Ignite Creation Date:
2024-05-06 @ 8:36 AM
Last Modification Date:
2024-10-26 @ 12:02 PM
Study NCT ID:
NCT02776397
Status:
UNKNOWN
Last Update Posted:
2016-05-19
First Post:
2016-05-12
Brief Title:
Relationship of Haptoglobin Phenotype to Vascular Function and Response to Vitamin E Supplementation in Patients With Diabetes Mellitus Type 2 The EVAS Trial
Sponsor:
Tan Tock Seng Hospital
Organization:
Tan Tock Seng Hospital
Study Overview
Official Title:
Relationship of Haptoglobin Phenotype to Vascular Function and Response to Vitamin E Supplementation in Patients With Diabetes Mellitus Type 2 The EVAS Trial
Status:
UNKNOWN
Status Verified Date:
2016-05
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
EVAS
Brief 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 11 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
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 11 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
Detailed Description:
10 Background and Clinical Significance 11 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 170-1000 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 monocytemacrophages 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 CD163pAkt 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 p0002 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 Hp10330Hp20670Hp0 0029 MalaysHp10298Hp20702 Hp00004 Indians Hp1 0167Hp20833Hp00009 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
12 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
13 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 consensusTwo 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 -01 to 01
14 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 84 -23 mlmmHg versus 126 -41 mlmmHg x 100 p00001 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
15 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
16 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 Lpa concentrations putting these patients at higher cardiovascular risk Detailed phenotyping of plasma lipids using proteomics to look for novel associations will be done
17 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 2008411162-7
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
18 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 inflammationCurrently 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
20 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 1434 DM individuals or 55 years of age with the Hp 2-2 phenotype were randomised to vitamin E 400 IUdayplacebo The primary composite outcome was significantly reduced in individuals receiving vitamin E 22 compared to placebo 47 p0001 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 patients 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
21 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 15mgday The highest safe level of vitamin E supplements for adults is 1500 IUday for natural forms of vitamin E and 1000 IUday 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 1320 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 2000 IU have been used in clinical trials The institute of medicine USA suggests a recommended dietary intake RDA of 15-1000 mgday 1 mg 15 IU 225-1500 IUday 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
30 Statistical Considerations
31 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 differencepooled-standard error of 05 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 025 units with corresponding standard deviation SD of 03 - yielding a standardized effect size of 02503 083 as minimal difference in RHI seen in another study We did not adjust for multiple testingcomparisons 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 05 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 11 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
32 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
50 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
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 170-1000 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 monocytemacrophages 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 CD163pAkt 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 p0002 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 Hp10330Hp20670Hp0 0029 MalaysHp10298Hp20702 Hp00004 Indians Hp1 0167Hp20833Hp00009 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
12 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
13 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 consensusTwo 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 -01 to 01
14 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 84 -23 mlmmHg versus 126 -41 mlmmHg x 100 p00001 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
15 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
16 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 Lpa concentrations putting these patients at higher cardiovascular risk Detailed phenotyping of plasma lipids using proteomics to look for novel associations will be done
17 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 2008411162-7
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
18 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 inflammationCurrently 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
20 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 1434 DM individuals or 55 years of age with the Hp 2-2 phenotype were randomised to vitamin E 400 IUdayplacebo The primary composite outcome was significantly reduced in individuals receiving vitamin E 22 compared to placebo 47 p0001 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 patients 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
21 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 15mgday The highest safe level of vitamin E supplements for adults is 1500 IUday for natural forms of vitamin E and 1000 IUday 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 1320 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 2000 IU have been used in clinical trials The institute of medicine USA suggests a recommended dietary intake RDA of 15-1000 mgday 1 mg 15 IU 225-1500 IUday 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
30 Statistical Considerations
31 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 differencepooled-standard error of 05 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 025 units with corresponding standard deviation SD of 03 - yielding a standardized effect size of 02503 083 as minimal difference in RHI seen in another study We did not adjust for multiple testingcomparisons 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 05 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 11 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
32 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
50 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
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None