Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT02776397 |
| Other study ID # |
EVAS001 Version 4 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
May 12, 2016 |
| Last updated |
May 18, 2016 |
| Start date |
June 2015 |
| Est. completion date |
July 2018 |
Study information
| Verified date |
May 2016 |
| Source |
Tan Tock Seng Hospital |
| Contact |
Rinkoo Dalan, MBBS, FRCP (Edin) |
| Phone |
63571000 |
| Email |
rinkoo_dalan[@]ttsh.com.sg |
| Is FDA regulated |
No |
| Health authority |
Singapore: Domain Specific Review BoardsSingapore: Health Sciences Authority |
| Study type |
Interventional
|
Clinical Trial Summary
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.
Description:
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.
