Single dose pharmacokinetics of intravenous 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid in rats.

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (3-HPAA) are intestinal metabolites of the dietary flavonoid quercetin. DOPAC reportedly showed anxiolytic activity after i.p. administration in rats. The fate of these metabolites after consumption, and the pharmacological properties of 3-HPAA in the body are largely unknown. The aim of the current study was to characterize pharmacokinetic properties of DOPAC and 3-HPAA after intravenous bolus application in rats. UHPLC-MS/MS methods for quantification of DOPAC and 3-HPAA levels in lithium heparin Sprague Dawley rat plasma were developed and validated according to international regulatory guidelines. Non-compartmental and compartmental analyses were performed. Pharmacokinetic profiles of DOPAC and 3-HPAA followed a two-compartment body model, with a fast distribution into peripheral tissues (half-lives of 3.27-5.26 min) and rapid elimination from the body (half-lives of 18.4-33.3 min).

A Mixture of Phenolic Metabolites of Quercetin Can Decrease Elevated Blood Pressure of Spontaneously Hypertensive Rats Even in Low Doses.

Quercetin is proven to decrease arterial blood pressure when given orally. Its bioavailability is, however, low and, therefore, its metabolites could rather be responsible for this effect. In particular, the colonic metabolites of quercetin, 3,4-dihydroxyphenylacetic acid (DHPA), 4-methylcatechol (4MC), and 3-(3-hydroxyphenyl)propionic acid (3HPPA), have been previously shown to decrease the blood pressure in spontaneously hypertensive rats (SHR). Interestingly, the mechanisms of action of these three metabolites are different. The aim of this study is hence to investigate if these metabolites can potentiate each other and thus decrease blood pressure in reduced doses. Three double-combinations of previously mentioned metabolites were administered to SHR as infusions to mimic a real biological situation. All combinations significantly decreased the blood pressure in SHR but there were important differences. The effect of DHPA and 4MC was mild and very short. A combination of DHPA with 3HPPA caused more pronounced effects, which were also rather short-lived. The last combination of 3HPPA and 4MC caused a long-lasting effect. In conclusion, certain combinations of quercetin metabolites have a more pronounced antihypertensive effect than single metabolites.

Pharmacokinetic, Antiproliferative and Apoptotic Effects of Phenolic Acids in Human Colon Adenocarcinoma Cells Using In Vitro and In Silico Approaches.

Colon cancer is the second most common cause of cancer deaths in the USA and Europe. Despite aggressive therapies, many tumors are resistant to current treatment protocols and epidemiological data suggest that diet is a major factor in the etiology of colon cancer. This study aimed to evaluate the antioxidant activity and the influence of 3,4-dihydroxyphenylacetic (3,4-DHPAA), p-coumaric (p-CoA), vanillic (VA) and ferulic (FA) acids on cell viability, cell cycle progression, and rate of apoptosis in human colon adenocarcinoma cells (HT-29). The results showed that all compounds tested reduce cell viability in human colon cancer cells. 3,4-DHPAA promoted the highest effect antiproliferative with an increase in the percentage of cells in G0/G1 phase, accompanied by a reduction of cells in G2/M phase. Cell cycle analysis of VA and FA showed a decrease in the proportion of cells in G0/G1 phase (10.0 µM and 100.0 µM). p-CoA and FA acids increased the percentage of apoptotic cells and non-apoptotic cells. 3,4-DHPAA seems to be the substance with the greatest potential for in vivo studies, opening thus a series of perspectives on the use of these compounds in the prevention and treatment of colon cancer.

Gender differences in plasma and urine metabolites from Sprague-Dawley rats after oral administration of normal and high doses of hydroxytyrosol, hydroxytyrosol acetate, and DOPAC.

PURPOSE: To date, several in vitro and in vivo studies have shown phenolic compounds occurring naturally in olives and olive oil to be beneficial to human health due to their interaction with intracellular signaling pathways. However, the bioavailability of the most important of these compounds, hydroxytyrosol (HT), and its transformation into derivatives within the organism after oral intake are still not completely understood, requiring further in vivo research. This study deals with the differential bioavailability and metabolism of oral HT and its derivatives in rats. METHODS: Hydroxytyrosol (HT), hydroxytyrosol acetate (HTA), and 2,3-dihydroxyphenylacetic acid (DOPAC) were administered at doses of 1 and 5 mg/kg to Sprague-Dawley rats (n = 9 per treatment) by oral gavage. Their plasma kinetics and absorption ratio, assessed as their excretion in 24-h urine, were determined by UHPLC/MS/MS. RESULTS: Plasma and urine levels indicated that although the three compounds are efficiently absorbed in the gastrointestinal tract and show similar metabolism, the bioavailability is strongly dependent on the derivative considered, dosage, and gender. Inter-conversion among them has been described also, suggesting an interaction with internal routes. Microbiota metabolites derived from these phenolics were also taken into account; thereby, homovanillic alcohol and tyrosol were identified and quantified in urine samples after enzymatic de-conjugation, concluding the metabolic profile of HT. CONCLUSIONS: Our results suggest that different dosages of HT, HTA, and DOPAC do not provide a linear, dose-dependent plasma concentration or excretion in urine, both of which can be affected by the saturation of first-phase metabolic processes and intestinal transporters.

Phenolic acid concentrations in plasma and urine from men consuming green or black tea and potential chemopreventive properties for colon cancer.

SCOPE: Tea polyphenols are metabolized by the colonic microflora yielding phenolic metabolites, which may contribute to the health benefits of tea. We determined the serum and urine concentrations of phenolic acids, hippuric acid, and polyhydroxyphenyl-γ-valerolactones during green tea (GT) and black tea (BT) administration. The effects of (-)-epigallocatechin gallate (EGCG) and 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) alone and in combination on bioavailability, intracellular metabolism, and antiproliferative activity were determined in HCT-116 colon cancer cells. METHODS AND RESULTS: The concentration of phenolic metabolites was quantified by HPLC with electrochemical detection and MS. Urine concentrations of 4-hydroxyphenylacetic acid (4-HPAA), 3-hydroxyphenylacetic acid (3-HPAA), and polyhydroxy-γ-valerolactones were increased significantly in men drinking GT compared to control. Urine concentration of 3-O-methylgallic acid (3OMGA) was significantly increased in men drinking BT compared to control. Serum 3,4-DHPAA was significantly increased after consumption of GT and BT and 4-HPAA after GT consumption. In vitro treatment of HCT-116 colon cancer cells with 3,4-DHPAA and EGCG exhibited an additive antiproliferative effect, while methylation of 3,4-DHPAA was significantly decreased. 3OMGA exhibited the strongest antiproliferative activity among the phenolic acids. CONCLUSION: The consumption of both, GT and BT, was associated with a significant increase in urinary and serum phenolic acids.

Norepinephrine alters the expression of genes involved in neuronal sprouting and differentiation: relevance for major depression and antidepressant mechanisms.

Recent research into depression has focused on the involvement of long-term intracellular processes, leading to abnormal neuronal plasticity in brains of depressed patients, and reversed by antidepressant treatment. Given a suggested decrease in noradrenergic transmission in depression, and an antidepressant induced increase in norepinephrine (NE) level, a possible role for NE in mediating alterations in neuronal morphology and plasticity was examined. Human neuroblastoma SH-SY5Y cells treated with 10-5 m NE presented an elongated granule-rich cell-body and increased number of neurites, when compared with non-treated cells. Moreover, cell survival was enhanced in the presence of NE, while proliferation was inhibited. The above effects suggest a role for NE in cell differentiation. Indeed similar effects on cell survival and neurite outgrowth were induced in SH-SY5Y cells by retinoic acid (RA), an established differentiating agent. Finally, NE treatment resulted in a progressive decrease in the pluripotent marker Oct4 and an increase in the neuronal growth cone marker, growth-associated-protein 43 (GAP-43). Alongside these effects, NE-treated cells presented alterations in the expression of 44 genes as observed in a neurobiology cDNA microarray. Among the altered genes, an increase in the expression level of two neurite-outgrowth promoting genes, neural cell adhesion molecule L1 and laminin, was confirmed by RT-PCR. Taken together, the results support a role for NE in processes of synaptic connectivity, and may point to a role for this neurotransmitter in mediating the suggested neuronal plasticity in depression and in antidepressant treatment.

Circadian phase dependent pharmacokinetics of L-dopa, its main metabolites (3-OMD, HVA, DOPAC) and carbidopa in rats.

This study aims to evaluate whether or not the kinetics of L-dopa, its main metabolites (3-O-methyldopa, 3-OMD, homovanilic acid, HVA and 3,4-dihydroxyphenylacetic acid, DOPAC) and carbidopa, vary according to the 24-hour scale in rats. Four groups of seven adult male Wistar AF EOPS rats were used for these experiments; each group received L-dopa (200 mg.kg-1 ip) and carbidopa (20 mg.kg-1 ip) at 1000, 1600, 2200 or 0400 hours. L-dopa, 3-OMD, DOPAC, HVA and carbidopa were simultaneously determined by specific ion-pair reversed-phase high performance liquid chromatography with electrochemical detection. A temporal variation of the kinetics of both L-dopa and carbidopa was demonstrated with higher plasma clearance and lower area under concentration curve after the administration at 2200 hours. Moreover, a temporal variation of the metabolism of L-dopa was indirectly documented by temporal variation in kinetics of 3-OMD, DOPAC and HVA.

Quantitative microdialysis for studying the in vivo L-DOPA kinetics in blood and skeletal muscle of the dog.

In this study the microdialysis technique, using alpha-methyldopa as internal standard (IS), is introduced for the in vivo determination of L-DOPA, dopamine (DA), and their metabolites dihydroxyphenylacetic acid (DOPAC) and 3-O-methyldopa (3-OMD) in blood plasma and skeletal muscle extracellular fluid (ECF), in anaesthetised beagle dogs, after i.v. administration of L-DOPA. In a first calibration experiment, the in vivo relative losses (RL) of the compounds and the IS were determined. These were lower in skeletal muscle than in blood plasma. K was defined as the ratio of the RL of the IS to the RL of the compound of interest and was shown to be constant for a certain compound within one tissue. However, except for DA, a significant difference was seen in K values between blood plasma and skeletal muscle. In a second step, the method was validated in blood plasma. The AUC0-->3 values for the non-protein bound L-DOPA did not differ significantly between the dialysis (141.3 +/- 16.0 nmol.h/ml) and traditional whole blood sampling (145.3 +/- 18.7 nmol.h/ml), confirming that microdialysis combined with accurate calibration is a reliable technique for studying the kinetics of drugs in vivo in different tissues.

In vivo depigmentation by hydroxybenzene derivatives.

Certain mono- and dihydroxybenzene derivatives are selectively cytotoxic for melanocytes in vivo, and can cause depigmentation of skin and hair. We produced selective melanocytotoxicity/hair depigmentation in C57Bl mice by injection of 0.032-1.0% p-t-butylcatechol (tBC) or p-hydroxyanisole (MMEH) in physiological saline. No depigmentation occurred on injection of 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenylacetic acid (DOPAC). Light- and electron-microscopic examination of biopsy specimens taken from depigmented areas indicates selective melanocyte damage as early as 2 h post-injection. Melanocytes from anagen hair are most susceptible to depigmentation. All four compounds are substrates for tyrosinase, but only tBC and MMEH generate their respective isolable 1,2-benzoquinones, tBCQ and MMEHQ. These caused depigmentation in C57Bl mice to a comparable degree to the parent compounds. DOPA- and DOPAC-quinones (DOPAQ and DOPACQ) are not spectroscopically detectable in solution, suggesting extremely low steady-state levels of these compounds. The net observed rate of reaction of the respective 1,2-quinone with 300 microM bovine serum albumin (BSA) in vitro varies widely, with tBCQ >> MMEHQ = DOPACQ >> DOPAQ. The results are consistent with a mechanism involving attack of -SH on melanosomal proteins and/or enzymes by tyrosinase-generated 1,2-quinones. This mechanism evidently differs from that involved in in vitro hydroxybenzene melanocytotoxicity of melanoma cells, in which active oxygen intermediates generated by hydroxybenzene autoxidation play a significant role. The most reliable prognosticator of in vivo depigmentation appears to be the ability of the depigmenter to form a spectroscopically stable 1,2-quinone which is capable of reacting with protein -SH.

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.

Studies on the role of sodium on the synthesis of dopamine in the rat kidney.

The accumulation of Ldopa, dopamine (DA) and 3,4-dihydroxyphenylacetic (DOPAC) in kidney slices loaded with Ldopa (10-100 microM) was found to be dependent on the concentration of sodium in the medium (0-160 mM). The constant rate of accumulation did not depend on the concentration of Ldopa used and was about 0.0025, 0.0035 and 0.0065 for Ldopa, DA and DOPAC, respectively. In experiments performed in the presence of 120 and 160 mM sodium, but not with 20 mM sodium in the medium, ouabain (500 and 1000 microM) and amphotericin B (10 and 50 micrograms/ml) significantly reduced the accumulation of both DA and DOPAC (6-21 and 29-56% reduction, respectively). Amiloride (5-100 microM) produced an increase in the accumulation of DA and drastically reduced the formation of DOPAC. This effect was found to be due to inhibition of monoamine oxidase. During monoamine oxidase inhibition ethylisopropylamiloride (1, 5 and 10 microM), but not amiloride (10 and 50 microM), increased the accumulation of newly formed DA in kidney slices and reduced the outflow of the amine into the incubation medium. In conclusion, the results presented here show that the formation of DA in kidney slices loaded with Ldopa is dependent on the concentration of sodium in the medium and sensitive to the inhibition of the enzyme Na(+)-K+ adenosine triphosphatase and activation of mechanisms which bypass the tubular transport of sodium.

Conjugation and deamination of circulating dopamine: relationship between sulfated and free dopamine in man.

In order to assess the importance of conjugation in dopamine metabolism, we infused dopamine (20 nmol/kg/min, 1 h) to seven male subjects, then measured the kinetics of free and conjugated dopamine and DOPAC (deaminated dopamine) in plasma both during and after the infusion. Dopamine increased as early as 2 min after the start of the infusion, then reached a plateau and remained elevated on a level of 25 times the basal value. Dopamine sulfate and DOPAC followed very similar patterns and increased continuously from the 30th and 20th min, respectively, until the end of the infusion (5.18- and 5.76-fold, respectively), but only dopamine sulfate remained elevated (1.65-fold) on the first day following infusion. DOPAC glucuronide increased moderately (2.58-fold) at the end of the experiment, but neither dopamine glucuronide nor DOPAC sulfate were increased despite high precursor circulating levels. Free DOPAC and dopamine sulfate levels during infusion were correlated with dopamine levels when all subjects were pooled. In addition, within a subject dopamine sulfate to dopamine correlations were found with slopes varying in a range of 1 to 15. Our results show that sulfation is significantly involved in the metabolism of circulating dopamine and is not easily saturated, suggesting that dopamine sulfate may be an index of endogenous dopamine release.

Bioavailability and pharmacokinetics of oral dopamine in dogs.

The bioavailability and pharmacokinetics of oral dopamine (DA) were studied in dogs. Plasma concentrations of DA and its main metabolites, such as dopamine-3-O-sulfate (DA-SO4) and 3,4-dihydroxyphenyl acetic acid (DOPAC) were determined after intravenous or oral administration of DA using high-performance liquid chromatography with electrochemical detector (HPLC-ECD). Following the intravenous administration, plasma DA-SO4 and DOPAC concentrations were lower than the plasma DA concentration. On the other hand, following the oral administration, plasma DA-SO4 and DOPAC concentrations were much higher than the plasma DA concentration. The absolute bioavailability of DA after oral administration was calculated to be approximately 3%. Intraduodenal and mesenteric venous administration of DA revealed that DA-SO4 was mainly produced in the intestine and DOPAC was produced in the intestine and liver. On the basis of these observations, the bioavailability and pharmacokinetics of oral DA are discussed in connection with the metabolic inactivation due to first-pass metabolism.