Clinical Chemistry 43: 740-744, 1997;
(Clinical Chemistry. 1997;43:740-744.)
© 1997 American Association for Clinical Chemistry, Inc.
Microdialysis-HPLC for plasma levodopa and metabolites monitoring in parkinsonian patients
Sophie Dethy1,2,a,
Marie Aline Laute1,
Nadège Van Blercom1,
Philippe Damhaut2,
Serge Goldman2 and
Jerzy Hildebrand1
1
Service de Neurologie and
2
PET/Biomedical Cyclotron Unit, ULB-Hôpital Erasme, Brussels, Belgium.
a Address correspondence to this author at: Service de Neurologie, ULB-Hôpital Erasme, 808, route de Lennik, B-1070 Brussels, Belgium. Fax (32–2) 555-4701; e-mail sdethy{at}ulb.ac.be
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Abstract
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We used in vitro microdialysis-HPLC to determine
L-3,4-dihydroxyphenylalanine (L-DOPA) and its
metabolites in plasma of patients with advanced Parkinson disease.
Blood samples and clinical evaluations were obtained 0, 30, 60, 90,
120, and 150 min after oral administration of
carbidopa/L-DOPA (25/100 mg, 12.5/125 mg, and 50/200 mg).
In vitro recoveries for L-DOPA and metabolites ranged from
22% to 36%. Linear correlation was found between metabolite
concentrations in the dialysate and in the surrounding medium. There
was a significant positive correlation between L-DOPA dose
and plasma concentration of L-DOPA and homovanillic acid
(P <0.04). Clinical response was maximum 60 min
after L-DOPA administration. Threshold L-DOPA
plasma concentration averaged 7.74 ± 3.3 µmol/L. Motor effect
is longer with the highest L-DOPA peak concentration
(P <0.01). Microdialysis-HPLC is readily applicable,
reproducible, and allows monitoring of plasma L-DOPA
and metabolites in parkinsonian patients.
Key Words: indexing terms: Parkinson disease • levodopa • catecholamines
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Introduction
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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.
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Materials and Methods
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patients
Seven patients with idiopathic Parkinson disease and disabling
fluctuations in response to L-DOPA were examined. The
mean age ± SD was 60 ± 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 ± 3 (mean ±
SD) during "off" periods and 16 ± 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 µL (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 C18/Corasil 37–50 µm;
Waters) was used in conjunction with a C18 column (ODS,
250 x 4.8 mm, 5 µm; Beckman, Fullerton, CA). The mobile phase
consisted of 80% (by vol) 70 mmol/L NaH2PO4,
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, Na2EDTA,
NaH2PO4, and
Na2S2O5 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 HClO4 and stored at
-4 °C in the dark. Further dilution was made in antioxidant
solution (10 mmol/L HCl, 1 g/L
Na2S2O5, 0.1 g/L
Na2EDTA).
Venous blood samples from parkinsonian patients were collected into
prechilled polypropylene tubes containing
Na2S2O5 and
Na2EDTA and centrifuged (10 min, 700g) to
separate the plasma. Plasma samples were kept at -80 °C. In
microfuge tubes, 450 µL of standard mixture or plasma sample were
mixed with 50 µL of the antioxidant solution containing isoHVA (310
µg/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 µL/min. One
dialysate for each plasma sample was collected over 20 min in a vial
containing 80 µL of the antioxidant solution. A volume of 100 µL
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 µg/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 µmol/L corrected for the relative recovery
of the probe.
statistical methods
Experimental data were analyzed by two-factor ANOVA with repeated
measures.
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Results
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In vitro recoveries of L-DOPA, DA, DOPAC, and HVA
were 35.00% ± 9.15%, 27.88% ± 9.46%, 35.96% ± 6.34%, and
21.95% ± 9.76%, respectively (mean ± 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, r2 = correlation
coefficient): L-DOPA: y = 0.118x
+ 20.957, r2 = 0.999; DA:
y = 0.237x + 25.217,
r2 = 0.985; DOPAC: y =
1.330x + 53.217, r2 = 0.998;
HVA: y = 0.233x + 40.696,
r2 = 0.996.
Figure 1
shows typical chromatograms of standard solution and dialysate
from human plasma.
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Figure 1. Typical chromatograms of (left) a standard
mixture containing (1) L-DOPA (3.2 ng),
(2) DOPAC (2.1 ng), (3) DA (2.1 ng),
(4) HVA (2.4 ng), and (5) isoHVA (5 ng) and
(right) a dialysate from a patient's plasma
[(1) L-DOPA, (2) DOPAC,
(3) DA, (4) HVA, and (5) isoHVA].
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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 (Tmax) 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).
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Table 1. Basal and peak concentrations (µmol/L) of L-DOPA, DA, DOPAC, and HVA in dialysates from plasma at
three doses of exogenous L-DOPA administration and time to
reach peak concentration in plasma (Tmax)
(n = 7).
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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 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
± 3.3 µmol/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.
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Discussion
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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 µmol/L and is equivalent to
the range of 3 to 15 µmol/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 µL) 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.
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Acknowledgments
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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.
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Footnotes
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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. 
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Top
Abstract
Introduction
), 125 mg
(
), and 200 mg (
) in one patient.