Haptoglobin 在臨床上之重要性

Abstract

Haptoglobin (Hp) 最早被認知的重要生理功能是:結合並代謝掉自紅血球破裂後釋放出的血紅素, 以預防血紅素造成的氧化壓力並且降低其對組織的傷害。 同時 Hp 是一個急性期 (acute phase) 會大量表現的蛋白質, 使得個體在遭受細菌感染時 Hp 在血液中的濃度增加,負責將細菌增殖所需的鐵離子清除, 抑制細菌的生長 。 最近本實驗室更進一步發現 Hp 是一個很強的抗氧化蛋白質, 其強度更甚於抗氧化最強藥物之一的 probucol (抗動脈硬化藥物) 。 如同血型一般 Hp 也分有三個型, 每個人擁有自己特有的一個型。 分別是: Hp 1-1, Hp 2-1 以及 Hp 2-2。 此現象是由於在人的 16 號染色體上有兩個基因座 (allele) 表現 Hp 蛋白質 (Hp 1, Hp 2) 。 Hp 1-1 和 Hp 2-2 是同型結合子 (homozygous) 的基因表現, Hp 2-1是異型結合子(heterozygous) 的基因表現。 蛋白質結構方面: Hp 1-1 由兩個單體共價鍵結合形成的二聚體 (dimmer), Hp 2-1 及 Hp 2-2 分別是線型 (linear form) 及 環型 (cyclic form) 的結構, 以各種不同數目的單體據合成一系列不同大小的蛋白質結構。 不同 type 的 haptoglobin 由於在蛋白質體結構上的差異, 因此在生理上具有不同的意義及其重要性。 在流型病學上, 不同的 phenotype 在某些疾病上有不同的發生率及致死率。 例如: 心血管疾病、自體免疫、及癌症等。 同時流行病學及人類學家更進一步的發現, 如同不同疾病的發生率再族群及區域上的差異, Hp 1-1 在族群及種族之間有極大差異。 更進一步, 本論文發現在 Hp 1-1 的個體, 其血漿中 Hp 濃度遠大於其他兩個 type 的濃度。 因此 Hp 不同 type 在濃度上的差異, 在生理上應該扮演重要的功能。 但是, 因為定量上的困難, 在過去數十年有關Hp的研究, 有關濃度在生理上的重要性, 一直被忽略。 本論文將就血紅素及 Hp 在生理上的重要性、Hp 的 phenotypes、 濃度之間的差異在生理功能的意義 及 Hp 1-1 在不同族群、區域的差異所導致跟疾病之間的關係做進一步的探討。

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.