Lorenz W, Thon K, Neugebauer E, Stöltzing H, Ohmann C, Weber D, Schmal A, Hinterlang E, Barth H, Kusche J
Agents Actions. 1987 Jun;21(1-2):1-25. doi: 10.1007/BF01974915.
Histamine, among various "biologic-physiologic" abnormalities, is considered as a pathogenetic factor in chronic duodenal ulcer disease. The 10-30 per cent difference between its concentration in gastric and duodenal mucosa of patients compared to healthy controls, however, has to be demonstrated to be specific for the disease. It has to be shown to be neither a methodological artefact nor a common effect, concomitant factor or consequence. This study, after a series of pathogenetic trials examines systematic errors (biases) in the fluorometric-fluoroenzymatic histamine assay under the conditions of field studies including tests on specificity over a time period of 10 years. It concentrates on sensitivity (detection limits) and specificity of a standard technique described herein. A modified Shore procedure for large scale assays in human biopsies was developed including reference luminescence values for all reagents, cleaning material and glassware, reduction of OPD concentration to 0.05%, purification of n-heptan, omission of centrifugation steps in the extraction procedure and use of 2 ml 1 M HClO4 in the homogenization step to prevent losses of histamine due to adherence to the mechanical homogenizer. This assay was sensitive enough to measure histamine without difficulty in any biopsy taken. The detection limit was 3 ng/biopsy, but the smallest quantities of the amine ever obtained were 10.6 and 18.3 ng/biopsy (depending on both histamine content and biopsy weight). A series of problems had to be solved both in achieving and demonstrating specificity. It had to be defined not only for the assay in general, but also for assessing the difference in histamine content between ulcer patients and healthy controls. Exogenous more than endogenous fluorescing material interfering with the determination had to be excluded. A series of pitfalls were detected which had to be overcome in demonstrating the specificity of the assay by physicochemical and enzymatic tests. The specificity of the identification tests was more often impaired than the histamine assay itself. Fluorescing material interfering with the assay occurred in the homogenization, extraction and condensation steps, was found in water, OPD, the organic solvents, the cleaning material and in all kinds of plastic vessels. Plasticizers were shown by physicochemical characteristics including fluorescence spectra to be most likely responsible for this interfering material. Rules were developed to exclude such hazards in specificity in longterm pathobiochemical studies. Enzymatic identification test were applied to exclude endogenous fluorecing substances interfering with the standard technique. Simil
在各种“生物 - 生理”异常情况中，组胺被认为是慢性十二指肠溃疡病的发病因素。然而，与健康对照组相比，患者胃和十二指肠黏膜中组胺浓度10% - 30%的差异，必须证明对该疾病具有特异性。必须证明它既不是方法学假象，也不是常见效应、伴随因素或结果。本研究在进行了一系列发病机制试验后，在现场研究条件下，对荧光 - 荧光酶法组胺测定中的系统误差（偏差）进行了检查，包括在10年期间对特异性的测试。它集中于本文所述标准技术的灵敏度（检测限）和特异性。开发了一种用于人体活检大规模测定的改良肖尔程序，包括所有试剂、清洁材料和玻璃器皿的参考发光值，将邻苯二胺（OPD）浓度降至0.05%，正庚烷的纯化，在提取过程中省略离心步骤，以及在匀浆步骤中使用2 ml 1 M高氯酸以防止组胺因附着于机械匀浆器而损失。该测定法灵敏度足够高，能够轻松测量任何活检样本中的组胺。检测限为3 ng/活检样本，但所获得的该胺类物质的最小量为10.6和18.3 ng/活检样本（取决于组胺含量和活检样本重量）。在实现和证明特异性方面必须解决一系列问题。不仅要为一般测定定义特异性，还要为评估溃疡患者和健康对照之间组胺含量的差异定义特异性。必须排除干扰测定的外源性荧光物质多于内源性荧光物质的情况。通过物理化学和酶学测试证明测定特异性时，发现了一系列必须克服的陷阱。鉴定测试的特异性比组胺测定本身更容易受到损害。干扰测定的荧光物质出现在匀浆、提取和浓缩步骤中，在水、OPD、有机溶剂、清洁材料以及各种塑料容器中均有发现。通过包括荧光光谱在内的物理化学特性表明，增塑剂最有可能是这种干扰物质的来源。制定了规则以排除长期病理生化研究中特异性方面的此类危害。应用酶学鉴定测试以排除干扰标准技术的内源性荧光物质。类似地
