Reliability and practicability of the fluorometric-fluoroenzymatic histamine determination in pathogenetic studies on...

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