以微量體液化學發光法定量早產兒血液中活性氧及抗氧化狀態來評估與視網膜病變嚴重度之相關性(2/2)

Abstract

新生嬰兒的生產過程中，常會合併有氧化自由基的侵襲；因為胎兒在母體的子宮內是處 於相對性的缺氧狀況，血氧分壓為20 到50 毫米汞柱，但出生後立即暴露於相對高氧狀態， 血氧分壓為100 毫米汞柱，肺上皮細胞更直接接觸於氧分壓為140 毫米汞柱的空氣中，因此 新生兒一誕生，需馬上面對氧化侵襲的嚴苛考驗。 近年來的研究認為氧氣、一氧化氮等自由基的過量產生可能會造成新生兒，尤其是早產 兒的各種疾病，包括新生兒慢性肺疾，早產兒視網膜病變，壞死性腸炎，溶血性黃疸等。在 懷孕週數低於28 週的早產兒，視網膜病變發生率甚至可高達80％，其中的百分之二十會有 進行性的變化，嚴重者甚至造成視網膜剝離或失明。針對視網膜病變的致病機轉，近年來的 研究傾向於和早產兒體內的活性氧及抗氧狀態有相關性，但是仍缺乏一致性的結論。無法下 定論的其中原因，也許是新生兒單單早產本身就會出現視網膜病變，另一方面也有可能和研 究方法有關，大部分檢查以測試蛋白質或脂類的氧化產物作為間接證據，僅有少數研究直接 測試glutathione 的氧化還原狀態來評估與視網膜病變之相關性，此外，檢驗項目的特異性 值得商榷。台大醫院醫學研究部近年來發展出新的化學發光測定法，僅需採用微量體液即可 分析血中的活性氧化自由基，而且不易造成早產兒的醫源性貧血。針對住在加護病房的早產 兒，在接受氧氣的治療後，可能引起氧化自由基的上昇，造成進一步早產兒的各種病變，值 得作相關的研究。我們希望採用這種化學發光測定法研究活性氧自由基與早產兒視網膜病變 之嚴重度的相關性。 在兩年的研究期間，我們針對早產兒共收集45 例，其中4 例最後出現嚴重的視網膜病 變，需接受開刀治療。出生體重分別為2119 ± 997 公克及1195 ± 598 公克(p = 0.042)，懷 孕週數分別為33.71 ± 4.77 週及28.50 ± 4.04 週(p = 0.041) 。針對這些早產兒分別抽取臍 帶血及出生後第三天例行篩檢時的微量血液作檢驗，比較兩組間之差異；我們發現出生三天 後血中的活性氧自由基比起臍帶血明顯的上昇，確認出生後新生兒對於新環境中相對高氧濃 度的反應。但是兩組間之差異僅於出生後第三天的Luminol-dependent ROS 有統計上的意 義。此外針對這些早產兒分別於出生後第7、14、21、28、35、42、49、56 天分別留左右眼 收集淚液來測定淚液中的自由基；並且由視網膜專家定期追蹤與評估早產兒視網膜病變的發 生率與嚴重度，探討氧化自由基及抗氧化狀態與早產兒視網膜病變之嚴重度的相關性我們發 現絕大部份兩組間之差異並不明顯僅在第八週發生視網膜病變的早產兒淚液中的活性氧自 由基比未發生視網膜病變的早產兒具統計上有意義較高的現象，但是由於眼淚收集的不容易 導致收集量少亦可能影響研究的結果。

Neonates undergo a dramatic change during the process of childbirth by an increase in oxidative aggression. The fetus exchange a low oxygen intrauterine environment, with PO2 of 20-25 mmHg and a low presence of free radicals, for another with a relative high oxygen extrauterine environment, with PO2 of 100 mmHg. This change results in considerable oxidative stress. Damage due to free radicals, including reactive oxygen species and reactive nitrogen species, is thought to be one of the common mechanisms for several neonatal diseases especially in preterm infants. Chronic lung disease of neonate, retinopathy of prematurity, necrotizing enterocolitis, and hemolytic diseases of neonate are accepted as caused by the excessive production of oxygen or nitrogen free radicals. The oxidative aggression suffered by the neonate is counteracted by the maturation of complex antioxidant defense systems, including both enzymatic systems (superoxide dismutase, catalase, glutathione peroxidase, etc.), and non-enzymatic systems (vitamins E, A, C ). Despite the knowledge that has been advanced in recent years regarding the oxidative stress and antioxidant mechanism, there have remained questions about the maternal-fetal transfer of antioxidant defense mechanism and the free radical and antioxidant status in neonates during perinatal period. Retinopathy of prematurity (ROP) is a disease of the incompletely vascularized immature reteina characterized by retinovitreal neovascularization. It develops in more than 80% of premature survivors born at <28 weeks gestation. Some evidences, by estimating the associations between protein oxidation and lipid peroxidation products and retinopathy of prematurity, indirectly suggest that reactive oxygen species and reactive nitrogen species may play important roles in the pathogenesis of ROP. However, direct evidence that oxidation plays a causative role in ROP is limited. It is not always clear whether the association between oxygen and ROP just reflect a higher disease incidence in the very-low- birth-weight infants who receive more oxygen. Besides, the assay procedures of protein oxidation and lipid peroxidation products may be different and nonspecific, newer and more sensitive tests is warranted. We have developed to use a characteristic emission spectrum analysis of the chemiluminescence (CL-spectrum) with small amount of body fluids for the first time to evaluate the specific reactive oxygen species (ROS) activity including H2O2 and HOCl in the plasma and to adapt a chemiluminescence-high performance liquid chromatography (CL-HPLC) for measurement of PCOOH before and after the dialysis session, in the absence and presence of antioxidant treatment. We hypothesize that the contents of free radicals and the antioxidant defense system are III different between the preterm infants with ROP and those without ROP. We propose a two-year research project. During the first year of our study, 10 preterm and 14 term infants had been enrolled. We found that the plasma ROS level was significantly higher then those of the cord blood. However, the levels were not affected by the gestational age, the gender and the mode of delivery. The elevation was supposed due to the oxidative stress after the birth. During the second year of our study, we continually collected the tears from the preterm infants. Totally 45 infants were enrolled including 41 infants without retinopathy of prematurity and 4 infants with retinopathy of prematurity. We found that the plasma ROS levels at the age of three says were also significantly elevated after birth when compared with those of the cord blood both in the preterm infants with ROP and those without ROP. This result was compatible with our first year study. Again, we believed that the neonates suffered from oxidative stress after birth. In the analysis of ROS in tears of the preterm infants, we found that both of the Luminol-dependent ROS and Lucigen-dependent ROS were not significantly different at the 7, 14, 21, 28, 35, 42 and 49 days of age. The difference was significant at the eighth week of age. We speculated that the difference was associated with the development of ROP. However, that collection of the tears from the preterm infants was not satisfactory and the amount of the tears was usually limited. The limitation of the samples collection may substantially affect the results of our study. Further improved methods for the analysis of tears ROS is necessary for the interpretation of the pathophysiology of ROP.