- 學位論文
肝癌患者的維生素B-6及穀胱甘肽的變化與氧化壓力及抗氧化能力的相關性
The Associations of Vitamin B-6 and Glutathione Changes with Oxidative Stress and Antioxidant Capacities in Patients with Hepatocellular Carcinoma
摘要
肝細胞癌(hepatocellular carcinoma)，簡稱肝癌，為最常見的原發性肝腫瘤。肝癌發生率雖在全球男性癌症排名第五位，女性排名第九位，但卻高居全球癌症死因的第二位。當肝細胞暴露在病毒、藥物、酒精及內毒素的環境中，這些物質不僅會造成肝細胞損傷也會進而引起一連串的免疫及發炎反應。在發炎反應過程中會增加活性氧及活性氮的生成，使得體內氧化還原機制失去平衡，增加體內氧化壓力，進而導致細胞不正常的增生及肝癌細胞的形成。除了醫療介入之外，若能降低肝癌患者的氧化壓力，或是提供抗氧化營養素的補充以改善肝癌患者體內的抗氧化-氧化平衡機制，或許能提高存活率以及降低肝癌的復發。 維生素B-6因其化學結構所含的氫氧基（－OH）及胺基（－NH2）被認為可直接與過氧化物自由基結合而清除自由基及降低脂質過氧化。此外，維生素B-6參與同半胱胺酸的轉硫代謝，間接合成穀胱甘肽，穀胱甘肽及其相關的抗氧化酵素是體內重要的抗氧化防禦系統。因維生素B-6與與穀胱甘肽主要在肝臟代謝，若肝功能受損，可能會影響維生素B-6與穀胱甘肽所擔任的抗氧化的生理功能。目前有關維生素B-6及穀胱甘肽在肝癌患者接受腫瘤切除前後的變化，以及與氧化壓力和抗氧化能力的關係尚未被全面檢視，且維生素B-6應用於腫瘤切除後的肝癌患者之臨床介入研究並不多。因此，本研究以橫斷面研究模式，觀察及比較肝癌患者接受腫瘤切除手術前後與健康受試者的維生素B-6營養狀態、同半胱胺酸濃度與氧化壓力及抗氧化能力指標的差異。結果發現：肝癌患者接受腫瘤切除手術前較健康受試者有顯著較低的血漿磷酸比哆醛(維生素B-6的輔酶型式)濃度、穀胱甘肽過氧化酶及超氧岐化酶活性；但有較高的氧化壓力指標值及穀胱甘肽硫轉移酶活性。不過，肝癌患者血漿磷酸比哆醛濃度與血漿同半胱胺酸濃度、氧化壓力及抗氧化能力指標數值皆無顯著相關性。當肝癌患者長期處於較高的氧化壓力狀態，可能會增加血漿磷酸比哆醛的消耗與代謝，以及降低肝臟維生素B-6的儲存。因此，進一步以雙盲隨機介入研究模式，給予接受腫瘤切除手術後之肝癌患者高劑量維生素B-6 的補充劑，探討對同半胱胺酸、氧化壓力及抗氧化能力的影響。結果發現：每天50毫克的維生素B-6補充12週後，血漿同半胱胺酸和穀胱甘肽濃度，還原型和氧化型穀胱甘肽比值以及穀胱甘肽還原酶活性顯著降低，但總抗氧化能力顯著上升。以多重迴歸分析顯示：維生素B-6的補充會顯著降低肝癌術後患者的血漿同半胱胺酸濃度(훽 = -2.4, 푝 = 0.02)，而血漿同半胱胺酸濃度的降低會進而增加總抗氧化能力(훽 = -162.0, 푝 = 0.03)。維生素 B-6的介入，似乎不會直接增加肝癌術後患者的總抗氧化能力，而是間接透過降低血漿同半胱胺酸濃度進而增加總抗氧化能力。維生素B-6因間接參與穀胱甘肽合成，理論上維生素B-6濃度的變化應該會影響穀胱甘肽濃度及其相關抗氧化酵素活性。但是，在此研究中卻無法解釋高劑量的維生素B-6補充後，為何肝癌術後患者還原態與氧化態穀胱甘肽的比值以及榖胱甘肽還原酶活性顯著降低，但氧化態穀胱甘肽以及榖胱甘肽過氧化酶活性仍維持沒有顯著變化。因此，以橫斷面研究模式，觀察及比較肝癌患者腫瘤及鄰近正常組織以及接受腫瘤切除手術前後之穀胱甘肽及其相關的抗氧化酵素活性與氧化壓力之差異性及關係。結果顯示：肝癌患者腫瘤切除前較腫瘤切除後有顯著較高的氧化壓力指標數值以及顯著較低的血漿穀胱甘肽及其相關的抗氧化酵素活性。但是，肝臟腫瘤組織卻較鄰近正常組織有顯著較低的氧化壓力指標數值以及高的穀胱甘肽的濃度及其相關的抗氧化酵素活性；且血漿穀胱甘肽濃度與腫瘤組織的穀胱甘肽濃度及總抗氧化能力呈現顯著相關性。比起鄰近正常組織，肝臟腫瘤組織似乎更能將血漿的穀胱甘肽保留在腫瘤組織中，進一步保護腫瘤組織抵抗氧化壓力。 總結上述三個研究，維生素B-6濃度變化雖然與肝癌患者體內氧化壓力及抗氧化能力指標無直接顯著的相關性，但可藉由補充高劑量的維生素B-6，降低肝癌腫瘤切除後患者的血漿同半胱胺酸濃度，進而間接增加其總抗氧化能力。此外，肝臟腫瘤組織會藉由將血漿穀胱甘肽保留在腫瘤組織中，使腫瘤組織得以在高氧化壓力環境中生存。
並列摘要
Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Although HCC is the fifth most prevalent cancer in men and the ninth in women, it is the second most common cause of cancer-related death worldwide. Viruses, drugs, alcohol, and endotoxins not only cause liver damage, but also initiate a series of immune and inflammatory cascades. In addition to inflammatory responses, oxidative stress would be increased due to imbalance between production of free radicals like reactive oxygen species and reactive nitrogen species, and the innate antioxidant ability, which might lead to abnormal cell proliferation, oncogenesis, and tumor-progression. In addition to medical interventions, it might be possible to reduce the recurrence rate and improve survival parameters of patients with HCC by reducing oxidative stress or providing antioxidant nutrient supplementation. Vitamin B-6 is capable of directly binding and scavenging peroxy radicals. This can lead to reduced lipid peroxidation through the substitution of hydroxyl (-OH) and amine (-NH2) groups on a pyridine ring in vitamin B-6 compounds. Pyridoxal 5-phosphate (PLP, a coenzyme form of vitamin B-6) serves as a coenzyme in the transsulfuration of homocysteine and is indirectly involved in the synthesis of glutathione (GSH). Glutathione and its dependent antioxidant enzymes play a fundamental role in cellular defense against reactive free radicals and other oxidant species in the human body. Liver is the organ responsible for metabolism and turnover of Vitamin B-6, and hepatic dysfunction might affect the physiological function of vitamin B-6 and GSH, possibly leading to dysregulation of vitamin B-6 and GSH-dependent antioxidant system. To date, the effects of disrupted vitamin B-6 and GSH metabolism on oxidative stress and antioxidant capacities in patients with HCC before and after tumor resection have not been fully investigated. In addition, few intervention studies have been done to determine the application of vitamin B-6 supplementation in HCC patients after receiving tumor resection. Therefore, we conducted a cross-sectional observational study to compare the changes in vitamin B-6, homocysteine, oxidative stress, and antioxidant capacities in HCC patients before and after tumor resection, with those of healthy controls. Results revealed that patients with HCC before tumor resection had significantly lower plasma PLP, glutathione peroxidase (GSH-Px), and superoxide dismutase activities but higher oxidative stress indicators and glutathione S-transferase activity when compared to healthy controls. There was no significant association of plasma PLP level with plasma homocysteine, indicators of oxidative stress, and antioxidant capacity. If HCC patients have increased plasma homocysteine and oxidative stress, and decreased antioxidant capacities, this may possibly accelerate the metabolic turnover of plasma PLP and subsequently decrease vitamin B-6 reserves in the liver. A randomized, double-blind intervention study was further conducted to investigate the effects of high dose vitamin B-6 supplementation (50 mg/day) on homocysteine, oxidative stress, and antioxidant capacities in HCC patients following tumor resection. Results indicated that the plasma homocysteine and GSH concentrations, the ratio of reduced and oxidized GSH (GSH/GSSG ratio), and activity of glutathione reductase (GSH-Rd) in the vitamin B-6 group were significantly decreased at week 12, while total antioxidant capacity (i.e., trolox equivalent antioxidant capacity, TEAC) level was significantly increased at the end of the intervention period. Multiple linear regression analysis showed that vitamin B-6 supplementation significantly lowered plasma homocysteine levels (β = -2.4, p = 0.02), but not the change of TEAC levels. However, the changes in plasma homocysteine concentrations had a significant effect on the change of TEAC levels (β = -162.0, p = 0.03) after adjusting for potential confounders. Vitamin B-6 supplementation seemed to mediate antioxidant capacity indirectly by reducing plasma homocysteine rather than by having a direct antioxidant effect in HCC patients who underwent tumor resection. Theoretically, the change in vitamin B-6 concentration might have an influence on plasma GSH level and activities of its-dependent antioxidant enzymes, since vitamin B-6 has an indirect role in GSH synthesis. However, we could not explain why the GSH/GSSG ratio and GSH-Rd activity were reduced, while the GSSG level and GSH-Px activity remained constant after a large dose of vitamin B-6 supplementation. A cross-sectional study was then conducted to determine the changes in oxidative stress, GSH level, as well as its dependent antioxidant enzyme activities in patients with HCC at pre- and post-tumor resection. We investigated the association of oxidative stress with GSH and its dependent antioxidant enzyme activities in plasma and tissues. Our results showed that HCC patients had significantly increased oxidative stress indicators but decreased plasma GSH level and GSH-dependent antioxidant enzyme activities before tumor resection. In contrast, HCC tissue had significantly increased GSH and TEAC levels and activities of GSH-Px when compared with the adjacent normal tissue. Plasma GSH correlated significantly with tumor GSH and TEAC levels. It is possible that the hepatocellular tumor may take up more GSH from circulation than adjacent normal tissue, to protect itself against increased oxidative stress. In summary, results from our three studies in HCC patients showed that changes in vitamin B-6 concentration had no direct association with indicators of oxidative stress and antioxidant capacities, but a large dose of vitamin B-6 supplementation could increase antioxidant capacity indirectly by reducing plasma homocysteine, rather than having a direct anti-oxidant effect in HCC patients. In addition, the hepatocellular tumor could possibly protect itself against increased oxidative stress by the redistribution of GSH from plasma to HCC tissue during HCC progression.