fortification, agronomic fortification or biofortification. Once fortified, the efficacy of such foods to increase selenium status should be assessed. Stakeholders such as universities; governmental and non-governmental institutes; food industry; and legislation bodies should be involved in an early stage in order to be effective and successful. ix

10 1. Background 1.1 HIV/AIDS and nutritional status The global distribution of the human immunodeficiency virus (HIV)- infection has been stabilized for the first time since the discovery of the symptoms of acquired immunodeficiency syndrome (AIDS) 25 years ago. However, the HIV/AIDS-pandemic is still becoming increasingly prevalent in Sub-Saharan countries. In these countries, almost 26 million people were living with HIV/AIDS by the end of 2005 (1). In Sub-Saharan Africa, HIV/AIDS often coincides with micronutrient malnutrition. Micronutrient malnutrition can exist even when people have enough to eat but lack the resources to buy fresh vegetables, fruits, meat and other foods rich in vitamins and minerals. More than a third of the people in Sub-Saharan Africa suffer from micronutrient malnutrition. Diets poor in micronutrients cause illness, blindness, premature death, reduced productivity and intellectual capacity and impaired immune response (2;3). HIV/AIDS affects the body in a similar way as malnutrition: both conditions weaken the immune system and cause impaired growth and development. HIV infection on top of pre-existing malnutrition therefore creates a tremendous burden on an individual s health status. Optimal nutrition can improve the quality of life of people living with HIV/AIDS, as it slows down progression of HIV-infection to AIDS and increases tolerance to antiretroviral therapy (4). A failing immune system due to lack of antioxidant micronutrients such as vitamins A, C and E, iron, zinc and selenium is an important determinant of HIV infection that is often ignored (5;6). This report focuses on one of these antioxidants: selenium. Selenium plays an essential role in the immune system and can potentially protect against HIV progression through various mechanisms. If selenium is indeed a health issue in Southern Africa, strategies are needed to improve selenium status in the population. Various food-based strategies that can be applied to increase the Se content of foods are addressed later in this report. 1.2 HIV/AIDS and the immune function HIV is a retrovirus *, which is defined as a virus with genetic material in the form of RNA that can be translated into DNA * of the host cells. When this DNA is copied, many new virus particles (viraemia * ) are produced in the host cells. Humans can be infected by two species of the virus: HIV-1 and/ or HIV-2. The first is most virulent * and easily transmitted and causes the 10

11 majority of HIV-infections in the world. HIV-2 is less transmittable and largely restricted to West Africa. This review will be focused on HIV-1. HIV causes AIDS, a syndrome characterized by a failing immune system. The immune system is categorized in innate * and acquired * (or adaptive) immunity. Innate immunity is a non-specific way of removing foreign particles, known as antigens *, from the body by phagocytes * such as neutrophils *, macrophages * and leukocytes *. Natural killer cells * assist in eliminating the antigens (7). Acquired immunity can be cell-mediated * or humoral *. Cell mediation gives a specific immune response facilitated by T- and B-lymphocytes *, and is characterized by its memory function. As shown in Figure 1.1, activation of two major cell types, i.e. T-helper (CD4+) and cytotoxic * T (CD8+) cells, will take place when antigens * are presented to T-cells. CD8+ T-cells are cytotoxic to infected cells (8). Humoral immunity deals with antibody * production in B-lymphocytes. Antibodies bind with matching antigens of invading microbes or viruses and destruct these. The HIV-life cycle consists of several stages. The first or acute stage starts with cell entry by the virus and is characterized by high plasma viraemia and low levels of CD4+ cells. In the second, chronic, stage of HIV-infection viraemia levels are decreased due to the cytotoxic effect of CD8+ cells. Symptoms may be absent during this chronic phase of HIV-infection, but the virus will be replicated continuously. In the final stage of full-blown AIDS, levels of CD4+ cells decrease dramatically and a high level of viraemia affects the immune system (9). Time span of the chronic phase of HIV-infection, without antiretroviral treatment, is on average about years, but AIDS can be developed within 5 years or over 20 years as well. 1.3 Oxidative stress Oxidative stress is a condition in which cellular damage is caused by oxygen and oxygen-derived oxidants. These are normally produced as byproducts of metabolism, resulting in a low background level of oxidative stress * in all living cells. Oxidative stress can be increased under certain conditions, such as exposure to toxins, cigarette smoke, specific foods, physical injury, or disease. As a result, cells will be damaged. Super oxide (O2 - ) and hydrogen peroxide (H 2 O 2 ) are the most common reactive oxygen species * (ROS) and are assumed to contribute to the pathogenesis of several human diseases. Antioxidants * are substances that inhibit or delay oxidation of DNA, membranes, lipids and proteins (10-14). Examples of antioxidants are retinol (vitamin A), ascorbic acid (vitamin C), tocopherol (vitamin E), zinc, iron and also selenium (Se). 11

12 Figure 1.1 Overview of the acquired immune system ( ) Cell entry by viruses disturbs the normal biochemistry of the endoplasmatic reticulum * and mitochondria *, causing ROS generation and depletion of antioxidants (15). Early in the path of infection, phagocytes release an increased amount of ROS, resulting in increased lipid peroxidation of cell membranes and oxidative DNA damage (15). Lipid peroxidation * contributes to disease progression by reduction of antioxidant activity, therewith supporting further viral replication (14). 1.4 Why selenium? Se plays an essential role in the immune system. It affects antioxidant defence systems, thyroid hormone metabolism and redox control of enzymes and proteins (16). Moreover, plasma Se is a strong predictor of HIV-progression, and it has been suggested that low plasma Se is a greater risk factor for mortality than other antioxidants or a low CD4+ count (17). 12

13 Se is naturally present in soil and gets into the food chain through plants. There are indications that parts of Sub-Saharan Africa lack sufficient concentrations of Se in soil, crops and animals (18-20). Currently, only scattered data of Se status of soils, crops, animals and humans are available from these areas. In this desk research, state of the art knowledge on the role of Se as a key nutrient in the defence against HIV/AIDS is described. Moreover, this report provides the basis for mapping Se content in soils and crops in Southern Africa. Furthermore, the most promising food-based strategies are identified that could increase Se content of soil, crops and animal products and that could help to mitigate the impact of HIV/AIDS in Sub- Saharan Africa. 2. Evidence for a beneficial role of selenium in HIV/AIDS Observational studies on Se and HIV/AIDS consistently show a positive association between Se status and delayed disease progression or increased mortality. Although only a few small clinical trials have been conducted so far, positive effects of Se supplementation have been found. No negative or neutral findings have been published. Currently, two large clinical trials with Se supplements are ongoing in Southern Africa. The positive effect of Se can be explained by various mechanisms. In order to understand these mechanisms, a brief explanation of the function of Se in the human body and its role in the immune system is given first. 2.1 Selenoproteins Se forms an integral part of selenoproteins * in the human body (21). Among the selenoproteins, a distinction can be made between different families of Se-containing enzymes. The first group, including glutathione peroxidase * (GSHPx), is involved in control of tissue concentrations of highly reactive oxygen-containing species * (ROS) and is therefore essential for maintaining cell-mediated immunity * against infections. GSHPx is present in blood cells and blood platelets. The activity of GSHPx enzymes decreases rapidly at early stages of Se deficiency (22). A second group of selenoproteins includes thioredoxin reductase *, a major component of redox systems, which are amongst others involved in disposal of products of oxidative metabolism and regulation of enzyme 13

14 transcription factors * and receptors *. Thioredoxin stimulates expression of a subunit of the interleukin-2 * (IL-2) receptor: IL-2 is a cytokine * responsible for an early clonal expansion * of cytotoxic * T-lymphocytes * (21;23;24). Supplementation with Se resulted in a significant increase in the number of high affinity IL-2 binding sites (25), whereas Se deficiency had the opposite effect (26). A third group of selenoproteins is that of the iodothyronine deiodinases *. Se deficiency reduces the activity of the deiodinase enzymes, which are responsible for the production of triiodothyronine * (T 3 ), the active thyroid hormone, from thyroxine * (T 4 ). The thyroid hormone is involved in processes of growth, development and metabolism (27). Co-occurrence of Se deficiency and iodine deficiency has been suggested to be causal for myxedematous cretinism (28;29). A third factor, thiocyanate overload, could also be involved in this (30). A fourth selenoprotein is selenoprotein P, which is necessary for Se transport and distribution and is suggested to play a role in cell membranes. Selenoprotein P possibly participates in antioxidant defence (31). 2.2 Effects on the immune system Selenium Se has an effect on several cells of both the innate * and the acquired * immune system. Se deficiency impairs macrophage * activity, leading to a decreased intracellular killing of pathogens. Se deficiency also influences antibody * production, resulting in decreased maturation of T-lymphocytes * and natural killer cell * activity. Supplementation with Se has a positive effect on these cells. In general it can be stated that the effect of Se on immune cells is dependent of dose (13). There appears to be a Se threshold below which Se deficiency results in a weakened immune system and less efficient protection against HIV- infection (18). Whole blood Se concentrations >85 µg/l ( 1.08 µmol/l) are considered to be adequate for functioning of the immune system, whereas Se deficiency is defined as a Se concentration below this value (32-34). However, selenium concentrations were inversely related to immune activation in 244 HIVinfected patients in the USA despite their adequate Se status (35), suggesting that optimal concentrations of Se in whole blood might be well above the current threshold. Se can up-regulate expression of IL-2 * receptors on the surface of activated lymphocytes and natural killer cells. Low IL-2 levels hinder the maturation processes of lymphocytes in the thymus, resulting in a lack of replacement of T-cells (36). Since CD4+ T-cells form a key component in stimulating B- cells to synthesize antibodies * (see Figure 1.1), this may explain the 14

15 stimulatory effects of Se on antibody production. Se supplementation could partially reverse age-related decreases in cell-mediated immunity * by increased responsiveness to IL-2. Increased T-cell response due to Se supplementation resulted in reduced oxidative stress * -induced damage to immune cells (21). Activated T-cells showed an improved selenophosphate synthetase activity (17), which is a crucial precursor for synthesis of selenocysteine (SeCys) during selenoprotein synthesis (37) Interaction between selenium and other nutrients Se is not the only element that is important in antioxidant * defence. Interaction with other nutrients is of great importance to the immune system. Se functions in close relationship with vitamin E and it is often the case that a combination of these two nutrients achieves the most optimal effect, as both elements are important in maintaining the efficiency of antioxidant systems (13). Maintenance of glutathione status is affected by pyridoxine (vitamin B 6 ) and riboflavin (Vitamin B 2 ). Vitamin B 6 is a cofactor in the cysteine-synthesis and therewith a limiting factor for glutathione biosynthesis; vitamin B 2 is a cofactor for glutathione reductase. Deficiencies in these two vitamins will produce functional disturbances in the immune response (38). GSHPx-activity is further affected in a negative way by deficiencies in iron, zinc, copper and magnesium (13). Zinc is able to up-regulate gene expression of GSHPx. Zinc and copper interact with Se in antioxidant defence by conversion of super oxide to oxygen and hydrogen peroxidase, which in turn can be reduced by GSHPx (39). Absorption and erythrocyte concentration of Se are decreased in magnesium-deficient individuals, leading to a lower bioavailability * of Se (40). Increased vitamin A and ascorbic acid levels, however, promote absorption of Se (13). The antioxidant role of Se can further be affected by factors that increase oxidative stress, such as smoking (19), high intake of polyunsaturated fatty acids * (PUFA) and extreme exercise (41). Requirement of antioxidants including Se - are increased under these conditions. In addition to that, smoking is shown to be associated with inadequate dietary intake of Se (16;41). Low dietary intake, increasing age, and diseases other than HIV can all be factors that result in low Se levels (16). 2.3 Mechanisms of selenium deficiency on HIVprogression Characteristic for HIV-disease progression is a reported decline in Se parallel with the loss of CD4+ T-cells, even in the early stages of the disease. Various mechanisms have been suggested by which lack of Se would lead to enhanced progression of HIV-disease. 15

16 2.3.1 GSHPx activity Due to Se deficiency the immune system is lacking the possibility to produce an adequate level of GSHPx proteins. GSHPx activity is directly involved in the control of tissue concentrations of ROS *. GSHPx-activity gives a reliable impression of long-term Se status and decreases when deficiency is reached (42). Intracellular GSHPx-activity correlates well with whole blood Se concentrations below a certain threshold value. This value lies within a range of µmol Se/L ( µg Se/L) of whole blood (16;41;43-45). In blood plasma, full expression of GSHPx-activity is expected at plasma Se concentrations between 1.23 and 1.69 µmol/l ( µg Se/L). This range is confirmed in a Se supplementation study in Finnish men, which showed that plasma Se concentration required for maximal GSHPx-activity was µmol Se/L (46). Duffield et al. (47) found that a plasma concentration of 1.14 µmol Se/L was adequate to achieve full GSHPx-activity. Maximal activity of GSHPx is often used as a marker for Se status, as it can be linked easily to functioning of the immune system. However, consequences of less than maximal GSHPx-activities have not been ascertained as yet (16;47). Se in the body is used competitively by selenoproteins. Maximal activity of selenoproteins other than GSHPx have been shown to occur at lower plasma Se concentrations (41;48;49). Under conditions of Se deficiency, GSHPx activity decreased dramatically as compared to activity of other selenoproteins (50). GSHPx is lowest in the hierarchy of competitive Se use by selenoproteins and is therefore a sensitive marker of Se deficiency. In HIV-infected individuals, glutathione is decreased in plasma, lung epithelial lining fluid and T-lymphocytes. Staal et al. (51) showed that GSHPx levels in CD4+ and CD8+ T-cells of HIV-positive individuals (n=134) were significantly lower than GSHPx levels of HIV-uninfected controls (n=31). Sappey et al. (52) confirmed this in vitro by showing an effective increase of GSHPx-activity in infected T-cells after Se supplementation. Increased GSHPx-activity was also shown in 45 HIVinfected patients after Se supplementation, whereas a decline of activity was shown in the control group (53). Repletion of glutathione concentrations by N-acetyl-cysteine has been shown to inhibit HIVtranscription and replication (14). Diminished GSHPx activity thus facilitates HIV-replication and likely accelerates disease progression, and sufficient Se is required to maintain adequate GSHPx activity Cytokine production Exposure to foreign particles stimulates cellular inflammatory responses and the release of mediators, such as tumour necrosis factor (TNF-α) * and 16

17 prostaglandin E 2 * (54). TNF-α plays a central role in the development of chronic inflammation. Se supplementation has been shown to stimulate GSHPx-activity, thereby inhibiting the production of TNF-α and HIVreplication induced by this cytokine * (25;32). Plasma Se levels were inversely correlated with levels of TNF receptors, and high Se concentrations decreased the effects of high TNF-α circulating levels (36). Prostaglandin E 2 functions as an immunomodulator by inhibiting IL-2 production and proliferation of T-cells, and by suppression of natural killer-cell * activity (55). Antioxidant deficiency is associated with increased inflammation and decreased lymphocyte activity due to overproduction of prostaglandin E 2 (56). Se concentrations were also inversely correlated with interleukin-8 * (IL-8). Elevated IL-8 levels caused increased oxidative stress by depletion of GSHPx, thereby exhausting the Se pool. Se supplementation inhibited the release of IL-8 from endothelial cells (12;25) Viral replication Low Se levels result in increased oxidative stress and apoptosis of infected cells, thereby activating the virus to replicate at higher rates (17). HIVreplication is regulated by nuclear factor kappa B * (NFκB), which in turn is activated by hydrogen peroxide. Cells supplemented with Se show reduced NFκB activation due to protection against the effects of H After activation, NFκB is transferred to the nucleus, where it binds to the two binding sites of the HIV-1 viral genome and stimulates gene-transcription. The level of NFκB was increased in infected T-cells, resulting in HIVstimulation and viral replication (57) Viral mutations Decreased GSHPx-activity is suggested to be one of the driving forces of nucleotide changes in viral RNA and therefore responsible for mutation of the virus (58). Mutations in the viral genome might lead to increased virulence * (59). Se deficiency could contribute to the emergence of new viral strains that may be capable of promoting new epidemics (23). Investigations into possible mechanisms of such mutations in mice suggested either a selection mechanism or involvement of increased oxidative stress. Mutations showed up at each viral replication cycle, but only modified viral variants leading to more efficient pathogenic characteristics were replicated DNA damage Se deficiency leads to a decreased effectiveness of DNA methylation with selenomethionine (SeMet) (60). SeMet might be protective against DNA damage and it has a positive effect on DNA repair mechanisms. 17

18 2.3.6 Depletion of the Se pool HIV has the capacity to incorporate Se into its viral selenoproteins at the expense of its host and thus to take control over the Se supply. As a result, the ability of the host to give an effective immune response will be reduced (32). It is questioned whether this viral selenoprotein production is responsible for significant depletion of the Se pool of the host (61). If this is the case, infected individuals will not only be left without sufficient selenoproteins, but without its basic components (Se, cysteine, glutamine and tryptophan) as well. Deficiencies of these nutrients contribute to development of symptoms of AIDS: Se deficiency affects the CD4+ T- lymphocyte production; shortage of cysteine stimulates abnormal immune function and psoriasis; lack of glutamine results in diarrhoea and muscle wasting and tryptophan depression facilitates dementia and dermatitis (62). 2.4 Effect of selenium status on HIV-progression It has been assumed that a low intake of dietary Se increases the probability of an individual to become infected with HIV-1, and that poor Se status is associated with greater risk of transmission of the virus to others (18). There is no hard evidence to support this assumption. Longterm cohort or supplementation studies would be required to address this issue. However, HIV-infected persons with low Se status have a higher risk of morbidity and mortality as shown in a number of observational and clinical studies, which will now be discussed Observational studies Se status was a predictive measure of survival time of HIV-infected persons and AIDS patients (63). Studying the contribution of Se, zinc and iron on disease progression and survival in predominantly black HIVinfected children (n=24) in the USA, Se deficiency was the only nutritional factor that was found to be significantly correlated with mortality (32). Baum et al. (33) found similar results in an American study (n=125) on the effect of nutritional and immunological factors on survival in HIV-infected drug-users: only CD4+ counts and Se concentration were significantly associated with mortality. Even when controlled for poor nutritional status in general, vitamin A-, vitamin B 12 - and zinc-deficiencies, Se deficiency was the only independent predictor of survival. Kupka et al. (6;64;65) concluded that low plasma Se levels were significantly correlated to increased risk of mortality in HIV-infected pregnant women (n=949), foetal death, intrapartum HIV transmission and child death in Tanzania. 18

19 2.4.2 Clinical trials Both the number and size of intervention studies of the effects of Se supplementation on HIV are small, but do show beneficial effects of the element. Supplementation of HIV-infected adults with SeMet for 1 year (n=14) resulted in increased GSHPx activity as compared to placebo (n=18) (53). A combination of N-acetylcysteine and sodium selenite supplementation in HIV-infected adult patients (n=13) resulted in an enhanced percentage of CD4+ lymphocytes and an increase in the CD4+/CD8+ ratio (66). This ratio can be used in HIV-infected humans to predict the stage of HIV-disease progression. Patients with a high CD4+/CD8+ ratio are assumed to progress to AIDS slower than infected individuals with a lower ratio (67). HIV-infected patients with Se that were treated (n=186) had a lower rate in hospital admission and hospitalisation due to infections compared to the placebo group (68) Null and negative findings Published studies indicating no or negative effects of Se supplementation below toxic levels with respect to immune function and viral infections have not been found Ongoing studies The findings described above should be confirmed, preferably by doubleblind *, placebo-controlled * Se intervention studies performed in areas where HIV infection is highly prevalent, such as in sub-saharan Africa. The scientific attention for the association of Se with the health status of HIV-patients is increasing and supplementation studies are in progress: Kupka and colleagues (Harvard School of Public Health, Boston, USA) are currently performing a study in which the effect of Se supplementation on mother-to-child-transmission of HIV in 915 pregnant women from Tanzania will be determined; Baum and colleagues (University of Miami School of Medicine, USA) will study the effects of multivitamins and Se on HIV-disease progression in 869 drug abuse patients from Botswana; and the effects of Se on immune function in general will be investigated in 144 elderly from the United Kingdom. The latter two studies are currently recruiting patients ( April 10, 2007). Furthermore, a clinical trial in a South African prison treating HIV-positive prisoners with Se supplements or placebo is about to start. This intervention study will be coordinated from Surrey University (United Kingdom) as part of a PhDproject (McCourtScott, personal communication). 19

20 2.5 Conclusion At present, scientific evidence of a beneficial effect of Se on HIV/AIDS is emerging but not yet conclusive. Observational studies show a positive association between Se status and HIV disease progression and survival. These findings have been supported by a few small clinical studies. Moreover, in the scientific literature various plausible mechanisms have been raised that further support a beneficial role of Se in HIV/AIDS. Larger clinical trials conducted in Sub-Saharan Africa are required to justify public health action. Several of such trials are currently ongoing and results are expected to be available in 2008 at earliest. 3. Selenium in the food chain in Sub-Saharan Africa Se is present in soil and enters the food chain through plants. Inorganic Se exists in four natural oxidations states: elemental Se (0), selenide (-2), selenite (+4), and selenate (+6), whereas the predominant forms of organic selenium are selenocysteine (SeCys) and selenomethionine (SeMet). 3.1 Soil In general, soil contains mg Se/kg, but the concentration can range up to 1200 mg Se/kg in areas of high Se content. Se deficiency of soil is defined as a concentration of <0.6 mg Se per kg of soil (69). Soil Se deficiency appears to be a problem in sub-saharan Africa, although data on soil Se concentrations are largely lacking. Areas with relatively young rock or subjected to high rainfall are considered as problem areas, with only low soil Se levels present. As changed methodology and knowledge affect comparability of research conclusions, it is difficult to give a generalized overview of mineral concentrations in Africa (70). Furthermore, geographical and environmental conditions differ between and within countries. Mining activities for instance can increase soil Se concentrations locally to very high levels. 3.2 Plants Accumulators and non-accumulators Plants can be divided in Se accumulators and non-accumulators (Table 3.1). Non-accumulator plants take up Se proportionally with the concentration in the soil up to a maximum of 100 mg Se/kg plant tissue. 20

21 Table 3.1. Characteristics of non-accumulators and accumulators with respect to Se Nonaccumulators Secondary accumulators Primary accumulators Se concentration ,000 (mg Se/kg plant tissue) Se species Selenoproteins Seleno-amino acids Seleno-amino acids Se distribution in plant parts Example crops Similar Se concentrations in grain and roots, smaller amounts in stems and leaves Many vegetables and fruits; Grains (wheat> rice> maize> barley> oats); Forage and grasses Atriplex Accumulation in young leaves during early vegetative stage of growth; high levels in seeds during reproductive stage Allium (Onions, leek, garlic, oilseed rape); Brassica (broccoli, cabbage, Brussels sprouts, cauliflower, Indian mustard); Astragalus Aster Atriplex Castilleja Comandra Grayia Grindelia Gutierrezia Machaeranthera B. napus (canola) Accumulation in young leaves during early vegetative stage of growth; high levels in seeds during reproductive stage Lecythidaceae (Brazil nut, Coco de Mono, Sapucaia nut); Astragalus Stanleya Morinda Neptunia Oonopsis Xylorhiza Such plants typically have Se concentrations in a range of mg Se/kg plant material. Examples of non-accumulators are grain (wheat, barley), most crop plants (alfalfa), grasses (rye grass) and forage (71;72). In non-accumulator plants Se can be found incorporated in proteins (13). It has been suggested that values of <0.01 mg/kg (dry weight) for grain Se can be used to define deficient areas (22). Non-accumulators have a lower ratio of Se to sulphate in their shoot tissues than accumulator plants (73). Accumulators take up Se against a concentration gradient, suggesting that the transporters responsible for the uptake and transport of Se are selective for selenate in these plants at the expense of sulphate. Secondary accumulators can contain up till 1,000 mg Se/ kg plant tissue even when grown on low or medium Se concentrated soils (71). Species of Brassica (broccoli, cabbage, Indian mustard) and Allium (garlic, leek, onion) are assumed to accumulate Se many-fold further than Se concentrations in the soil and concentrations can go up to 500 mg Se/kg in high-se-broccoli 21

22 (74). Primary accumulators, such as species from the Lecythidaceae family (Brazil nut) can build up even higher amounts of Se when grown on Se rich soils and can contain up to 40,000 mg Se/kg of plant tissue. In accumulators, Se is incorporated into seleno-amino acids but not integrated into selenoproteins (13). Se toxicity can occur in animals and humans after consuming accumulator plants. Crops that are common in sub-saharan countries, such as maize, millet, sorghum, cassava, and legumes have hardly been investigated with respect to Se accumulation so far. Among legumes, soybeans were reported to have higher Se content than others (69). It is not yet clear what the role of Se is in the life cycle of a plant, but levels of the anti-oxidants super oxide dismutase and glutathione peroxidase increase with elevated Se exposure, suggesting that Se in plants plays a role in prevention from oxidative stress (Xue et al., 2001). Se accumulation may be protective against infections with Fusarium and Alternaria and against herbiovory by aphids and caterpillars, but not by snails (73) Uptake, transport and distribution Soil characteristics, such as porosity and ph of the soil affect the uptake of Se. It can be stated that acidic soils reduce the availability of Se (43). Small decreases in shoot accumulation of Se with increasing salt levels have been reported (75). Decrease of redox potential (Eh), e.g. due to long term waterlogged conditions, results in reduction to elemental Se with low availability for plants (13;71). Se uptake decreases as the content of clay and organic matter in the soil increases (76). Both inorganic and organic forms of Se are taken up by plants, but organic selenate is the main form accumulated because selenite binds stronger to soil particles (13;71). Selenate is taken up via sulphate transporters in the plant roots and therefore Se competes with sulphate. The amount of Se or sulphate taken up by plants depends on the level of sulphate transporters. Expression of these transporters is reduced by a high sulphur status of the plant and high glutathione levels. However, Se accumulators take up selenate preferentially over sulphate. Transport and distribution of Se depends on its form and concentration available for uptake, kind of plant species, phase of development and physiological conditions. In accumulators, Se is found in leaves during growth and in seeds during reproductive stage. Non-accumulating cereal crops have similar levels of Se in both grain and roots and accumulate lower amounts in leaves and stem. After uptake of inorganic forms of Se, plants metabolize Se to SeMet and to SeCys. Se in plants is non-specifically incorporated into seleno-amino acids and proteins via the sulphur assimilation pathway. Incorporation of 22

23 Se into proteins in non-accumulators starts when selenate enters chloroplasts inside the leaf. In a number of steps selenate will be activated and reduced to selenite, which is in turn reduced through selenodiglutathione to selenide. Selenide is incorporated into SeCys, SeMet and proteins. In general SeCys exists in lower concentrations in plants than SeMet. Se accumulators have a similar pathway to convert inorganic forms of Se into SeCys, but do not incorporate SeCys in protein particles (71;75;77) Selenium toxicity within plants Se toxicity occurs when plants are exposed to high Se concentrations, leading to stunted growth, decreased protein synthesis, dry leaves and death of the plant. Concentrations that non-accumulators can handle without showing symptoms of toxicity differ among species. Two mg Se/kg in rice resulted in a 10% reduction of yield, whereas in white clover this reduction was only reached at a concentration of 330 mg Se/kg plant material (75). Se accumulators can contain up to 4,000 mg Se/kg plant material without negative effects on growth, due to the accumulation of Se in non-protein seleno-amino acids instead of incorporation into proteins. An excess of proteins can have damaging effects on plant function. Mature plants turned out to be less susceptible to Se toxicity than younger plants and high sulphate concentrations will lead to increased tolerance to Se as well. Inorganic forms are absorbed more readily and are, therefore, more toxic to plants than organic Se (75;77). 3.3 Animals Chicken Organic Se (SeMet) is actively absorbed in the intestine of chicken as an amino acid, in contrast to inorganic Se. SeMet and methionine are used interchangeably in protein synthesis, which makes it possible to build Se reserves in muscle tissue ((78-80), cited by (13)). There is little if any Se reserve in the chicken body when inorganic Se is used (13). Moreover, organic Se is less toxic to chicken than inorganic Se ((81), cited by (13)). Se deficiency related diseases in chicken are mainly characterized by lipid peroxidation and is associated with development of diseases targeting muscles, heart-vascular, and nervous systems and reproductive performance (13). Se deficiency is also associated with impaired immuno competence and reduced egg production. Chicken kept on a low Se diet had an egg production of 56%, whereas it was maintained at about 77% on a sufficient Se diet ((82), cited by (13)). When organic Se in the form of Seenriched yeast was fed to growing broilers at 0.2 ppm, Se concentrations in 23

24 breast muscle increased more than twice in comparison to a similar dose of selenite ((83), cited by (13)). The quality of chicken meat after long-term storage was shown to be improved with increased dietary Se (13). A combination of Se and vitamin E would be even more beneficial in preventing lipid peroxidation, membrane deterioration, peroxide accumulation and maintaining meat freshness and quality. Egg quality maintenance during storage has been shown to improve by Se supplementation of chicken. After inclusion of organic selenium in the diet at 0.3 ppm Se/ kg, egg freshness increased up to over 7 days of storage ((84), cited by (13)). Similarly, it was shown that addition of organic Se in the diet of hens has a positive effect on yolk colour, egg weight and egg shell quality ((85-88), cited by (13)), hatchability of stored eggs, GSH-Px activity and protection against lipid peroxidation ((89-92), cited by (13)) Dairy cows Absorption of Se in cows takes place mainly in the duodenum ((93), cited by (13)). Several factors affect the efficiency of Se absorption such as: form of the element; the amount ingested; and other dietary factors such as sulphur, calcium, arsenic, nitrate, and cobalt intake that may decrease Se absorption by 50% or more. Excessive intake of copper, zinc or iron also reduced Se availability ((94), cited by (13)). Absorption of inorganic selenium in ruminants is much lower than in monogastric animals ((95;96), cited by (13)). It is likely that sodium selenite is converted into insoluble forms such as elemental Se or selenides in the rumen, which cannot be absorbed ((97), cited by (13)). Se absorption measured by the balance technique ranged from 17 to 50% of Se intake in non-lactating dairy cows with or without Se supplementation ((98), cited by (13)). Absorption of inorganic 75 Se in steers was estimated to be only 13% ((99), cited by (13)) and 11% in non-lactating cows ((100), cited by (13)). In ruminants, Se enters the blood stream bound to alpha- and gammaglobulins or in a free form, is delivered to the liver and after metabolic changes forms selenoproteins, such as glutathione peroxidase. After absorption, Se metabolism in ruminants is similar to that in nonruminants. Se deficiency in dairy cows is amongst others associated with diseases such as nutritional muscular dystrophy, compromised immune system, reduced growth and reproduction rates and peripartum diseases such as retained placenta, metritis and mastitis (13). Mastitis causes a decreased quantity and quality of milk. Optimal dietary Se supplementation is an effective means of prevention of mastitis in cows (( ), cited by (13)). Se deficiency in cattle has been confirmed under natural grazing conditions in 24

25 many countries all over the world. Grant and Sheppard (106) have shown that Se deficiency occurs in flocks grazing pastures that have Se concentrations <0.030 mg Se/ kg dry matter. Overt signs of inadequacy such as nutritional muscular dystrophy occur primarily in young calves born to selenium deficient mothers. Introduction of organic Se in the feed of cows is potentially a better way to improve Se status than inorganic Se. Weiss (107) compared 8 studies in which the effects of Se from selenite or Se yeast were compared. From these studies, 5 reported various benefits of Se-yeast, while 3 studies reported no difference between the two Se sources. Seven studies reported larger increases in milk Se concentrations after Se-yeast supplementation as compared to sodium selenite. In an experiment with 100 Se-deficient dairy cows, relative bioavailability of Se from yeast or from selenite was 1.9 based on Se concentration in blood, 2.7 based on Se concentration in milk and 1.4 based on GSHPx activity ((104), cited by (13)). Similar results are reported by other authors (13). Pure selenomethionine was shown to be less effectively transferred to the milk than Se in the form of yeast (108). In cows fed Se-yeast, the efficiency of Se transfer to the milk ranged from 9.9 to 12.5%, compared with % for cows fed sodium selenite ((109), cited by (13)). Moreover, addition of sodium selenite into drinking water was not effective in increasing Se level in the milk. Se concentration in milk increased linearly with Se intakes from about 2 to 6 mg/ day. However, little further increases in milk Se concentration were seen at higher Se intake. Se supplementation of cows may prevent oxidation of milk and the development of a rancid flavour. The US National Research Council set the Se requirement at 0.3 ppm for cows (110). The current US Food and Drug Administration regulation allows ruminant diets to be supplemented with 0.3 ppm Se from either sodium selenite or selenate. In 2003, an organic source of Se (Sel-Plex TM ) was cleared for dairy and beef cattle. 3.4 Humans Total body values of Se show a wide range of 3 mg (New Zealanders) to 14 mg (some Americans) (22). Se is distributed throughout the body with 30% of total body Se stored in the liver, 15% in the kidneys, 30% in muscle and 10% in blood plasma (111) Absorption and metabolism Absorption of Se compounds takes place in the duodenum and is in general a very efficient process. Various studies showed similar Se absorption levels from food: absorption of selenite is assumed to be >80%, and absorption of SeMet and SeCys over 90% (16;19;22). Absorption is, however, not similar to bioavailability for the body; conversion of the 25

26 absorbed Se to metabolically active forms is the limiting step (22). Organic forms are supposed to be re-used by the body more efficiently than inorganic forms (21). SeMet itself shows no catalytic activity and is not available for functional forms until it is catabolized and converted into SeCys (16). Inorganic forms of Se are highly soluble and absorbed by passive diffusion. However, storage of selenite and selenate in tissues is low, and due to excretion via urine a relatively low level of inorganic Se is available after consumption. SeMet is absorbed in the same way as methionine (Met) (13). This means that it is actively transported through the intestinal membranes and accumulated into tissues, e.g. muscle and liver. Little is known about the absorption of SeCys. Absorption studies showed that organic and inorganic Se is absorbed independently (112), but possible active transport systems of SeCys are not yet identified. Figure 3.1 Metabolic pathway of selenium (113) For bioactivity of the element, Se must be present as an intermediate (selenide, HSe - ) that can be incorporated in SeCys residues, at the active site of selenoproteins. Selenide is more readily formed from inorganic than from organic Se. Selenite in the bloodstream is taken up by erythrocytes and reduced to selenide by glutathione (21;113), whereas selenate is taken up by the liver and reduced in the hepatocytes. The metabolism of organic 26

27 Se compounds into selenide is more complex, as they are converted to selenide through cleavage of the C-Se binding bond by lyase reactions. SeMet has to be converted to SeCys before it can enter the Se pool and be converted into selenide (113). Despite this extensive metabolism, organic forms are preferred in interventions because of their lower acute toxicity (17). Absorbed SeMet will be present in GSHPx after conversion to SeCys, but can also be incorporated into other proteins, like haemoglobin and plasma albumin, without being distinguished from Met. These proteins contribute to the body reserve of Se and can be used in conditions of oxidative stress when Se requirement is increased. Proteins including SeMet are called Secontaining proteins, whereas proteins including incorporated SeCys are called selenoproteins (16;113). Studies with stable isotopes showed that the size of the functional Se pool responds to changes in dietary Se intake. This pool does not include the protein-bound Se in SeMet, which is considered to be a storage pool (114). A schematic overview of Se metabolism is given in Figure Markers of selenium status A series of functional markers of Se status are available, but choice of the marker depends on which specific function of Se is investigated. When assessing Se status in relation to disease risk, possible interactions with other antioxidants in the body should be considered. For international comparisons of Se status, concentration of Se in plasma is used most often and reflects short-term changes in dietary Se intake. Since these values have large variations among countries, no universal normal reference ranges have been defined for plasma Se concentrations. Whole blood Se concentration is an index of long-term Se intake, corresponding with the 120-day life span of erythrocytes, and is relatively constant over time. Study results allowing comparisons of Se concentrations of plasma and whole blood showed that the plasma Se concentrations are 75-80% of Se concentrations in whole blood (19;42). Only a maximum of 15% of blood Se is incorporated in GSHPx. The major part of Se is in the form of SeMet incorporated into haemoglobin, which can be seen as a Se storage pool. The relation of blood Se with intake is complex. Responses on low Se diets or on supplementation are shown after a period of months, as incorporation into erythrocytes requires a long time period. There are several factors influencing both plasma and whole blood Se concentrations, such as pregnancy, disease state and genetic factors. Possible effects of age, gender or race on whole blood concentrations are still points of discussion, partly because the analysis of erythrocyte Se is difficult. Changes observed in erythrocytes due to change 27

28 of dietary intake are in the same range of changes in plasma Se (16;41;115). Other markers of Se status are urinary Se since homeostatic regulation of Se is controlled by excretion in the urine; GSHPx and selenoprotein P; and hair and toenail Se concentrations, which are both indices of long-term Se status (16;41) Recommendations for selenium intake Since soil concentrations of Se differ between (and within) countries, realizing a certain intake can be a challenge in one and not difficult at all in another country. Se recommendations can be linked to full expression of GSHPx-activity, but for human health two-thirds of full activity is assumed to protect against oxidative stress (116). A formal Recommended Daily Allowance (RDA) Committee set recommendations for Se intake based on intervention trials designed to estimate Se requirements for maximal GSHPx-activity. After adjustment for body weight, RDAs for healthy adults (>14 y) were set at 55 µg/day (116). The Scientific Committee for Food recommends 40 µg Se/day for the European Community, and a recommended 30 µg Se/day should be sufficient for women. This proposed intake was based on results showing that 41 µg Se/day was adequate to express two-thirds of GSHPx-activity in healthy men with an average body weight of 60 kg (111) Selenium toxicity in humans Although Se is an essential element in normal body function, it is highly toxic as well. The range between deficient and excessive doses is relatively narrow. In humans, clinical signs of toxicity (selenosis) appear when dietary intake exceeds 900 µg Se/day, an intake that is unlikely to be surpassed in countries, even if high concentrations of Se are found in the soil. Intake up to 450 µg Se/day (the Tolerable Upper Intake Level) is regarded as safe (21) and still not likely to be attained through dietary intake alone in most industrialized countries. In the United States of America the toxic threshold is set at 800 µg/day (16). Humans can suffer from both acute and chronic selenosis, e.g. due to occupational exposure. Living in areas with a Se-rich soil is also assumed to be one of the causes of chronic selenosis. Symptoms of acute selenosis vary from irritation of the respiratory system to gastrointestinal distress and neurological changes, depending on the form of exposure to Se. Chronic selenosis will be accompanied by symptoms as a garlic-like odour of the breath, depression, fatigue, loss of hair, breaking of the nails and dermatitis (117). 28

29 3.5 Foods Selenium content of foods Dietary intake of Se is determined by Se content in food and by the amount of food consumed. Average values of Se in food products differ per region. Se concentrations in cereals can range from 1-55 µg/100 g fresh weight; Se levels in meat, fish and eggs can vary between 1 and 36 µg/ 100 g fresh weight; Se in dairy products fluctuates from 0.1 µg to 17 µg/100 g fresh weight; fruits in general contain only µg Se/100 g fresh weight (42), see Table 3.2 for an overview of Se concentrations of common foods from Malawi and Mauritius (118;119). Se content should preferably be determined in local foods of separate countries or areas since geographic variation in Se content of foods can be substantial (120). In general, fish and organs contain the highest concentrations of Se, followed by meat and eggs (71). Still, products within both food groups vary greatly in Se content. Brazil nuts are known for their high Se content, although levels depend on the Se content of the soil in which they are grown. One single Brazil nut can exceed the recommended daily allowance for Se as stated in the USA (55 µg). However, these nuts are not widely consumed. Several African wild plants have been reported to contain a substantial amount of Se in their leaves, such as Ximenia americana, Amaranthus viridus, Corchorus tridens, Hibiscus sabdarifa, Maerua crassifolia, Moringa oleifera, and Leptadenia hastada (range: µg Se/g dry weight) (121). Almost all Se in foods occurs in proteins in the form of SeCys in selenoenzymes and SeMet in general proteins. Foods from plant sources contain a greater portion of SeMet as compared to animal sources, in which Se occurs in a variety of forms. Vegetables from the Brassica genus, such as broccoli, Brussels sprout, cabbage, cauliflower and mustard, as well as onions and garlic convert a large amount of Se into seleno-amino acids (42). Selenite and selenate are mainly found in drinking water (19; 113). Table 3.2 Se content of staple foods from Malawi and Mauritius English name Scientific name Se content (µg/ 100 g) Cereals Maize flour, 95% extraction Maize flour, 65% extraction uncooked cooked Zea mays Zea mays Zea mays Millet flour Eleusine coracana 8.0 Rice Oryza sativa 2.4 Sorghum Sorghum bicolor 12.9 Sorghum Flour Sorghum bicolor

30 Table 3.2 Se content of staple foods from Malawi and Mauritius English name Scientific name Se content (µg/ 100 g) Vegetables Cassava leaf Manihot esculenta 0.7 Okra leaf Abelmoschus esculentus 0.7 Pumpkin fruit Cucurbita maxima 0.3 Pumpkin leaf Cucurbita maxima ND 1 Turnip leaf Brassica chinensis ND 1 Wild blite Amaranthus thunbergii 0.7 Fruits Banana Musa paradisiaca 0.5 Custard apple Annona squamosa 0.9 Guava Psidium guajava 0.6 Indian plum Flacourtia indica 1.2 Mango Mangifera indica 0.3 Mango, green Mangifera indica 0.3 Passion fruit Passiflora edulis 0.2 Legumes Bengal bean Stizolobium aterrimum 2.5 Hyacinth bean and pod Lablab purpureus 1.3 Pigeon pea Pigeon pea, dried Pigeon pea, immature Cajanus cajan Cajanus cajan Cajanus cajan Chickpea, whole yellow Chickpea, black Chickpea, split Soybean Lablab bean Lentil, brown Lentil, red Lentil, blonde Lima bean Lima bean, immature Bean, red kidney Bean, white Bean, red Bean, small green Bean, canned red Bean, canned white Bean, canned green Cicer arietinum Cicer arietinum Cicer arietinum Glycine max Lablab purpureus Lens culinaris Lens culinaris Lens culinaris Phaseolus lunatus Phaseolus lunatus Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Pea Pisum sativum 2.7 Urad dhall Phaseolus ureus 1.0 Green gram Vigna radiate 20.0 Cowpea Vigna unguiculata 10.2 Bokla Vici faba 37.3 Mung dhall Phaseolus areus 0.9 Kabuli gram Canjanus cajan 92.8 Pigeon pea Arachis hypogaea 6.4 Nuts Groundnut Arachis hypogaea

31 Table 3.2 Se content of staple foods from Malawi and Mauritius English name Scientific name Se content (µg/ 100 g) Peanut Voandzia subterran 4.8 Peanut Voandzia subterran 22.3 Almond Prunus amygdalus 16.0 Animal products Catfish, dried Clarias gariepinus 24.6 Chambo, dried Oreochromis shiranus 26.2 Chambo, smoked Oreochromis shiranus 10.8 Duck egg, boiled 19.5 Grasshopper, roasted 8.9 Matemba, dried Barbus paludinosus 48.0 Matemba, fresh Barbus paludinosus 11.1 Usipa, dried Engraulicypris breianalis Not detectable, limits of detection was 0.1 µg/ 100 g fresh weight Data are compiled from Subratty et al., 2004 (119); and Donovan et al., 1991 (118) Effect of processing on the selenium content of foods Milling of cereal products reduced the amount of Se in some final readyto-use products: Feretti and Levander (122) showed that milling of oats and rice resulted in only a little loss of Se, but processing of wheat and corn into flours using traditional stone mills resulted in Se losses of 14 and 6%, respectively. This can be explained by removal of the bran during milling; wheat bran and rice bran have both been reported to contain relatively high amounts of Se. Hulls from rice, however, did not contain much Se. Processing of wheat flour to breakfast cereals showed no further Se losses (122). The effects of cooking and other preparation methods on Se levels in foods are still unclear and data are sparse. Loss of Se during ordinary cooking is assumed to be negligible (123). Higgs et al. (124) showed that only little or no loss of Se occurred as a result of broiling of meat, baking of sea foods, frying of eggs and boiling of vegetables. However, dry heating of cereals resulted in a loss of 7-23% of Se. After boiling, mushrooms and asparagus that both contain relatively high amounts of Se, lost 29 and 44% of their Se content respectively. Data from the US National Nutrient Database reported 6.2 µg Se/100 g for boiled asparagus and 9.3 µg Se/ 100 g of raw white mushrooms (125). Canning of foods had small effects, but grilling and frying caused greater losses of Se. Fish products lost a relatively large amount of Se after preparation (36-46%), followed by cereals (20-30%), vegetables (12-37%) and peas/beans/cereal products (6-10%) (123). Skim milk contained 93% of total milk Se, mainly protein bound and associated with the casein fraction or whey proteins ((126), cited by (13)). Pasteurisation and sterilization (UHT) of milk and the preparation of milk powder did not lead to substantial Se losses. Production of cheese 31

32 increases the Se concentration in dry matter by a factor of 1.4 in comparison to the starting milk (127). It can be concluded that processing of food in both industrial processes and domestic preparation has mild or little effect on the Se content of foods. 3.6 Bioavailability Factors affecting selenium bioavailability The efficiency by which Se is used in the body, i.e. bioavailability, is dependent on the marker for Se status in which it is expressed, e.g. incorporation into selenoproteins, plasma Se, whole blood Se, or GSHPxactivity. This complicates the comparability of studies. Bioavailability of Se also depends on physiochemical characteristics of the food, i.e. ph and Eh. Dietary factors, such as lipids and metals, can result in formation of complexes with Se, thereby hampering bioavailability of Se (13;71). Bioavailability of Se and distribution to tissues also depends on the form of the ingested Se (17). Organic Se appears to be more bio-available than inorganic forms when maximal GSHPx-activity is taken as outcome measure (128). Supplemental SeMet raised GSHPx-activity almost twice as high as compared to selenite supplementation (129). However, in another study supplementation with sodium selenite and SeMet showed GSHPxactivities of 81% and 80% respectively, although a difference between inorganic and organic forms was expected (130). A study carried out on rats showed that Se from beef is more available than inorganic selenite with respect to GSHPx-activity, but slightly lower available than SeMet (131). Se in spring onions, broccoli and garlic occurs in the form of Se-methyl SeCys (SeMC) instead of SeMet in wheat and meat. Where SeMet will be incorporated non-specifically into proteins, SeMC is available for incorporation into selenoproteins and shows metabolic activity. Nevertheless, Se availability from broccoli is assumed to be low (42;132). Vegetarians show higher Se plasma concentrations than non-vegetarians, probably due to the fact that SeMet in vegetable foods is more bioavailable than SeCys found in animal products (133) Bioavailability studies in animals Based on experiments with chickens in which GSHPx-activity was measured, Se from wheat and yeast was % available as compared to Se from other plant sources. Bioavailability of Se from barley and oats was 78-85% and 41-45%, respectively. Bioavailability of Se from Brazilian nuts was approximately 90% in combination with a high Se content; therefore, these nuts form an excellent source of Se (13;123). Milk, meat and eggs are 32

33 sources of highly available Se as well. Although fish contains a high amount of Se, availability is assumed to be relatively low (tuna: 20-60%; herring: 25%) possibly due to negative interaction of heavy metals with Se 1 (22). However, in a well-conducted trial, Se bioavailability from trout was reported to be high, whereas Se from yeast was less bio-available (134). Pork (86%), beef (80%), chicken and veal (both 77%) are all sources with relatively high Se availability. Bioavailabity of Se from organs was higher than from lean meat: beef kidney showed an availability of 90% in rats with respect to GSHPx-activity compared to 80% for lean beef (22) Bioavailability studies in humans Only a few metabolic Se studies in humans are published, mostly including small samples. Xia et al. (129) found that in Se deficient subjects with an average Se intake of 10 µg/day, supplements in the form of SeMet led to maximum plasma GSHPx-activity at a dose of 37 µg/day. In case of selenite-supplementation a dose of 66 µg/day of selenite was required to express the GSHPx completely, almost twice as much as compared to SeMet-supplementation. Assumed low Se availability from fish was confirmed in a study on 32 healthy subjects. After six weeks of consumption of wheat, fish or control diet, the wheat-group showed a significant increase of 17% in serum and 30% in whole blood Se level, whereas the fish-group only showed small insignificant increases in Se levels. The amount of Se in wheat and fish were similar (135). In a Dutch study (n=28), differences between the effects of a 9-week intervention with wheat bread rolls, minced meat or low-se bread (control) were determined. Based on whole blood Se levels, bioavailability of Se from bread and meat was similar. Moreover, increases in GSHPx-activity were similar for both bread and meat and were significantly different from the control group (136). In a similar study (n=50) from Finland, supplementation with both Se-rich yeast and wheat for 11 weeks resulted in a significant rapid increase of GSHPx-activity. Se availability from these sources was higher than that of selenate, considering plasma and whole blood Se concentrations as endpoint. After supplementation had ended, wheat and yeast were better in maintaining GSHPx-activity as compared to selenate (137). Bioavailability of Se from milk was reported to be reasonable (138;139). 1 Se plays a role in metal detoxification by binding heavy metals, especially mercury (Hg). The HgSe-complex has low availability and will be further metabolized in liver and kidney to reduce toxic effects of the heavy metals. 33

34 3.7 Selenium status of populations in Sub-Saharan Africa Selenium intake in Sub-Saharan Africa Almost all Se in foods occurs in proteins. Due to protein malnutrition in sub-saharan countries, Se intake in Southern Africa can be assumed to be low. Assessment of micronutrient intake in HIV-positive women (n=249) and uninfected controls (n=239) in Manguang (Free State, South Africa) showed that the median intake of all subjects was lower than the RDA of 55 µg/day and about half of the subjects consumed less than 67% of RDA. Consumption of maize products as staple foods, which are assumed to be poor sources of Se, might contribute to this deficient Se intake (140). Levels of dietary Se intake in HIV/AIDS patients (n=35) in the African community of Bloemfontein (Free State, South Africa) were on average higher than the RDA, but median Se intake among females was less than the RDA and almost half of the women had an Se intake <67% of the RDA (141). Results of the South African National Food Consumption Survey of 1999 showed that Se intake, assessed by a 24hr recall to measure consumption levels of children, was constantly low in all age groups and all provinces: 60% of all children had an intake of less than 50% of the recommended daily intake. Northern Province, Free State and Mpumalanga showed the lowest Se ingestion level (142). A report from Malawi indicated that 43% of children aged 4 to 6 years had a Se intake below the RDA (20 µg/day) (143) Blood concentrations of selenium in Sub-Saharan Africa Characterization of antioxidant micronutrient status among 500 adults from Malawi showed that 88% of all subjects was Se deficient (plasma Se concentration <0.89 µmol/l). Non significant differences between HIVpositive (n=370) and HIV-negative subjects (n=130) were shown. A study done in black South Africans in Soweto (Gauteng Province, n=29) with chronic pancreatitis as clinical endpoint showed Se deficiency in patients (0.85 µmol Se/L plasma), but healthy controls had an adequate Se status (1.33 µmol Se/L plasma) (144). Jaskiewicz et al. (145) showed that Se deficiency occurs in South Africans living in Ciskei and Transkei (Eastern Cape), but not in inhabitants of Cape Town (Western Cape). Combs (19) reported on blood Se concentrations in sixty-nine countries to get an impression of Se inadequacy in the world. Most of the data were obtained from healthy adults. Data from Burundi, Zambia, Nigeria, Niger and South Africa showed mean serum plasma concentrations ranging from 15 to 117 µg Se/L; Se deficiency was found in half of the African 34

35 populations included. In Zaire, serum Se concentrations were shown to be adequate in Bas-Zaire ( µg/l), sub-optimal in the regions of Badundu and Kasai (55-80 µg/l) and inadequate in Kivu, Haut-Zaire, Equateur and Shaba (< 55µg/L) (146). Apparently, Se adequacy and inadequacy go hand in hand in Africa and can both occur within a country, dependent on the target population. Larger scale surveys should indicate exactly how widespread the problem of Se deficiency is. There is no doubt that improvement of the dietary intake of Se in groups at risk for Se deficiency is advisable. 3.8 Risk maps of selenium deficiency in South Africa Risk maps reflecting Se status in Sub-Saharan Africa could be helpful to determine areas where Se intake is inadequate. Van Ryssen has compiled data on Se status of grazing herbivores in South Africa to get an impression of Se status in South African soil and plants (20). In general, human blood Se levels are comparable to those of livestock in the same region (111). Deficient regions were defined as areas with whole blood concentrations below 50 µg Se/L, but Se concentrations of µg Se/L blood ( marginally deficient ) are included in deficient areas as well. Adequate levels include Se blood concentrations of µg Se/L. Although no information on Se status could be found for North-Eastern KwaZulu-Natal and its coastal areas, North West Province, Limpopo and the Southern part of Eastern Cape, enough data were gathered from other areas to compile a map of Se distribution (Figure 3.2). Gauteng Province, Mpumalanga and Northern Free State showed variable status between sufficient and deficient, as well as the south-western coastal part of Western Cape and the South of Kruger National Park. Se status in the upper part of Kruger National Park appears to be adequate, just as the main part of Northern Cape and central Karoo (Northern part of Western and Eastern Cape). Coastal regions in Western Cape and a dominating part of KwaZulu Natal show Se deficiency (20). Another risk map of South Africa (Figure 3.3), showing areas at risk of having Se deficiency in local foodstuff, illustrates a similar pattern. The map was compiled by applying geochemical principles to global satellite images and world-wide datasets of climate and soil characteristics, and this information was converted to assess average Se concentrations in plant material (Grundl, personal communication). Recent data from Tanzania showed concentrations of Se in top soils ranging from mg/kg, which on average can be considered to be adequate (147). Moreover, Se concentrations in grass, cattle and sheep did not indicate deficiency in districts surrounding Karatina, Kenya (148). 35

36 Figure 3.2 Geographical distribution of Se status of herbivores in South Africa (20). Figure 3.3 Risk map of soil Se status in Southern Africa. Risk of Se deficiency decreases in the order of orange (<0.05 mg Se/kg dry matter plant material), green ( mg/se kg dry matter plant material), blue and white areas (Se content unknown) (Grundl, unpublished data). 36