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Microdialysis as a Tool in Studies of L-Dopa and Metabolites in Malignant Melanoma and Parkinson’s Disease
Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences.
1999 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A model with human melanoma xenografts transplanted to athymic mice has been adopted for in vivo studies of 5-S-cysteinyldopa (an intermediate pigment metabolite), glutathione, and cysteine. L-Dopa is an intermediate metabolite in pigment formation and is also important in the treatment of Parkinson's disease, and therefore 1 have also studied the pharmacokinetics of this compound.

We were first to describe in vivo microdialysis in melanoma tissue and showed that dialysis membranes of cuprophane or polyamide are suitable for studies of interstitial 5-S-cysteinyldopa and selected thiols. Analytical procedures were also improved for quantitation of 5-S-cysteinyldopa, L-dopa, glutathione, cysteine, and N-acetylcysteine (NAC). In the melanoma xenografts the interstitial concentration of 5-S-cysteinyldopa reflected the high intracellular production of this intermediate metabolite. For in vivo manipulation of glutathione in the melanoma tissue we gave intraperitoneal injection of buthionine sulphoximine to the animals and thus reduced the glutathione concentrations substantially. We showed that restitution of glutathione in melanoma tissue occurs spontaneously and is not much improved by treatment with the cysteine deliverers NAC and L-2-oxothiazolidine-4-carboxylate (OTC). 5-S-Cysteinyldopa was not substantially affected by great variations in glutathione concentrations. Transport of NAC from intraperitoneal injection to melanoma tissue occurred rapidly and deacetylation to cysteine in vivo could be detected soon after NAC injection. In vivo formation of cysteine was slower from OTC than from NAC.

Pharmacokinetic studies of L-dopa in human subjects indicated a slight to moderate protein binding. Plasma free L-dopa had similar elimination T½ as interstitial L-dopa, but in some cases the elimination of total L-dopa was slower. Difficulties in intestinal absorption of L-dopa were revealed by microdialysis in blood and subcutaneous tissue. Studies showed that this was due to delayed emptying of the stomach. L-Dopa intake increased 5-S-cysteinyldopa concentrations in blood within 30 min in patients with Parkinson's disease and a history of melanoma. No melanoma activation occurred during long-term treatment with L-dopa.

Microdialysis is thus a safe and easily applied method for in vivo studies of both pigment metabolites from human melanoma tissue transplanted to nude mice and for pharmacokinetic studies of L-dopa.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 1999. , 51 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 588
Keyword [en]
Microdialysis, L-Dopa, Malignant Melanoma, Parkinson's Disease, in vivo
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-5009ISBN: 91-7219-332-8 (print)OAI: oai:DiVA.org:liu-5009DiVA: diva2:20914
Public defence
1999-04-09, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Note
On the day of the public defence the status of the articles IV, V and VI was: Submitted.Available from: 1999-05-21 Created: 1999-05-21 Last updated: 2012-01-24Bibliographically approved
List of papers
1. Microdialysis of 5-S-cysteinyldopa from interstitial fluid in cutaneous human melanoma transplanted to athymic mice
Open this publication in new window or tab >>Microdialysis of 5-S-cysteinyldopa from interstitial fluid in cutaneous human melanoma transplanted to athymic mice
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1991 (English)In: Melanoma Research, ISSN 0960-8931, Vol. 1, no 1, 23-32 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13524 (URN)
Available from: 1999-05-21 Created: 1999-05-21
2. A high-sensitivity fluorometric high-performance liquid chromatographic method for determination of glutathione and other thiols in cultured melanoma cells, microdialysis samples from melanoma tissue, and blood plasma.
Open this publication in new window or tab >>A high-sensitivity fluorometric high-performance liquid chromatographic method for determination of glutathione and other thiols in cultured melanoma cells, microdialysis samples from melanoma tissue, and blood plasma.
1991 (English)In: Melanoma Research, ISSN 0960-8931, Vol. 1, no 1, 33-42 p.Article in journal (Refereed) Published
Abstract [en]

A high-performance liquid chromatographic method with fluorometric detection is described which is suitable for determination of glutathione in small samples. Reduced glutathione (GSH) and total glutathione obtained as GSH after reduction with glutathione reductase is derivatized with N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM) and subjected to chromatography. The detection limit for the GSH-DACM derivative was 5-10 fmol/injection, and analytical recovery was quantitative. The method is suitable for determination of both reduced and total glutathione in samples from microdialysis of melanoma tumours, and cysteine can be quantified in the same chromatogram. Application is shown also for glutathione determinations in cultured melanoma cells, melanoma homogenates and plasma.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13525 (URN)
Available from: 1999-05-21 Created: 1999-05-21 Last updated: 2009-05-08
3. Effects on interstitial glutathione, cysteine and 5-S-cysteinyldopa of buthionine sulphoximine in human melanoma transplants
Open this publication in new window or tab >>Effects on interstitial glutathione, cysteine and 5-S-cysteinyldopa of buthionine sulphoximine in human melanoma transplants
1997 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 7, no 4, 322.-328 p.Article in journal (Refereed) Published
Abstract [en]

Using microdialysis of human melanoma transplants in athymic mice we have shown that interstitial glutathione levels decreased during treatment with buthionine sulphoximine (BSO) and recovered after cessation of treatment. The cysteine concentrations also decreased, while 5-S-cysteinyldopa tended to increase during BSO treatment. Restoration of the glutathione levels was not seen after either N-acetylcysteine (NAC) or L-2-oxothiazolidine-4-carboxylate (OTC) injections, given on the third day of BSO treatment. These results were to be expected since NAC and OTC were given during the BSO treatment, and BSO is a specific and potent inhibitor of glutathione synthesis. Cysteine levels, however, increased after the NAC injection but remained unaltered after the OTC injection, while 5-S-cysteinyldopa remained unaltered after both the NAC and the OTC injections.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13526 (URN)
Available from: 1999-05-21 Created: 1999-05-21 Last updated: 2017-12-13Bibliographically approved
4. Comparison of N-acetylcysteine and l-2-oxothiazolidine-4-carboxylate as cysteine deliverers and glutathione precursors in human malignant melanoma transplants in mice
Open this publication in new window or tab >>Comparison of N-acetylcysteine and l-2-oxothiazolidine-4-carboxylate as cysteine deliverers and glutathione precursors in human malignant melanoma transplants in mice
2000 (English)In: Cancer chemotherapy and pharmacology, ISSN 0344-5704, Vol. 45, no 3, 192-198 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: Glutathione is an important cellular compound which affects detoxification of electrophiles and may have direct or indirect effects on pigment formation. It is therefore of importance to study interstitial concentrations in melanoma tissue while decreasing its formation with an enzyme inhibitor and increasing its amount with cysteine deliverers. Method: Glutathione formation was inhibited by intraperitoneal (i.p.) injection of BSO. N-Acetylcysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) were then given i.p. to subgroups of the animals. Intratumoral microdialysis was performed during BSO treatment, during BSO treatment combined with NAC or OTC and after discontinuation of BSO but ongoing NAC or OTC treatment. Results: Glutathione formation was inhibited during BSO treatment. The dialysate concentrations of both glutathione and cysteine decreased during concomitant treatment with BSO and NAC or OTC. Recovery of the amounts of the two compounds was seen in both groups after discontinuation of BSO treatment. In the NAC group we also observed an acute increase in dialysate concentrations of cysteine after NAC injection. The 5-S-cysteinyldopa concentrations were unaffected by variations in glutathione and cysteine concentrations. Conclusions: 5-S-Cysteinyldopa in melanoma is not formed from glutathione in vivo to any appreciable extent. The intracellular amount of cysteine is probably not a limiting factor for cysteinyldopa formation. It seems that both NAC and OTC can be used as cysteine deliverers to melanoma cells in vivo to produce recovery of glutathione levels after synthesis inhibition by BSO treatment.

Keyword
N-Acetylcysteine, Athymic mice, Buthionine 4-sulphoximine, 5-S-Cysteinyldopa, Microdialysis, Glutathione, l-2-Oxothiazolidine-4-carboxylate
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13527 (URN)10.1007/s002800050029 (DOI)
Available from: 1999-05-21 Created: 1999-05-21 Last updated: 2009-08-17
5. Human pharmacokinetics of L-3,4-dihydroxyphenylalanine studied with microdialysis
Open this publication in new window or tab >>Human pharmacokinetics of L-3,4-dihydroxyphenylalanine studied with microdialysis
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1999 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 45, no 10, 1813-1820 p.Article in journal (Refereed) Published
Abstract [en]

Background: Intravenous and subcutaneous microdialysis was performed to compare the free concentrations and pharmacokinetics of L-3,4-dihyroxyphenylalanine (L-dopa) in blood and tissue in healthy subjects and in patients with Parkinson disease.

Methods: Nine healthy volunteers and 10 patients with Parkinson disease, stage 1.5–2 according to the Hoehn-Yahr rating scale, took part of the study. In the patient group subcutaneous microdialysis and ordinary blood sampling were performed, whereas in the control group intravenous microdialysis was also performed. Microdialysis samples were collected in fractions of 15 min. The first two fractions were collected for analysis of basal concentrations. A blood sample was also taken. The patients were then given one tablet of Madopar® (100 mg of L-dopa and 25 mg of benserazide), and the microdialysis was continued for another 210 min. Blood samples were obtained at 30-min intervals.

Results: The serum samples gave a significantly higher mean area under the curve (AUC; 491 ± 139 µmol · min/L) than that for intravenous dialysates (235 ± 55.3 µmol · min/L), suggesting a protein binding of 50%. The L-dopa concentrations from the subcutaneous dialysates matched those from the intravenous dialysates, indicating rapid distribution of L-dopa to the tissues.

Conclusions: Parkinsonian patients in early stages of the disease have a pharmacokinetic pattern of free L-dopa similar to that of healthy subjects. Comparison of AUCs from microdialysis with ordinary serum analysis revealed data indicating significant protein binding. Microdialysis is a suitable and easily applied tool in pharmacokinetic studies.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13528 (URN)
Available from: 1999-05-21 Created: 1999-05-21 Last updated: 2017-12-13Bibliographically approved
6. L-dopa pharmacokinetics studied with microdialysis in patients with Parkinson's disease and a history of malignant melanoma
Open this publication in new window or tab >>L-dopa pharmacokinetics studied with microdialysis in patients with Parkinson's disease and a history of malignant melanoma
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1999 (English)In: Acta neurologica Scandinavica, ISSN 0001-6314, Vol. 100, no 4, 231-237 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: The pharmacokinetics of free L-dopa in blood and tissue of five parkinsonian patients with malignant melanoma was studied with microdialysis. In one case the effect of L-dopa treatment on 5-S-cysteinyldopa and the melanoma was studied. Gastric emptying and its effects on free L-dopa in blood were also investigated in one of the patients.

METHODS: Five patients were given 100 mg L-dopa with 25 mg benserazide. Blood and dialysates from the circulation and fatty tissue were collected for analysis. [13C]-Octanoic breath test was used for analyzing gastric half-emptying time.

RESULTS: Four of the patients had similar pharmacokinetic patterns for L-dopa and a significant (P < 0.05) increase of serum 5-S-cysteinyldopa occurring 30 min after L-dopa intake. Delayed L-dopa peaks and slow gastric half-emptying time were found in 1 patient. A dose-dependent increase of 5-S-cysteinyldopa occurred but no melanoma metastases were seen during long-term L-dopa therapy.

CONCLUSION: L-dopa therapy increases 5-S-cysteinyldopa levels but does not seem to cause progress of melanomas. Gastric emptying impacts L-dopa pharmacokinetics.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13529 (URN)10.1111/j.1600-0404.1999.tb00386.x (DOI)
Available from: 1999-05-21 Created: 1999-05-21 Last updated: 2009-08-17

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