Introduction

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Dobutamine (Fig. 1) is an ionotropic synthetic catecholamine commonly used alone or with other adrenergic agents for temporary support of patients with low cardiac output states and shock (Latifi et al., 2000). Given as a racemic mixture of (+)- and ()-isomers with differing activity at - and -adrenergic receptors, this compound enhances cardiac output primarily by increasing stroke volume with usually only modest chronotropic effects. Dobutamine is given by intravenous infusion because it disappears rapidly from the systemic circulation.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Chemical structures of dobutamine (A) and 3-O-methyldobutamine (B).

The metabolic disposition of infused dobutamine has yet to be defined in humans. Catecholamines introduced directly into the bloodstream are generally metabolized by catechol-O-methyltransferase (COMT¹) and monoamine oxidase in most animal species, whereas dietary or orally administered catecholamines are commonly conjugated with sulfate (Kopin, 1985; Eisenhofer et al., 1999; Dooley et al., 2000). However, formation of 3-O-methyldobutamine (Fig. 1) via the action of COMT followed by conjugation with glucuronide constitutes the major metabolic route of dobutamine disposition in the dog (Murphy et al., 1976). Whether 3-O-methyldobutamine and/or its conjugates contribute to the overall cardiovascular effects of dobutamine or whether these compounds interact with other drugs in human patients requires elucidation, especially because 3-O-methyldobutamine can act as a potent inhibitor of ₁-adrenoreceptors in isolated in vivo systems (Ruffolo et al., 1985).

Herein, we report the isolation of 3-O-methyldobutamine from urine samples of pediatric patients treated with racemic dobutamine. Moreover, about 80% of dobutamine administered intravenously at steady state to a child with heart failure was detected in the urine, largely as 3-O-methyldobutamine-related derivatives (47%) and dobutamine metabolites (32%). Sulfate conjugates of both 3-O-methyldobutamine and dobutamine predominated, comprising 33 and 16% of the infused dopamine. In vitro experiments disclosed that crude sonicates of mononuclear cells isolated from human blood catalyzed the formation of 3-O-methyldobutamine from dobutamine and S-adenosylmethionine.

	Materials and Methods

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Chemicals. S-Adenosyl-1-methionine (SAM), Tris (hydroxymethyl) aminomethane base, bovine serum albumin, adenosine deaminase (EC 3.5.4.4; 78 U/mg of protein) from bovine spleen, -glucuronidase (EC 3.2.1.31; 1 × 10⁶ U/mg of protein) from bovine liver, and partially purified sulfatase (EC 3.1.6.1; 2-5 U/mg of protein) from Aerobacter aerogenes were obtained from Sigma Chemical (St. Louis, MO). Sodium octylsulfate was purchased from Eastman Kodak (Rochester, NY), partially purified porcine liver COMT (EC 2.1.1.6, 2200 U/mg of protein) from Calbiochem (La Jolla, CA), and (±)-dobutamine from Sigma/RBI (Natick, MA). HPLC-grade acetonitrile, ethyl acetate, and methylene chloride were obtained from Burdick and Jackson Labs (Muskegon, MI) and acid-washed alumina from Bioanalytical Systems (West Lafayette, IN). Deuterated chloroform was purchased from Merck (Whitehouse Station, NJ). HCl, HClO₄, NaH₂PO₄, and HPLC-grade 85% phosphoric and glacial acetic acids and other reagent-grade chemicals were supplied by Fisher Scientific (Pittsburgh, PA).

Collections from Patients. At this institution urine samples are routinely collected and subjected to various clinical laboratory tests for each patient's benefit before being discarded. Accordingly, although patient confidentiality was maintained, parental permission was not routinely sought to save urine samples used for the experimental determinations described herein. Urine from several pediatric patients undergoing intravenous therapy with racemic dobutamine for cardiac support was saved for isolation and purification of the 3-O-methyldobutamine standard. These collections were pooled and stored at 70°C before further processing. For the partial balance study done in a 9-year-old white male with cardiac failure (Table 3) urine was collected both before (control) and during a timed 5.5-h (experimental) period beginning 48 h after a continuous dobutamine intravenous infusion prescribed at 3 µg min¹ kg¹. A sample of the infusate was stored at 70°C for later determination of dobutamine. Control and experimental urine samples also were stored at 70°C for later analyses of free dobutamine and total dobutamine released by acid hydrolysis, dobutamine sulfate and dobutamine glucuronide, free 3-O-methyldobutamine and total 3-O-methyldobutamine released by acid hydrolysis, 3-O-methyldobutamine sulfate, and 3-O-methyldobutamine glucuronide. The total volume and pH of collected urine were recorded before sample storage.

Equipment. Liquid chromatography with electrochemical detection (HPLC-EC) was carried out with an LC-400 Bioanalytical Systems liquid chromatograph equipped with a carbon/carbon electrode and interfaced with a Varian Instruments (Sunnydale, CA) model 2510 pump. The potential of the working electrode was maintained at +700 mV versus a Ag⁺/AgCl reference electrode. Separations were achieved in a reversed phase system with a Bioanalytical Systems phase II ODS stainless steel prepacked column used as the stationary phase (100 × 3.2-mm i.d.; particle size 3 µm). The mobile phase consisted of 880 ml of 0.069 M acetic acid, 2 mM Na₂EDTA adjusted to pH 4.5 with 5 M NaOH before addition of 120 ml of acetonitrile; the resulting solution was passed through nitrocellulose filters and degassed for 15 min with nitrogen. After manual injection of 0.1 ml of sample, the flow rate used for chromatography was 0.8 ml/min at 25°C.

Preparation of Reference 3-O-Methyldobutamine. To establish conditions for chromatographic separation of 3-O-methyldobutamine from human urine, the reference metabolite was generated enzymatically from dobutamine, SAM, and partially purified porcine COMT essentially as described by Allen et al. (1992). Reaction mixtures (0.55 ml) contained 0.1 M NaH₂PO₄ buffer, pH 7.4, 0.125 mM dobutamine, 218 µM SAM, 10.9 mM MgCl₂, and 3 U (38 µg of protein) of adenosine deaminase. Reactions were initiated by addition of 25 units of COMT (~10 µg of protein) and proceeded for 45 min at 37°C when they were stopped by addition of 0.5 ml of 1.33 M sodium borate buffer, pH 11, containing 1% (w/v) Na₂EDTA, followed immediately by 5 ml of methylene chloride. After vigorous vortex mixing (30 s) the lower organic phase was removed and evaporated to dryness under vacuum. The residue was reconstituted in 0.2 ml of mobile phase solution, filtered through a nylon microfilter (0.2-µm pore size), and 0.1 ml was injected into the HPLC system for chromatographic analysis.

Preparation of 3-O-Methyldobutamine Standard from Human Urine. 3-O-Methyldobutamine used as the external standard was isolated and purified from acid-hydrolyzed pooled samples of urine from dobutamine-treated patients. For acid hydrolysis, 5 ml of thawed urine adjusted to pH 3 with 6 M HCl was mixed with 0.2 ml of 12 M HCl and incubated at 90°C for 30 min after which the mixture was cooled and adjusted to pH 6.5 with 3 M NaOH. Subsequent extraction and processing for reverse phase HPLC followed the same procedure used for enzymatically prepared reference 3-O-methyldobutamine. Fractions eluting with the same retention time as reference 3-O-methyldobutamine were pooled and evaporated to dryness with a Savent Speed Vac concentrator (Farmington, NY). For further purification, residues from many chromatographic separations were taken up in 0.5 ml of mobile phase solution and chromatographed again. Fractions containing a single large peak eluting at the 3-O-methyldobutamine reference retention time were combined, evaporated to dryness, dissolved in 0.5 ml of purified water, extracted into 5 ml of methylene chloride, back extracted with 1 ml of 0.1 N HCl, and vortex mixed. After centrifugation, the upper aqueous layer was removed, evaporated to dryness, taken up in 0.5 ml of water, added to 0.5 ml of 1.33 M sodium borate buffer, pH 11, containing 1% (w/v) Na₂EDTA, extracted into 4 ml of chloroform, and dried in the Savant Speed Vac concentrator. The repurified material was used as the 3-O-methyldobutamine standard.

Determinations of Free Dobutamine and Urinary Total Dobutamine, Dobutamine Sulfate, and Dobutamine Glucuronide. Thawed samples of infusate and urine analyzed for free dobutamine were not subjected to acid hydrolysis, whereas urine samples analyzed for total dobutamine (i.e., dobutamine plus its acid-hydrolyzed metabolites) were first subjected to the acid hydrolysis and neutralization steps described above for preparation of the 3-O-methyldobutamine standard from human urine. Before analysis all urinary samples were diluted 10-fold with Millipore water. Then 50 mg of acid-washed alumina was added to 0.2 to 2.0 ml of diluted sample in a 12 × 75-mm polypropylene tube and the mixture was stoppered and vortexed for 3 s. After addition of 1 ml of 1 M Tris-Cl buffer, pH 8.65, the contents were mixed on a roto-torque (setting 5.5) for 10 min and then centrifuged at 500g for 30 s. The supernatant was discarded and the alumina was washed three times with 2 ml of Millipore water. The slurry with the last wash was transferred to a microfilter tube before centrifugation at 500g for 3 min. The filtered water wash was discarded, a dry receiver tube was substituted, and O.2 ml of 0.1 M HClO₄ was added. This mixture was vortexed for 2 s, allowed to stand for 5 min, vortexed again for 2 s, and centrifuged at 500g for 3 min. Exactly 0.1 ml of the filtered extract containing dobutamine (about 0.2 ml) was injected for the HPLC analysis done as described for 3-O-methyldobutamine. Under these conditions, dobutamine eluted as a single peak at 10 min and recovery of a dobutamine standard was 70%.

Determinations of Free and Total 3-O-Methyldobutamine, 3-O-Methyldobutamine Sulfate, and 3-O-Methyldobutamine Glucuronide in Urine. To determine total 3-O-methyldobutamine in the urine, both control (before dobutamine therapy) and experimental (during dobutamine therapy) urine samples were thawed, subjected to acid hydrolysis, and chromatographed as described for the purified urinary 3-O-methyldobutamine standard. Processing of samples used to quantitate free 3-O-methyldobutamine in the urine was identical except that the acid hydrolysis step was omitted. For 3-O-methyldobutamine sulfate analysis, 0.05 ml of a thawed urine sample was added to 0.45 ml of 0.01 M Tris-Cl buffer, pH 7.5, and incubated with 2.2 mg of partially purified A. aerogenes sulfatase for 6.5 h at 37°C. The reaction was stopped by chilling on ice and the sample was extracted and chromatographed as described for 3-O-methyldobutamine. For determination of 3-O-methyldobutamine glucuronide, 0.05 ml of a thawed urine sample was added to 0.45 ml of 0.1 M potassium citrate buffer, pH 5.0, and incubated at 37°C with 5 mg of partially purified bovine -glucuronidase for 16 h. The reaction was terminated by chilling on ice, and the pH of the sample was adjusted to 6.5 before extraction and chromatographic analysis as outlined for 3-O-methyldobutamine.

Assays for Catechol-O-Methyltransferase Activity in Human Blood Mononuclear Cells. Formation of 3-O-methyldobutamine from dobutamine and SAM was assayed as described above for enzymatic formation of reference 3-O-methyldobutamine except that human mononuclear cell sonicates (0.30-0.44 mg of protein/0.55-ml reaction mixture) were substituted for porcine COMT. Human mononuclear cells from 3 ml of blood were separated, washed, sonicated, and stored at 70°C as described by Allen et al. (1992); protein concentrations in the sonicates were determined by the method of Lowry et al. (1951), with bovine serum albumin used as the standard. Reactions were run for 45 min after which methods for terminating the reaction, and extracting and chromatographing the product were identical to those used to generate the reference 3-O-methyldobutamine with porcine enzyme. Recovery and quantitation of the 3-O-methyldobutamine product was achieved by spiking control and experimental reaction mixtures with known amounts of 3-O-methyldobutamine standard repurified from urine of dobutamine-treated patients.

	Results

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3-O-Methyldobutamine generated enzymatically from dobutamine with partially purified porcine COMT served as the reference used to establish optimal conditions for the extraction and chromatographic separation of this metabolite from urine. Conditions for 3-O-methyldobutamine generation were those used by Allen et al. (1992), to assay human blood mononuclear cell COMT activity with dopamine as the substrate. However, ethyl acetate, the organic solvent used to extract the dopamine reaction products, coextracted over 10% of the dobutamine substrate, which interfered with the subsequent HPLC product analysis (Table 1). Substitution of methylene chloride for ethyl acetate selectively reduced the amount of coextracted dobutamine (<0.4%) without affecting the recovery of a reaction product subsequently identified as 3-O-methyldobutamine (36.7 ± 3.5%) (Table 1). When subjected to HPLC, the latter was detected as a large peak in fractions eluting at 21 min that depended on the presence of dobutamine, SAM, and active porcine enzyme in the original reaction mixture (Fig. 2, peak B). Mass spectral analysis of material in the 21-min fraction revealed a molecular ion signal at 315 m/z, consistent with its identity as 3-O-methyldobutamine (data not shown). The substance eluting in a minor peak at 30 min (Fig. 2, peak C) was not identified but may have been 4-O-methyldobutamine (see Discussion).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   HPLC-EC chromatogram of metabolites formed by porcine liver COMT incubated with substrates (see text). A, dobutamine; B, major metabolite of dobutamine; and C, minor metabolite of dobutamine.

Chromatographic conditions that separated the enzymatically formed reference 3-O-methyldobutamine from dobutamine were used to isolate and purify this metabolite from acid-hydrolyzed urine samples obtained from pediatric patients treated with dobutamine. Repurified material eluting at 21 min from multiple chromatographic separations (Fig. 3) was subjected to both mass spectral and proton NMR analysis (see Materials and Methods).

View larger version (8K): [in this window] [in a new window]   Fig. 3.   HPLC-EC chromatogram of repurified major dobutamine metabolite isolated from urine of pediatric patients treated with dobutamine.

The largest ion (M⁺) in the mass spectrum of the repurified material dissolved in methanol was noted at 315.18304, which corresponds closely to the calculated molecular weight of 3-O-methyldobutamine (315.1834) (Fig. 4). The mass spectrum also exhibited several characteristic ion fragments at m/z 107, 137, 178, and 194, which, taken together, are consistent with the identity of the purified material as 3-O-methyldobutamine.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Mass spectrum of repurified major dobutamine metabolite isolated from urine of pediatric patients treated with dobutamine. The M+ ion signal is seen at 315.183 m/z. Assignments of ion fragments at 107, 137, 178, and 194 m/z are shown in the diagram.

Figure 5 shows the proton NMR spectrum of the repurified urinary metabolite, and Table 2 lists the chemical shifts of the proton signals and their proposed assignments relative to CDCl₃ at 7.3 ppm. Signals of protons on the two aromatic rings were noted at 6.61 to 6.92 ppm, whereas those due to the three protons of the hydroxymethyl group occurred at 3.90 ppm. Signals from most of the remaining saturated hydrocarbon protons were seen at 1.08 to 2.98 ppm, although a few could not be detected because of resonance broadening and/or overlap. The proton NMR spectrum also is consistent with the identity of the repurified urinary material as 3-O-methyldobutamine.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Proton NMR spectrum of repurified major metabolite of dobutamine isolated from urine of pediatric patients treated with dobutamine. Assignments of proton signals (a-i) relative to CDCl3 at 7.3 ppm are shown in the diagram; chemical shifts of these signals are depicted in Table 2.

The repurified urinary metabolite was used as an external standard for extraction, isolation, and quantitation of 3-O-methyldobutamine both in urine and enzymatic reaction mixtures. The extinction coefficient, , of this standard dissolved in water was 5.73 optical density units and its recovery from urine was about 36% (Table 1). The sensitivity of the method was 22 ± 5 ng/ml and the between day variation for determinations on different frozen aliquots of the same urine sample was 11.8%.

Methodology devised to measure dobutamine and 3-O-methyldobutamine in addition to their total acid-hydrolyzed derivatives and their sulfate and glucuronide conjugates permitted a partial balance study of a patient in whom the rate of dobutamine infusion at steady state was compared with the rates of elimination of these entities in the urine (Table 3). Steady-state conditions were achieved in a 9-year-old, 31-kg male child with cardiac failure because dobutamine and its metabolites have very short plasma half-lives relative to the 48-h period of dobutamine infusion (Latifi et al., 2000). Dobutamine was given intravenously at a constant ordered rate of 3 µg min¹ kg¹ for 48 h both before and during the 5.5-h experimental period. Analysis of the infusate for dobutamine showed that its actual concentration was 3.13 µg kg¹ rather than the prescribed 3 µg kg¹ (Table 3). Assays of the 200 ml of urine, pH 5.9, collected over the 5.5-h experimental period indicated that ~80% of the infused dobutamine was detected, about 33% as total dobutamine released by acid hydrolysis, and another 47% as total 3-O-methyldobutamine released after the same procedure. Further examination revealed that 70% of the total dobutamine found in acid-hydrolyzed urine could be explained by the presence of free dobutamine (3%), dobutamine sulfate (49%), and dobutamine glucuronide (18%). Over 80% of the total 3-O-methyldobutamine measured in acid-hydrolyzed urine was accounted for by free 3-O-methyldobutamine (4.6%), 3-O-methyldobutamine sulfate (70.9%), and 3-O-methyldobutamine glucuronide (5.3%). Thus, most of the urinary dobutamine and 3-O-methyldobutamine found after acid hydrolysis consisted of sulfate conjugates (Table 3).

In vitro experiments revealed that crude preparations of human blood mononuclear cells catalyzed the formation of 3-O-methyldobutamine from dobutamine and SAM. Detection of a product peak eluting at 21 min upon HPLC of the reaction mixture depended on the presence of dobutamine, SAM, and active enzyme in the original reaction mixture. Moreover, a molecular ion signal at 315 m/z was consistent with the presence of 3-O-methyldobutamine in the fraction eluting at 21 min (data not shown). Conditions found optimal for assaying this crude blood mononuclear cell enzyme with dobutamine were similar to those described previously for dopamine (see Materials and Methods; Allen et al., 1992). Thus, at pH 7.4 and 37°C with 0.125 mM dobutamine, 218 µM SAM, 10.9 mM MgCl₂, and 3 units of adenosine deaminase in 0.55 ml of reaction mixture, formation of 3-O-methyldobutamine was linear with added mononuclear cell protein concentration and with time up to 60 min.

	Discussion

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To our knowledge this is the first time that 3-O-methyldobutamine has been identified as a major metabolite of infused dobutamine in humans. This compound was isolated from the urine of dobutamine-treated pediatric patients and identified by its chromatographic behavior and spectral properties. 3-O-Methyldobutamine and its derivatives also comprised most of the drug forms detected in the urine of a patient infused with dobutamine at steady state. Moreover, COMT activity in crude preparations of human blood mononuclear cells was shown to catalyze the formation of 3-O-methyldobutamine from dobutamine in vitro.

Two lines of evidence suggest that our isolated urinary dobutamine metabolite is 3-O-methyldobutamine rather than 4-O-methyldobutamine, even though the latter was not available for direct comparison. First is that COMT catalyzes the transfer of the methyl group from SAM predominantly to position 3 of dihydroxyphenyl derivatives (Kopin, 1985; Lotta et al., 1995; Männisto and Kaakkola, 1999). Thus, porcine COMT generated a major dobutamine metabolite (Fig. 2, peak B) that eluted before a small peak due to a minor unidentified dobutamine metabolite, possibly 4-O-methyldobutamine (Fig. 2, peak C). Material in the major peak fraction had the same retention time (21 min) and molecular ion (m/z 315) as our purified urinary metabolite standard that exhibited both mass and proton NMR spectra consistent with the 3-O-methyldobutamine structure. The second observation supporting the 3-O-methyl structure for our dobutamine metabolite is that 3-methoxytyramine, a major metabolite of dopamine, elutes before 4-methoxydopamine under similar chromatographic conditions (Allen et al., 1992). Tandem mass spectrometry of our standard urinary dobutamine metabolite could unequivocally establish the position of its O-methyl group by further fragmentation of the 137 m/z ion (Fig. 4).

That 47% of dobutamine infused at steady state appeared as 3-O-methyldobutamine in acid-hydrolyzed urine of our patient (Table 3) and that appreciable quantities of this metabolite were isolated from the urine of other pediatric patients treated with dobutamine argue that 3-O-methyldobutamine is a major metabolite of infused dobutamine in humans. COMT with its preference for catechol substrates and wide tissue distribution presumably accounts for this finding but the phenotype/genotype of this enzyme was not determined in our patient. An enzyme that exists in both soluble and membrane-bound forms encoded by a single gene, COMT exhibits a genetically balanced polymorphism with a trimoidal distribution of low, intermediate, and high activities in human tissues (ratio 1:2:1) (Männisto and Kaakkola, 1999; Weinshilboum et al., 1999). Low activity is due to a thermolabile form of the enzyme with a single G  A transition that results in a change from valine to methionine at codon 108 in soluble COMT and at codon 158 in membrane-bound COMT. Future population studies should reveal whether different COMT genotypes account for large differences in the elimination of 3-O-methyldobutamine and its metabolites in the urine of dobutamine-treated patients.

Most of the dobutamine infused into our patient at steady state was detected in the urine as unidentified sulfate conjugates of either 3-O-methyldobutamine (33%) or dobutamine (16%) (Table 3). Because both sulfation and glucuronidation were established indirectly by enzymatic hydrolysis in the present study, the sites of sulfation and glucuronidation on 3-O-methyldobutamine and dobutamine await direct isolation and characterization of the individual metabolites [i.e., theoretically, conjugation could take place on the catechol ring, the monophenolic ring, or both (Fig. 1)]. Nonetheless, the unique human catecholamine sulfotransferase SULT 1A3 is an especially attractive candidate to explain most of the sulfation observed herein because of all the human SULTs, this enzyme has the most marked affinity for catecholamine substrates (e.g., dopamine K_m, ~1 µM) and a wide tissue distribution. One of many human cytosolic sulfotransferases that use phosphoadenosine phosphosulfate as the activated sulfate donor, SULT 1A3 has been cloned, sequenced, expressed, and characterized in various systems and crystallized at a resolution of 2.4 A (Brix et al., 1999; Bidwell et al., 1999; Dajani et al., 1999; Dooley et al., 2000). This thermolabile sulfotransferase is highly expressed in intestine and because its postprandial activity increases dramatically it has been suggested to serve as a "gut-blood" barrier for detoxifying dietary biogenic amines (Eisenhofer et al., 1999; Dooley et al., 2000). However, much still needs to be learned about the number and genetics of the human sulfotransferases and the presence of various sulfatases makes the physiological role of particular sulfotransferases difficult to elucidate (Dooley et al., 2000; Iida et al., 2001).

In the child treated with dobutamine at steady state our partial balance study accounted for about 80% of the infused drug in the urine but left the remaining 20% unexplained (Table 3). Unidentified urinary metabolites of dobutamine might have contributed to this discrepancy because the urinary analysis was limited to quantifying dobutamine, 3-O-methyldobutamine, and their acid-hydrolyzed derivatives. But dobutamine also might undergo a significant enterohepatic circulation due to its chemical structure as a weak organic base and partial biliary elimination with or without conjugation/deconjugation recycling. Indeed, dogs infused with C¹⁴-labeled dobutamine excreted 20% of the radiolabel in the stool compared with 67% in the urine and animals with cannulated bile ducts eliminated 30 to 35% of a radiolabeled dose in the bile (Murphy et al., 1976). An enterohepatic circulation of dobutamine in humans might be detected by analyzing stool samples from dobutamine-treated patients or by testing bile from dobutamine-treated patients with biliary fistulas for dobutamine and its metabolites.

That crude sonicates of human blood mononuclear cells could substitute for porcine COMT in catalyzing the methylation of dobutamine in vitro is consistent with previous work that defined optimal conditions for this preparation with dopamine as the catecholamine substrate (Allen et al., 1992, 1997). Those studies also provided evidence that COMT activity in human blood mononuclear cell preparations reflects that found in human red blood cells, which in turn have been used extensively to characterize the genetics of human COMT (Weinshilboum et al., 1999). Recent kinetic studies with the human mononuclear cell preparation reveal that dobutamine and dopamine act as competitive inhibitors of each other for COMT activity and that dobutamine serves as the better substrate, largely due to its lower apparent K_m (0.05 versus 0.44 mM) (Yan et al., 2002). These observations are in accord with a recent structure-activity kinetic study that showed that both catecholamines serve as substrates for a human recombinant COMT enzyme (Lautala et al., 2001).

In summary, we present evidence, both in vivo and in vitro, that formation of 3-O-methyldobutamine is a major pathway of dobutamine metabolism in humans. In a pediatric patient treated intravenously with dobutamine at steady state, about 47% of infused dobutamine was found in the urine as 3-O-methyldobutamine and its derivatives. About 49% of the infused drug consisted of urinary sulfate conjugates of 3-O-methyldobutamine (33%) and dobutamine (16%).