Orientador: Prof. Dr. Adelar Bracht

Data da Defesa: 26/02/2016



INTRODUCTION. Citrus aurantium (bitter orange) extracts have been used in products that allegedly can induce weight loss. Several alkaloids with adrenergic activity have been found in C. aurantium, such as p-synephrine and p-octopamine. The alkaloids p-synephrine and p-octopamine present structural similarities to endogenous neurotransmitters and ephedrine. A lipolytic activity of p-synephrine and p-octopamine has been found in both rat and human adipocytes. In this respect p-synephrine is more active in rat than in human adipocytes. Studies have also shown that p-synephrine and p-octopamine are able to induce many modifications in both metabolism and hemodynamics in the perfused rat liver. The modifications include stimulations of glycogenolysis, glycolysis, gluconeogenesis and oxygen consumption. Similar stimulations were found when the aqueous extract of C. aurantium was infused.
The bioavailability of orally ingested p-synephrine depends on the rate of absorption in the gastrointestinal tract but also on the rate by which the compound is transformed in the liver, the first organ that receives the compound via the portal vein. The biotransformation of p-synephrine and p-octopamine is not as well known as that of other adrenergic amines such as epinephrine and norepinephrine, but it is known that gastric absorption is complete. In the present work, the transformation of p-synephrine and p-octopamine was investigated in the isolated perfused liver in which arterio-venous differences can be accurately measured. It is hoped that valuable and still lacking information about the biotransformation of p-synephrine and p-octopamine can be obtained. Special attention was devoted to the single pass extraction of each compound as well as to the kinetics of uptake. For comparative purposes the transformation of epinephrine and norepinephrine was also measured. In addition to this, two important questions related to the actions of p-synephrine and p-octopamine were investigated. One of these questions was if these compounds also exert lipolytic activity in the liver. The other question was if the lipolytic and glycogenolytic effects of these amines that were detected in vitro can be demonstrated to occur in vivo.
MATERIAL AND METHODS. Male Wistar rats weighing 250-280 g (mean weight 265 g) and male Swiss mice weighing 40-50 g (mean weight 45 g) were used in the experiments. Animals were fed ad libitum with a standard laboratory diet (Nuvilab®, Colombo, Brazil) and maintained on a regulated light-dark cycle. All experiments were done in accordance with the world-wide accepted ethical guidelines for animal experimentation. The experimental protocol was approved by the Ethics Committee of Animal Experimentation of the University of Maringá (Protocol n. 108/2014).
Biotransformation, fatty acid metabolism and fatty acid release were investigated in the isolated perfused rat liver. Non-recirculating hemoglobin-free perfusion was done using the Krebs/Henseleit-bicarbonate (pH 7.4) as perfusion fluid. Oxygen uptake was measured polarographically. The non-esterified fatty acids, -hydroxybutyrate and acetoacetate concentrations were measured by standard enzymatic assays. The 14CO2 production deriving from [1-14C]octanoate or [1-14C]oleate infusions was measured by liquid scintillation spectroscopy after trapping in phenylethylamine. The activity of the hepatic triacylglycerol lipase was measured after homogenizing the perfused liver in Tris-HCl buffer (pH 8.0) using a commercial kit.
The assay of p-synephrine, p-octopamine, epinephrine and norepinephrine in the outflowing perfusate was done by means of high performance liquid chromatography (HPLC) with either spectrophotometric or fluorometric detection, using preestablished techniques and procedures.
In vivo experiments consisted in the oral or endovenous administration of p-synephrine to rats (200, 300, 400 mg/kg) or oral administration of p-synephrine to mice (200 mg/kg). Blood was sampled for measuring glucose (by means of a glucometer) and non-esterified fatty acids (enzymatic assay).
RESULTS. The main results can be summarized as follows:
1. Uptake of p-synephrine and analogs reached steady-state conditions before the 5th minute after the onset of the infusion; in relation to the [3H]water on-kinetics, the p-synephrine curve presented a slight delay, suggesting a distribution space that slightly exceeds that of the water space.
2. The single pass extraction of p-synephrine was higher than 90% at the portal concentration of 10 M. It declined with the concentration, but was still around 30% at the concentration of 500 M.
3. The single pass extraction of p-octopamine was comparable to that of epinephrine at low concentrations (approximately 60% at 10 M). The lowest single pass extraction was that of norepinephrine (25% at 10 M).
4. Rates of uptake of p-synephrine showed a clear saturation curve (rate versus concentration) of the Michaelian type in the range up to 500 M, with a KM value of 290.732.1 M and a Vmax of 0.7620.042 mol min1 g1. The rates of uptake of p-octopamine did not present clear saturation to the point that the whole curve of rate versus concentration could be approximated by a linear relationship with a first order rate constant of 1.5 min1.
5. The rates of uptake of epinephrine and norepinephrine could be measured only for concentrations up to 100 M, as the strong vasoconstrictive action of these amines, which caused pronounced leakage of the perfusion fluid and even a partial disintegration of the hepatic tissue, did not allow the infusion of higher concentrations. The apparent “substrate inhibition” in the uptake rate versus epinephrine concentration curve is probably caused by the hemodynamic effects of the catecholamine.
6. In the perfused rat liver p-synephrine and p-octopamine, at the concentrations of 100 M, were found to stimulate the hepatic triacylglycerol lipase by 40 and 51%, respectively. These seem to be the maximal stimulations possible in the liver. In the perfused liver, p-synephrine, when present at an initial concentration of 500 M, was able to increase the non-esterified fatty acid release after one hour of recirculating perfusion.
7. The effects of p-synephrine on the oxidation of exogenously supplied [1-14C]octanoate and [1-14C]oleate were minimal. Only oxygen uptake, already stimulated by octanoate or oleate, was additionally increased by the infusion of p-synephrine. These results contrast with those obtained in a previous study with p-octopamine, which increased the production of 14CO2 from both [1-14C]-octanoate and [1-14C]oleate.
8. The oral administration of 200 mg p-synephrine per kg to mice increased the blood glucose levels at 60 minutes (+58%), an effect that vanished after 140 minutes. No modifications in the blood levels of non-esterified fatty acids was found in these animals.
9. The oral administration of 200 mg p-synephrine per kg to rats increased the blood glucose levels at 60 minutes (+34%), an effect that vanished after 140 minutes. The modifications in the blood levels of non-esterified fatty acids could not be differentiated from those found in control rats.
10. The oral administration of 400 mg p-synephrine per kg to rats increased the blood glucose levels during 2 hours with a peak increment of 75%. No changes were found in the blood levels of non-esterified fatty acids.
11. Endovenous injection of 300 mg p-synephrine per kg to rats increased the blood glucose levels starting at 30 minutes after injection. No changes were found in the blood levels of non-esterified fatty acids.
DISCUSSION AND CONCLUSIONS. The fast transformation of p-synephrine (more than 90% at the concentration of 10 M) means not only that the enzymatic system in the liver cells is highly efficient in transforming the compound but also that its transport across the cell membrane must be very fast. The on-kinetics even suggests that the cell membrane permeation is flow-limited rather than barrier-limited, a point to be clarified by future investigations. When comparing the single pass extractions of the various amines, two factors determining the rates of transformation can be divised: the catechol ring diminishes transformation (epinephrine < p-synephrine; norepinephrine < p-octopamine), but the methylated amine enhances transformation (p-synephrine > octopamine; epinephrine > norepinephrine). The rapid hepatic transformation of p-synephrine and p-octopamine has implications because these compounds are ingested orally. A considerable part of the compounds absorbed in the intestine comes to the liver via the portal vein, but transformation reduces the amount that reaches the other tissues. The metabolic effects of the compounds will, thus, be exerted predominantly in the liver.
The action of p-synephrine on lipid metabolism in the liver was less pronounced than that of p-octopamine. Whereas p-octopamine, as shown by a previous study, enhances the oxidation of exogenous fatty acids, p-synephrine was ineffective. Apparently only the oxidation of endogenous fatty acids is stimulated by p-synephrine. On the other hand, both p-synephrine and p-octopamine stimulate the hepatic triacylglycerol lipase to a much lesser extent than the adipocyte lipase.
The answer to the question if glycogenolysis and lipolysis stimulation by p-synephrine can be demonstrated in vivo was positive for the former but negative for the latter. Glycogenolysis stimulation by p-synephrine, as indicated by a hyperglycemic effect, was demonstrated in both mice and rats. It was also demonstrated for both oral and endovenous administration of p-synephrine. No signs of enhanced lipolysis could be detected in the plasma in the form of increased levels of non-esterified fatty acids. The reasons for this are not clear, but they could be related to the fast transformation of p-synephrine, especially in the liver in the case of the oral administration, and/or to vasoconstrictive effects difficulting the access of the compound to the adipose tissue. Alternatively, it could be that stimulation of lipolysis occurs simultaneously with an enhancement of the oxidation of the released fatty acids so that no net changes in the plasma levels can be observed. Irrespective of the correct interpretation, it is clear that p-synephrine is at least able to act on hepatic glycogenolysis. Furthermore, it is reasonable to assume that if the compound acts on glycogenolysis it will equally affect glycolysis and oxygen consumption considering the fact that these variables are stimulated at concentrations similar or even smaller than those that stimulate glycogenolysis.
Key words: Citrus aurantium; p-synephrine; p-octopamine; biotransformation; lipolysis; glycogenolysis.

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