CN1158389C - 一种用于鉴别有血管及癌症疾病患病风险的个体的方法 - Google Patents
一种用于鉴别有血管及癌症疾病患病风险的个体的方法 Download PDFInfo
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Abstract
本发明目的是鉴别有血管疾病及癌症患病风险的个体的方法和试剂盒，该方法包括以下几步，定量测定该个体血清胃蛋白酶I(PGI)分析物的浓度，确定该分析物的方法特异性阈值，然后将上述方法测定的分析物浓度与该分析物的方法特异性阈值比较，并测定该个体血清样品中同型半胱氨酸的浓度。
Description
发明领域
本发明涉及一种联合诊断方法，其用于鉴别有冠脉，血管及癌症疾病患病风险的个体。
本发明的方法是基于将两种在血液或血清标本上进行的检测组合，以鉴别易出现或已表现出升高的血清同型半胱氨酸水平的个体，这些个体由于血清同型半胱氨酸水平增高而存在发生某些病患的可能，例如心脑血管疾病，包括动脉粥样硬化和缺血性发作，及癌症。
发明背景
许多不同的胃部疾病发病在胃癌之前，例如慢性萎缩性胃炎，恶性贫血，胃窦部溃疡，胃息肉和Ménétrier病(巨大肥厚性胃炎)。明显可识别的粘膜改变是发育异常和粘液腺瘤。已知在几乎所有疾病中，其患病风险是由慢性萎缩性胃炎引起的。
慢性萎缩性胃炎是一种胃粘膜的长期炎症性疾病。此病可粗略地分为浅表性胃炎和萎缩性胃炎。在浅表性胃炎中，炎症细胞浸润集中在表层上皮细胞下。一旦炎症进展并蔓延至特定的胃分泌腺之间，就称为慢性萎缩性胃炎。在这种情况下，胃粘膜正常的腺体结构至少部分被恶性变化取代。
从芬兰的癌症统计数据得出，胃体部萎缩性胃炎病人患胃癌的相对危险性大约是胃粘膜正常的人的4-5倍。此外，这些病人因内因子缺乏和维生素B12吸收障碍而易患恶性贫血。在严重的胃窦部萎缩性胃炎病人中，所述风险甚至达到18倍。如果在胃窦部和胃体部都出现萎缩性改变(全胃炎)，所述风险将上升至90倍。
WO 96/15456公开了一种筛查胃癌患病风险的方法，根据该方法，胃体部和/或胃窦部粘膜的萎缩可通过测定血清胃蛋白酶I(PGI)和胃泌素-17(G-17)水平，并与各自的方法特异性分析物阈值比较来判定。优选还与各自的方法特异性分析物参考值比较。
血清PGI水平低于PGI特异性阈值提示胃体部患有萎缩性胃炎。如果血清G-17水平低于其阈值，则提示胃窦部患有萎缩性胃炎。在全胃炎患者中，血清PGI水平低于阈值且血清G-17水平处于其参考值的低限。
亚甲四氢叶酸还原酶(MTHFR)是一种细胞内酶，它是同型半胱氨酸再甲基化为蛋氨酸必需的。此酶功能障碍是由于MTHER基因结构缺陷，或诸如叶酸，维生素B6和/或维生素B12营养缺乏所引起的。MTHER酶功能障碍导致血清/血浆同型半胱氨酸水平增高(同型半胱氨酸血症)及同型半胱氨酸尿。许多研究显示，增高的血清/血浆同型半胱氨酸水平与各种冠脉及血管疾病增高的患病风险相关，超过参考值的较高血清/血浆同型半胱氨酸水平是患冠脉及血管疾病和缺血性发作的重要独立危险因子1-5。
同型半胱氨酸致动脉粥样化作用被认为是基于反应性氧物质生成增加，它能使脂质过氧化。充足的维生素B12供应是叶酸代谢和正常血生成及维持神经细胞功能必需的。维生素B12与蛋白质结合形成一种复合物，即内因子，它是由胃体部粘膜产生的，这一复合物在回肠较低部被重吸收。这是维生素B12优选的重吸收方式。
由于萎缩性胃炎或胃癌引起内因子缺乏，特别是在胃体部的缺乏，最终将导致体内维生素B12缺乏，继而引起同型半胱氨酸浓度升高。这对于鉴别那些因萎缩性胃炎而已患或易患维生素B12缺乏症的患者，以及那些因此而血清/血浆同型半胱氨酸水平增高的患者，具有很高价值。在这些人中，尽早补充维生素B12对于预防血管疾病是有益的。对于鉴别那些因细胞内氧自由基过量生成而易发生癌症的个体，以及那些能从补充维生素B12及其他治疗中获益的患者，此方法也是很有价值的。
发明概述
本发明涉及，将测定血清样品中萎缩性胃炎的标记物的测定法以及检测同型半胱氨酸的测定法组合，来辅助诊断或判定某个体患血管及癌症疾病的风险，术语“血管”应理解为泛指包括因同型半胱氨酸的致动脉粥样化影响而造成的任何冠脉或血管疾病。
本发明方法是一种用来鉴别有血管及癌症疾病患病风险的个体的方法，此方法包括以下几步
-定量测定该个体血清样品中胃蛋白酶I(PGI)分析物的浓度，
-针对该分析物选择方法特异性阈值
-将如此测定的分析物浓度与此分析物的方法特异性阈值比较，并且
-测定该个体血清样品中同型半胱氨酸浓度，并将其与同型半胱氨酸的方法特异性参考值比较。
本发明因此可将，血清胃蛋白酶I浓度低于血清胃蛋白酶I的方法特异性阈值且血清同型半胱氨酸浓度高于同型半胱氨酸的参考值，的个体鉴定为有血管和/或癌症疾病高患病风险或易感体质的个体。
血清胃蛋白酶I及同型半胱氨酸浓度的测定可不分先后顺序，以期同时了解血清胃蛋白酶I和同型半胱氨酸浓度，从而辅助诊断或评估患血管疾病及癌症的风险。但根据本发明的实施方案，先测定血清胃蛋白酶I浓度，再与其确定或选择的方法特异性阈值比较，之后选出血清胃蛋白酶I浓度低于该方法特异性阈值的个体进行进一步诊断。在此实施方案中，只选择那些表现出低血清蛋白酶I水平而提示胃体部萎缩的个体进行血清同型半胱氨酸检测。
按照本发明的一个实施方案，还测定了所述个体的维生素B12浓度，并与维生素B12的方法特异性参考值比较。
本发明包括将所测分析物浓度与该分析物的方法特异性阈值或参考值比较的步骤。对这些值的选择是本领域技术人员已知的，并取决于检测该分析物浓度的方法所选择的特异性和敏感度，参见例如William J Marshall，Clinical Chemistry(临床化学)，第三版，1995，Mosby。
因此按照优选实施方案，本发明尤其旨在筛选那些尚未表现出维生素B12缺乏，即显示基本正常的维生素B12水平，但因低胃蛋白酶I水平而被诊断为胃体部萎缩并且还具有较高血清同型半胱氨酸水平的个体。这一鉴别方法使得有可能尽早采取预防措施，例如补充维生素B12，以拮抗与血清同型半胱氨酸水平增高相关的疾病的发生。
发明详述
1.胃蛋白酶I(PGI)浓度的测定
测定血清样品中PGI水平的方法可按WO 96/15456所述的进行，该出版物在此作为参考。
该方法优选包括在测定胃蛋白酶I的免疫学方法中使用抗胃蛋白酶I多克隆或单克隆抗体的步骤。反应优选在适合的支持物上进行，例如塑料，玻璃或纤维素支持物，比如在微量培养板上。免疫学方法可采用已知的方式，例如应用吸光率，发光或荧光技术来测定样品中的胃蛋白酶I浓度。
如果血清胃蛋白酶I浓度低于阈值，即20-30μg/l(相当于约450-690pmol/l)，则表明胃体部有萎缩，其中所述阈值取决于所述方法认可的特异性和敏感度。PGI正常值或参考值范围是25-120μg/l。
2.同型半胱氨酸的测定
血清同型半胱氨酸水平可根据本身已知用于此目的、且已有商品如试剂盒的任何方法来测定。现有定量血浆或血清同型半胱氨酸总水平的方法是高效液相色谱法合并放射性，荧光或电化学检测。还开发了酶免疫分析(EIA)(Bio-Rad Laboratories；Axis-Shield A/S)以及荧光极化免疫分析(FPIA；Abbott Laboratories)，该免疫分析包括用二硫苏糖醇和腺苷预处理标本，接着通过酶促步骤形成s-腺苷-L-同型半胱氨酸，而同型半胱氨酸总水平可使用抗S-腺苷-L-同型半胱氨酸单克隆抗体检测，参见US 5,631,127。
同型半胱氨酸的参考值在某种程度上是方法特异性的，但通常在约5-15μmol/l之间变化。高于该方法特异性参考值的血清同型半胱氨酸水平被视为构成一个危险因子，如上文的解释。同型半胱氨酸水平高于15μmol/l在大多数情况下可被认为是一个明确的危险因子。
3.维生素B12的测定
按照本发明，诊断方法包括一种任选的检测血清样品中维生素B12浓度的方法。维生素B12(钴胺素)浓度可按照就此目的而言的任何已知的方法进行测定。这些已知方法包括，利用需要钴胺素才能生长的生物，如纤细眼虫(Euglena gracilis)或乳乳杆菌(Lactobacillus leichmannii)测定血清B12的微生物学测定法。也可使用测定维生素B12的放射性同位素稀释法，这类检测技术在文献中已被详细记载，例如Lau等，利用放射性同位素稀释法和包被的炭测定血清维生素B12水平，Blood，26(1965)，202。放射性同位素稀释法与诸如眼虫测定法相比，更快速且结果具有可比性。前提是其中的结合蛋白对生物活性钴胺素是特异的。标准纯的或纯化的内因子制剂由于能特异性结合真正的钴胺素而不是钴胺素类似物因此是最令人满意的结合蛋白。
测定维生素B12的放射性同位素稀释法通常包括使内源性B12从其天然结合蛋白中释放的步骤，这可通过在指定pH值下煮沸，之后添加可检测的量的放射性同位素57Co-B12，及有限量的结合蛋白。由于加入的放射性同位素B12的量足以结合少量的蛋白质，所有结合蛋白将被某种形式的维生素B12结合。当天然B12和放射性B12竞争性地与结合蛋白结合时，结合B12的蛋白的放射量被抑制的程度提示了标本中B12的量。Lau，出处同上改进了这一方法，其用蛋白包被的炭将未结合的B12与蛋白结合的B12分离，然后计数含有结合型放射性B12和结合型非放射性B12的混合物的上清液中的放射活性。然后从该数值计算血清B12浓度，通常是与标准图谱比较。已有放射分析试剂盒的商品可用于实施该方法。
维生素B12缺乏还可以应用例如化学发光受体测定法(Wentworth，S.等.，Clin.Chem.，vol 40 537-540)，放射免疫分析(Endres，D.B.，等Clin.Chem.，Vol.24，460-465)及非同位素结合测定法，CEDIA，克隆的酶供体免疫分析(van der Weide，J.Et al.Clin.Chem.，Vol.38，766-768)来检测。
B12的参考值范围在200-900ng/l之间，相当于大约170-700pmol/l。
在我们最近未公开的工作中，我们发现那些血清维生素B12浓度低于参考值下限(＜170pmol/l)的胃体部萎缩性胃炎(SPGI＜25μg/l)患者中，50％的个体的血清同型半胱氨酸浓度有明显升高(平均值33.3μmol/l，16-157μmol/l)。此外，那些血清维生素B12浓度在180-230pmol/l之间(参考值范围为170-700pmol/l)的患者中，22％的个体也出现血清同型半胱氨酸浓度升高(平均值18.9μmol/l，16-25μmol/l)。
本发明还涉及用于本发明方法的试剂盒，其包括
-用于测定血清样品中PGI浓度的工具(means)
-用于测定血清样品中同型半胱氨酸浓度的工具
按照本发明，该试剂盒包含需要定量测定血清样品中胃蛋白酶I和同型半胱氨酸浓度的单个成分的混合物。为此，该试剂盒可包含不同的小瓶或容器，以便容纳必要成分如抗生素及用于测定所述分析物的底物。
参考文献
1.Boushey，C.J.，Beresford，S.A.A.，Omenn，GS.，Motulsky，A.G.，JAMA1995；274：1049-57
2.Grahan，I.M.，Daly，L.E.，Rafsum，H.M.et.al.，The European ConcertedAction Project，JAMA 1997；277：1775-81
3.Jacobsen，O.W，Clin Chem 1998；44：1833-43
4.Mogadashian，M.H.，McManus，B.M.，Frohlich，J.J.，Arch Intern Med1997；157：2299-2308
5.Rafsum，H.，Ueland，P.M.，Nygard，O.，Vollset，S.E.，Rev Medicine 1998；49：31-62。
Claims (5)
1.一种用于鉴别有血管及癌症疾病患病风险的个体的方法，该方法包括以下步骤
-定量测定该个体血清样品中胃蛋白酶I(PGI)分析物的浓度，
-选择该分析物的方法特异性阈值，
-将如此测定的分析物浓度与此分析物的方法特异性阈值比较，
-测定该患者血清样品中同型半胱氨酸浓度，并将其与同型半胱氨酸的方法特异性参考值比较，若血清胃蛋白酶I浓度低于其方法特异性阈值且血清同型半胱氨酸浓度高于其方法特异性参考值，则可以判定此个体患血管及癌症疾病的患病风险增加。
2.权利要求1的方法，包括测定血清PGI浓度，并选出血清PGI浓度低于其阈值的个体以便进一步测定同型半胱氨酸浓度。
3.权利要求1或2的方法，包括进一步测定样品中维生素B12浓度，并将其与方法特异性参考值比较。
4.实施权利要求1的方法所用的试剂盒，其包括
-用于测定血清样品中胃蛋白酶I浓度的工具，
-用于测定血清样品中同型半胱氨酸浓度的工具。
5.权利要求4的试剂盒，其中用于测定胃蛋白酶I及同型半胱氨酸浓度的工具包含免疫学工具。
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