RESUMO
Microdialysis was investigated as a tool for the determination of the extracellular concentration of the pigment metabolite 5-S-cysteinyldopa in human melanoma transplanted to athymic mice. Histology of the tumour with the microdialysis probes in situ showed no tissue damage. With probes equipped with polycarbonate membranes (20 kD) extraction (relative recovery) was approximately 50% at pH 4.0 and flow rates of 1 microliter/min, but at pH 7.0 recoveries were markedly lower, particularly from serum. In a first series of human melanomas transplanted to athymic mice low concentrations of 5-S-cysteinyldopa were detected in only two out of ten dialysates and were not detected in the other eight. Utilizing devices constructed for comparison of membrane characteristics in vitro we found about 4-fold higher recoveries with cuprophane and polyamide membranes than with polycarbonate membranes. Therefore newly constructed microdialysis probes (CMA/11) with cuprophane membranes were tested in vitro and gave recoveries of 38-48% from Ringer-Acetate solutions and 22-31% from serum, and the pH effects were low. When these probes were utilized in a second series of melanomas transplanted to athymic mice, 5-S-cysteinyldopa could easily be quantified in 10/10 experiments. A steady-state level of the dialysate 5-S-cysteinyldopa concentration was reached after 45 min.
Assuntos
Biomarcadores Tumorais/isolamento & purificação , Cisteinildopa/isolamento & purificação , Diálise , Melanoma/química , Microquímica , Neoplasias Cutâneas/química , Animais , Líquidos Corporais/química , Celulose/análogos & derivados , Diálise/instrumentação , Masculino , Membranas Artificiais , Camundongos , Camundongos Nus , Microquímica/instrumentação , Transplante de Neoplasias , Nylons , Cimento de PolicarboxilatoRESUMO
A phenylboronate affinity gel has been investigated for use in the prepurification of urine before "high-performance" liquid chromatography and electrochemical determination of 5-S-L-cysteinyl-L-dopa. At pH 5.6 this naturally occurring 5-S-cysteinyldopa was adsorbed on a phenylboronate column and was quantitatively eluted with trichloroacetate, pH 3.0. This pretreatment of urine before "high-performance" liquid chromatography produced satisfactory chromatographic separations, and the results were further improved when the purification procedure also included treatment on a cation exchanger. By using 5-S-D-cysteinyl-L-dopa--a diastereomer to 5-S-L-cysteinyl-L-dopa--as an internal standard, we have developed a practical routine method for the quantitative determination of urinary 5-S-L-cysteinyl-L-dopa. The precision (CV = 2.4%) and analytical recovery (96.9%, SD 5.3%) were satisfactory and the results obtained correlated well with a previously described method.
Assuntos
Ácidos Borônicos , Cromatografia Líquida de Alta Pressão , Cisteinildopa/urina , Di-Hidroxifenilalanina/análogos & derivados , Cromatografia de Afinidade , Cromatografia em Gel , Cisteinildopa/isolamento & purificação , Eletroquímica , Feminino , Humanos , Masculino , Padrões de Referência , Estatística como Assunto , EstereoisomerismoRESUMO
A high-performance ion-pair liquid chromatographic method with electrochemical detection is described, which is suitable for routine determination of urinary 5-S-cysteinyldopa. The clean-up procedure includes a first purification step on the cation exchanger AG 50 W (H +). After desorption from the resin at moderately raised pH the catecholic amino acid is adsorbed on alumina at pH 8.6, washed and finally desorbed by elution with perchloric acid. By the combined clean-up procedures, easily oxidized compounds are eliminated, which otherwise cause a number of interfering peaks in the chromatography. The synthesis of 5-S-cysteinyl-L-3,4-dihydroxyphenyl [2,3-3H]alanine is described, and this tritium-labelled 5-S-cysteinyldopa is used to determine the recovery in the sample. The precision (C.V. = 5.7% at low and C.V. = 4.9% at high 5-S-cysteinyldopa concentration) and recovery (105.0 +/- 8.6%) were satisfactory. The mean urinary excretion was 0.34 +/- 0.13 (S.D.) mumol per 24 h (range 0.02-0.58 mumol per 24 h) in healthy subjects (n = 24) and in patients with melanoma metastates (n = 13) the excretion ranged from 0.9 to 4.8 mumol per 24 h.
Assuntos
Cisteinildopa/urina , Di-Hidroxifenilalanina/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Cisteinildopa/síntese química , Cisteinildopa/isolamento & purificação , Eletroquímica , Feminino , Humanos , Masculino , Melanoma/urinaRESUMO
Diastereomers of 5-S-cysteinyldopa formed from D-dopa and L-cysteine or from L-dopa and D-cysteine can be separated from 5-S-cysteinyldopa formed from L-dopa and L-cysteine by liquid chromatography. The diastereomers have a great potential as internal standards in the analysis of 5-S-cysteinyldopa. They can be used as reference substances in the differentiation of stereospecific enzymatic oxidation of dopa from non-specific oxidation.
Assuntos
Cisteinildopa/isolamento & purificação , Di-Hidroxifenilalanina/análogos & derivados , Cromatografia Líquida , EstereoisomerismoRESUMO
The tapetum lucidum of the alligator gar Lepisosteus was shown by t.l.c. to contain a new phenolic amino acid, which is apparently a major constituent of the reflecting material. It was isolated in a yield of 0.5 mg/eye and its physical and chemical characteristics, especially reductive hydrolysis with hydriodic acid giving dopa (3,4-dihydroxyphenylalanine) and cysteine, suggested that it might to SS-dicysteinyldopa. Tyrosinase oxidation of L-dopa in the presence of an excess of L-cysteine yielded, in addition to known 5- and 2-S-cysteinyldopa, the same amino acid as that isolated from the eye of the gar, thus confirming the gross structure. The position of the two cysteine residues was established by the fact that tyrosinase oxidation of catechol and cyteine gave 3-S-cysteinylcatechol and 3,6-SS-dicysteinylcatechol. The natural amino acid is therefore formulated as 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine (2,5-SS-dicysteinyldopa), which may be formed by two consecutive additions of cysteine, first to dopaquinone and then to 5-S-cysteinyldopaquinone. The enzymic synthesis of 2,5-SS-dicysteinyldopa in vitro suggests that it may also be involved in the biosynthesis of phaeomelanin.