Jean-Pierre Garnier

Determination of the l-DOPA/l-tyrosine ratio in human plasma by high-performance liquid chromatography: Usefulness as a marker in metastatic malignant melanoma

A first procedure was devised for determining 3,4-dihydroxyphenylalanine (L-DOPA) in human plasma by isocratic RP-HPLC coupled with electrochemical detection. A second procedure was devised for determining 3-hydroxyphenylalanine (L-tyrosine) in human plasma by isocratic RP-HPLC coupled with fluorescence detection. These methods were used to ascertain the L-DOPA/L-tyrosine ratio in plasma of patients with melanoma. Reference values were established by analysis of the L-DOPA/L-tyrosine ratio in the plasma of 35 normal healthy subjects. For 29 patients diagnosed as having melanoma without metastasis, the L-DOPA/L-tyrosine (11.96 x 10(-5) +/- 2.69 x 10(-5)) level was not significantly different from that of 35 normal controls (11.20 x 10(-5) +/- 2.92 x 10(-5)). However, this level was significantly increased (p < 0.05) in the plasma of 17 patients with developing metastasis (21.02 x 10(-5) +/- 4.68 x 10(-5)).

High Performance Liquid Chromatography of Tryptophan and Serotonin Metabolites

Abstract Serotonin is a neurotransmitter in cerebral centers and its perturbations can produce humor and behavior disorders. In addition, exploration of tryptophan and serotonin metabolism is extremely important for early detection and supervision of treatment in carcinoid tumors. High performance liquid chromatography (HPLC) enabled us to separate and titrate different metabolites (5-hydroxyindolylacetic acid, serotonin, tryptophan, 5-hydroxy-tryptophan and N-acetyl tryptophan (NAT) as internal standard) in several types of biological tissues: blood, urine, brain and cerebrospinal fluid. This method is original because it connects HPLC with fluorescence in continuous flow, which allows to change the conditions of pH and buffer. This technique is highly sensitive (below 100 picograms) and very quick (ten minutes).

Evaluation of standard tyrosinase RT-PCR in melanoma patients by the use of the LightCycler system.

BACKGROUND Haematogenous spread influences outcome in melanoma patients. The clinical relevance of detecting circulating melanoma cells in peripheral blood by tyrosinase mRNA RT-PCR is, however, questioned as rates of positivity considerably vary between studies. Standard tyrosinase-nested RT-PCR was here compared with a real-time PCR technique. METHODS Forty-three blood samples from 20 stage III--IV melanoma patients were analyzed. Mononuclear cells were isolated using a Ficoll Hypaque gradient technique. Total RNA extracted by the acid guanidinum thiocyanate-phenol-chloroform method was reverse transcribed using random hexamers or specific primers. Standard tyrosinase-nested PCR was performed on Touchdown machine (Hybaid) and real-time PCR on a LightCycler instrument (Roche). RESULTS Only two samples from stage IV patients (one from random hexamers, one from antisense primers) were found tyrosinase positive with a 100% agreement between the two PCR techniques. A 10-fold dilution of the first-round products improved the PCR kinetic and the final amount of amplified product of positive samples, but not the rate of positivity. CONCLUSIONS Efficiency of the PCR reaction can be monitored in an online fashion by the LightCycler instrument allowing technical improvements. However, tyrosinase mRNA RT-PCR cannot be yet considered as a useful technique in the monitoring of melanoma patients.

Modern Aspects Of Fluorometric Detection In Liquid-Phase Chromatography

Recent advances are described in the combined use of fluorometric derivatization and high performance liquid chromatography (HPLC) for clinical chemistry determinations. Derivatives (especially dansyl derivatives) can be formed prior to chromatography in the case of estrogens, amino acids, and catecholamines. In post-column reactions, we preferred to use air-segmented reactions as they conform better to all the optimized chromatographic and spectrofluorometric parameters. Fluorescent derivatives produced from cate-cholamines, tryptophan and its metabolites, hydroxyindoles, tryptamine, amino acids, sugars, polyamines, and other substances are often sufficiently sensitive to be detected in picogram quantities by HPLC. Their reaction principle and some of their applications to samples are described. Recently, chemical excitation of fluorophore-like dansyl amino acid was proposed as a detection system for HPLC. By a post-column reaction, a fluorophore can be made to emit light by its reaction with trichlorophenyl oxalate (TCPO) and hydrogen peroxide. The detection limit of this system is about 10 fmol for each dansyl amino acid. Application of this new reaction to catecholamines opens up new prospects for fluorometric detection.