Features and Comparative Characteristics of Fucosylated Glycans Expression in Endothelial Glycocalyx of Placental Terminal Villi in Patients with Preeclampsia Treated with Different Antihypertensive Regimens.

Antihypertensive therapy is an essential part of management of patients with preeclampsia (PE). Methyldopa (Dopegyt®) and nifedipine (Cordaflex®) are basic medications of therapy since they stabilize blood pressure without affecting the fetus. Their effect on the endothelium of placental vessels has not yet been studied. In this study, we analyzed the effect of antihypertensive therapy on the expression of fucosylated glycans in fetal capillaries of placental terminal villi in patients with early-onset PE (EOPE) and late-onset PE (LOPE), and determined correlation between their expression and mother's hemodynamic parameters, fetoplacental system, factors reflecting inflammatory response, and destructive processes in the endothelial glycocalyx (eGC). A total of 76 women were enrolled in the study: the comparison group consisted of 15 women with healthy pregnancy, and the main group comprised 61 women with early-onset and late-onset PE, who received one-component or two-component antihypertensive therapy. Hemodynamic status was assessed by daily blood pressure monitoring, dopplerometry of maternal placental and fetoplacental blood flows, and the levels of IL-18, IL-6, TNFα, galectin-3, endocan-1, syndecan-1, and hyaluronan in the blood of the mother. Expression of fucosylated glycans was assessed by staining placental sections with AAL, UEA-I, LTL lectins, and anti-LeY MAbs. It was found that (i) expression patterns of fucosylated glycans in eGC capillaries of placental terminal villi in EOPE and LOPE are characterized by predominant expression of structures with a type 2 core and have a similar pattern of quantitative changes, which seems to be due to the impact of one-component and two-component antihypertensive therapy on their expression; (ii) correlation patterns indicate interrelated changes in the molecular composition of eGC fucoglycans and indicators reflecting changes in maternal hemodynamics, fetoplacental hemodynamics, and humoral factors associated with eGC damage. The presented study is the first to demonstrate the features of placental eGC in women with PE treated with antihypertensive therapy. This study also considers placental fucoglycans as a functional part of the eGC, which affects hemodynamics in the mother-placenta-fetus system.

Lisdexamfetamine: chemistry, pharmacodynamics, pharmacokinetics, and clinical efficacy, safety, and tolerability in the treatment of binge eating disorder.

INTRODUCTION: The indications for lisdexamfetamine (LDX), a central nervous system stimulant, were recently expanded to include treatment of moderate to severe binge eating disorder (BED). Areas covered: This review aims to describe the chemistry and pharmacology of LDX, as well as the clinical trials investigating the efficacy and safety of this medication for the management of BED. Expert opinion: LDX is the first medication with United States Food and Drug Administration approval for the treatment of BED. It is an inactive prodrug of d-amphetamine that extends the half-life of d-amphetamine to allow for once daily dosing. D-amphetamine acts primarily to increase the concentrations of synaptic dopamine and norepinephrine. Metabolism of LDX to d-amphetamine occurs when peptidases in red blood cells cleave the covalent bond between d-amphetamine and l-lysine. D-amphetamine is then further metabolized by CYP2D6. Excretion is primarily through renal mechanisms. In clinical trials, LDX demonstrated statistical and clinical superiority over placebo in reducing binge eating days per week at doses of 50 and 70 mg daily. Commonly reported side effects of LDX include dry mouth, insomnia, weight loss, and headache, and its use should be avoided in patients with known structural cardiac abnormalities, cardiomyopathy, serious heart arrhythmia or coronary artery disease. As with all CNS stimulants, risk of abuse needs to be assessed prior to prescribing.

Dopamine replacement modulates oscillatory coupling between premotor and motor cortical areas in Parkinson's disease.

Efficient neural communication between premotor and motor cortical areas is critical for manual motor control. Here, we used high-density electroencephalography to study cortical connectivity in patients with Parkinson's disease (PD) and age-matched healthy controls while they performed repetitive movements of the right index finger at maximal repetition rate. Multiple source beamformer analysis and dynamic causal modeling were used to assess oscillatory coupling between the lateral premotor cortex (lPM), supplementary motor area (SMA), and primary motor cortex (M1) in the contralateral hemisphere. Elderly healthy controls showed task-related modulation in connections from lPM to SMA and M1, mainly within the γ-band (>30 Hz). Nonmedicated PD patients also showed task-related γ-γ coupling from lPM to M1, but γ coupling from lPM to SMA was absent. Levodopa reinstated physiological γ-γ coupling from lPM to SMA and significantly strengthened coupling in the feedback connection from M1 to lPM expressed as ß-ß as well as θ-ß coupling. Enhancement in cross-frequency θ-ß coupling from M1 to lPM was correlated with levodopa-induced improvement in motor function. The results show that PD is associated with an altered neural communication between premotor and motor cortical areas, which can be modulated by dopamine replacement.

Multi-spectroscopic and molecular modeling studies on the interaction of antihypertensive drug; methyldopa with calf thymus DNA.

The interaction of methyldopa [(S)-2-amino-3-(3,4-dihydroxyphenyl)-2-methyl propanoic acid] (MDP), antihypertensive drug, with calf thymus DNA (ct-DNA) was investigated by spectroscopic and viscometric techniques. According to the results arising from the fluorescence spectra, viscosity measurements and molecular modeling studies; we concluded that MDP is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide (EB) displacement studies revealed that MDP did not have any effect on EB bound DNA which is indicative of groove binding. This was substantiated by displacement studies with Hoechst 33258, a known minor groove binder. In addition, the thermodynamic and docking parameters showed that hydrophobic interaction via drug aromatic rings inside the DNA minor groove plays a major role in this binding.

[Methyldopa-induced acute reactive hepatitis in pregnancy, drug-metabolizing capacity of the liver]. / Methyldopa által indukált akut reaktív hepatitis terhességben, a máj gyógyszer-metabolizáló képességének alakulása.

Alpha-methyldopa is a regularly used antihypertensive drug during pregnancy. Methyldopa, which decreases the sympathoadrenal system, is the first drug of choice since decades. The reactive hepatitis is not frequent, but known serious side effect of alpha-methyldopa. In non-pregnant women the estimated rate of manifest hepatotoxicity is 2.5-10%. In our case, gestation hypertension developed at the 21st gestation week of a 35 year-old pregnant woman. Oral methyldopa, a central alpha adrenergic blocker therapy was introduced. On the 23rd gestation week acute hepatitis developed. During differential diagnosis of hepatitis, the etiology of methyldopa was taken into account. Viral and autoimmune origin was rolled out. No fetal aberration was found during ultrasound examination. The function of drug metabolizing function from blood was measured by CYP phenotyping (CYP gene expression analysis). CYP3A4 enzyme plays a primary role in the metabolism of nifedipine. Antihypertensive therapy was changed from methyldopa to nifedipine. Nifedipine dosage was based on the value of CYP3A4 gene expression. With the reduced nifedipine therapy (30 mg daily), blood pressure was successfully under control. The diagnosis of alpha-methyldopa induced hepatitis was based on anamnesis, clinical picture and the results of chemical and radiological examination and confirmed by the level of drug-metabolizing capacity. The gestation hepatotoxicity of alpha-methyldopa was reported first in 1969 by Elkington Smith, who suggested the monitoring of serum aminotransferase during alpha-methyldopa therapy in pregnancy in their case report. Our case report confirms that monitoring of serum aminotransferase level is still valuable when treating a pregnant woman with alpha-methyldopa.

Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa.

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.

Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates.

Cryptococcus neoformans produces pigments in vitro in the presence of exogenous substrate. We characterized acid-resistant particles isolated from pigmented cells grown in L-dopa, methyl-dopa, (-)-epinephrine or (-)-norepinephrine. The goals of this study were to determine whether pigments made from each of these substrates were melanins and the consequences of pigmentation on related cell characteristics. The greatest yield of acid-resistant particles occurred with methyl-dopa followed by L-dopa. Electron microscopy indicated that L-dopa and methyl-dopa produced particles with thicker shells. The mAb 6D2 reacted with all particles, but a lower reactivity was observed with epinephrine-derived particles. ESR analysis revealed that epinephrine-derived particles failed to produce a stable free radical signal typical of melanins. Growth of C. neoformans in different substrates affected cell and capsule size but not capsule induction. Hence, the type of pigment produced by C. neoformans is dependent on the substrate and not all pigments meet the criteria for melanins.

Mediated exodus of L-dopa from human epidermal Langerhans cells.

L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, alpha-methyl-dopa (alpha-m-dopa), D-dopa and the dipeptide, met-ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5-7 min and a complete exodus of L-dopa was attained at about 20 min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of alpha-m-dopa, D-dopa and met-ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.

Catechol-O-methyltransferase inhibition in erythrocytes and liver by BIA 3-202 (1-[3,4-dibydroxy-5-nitrophenyl]-2-phenyl-ethanone).

The present study evaluated the relationship between the degree of catechol-O-methyltransferase (COMT) inhibition in erythrocytes and liver by BIA 3-202 (1-[3,4-dihydroxy-5-nitrophenyl]-2-phenyl-ethanone) and determined its effects upon the O-methylation of L-DOPA in rats orally treated with L-DOPA plus benserazide. The soluble form of COMT (S-COMT) in erythrocytes was endowed with the same affinity as liver S-COMT for the substrate adrenaline. BIA 3-202 inhibited erythrocytes and liver S-COMT with ED50's of 1.9 (0.7, 3.1) and 1.9 (0.5, 3.2) (95% confidence limits) mg kg(-1), respectively. BIA 3-202 reduced the L-DOPA-induced rise of 3-O-methyl-L-DOPA in the peripheral circulation, striatal dialysate levels and striatum, and increased dopamine striatal levels. In BIA 3-202-treated rats the increase in L-DOPA in peripheral blood and striatal dialysates was significantly greater than in vehicle-treated rats. It is concluded that S-COMT activity in erythrocytes may provide important information on the pharmacodynamic profile of COMT inhibitors. The novel COMT inhibitor BIA 3-202 is a potent COMT inhibitor that enhances the availability of L-DOPA to the brain by reducing its O-methylation, which may prove beneficial in patients with Parkinson's disease treated with L-DOPA.

Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice.

Catechol-O-methyltransferase (COMT) catalyses the O-methylation of compounds having a catechol structure and its main function involves the elimination of biologically active or toxic catechols and their metabolites. By means of homologous recombination in embryonic stem cells, a strain of mice has been produced in which the gene encoding the COMT enzyme is disrupted. We report here the levels of catecholamines and their metabolites in striatal extracellular fluid in these mice as well as in homogenates from different parts of the brain, under normal conditions and after acute levodopa administration. In immunoblotting studies, COMT-knockout mice had no COMT protein in brain or kidney tissues but the amounts of catecholamine synthesizing and other metabolizing enzyme proteins were normal. Under normal conditions, COMT deficiency does not appear to affect significantly brain dopamine and noradrenaline levels in spite of relevant changes in their metabolites. This finding is consistent with previous pharmacological studies with COMT inhibitors and confirms the pivotal role of synaptic reuptake processes and monoamine oxidase-dependent metabolism in terminating the actions of catecholamines at nerve terminals. In contrast, when COMT-deficient mice are challenged with l-dihydroxyphenylalanine, they show an extensive accumulation of 3,4-dihydroxyphenylacetic acid and dihydroxyphenylglycol and even dopamine, revealing an important role for COMT under such situations. Notably, in some cases these changes appear to be Comt gene dosage-dependent, brain-region specific and sexually dimorphic. Our results may have implications for improving the treatment of Parkinson's disease and for understanding the contribution of the natural variation in COMT activity to psychiatric phenotypes.

Reaction of dopa decarboxylase with alpha-methyldopa leads to an oxidative deamination producing 3,4-dihydroxyphenylacetone, an active site directed affinity label.

Dopa decarboxylase (DDC) catalyzes the cleavage of alpha-methylDopa into 3,4-dihydroxyphenylacetone and ammonia, via the intermediate alpha-methyldopamine, which does not accumulate during catalysis. The ketone has been identified by high-performance liquid chromatography and mass spectroscopic analysis, and ammonia by means of glutamate dehydrogenase. Molecular oxygen is consumed during the reaction in a 1:2 molar ratio with respect to the products. The kcat and Km of this reaction were determined to be 5.68 min-1 and 45 microM, respectively. When the reaction is carried out under anaerobic conditions, alpha-methyldopamine is formed in a time-dependent manner and neither ammonia nor ketone is produced to a significant extent. The reaction is accompanied by a time- and concentration-dependent inactivation of the enzyme with kinact of 0. 012 min-1 and Ki of 39.3 microM. Free 3,4-dihydroxyphenylacetone binds to the active site of DDC and inactivates the enzyme in a time- and concentration-dependent manner with a kinact/Ki value similar to that of alpha-methylDopa. d-Dopa, a competitive inhibitor of DDC, protects the enzyme against inactivation. Taken together, these findings indicate the active site directed nature of the interaction of DDC with 3,4-dihydroxyphenylacetone and provide evidence that the ketone generated by the reaction of DDC with alpha-methylDopa dissociates from the active site before it inactivates the enzyme. Inactivation of the enzyme by ketone followed by NaB3H4 reduction and chymotryptic digestion revealed that the lysine residue which binds pyridoxal 5'-phosphate (PLP) in the native enzyme is the site of covalent modification. Together with the characterization of the adduct released from the inactivated DDC, these data suggest that the enzyme is inactivated by trapping the coenzyme in a ternary adduct with ketone and the active site lysine. As recently reported for serotonin (5-HT) [Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem. 271, 23954-23959], the conversion of dopamine (DA) into 3,4-dihydroxyphenylacetaldehyde and ammonia catalyzed by DDC is accompanied by irreversible loss of decarboxylase activity. However, the comparison between the absorbance, fluorescence, and CD features of DDC after 5-HT- or 3, 4-dihydroxyphenylacetone-induced inactivation shows that a different covalent adduct is formed between either of these two molecules and DDC-bound PLP.

HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro-in vivo correlation with permeability data from rats and humans.

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff > or = 10 x 10(-6) cm/s and drugs that are absorbed 40-70% with Peff = 0.1-1 x 10(-5) cm/s. A permeability of Peff < 0.1 x 10(-5) cm/s is suggested for low absorbable drugs.

Intestinal absorption studies on peptide mimetic alpha-methyldopa prodrugs.

Two dipeptide mimetic prodrugs, 1 and 2, and two tripeptide mimetic prodrugs, 3 and 4, of L-alpha-methyldopa were evaluated for intestinal absorption by in-situ single pass rat jejunal perfusion studies and by in-vitro uptake experiments in brush-border membrane vesicles (BBMVs) prepared from rat intestine. In the perfusion studies, compound 1 demonstrated a 3.5-fold increase in permeability (Pm* = 2.27) as compared with that of alpha-methyldopa (Pm* = 0.65), indicating that this prodrug was better absorbed in the intestine than its parent drug. Other prodrugs showed no significant improvement in intestinal permeability. The results correlated with the results of BBMV uptake studies. In the presence of an inward proton gradient, compound 1 showed Michaelis-Menton saturable kinetics of BBMV uptake with a low value of K(m) (0.06 +/- 0.13 mM) and a high value of Vmax/K(m)(36.38 nmol (mg protein)-1/30s mM-1) at a low concentration range and a linear uptake at high concentrations with Kd = 0.14 +/- 0.02 mM. Compounds 2 and 3 were mainly taken up in BBMVs via passive diffusion. Compound 4 was taken up in BBMVs basically via the carrier-mediated transport system, while the rate of uptake was much lower than that of compound 1. The uptake of compounds 1 and 4 was significantly inhibited by dipeptides L-Gly-L-Pro and L-Gly-L-Phe, and cephradine, a beta-lactam known to be transported via the dipeptide carrier system, indicating that both compounds were taken up in BBMVs via the H(+)-coupled dipeptide-mediated transport system. In contrast to the complicated uptake profile of alpha-methyldopa, the higher rate of BBMV uptake with less variation demonstrated on compound 1 suggested that the attached nonessential amino acid moiety, D-phenylglycine, is a feasible delivery tool in carrying the parent drug through the intestine.

Early L-dopa treatment initially retards but later enhances dopamine receptor supersensitivity following unilateral dopamine denervation.

Rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of nigrostriatal dopamine neurons were administered 25 mg/kg L-DOPA methyl ester/2 mg/kg carbidopa once or twice per day or saline in separate treatment groups for 13 days. Treatment was initiated within 18-20 h postoperative, well-before the onset of denervation supersensitivity. Contralateral rotation emerged as a response to L-DOPA on day 7 postoperative first in the L-DOPA once/day group and then on day 9 for the L-DOPA twice/day group. Thus, early postoperative L-DOPA treatment retarded but did not prevent the development of dopamine receptor supersensitivity. Following a 14-day withdrawal period, these same L-DOPA treated groups exhibited substantially higher rates of contralateral rotation to an L-DOPA challenge as compared to a drug-naive group with comparable 6-OHDA lesions. HPLC-EC measurements of L-DOPA in striatal and cortical tissue samples showed no differences in concentration across the L-DOPA treatment groups. There were several differences, however, in the neurochemical impact of L-DOPA on frontal cortex vs. striatum. In the striatum but not the cortex, L-DOPA concentrations were higher in the 6-OHDA than the intact hemisphere and, L-DOPA increased dopamine concentrations in cortex but not in the striatum. Behaviorally, L-DOPA exerted two diametrically opposite effects linked to the state of dopamine receptors. Prior to the onset of dopamine receptor supersensitivity L-DOPA suppressed locomotor behavior and delayed the emergence of denervation supersensitivity. Once denervation supersensitivity developed, however, the L-DOPA engaged sensitization mechanisms and enhanced locomotor behavior and dopamine receptor supersensitivity. These findings suggest that the initiation of antiparkinsonian treatment prior to the onset of denervated dopamine receptor supersensitivity in the 6-OHDA model is a valuable strategy to determine if a drug treatment retards or facilitates the development of dopamine receptor supersensitivity.

Metabolism of L-alpha-methyldopa in cultured human intestinal epithelial (Caco-2) cell monolayers. Comparison with metabolism in vivo.

Our objective was to evaluate the utility of an in vitro cell culture system (Caco-2) to study the pathways for the metabolism of L-alpha-methyldopa (MD) in the intestinal mucosa during its transepithelial transport. Caco-2 cell monolayers grown onto polycarbonate membranes in Transwells have been reported to exhibit morphological and biochemical properties similar to those of the human intestinal mucosa. After oral administration, MD has been reported to undergo extensive first-pass metabolism during its transport across the intestinal mucosa. In earlier studies, our laboratory showed that MD was rapidly transported across Caco-2 cell monolayers consistent with a carrier-mediated mechanism involving the large neutral amino acid transporter (Hu and Borchardt, 1990). Reversed-phase, ion-pair HPLC coupled to an electrochemical (amperometric) detector operating in the oxidative mode was used in this study to detect MD and its metabolites in the transport and cellular media. After rapid cellular uptake from the apical surface of Caco-2 cell monolayers, MD was predominantly metabolized to form MD-sulfate and MD-glucuronide and 3-O-methyl-methyldopa and its sulfated conjugate. Although methyldopamine was detected in minor quantities, it was present mainly as the sulfate and glucuronide conjugate. Although the cellular levels of MD and its metabolites rapidly reached steady-state, the corresponding basolateral levels continuously increased after an initial small lag-time. We observed that the 25-day-old Caco-2 cell monolayers used in this study were able to metabolize MD to a greater extent than those cultured for 11 days (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of pH on the suicide inactivation of frog epidermis tyrosinase.

This paper presents a new reaction mechanism for the effect of the pH on the suicide inactivation of the diphenolase activity of tyrosinase. The applicability of the mechanism is supported by the experimental characterization of the kinetic behaviour of the frog epidermis enzyme acting on catechol, L-dopa and alpha-methyldopa at several pH values. Two enzyme froms 'met-' and 'oxy-' tyrosinase, but no their corresponding enzyme-diphenol complexes, present one ionizable group with very similar value of Ka which has been determined. The highest values of catalytic and inactivation efficiencies correspond to alpha-methyldopa and catechol, respectively. These kinetic studies have been carried out by using the transient phase approach previously developed, with negligible substrate consumption during the assay time. That illustrate the usefulness of the method for multisubstrate enzyme reactions.

Inhibition of human glutathione S-transferases by dopamine, alpha-methyldopa and their 5-S-glutathionyl conjugates.

The reversible and irreversible inhibition of human glutathione S-transferases (GST) by dopamine, alpha-methyldopa and their 5-S-glutathionyl conjugates (termed 5-GSDA and 5-GSMDOPA, respectively) was studied using purified isoenzymes. The reversible inhibition, using CDNB as substrate and expressed as I50, ranged from 0.18-0.24 (GST M1a-1a), 0.19-0.24 (GST M1b-1b) to 0.5-0.54 mM (GST A1-1) for 5-GSDA and 5-GSMDOPA, respectively. About 20% inhibition was observed for GST A2-2 and P1-1, using 0.5 mM of both 5-GSDA and 5-GSMDOPA. No significant reversible inhibition was observed with dopamine and alpha-methyldopa. Tyrosinase was used to generate ortho-quinones from dopamine and alpha-methyldopa which may bind covalently to GST and thereupon irreversibly inhibit GST. In this respect, GST P1-1 was by far the most sensitive enzyme. The inhibition (expressed as a % of control) after incubating 0.5 microM GST in the presence of 100 units/ml tyrosinase with 5 microM of the catecholamines for 10 min at 25 degrees, was 99% and 67% for dopamine and alpha-methyldopa, respectively. Moderate irreversible inhibition of GST A1-1 by both dopamine and alpha-methyldopa (33% and 25%, respectively), and of GST M1b-1b by dopamine (45%) was also observed. GST P1-1 is also the only isoenzyme susceptible to irreversible inhibition by 5-GSDA (33% inhibition), while no significant inhibition was observed with 5-GSMDOPA. A minor part of the inhibition by dopamine (23%), and the complete inhibition by 5-GSDA was restored by reduction with dithiotreitol. This suggests that GST P1-1 is inhibited by disulfide formation in the case of 5-GSDA, while this oxidative pathway also substantially contributes to the inactivation by dopamine. This was supported by the HPLC-profile of the GST P1-1 subunit which was strongly affected by dopamine, while for 5-GSDA after reduction with dithiotreitol the original elution profile of the subunit returned.

Simultaneous monitoring of levodopa, dopamine and their metabolites in skeletal muscle and subcutaneous tissue in different pharmacological conditions using microdialysis.

Microdialysis, in combination with ion-pair reversed-phase liquid chromatography and electrochemical detection is described for the simultaneous determination of levodopa, dopamine, 3-O-methyldopa and 3,4-dihydroxyphenylacetic acid in the extracellular space of skeletal muscle and subcutaneous tissue in vivo in beagle dog. The relative recoveries in vitro for levodopa, dopamine, 3-O-methyldopa and 3,4-dihydroxyphenylacetic acid with a 16 mm probe at a flow rate of 5 microliters min-1 were 29.1, 25.1, 34.7 and 30.1%, respectively. This technique was then applied for three types of pharmacological experiments. In the first experiment L-dopa was administered without carbidopa pretreatment, in the second one, L-dopa was administered following carbidopa pretreatment, and in the last experiment, following pretreatment with both carbidopa and the catechol-O-methyltransferase inhibitor, OR-611. After the administration of levodopa without carbidopa pretreatment, all four compounds could be detected in dialysates from skeletal muscle, whereas dopamine and 3,4-dihydroxyphenylacetic acid were not found in dialysates from subcutaneous tissue. After the administration of levodopa following carbidopa pretreatment and following pretreatment with both carbidopa and OR-611 all compounds could be measured except for dopamine. This method enables the pharmacokinetics and metabolism of levodopa to be studied in subcutaneous tissue and skeletal muscle simultaneously.