臺灣博碩士論文加值系統

急性鎘中毒試驗(14~38 ppm)中，吳郭魚和烏魚體內鎘的蓄積量，初期以魚鰓內最高，其次為肝臟；魚鰓及肝臟內鎘蓄積量隨曝露時間延長而增加，魚鰓於曝露50小時後蓄積量達最高，肝臟於曝露50~90小時內大幅增加；血液及肌肉中，鎘蓄積情形均不明顯。
吳郭魚曝露在含鎘19、28及38 ppm水中，隨鎘濃度增加及曝露時間延長，魚體血液值有變異現象，在曝露49小時後，其白血球數目(WBC)、紅血球數目(RBC)、血比容積(Hct)、血色素濃度(Hgb)，及平均血色素濃度(MCHC)均下降，在曝露19小時，降至最低，紅血球有變大現象。
在體內試驗中，鎘誘發魚鰓細胞中脂氧合(lipoxygenase, LOX, E.C. 1.13.11.12)活性。吳郭魚鰓LOX活性隨曝露於19或28 ppm鎘之時間延長而上升，在曝露19或20小時後，達到最高，以5-及12-LOX活性明顯增加，但15-LOX則隨曝露於鎘時間延長而下降，在曝露於含鎘14 ppm 水中20小時後，烏魚鰓5-, 12-及15-LOX活性均達最高，吳郭魚或烏魚鰓中有類似dihydroxyeicosatetraenoic acid (di-HETE)之產物或白三烯素(leukotriene, LT)之形成。
試管試驗中，鎘抑制魚鰓LOX活性。LOX抑制劑，nordihydroguaiaretic acid及diphenyl sulfide，同時降低5-LOX催化花生四烯酸(C20:4)形成之5-氫過氧化物(hydroperoxy eicosatetraenoic acid, 5-HpETE）產量與LT合成催化5- HpETE形成LTs之產量，如同在老鼠嗜鹼性白血球中分離出之單一蛋白質具有此二種酵素活性，顯示吳郭魚、烏魚鰓之5-LOX與LT 合成亦可能為同一蛋白質。
分別以迴流加熱90分鐘及沸水浸泡5分鐘所得含固形物47~57 ppm 之綠茶萃取液(green tea extracts, GTE)，對吳郭魚及烏魚鰓5-，12-及15- LOX有抑制作用，兩種萃取液之抑制效果相當，以GTE與C20:4混合恆溫靜置後，再與吳郭魚LOX作用產物與發光劑luminol反應，產生之冷光強度減少98.5 %；GTE對吳郭魚鰓LOX活性之抑制率為90.2 %，其抑制率隨GTE濃度增加而上升。GTE亦抑制血紅素催化亞麻油酸(C18:2) 的氧化作用，其抗氧化效果較83 ppm維生素E為高，在模式試驗中，添加GTE於吳郭魚或烏魚紅血球中達固定血比容積後，其血液流體行為符合冪次方程式 =K n + C，GTE可降低吳郭魚及烏魚血液黏度，與使流體特性更趨於牛頓型，並增加血液的流動性，多飲用綠茶可能有助於心血管功能正常。

When tliapia (Oreochromis niloticus  O. aureus) and grey mullet (Mugil cephalus) were exposed to acute toxicity concentration of 14~38 ppm cadmium (Cd), gill followed by liver accumulated the highest concentrations of Cd which increased as the exposure time and Cd concentration in water increased. Maximal accumulation reached when the fish were exposed to Cd for 50 h. Prolonged exposure from 50 to 90 h, Cd accumulation increased apparently in liver. Muscle and blood showed no significant increase in Cd.
Acute toxicity of 19, 28 and 38 ppm Cd caused conspicuous hematological anomalies in tilapia. Tilapia exposed to Cd for up to 49 h showed changes in white blood cell count (WBC), red blood cell count (RBC), hematocrit (Hct), hemoglobin (Hgb), and mean corpuscular hemoglobin concentrations (MCHC), all of which decreased apparently and erythrocytes become macrocytie after19 h.
In-vivo studies showed that lipoxygenases (LOX, E.C.1.13.11.12) of tilapia gill were induced to the highest activities when the fish were exposed to 28 ppm Cd for 19 h, or 19 ppm for 20 h, while 5-, and 12-LOX increased in activities and 15-LOX decreased. Gill 5-LOX of grey mullet was induced to the highest activity when the fish were exposed to 14 ppm Cd for 20 h, but activities of 12-, and 15-LOX induced to the highest after 50 h exposure. Dihydroxy- eicosatetraenoic acid (di-HETE) or leukotrienes (LTs) were detected in gill tissues of both tilapia and grey mullet.
In-vitro studies showed that Cd inhibited the tilapia gill LOX. Inhibitors of LOX, nordihydroguaiaretic acid and diphenyl sulfide inhibited 5-LOX-catalyzed arachidonate oxidation producing 5-hydroperoxyeicosatetraenoic acid (5-HpETE) and LT synthase-catalyzed 5-HpETE reaction producing LTs both in tilapia and grey mullet gill. It seemed that 5-LOX or LT synthase activities resided on a single protein in tilapia and grey mullet gill similar to the protein possessing both activities characterized from rat basophilic leukemia (RBL-1) cells.
Green tea extracts (GTE) obtained by refluxing for 90 min or by soaking in 100 oC hot water for 5 min, containing a dry weight of 47-57 ppm inhibited 5-, 12-, and 15-LOX activities of fish gill. The two extracts had similar inhibitory effects. GTE was found to scavenge active oxygen radical. Similar inhibitory effects were found on LOX of tilapia gill and grey mullet gill. GTE was a stronger inhibitor of the hemoglobin-catalyzed oxidation of linoleic acid than vitamin E. Mixing GTE with tilapia or grey mullet red blood cells in-vitro resulted in a blood thinning effect. The rheological data fit power-law model,  =K n + C, where  = shear stress,  = shear rate, K = fluid consistency, n = flow behavior index. The addition of GTE reduced tilapia and grey mullet blood fluid consistency and caused a thinning consistency making the blood flow behavior becoming closer to Newtonian type.
These results suggest the antioxidants naturally occurring in green tea inhibited both hemoglobin-catalyzed oxidation of linoleic acid, and an inhibition of LOX activities in fish gill. Drinking of green tea may have preventive effect in blood thinning and atherosclerosis.

頁數
中文摘要--------------------------------------------- I
英文摘要--------------------------------------------- II
壹、 前言--------------------------------------------- 1
貳、 文獻整理-------------------------------------- 3
一、養殖魚的緊迫------------------------------------- 3
二、重金屬鎘對魚類的影響----------------------------- 3
1. 鎘在魚體的分布與蓄積------------------------ 4
2. 中毒機制------------------------------------ 4
3. 對血液的影響-------------------------------- 5
三、脂氧合之生理作用與功能------------------------- 5
1. 化學作用------------------------------------ 5
2. 在植物體之生理功能-------------------------- 6
3. 在動物體之生理功能-------------------------- 6
四、白三烯素之特性----------------------------------- 7
1. 命名來源------------------------------------ 7
2. 合成機制------------------------------------ 7
3. 生理功能------------------------------------ 7
五、發炎及過敏反應之抑制----------------------------- 8
六、脂氧合與動脈粥狀硬化的關係--------------------- 9
七、脂氧合及血液黏度的關係------------------------- 11
八、抗氧化劑對脂氧合與血液黏度的影響--------------- 12
九、綠茶之化學組成及生理功效------------------------- 13
參、 材料與方法-------------------------------------- 15
一、材料--------------------------------------------- 15
(一). 吳郭魚--------------------------------------- 15
(二). 烏魚----------------------------------------- 15
(三). 綠茶----------------------------------------- 15
(四). 化學藥品------------------------------------- 15
二、方法--------------------------------------------- 16
(一). 鎘急性毒試驗--------------------------------- 16
(二). 綠茶萃取液的製備----------------------------- 16
(三). 綠茶湯之脫色--------------------------------- 16
(四). 魚體一般生理指標之測定----------------------- 17
(五).血液值分析------------------------------------ 17
1. 採血---------------------------------------- 17
2. 紅血球個數(erythrocyte count)和血比容積(hematocrit,
Hct)---------------------------------------- 17
3. 白血球個數(leukocyte count)和血色素(hemoglobin, Hgb)
-------------------------------------------- 17
4. 平均血色素濃度(mean corpuscular hemoglobin
concentration, MCHC)------------------------ 17
(六). 鎘之定量------------------------------------- 18
(七). 綠茶液抗氧化性測定--------------------------- 18
(八). 魚血液流變性之測定--------------------------- 18
1. 採血---------------------------------------- 18
2. 黏度---------------------------------------- 19
(九). 蛋白質定量----------------------------------- 19
(十). 魚鰓脂氧合酵素液製備----------------------- 19
1. 去血鰓絲製備-------------------------------- 19
2. 魚鰓脂氧合之萃取-------------------------- 19
3. 脂氧合之部分純化-------------------------- 19
(十一). 脂氧合活性測定--------------------------- 20
1. 氧電極法(Oxygen consumption) --------------- 20
2. 吸光度法 ----------------------------------- 21
3. 冷光測定法 (chemiluminometric assay) ------- 21
4. 逆向高效液相層析法 (HPLC)------------------- 22
(十二). 白三烯素合成活性測定---逆向高效液相層析法-- 22
(十三). 抑制劑對5-脂氧合與白三烯素合成之作用----- 23
肆、 結果與討論-------------------------------------- 24
(一) 緩衝液之選擇---------------------------------- 24
1. Tris-HCl 緩衝液----------------------------- 24
2. 磷酸鉀緩衝液-------------------------------- 24
(二) 化學冷光法檢測脂氧合活性之最適條件---------- 25
1. 緩衝液pH值---------------------------------- 25
2. 花生四烯酸濃度------------------------------ 25
3. 標準品定量---------------------------------- 26
(三) 鎘在魚體內之蓄積與外觀變化-------------------- 26
(四) 鎘對魚鰓脂氧合活性之影響-------------------- 27
1. In-Vitro----------------------------------- 27
2. In-Vivo------------------------------------ 27
(五) 鎘對魚鰓白三烯素合成活性之影響-------------- 29
(六) 魚鰓脂氧合與白三烯素合成之關係------------ 30
(七) 鎘對魚血液特性之影響-------------------------- 32
1. 白血球-------------------------------------- 32
2. 紅血球-------------------------------------- 32
(八) 綠茶的抗氧化性-------------------------------- 33
1. 對魚鰓脂氧合作用之影響-------------------- 33
2. 對血色素催化自氧化之影響-------------------- 35
(九) 綠茶液對魚血液流變性之影響-------------------- 37
伍、 結論-------------------------------------------- 40
陸、 參考文獻---------------------------------------- 41
柒、 表---------------------------------------------- 53
捌、 圖---------------------------------------------- 64

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