The present invention relates to the Millon's reagent which can be conveniently used in the screening diagnosis of cancers by detecting phenolic metabolites present in an increased amount in the urine of cancer patients.
Cancer is still an incurable, fatal disease which cannot be cured even by modern medicinal technology. Although its early discovery is absolutely necessary for treatment, most cancers are discovered only after they are either considerably developed or metastasized. Therefore, the opportunity for any early cure is often missed. This is due to low accuracy of conventional diagnostic methods and use of expensive equipments, such as NMRS, tomographs, etc., which can be a financial burden for patients. Furthermore, patients must be hospitalized to receive accurate assays, such as tissue assay. Because of those disadvantages, conventional diagnostic methods could not be utilized for early diagnosis of cancer.
Recently, a diagnostic method which utilizes tumor markers, such as CEA, .alpha.-Fetoprotein, etc., and monoclonal antibodies, has been introduced. However, its results vary greatly depending on the kind of cancer. Furthermore, under the said method, smokers and pregnant women were also diagnosed as positive. Because of such low accuracy and the economical disadvantage owing to its high cost, the above method is yet too premature to be utilized in early cancer screening diagnosis.
The present invention eliminates the disadvantages and problems caused by the Millon's reagent for cancer diagnosis of Korean Patent No. 21558, and provides a diagnosing reagent which can be utilized more conventiently and rapidly for cancer screening diagnosis than the conventional diagnosis methods.
A urine test is the most frequently used diagnostic technique for any diseases because collection of urine sample is very convenient. Various human metabolites are excreted through urine. It is a well known fact that excretion of a particular metabolite increases in a patient's urine according to the nature of the disease. In the case of cancer patients, it is supposed that phenolic metabolites from tyrosine, peptides and proteins having terminal tyrosine, which react with the Millon's reagent, are excreted excessively. Accordingly, it is very interesting to note that in the prior techniques, the urine of patient was allowed to react with the Millon's reagent to diagnose cancer.
Accordingly, in order to utilize the Millon's reagent clinically for the diagnosis of cancers, the present inventor, first, examined the Millon's reagent of Korean Patent No. 21558. As a result, the reagent made in a gel type by adding gelatine to a stock solution or a dilute solution of the solution prepared by dissolving mercuric sulfate in 15% sulfuric acid did not show any color reaction with tyrosine standard and even cancer patients' urine. However, the gel-type reagent, when made by adding gelatine to a dilute solution of the reagent prepared by using metallic mercury and concentrated nitric acid, showed the color reaction when added to cancer patients' urine. However, the color obtained was very weak and the reaction time was relatively long. Because of these shortcomings, it was very difficult to establish an objective criteria for distinguishing cancer patients from noncancer patients. Thus, this type of Millon's reagent was not suitable for the clinical assay.
The Millon's reagent is a non-specific confirming reagent used in the color confirmation tests to verify the presence of 3,5-unsubstituted p-hydroxyphenol derivatives, such as tyrosine. The Millon's reagent is used in the following two methods. One of the methods comprises dissolving metallic mercury in concentrated nitric acid or fuming nitric acid, and diluting the resulting solution with distilled water to a desired concentration for use. The other involves the addition of sulfuric acid solution of mercuric sulfate to the sample to be tested. The whole mixture is then heated in a water bath and finally either sodium nitrate or potassium nitrate is added to develop color. However, in the former case upon dissolving metallic mercury in concentrated nitric acid or fuming nitric acid large quantities of nitrogen dioxide gas are generated. This brings up the issues of environmental pollution and the safety of the operator handling the poisonous metallic mercury. In addition, if it is used as a reagent, formation of the mercuric salts and standardization of the acid concentration are very difficult. In the latter case, necessity of a heating operation makes the process complicated, and further, hydrolysis of proteins in the urine sample isolates substances interfering color developing process, such as tryptophan.