| Summary: | 博士 === 國立交通大學 === 生物科技系所 === 96 === Increased hemolytic hemoglobin (Hb) is found to be associated with patients undergoing hemo- and peritoneal dialysis. Rupture of red-blood-cell trauma is often seen at turbulent sites of vessels with hemorrhagic atheromatous plaques, which have been found in acute coronary syndromes and during invasive procedures of thrombolytic therapy for acute myocardial infarction. Cardiovascular mortality in these patients is 10-20 times greater than those in general population. In the present studies, we show that cell free Hb is an extremely active oxidant of poly unsaturated fatty acid (including low-density lipoproteins, linoleate, and human umbilical vein endothelial cell lipid extract) and peroxidase activity, a phenomenon explained so far by different mechanisms. We found that Hb could induce the formation of H2O2 by reacting with LDL, linoleic acid, and cell membrane lipid extract. In the presence of NaN3 (a peroxidase inhibitor), LDL oxidation and peroxidase activity was significantly inhibited and H2O2 concentration was accumulated. As well as, it was found that Hb dramatically stimulated the expression of intercellular and vascular cell adhesion molecules-1 (ICAM-1 and VCAM-1) in a concentration- (1.25-50 μM of Hb) and time- (0.33-30 h) dependent manner. Interestingly, the stimulation completely inhibited by a potent antioxidant probucol (0.625-20 μM). We found that 2,2`azo-bis-(2-amindinopropane) hydrochloride (AAPH) could induce LDL oxidation and stimulated the expression of ICAM-1 and VCAM-1, but could not be inhibited by NaN3. Concluding above, Hb could induce atherogenic oxidative stress and inflammatory response.
Haptoglobin (Hp) is known as an acute phase protein with a key physiologic function to captures the free hemoglobin in plasma allowing hepatic clearance of hemoglobin and preventing its oxidative damage from the tissues. Hp also acts as an antibacterial agent for depleting the iron required for bacterial growth in an acute phase response.
Similar to blood types, there is a specific Hp phenotype 1-1, 2-1, or 2-2 for each individual attributed by two common alleles (Hp 1 and Hp 2). Hp1-1 or 2-2 forms a homozygous dimer or a series of cyclic polymers respectively, while a heterozygous Hp 2-1 gives a series of linear polymers. Several functional differences among Hp phenotypes have been demonstrated which appear to have various important biological and clinical consequences.
Possession of a particular phenotype is found to be associated with the prevalence of some common disorders (e.g. cardiovascular disease, autoimmune disorders, and malignancy). It remains controversial at risk association between Hp phenotypes and coronary artery diseases (CAD). We admitted patient between years 1999 to 2004 for coronary arteriographic examination at the Cardiology Division of Taipei Veterans General Hospitals were investigated into this study. Significant CAD was defined by fixed stenotic lesion with luminal narrowing ≧ 50% in at least one of the major or minor coronary arteries. A total of 1132 subjects were examined, consisting of 559 non-significant CAD and 573 significant CAD patients. The association between the individual Hp phenotype and risk of CAD were analyzed using stepwise multivariate adjustment test. After adjustment with plasma concentrations of apoA-I, Hp 1-1 subjects presented positive association with significant CAD in an OR of 2.14 versus Hp 2-1 (95% CI, 1.11 to 4.12), and an OR of 2.19 versus Hp 2-2 (95% CI, 1.13 to 4.13). When quoted the data for surveillance of cardiovascular diseases from the collaborating center of World Health Organization (WHO), a strong linear correlation (male, r = 0.95, p < 0.001; female, r = 0.92, p < 0.001) between the population of Hp 1-1 and the mortality of age-standardized assessment of ischemic heart disease (IHD) was elucidated among ethnically different populations.
In fact, plasma Hp concentrations of Hp 1-1 individuals are two times greater than that of Hp 2-2. This concentration difference may even play a more crucial role in regulating its physiologic functions, but it has been fundamentally ignored in the last decades.
Since the biochemical structure is rather heterogeneous among the phenotypes, it is exceptionally difficult to use immunoassays in determining the plasma Hp levels. Our studies shown that, in immunodiffusion and immunoturbidimetric assays, the immunoreactivity of Hp 1-1 was markedly higher than 2-1 and 2-2, while an opposite result was observed using an ELISA. The latter was primarily due to the repeated antigenic epitopes in polymeric 2-1 and 2-2. Thus, Hp levels could be significantly over- or underestimated depending on the method. An accurate ELISA could be achieved when using each typespecific Hp calibrator matched to each type subject. We show the mean levels of Hp 1-1 subjects (n=16; 184±42 mg/dL) to be significantly and differentially greater than 2-1 (n=28; 153±55 mg/dL) (pb0.05) and 2-2 (n=24; 93±54 mg/dL) (pb0.01) subjects. Due to the diverse immunochemical structure among the Hp types, phenotyping should be performed in all the patients and a type-matched Hp calibrator should be used in clinical Hp determination.
To summarize, (1) Hp phenotypes and its circulation levels could therefore be applied in the diagnosis of diseases and allow treatment to be better adapted to suit patient needs. (2) Hb extracts hydrogen from poly unsaturated fatty acids (including LDL, cell membrane, and linoleate) to generate and it produce ROS generation and stimulate ICAM-1/VCAM-1 expression. Thus, use of antioxidant reagents and peroxidase inhibitor may be the possible solution to Hb-induced atherogenic problems. (3) Hp 1-1 phenotype was an independent risk factor for significant CAD with two-times greater OR than either Hp 2-1 or 2-2 phenotype. (4) Phenotyping should be performed prior to the determination process of plasma Hp levels to ensure accuracy in use for clinical diagnosis.
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