Measuring L-Dopa in Plasma and Urine to Monitor ... - Clinical Chemistry

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39, No. 4, 1993. 629. Measuring L-Dopa in Plasma and Urine to Monitor Therapy of Elderly Patients with. Parkinson Disease Treated with L-Dopa and a Dopa ...

CHEM. 39/4,

CLIN.

Measuring Parkinson J. Dutton,’ have

We

plasma

629-634

(1993)

L-Dopa in Plasma and Urine to Monitor Therapy of Elderly Patients Disease Treated with L-Dopa and a Dopa Decarboxylase Inhibitor L. G. Copeland,2

established

and urine,

and L-dopac,

J. R. Ptayfer,2

a method

including

and

for measuring

using separation

by ion-pair

reversed-phase

with an electrochemical

The

to the therapeutic

was

applied

in

dopamine

HPLC and quantification assay

N. B. Roberts”3

L-dopa

the metabolites

detector.

monitoring

of

elderly patients with established Parkinson disease being treated with L-dopa plus a dopa decarboxylase inhibitor. Plasma L-dopa was evaluated in relation to dosage and postdose sampling time in 71 outpatients with Parkinson disease. L-DOpa concentrations were greatest in the patients taking the highest dosages prescribed and decreased significantly with increasing time after postdose sampling. Comparison of plasma L-dopa concentrations with a published therapeutic range established by intravenous

administration

of L-dopa

was helpful

in assessing

the suitability of each patient’s drug dosage, assessing patients’ compliance, and avoiding overdosage but was not useful in the overall clinical assessment of progression of disease or of the long-term therapeutic response. Urine measurements confirmed the plasma concentrations but showed no further advantage. The recommended time for sample collection is between 1.5 and 3 h after the first morning dose. Plasma is the preferred matrix but if blood sampling is difficult, particularly from elderly! infirm used.

individuals,

Indexing phase

.

L-Dopa

an

untimed

Terms: genatric electrochemical

detection

is the

mainstay

urine

chemistr y

collection

could

be

reversedcompliance

chromatography,

monitoring

of treatment

for patients

with

Parkinson disease (1); indeed, a positive response to L-dopa therapy may help confirm the diagnosis (2). Pharmacokinetic studies indicate that the maximum clinical response is related to the blood concentration of L-dopa (3), and drug infusion studies have shown that maintenance 1.6 mg/L

of L-dopa concentrations between 0.3 and the most consistent response (4). However, many tissues in the body have the ability to enzymatically decarboxylate L-dopa to produce dopamine (5). The introduction of a selective L-dopa decarboxylase inhibitor, either carbidopa or benzerazide, has been included in L-dopa therapy to inhibit this process and thereby reduce the associated peripheral side effects of increased dopamine production, such as postural hypotension and nausea, and also to increase the bioavailability of the parent drug to the brain (6). Because

gives

L-dopa

is

a pro-drug

Departments of’ Clinical Chemistry oyal Liverpool University Hospital, 3Author for correspondence. Received January 6, 1992; accepted

that

must

enter

the

and 2 Geriatric Medicine, Liverpool L7 8XW, UK. November

10, 1992.

with

central within effects,

nervous system and undergo decarboxylation the striatum before it produces pharmacological routine monitoring of drug and metabolite

centrations in plasma has We, however, believe that important cause of variation larly in elderly patients

therapy.

can often take less than and a few increase their dosage inappropriately to toxic concentrations. We have therefore investigated the development of a procedure for quantifying L-dopa and its major metabolites dopainine and L-dopac in plasma. We applied the assay to samples from patients with Parkinson disease seen in a routine outpatient clinic and assessed the usefulness of the drug measurement by examining individual responses to therapy. We also measured L-dopa and metabolites in urine to determine whether this would add any further information to assist in the interpretation and management of therapeutic drug control. Various studies have recently demonstrated that the renal decarboxylase enzyme converting L-dopa to dopemine (dopa decarboxylase; aromatic-L-amino-acid detheir

Many

prescribed

such

con-

not been considered useful. drug compliance may be an in drug response, particureceiving long-term L-dopa

patients

dose,

carboxylase, EC 4.1.1.28) is inhibited by carbidopa and is associated with significant decreases in urinary sodium, in both an experimental rat model (7) and healthy humans (8, 9). It is of considerable interest, therefore, to know whether long-term therapy with L-dopa, in combination with a dopa decarboxylase inhibitor, would continue to block the renal conversion of L-dopa to dopaniine and what effect this might have on the subsequent urinary excretion of sodium and water.

MaterIals

and Methods

The chemicals used were ANALAR-grade (Sigma Chemical Co., Poole, Dorset, UK), and the solvents for chromatography were HPLC-grade (Rathburn, Walkerburn, UK). De-ionized water (Spectrum System, Elga, High Wycombe, UK) was used throughout. The catechol compounds were separated on a 25 X 0.45 cm column of Ultratechsphere ODS or Spheriaorb ODS (5-j.tm particles; HPLC Technology, Macclesfield, UK) by ion-pair chromatography. The mobile phase contained, per liter, 75 mmol of citric acid, 58.5 mmol of sodium dihydrogen orthophosphate, 0.2 mmol of disodium EDTA, and 4.4 mmol of heptanesulfonic acid, carefully adjusted to pH 3.4 and made to a final volume of 2.0 L with de-ionized water. To this we added 200 mL of methanol, then filtered the solution through a 0.45-tim (pore-size) filter and degassed it before use. The HPLC system included a Kratos pump (Model 770; Applied

Biosystems CLINICAL

Inc.,

Warrington,

CHEMISTRY,

UK)

operated

Vol. 39, No. 4, 1993

at a 629

flow rate of 1.0 mL/min and fitted with a pulse-dampening device. The catechol compounds were detected electrochemically with a Coulochem electrochemical detector (ESA Analytical Ltd., Huntingdon, UK). The instrumental settings adopted were as follows: conditioning cell oxidation potential + 0.35 V, first electrode oxidation potential +0.05 V, and the second electrode reduction potential -0.35 V, with gain settings at 100 x 2 for patient monitoring (plasma and urine) and 100 x 9 for untreated controls (plasma). The individual catecholamine compounds were identified by comparing their retention time (we used a computing integrator CI 10; LDC, Stone, UK) with that of standards. The computing integrator settings were 10 mV full-scale, attenuation 3. The unconjugated (free) catechol compounds from plasma or urine were selectively extracted onto alumina (10) before quantification from 0.2 mL of plasma (undiluted) and 0.2 mL of urine (diluted 50-fold with sodium chloride, 9 g/L) for patients taking L-dopa and from 2.0 mL of plasma and 1.0 mL of urine for the healthy control subjects not receiving therapy. Extraction losses were corrected for by use of the internal standard, dihydroxybenzylamine (10). Calibration of the assay for each compound was carried out with standards prepared in human plasma or urine. The assay was calibrated between 0 and 4 gfL for the estimation of plasma concentrations (to include norepinephrine and epinephrine) in nontreated control subjects and between 0.01 and 2.0 mgfL in plasma and urine for the patients receiving L-dopa therapy and for the assay of urine samples from the untreated group (Figure 1). Aliquots of urine were analyzed for sodium and potassium by flame emission spectrometry and for urea and creatinine by standard procedures (Technicon RAXT; Technicon, Tarrytown, NY). The urine data were expressed as mass of catechol per mole of creatinine. We studied 71 patients with Parkinson disease; their clinical status and drug regimen are outlined in Table 1. Each patient received a full clinical examination; muscular rigidity, tremor, and bradykinesia were each assessed on a five-point rating scale. Patients were scored on the Hoehm and Yahr symptom severity scale (11). All observations were made without knowledge of the plasma L-dopa concentration. An overall impression of whether the patient was responding to his or her medication was then made and the response was reported as very good to poor. The patients’ drug dosage and the time of the morning dose was noted, and a timed 10-mL sample of blood taken, usually after an overnight fast, into lithium heparin tubes that were kept in ice-cold water. The plasma was separated within 30-60 mm and stored at -30 to -40 #{176}C. A midmorning urine sample was also collected, the time noted, and maintained in ice-cold water before storage in the laboratory at -30 to -40 #{176}C. The compounds collected and stored under these conditions have been shown to be stable for at least 3 months (10). The group of control patients was 12 elderly patients

630

CLINICAL CHEMISTRY,

Vol. 39, No. 4, 1993

Table

1. ClinIcal

DetaIls of PatIents DIsease

years6

Duration of disease,

Parkinson

Women

Men

Both sixes

37

34

71

Number Age,

wIth

78±6.7

73±7

(58-90)

(60-84)

yearsa

6

±

3.3

(1-15)

Hoehn-Yahr

score”

II

16

11

III

14

15

IV

2

8

L-Dopa

dosage,

mg/day

150-200(22)

()C

300-400 500

(33)

(2)

600-700(10) 800-1250 (4) a

Mean

±

SD (and range).

“Hoehn and Vahr scores (11): II, bilateral midline involvement without impairment of balance; Ill, mild to moderate disability with only slight restriction in activities and able to lead an independent life; IV, severely disabling disease, markedly incapacitated but still able to walk or stand unassisted. c Patients on a specified drug dosage for at least 1 year.

(ages

65-85

years;

7 men,

5 women)

attending

an

outpatient clinic for various conditions, e.g., angina, ischemic heart disease, and diabetes. None was taking L-dopa or had any previous history of Parkinson disease or any other significant central neurological dysfunction. Healthy laboratory control subjects (n = 19, 10 men, 9 women; ages 20-45 years) had no evidence or history of previous disease, medication, e.g., for diabetes normal renal function and Technicon SMAC).

were not on any form of or lipid disease, and had biochemical proffles (by

Results The

simultaneous

detection

of

L-dopa,

L-dopac,

and

dopamine in samples of plasma or urine was established after an alumina cleanup and separation of the ion-pair complexes by reversed-phase HPLC (Figure 1, top). Table 2 shows the retention times for these and other compounds and confirms that the chromatographic procedure can be used for their accurate quantification. Each catechol compound responded differently under the electrochemical conditions used and therefore had to be calibrated separately. Figure 1 (bottom) shows a typical calibration graph adapted for application to L-dopa monitoring in patients taking this drug. The assay performance characteristics (Table 3) indicate acceptable precision with plasma or urine for both withinand between-batch assays. The precision of analysis (CV) for each compound throughout the range of assay varied between 5% and 11% for within-batch analysis and between 7.0% and 12.0% for between-batch analysis.

Analytical

recoveries

of

L-dopa,

dopac,

dopamine from plasma (n = 35) and urine (n = 35), 0.2 mgfL of each compound added, were (mean ± SD, as follows: plasma, 103 ± 6, 92 ± 10, and 108 ± 11; urine, 108 ± 4, 95 ± 12, and 112 ± 8, respectively. 1.0 mgIL added these were: plasma, 96 ± 10, 80 ±

and

for %) an Fo 15

Table 2. Chromatographic Retention Times of Electroactlve Compounds and Metabolltes Related the Catecholamines

4

Ren

d

a

time,

Compound

Dihydroxyphenylglycol 3-Methoxy-4-hydroxymandelic L-Dopa-3,4-dihydroxyphenylalanine Iso-3-methoxy-4-hydroxymandelic

a

d 0

Tb S

is

0

0 m

ren mine

tlm.b

4.23

0.41

4.55

0.43

5.37 5.73

0.51

Norepinephnne

6.00

0.57

Metanephnne

6.95

0.66

3-Methoxy-4-hydroxyphenylglycol

6.98

0.663

Epinephrine 3-Methoxytyrosine

8.18

acid acid

0.54

lso-3-methoxy-4-hydroxyphenylglycol

10.10

0.78 0.88 0.96

Methoxytyrosine

10.52

0.99

Dihydroxybenzylamine Normetanephrine Dihydroxyphenylacetic acid

10.53

1.00

11.62

1.10

9.30

Dopamine

20

to

5-Hydroxyindoleacetic

acid

12.45

1.18

15.89 23.91

1.51 2.27

>30.00 5-Hydroxytryptamine 6Determined with a25 x 0.46 cm Ultratechsphere ODS column. b Relative to the internal standard dihydroxybenzylamine.

ii

250

oo

Table 3. Intra- and Interassay PrecisIon of AnalysIs for L-DOpa and Other Catecholamines In Serum (n = 6)

0

l_

0

50 (0

Mien,

I

cv, s

mL

C

L-Dopa

0.41 0.92

7.6 5.1

Dopamine

0.34

5.8

8.9

6.3

Dopac

1.01 0.25

10.1 10.1

00

50

0.81

Norepinephnne6

0 0.0

0.4

0.8

1.6 Concentration

2.0

mg/L

Example of chromatographic separation of catecholamine compounds on reversed-phase HPLC; (bottom) calibration relationships for L-dopa, L-dopac, and dopamine suitable for theraFig. 1. (Top)

0.3 x iO 0.15 x iO

6.4 5.8

9.6 9.1

9.9

6.1 11.3

7.6 12.4 Epinephnne6 n = 6 each. A serum was supplemented to contain two different concentrations to cover the therapeutic limits expected for patients receiving L.dOpa therapy. Bio-Rad control serum.

peutic drug monitoring of L-dopa Top (a) L-dopa, (b) norepinephrine, (c) epinephnne, ( dopamine, (IS) internal standard (dihydroxybenzylamine) at concentrations of 30,8.5,4.6, and 30 ug/t. (left) and 300, 85, 46, and 300 p.g/L (nght), respectively. L-Dopac elutes at --12.5 mm, between the IS and dopamine. Full-scale deflection, 5 mV. Bottom: the data are expressed as a percentage of the IS, which is added in concentrations sufficient to give a similar electrochemical response to that of the

highest-concentration

dopamine

standard

and

115± 12;andurine, 101 ± 6,98 ± 10, and 108±9. Plasma L-dopa concentrations for 104 analyses of samples from the 71 patients varied from 1.6 mg/L) suggested overdosage. Plasma L-dopa concentrations varied from 0.06 to 2.28 mg/L in nine patients who had had a good response to L-dopa on 375-750 mg/day; however, these values were not different from the concentrations of 0.1-0.9 mg/L observed in three patients, taking 375-825 mg/day, who had poor drug responsiveness. Thus, measurement of L-dopa was helpful in assessing adequate therapeutic concentrations but not in determining the patients’ long-term drug responsiveness. Plasma and urine L-dopa concentrations were signifCLINICAL

CHEMISTRY,

Vol. 39, No. 4, 1993

631

20

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