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Two tyrosinase isozymes were purified from pigmented hamster melanoma and injected i.v. into rats. It was shown that while T1 (sialylated) isozyme survived in the circulation, native asialo (T2) isozyme and neuraminidase-desialylated T1 isozyme disappeared from the circulation in a few minutes. Desialylated fetuin had a marked inhibitory effect on the removal of asialo-T1 tyrosinase. These data indicate that the enzyme tyrosinase shares the common pattern of clearance from circulation known for the majority of serum glycoproteins. The electrophoretic pattern of tyrosinase isozymes partially purified from the sera of melanoma-bearing animals were compared with those from the soluble fraction of the tumors. In hamsters, melanoma tissue revealed both T1 and T2 isozymes while serum exhibited T1 and very weak T2, supporting the mechanism of clearance demonstrated in rats. In mice bearing Cloudman S-91 or B-16 melanomas, only T1 isozyme was seen in sera and in tumors.

Little has been known about the excretion of melanogens (melanogenuria) in animals with experimental melanoma. In this report the first information about melanogenuria in hamsters with transplantable melanotic and amelanotic melanoma is given. In hamsters with melanotic melanoma apparently increased concentration and excretion of so called Thormahlen positive melanogens in urine was found in comparison with hamsters with amelanotic melanoma or hamsters without tumours. Similar results were obtained when the excretion of all diazo positive compounds in urine was measured. Thin-layer chromatography of ethyl acetate extracts of hamster urine proved the presence of at least 15 phenolic or indolic acids. Some of them remained unidentified. However, such compounds as p-hydroxyphenylacetic acid, homovanillic acid, vanilmandelic acid or 5-hydroxyindole-acetic acid were identified. Only in the urine of hamsters with melanotic melanoma were we able to detect the isomeric 5-hydroxy-6-methoxyindolyl-2-carboxylic and 6-hydroxy-5-methoxyindolyl-2-carboxylic acids.

Thormahlen positive melanogens (TPM) were determined in the urine of 585 primary and/or metastatic cutaneous melanoma patients, in 105 primary and/or metastatic ocular melanoma patients, in 100 patients suffering from other diseases with the exception of melanoma and in 72 healthly persons. On the basis of our results we can recommend the determination of the sum of TPM as sufficient for routine monitoring of patients in the course of malignant melanoma. The determination of TPM urinary excretion cannot substitute histological and clinical examinations in an early diagnosis of malignant melanoma, but it is very important and useful for a differential diagnosis and prognosis of malignant melanoma. TPM urinary excretion can be followed in melanoma patients also for the indication of further treatment and the evaluation of the response on chemotherapy or immunotherapy.

Two new isomeric metabolites, 5-hydroxy-6-methoxyindole-2-carboxylic (a) and 5-methoxy-6-hydroxyindole-2-carboxylic (b) acids have been identified in melanotic urine: Their constitution was identified by Chromatographic and spectral comparison of the two isomers isolated from urine with the same compounds prepared synthetically.

1. Tyrosinase was purified from melanosomal fraction of hamster melanoma. 2. A radioimmunoassay was developed to quantitate the tyrosinase protein in hamster serum and hamster melanoma tissue using polyclonal anti-tyrosinase antibodies and 125I-labeled enzyme. 3. The serum tyrosinase levels were found to be about 0.24 micrograms and 1.14-4.48 micrograms/ml in normal hamsters and melanoma-bearing hamsters, respectively. 4. Tyrosinase protein in serum correlated significantly with the enzyme activity in hamsters with melanoma (r = 0.733). 5. In the cytosol fraction of hamster melanoma, a level of 2.2 micrograms of tyrosinase/mg protein was determined. 6. The usefulness and possible applications of the tyrosinase radioimmunoassay are discussed.