以串聯式質譜分析方法研發乾濾紙血片乳酸和丙酮酸以及尿液胺基多醣雙醣之臨床應用

Development and Clinical Application of Tandem Mass Methods for Lactate and Pyruvate Quantifications in Filter-Paper Dried Blood Spot and Urinary Glycosaminoglycan Identifications

莊志光(Chih-Kuang Chuang)

指導教授 ： 劉宣良

國立臺北科技大學/工程學院/工程科技研究所/博士(2013年)

https://doi.org/10.6841/NTUT.2013.00192

摘要

現代醫學臨床實驗診斷強調低污染、快速、準確、低侵襲性及少量檢體之功能需求，質譜分析完全符合以上條件，是二十一世紀分析技術發展的主流。應用質譜分析設備之高敏感度、高特異性、高通運量、分析速度快、以及具備有多元應用之特質，讓我們可以突破傳統分析設備所無法全面施展的困境，充分提昇分析檢測之深度和廣度。質譜分析技術可以偵測到人體體液中微量的物質，包括荷爾蒙、藥物、代謝物、營養成分、以及一些微小物質，是作為轉譯醫學臨床診斷用途之最佳利器。利用串聯式質譜乾濾紙血片乳酸和丙酮酸定量檢測作為嬰幼兒乳酸性酸中毒之新診斷指標以及利用串聯式質譜黏多醣症分型檢測是兩個成功的範例，係屬創新醫療科技，極具臨床診斷價值，值得推展。本論文將分兩個單元就串聯式質譜分析方法的創新性、實用性、與臨床應用價值分別敘述。

乳酸血症是罹患嚴重小兒疾病最常見之合併症，病人常因為細胞間氧化反應的退化，引發形成一些不可逆的病徵。血液乳酸和丙酮酸的定量分析有助於疾病的診斷以及後續的治療追蹤。傳統酵素學檢測方法需要大量血液檢體，且容易受到血液中干擾物質的影響出現檢測結果的誤差，造成判讀的錯誤。研發串聯式質譜乾濾紙血片乳酸和丙酮酸定量檢測方法可以填補傳統方法的不足與可能發生的誤差結果。乾濾紙血片檢體的來源，包括580位足月新生兒、120位早產兒(懷孕週數介於24-36週)、以及65位懷疑併發有乳酸血症之病人；所有的檢體都取得家屬簽名之書面同意函。本方法極具高的準確度，乳酸和丙酮酸之within-run與between-run分析分別為1.9%和3.9%，以及5.7%和7.3% (n=20)；根據內標準品測得之線性分析結果相當理想，線性r值分別為0.9986和0.9973；另外，串聯式質譜分析方法與傳統酵素學檢測方法之迴歸分析(r2)說明兩種方法所測得的結果具備有高的一致性與接受度，乳酸和丙酮酸r2分別為0.9405和0.9447。本方法同時確定乳酸和丙酮酸在乾濾紙血片中高的穩定性。新研發串聯式質譜乾濾紙血片乳酸和丙酮酸定量檢測方法具備有高的敏感度、特異性、和可再現性，以及低血量檢體需求，尤其適用在小兒科之臨床診斷用途。

胺基多醣是一種結合有負電荷異質型黏多醣和少量蛋白質結構之大分子化合物，它的代謝需要仰賴多種特定之溶?小體酵素以逐步分解的方式進行。當缺乏某特定溶?小體酵素活性，胺基多醣的代謝會被阻斷，導致大量屯積在細胞、組織、和器官中，引發不可逆性的病變，例如黏多醣症。目前，黏多醣症的診斷主要以尿液二次元電泳法作為分型診斷的憑據標準；然而二次元電泳法的實驗步驟繁複、冗長，而且結果的判讀會因人為主觀意識或偏差，徒增診斷的不易性和不可靠性。研發尿液串聯式質譜定量檢測硫酸皮膚素(DS)和硫酸乙醯肝素(HS)方法，可以決定相關黏多醣症之型別以為診斷之憑據指標。本研究共計收集56支尿液檢體分別取自4位黏多醣症第一型病人、10位第二型、5位第三型、5位第六型、以及32位正常人。尿液檢體首先沉澱氨基多醣後再以鹽酸甲醇水解，使形成糖醛酸-氨基已糖結合帶有甲氧官能基之二聚物；此衍生物質可在液相層析串聯式質譜分析的條件下進行定量分析、鑑定等工作。正常人尿液中只有微量硫酸皮膚素和硫酸乙醯肝素的存在，而黏多醣症各型(I、II、III、VI型)之尿液其硫酸皮膚素或硫酸乙醯肝素的量均出現顯著的增加。黏多醣症各型在持續接受酵素替代療法治療後，黏多醣第一型治療效果最佳，硫酸皮膚素量和硫酸乙醯肝素的下降幅度最顯著。串聯式質譜尿液硫酸皮膚素和硫酸乙醯肝素定量檢測可以取代傳統黏多醣症之檢查方法，具備有高敏感度、高特異性、方便操作、以及高準確度等特質，並且適用於一般實驗室，提供黏多醣症正確分型診斷與後續治療追蹤之臨床服務。

關鍵字

轉譯醫學 ； 液相層析串聯式質譜分析 ； 乾濾紙血片 ； 乳酸 ； 丙酮酸 ； 乳酸性酸中毒 ； 黏多醣症 ； 胺基多醣 ； 溶小體酵素 ； 甲醇水解 ； 硫酸皮膚素 ； 硫酸乙醯肝素

並列摘要

Current methods of clinical laboratory diagnosis accentuate the requirements in low radioactive contamination, automation, high-speed, high accuracy, less invasive sampling, and small amount of sample collection. Mass spectrometry analysis can completely fulfill these requirements and has become the mainstream of analytic technology evolution in the twenty-first century. The analytic properties of mass spectrometry, such as high sensitivity, high specificity, high throughput, easy performance and multiple applications, are extraordinary and can be used for quantitative analysis of small molecules in physiological fluids of human, including hormones, drugs, toxins, metabolites, and nutrient components. Mass spectrometry is thus a powerful tool for clinically diagnostic purpose in translational medicine. Two novel examples, “A Method for Lactate and Pyruvate Determination in Filter-Paper Dried Blood Spots” and “The Tandem Mass Study of Mucopolysaccharidoses and Its Clinical Application”, will be presented in this doctoral dissertation separately in details, particularly focusing on method innovation, performance, result interpretation, diagnostic value, and clinical applications.

Lactic acidemia is commonly associated with severe diseases in pediatric patients. Quantification of blood lactate and pyruvate is important for the diagnosis and clinical management. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using dried blood spots (DBS) was developed and could be used for simultaneous quantification of blood lactate and pyruvate. Lactate and pyruvate were extracted from DBS obtained from 580 full-term, 120 pre-term infants (gestations ranging from 24 to 36 weeks), and 76 patients with suspected lactic acidemia. An API-2000 LC-MS/MS system with multiple reaction monitoring (MRM) mode was applied. The within-run and between-run precisions (CV %) and the linearity of lactate and pyruvate based on the IS were excellent. The r2 of linear regression between both methods was good, and the agreement between these methods was consistent and acceptable. The stability of lactate and pyruvate on DBS was also confirmed. The LC-MS/MS method is a specific, sensitive, and reproducible for blood lactate and pyruvate measurements. The use of DBS in this method makes it particularly attractive for pediatric patients.

The glycosaminoglycan (GAG) stepwise degradation requires a numbers of lysosomal enzymes. While one or more specific enzyme activities are deficient, the GAG catabolism is block and results in an irreversible mucopolysaccharidosis (MPS). Conventionally, two-dimensional electrophoresis of urinary GAG is the most common method used for MPS phenotype determination; however, time-consuming and ambiguous interpretation adds to the diagnosis more difficult. Identification of acid MPS by LC-MS/MS method of predominant disaccharide component of GAGs (dermatan sulfate, DS; heparin sulfate, HS) after methanolysis is validated and applicable for MPS classification. A total of 56 urine samples were collected and analyzed including 4 MPS I patients, 10 MPS II, 5 MPS III, 5 MPS VI, and 32 normal controls. Urinary GAG was first precipitated followed by a treatment of 3N HCl methanol. An AB 4000 Q TRAP LC-MS/MS system with MRM mode was applied for the protonated species of the methylated disaccharide products. One particular disaccharide for each GAG was selected, in which the parent ion and its assumed daughter ion after collision were m/z 426.1→236.2 for DS (m/z 432?239 for dimmers derived from [2H6] CS and [2H6] DS) and m/z 384.2→161.9 for HS (m/z 390.4?162.5 for the [2H6] HS dimmer). The results were correspondent well when comparing the LC-MS/MS method and 2-D EP method. The quantities of DS and HS were determined, which were varied from one MPS phenotype to the others, and the results can be used to evaluate the severity of MPS subgroups, as well as the amelioration of follow-up therapy. The modified LC–MS/MS method for MPS phenotype determination is validated and applicable for simultaneous quantification of urinary DS and HS. By using this method, a precise MPS diagnosis can be successfully achieved; besides, this method can also be used to evaluate the effectiveness of follow-up ERT.

並列關鍵字

liquid chromatography-tandem mass spectrometry ； dried blood spots ； lactate ； pyruvate ； lactic academia ； glycosaminoglycan ； mucopolysaccharidoses

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