Bernard Bousquet

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)).

Urinary free light chain analysis by the Freelite® immunoassay: a preliminary study in multiple myeloma

Evaluate the Freelite free light chain immunoassay for urine analysis in myeloma. Urine specimens from 20 patients were analyzed by Freelite (The Binding Site) and SDS-agarose gel electrophoresis (Hydragel protéinurie, Sebia). Using the kappa/lambda ratio, Freelite was more sensitive than electrophoresis to detect free light chains, but concentration was overestimated in 75% of cases. Despite high sensitivity and full automation, Freelite inaccurately measures monoclonal free light chains in urine.

Melanoma progression and serum L-dopa/L-tyrosine ratio: a comparison with S100B.

The challenge to find a reliable tumour marker for the management of melanoma patients still remains. In this study, the serum l-dopa/l-tyrosine ratio was compared with serum S100B as a reference marker. A total of 89 melanoma patients were sampled and staged according to the American Joint Committee on Cancer (AJCC) classification. Of these, 19 stage III and 28 stage IV patients were evaluated for disease progression at 1.5 years and 6 months post-sampling, respectively. Serum l-dopa and l-tyrosine were measured by high performance liquid chromatography (HPLC) (normal value for ratio < 16 × 10−5) and S100B using the LIA-mat Sangtec 100 assay (normal value < 0.10 μg/l). Non-parametric tests (Kruskal–Wallis analysis of variance, Dunns and Spearman) were used for the statistical analysis. The median serum l-dopa/l-tyrosine ratio was 16.0 × 10−5 (range 2.7–545.1 × 10−5 and the median S100B level was 0.15 μg/l (range < 0.10–13.8 μg/l), with a sensitivity of 51% for the ratio and 66% for S100B. There was a 47% discordance and no correlation between the two markers (r = 0.149). The ratio was higher in stage IV than in other stages (P < 0.05), as was the S100B level (P < 0.0001). Both markers were higher in patients with evolutive disease (n = 23) than in stable patients (n = 24), with values of 20.8 × 10−5 versus 13.1 × 10−5 for the ratio (P < 0.05) and 0.89 μg/l versus 0.16 μg/l for S100B (P < 0.001); for the ratio, this difference was more pronounced in stage III than in stage IV patients. The overall sensitivity and specificity of the markers to predict disease progression were 78% and 67%, respectively, for the ratio, and 74% and 83%, respectively, for S100B (using an ROC cut-off of 0.38 μg/l). In conclusion, the serum l-dopa/l-tyrosine ratio correlates with melanoma progression and has predictive value, especially in stage III patients. This tumour marker, like S100B, could serve as an additional tool in the management of melanoma.

Urinary carboxyterminal telopeptide of collagen I as a potential marker of bone metastases chemotherapy monitoring in breast cancer

Breast cancers frequently have osteoclastic bone metastases that are difficult to monitor and treat. Bone scintigraphy with 99mTc-labeled biphosphonates is still the reference method for detecting and localizing bone involvement. Classical biochemical markers such as urinary calcium have poor sensitivity for detecting and monitoring metastases of breast cancers. New biochemical markers for the study of bone remodeling have recently been developed, including a degradation product of the C-terminal end of the telopeptide of type I collagen (CTX). We used an immunoenzymatic assay technique for urinary CTX in 84 pre- and post-menopausal women and demonstrated a correlation between scintigraphic scores and urinary CTX concentrations. CTX values are significantly different between the control group and patients with bone metastasis, except those with score 0. There is a regular increase in urinary CTX concentration from score 0 (no abnormal uptake) to score 4 (diffuse carcinomatosis). There is no significant variation between control population and score 0 to 3 for urinary calcium. Only women with scintigraphic score 4 have significantly increased urinary calcium concentrations. Measuring CTX in pre- and post-menopausal patients during breast cancer chemotherapy might be of great interest for monitoring the development of metastases and the therapeutic efficacy of chemotherapy.

Micellar electrokinetic capillary chromatography quantification of cytosine arabinoside and its metabolite, uracil arabinoside, in human serum

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6-O-methylguanine as an internal standard. The assay was linear from 6.25 to 200 microg/ml with a quantification limit between 3 and 6 microg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m2 body surface).

Comparison of urinary markers for bone resorption in multiple myeloma.

Multiple myeloma causes extensive bone remodeling. Classical biochemical markers such as urinary calcium have poor sensitivity for detecting multiple myeloma bone remodeling. New biochemicals have been developed including a carboxyterminal telopeptide of collagen I (CTX). We used an immunoenzymatic assay to determine urinary CTX in 60 patients with multiple myeloma. This marker was evaluated with regard to total pyridinolines, urinary calcium, radiological features, pain and response to treatment with bisphosphonates. In patients with bone involvement, CTX concentrations were significantly higher (+230%) than those of deoxypyridinoline (DPD) (+175%) and pyridinolines (PYD) (+130%). In all patients we have found a close correlation between CTX and DPD but not between CTX and PYD. Compared to radiological features, CTX was more sensitive (97%) and specific (96%) than DPD. After treatment by bisphosphonates, the fall in CTX concentrations was paralleled to urinary calcium and more marked than pyridinolines. Although our results need to be confirmed, CTX appears to be a potential marker to explore bone involvement in multiple myeloma.

Determination of 4-hydroxyifosfamide concomitantly with ifosfamide and its dechloroethylated metabolites using gas chromatography and a nitrogen phosphorus-selective detector

A sensitive gas chromatographic (GC)/nitrogen phosphorus detection (NPD) system was developed for the determination of the antitumor drug ifosfamide (Ifos) and its 2-dechloroethylifosfamide (2-Difos), 3-dechloroethylifosfamide (3-Difos) and 4-hydroxyifosfamide (4-OHIfos) metabolites in human blood. 4-OHIfos was analyzed after coupling with a trapping agent and was used as an indicator of isophosphoramide mustard (IPM). Ifos and its metabolites 2-DIfos, 3-DIfos, 4-OHIfos and the internal standard (trofosfamide) were extracted into chloroform and then resolved by gas chromatography using a Hewlett Packard HP5 capillary column cross-linked with 5% phenyl methyl silicone (30 m; 530 microm I.D.; 2.65 microm film thickness). Precision and accuracy of the assay were determined over a three-day period and a concentration range of 3.25-50 microg/ml for Ifos, 0.8-14 microg/ml for 2D-Ifos, 0.6-10 microg/ml for 3D-Ifos and 0.08-1.40 microg/ml for 4-OHIfos. The limit of quantitation was set at 3.25, 0.80, 0.62 and 0.08 microg/ml, respectively, for Ifos, 2-DIfos, 3-DIfos and 4-OHIfos. The intra- and inter-day coefficients of variation and accuracies were less than 20%, except for a low concentration 4-OHIfos. This assay was then used to provide pharmacokinetic data on antitumor and toxicologic effects following intravenous infusion of Ifos.

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).

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.

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.