Relationship of Haptoglobin Phenotype to Vascular Function and Response to Vitamin E Supplementation in Patients With Diabetes Mellitus Type 2: The EVAS Trial
Relationship of Haptoglobin Phenotype to Vascular Function and Response to Vitamin E Supplementation in Patients With Diabetes Mellitus Type 2: The EVAS Trial
Relationship of haptoglobin phenotype to vascular function and response to Vitamin E
 supplementation in Patients with Diabetes Mellitus Type 2: The EVAS Trial

 Specific Aims:

 The phenotype haptoglobin 2-2 (Hp 2-2) is associated with higher oxidative stress,
 inflammation, LDL peroxidation and higher cardiovascular risk in patients with diabetes. We
 aim to determine whether Hp 2-2 phenotype is associated with surrogate markers of
 cardiovascular risk, inflammation, lipids and lipoprotein profile, oxidative stress, and
 endothelial cell (EC) apoptosis (in vitro study) in patients with diabetes in our population
 and whether vitamin E supplementation mitigates this risk.

 Methods:

 Screening Phase:

 We will recruit 300 patients with diabetes mellitus type 2 (100 Chinese, 100 Malays and 100
 Indians) and assess their Hp phenotype, surrogate markers of cardiovascular risk,
 inflammation, vascular biomarkers and lipids phenotype.

 In vitro Study:

 Plasma from 20 patients with Hp 2-2 phenotype and 20 patients with non Hp 2-2 phenotype will
 be studied in vitro using a haemodynamic lab-on-chip system to determine whether there is a
 difference in EC apoptosis between the two groups.

 Randomisation Phase 200 patients will be recruited to a pilot randomized controlled trial
 (RCT), stratified by Hp 2-2 phenotype status (100 Hp 2-2 and 100 non-Hp 2-2), and randomly
 allocated in a 1:1 ratio to either vitamin E 400 IU supplementation daily for 6 months or a
 placebo group. The trial will determine whether vitamin E improves the aforementioned
 surrogate markers in the Hp phenotype strata.

 Importance of proposed research to science and medicine:

 This study allows us to understand the possible mechanism of cardiovascular risk in patients
 with Hp 2-2 phenotype and to see whether vitamin E supplementation reduces this risk in a
 pharmacogenomic targeted manner.
1.0 Background and Clinical Significance 1.1 Introduction The incidence of Diabetes Mellitus
 type 2 (DM2) is growing rapidly globally and in Singapore. The main cause of increased
 morbidity and mortality in patients with DM2 is the development of microvascular and
 macrovascular complications. Although strict glycaemic control has been proven to reduce
 microvascular complications, the evidence is still lacking with regards to macrovascular
 complications. Accelerated atherosclerosis is the leading cause of increased mortality and
 morbidity in these patients. It is of paramount importance to assess novel targets to control
 atherosclerosis in patients with DM2 on top of conventional treatment. There is an unmet need
 to find new targets or markers predicting increased risk in patients with diabetes mellitus
 and we need to consider alternative treatments on top of conventional targets to reduce risk
 in these high risk group patients.

 Patients with DM2 are not only at an increased risk for atherosclerosis, they also carry a
 greater extent of the disease burden. Endothelial dysfunction is considered the hallmark of
 the pathological insult inflicted on the blood vessels. Hyperglycaemia affects mitochondrial,
 enzymatic, and non-enzymatic pathways associated with the generation of reactive oxygen
 species (ROS), leading to decreased nitric oxide bioavailability and endothelial dysfunction,
 commonly demonstrated by reduced endothelium dependent vasodilatation and increased plasma
 levels of endothelium derived regulatory proteins. Moreover, DM2 patients have compromised
 antioxidant defenses in the form of low levels of the antioxidant enzymes and
 alpha-tocopherol (vitamin E), which may impede an adequate compensation for the increase in
 oxidative stress. Another role of oxidative stress in mediating the development of
 atherosclerosis has also been demonstrated in the oxidative hypothesis. In this model the
 most prominent target for oxidative modification is the LDL molecule. Oxidised LDL is not
 recognized by the LDL receptor but is readily taken up by the CD36 scavenger receptor pathway
 in macrophages leading to appreciable cholesteryl ester accumulation and foam cell formation.
 Oxidized LDL is proinflammatory, it causes inhibition of endothelial NO synthetase, promotes
 vasoconstriction and monocyte adhesion, and promotes platelet aggregation and thrombosis.

 Hp Phenotype and Oxidative Tissue Damage

 The haptoglobin (Hp) protein is an antioxidant due to its ability to neutralize the oxidative
 activity of haemoglobin (Hb). In humans, Hp is characterised by a genetic polymorphism with
 three structurally different phenotypes (Hp1-1, Hp 2-1 and Hp 2-2 which result from
 expression of two different alleles (Hp 1 and Hp 2) of the haptoglobin gene located on
 chromosome 16q22. The protein product of the Hp2 allele is an inferior antioxidant compared
 to Hp1 allele product. Hp 1-1 is a small molecule (86kDa) of well-defined structure, whereas
 Hp 2-1 is characterised by heteropolymers (86-300 kDA) and Hp 2-2 forms large macromolecular
 complexes (170-1,000 kDa).

 The function of Hp is to bind free Hb released from red blood cells, which is released into
 the blood during the natural turnover of red blood cells. Free Hb is capable of causing
 considerable oxidative tissue damage as a result of its heme iron. However, whenever Hb is
 released into the circulation it immediately binds to Hp with extremely high affinity to form
 an Hp-Hb complex. This binding serves to inhibit the oxidative potential of Hb by preventing
 the release of heme iron from Hb. Hp is normally found in the blood in a more than 400-fold
 molar excess to free Hb and therefore Hp is capable of binding all of the Hb that is released
 during normal red blood cell turnover. Once Hb is bound to Hp it is rapidly cleared from the
 blood stream via the CD163 scavenger receptor expressed on monocyte/macrophages, however,
 formation and clearance of Hp-Hb complexes are impaired in Hp 2-2 phenotypes.

 Iron derived from Hb can catalyse a number of oxidative reactions which can be inhibited by
 Hp .

 1. Ferrous heme iron (Fe2+) can react with hydrogen peroxide to yield ferric Hb (Fe3+) and
 the highly reactive hydroxyl radical species. By abstracting a hydrogen atom from
 polyunsaturated fatty acids, hydroxyl radicals may initiate the process of lipid
 peroxidation.

 2. Ferrous Hb (Fe2+) can also react with hydrogen peroxide to produce ferryl Hb (Fe4+), a
 highly unstable molecule which readily reacts with a second molecule of hydrogen
 peroxide to yield ferric Hb (Fe3+) and superoxide anion. The damaging effects of
 superoxide anion are two-fold: reduction of ferric iron (Fe3+) in Hb to ferrous iron
 (Fe2+), allowing for the production of additional hydroxyl radical as described in
 reaction 1, and dismutation of superoxide anion to produce hydrogen peroxide, again
 promoting the production of ROS.

 3. Ferric Hb (containing Fe3+), also known as methaemoglobin, can spontaneously transfer
 its heme moiety resulting in heme entry into diverse lipophilic environments such as LDL
 or cell membranes. Once intercalated into its new lipid environment, heme iron can
 undergo reactions with hydrogen peroxide as described above or with adjacent lipid
 peroxides generating a free radical cascade and leading to extensive lipid oxidation.

 As a part of the Hp-Hb complex, Hp stabilizes heme in the heme pocket of Hb, and
 prevents Hb from causing oxidative injury . However, the degree to which Hp neutralizes
 the redox activity of heme iron differs among Hp types. This has been shown in a number
 of systems both in vitro and in vivo. For example, studies using linolenic acid showed
 that Hp 1-1 prevented oxidation (diene formation) as measured by an increase in
 absorbance at 232 nm to a greater extent than Hp 2-2 . Another study examined LDL
 oxidation due to heme transfer from Hb to LDL. Heme transfer was measured by quenching
 of the fluorescence signal emitted by dansylated LDL. It was found that Hp 1-1 was
 superior in preventing heme transfer from Hb as compared to Hp 2-2.

 Hp Phenotypes and Cardiovascular Risk Studies have shown that Hp 2-2 Hb complexes are
 also cleared less efficiently than non Hp 2-2 Hb complexes. In DM2 patients this
 phenomenon is more pronounced due to the downregulation of CD163, particularly in Hp 2-2
 individuals. An impairment in anti-inflammatory macrophage signalling through a
 CD163/pAkt /IL-10 axis is also seen in Hp 2-2 patients.

 Hp-Hb deficient clearance in Hp 2-2 DM2 individuals results in increased Hp-Hb binding
 to Apo A1 on high-density lipoprotein (HDL-C), thereby tethering the pro-oxidative heme
 moiety to HDL. This renders it deficient in its ability to reverse transfer cholesterol
 from macrophages.

 The Hp 2-2 protein is less efficient at blocking heme transfer from Hb compared to Hp
 1-1. Furthermore, the increase in heme transfer when Hb is glycosylated may provide a
 mechanistic explanation for the increase in cardiovascular disease seen in Hp 2-2 DM2.

 Hence Hp 2-2 phenotype is associated with decreased ability to bind with Hb, decreased
 clearance of Hp 2-2 Hb complexes, impairment in anti-inflammatory signalling pathway,
 increased LDL oxidation, renders HDL-Cholesterol inefficient and less efficient in
 blocking heme transfer from Hb to Hp 1-1 all leading to a higher cardiovascular risk. In
 diabetes patients where some of these pathways are also affected the synergetic effect
 of hyperglycaemia and haptoglobin phenotype is exacerbated leading to higher risk.

 In longitudinal studies done in other populations, it has been seen that Hp 2-2 genotype
 is associated with a 2-5 fold increased risk of incident CVD in individuals with DM. In
 particular the strong heart study the odds ratio of having CVD in DM with the Hp 2-2
 phenotype was 2-5 times greater than in DM with Hp 2-1 phenotype (p=0.002). In the
 Munich Stent study a consecutive series of 935 treated diabetic individuals were
 followed up for one year after stenting for major adverse cardiac events. In this study
 the haptoglobin 2-2 phenotype was seen to be an independent predictor of major adverse
 cardiac events. In addition it has been seen that vitamin E provides substantial
 cardiovascular benefit to Hp 2-2 DM patients in one population (Israel-ICARE STUDY) and
 post-hoc analysis of the WHS (Women Health Study,) and HOPE study to see whether Vitamin
 E supplementation in subgroup of patients with the haptoglobin 2-2 phenotype influenced
 mortality showed a non-significant reduction in total mortality. This has not been
 widely adopted as larger trials, and on multiple populations, are needed to substantiate
 the association and benefits.

 Hp Phenotype and Ethnicity

 In a local study done in Singapore, it has been seen that the frequency of the Hp genes
 vary in the different ethnicities as follows :Chinese Hp1:0.330;Hp2:0.670;Hp0: 0.029;
 Malays:Hp1:0.298;Hp2:0.702 ;Hp0:0.004; Indians Hp1: 0.167;Hp2:0.833;Hp0:0.009. The
 distribution of the Hp frequencies has been seen to be at Hardy-Weinberg equilibrium in
 our population hence the expected prevalence of Hp 2-2 is around 30-40%. The Hp
 phenotypes will be determined by TaqMan analysis at the TTSH Research Laboratory.

 1.2 Haptoglobin genotypes and endothelial function

 Endothelial dysfunction has received increasing attention as a potential contributor to
 pathogenesis of vascular disease in DM. In DM2, the natural delicate balance in the
 release of contracting and relaxing factors by the endothelium is altered which
 contributes to further vascular and end-organ damage. Impaired endothelial function has
 been postulated to provide a final common pathway by which multiple risk factors exert
 their deleterious effects on cardiovascular health and has been established as a
 powerful surrogate marker for cardiovascular risk with one study showing even better
 predictability than the Framingham risk score . The EndoPAT 2000 device will be used as
 this has been established for estimation of endothelial function in a non-invasive
 manner.

 There are no direct studies done on looking at an association of Hp genotypes to
 endothelial function, in one pilot study wherein endothelial function was assessed using
 post-ischemic reactive hyperaemia and strain gauge plethysmography and expressed as
 maximal flow after an ischemic period, it was seen that Hp 2-2 patients with diabetes
 had worse endothelial function compared to non Hp 2-2 patients (450 +-50 versus
 600+-40).

 1.3 Haptoglobin genotypes and CIMT (Carotid intima media thickness)

 In the Diabetes Heart study, genetic analyses of Hp genotypes showed an association
 between Hp 2-2 genotype and carotid intima media thickness (CIMT). These measurements
 will be made with the subject lying down, with the head extended and slightly turned
 opposite to the carotid examined, following the recommendations of the Mannheim CIMT
 consensus.Two investigators have estimated the CIMT in 23 individuals and a Bland-Altman
 plot was plotted and the limits of inter-user agreement was found to be within -0.1 to
 +0.1.

 1.4 Haptoglobin genotypes and aortic artery stiffness

 Although there are no direct studies done comparing Hp genotypes and aortic artery
 stiffness, one study was done wherein they evaluated the arterial elasticity of large
 and small arteries using pulse wave contour analysis method. The large artery elasticity
 index was lower in patients with Hp 2-2 compared with Hp 1-1 (8.4 +-2.3 ml/mmHg versus
 12.6 +-4.1 ml/mmHg x 100; p<0.0001). In this study the small artery elasticity index was
 also significantly lower in patients with Hp 2-2 phenotype.

 Increased vascular stiffness has been seen early in the course of Diabetes Mellitus Type
 2 using sphygmocor device. It is likely that this stiffness is related to endothelial
 dysfunction rather than structural vascular alterations-this in turn suggests that it is
 reversible. Aortic pulse wave velocity (PWV), a measure of aortic distensibility, has
 also been seen to predict mortality in patients with diabetes independently of known
 confounding factors. The SphygmoCor Xcel device to estimate the aortic artery stiffness
 using the carotid to femoral pulse wave velocity and central aortic pressure will be
 used. The investigators will estimate the pulse wave velocity in 20 individuals in order
 to establish the limits of agreement using the Bland-Altman plot before starting the
 study.

 1.5 Haptoglobin genotypes and vascular markers

 Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1
 (ICAM-1) are proteins expressed on the surface of activated endothelial cells (ECs) and
 expressed in early atherosclerosis. These markers have been evaluated and considered
 good markers of endothelial dysfunction because part of the protein is shed in the
 circulation and can be detected in peripheral plasma.

 1.6 Haptoglobin genotypes and phenotyping of plasma lipids

 Hp 2-2 phenotype has been associated with higher oxidised LDL concentrations, which is
 primarily involved in atherosclerosis. The concentrations of Apo-A1 HDL is also known to
 be higher in this group of patients. Hp 2-2 phenotype may also be associated with higher
 Lp(a) concentrations putting these patients at higher cardiovascular risk. Detailed
 phenotyping of plasma lipids using proteomics to look for novel associations will be
 done.

 1.7 Haptoglobin genotypes and Oxidative Stress

 It is known that Hp 2-2 genotype confers a higher oxidative stress on the endothelium.
 The total oxidative potential will be calculated as the Oxidative-INDEX. Two tests will
 be done as follows to calculate this index. This index has been established as a good
 estimate of the overall oxidative stress.

 1. d-ROMs test: This test will be performed on serum samples by using automated d-ROMs
 method. (Vassalle C, Pratali L, Boni C, Mercuri A, Ndreu R. An oxidative stress
 score as a combined measure of the pro-oxidant and anti-oxidant counterparts in
 patients with coronary artery disease. Clin Biochem 2008;41:1162-7)

 2. FRAP (ferric reducing ability of plasma - a measure of the ability of the plasma to
 prevent damage to vessels) and d-ROMs (derivatives for the Reactive Oxygen
 Metabolites) test will be used to calculate the oxidative stress score.

 Other markers of oxidative stress will also be measured such as glyoxal, methylglyoxal,
 asymmetric dimethylarginine and homoarginine.

 1.8 Haptoglobin genotypes and retinal Arteriovenous index

 The presence of retinal microvascular abnormalities especially arterial constriction and
 venular dilatation has been associated with an increased cardiovascular risk and has
 been associated with endothelial dysfunction and inflammation.Currently there are no
 studies looking at the relationship between Hp genotypes and retinal arteriovenous (AV)
 index. . Prior to the investigations, eye drops will be instilled to dilate pupils for
 fundus examination and to lubricate the cornea. Retinal imaging will be done for the
 subjects at the eye clinic. If patients do not wish to participate in the retinal
 imaging, they may choose to opt out from the retinal imaging test.

 2.0 Vitamin E, haptoglobin phenotype and cardiovascular risk reduction

 While functional differences between Hp1 and Hp2 allelic protein products particularly
 in DM can explain the differences in susceptibility to complications in the Hp 2-2
 individuals from non Hp 2-2 individuals, the main reason of unique benefit from vitamin
 E is that redox active Hb is associated with HDL only in Hp 2-2 DM individuals. In
 patients with DM decreased Hp-Hb complexes results in increased Hp-Hb binding to Apo-A1
 on high-density lipoprotein (HDL), thereby tethering the pro-oxidative heme moiety to
 HDL. HDL in Hp 2-2 DM individuals is deficient in its ability to stimulate the reverse
 transfer of cholesterol from macrophages. Besides these, the Hp phenotype 2-2 is
 associated with increased oxidative stress due to deficient clearance of free radicals
 and increased LDL peroxidation.

 Vitamin E is a potent antioxidant with anti-inflammatory properties. It significantly
 alleviates the condition of oxidative stress by both its potent free radical scavenging
 properties and by interacting directly and strongly with the antioxidant enzymes.
 Vitamin E supplementation in humans and animal models has shown to decrease lipid
 peroxidation, superoxide production and decreasing the expression of scavenger receptors
 (SR-A and CD36) which are particularly important in the formation of foam cells.
 Although vitamin E has not been proven to be useful in reducing cardiovascular risk in
 the general population, it has been useful in patients with Hp 2-2 phenotype with DM,
 both conditions which increase oxidative stress substantially in studies done in one
 population.

 There have been only three interventional randomized controlled trials (RCTs) in which
 the only antioxidant which the DM participants received was vitamin E and in which the
 Hp type of study participants was determined. The ICARE study was the only RCT aimed to
 evaluate vitamin E in DM patients for which Hp genotype was prospectively collected. In
 this study, 1,434 DM individuals > or = 55 years of age with the Hp 2-2 phenotype were
 randomised to vitamin E (400 IU/day/placebo). The primary composite outcome was
 significantly reduced in individuals receiving vitamin E (2.2%) compared to placebo
 (4.7%, p=0.001) at 18 months after initiation of the trial when it was terminated.
 Additionally, blood samples from a subset of patients recruited for the WHS and HOPE
 studies were analysed for Hp polymorphism, and the outcomes reassessed according to the
 patient's Hp type. In all these studies, a higher risk of cardiovascular events was seen
 in Hp 2-2 individuals and a benefit to vitamin E supplementation was seen in this group.

 2.1 Justification for dose and duration of vitamin E (alpha-tocopherol).

 We will be using vitamin E 400 IU per day and matched placebo. We will commission the
 company Beacons who will prepare the vitamin E capsules (400 IU) and the matching
 placebo capsules. Vitamin E preparation will be the natural tocopherol which occurs in
 the RRR-configuration. We will give vitamin E 400 IU for 6 months as most studies using
 surrogate markers of cardiovascular risk has seen an improvement in 6 months after
 supplementation. Moreover, in the ICARE study mentioned above an improvement in
 cardiovascular outcome was seen at 18 months thus suggesting that 6 months should be
 adequate duration to see an improvement in surrogate markers of cardiovascular risk.

 The eight forms of vitamin E are divided into two groups; four are tocopherols and four
 are tocotrienols. They are identified by prefixes alpha- (α-), beta- (β-), gamma- (γ-),
 and delta- (δ-). alpha-tocopherol is the most abundant form in nature, known to have the
 highest biological activity based on fetal resortion assays and reverses vitamin E
 deficiency symptoms in humans. Natural tocopherols occur in the RRR-configuration only.
 The synthetic form contains eight different stereoisomers and is called
 'all-rac'-α-tocopherol.

 Vitamin E is found in its natural form in vegetable oils (wheat germ, sunflower,
 safflower, corn and soybean oils), nuts (almonds, peanuts and hazelnuts), seeds
 (sunflower seeds), green leafy vegetables (spinach and broccoli) and fortified breakfast
 cereals, fruit juices, margarine and spreads. The institute of Medicine recommended
 intakes for individuals is about 15mg/day. The highest safe level of vitamin E
 supplements for adults is 1,500 IU/day for natural forms of vitamin E, and 1,000 IU/day
 for the man-made (synthetic) form. Popular vitamin E supplements available includes,
 D-alpha tocopherol which is derived from natural oils. Commercially available vitamin E
 supplements usually contain only alpha-tocopherol provided either unesterified or as the
 ester of acetate, succinate or nicotinate. In humans, free and esterified
 alpha-tocopherol have the same bioavailability. Supplements can contain either the
 natural RRR-or synthetic (all-rac) alpha-tocopherol. The biological activity of natural
 RRR alpha-tocopherol is higher than that of synthetic all-rac-alpha-tocopherol and other
 natural forms of vitamin E.

 Both oxidative stress and individual genetic makeup contribute to vitamin E homeostasis
 in humans and this may be responsible for the variable clinical effects seen in
 improvement of clinical variables in clinical trials. Vitamin E is absorbed in the
 intestine, enters the circulation via the lymphatic system where absorbed together with
 lipids, it is packed into chylomicrons and transported to the liver. After passage
 through the liver, only alpha-tocopherol preferentially appears in the plasma and most
 of the other forms of vitamin E is preferentially metabolised and either secreted in the
 bile or not taken up and excreted in the faeces. In the liver, hepatic alpha-tocopherol
 transfer protein (α-TTP) specifically sorts out the α- form with the 2R-stereoisomers.
 Plasma RRR-α-tocopherol incorporation is a saturable process. Plasma levels of
 RRR-α-tocopherol cease to increase at approximately 80 μM despite increasing dosages of
 vitamin E supplementation of up to 1,320 mg all-rac-α-tocopherol per day. This is likely
 secondary to the rapid replacement of circulating with newly absorbed α-tocopherol and
 kinetic analyses demonstrates that the entire pool of α-tocopherol is replaced daily. In
 humans, the preferential accumulation of α-tocopherol in the body is dependent upon both
 a functional α-TTP and increased metabolism and excretion of non-α-tocopherols. The
 alpha-tocopherol transfer protein regulates whole-body distribution and concentrations
 of vitamin E by controlling the secretion of vitamin E from the liver. It has been seen
 that the expression of the alpha-tocopherol transfer protein gene can be induced by
 oxidative stress and hypoxia, by agonists of the nuclear receptor PPARα and RXR, and by
 increasing cAMP levels. This is mediated by an already present transcription factor
 called cAMP response element-binding (CREB) transcription factor. Single-nucleotide
 polymorphisms that are commonly found in healthy people drastically affect promoter
 activity.

 Various doses of vitamin E ranging from 400 IU to 2,000 IU have been used in clinical
 trials. The institute of medicine, USA suggests a recommended dietary intake (RDA) of
 15-1,000 mg/day (1 mg =1.5 IU; 22.5-1,500 IU/day). We are using a dose of 400 IU for six
 months. There is no evidence of adverse effects if taken within the RDA, However there
 may be haemorrhagic toxicity in high doses especially in patients on anticoagulants.
 Hence we will be excluding patients on anticoagulants.

 3.0 Statistical Considerations

 3.1 Sample size calculation: The overall estimated sample size for the study is 300
 patients. The required sample size of 100 for each Hp phenotype stratum of the RCT phase
 is based on: 5% type I error; 90% power; the assumption that vitamin E is expected to
 have at least a moderate effect, represented by a standardized effect size (mean
 difference/pooled-standard error) of 0.5, on each risk marker; two sample t-test with
 equal variance; and a 15% drop out rate. Assuming 35% prevalence of Hp 2-2 in our
 population, we need to screen 300 patients to recruit 100 Hp 2-2 phenotype patients.

 Assuming a mean difference on RHI of 0.25 units with corresponding standard deviation
 (SD) of 0.3 - yielding a standardized effect size of 0.25/0.3 = 0.83 as minimal
 difference in RHI seen in another study. We did not adjust for multiple
 testing/comparisons due to the pilot nature of the RCT, as well as the exploratory
 nature of the study in general.

 The sample size for the in vitro study is constrained by limited resources.
 Nevertheless, simulation results based on a two-sided Wilcoxon Signed-Ranked test, 5%
 type I error, a correlation between pairs of 0.5, and 5000 Monte Carlo simulation
 samples indicate that 20 pairs provide adequate power to detect moderate to large
 standardized effect sizes (M1).

 Randomisation will be done electronically through the web - a centralized
 password-protected intranet website to ensure that the patients are randomised the
 moment they are eligible for the trial (strictly sequential). A blocked randomisation
 schedule will be employed, in blocks of 10, for the study based on a 1:1 allocation
 ratio. The dedicated password-protected site will then allocate a unique patient trial
 number which will correspond to the treatment numbers labelled in the medication boxes.
 Following randomisation, the first dose will be administered to the patient.

 3.2 Data handling and statistical analyses: Data Handling All relevant will be collected
 using appropriate well-designed study data-collection forms at each visit and telephone
 follow up assessment. All study data will be stored in a study database assessable only
 to data entry and data validation study personnel.

 Statistical Analysis Plan Data on baseline demographic and clinical variables as well as
 risk markers will be summarized by Hp phenotypes and overall to provide insight on
 potential associations. Binary data will be summarized using frequency and proportions.
 Chi-square test and Fisher exact test will be used to evaluate relevant associations
 (including benefits of vitamin E), and if necessary logistic regression will be used to
 characterize associations while adjusting for potential confounders. Continuous
 variables will be summarized using means (standard deviations) or median (range) as
 deemed appropriate. Two sample t-test or Mann-Whitney test will be used to evaluate
 relevant associations and generalized linear models will be used to characterize
 associations while adjusting for potential confounders. Generalized linear models are
 considered as they can accommodate non-normal (asymmetric) data or log-normal data (such
 as laboratory data) if necessary. Separate tests and models will be performed for each
 relevant outcome.

 Data from the in vitro study will be summarized similarly as described in the preceding
 paragraph, by Hp 2-2 phenotype status and vitamin E concentrations. Wilcoxon
 Signed-Ranked test will be used to evaluate the benefits of vitamin E between Hp
 phenotype groups by concentrations. Mann-Whitney and Jonckheere-Terpstra test will be
 used to evaluate the concentration-benefit relationship of vitamin E by Hp phenotype
 groups. Generalized linear mixed models will be employed to explore various trends and
 associations while accounting for (i) the matching of Hp 2-2 and non Hp 2-2 patients,
 (ii) repeated assessment within a patient by vitamin E concentration, (iii) adjusting
 for potential confounders, and (iv) adjusting for potential non-normality (asymmetry) of
 the data by using other appropriate distributions such as log-normal or gamma
 distributions. An overall analysis of the data from in vitro study will be done.

 Bland-Altman analysis will be used to estimate and evaluate the limits of agreement for
 the agreement and reliability studies. Where appropriate, a mixed model approach will be
 used to estimate and evaluate the relevant reliability coefficients.

 5.0 Clinical Significance

 If an association is seen between Hp 2-2 phenotype with cardiovascular risk, this group
 of patients can be targeted for vitamin E treatment on top of statins and other
 conventional treatment to reduce the cardiovascular risk. Future large scale nation-wide
 RCT can be planned to see whether vitamin E treatment helps to reduce the risk in this
 group of patients. Conducting such studies in a multi-ethnic population is imperative as
 it provides insight on the consistency and generalizability of the expected benefits.
Inclusion Criteria:

 Study patients should meet the following criteria for inclusion in the study:

 1. 100 Chinese, 100 Malays, 100 Indian patients with DM2

 2. Age 21-80 years

 3. Able to give informed consent

 4. Stable diabetes, blood pressure and hyperlipidaemia medications (a 25% dose adjustment
 is allowed) in the last three months

 5. For eligibility to be randomized: HbA1c should be 10% inclusive or below at time of
 randomisation

 6. Blood Pressure should be less than 180/120 mm Hg at time of recruitment

 7. Non-smokers or discontinued smoking at least 6 months ago

 8. No h/o previous myocardial infarction, previous cerebrovascular accident inclusive of
 haemorrhage and infarction, or h/o of peripheral amputation or bypass procedures

 Exclusion Criteria:

 1. Inability to give informed consent

 2. Pregnant subjects

 3. Patients hospitalized for any condition less than 1 month from enrolment

 4. Patients having any recent infections or symptoms suggestive of any systemic infection
 in the last 2 weeks

 5. Myocardial Infarction or stroke within 6 months before enrolment

 6. Patients with creatinine concentrations >200 µmol/L or eGFR<30 µmol/L

 7. Patients on anticoagulants such as warfarin

 8. Known allergy to vitamin E

 9. Current smokers

 10. h/o previous myocardial infarction, previous cerebrovascular accident inclusive of
 haemorrhage and infarction, or h/o of peripheral amputation or bypass procedures

 11. Patients on immunosuppressive agents or corticosteroids for other conditions

 12. Presence of concomitant malignancies or rheumatological conditions at the time of
 recruitment

 13. Patients taking orlistat & cholestyramine