Methyldopa kinetics before and after ingestion of methyldopa for eight weeks.

Methyldopa urine and plasma levels and urine metabolite levels were assessed following intravenous (IV) and oral (PO) methyldopa before, and after ingestion of methyldopa (500 mg) daily for eight weeks. There was no increase in (estimated) methyldopa absorption (8.4%) or renal clearance (PO 13.9%, IV 2.33%) after the eight weeks of methyldopa ingestion. However, the initial methyldopa absorption and renal clearance values in this study were higher than that in previous studies. There was an inverse relation between the initial methyldopa absorption and the change in absorption (r - 0.605) and between the initial methyldopa renal clearance and the change in renal clearance (PO r -0.874, IV r -0.891). Overall, this study did not confirm our previous studies showing induction of methyldopa absorption and renal clearance, possibly due to prior up regulation of transporter function. Consistent with methyldopa inducing drug transporters, those with low initial absorption and renal clearance values had the greatest increases.

Increases in methyldopa absorption and renal excretion after multiple doses.

A retrospective analysis of previous studies examining methyldopa absorption suggested the possibility that the absorption of methyldopa might increase on repeat methyldopa ingestion. A prospective study was undertaken to determine the effect of repeated oral doses of methyldopa on methyldopa absorption. Thirteen healthy subjects ingested single 250 mg methyldopa doses on days 0, 7, 14, 28, 56, and 112; 24 urine samples were collected and analyzed for methyldopa and its major metabolites on each study day and methyldopa plasma levels were measured over 8 hours at days 0 and 56. There were significant increases in the absorption of methyldopa (as estimated by the urinary excretion of methyldopa and the measured metabolites over 24 hours) at day 56 (33.4 +/- 8.9%, P less than .025) compared with day 0 (26.0 +/- 10.8%). There was also a significant increase in renal clearance of unmetabolized methyldopa (62.7 +/- 13.6 vs. 99.3 +/- 29.1 mL/min, P less than .01) and a decrease in the plasma half-life of methyldopa at day 56 (2.22 +/- 0.91 vs. 1.56 +/- 0.68 hr, P less than .05). There was a tendency toward increases in methyldopa absorption at day 7, 14, 28, and 112. Several possible explanations for the changes in methyldopa disposition are discussed.

High-performance liquid chromatographic determination of L-3-(3,4-dihydroxyphenyl)-2-methylalanine (alpha-methyldopa) in human urine and plasma.

A procedure is described for the determination of alpha-methyldopa (MD) [L-3-(3,4-dihydroxyphenyl)-2-methylalanine], its metabolite and catecholamines in the urine and plasma of patients undergoing MD therapy, by high-performance liquid chromatography with dual working electrode coulometric detection. An efficient sample preparation procedure is presented for the isolation of endogenous MD, its metabolite and catecholamines from plasma or urine. After deproteinization of a plasma sample with methanol containing 2% of 0.5 M perchloric acid and dilution of a urine sample (1:200), MD, dihydroxyphenylacetic acid (DOPAC), 3-O-methylmethyldopa (3-OMMD) and homovanillic acid (HVA) were separated with a Supelcosil LC-18 column. Catecholamines were extracted from the supernatant of deproteinized plasma or from urine by ion exchange on a Sephadex CM-25 column and subsequent adsorption on alumina. The use of the same mobile phase for the concurrent assay of MD, its metabolite and catecholamines increased considerably the efficiency of sample separation. Recoveries were close to 100% for MD, DOPAC, 3-OMMD and HVA and 70% for catecholamines. The effects of various experimental parameters related to mobile phase composition on chromatographic performance are reported. The purity of the eluted compounds was tested by recording both the first detector response (oxidation current) and the second detector response (reduction current). The ratio of the detector responses yielded a chemical reversibility ratio for the detected compound. A number of applications such as monitoring data from patients under MD therapy are presented.

Fully automated high-performance liquid chromatographic assay for the analysis of free catecholamines in urine.

A totally automated and reliable high-performance liquid chromatographic method is described for the routine determination of free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines were isolated from urine samples using small alumina columns. A standard automated method for pH adjustment of urine before the extraction step has been developed. The extraction was performed on an ASPEC (Automatic Sample Preparation with Extraction Columns, Gilson). The eluate was collected in a separate tube and then automatically injected into the chromatographic column. The catecholamines were separated by reversed-phase ion-pair liquid chromatography and quantified by fluorescence detection. No manual intervention was required during the extraction and separation procedure. One sample may be run every 15 min, ca. 96 samples in 24 h. Analytical recoveries for all three catecholamines are 63-87%, and the detection limits are 0.01, 0.01, and 0.03 microM for norepinephrine, epinephrine and dopamine, respectively, which is highly satisfactory for urine. Day-to-day coefficients of variation were less than 10%.

Sulfate and methyldopa metabolism: metabolite patterns and platelet phenol sulfotransferase activity.

Sulfate conjugation catalyzed by phenol sulfotransferase (PST) is the major metabolic pathway for methyldopa. Methyldopa is also O-methylated in a reaction catalyzed by catechol-O-methyltransferase (COMT). Our studies were performed to determine whether sodium sulfate alters methyldopa metabolism. Methyldopa powder, 3.5 mg/kg, was taken with and without sodium sulfate, 13.25 mg/kg, by 24 subjects in a randomized, crossover design. Compared with results obtained when only methyldopa was taken, sodium sulfate taken with methyldopa increased the proportion of drug excreted as methyldopa sulfate expressed as the percentage of all urinary metabolites (66.0% +/- 5.3% and 50.1% +/- 7.5%; means +/- SD). The percentage of free methyldopa excreted also decreased (17.1% +/- 3.7% and 27.3% +/- 5.5%). Platelet PST and red blood cell COMT activities were measured in blood samples from these subjects. When sodium sulfate was taken with methyldopa, there was a significant correlation between platelet PST activities and percentages of metabolites excreted as methyldopa sulfate (r = 0.545; P less than 0.01). This correlation was not significant when methyldopa was taken alone (r = -0.340; P greater than 0.10). There was a significant correlation between red blood cell COMT activities and the proportion of urinary metabolites excreted as 3-O-methyl-alpha-methyldopa when methyldopa was taken alone (r = 0.532; P less than 0.01) but not when it was taken with sodium sulfate (r = 0.153; P greater than 0.20). Our data support the conclusion that variation in sulfate availability may be one factor responsible for individual differences in the metabolism of clinically used doses of methyldopa.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitive high-performance liquid chromatographic assay using electrochemical detection for a novel prodrug ester of methyldopa, pivaloyloxyethyl 3-(3,4-dihydroxyphenyl)-2-methylalaninate, in plasma and urine.

The pivaloyloxyethyl ester of methyldopa is an antihypertensive prodrug possessing improved bioavailability properties over methyldopa. A sensitive cation-exchange, high-performance liquid chromatographic assay using electrochemical detection has been developed for the ester in plasma and urine in order to determine the extent of its hydrolysis after oral administration. The chromatographic conditions involve two Altex Partisil 10 SCX columns (25 cm X 4.6 mm) in series; a mobile phase consisting of methanol, potassium phosphate buffer, pH 3.0, and EDTA disodium dihydrate; and an electrochemical detector set at 0.5 V. The pivaloyloxyethyl ester in plasma or urine is extracted into ethyl acetate, back-extracted into 0.1 M sulfuric acid, and analyzed directly by high-performance liquid chromatography. For urine, the ethyl acetate extract is washed with a buffer (pH 8.0) prior to the back-extraction step. The assay gives a linear response over the concentration range of 10-160 ng/ml in plasma and 20-400 ng/ml in urine.

Metabolism of methyldopa in man after oral administration of the pivaloyloxyethyl ester.

In a crossover study, the pivaloyloxyethyl ester (POE) of methyldopa, labeled either with 3H in the methyldopa moiety or 14C in the pivalic acid moiety, was administered orally to four volunteers in 1000-mg single doses (equivalent to 500 mg of methyldopa). The majority (93%) of either the 3H- or 14C-labeled dose was excreted in the urine. Methyldopa, which was assayed by a fluorometric technique, peaked (approximately 6 micrograms/ml) at 1 hr in the plasma. Forty-five per cent of the dose was excreted as methyldopa as opposed to 18% normally seen after oral methyldopa dosages. Intact POE was absent in the urine of three volunteers and present in only trace amounts in urine from a fourth volunteer. Thus, the oral dose of POE was well absorbed and rapidly hydrolyzed to methyldopa. After oral administration of methyldopa, methyldopa sulfate is the principal urinary metabolite in man. However, after administration of POE, a relatively small fraction (13%) of the dose was excreted as methyldopa sulfate. The major urinary metabolite of POE, other than methyldopa, was 3-OCH3 methyldopa. Methyldopamine was a minor metabolite. It was concluded that a shift from sulfation to methylation occurred in the metabolic profile of methyldopa when it was administered as POE and that the metabolites of POE (including conjugated pivalic acid) were rapidly eliminated from the body.

A study of conjugation and drug elimination in the human neonate.

1 An investigation has been made of the excretion of alpha-methyldopa, alpha-methyldopa sulphate, lorazepam and lorazepam glucuronide in the urine of neonates. 2 The rate of elimination of both the drugs in the newborn is slow compared with the adult rate, and apparent half-life being 3 to 4 times longer than the reported adult values. 3 The newborn appear able to readily metabolise alpha-methyldopa to alpha-methyldopa sulphate and to slowly conjugate lorazepam with glucuronic acid. alpha-Methyldopa tends to be conjugated to a greater extent and lorazepam to about the same or slightly lesser extent in the newborn than in adults. 4 It is postulated that elimination in the neonate is mainly controlled by the rate of renal excretion in the case of alpha-methyldopa and by the rate of conjugation in the case of lorazepam.

A study of the disposition of alpha-methyldopa in newborn infants following its administration to the mother for the treatment of hypertension during pregnancy.

1 The nine infants participating in this study were born to mothers who received continuous therapy with alpha-methyldopa (0.75-2.0 g/day) for several weeks extending to the time of delivery. 2 The concentration of free and total (free plus conjugated) alpha-methyldopa was determined by gas chromatography-mass spectrometry in amniotic fluid, umbilical cord plasma and maternal plasma at delivery; also in urine collected over time intervals from neonates during the first days after birth. 3 The results indicate that alpha-methyldopa administered to the mother is present in the infant at birth at a level comparable to the maternal level and persists for some days. The ratio of conjugated to free drug increases with time after birth. 4 The excretion of free and conjugated alpha-methyldopa in the urine indicated that the drug is slowly eliminated in the neonate by excretion in the urine and apparently by metabolism, mainly to the sulphate conjugate. 5 The concentration of free and conjugated alpha-methyldopa in amniotic fluid tended to be higher than in umbilical cord plasma but lower than in neonatal urine, conjugated drug predominated.

Urinary free methyldopa determined by reversed-phase high-performance liquid chromatography.

We describe a chromatographic method, in which 3,4-dihydroxybenzylamine is used as the internal standard, for determining free methyldopa in human urine. The drug was adsorbed onto alumina, eluted, and the eluate directly injected onto a reversed-phase column (octadecyl-bonded silica stationary phase), with dilute acetate buffer (pH 2.7) as the mobile phase and ultraviolet detection at 280 nm facilitated. Methyldopa is well separated from other urinary biogenic amines present in the alumina extract, and other commonly used antihypertensives and diuretics do not interfere with the analysis. The sensitivity of the method is adequate to quantify 8.0 mg of methyldopa per liter in 30 ml of sample; the lower limit of detection is 25 ng. Analytical recovery for methyldopa varied from 95 to 102% with within-run and day-to-day coefficients of variation of 2.7 (n = 10) and 3.8% (n = 5), respectively. This procedure is readily adaptable for use in studies of the pharmacokinetics of methyldopa and to routine clinical laboratory use.

Noneffect of methyldopa on urine glucose tests.

Methyldopa has been reported to cause a false-positive urine glucose test by the copper reduction method (Clinitest). We investigated the effect of methyldopa on urine glucose tests in 30 patients by comparing commonly used glucose oxidase methods to Clinitest. The results of our study indicate that methyldopa does not interfere with the copper reduction method for urine glucose determination.

Identification of selected antihypertensive drugs by thin-layer chromatography.

A thin-layer chromatographic procedure is described for the qualitative identification of several antihypertensive drugs including certain thiazide diuretics spironolactone, triamterene, methyldopa and their metabolites. Utilization of new solvent developing systems and spray detecting reagents provides a method useful for the identification of these compounds in biologic fluids at low therapeutic concentrations. Sensitivity limits for these antihypertensive drugs are given, and alternate techniques to provide confirmatory analyses are also presented.

Clinical and cardiovascular effects of alpha methyldopa in combination with decarboxylase inhibitors.

The peripheral decarboxylase inhibitor carbidopa, given (100 mg/day) for 6 wk in a double-blind trial, lowered supine diastolic pressure of 10 patients with essential hypertension treated with alpha methyldopa by a small (6 mm Hg) but significant (p less than 0.05) amount. Large doses of benserazide (1.5 gm) did not modify the hypotensive effect of 1.0 gm of alpha methyldopa in untreated hypertension but significantly reduced the central nervous side effects of sedation and dry mouth. These studies indicate that extensive peripheral decarboxylation is not necessary for alpha methyldopa to lower blood pressure and would be compatible with the central nervous site of hypotensive action of this drug.

Urinary free norepinephrine and dopamine determined by reverse-phase high-pressure liquid chromatography.

We used reverse-phase high-pressure liquid chromatography to measure free norepinephrine and dopamine simultaneously in human urine. Samples were treated with alumina, and the catecholamine(s) then eluted from it were directly injected onto a reverse-phase column (octadecyl-silica stationary phase), with 0.17 mol/liter acetic acid as the mobile phase and ultraviolet detection at 280 nm. The assay detects concentrations in urine as low as 5 mug/liter. Assay of 24-h urines (n = 10) gave within-run and day-to-day coefficients of variation of 3.7 and 4.7% for norepinephrine, and 2.6 and 3.5% for dopamine, respectively. Comparison studies with the traditional trihydroxyindole fluorometric method showed the liquid-chromatographic procedure to be more precise and subject to less interference. This relatively rapid procedure for urinary free norepinephrine and dopamine provides an efficient, reproducible method, readily adaptable to routine clinical use.

[Urinary excretion of 3,4-dihydroxyphenyl-alanine, 3-O-methyldopa, dopamine and homovanillic acid in man. Effects of an inhibitor of decarboxylase of aromatic amino acids (Benserazide) (author's transl)]. / Excretion Urinaire de 3,4-Dihyroxyphenyl-alanine, de 3-O-Methyldopa, de Dopamine et d'Acide Homovanillique chez l'Homme. Effet d'un Inhibiteur de la Decarboxylase des Acides Amines Aromatiques (Bensérazide)

After administration of a single and small (125 mg) dose of benserazide, 3,4-dihydroxyphenyl-alanine and 3-O-methyldopa are excreted in urine. Uturbancies last for at least 48 hours.

Detection of occult metastatic melanoma by urine chromatography.

By using ion-exchange column chromatography with effluent monitoring using the stable, free radical alpha,alpha-diphenyl-beta-picryhydrazyl as a colorimetric reagent, we have demonstrated the occurrence of elevated levels of five peaks in the urine of patients with metastatic disease. The tentative assignment of two of the peaks as 3,4-dihydroxyphenylalanine and as 3-methoxy-4-hydroxyphenylalanine has been made. Three remain unknown. The correlation of these peaks with the clinical status of melanoma patients shows that, while the individual excretion pattern of these compounds may be variable, the sustained occurrence of one or more of them in a patient's urine is evidence of recurrent or continuing disease. The excretion levels appear to be proportional to the tumor burden. The results with a group of 39 melanoma patientshaving Stage II or Stage III disease indicate that this chromatography technique provides earlier evidenc eof liver metastases than doses the liver scan, may detect occult metastases generally, and has detected tumor in clinically enlarged lymph nodes. This method, in its present form, does not detect small pulmonary lesions earlier than chest X-ray or tomography do or brain metastases earlier than do brain scan or computerized axial tomography. The technique is clinically useful for the diagnosis of melanoma patients and in their follow-up while under treatment.