新生兒高膽紅素血症之環境因子、自由基與基因多型性關係的探討及臨床意義

Abstract

新生兒黃疸是常見且讓醫師與父母擔心的問題。它的發生率與嚴重程度有種族上之差異，亞洲民族較歐、美、非民族來得高且嚴重，這意味著新生兒黃疸的發生與遺傳因子相關。 黃疸的發生是因為血液中紅血球遭破壞後，血質釋放出來，經一連串之代謝產生膽紅素堆積於體內，外顯出黃疸之症狀。除了外顯出黃疸以外，膽紅素本身也會造成神經之傷害。然而膽紅素也不全然是不好的東西。哺乳動物比低等動物多演化出膽紅素之形成，有其生理上之意義！目前所知，新生兒膽紅素的產生可以當作是生理上抗氧化物質來箝制過多的反應氧化物質。 胎兒是處在低氧環境且體內少抗氧化物質的平衡狀態；當胎兒被娩出成為新生兒時，環境間氧氣上升二至三倍，體內的抗氧化物質卻來不及因應，過多的活性氧(reactive oxygen species）因此而產生。大量增加的活性氧會破壞紅血球產生血質而進入血質代謝過程，而產生膽紅素。膽紅素的產生可能被當作是新生兒暫時性生理之抗氧化物質。因此，藉由臍帶血中反應氧化物質的量來預測新生兒高膽紅素血症便是我們研究的基本假設。 雙磷尿核苷-尿苷酸轉移酶是膽紅素代謝過程中最重要之酵素，它負責將非結合型之膽紅素轉換成結合型。文獻上雙磷尿核苷-尿苷酸轉移酶其基因（UGT1A1）之變異會導致一些與稽延性黃疸疾病，如Crigler-Najjar或是Gilbert''s 症候群的發生。文獻也發現在西方民族這基因啟動子上TATA盒之變異與新生兒高膽紅素血症是有相關的。然而近期對於日、韓與台灣之研究，卻發現在這基因的外顯子211位置的變異（211Gly71Arg）與新生兒高膽紅素血症較相關。這也代表不同種族間存在UGT1A1基因多型性之差異。 隨著母乳哺育之推廣與產後即早出院的趨勢，尋找出新生兒高膽紅素血症高危險群是臨床醫師努力之方向。目前廣被使用的是依出生後小時大相對應膽紅素之列線圖，但是其敏感度只有56%。 母乳哺餵有很多生理、心理與社會層面之好處，也是全世界在推廣之母嬰照護政策。但哺餵母乳與新生兒膽紅素血症之發生與嚴重性已有強烈之證據證明兩者之關係。然而，哺餵母乳與引起新生兒膽紅素血症的確切機轉則仍未明，較主流的說法是卡洛里之攝取不足、腸肝循環增加所致等。最近大規模的研究顯示是禁食而非母乳本身引起新生兒膽紅素血症。也有學者指出，在成人UGT1A1有基因TATA盒或是編碼區之變異者較會因禁食而引起血液中膽紅素值之上升。我們因此探討因母乳哺餵與UGT1A1基因多型性對出生一週內之新生兒高膽紅素血症之影響。 1. UGT1A1基因在211Gly71Arg 變異與哺餵母乳性黃疸之關係 方法：前瞻式地收集688個足月與幾近足月兒的臍帶血，之後將依其哺餵方式分成完全哺餵母乳組與添加餵食組，並紀錄出生後七天內每天體重之下降、膽紅素值與其他完整醫療資訊。UGT1A1基因序列研究以臍帶血分離去氧核醣核酸（DNA）並以變性高壓異象層析分析與直接定序分析啟動子與外顯子1的多型性。最後將兩組間基因多型性與高膽紅素血症發生狀況做比較。 結果：研究群體中在外顯子單條變異、啟動子單條變異、複合式單條變異、外顯子雙條變異與啟動子雙條變異的發生率分別是24.7%、16%、 3.5%、 2% 與2.2% 。在完全哺餵母乳組如果有外顯子211Gly71Arg單/雙條變異的新生兒，其最高膽紅素值與高膽紅素血症之發生率都會較其他變異來得高。有趣的是，在添加餵食組卻看不到同樣之現象。顯然外顯子211Gly71Arg單/雙條變異對於國人高膽紅素血症之影響較大，而且這基因多型性對新生兒高膽紅素血症的影響還需要有完全哺餵母乳這個因素存在。 結論：我們呈現了完全哺餵母乳組有較多的體重下降、較高的最高膽紅素值與高膽紅素血症之發生率，而且外顯子211Gly71Arg單/雙條變異對於完全哺餵母乳者的高膽紅素血症影響較大。母乳哺餵性黃疸與基因多型性存在著表現基因學上的關係。 2. 用臍帶血的氧化物質（過氧化氫，H2O2）來預測新生兒高膽紅素血症 方法：前瞻式地收集足月與幾近足月兒的臍帶血、三天大與五天大的腳跟血，檢測其全血中的過氧化氫、血比容與膽紅素值。並將研究族群依最高膽紅素值是否大於等於17 mg/dl區分成嚴重與較不嚴重高膽紅素血症兩組。比較兩組之間各數值之差異，並以接受者操作特徵曲線分析來取得最佳之過氧化氫訊號數值切點來預測嚴重型高膽紅素血症，同時計算其敏感度、特異度與陰性預測值。 結果：共有158個新生兒被納入研究。若依膽紅素13 mg/dL、15 mg/dL、17 mg/dL以上當定義，可得到新生兒黃疸的發生率分別為48.7% 、30.5%與13.3%。剛出生前5天，膽紅素逐漸上升，過氧化氫訊號也依相同之趨勢逐漸上升，兩者上升趨勢達統計上之相關性；再經離散分析，膽紅素值與過氧化氫訊號亦呈現相關性。如果取過氧化氫訊號2500 counts/10秒當作切點來預測嚴重高膽紅素血症的發生，其敏感度與陰性預測值分別為76.2% 與93.3%。 結論：我們證明了過氧化氫訊號與膽紅素值間呈現高度相關性，並以過氧化氫訊號2500 counts/10秒有相當之能力來預測嚴重高膽紅素血症的發生。這方法可以提供臨床上哪些新生兒需要被密切追蹤膽紅素值。 3. 探討其他可能影響新生兒高膽紅素血症之因子 我們探討了在推廣母嬰親善後(200年7月），比較在母乳哺餵率上升之後，新生兒黃疸發生率也隨之上升，相對地體重下降幅度較大、出生後前幾天之排便次數減少。 在2002年3月到2005年7月，我們也將完全哺餵母乳之新生兒，分析體重下降幅度與高膽紅素血症之關係，發現完全哺餵母乳之新生兒體重下降幅度達11%以上者，能預測接下來高膽紅素血症的發生。 最後用妊娠週數、體重降最大幅度與出生後頭72小時內的最高膽紅素值建立母乳哺餵新生兒高膽紅素血症預測模式，經2009年1到4月另一族群做外部確認，證實母乳哺餵新生兒高膽紅素血症預測模式之可行性。

Neonatal hyperbilirubinemia is a common problem with the incidence around 60~70%. The peak serum levels of unconjugated bilirubin in full-term Asian and American Indian neonates are almost double as those in Caucasian and black populations. The finding suggests that genetic factors are involved in the development of neonatal hyperbilirubinemia. Breastfeeding jaundice is common in exclusively breastfed neonates, but its pathogenesis is still unclear. The uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) gene polymorphism was shown to contribute to the development of neonatal hyperbilirubinemia. We hypothesize that the variation of UGT1A1 gene may contribute to neonatal breastfeeding jaundice. 211 G to A Variation of UDP-Glucuronosyl Transferase 1A1 Gene and Neonatal Breastfeeding Jaundice We prospectively enrolled 688 near term & term infants who were exclusively breast-fed (BF group) or were supplemented by infant formula partially (SF group) before onset of hyperbilirubinemia. Genotyping of the promoter and exon1 of UGT1A1 was performed in all neonates. Neonates in BF group had a significantly higher maximal body weight loss ratio, peak bilirubin level, and a greater incidence of hyperbilirubinemia than those in SF group. Neonates with nucleotide 211 GA or AA variation in UGT1A1 genotypes had higher peak serum bilirubin levels and higher incidence of hyperbilirubinemia than wild types (GG). This phenomenon was only seen in BF group but not in SF group when we do subset analysis. This suggests that neonates who carry the nucleotide 211 GA or AA variation within coding region in UGT1A1 gene are more susceptible to develop early-onset neonatal breastfeeding jaundice. Prediction of Severe Neonatal Hyperbilirubinemia Using Cord Blood Hydrogen Peroxide: A Prospective Study Given the relationship between bilirubin and hydrogen peroxide, which is one of reactive oxygen species, we hypothesized that cord blood hydrogen peroxide could be utilized to predict the severity of neonatal hyperbilirubinemia. We prospectively enrolled 158 term or near-term healthy neonates. Cord blood and capillary blood at three and five days of age were measured for hydrogen peroxide and bilirubin concentrations. Newborns were divided into severe or less severe hyperbilirubinemic groups. The rising patterns were similar among bilirubin concentrations and hydrogen peroxide levels during the first few days of life. There was a strong positive correlation between bilirubin concentrations and hydrogen peroxide levels after correlation analysis. It revealed that a cord blood hydrogen peroxide signal level of 2500 counts/10 seconds was an appropriate cut-off for predicting severe hyperbilirubinemia. Sensitivity and the negative predictive value were 76.2% and 93.3%, respectively. Our findings confirm that hydrogen peroxide levels and bilirubin concentrations in cord and neonatal blood are closely related. A cord blood hydrogen peroxide level above 2500 counts/10 seconds associated with a high predictive value for severe hyperbilirubinemia. Other factors associated with neonatal hyperbilirubinemia To evaluate the impact of breast-feeding promotion on the incidence of hyperbilirubinemia, on peak bilirubin level and on mean days of hospitalization. Total 1273 healthy full-term newborns born before and after the Baby Friendly Hospital Initiative Program was launched in July 2000. There was a significant association between monthly rates of breast-feeding and the incidence of hyperbilirubinemia, BW loss ratio, the bilirubin level on the third or fourth day, the peak bilirubin level, the mean hospital days, the mean days of phototherapy, as well as the frequencies of urination and defecation. A total of 874 neonates were exclusively breastfed and subsequently enrolled in this study. We analyzed the association between weight loss percentage and hyperbilirubinemia and investigated the best weight loss percentage cut-off value for prediction of subsequent hyperbilirubinemia before two weeks of age. Neonates with lower gestational age (GA) and greater weight loss percentage were associated with hyperbilirubinemia. By using weight loss > 8% of BBW after 48 hours and weight loss > 11% of BBW after 72 hours as the cut-off values for prediction of subsequent hyperbilirubinemia, respectively. We also retrospectively collected exclusively breast-feeding healthy term and near-term newborns born in our nursery between May 1 2002 to June 30 2005. Finally, 771 newborns were enrolled and 182 (23.6%) cases developed significant hyperbilirubinemia during 4th to 10th day of life. In the logistic regression analysis, gestational age, maximal body weight loss percentage and peak bilirubin level during the first 72 hours of life were significantly associated with subsequent hyperbilirubinemia. A prediction model was derived with the AUROC curve of 0.788. Model validation in the separate study (N=209) showed similar discrimination capability (AUROC=0.8340). Gestational age, maximal body weight loss percentage and peak serum bilirubin level during the first 3 days of life have highest predictive value of subsequent significant hyperbilirubinemia. We provide a good model to predict the risk of subsequent significant hyperbilirubinemia.