Intervention Protocol

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Vitamin E supplementation for preventing recurrent stroke and other vascular events in patients with stroke or transient ischaemic attack

  1. Malinee Neelamegam1,*,
  2. Irene Looi2,
  3. Kee Sing Ng3,
  4. Sharad Suryakant Malavade1

Editorial Group: Cochrane Stroke Group

Published Online: 22 OCT 2013

Assessed as up-to-date: 11 OCT 2013

DOI: 10.1002/14651858.CD010797


How to Cite

Neelamegam M, Looi I, Ng KS, Malavade SS. Vitamin E supplementation for preventing recurrent stroke and other vascular events in patients with stroke or transient ischaemic attack (Protocol). Cochrane Database of Systematic Reviews 2013, Issue 10. Art. No.: CD010797. DOI: 10.1002/14651858.CD010797.

Author Information

  1. 1

    University of South Florida, College of Public Health, Tampa, Florida, USA

  2. 2

    Hospital Seberang Jaya, Department of Medicine, Seberang Jaya, Malaysia

  3. 3

    Hospital Pulau Pinang, Department of Internal Medicine, Georgetown, Malaysia

*Malinee Neelamegam, College of Public Health, University of South Florida, 13201 Bruce B. Downs Blvd, MDC-056, Tampa, Florida, 33612, USA. nmalinee.79@gmail.com.

Publication History

  1. Publication Status: New
  2. Published Online: 22 OCT 2013

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Background

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
 

Description of the condition

Stroke is a cerebrovascular accident resulting in focal neurological deficit. It can be broadly classified as either ischaemic or haemorrhagic stroke. Diagnosis is based on the presence of acute onset of focal neurological deficit (i.e. hemiparesis or hemiplegia; hemisensory loss; homonymous hemianopia; loss of higher cortical functions such as dysphagia, visuospatial disturbances or reduced consciousness; and cerebellar dysfunction). Diagnosis is further confirmed by computed tomography (CT) scan or magnetic resonance imaging (MRI) of the brain.

Globally, approximately 5.54 million deaths are caused by stroke annually (WHO Report 2000), with 30% of stroke events being recurrent strokes (Goldstein 2006).

Stroke does not represent a well-defined entity. This is because the mechanisms underlying haemorrhagic and ischaemic stroke are different (Hossmann 2010; Tonk 2007). As a result, the standard treatments for ischaemic and haemorrhagic stroke are different. Medical treatment for ischaemic stroke emphasises the roles of antiplatelets to reduce prothrombotic events in cerebral circulation, intensive blood pressure control with antihypertensives, and statins. However, some components of standard treatments in ischaemic stroke might increase the incidence of haemorrhagic stroke, especially in the use of antiplatelets and statins.

Vitamin E has some unique properties that could be beneficial for stroke prevention, in particular ischaemic stroke. As vitamin E supplement is widely used and readily available, it is important to identify and assess the evidence, favourable or otherwise, for its use in people with stroke.

 

Description of the intervention

Vitamin E is an essential nutrient for the body. It is made up of four tocopherols and four tocotrienols (alpha, beta, gamma and delta respectively) (Traber 2006). After ingestion of naturally occurring vitamin E in food, the various forms are absorbed in the small intestine. They are transported to the liver for metabolism. Only alpha-tocopherol is preferentially resecreted by the liver via hepatic alpha-tocopherol transfer protein (Traber 2006). Other forms of vitamin E are metabolised and excreted by the liver (Traber 2007). Therefore, the concentration of alpha-tocopherol in the blood and cellular levels is higher than that of other forms of vitamin E (Dietrich 2006; Sen 2006). As a result, more research is conducted on alpha-tocopherols, although recently tocotrienols have received some scientific recognition, especially in many peer-reviewed research articles from 2009 to 2010.

Vitamin E is an antioxidant. Antioxidants protect cells from the damaging effects of free radicals. Vitamin E is a fat soluble antioxidant that is believed to reduce the production of oxygen free radicals generated during cellular metabolism. In addition to its antioxidant properties, vitamin E is also involved in some metabolic processes such as cell signalling and regulation of expression of genes (Traber 2006). It has also been shown that vitamin E can inhibit the activity of protein kinase C, an enzyme that plays an important role in cell proliferation and differentiation of smooth muscles, platelets and monocytes (Institute of Medicine 2000). Endothelial cells lining the interior surface of the blood vessels, if replete with vitamin E, can resist blood cell components adhering to this surface. In addition, vitamin E is involved in the dilatation of blood vessels and inhibition of platelet aggregation by increasing the expression of two enzymes that suppress arachidonic acid metabolism, thereby increasing the release of prostacyclin from the endothelium lining (Institute of Medicine 2000).

Due to the above unique properties, it is believed that Vitamin E could play a role in preventing stroke.

Numerous foods contain vitamin E. Vegetable oils, nuts and seeds contain ample amounts of alpha-tocopherol. It is also found in green leafy vegetables and fortified cereals. Gamma-tocopherol can be found in soy beans, canola, corn and some vegetable oils. Tocotrienols are found in some vegetable oils (for instance palm oil), wheat germs, barley, saw palmetto, and some nuts and grains. Tocotrienols typically occur at very low levels in nature. They are found with the highest level in crude palm oil and rice bran oil.

The subtypes of vitamin E, namely tocopherols and tocotrienols, are quite different. Tocotrienols have additional three double bonds to their side chains. This enables tocotrienols to have better tissue penetration, especially in brain cells. For further details of the difference between tocopherols and tocotrienols, please refer to  Table 1.

 

How the intervention might work

In recognition of the unique properties of vitamin E as an antioxidant, and its role in blood vessel dilatation, inhibition of platelet aggregation and healthy functions of endothelial cells, it is hoped that vitamin E could act as an effective treatment in addition to conventional medical therapy, for preventing recurrent stroke.

Recent studies suggest that Multidrug Resistance-Associated Protein 1 (MRP1) can act as a protective factor in acute ischaemic stroke (Park 2011). Natural vitamin E α-tocotrienol protects against ischaemic stroke by induction of MRP1. MRP1 is a key mediator in intracellular oxidised glutathione efflux from neural cells. This is because elevation of intracellular oxidised glutathione can trigger neural cell death. Besides, α-tocotrienol can confer neuroprotection by regulating microRNA expression in stroke-affected brain tissue.

In Leppala 2000, vitamin E was shown to be beneficial in ischaemic stroke but it increased the risk of subarachnoid haemorrhage. The exact mechanism for these findings is still unknown.

 

Why it is important to do this review

Globally, stroke remains a leading cause of death and disability (Donnan 2008).

Supplementation with vitamins has become popular, and they are relatively inexpensive compared with prescription medication. Research shows that 12.7% of the adult population taking supplements in the United States are taking vitamin E (Radimer 2004). Therefore, it can be said that the beneficial effects of this supplement may have a large-scale impact on a population. However, limited studies have been conducted on the effects of vitamin E in preventing stroke. These studies have produced mixed results. Vitamin E has been shown to be beneficial in people with ischaemic stroke but it increases the risk of subarachnoid haemorrhage (Leppala 2000).

We will systematically review trial data for vitamin E in preventing recurrent stroke, and its potential public health implications. At the same time, we hope that this review can help to clarify the effects of vitamin E in preventing ischaemic stroke and whether its use is associated with increased haemorrhagic stroke. Because a large number of people are taking vitamin E supplements, it is important to review the range of evidence for vitamin E in ischaemic and haemorrhagic stroke.

In addition, the findings in mice studies of the role of α-tocotrienol in preventing ischaemic stroke by inducing MRP1 (Park 2011) also contribute to the importance of this systematic review. If these findings could be translated into clinical benefits for adults, the benefits of vitamin E could be huge.

 

Objectives

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest

Primary objective: to assess the effects of vitamin E for preventing recurrent stroke.

Secondary objectives:

  1. to evaluate the adverse effects of vitamin E in people with stroke; and
  2. to examine the effects of vitamin E in different stroke types.

 

Methods

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
 

Criteria for considering studies for this review

 

Types of studies

All randomised controlled clinical trials. The intervention groups and comparison groups in the included studies must be truly randomised with both patients and investigators blinded.

 

Types of participants

Adults over the age of 18 years with a past history of stroke or transient ischaemic attack (TIA).

 

Types of interventions

  1. Intervention group: standard treatment with vitamin E at any dose, from the time of ischaemic stroke up to five years after the previous ischaemic stroke.
  2. Comparison group: standard treatment or placebo vitamin E, or both, from the time of ischaemic stroke up to five years after the previous ischaemic stroke.

We will not include diet modification to enhance intake of vegetable oils potentially rich in vitamin E.

 

Types of outcome measures

We will extract outcome data based on the longest follow-up period for the individual studies. The minimum period of follow-up is 30 days after the previous ischaemic stroke.

 

Primary outcomes

Recurrent stroke (fatal and non-fatal).

 

Secondary outcomes

  1. Composite vascular outcome (stroke, myocardial infarction or death from vascular causes) as defined in the studies.
  2. All-cause mortality.
  3. Adverse events, such as death. We will assess this based on the type of adverse event reported and the number of events reported.
  4. Special events of interest, such as cancer (any solid organ cancer).

 

Search methods for identification of studies

See the 'Specialized register' section in the Cochrane Stroke Group module. We will search for trials in all languages and arrange translation of relevant papers published in languages other than English.

 

Electronic searches

We will search the Cochrane Stroke Group Trials Register and the following electronic bibliographic databases and trials registers, to the most recent date:

  1. The Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library latest issue);
  2. MEDLINE (from 1946) (Ovid) (Appendix 1);
  3. EMBASE (from 1980) (Ovid);
  4. CINAHL (from 1982) (EBSCO);
  5. AMED (from 1985) (Ovid);
  6. ClinicalTrials.gov (www.clinicaltrials.gov/);
  7. EU Clinical Trials Register (www.clinicaltrialsregister.eu);
  8. Stroke Trials Registry (www.strokecenter.org/trials/);
  9. Current Controlled Trials (www.controlled-trials.com);
  10. WHO International Clinical Trials Registry Platform (http://apps.who.int/trialsearch/).

We developed the MEDLINE search strategy with the help of the Cochrane Stroke Group Trials Search Co-ordinator and will adapt it for the other databases.

 

Searching other resources

In an effort to identify further published, unpublished and ongoing trials we will:

  1. contact authors, experts in the field and researchers;
  2. contact relevant organisations and pharmaceutical companies;
  3. search reference lists of relevant studies and reviews;
  4. use Science Citation Index Cited Reference Search for forward tracking of important papers;
  5. identify and search relevant conference proceedings;
  6. search Google Scholar (http://scholar.google.co.uk/).

 

Data collection and analysis

 

Selection of studies

Two authors (MN, NKS) will screen the titles and abstracts of the records obtained from the electronic searches and exclude obviously irrelevant papers. We will then obtain the full-text articles for the remaining records and two authors (IL, SSM) will independently assess these for inclusion in the review based on the eligibility criteria (see Criteria for considering studies for this review). We will resolve any disagreements through discussion.

 

Data extraction and management

For the included studies, three authors (MN, NKS, SSM) will independently extract and record the following data on the study characteristics using a data extraction form:

  • source of data;
  • study eligibility;
  • methods;
  • details of participants;
  • interventions;
  • results and outcome.

We will resolve any differences in the extracted data through discussion, involving the third author (IL). Where there are insufficient or missing data, we will contact the study authors for additional information.

 

Assessment of risk of bias in included studies

Three authors (MN, NKS, SSM) authors will independently assess the risk of bias in the included studies. We will assess the risk of bias as 'high risk', 'low risk' or 'unclear risk', using the criteria described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2011), for the following domains: selection bias, performance and detection bias and reporting bias. In the event of missing or unclear details, or both, we will attempt to contact the study authors for further information. We will resolve any disagreements through discussion and consensus with the third author (IL). We will provide a narrative discussion of the risk of bias for those studies we find to have bias.

 

Measures of treatment effect

We will report the treatment effects for dichotomous data using risk ratio (RR), and for continuous data using mean difference (MD), each with their 95% confidence intervals (CI).

 

Unit of analysis issues

We do not anticipate any unit of analysis issues because no cluster randomised studies or crossover studies will be included.

 

Dealing with missing data

We will attempt to contact the investigators of studies with missing data. If we are unable to obtain missing data, we will use the guidelines from the Cochrane Handbook for Systematic Reviews of Intervention to impute data (Higgins 2011).

 

Assessment of heterogeneity

We will assess heterogeneity between the included studies by several methods: examining the forest plots and check for overlapping confidence intervals; using the Chi² test for heterogeneity with a 10% level of significance to detect inconsistency in study results that exceed chance; and using the I² statistic to denote the percentage of inconsistency in the results due to inter-trial variability, with a value above 50% indicating heterogeneity.

 

Assessment of reporting biases

If required, and if there are more than 10 included studies, we will evaluate the possibility of publication bias using funnel plots.

 

Data synthesis

We will analyse the data using Review Manager (RevMan 2012). If the data from trials used similar comparisons, we will synthesise data using the Mantel-Haenszel method to derive pooled, weighted RRs in a fixed-effect model. If heterogeneity is identified as significant (I² > 50%) and can not be explained by subgroup analyses, we will use a random-effects model for data synthesis. If substantial inter-trial variability in effect estimates not accounted for by chance is revealed (I² values 75% or more) or if the trials differ in clinical or methodological attributes, we will present the results of the trials in a forest plot without meta-analysis.

 

Subgroup analysis and investigation of heterogeneity

If data are available, we will perform the following subgroup analysis:

  1. participants (age group, less than 45 years of age, 45 to 65 years of age, more than 65 years of age);
  2. type of vitamin E (tocopherols and tocotrienols, natural or synthetic, mixed tocotrienols, isolated tocopherols or other individual analogues);
  3. low versus high doses of vitamin E;
  4. by risk factor profile (diabetes mellitus, hypertension);
  5. duration of treatment (time from treatment commencement to first event post treatment grouped as more than six months versus six months or less);
  6. ethnicity of participants (White, Black, Hispanic, Asian, Pacific Islander, American Indian);
  7. stroke type (ischaemic stroke versus intracerebral haemorrhage).

 

Sensitivity analysis

We will undertake sensitivity analyses to explore any assumptions we use in the analyses, such as studies with substantial missing data.

 

Acknowledgements

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest

We acknowledge Prof Jacqueline Ho of Penang Medical College, for her guidance in preparing this Cochrane protocol. We also acknowledge the Cochrane Stroke Group, namely Hazel Fraser and Brenda Thomas, for their guidance and support in the development of this protocol.

 

Appendices

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest
 

Appendix 1. MEDLINE search strategy

1. cerebrovascular disorders/ or exp basal ganglia cerebrovascular disease/ or exp brain ischemia/ or exp carotid artery diseases/ or exp cerebrovascular trauma/ or exp intracranial arterial diseases/ or exp intracranial arteriovenous malformations/ or exp "intracranial embolism and thrombosis"/ or exp intracranial hemorrhages/ or stroke/ or exp brain infarction/ or vasospasm, intracranial/
2. (stroke or poststroke or post-stroke or cerebrovasc$ or brain vasc$ or cerebral vasc$ or cva$ or apoplex$ or SAH).tw.
3. ((brain$ or cerebr$ or cerebell$ or intracran$ or intracerebral) adj5 (isch?emi$ or infarct$ or thrombo$ or emboli$ or occlus$)).tw.
4. ((brain$ or cerebr$ or cerebell$ or intracerebral or intracranial or subarachnoid) adj5 (haemorrhage$ or hemorrhage$ or haematoma$ or hematoma$ or bleed$)).tw.
5. ((transi$ adj3 isch?em$ adj3 attack$) or TIA$1).tw.
6. 1 or 2 or 3 or 4 or 5
7. vitamin e/ or tocopherols/ or alpha-tocopherol/ or beta-tocopherol/ or gamma-tocopherol/ or tocotrienols/
8. Vitamin E Deficiency/
9. (vitamin e or tocopherol$ or tocopheryl$ or tocotrienol$).tw,nm.
10. (aquasol e or austrovit e or auxina e or bio e or biopto e or biosan or bioweyxin or covitol or dagravit e or dal e or dalfatol or davitamon e or dermorelle or detulin or dumovit e).tw,nm.
11. (ecoro or e ferol or e mulsin or e perle or e recordati or e toplex or e vicotrat or e vimin or e vita or e viterbin or ecoferol or efer or eferol or elex verla or embial or enoulan forte or ephynal or eplonat or eprolin or epsilan m or epsylan m or equivit e or erevit or erevit spofa or esol or esorb or eterapion or eusovit or eviabit or evigen or eviol or evion or evit or evitol).tw,nm.
12. (godabion e or gonavit or hanobak or hydrovit e or ido e or juvela or juvele or lasar or livingpherol or malton e or micorvit e or mulsal e or natopherol or optovit e or phytoferol or pletocol or puncto e or richtavit e or sanavitan s or socopherol or spondyvit or toco 500 or tocoferolo bioglan or tocolion or tocomine or tocopa or tocopharm or tocopherex or tocophrine or tocovigor or tocovital or toferol or topherol).tw,nm.
13. (unique e or unovit or uno-vit or vi dom e or vi e caps or vi ea or vi etal or vibolex e or vidom e or viea or vietal or viprimol or vita plus e or vitaplus e or vit e or vita e or vitazell or vita e gelucaps or vitamin emp or viteolin or viteoline or wandervit e).tw,nm.
14. 7 or 8 or 9 or 10 or 11 or 12 or 13
15. 6 and 14
16. Randomized Controlled Trials as Topic/
17. random allocation/
18. Controlled Clinical Trials as Topic/
19. control groups/
20. clinical trials as topic/ or clinical trials, phase i as topic/ or clinical trials, phase ii as topic/ or clinical trials, phase iii as topic/ or clinical trials, phase iv as topic/
21. double-blind method/
22. single-blind method/
23. Placebos/
24. placebo effect/
25. Drug Evaluation/
26. Research Design/
27. randomized controlled trial.pt.
28. controlled clinical trial.pt.
29. (clinical trial or clinical trial phase i or clinical trial phase ii or clinical trial phase iii or clinical trial phase iv).pt.
30. random$.tw.
31. (controlled adj5 (trial$ or stud$)).tw.
32. (clinical$ adj5 trial$).tw.
33. ((control or treatment or experiment$ or intervention) adj5 (group$ or subject$ or patient$)).tw.
34. (quasi-random$ or quasi random$ or pseudo-random$ or pseudo random$).tw.
35. ((singl$ or doubl$ or tripl$ or trebl$) adj5 (blind$ or mask$)).tw.
36. placebo$.tw.
37. controls.tw.
38. (RCT or RCTs).tw. or trial.ti.
39. or/16-38
40. 15 and 39
41. exp animals/ not humans.sh.
42. 40 not 41 

 

Contributions of authors

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest

MN & NKS were involved in drafting the protocol and the final write-up of the protocol. IL edited the draft protocol and was involved in the write-up of the final protocol.

 

Declarations of interest

  1. Top of page
  2. Background
  3. Objectives
  4. Methods
  5. Acknowledgements
  6. Appendices
  7. Contributions of authors
  8. Declarations of interest

None known

References

Additional references

  1. Top of page
  2. Abstract
  3. Background
  4. Objectives
  5. Methods
  6. Acknowledgements
  7. Appendices
  8. Contributions of authors
  9. Declarations of interest
  10. Additional references
Dietrich 2006
  • Dietrich M, Traber MG, Jacques PF, Cross CE, Hu Y, Block G. Does γ-tocopherol play a role in the primary prevention of heart disease and cancer? A review. Journal of the American College of Nutrition 2006;25:292-9.
Donnan 2008
Goldstein 2006
  • Goldstein LB, Adams R, Alberts MJ, Appel LJ, Brass LM, Bushnell CD, et al. Primary prevention of ischemic stroke: a guideline from the American Heart Association/AmericanStroke Association Stroke Council: co-sponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: the American Academy of Neurology affirms the value of this guideline. Stroke 2006;37(6):1583–633.
Higgins 2011
  • Higgins JPT, Green S (editors). Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [updated March 2011]. The Cochrane Collaboration, 2011. Available from www.cochrane-handbook.org.
Hossmann 2010
  • Hossmann KA, Heiss WD. Neuropathology and pathophysiology of stroke. In: Brainin M, Heiss WD editor(s). Textbook of Stroke Medicine. Cambridge University Press, 2010:1-27.
Inokuchi 2003
Institute of Medicine 2000
  • Institute of Medicine. Food and Nutrition Board. Dietary Reference Intakes: Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington DC: National Academic Press, 2000.
Leppala 2000
  • Leppala JM, Virtamo J, Fogelholm R, Huttunen JK, Albanes D, Taylor PR, et al. Controlled trial of alpha-tocopherol and beta-carotene supplements on stroke incidence and mortality in male smokers. Arteriosclerosis, Thrombosis, and Vascular Biology 2000;20(1):230-5. [PUBMED: 10634823]
Park 2011
  • Park H-A, Kubicki N, Gnyawali S, Chan YC, Roy S, Khanna S, et al. Natural Vitamin E α-Tocotrienol protects against ischaemic stroke by induction of Multidrug Resistance-Associated Protein 1. Stroke 2011;42:2308-14.
Parker 1993
  • Parker RA, Pearce BC, Clark RW, Gordon DA, Wright JJ. Tocotrienols regulate cholesterol production in mammalian cells by post transcriptional suppression of 3-hydroxy-3methylglutaryl-coenzyme A reduction. Journal of Biological Chemistry 1993;268(15):11230-8.
Pruthi 2001
Qureshi 1996
  • Qureshi AA, Pearce BC, Nor RM, Gapor A, Peterson DM, Elson CE. Dietary alpha-tocopherol attenuates the impact of gamma-tocotrienol on hepatic 3-hydroxy-3methylglutaryl coenzyme A reductase activity in chickens. Journal of Nutrition 1996;126(2):389-94.
Radimer 2004
  • Radimer K, Bindewald B, Hughes J, Ervin B, Swanson C, Picciano MF. Dietary supplement use by US adults: data from the National Health and Nutrition Examination Survey, 1999-2000. American Journal of Epidemiology 2004;160(4):339-49. [PUBMED: 15286019]
RevMan 2012
  • The Nordic Cochrane Centre, The Cochrane Collaboration. Review Manager (RevMan). 5.2. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2012.
Schaffer 2005
Sen 2006
Sontag 2007
  • Sontag TJ, Parker RS. Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherols-omega-hydroxylase. Journal of Lipid Research 2007;48(5):1090-8.
Stocker 2004
Suzuki 1993
  • Suzuki YJ, Tsuchiya M, Wassall SR. Structural and dynamic membrane properties of alpha-tocopherol and alpha-tocotrienol: implication to the molecular mechanism of their antioxidant potency. Biochemistry 1993;32(40):10692-9.
Tonk 2007
Traber 2006
  • Traber MG. Vitamin E. In: Shils ME, Shike M, Ross AC, Caballero B, Cousins R editor(s). Modern Nutrition in Health and Disease. 10th Edition. Baltimore MD: Lippincott Williams & Wilkins, 2006:396-411.
Traber 2007
WHO Report 2000
  • World Health Organization. The World Health Report 2000. The Stationary Office 2000.
Yoshida 2003