טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGoldenstein Hagit
SubjectWhat is the Main Mechanism by Which Haptoglobin Prevents
Hemoglobin Derived Oxidative Damage?
DepartmentDepartment of Medicine
Supervisor Professor Andrew Peter Levy
Full Thesis textFull thesis text - English Version


Abstract

In Diabetes mellitus (DM) there is an increase in intravascular hemolysis, and hemoglobin (Hb) is released to the plasma. Haptoglobin (Hp), an acute phase protein, binds extra corpuscular Hb and prevents it from mediating oxidative damage to cell membranes and plasma proteins.

The Hp gene has 2 common alleles, 1 and 2. Hp 2-2 phenotype is inferior as an antioxidant compared with Hp 1-1. Therefore individuals with DM and Hp 2-2 genotype have more oxidative stress and suffer from severe DM complications, such as cardiovascular diseases (CVD) and high density lipoprotein (HDL) dysfunction. As was found in epidemiological studies, supplementation with an antioxidant such as vitamin E helps to reduce the risk of developing CVD in this group.

Our hypothesis is that Hp 2-2 protein is an inferior antioxidant which, in the setting of diabetes, leads to excessive protein and lipid oxidation and increased vascular disease.

Our results showed a 45% decrease in vitamin E levels in HDL purified from patients with Hp 2-2 as compared to Hp 1-1. This result is explained by higher consumption of vitamin E which may have occurred in Hp 2-2 diabetic individuals due to increased exposure of their HDL to greater oxidation. This finding is consistent with the positive correlation that was found between cholesterol efflux ability and vitamin E levels.

Also, this study provides a mechanism of Hb oxidation, in which in the presence of H2O2, goblin radicals are formed and two unique cross links between Hb and proteins on HDL are formed. The first one, at the size of 43 kd, was present only on oxidized HDL, and disappeared almost entirely (97% decrease) in the presence of Hp 1-1 and in the presence of Hp 2-2 there was a 86% decrease. Its size, 43 kd, suggests that it composed of Hb monomer (16 kd) and Apo A1 (27 kd) which is the main protein on HDL. Since the binding sites of Hp and Apo A1 on HDL are adjacent, the possibility of Hb binding and interfering with Apo A1 function is relevant. This result was further supported by the significant reduction in cholesterol efflux ability that was noted for HDL oxidized with Hp 2-2, as compared to Hp 1-1.  The second Hb cross link, was at the size of 72 kd that was found also in native HDL, regardless of Hp typing. Our attempts to identify the proteins that are cross linked to Hb were unsuccessful, and for now, this question is still a mystery.   

These two unique findings are connected together by the following hypothesis. The presence of the 72 kd band on native HDL raised the possibility that it is a protective mechanism, which binds Hb and prevents it from participating in other oxidative reactions. Only when this protective mechanism is saturated, the additional Hb (16 kd) can create more cross links with other proteins in HDL, like Apo A1 (26 kd), the main protein on HDL. These cross- links might interfere with HDL function. In addition, the increase in vitamin E consumption due to oxidative stress results in further reduction of HDL function.