Sulfate and methyldopa metabolism: Metabolite ...

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Methyldopa powder, 3.5 mg/kg, was taken with and without sodium sulfate, 13.25 ... PST activities and percentages of metabolites excreted as methyldopa ...
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 0-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%; X ± 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 < 0.01). This correlation was not significant when methyldopa was taken alone (r = 0.340; P > 0.10). There was a significant correlation between red blood cell COMT activities and the proportion of urinary metabolites excreted as 3-0-methyl-a-methyldopa when methyldopa was taken alone (r = 0.532; P < 0.01) but not when it was taken with sodium sulfate (r = 0.153; P> 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. Platelet PST activity correlates with individual variations in the sulfate conjugation of methyldopa taken by mouth with sodium sulfate. (CLIN PHARMACOL THER 37:308315, 1985.)

Norman R. C. Campbell, M.D.,* Rajah S. Sundaram, M.D.,** Peter G. Werness, Ph.D., Jon Van Loon, B.A., and Richard M. Weinshilboum, M.D.*** Rochester, Minn. Methyldopa is a common antihypertensive. There are wide individual variations in methyldopa metabolism and in the dose of the drug required to control blood pressure in patients with hypertension.' The major metabolic pathway for methyldopa is sulfate conjugation catalyzed by phenol sulfotransferase (PST) .5 From the Clinical Pharmacology Unit, Department of Pharmacology, Mayo Foundation/Mayo Clinic. Supported in part by a grant from Merck Sharp and Dohme Research Laboratories and by National Institutes of Health Grant No. GM 28157. Received for publication Aug. 17, 1984; accepted Nov. 5, 1984. Reprint requests to: Dr. Richard Weinshilboum, Clinical Pharmacology Unit, Department of Pharmacology, Mayo Foundation/ Mayo Clinic, Rochester, MN 55905. *Merck Sharp and Dohme International Fellow in Clinical Pharmacology. **Canadian Heart Foundation Research Fellow. ***Burroughs Wellcome Scholar in Clinical Pharmacology.

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O-Methylation catalyzed by catechol-O-methyltransferase (COMT) and decarboxylation catalyzed by aromatic L-amino acid decarboxylase are quantitatively less important pathways for its biotransformation.''' O-Methylation results in the formation of 3-0-methyla-methyldopa (3-0MMD), while decarboxylation results in the formation of a-methyldopamine, which can also undergo sulfate conjugation to form a-methyldopamine sulfate.''"4 Previous studies of methyldopa metabolism have suggested that variations in the availability of the sulfate donor for the PST reaction, 3'phosphoadenosine-5'-phosphosulfate (PAPS), might be one factor responsible for individual differences in methyldopa metabolism.' For example, the percentages of methyldopa and a-methyldopamine excreted as sulfate conjugates decrease as the dose of methyldopa increases.' This observation is confirmed by the finding that decreasing percentages of methyldopa and a-meth-

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yldopamine are excreted as sulfate conjugates as absorption of the drug increases.' Salicylamide and acetaminophen are two other drugs metabolized by sulfate conjugation.9'12 Higher doses of acetaminophen and salicylamide are less sulfated than are lower doses.9'12 Dose-related changes in the metabolism of salicylamide and acetaminophen do not occur if sources of sulfate such as L-cysteine or sodium sulfate are taken orally with these drugs.9'12 Sulfate is required for the formation

Sulfate and methyldopa metabolism

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of PAPS." Our experiments were designed to determine whether oral inorganic sulfate might alter the metabolism of clinically used doses of methyldopa. We also studied the effect of acetaminophen on methyldopa, another drug that is metabolized by sulfate conjugation Finally, we determined whether oral inorganic sulfate might alter the relationship between methyldopa metabolism, platelet PST activity, and red blood cell (RBC) COMT activity. We have previously shown that in the absence of exogenous sulfate, platelet PST activity does not correlate with the extent of sulfation of methyldopa.' In the same study we found that RBC COMT activity correlates with the extent to which the drug is 0-methylated.' In that study and in the experiments reported here, it was not assumed that platelets or erythrocytes play important roles in drug metabolism. However, platelet PST and RBC COMT activities correlate with the activities of these enzymes in other tissues that do play a role in drug metabolism.2,18,19,21 Our results show that oral inorganic sulfate alters methyldopa metabolism in man.

METHODS Our subjects were 13 men and 11 women. Twentythree were white and one man was of East Indian origin. Average age was 26.8 ± 4.5 years (X ± SD) for the women and 29.1 ± 3.6 years for the men. None of the subjects was acutely or chronically ill, and none took any other medication during the study. All subjects fasted overnight. Blood was drawn between 7:30 and 9:30 AM the following morning. At that time, the drugs were given and 24-hour urine collections were initiated. Food was not permitted until noon on the day of the study. Platelet PST and RBC COMT activities were measured in the blood samples. Routine blood chemistry values (sodium, potassium, calcium, phosphorus, total protein, glucose, alkaline phosphatase, SGOT, bilirubin, uric acid, and creatinine) and routine hematology values were also determined. None of the subjects had abnormal hepatic or renal function. Subjects were randomized to take either methyldopa (3.5 mg/kg) or methyldopa (3.5 mg/kg) with sodium sulfate (13.25

MD

MD

SULFATE

a-MDA

a-MDA

3-0MMD

SULFATE

Fig. 1. Urinary excretion of methyldopa and four metabolites in 24 subjects after oral methyldopa alone or methyldopa with sodium sulfate. Each metabolite is expressed as a percentage

of methyldopa plus all measured metabolites excreted in urine (TC ± SE). The solid columns represent methyldopa and the open columns represent methyldopa plus sodium sulfate. < 0.005 compared with the same metabolite in the absence of sodium sulfate.

mg/kg) by mouth. All subjects were crossed over to the alternative treatment 1 week later. The methyldopa dose was chosen to approximate for a 70 kg subject the 250 mg dose used clinically. It was also the dose used in our earlier study of methyldopa metabolism.' The dose of sodium sulfate was chosen to be less than one fifth that recommended as a laxative.' Sodium sulfate at these dose levels is at least 50% bioavailable.° After completion of the initial protocol, the final 10 subjects studied were randomized to take methyldopa (3.5 mg/kg) and acetaminophen (10 mg/kg) or methyldopa (3.5 mg/kg), acetaminophen (10 mg/kg), and sodium sulfate (13.25 mg/kg) by mouth. These 10 subjects were then crossed over to the alternative treatment. The dose of acetaminophen was chosen to approximate for a 70 kg subject the drug dose most commonly used clinically. Finally, the same 10 subjects took a 250 mg tablet of methyldopa. All studies were performed 1 week apart. In all cases, 24-hour urine samples were collected. Methyldopa and its metabolites were measured in urine within 72 hours of collection. Previous experiments have shown that the metabolites of methyldopa we measured are stable in urine for at least 1 month when stored at 4° C.5 Platelets were isolated from blood samples anticoagulated with EDTA and platelet homogenates were prepared within 3 hours of venipuncture.1'2RBC lysates were prepared from blood anticoagulated with heparin as described by Raymond and Weinshilboum.15 Platelet

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Fig. 2. Correlation of the urinary recovery of methyldopa plus all measured metabolites with (A) the excretion of methyldopa (MD) sulfate expressed as a percentage of the sum of methyldopa plus all measured metabolites in urine and (B) the urinary excretion of a-methyldopamine (a-MDA) sulfate expressed as a percentage of all a-methyldopamine excreted.

PST activity was measured by the method of Foldes and Meek' as modified by Anderson and Weinshilbouml and by Reiter and Weinshilboum." Methyldopa and amethyldopamine are both sulfate conjugated in man by the thermolabile form of the enzyme.1416 One unit of PST activity represented the formation of 1 nmol dopamine sulfate per hour of incubation at 37° C. PST activity was expressed per 108 platelets, because this method for expressing the results produces less variation than expression of activity per milligram of platelet

Company); perchloric acid, 70% (E.I. Dupont de Nemours and Company); pargyline (Saber Laboratories Inc.); AG50W-X4 ion exchange resin (BioRad Laboratories); methyldopa and 3-0MMD (Merck Sharp and Dohme Research Laboratories); acetaminophen powder (City Chemical Corporation); sodium sulfate powder (Mallinckrodt Chemicals); and a-methyldopamine was a gift.*

protein.'

Average 24-hour urinary excretions of methyldopa and its metabolities by 24 subjects after either methyldopa alone or methyldopa with sodium sulfate are shown in Fig. 1. Because of wide individual variations in the absorption of methyldopa, each metabolite is expressed as a percentage of the sum of the parent drug plus all measured metabolites for that subject. When methyldopa was taken without sulfate, methyldopa sulfate averaged 50.1% ± 7.5% of all urinary metabolites (Fig. 1). Under the same circumstances, free urinary methyldopa accounted for an average of 27.3% ± 5.5% of the measured metabolites, while a-methyldopamine, a-methyldopamine sulfate, and 3-0MMD averaged 8.8% ± 1.7%,5.6% ± 4.1%, and 8.2% ± 2.9%. When the same 24 subjects took sodium sulfate with methyldopa, there was a significant change in the pattern of the urinary metabolites. The proportion of drug excreted as methyldopa sulfate rose 32%, from 50.1% ±_ 7.5% to 66.0% ± 5.3%. Excretion of free methyldopa decreased 35%, from 27.3% ± 5.5% to 17.1% ± 3.7%. After sulfate, there were also significant decreases in the percentages of urinary metabolites

RBC COMT activity was measured by the method of Raymond and Weinshilboum.' 3 ,4-Dihydroxybenzoic acid at a concentration of 1 mmol/L was used as the catechol substrate. One unit of COMT activity represented the formation of 1 nmol of methylated product per hour of incubation at 37° C.' Methyldopa and its metabolites were isolated from urine samples and were measured by HPLC as described previously." Mean values were compared by Student's t test for paired data. Pearson's product moment correlation coefficients were calculated for the correlation analyses. All calculations were performed with a Hewlett-Packard 9845B computer. Unless otherwise indicated, data are reported as X ± SD. The following materials were used: "S-PAPS (2.0 to 4.5 Ci/mmol) and '4C-S-adenosyl-L-methionine (52 tiCi/mmol; New England Nuclear Corp.); p-nitrophenol, dopamine, dithiothreitol, bovine serum albumin, alumina, D,L-3-methoxytyrosine, and 3,4-dihydroxybenzylamine (Sigma Chemical Co.); disodium EDTA, HPLC grade phosphoric acid, HPLC grade acetonitrile, and 1-heptane sulfonic acid (Fisher Scientific Company); sodium metabisulfite (Baker Chemical

RESULTS

*From Dr. Neil Castagnoli, University of California Medical School, San Francisco, Calif.

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