Relationship of Haptoglobin Phenotype to Vascular Function and Response to Vitamin E Supplementation in Patients With Diabetes Mellitus Type 2: The EVAS Trial

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.