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Microdialysis-HPLC for plasma levodopa and metabolites monitoring in parkinsonian patients.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective drug to relieve the symptoms of Parkinson disease. (1) After 4-5 years of treatment, however, the emergence of motor fluctuations and movement disorders such as dyskinesias complicates the therapeutic management. Because these complications are largely related to L-DOPA pharmacokinetic factors, monitoring of plasma L-DOPA concentrations in parkinsonian patients is of clinical interest [1][2][3][4][5][6]. Indeed, knowledge of motor fluctuations in relation to plasma L-DOPA measurements allows optimization of L-DOPA therapy in patients with advanced Parkinson disease [6][7]. Evaluation of new therapeutic strategies such as the use of controlled-release L-DOPA/carbidopa [8][9] or cathechol-O-methyltransferase inhibitors [10] also requires plasma L-DOPA monitoring. The plasma concentrations of dopamine (DA) metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), may be used as a predictor of DA turnover [11]. HPLC with electrochemical detection is a highly sensitive technique for the determination of catecholamines and their metabolites [12]. In vitro microdialysis combined with HPLC allows a very rapid determination of these metabolites in small plasma volumes [13][14]. This method therefore appears appealing for the monitoring of plasma L-DOPA and DA metabolites after drug administration since this pharmacokinetic analysis imposes numerous samples at short time intervals and rapid determination to help in treatment adaptation. The aim of the study was to validate in vitro microdialysis coupled with HPLC to monitor plasma concentrations of L-DOPA and its metabolites in patients with advanced Parkinson disease.

Materials and Methods

patients

Seven patients with idiopathic Parkinson disease and disabling fluctuations in response to L-DOPA were examined. The mean age [+ or -] SD was 60 [+ or -] 8 years. All patients exhibited prominent motor fluctuations characterized by mobile "on" intervals and immobile "off" periods. The Hoehn and Yahr stage during "off" periods was 3-4 and the motor ratings on the Unified Parkinson Disease Rating Scale (UPDRS) were 39 [+ or -] 3 (mean [+ or -] SD) during "off" periods and 16 [+ or -] 2 during "on" periods. All patients had taken antiparkinsonian medications including carbidopa/L-DOPA for at least 5 years with a drug regimen unchanged for at least 6 months. All medications had been discontinued for at least 14 h before the evaluation. Plasma samples were taken after overnight bed rest and fasting. Parkinsonian disabilities were assessed by the motor part of the UPDRS and the intensity of L-DOPA-induced involuntary dyskinesias was measured on a 0 to 4 scale. Blood samples (1 mL) were obtained 0, 30, 60, 90, 120, and 150 min after oral administration of L-DOPA. Each patient was tested 3 days consecutively at 3 different doses of L-DOPA administered as Sinemet (carbidopa/L-DOPA) pills: 25/100 mg on day 1, 12.5/125 mg on day 2, 50/200 mg on day 3. At the time each sample was obtained, the patient was evaluated with the UPDRS and the dyskinesia scale. All procedures were in accordance with the ethical standards of our institution's responsible committee.

apparatus and chromatographic conditions

The microdialysis sampling system consisted of a microinfusion pump CMA 100, microdialysis probes CMA 20 (10 mm in length), and in vitro stand CMA 130 (Carnegie Medicin, Stockholm, Sweden).

The HPLC system consisted of a model 590 pump (Waters, Milford, MA), an automatic injector with a standard loop of 150 [micro]L (Perkin-Elmer, Norwalk, CT), and an electrochemical detector model 460 (Waters) containing an electrochemical cell fitted with a glassy carbon working electrode and an Ag/AgCl reference electrode. A guard column (30 x 4 mm, Bondapak [C.sub.18]/Corasil 37-50 [micro]m; Waters) was used in conjunction with a [C.sub.18] column (ODS, 250 x 4.8 mm, 5 [micro]m; Beckman, Fullerton, CA). The mobile phase consisted of 80% (by vol) 70 mmol/L Na[H.sub.2]P[O.sub.4], 2.08 mmol/L octanesulfonic acid sodium salt (OSA), 0.08 mmol/L EDTA, pH 2.55, and 20% (by vol) methanol. The flow rate was set at 1 mL/min. The detector potential was +0.80 V vs the reference electrode. The limit of detection of the chemical assay was set at 1.1 nmol/L for L-DOPA, 0.4 nmol/L for DA, 0.4 nmol/L for DOPAC, and 0.7 nmol/L for HVA.

chemicals and reagents

L-DOPA, DA, DOPAC, HVA, isoHVA (internal standard), and OSA were purchased from Sigma (St. Louis, MO). HPLC-grade methanol, [Na.sub.2]EDTA, Na[H.sub.2]P[O.sub.4], and [Na.sub.2]S2O5 were purchased from Merck (Darmstadt, Germany).

sample preparation and assay

Standard stock solutions of L-DOPA, DA, DOPAC, HVA, and isoHVA were prepared each week at concentrations of 30 mg/L in a solution of 0.04 mol/L HCl[O.sub.4] and stored at -4[degrees]C in the dark. Further dilution was made in antioxidant solution (10 mmol/L HCl, 1 g/L [Na.sub.2][S.sub.2][O.sub.5], 0.1 g/L [Na.sub.2]EDTA).

Venous blood samples from parkinsonian patients were collected into prechilled polypropylene tubes containing [Na.sub.2][S.sub.2][O.sub.5] and [Na.sub.2]EDTA and centrifuged (10 min, 700g) to separate the plasma. Plasma samples were kept at -80[degrees]C. In microfuge tubes, 450 [micro]L of standard mixture or plasma sample were mixed with 50 [micro]L of the antioxidant solution containing isoHVA (310 [micro]g/L). At the beginning of each perfusion, microdialysis probes were rinsed in microvials containing Ringer solution for 5 min. Ringer solution was used to perfuse microdialysis probes at 2 [micro]L/min. One dialysate for each plasma sample was collected over 20 min in a vial containing 80 [micro]L of the antioxidant solution. A volume of 100 [micro]L from each dialysate was directly injected into the HPLC system with amperometric electrochemical detection for simultaneous determination of L-DOPA, DA, DOPAC, HVA, and isoHVA.

The probes were calibrated for in vitro assays. The probe was placed into different calibration mixture solutions (10 [micro]g/L to 8 mg/L) to check linearity. We assessed in vitro recovery of all metabolites. Analyte concentrations were measured in the dialysate and expressed as a percentage of the concentration in the surrounding medium. The results were expressed as [micro]mol/L corrected for the relative recovery of the probe.

statistical methods

Experimental data were analyzed by two-factor ANOVA with repeated measures.

Results

In vitro recoveries of L-DOPA, DA, DOPAC, and HVA were 35.00% [+ or -] 9.15%, 27.88% [+ or -] 9.46%, 35.96% [+ or -] 6.34%, and 21.95% [+ or -] 9.76%, respectively (mean [+ or -] SD, n = 4).

There was a linear correlation between metabolite concentrations in the dialysate and metabolite concentrations in the surrounding medium.

The calibration curves of the analytes in the described HPLC system were as follows (y = peak area, x = analyte concentration, [r.sup.2] = correlation coefficient): L-DOPA: y = 0.118x + 20.957, [r.sup.2] = 0.999; DA: y = 0.237x + 25.217, [r.sup.2] = 0.985; DOPAC: y = 1.330x + 53.217, [r.sup.2] = 0.998; HVA: y = 0.233x + 40.696, [r.sup.2] = 0.996.

Figure 1 shows typical chromatograms of standard solution and dialysate from human plasma.

[FIGURE 1 OMITTED]

Basal and peak plasma concentrations after L-DOPA administration (100 mg, 125 mg, and 200 mg) as well as time to reach peak concentration in plasma ([T.sub.max]) are given in Table 1 for L-DOPA, DA, DOPAC, and HVA. There is a significant positive correlation between the L-DOPA dose and the plasma concentration of L-DOPA (P <0.04) and HVA (P <0.04) but not between the L-DOPA dose and the plasma concentration of DA and DOPAC (P = 0.4).

Figure 2 shows evolution of plasma L-DOPA concentrations and UPDRS at different doses of L-DOPA in one patient. In this patient, the threshold dose of L-DOPA for clinical effect is 125 mg.

[FIGURE 2 OMITTED]

Figure 3 illustrates the evolution of mean plasma concentrations of L-DOPA and clinical scales at different doses of L-DOPA for the group of seven patients. The UPDRS is significantly reduced after each dose of L-DOPA (P <0.003). The maximum motor effect is reached 60 min after L-DOPA administration. Threshold plasma concentrations of L-DOPA average 7.74 [+ or -] 3.3 [micro]mol/L. The duration of the clinical response is related to the peak plasma concentration. The motor effect was longer with the highest L-DOPA peak concentration (P <0.01). No relation was found between plasma concentration of L-DOPA and the magnitude of clinical response or the severity of L-DOPA-induced dyskinesias. The clinical response lags behind the rise in the plasma L-DOPA concentration and persists after the plasma concentration has fallen.

[FIGURE 3 OMITTED]

Discussion

Our results demonstrate that in vitro microdialysis combined with HPLC is a suitable and reproducible technique for the determination of L-DOPA and its metabolites in plasma of parkinsonian patients. In vitro recoveries for L-DOPA and its metabolites range from 22% to 36% and are comparable with those found in other microdialysis studies [15]. In humans, in vivo microdialysis was validated in subcutaneous tissue and in muscle [16][17][18]. Microdialysis in subcutaneous tissue has been used for continuous long-term monitoring of glucose concentration in diabetic patients [18]. Cheng et al. reported that in vitro microdialysis combined with HPLC is a rapid, simple, and sensitive method for measurement of DOPAC, HVA, serotonin, and 5-hydroxyindolacetic acid in human plasma [13][14]. Minimal blood loss and lack of laborious and time-consuming cleanup procedures of sample are the major advantages of plasma microdialysis. Indeed, microdialysis purifies the plasma sample by excluding large molecules, and the dialysate is directly applied to HPLC. Variability introduced by extraction procedures is therefore avoided because of the microdialysis method.

Pharmacokinetics and pharmacodynamics of L-DOPA in our parkinsonian patients were similar to those reported in previous clinical studies [1][2][4][19]. We found a positive correlation between plasma L-DOPA concentrations and oral doses of L-DOPA. The duration but not the magnitude of the clinical response depends upon the peak plasma concentration of L-DOPA. Clinical response is very similar with L-DOPA minimum effective plasma concentrations and with greater concentrations. The minimum effective concentration of L-DOPA ranges from 3 to 12 [micro]mol/L and is equivalent to the range of 3 to 15 [micro]mol/L reported by Nutt and Woodward [4]. As described in the literature, we observed a delay between peak plasma L-DOPA concentration and clinical response [4]. This delay may reflect the time required for L-DOPA to pass from plasma to the central effector compartment. In addition, L-DOPA is not the active form of the drug and does not directly reflect striatal DA release. Concerning L-DOPA metabolites, only HVA plasma concentrations significantly correlate with L-DOPA doses. The peak plasma concentration of HVA lags 45 to 75 min behind the plasma peak concentration of L-DOPA. HVA is the major end product of DA metabolism in humans and is considered an index of DA synthesis [11]. Interestingly, plasma concentrations of HVA rise faster with higher doses and plasma concentration of L-DOPA. This observation may be due to an acceleration of DA turnover in remaining striatal dopaminergic projections of parkinsonian patients when the amount of exogenous L-DOPA increases.

Plasma DA concentrations do not correlate with L-DOPA doses, and plasma peak concentrations of DA appear 68 to 84 min later than plasma peak concentrations of L-DOPA. Thus plasma DA does not reflect striatal extracellular DA, since clinical response appears ~1 h before DA plasma peak concentration. This may result from the late appearance in plasma of DA derived from exogenous L-DOPA entered in a slow cellular pool [20][21].

In conclusion, in vitro microdialysis combined with HPLC offers adequate separation and sensitivity to monitor L-DOPA and its metabolites in the plasma of parkinsonian patients. These results open the way to the evaluation of in vivo microdialysis directly in a patient's blood vessel. For such in vivo analyses, microbore HPLC systems, as used in previous studies [13][14], would have some advantages in comparison with our classical HPLC system, since it would allow injection of very small volumes (<5 [micro]L) obtained during shorter periods of dialysis. In vivo microdialysis would have two substantial advantages over the in vitro method: (a) integration over time of plasma concentration determinations because of continuous analysis and (b) absence of blood withdrawal and processing. This in vivo method would allow prolonged monitoring of plasma L-DOPA concentrations over 24-h periods in patients with advanced Parkinson disease. Such a monitoring should enhance our understanding of the relation between plasma L-DOPA concentrations and complex clinical fluctuations. It should also help to define the L-DOPA threshold dose in individual patients.

Acknowledgments

We thank the Belgian National Fund for Scientific Research for financial support (grants no. FRSM-LN 9.4519.91, FRSM 3.4533.94). S.D. is a Research Assistant of the Belgian National Fund for Scientific Research.

The following articles in journals at HighWire Press have cited this article:

N. Dizdar, A. Kullman, B. Norlander, J.-E. Olsson, and B. Kagedal Human Pharmacokinetics of L-3,4-Dihydroxyphenylalanine Studied with Microdialysis Clin. Chem., October 1, 1999; 45(10): 1813-1820.

G. M. d'Eril, R. Moratti, and E. Perucca Total and Non-Protein-bound Fractions of 3,4-Dihydroxyphenylalanine Clin. Chem., April 1, 1998; 44(4): 895-895.

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[10.] Kaakkola S, Gordin A, Mannisto P. General properties and clinical possibilities of new selective inhibitors of catechol-O-methyltransferase. Gen Pharmacol 1994;25:813-824.

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[13.] Cheng FC, Yang LL, Chang FM, Chia LG, Kuo JS. Simultaneous measurement of serotonin, catecholamines and their metabolites in cat and human plasma by in vitro microdialysis-microbore high-performance liquid chromatography with amperometric detection. J Chromatogr 1992;582:19-27.

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[16.] Muller M, Schmid R, Georgopoulos A, Buxbaum A, Wasicek C, Eichler HG. Application of microdialysis to clinical pharmacokinetics in humans. Clin Pharmacol Ther 1995;57:371-380.

[17.] Lonnroth P, Strindberg L. Validation of the 'internal reference technique' for calibrating microdialysis catheters in situ. Acta Physiol Scand 1995;153:375-380.

[18.] Bolinder J, Ungerstedt U, Arner P. Long-term continuous glucose monitoring with microdialysis in ambulatory insulin-dependent diabetic patients. Lancet 1993;342:1080-1085.

[19.] Olanow CW, Gauger LL, Cedarbaum JM. Temporal relationships between plasma and cerebrospinal fluid pharmacokinetics of levodopa and clinical effect in Parkinson's disease. Ann Neurol 1991;29:556-559.

[20.] Melega WP, Luxen A, Perlmutter MM, Nissenson CHK, Phelps ME, Barrio JR. Comparative in vivo metabolism of 6-[[sup.18]F]fluoro-L-dopa and [[sup.3]H]L-dopa in rats. Biochem Pharmacol 1990;39:1853-1860.

[21.] Raftopoulos C, Dethy S, Laute MA, Goldman S, Naini AB, Przedborski S, Hildebrand J. Slow increase of homovanillic acid in cerebrospinal fluid after levodopa administration. Mov Disord 1996;11:59-62.

Footnotes

(1) Nonstandard abbreviations: L-DOPA, L-3,4-dihydroxyphenylalanine; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; UPDRS, Unified Parkinson Disease Rating Scale; and OSA, octanesulfonic acid.

Sophie Dethy (1,2), (a), Marie Aline Laute (1), Nadege Van Blercom (1), Philippe Damhaut (2), Serge Goldman (2) and Jerzy Hildebrand (1)

(1) Service de Neurologie and

(2) PET/Biomedical Cyclotron Unit, ULB-Hopital Erasme, Brussels, Belgium.

(a) Address correspondence to this author at: Service de Neurologie, ULB-Hopital Erasme, 808, route de Lennik, B-1070 Brussels, Belgium. Fax (32-2) 555-4701; e-mail sdethy@ulb.ac.be.
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Author:Dethy, Sophie; Laute, Marie Aline; Van Blercom, Nadege; Damhaut, Philippe; Goldman, Serge; Hildebran
Publication:Clinical Chemistry
Date:May 1, 1997
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