Study on the oxidation pathway of epinephrine and norepinephrine by successive kinetics

Wu Xinguo¹, Cai Ruxiu² and Lin Zhixin²
(¹School of Resources and Environmental Science, Wuhan University, Wuhan 430079,  China; ²School of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, China)

Received on Jun.27, 2005; Supported by National Science Foundation of China (Grant No 20377032).

Abstract This paper describes an extensive kinetic study of the oxidizing reactions of epinephrine and norepinephrine with various oxidizers. It is concluded that the reactions of the two catecholamines with different oxidizers can be characterized as successive reactions, and different intermediate products and final products are formed under different oxidizers, pH values and other experiment conditions. An oxidizing pathway is proposed to interpret the experiment results. A quantitative prediction of the kinetic behavior of the oxidizing reaction for the two catecholamines is well established by using successive kinetic models.
Keywords oxidation pathway, successive reaction, Epinephrine, Norepinephrine.

Epinephrine (EP) and Norepinephrine (NE) play important roles as neurotransmitters or hormones in the maintenance of homeostasis. The major metabolism route of these cateholamines are O-methylation and oxidative deamination, but there still remains a minor pathway in which they are oxidized to different stages until melanin is formed^[1]. What are the intermediate products still remains unclear. Kinetic study on the reaction of EP with H₂O_2, which catalyzed by Cu(II), reveals that two consecutive steps are involved^[2,3]:
    Adrenaline → Adrenochrome → other products
    ESR study reveals that free radical species are formed during enzymatic oxidation, and it may be a reason for heart disease^[4].
    This paper present the results of a detailed kinetic study of the reaction of EP and NE with different oxidizers, such as tris(1,10-phenanthroline)iron(III) complex [Fe(phen)₃³⁺], Potasium hexacyanoferrate(III) [K₃Fe(CN)₆], Oxygen (O₂), and Horsradish Peroxydase/Hydrogen Peroxide system (HRP/H₂O₂) etc.. The reactions are generally found to be successive reactions and the intermediate products varied with different experiment conditions. An oxidation pathway is proposed for EP and NE based on the experiment results. A mathematical model based on successive reactions describes the kinetic behavior of these systems very well.

1 EXPERIMENTAL
1.1 Instrumentation
The flow-injection-stopped-flow (FISF) instrument used for kinetic studies consisted of a UVIKON 941 spectrophotometer or a SFM25 spectrofluorimeter (Kontron Instrument, Zurich, Switzerland) and a masterflex peristaltic pump (Cole-Parmer Instrument, Chicago, IL, USA). The flow cell and sampling system were kept at a constant temperature by circulating water from a model TB-85 thermo bath (Shimadzu, Kyoto, Japan). The fluorescence versus time graph was recorded with a model 3056 pen recorder (Sichuan 4th Instruments Factory, Sichuan, China). The absorbances versus time data were collected on-line by UVIKON 941. Kinetic data were then transferred to an IBM compatible computer for processing with programmes written in VB6.0 language by the authors. Multipoint curve-fitting methods were used to process the signal vs. time data ^[5].
1.2 Reagents
Unless otherwise stated, all chemicals used were analytical-reagent grade and all water used was doubly distilled from quartz vessels.
    Stock solutions of 4.545×10^-3mol/L epinephrine hydrochloride and 6.250×10^-3mol/L norepinephrine bitartrate were prepared in 1×10^-2mol/L hydrochloric acid by using the two catecholamine standards (Test Institute of Medicine and Bioproducts, Health Ministry, China), and stored in a refrigerator. Working standard solutions were prepared fresh daily by diluting the stock solution with 1×10^-2mol/L hydrochloric acid.
    5×10^-3mol/L Fe(phen) were prepared before use by mixing 5ml 0.1mol/L NH₄FeSO₄ solution and 5ml 0.32mol/L phen solution in 100ml calibrated flask and diluting to volume with 1.5mol/L acetate buffer of desired pH. Other concentration of Fe(phen) in different pH are prepared similar as above.
    0.08 mol/L K₃Fe(CN)₆ solution were prepared before use by diluting 1 mol/L K₃Fe(CN)₆ with acetate buffer(pH 4.5).
    1.0 mg·mL^-1 HRP solution was prepared by dissolving 10.0mg HRP crystals(RZ∽3.0, activity>250m·mg^-1, Dongfeng Technology Company, Institute of Shanghai Biochemistry, Academy of P.R.China) in 10mL water, and stored in a refrigerator.
    5.6×10^-3mol·L^-1H₂O₂ was prepared by diluting 30% H₂O₂ with water (concentration was estimated according to e =43.6 L· mol^-1·cm^-1 at 240nm) 0.1mol· L^-1 phosphate buffer (pH 7.4) was prepared by dissolving 13.6g KH₂PO₄ and 3.2g NaOH in 1 L calibrated flask, adjusting the pH to 7.4, as measured by a pH meter, and diluting to volume with water.
1.3 Procedure
For studies on the reaction of EP and NE with Fe(phen), UVIKON941 was set in TIME DRIVE mode, detection wavelength was set at 510nm, measurement time was set at 3 min, sampling rate was set at 100 samples/min, thermobath was controlled at 45ºC. Suitable amounts of reagent and sample were placed in two tubes housed in the thermobath. After the system had reached the desired temperature, reagent and sample were sucked into mixing region from two streams by turn on the pump, and then stopped in the cell by turn off the pump. The kinetic curves were recorded and saved by UVIKON941. Twenty data points covering the whole kinetic curve were entered into the computer for calculation.
    For studies on the reaction of EP and NE with Fe (CN )₆^3-, the procedure was similar as described above, except that temperature was controlled at 40ºC and detection wavelength was set at 490 nm.
    For studies on the reaction of EP and NE with dissolved O₂ in alkaline solution, ^-, the procedure was similar as described above, except that the variation of fluorescence intensity with time was monitored at l_ex = 410 nm and l_em = 510 nm using the spectrofluorimeter and recorded by the pen recorder.
    For studies on the reaction of EP and NE with H₂O₂/HRP system, the procedure was similar as with Fe(phen), except that detection wavelength was set at 490nm, measurement time was set at 10 min, sampling rate was set at 50 samples/min, thermobath was controlled at 25ºC.

2 RESULTS AND DISCUSSION
2.1 Reaction with Fe(phen)
Epinephrine or norepinephrine was found to react with ferroin complex Fe(phen) in weakly acidic medium to form the red ferroin Fe(phen)₃²⁺complex, which yields an absorption peak at 510 nm. These reactions are quite rapid, but using the FISF technique, they can be followed by monitoring the reaction rate spectrophotometriclly at the absorption wavelength of the ferroin formed. Fig.1 shows the absorbance versus time graphs obtained for EP and NE on reaction with Fe(phen)in different pH values by using the FISF technique for mixing sample and reagents. As can be seen, the variation of pH values has changed not only the rate constants of the reactions, but also the sensitivity of the absorbance. Because the molar absorptivity of ferroin is constant in a wide range of pH and is about 1.15 × 10⁴ L mol^-1cm^-1, the changes in sensitivity must refers to that different amount of ferroin has been formed. Another feature is that several kinetic curves of both EP and NE, obtained at different pH values, are obviously divided into two parts, the starting phase are fast and the terminal phase are slow. All these observations make us realize that the reaction of EP and NE with Fe(phen) are successive reactions, and at different pH values, the intermediate product and final product are different as regard to the electrons lost or the numbers of ferroin molecules formed per molecule of EP or NE.

Fig.1 Kinetic curve of EP and NE on the reaction with Fe(phen) in different pH values. [Fe(phen)] = 5 × 10^-3 mol L^-1, temperature = 45℃, [EP[ = [NE] = 1 × 10^-5 mol L^-1(in the cell), upper panel—EP, lower panel—NE.


    The overall reactions can be represented as follows:
    A + mOB + mR                                      (1)
    B + nOC + nR                                       (2)
    Where A represent EP or NE, B represent the intermediate product of EP or NE, and C represent the final product of EP or NE. O is Fe(phen) which react with A or B to give R. m and n counts for the stoichiometry of the two reactions respectively, also refers to the electrons lost by A and B, m+n refers to the total electrons lost when A converted to C. k₁ and k₂ are pseudo-first order rate constants when O remain far excess amount over mA+nB through out the reaction. The kinetic equation of B and C is well known as follows^[6]:
[B]_t = [A]₀(e- e)
[C]_t = [A]₀ (1-(k₂e- k₁e))
from the fact that
[R]_t = m[B]_t + (m + n) [C]_t
we deduced the kinetic equations of R as follows:
[R]_t = [A]₀ ((m + n) +e+e)
where [B]_t, [C]_t, [R]_t are the concentration of B, C, R, at time t, [A]₀ is the initial concentration of A.
    In order to confirm the above mechanism, the experimental kinetic data obtained at different conditions are fitted to the kinetic equation of R, m and n are obtained by trying a series of integer numbers, and the best value is accepted at which other parameters are calculated in the smallest least squares. Some of the typical kinetic curves are given in a previous study^[7], some of the unreported are given here in Fig.2 It can be seen that kinetic data obtained at different conditions are all agreeing well with the theoretic kinetic equation of R. The parameters are summarized in Table1.


Fig.2 Experimental (■) and fitted (--) kinetic curves on reaction of EP and NE with Fe(phen). Upper panel, EP at pH 1.2; middle panel, NE at pH 1.2; lower panel, NE at pH 3.86. Other conditions as described in experimental section.

Table 1 Parameters for reactions of EP and NE with Fe(phen)

* [Fe(phen)] = 1.00 × 10^-2 mol L^-1
    The m and n values in Table 1 offers important information about the intermediate products formed in the oxidation reaction of EP and NE. According to the electrons lost, an oxidation pathway is proposed as in Scheme 1.
    This mechanism is different from literature^[8] in three aspects: (1) proposed intermediate products that lost 3, 5, 7 electrons given as 3, 5, 7, (2) produce products that lost 6 electrons (6) from products that lost 4 electrons (4), but not via 3,5,6-trihydroxyindole species, (3) The final product that lost 8 electrons (8).
    Although a common pathway is proposed, EP and NE behave differently due to the difference in structure. At pH 1.2, EP and NE lost 2 electrons first(2), then EP go on oxidation to form a product that lost 6 electrons(6), but the oxidation of NE is stopped. This is probably because in structure of NE, a hydrogen atom is substituted for methyl to connect with nitrogen atom, thus at acidity of pH 1.2, the protonation of nitrogen is so strong that the oxidation can not go on, while in the case of EP, because the methyl group connected to nitrogen have the effect of electron repelling and position hindering, the protonation of nitrogen is thus weaker and electron is easy to be lost, so the oxidation of EP at low pH value can go on.
    At pH 2, EP first form an intermediate product that lost 5 electrons(5), then go on oxidation to form a product that lost 8 electrons(8). NE pass the product that lost 3 electrons(3) and form an oxidation product that lost 4 electrons(4). At pH 3.86, the oxidation of NE pass the product that lost 4 electrons to give a final product that lost 8 electrons(8).
    At pH 4.25 the concentration of Fe(phen)₃³⁺ is doubled, EP first form a product with 6 electrons lost(6), then give a final product with 8 electrons lost. NE first form product that still lost 3 electrons(3), and the final product that only lost 7 electrons(7).
    At pH 4.6, the electrons lost by final products of both EP and NE are only 6, and the electrons lost by the first stage products of both EP and NE are 5.
    The structure of 2 to 8 in oxidation pathway were proposed by us only to fit the electrons they lost, other mesomeric structure may be favorable, for example, the structure of 2', 4', 6' and 7' has been proposed by others ^[9,10], and may be the mesomeric structure for 2, 4, 6 and 7 respectively. The need of acid to form the product of 6' may be used to explain our foundlings that at lower pH, final products with 8 electrons lost could be formed, but at higher pH, no such final product was formed. The detail mechanism for this observation is still unknown.


Fig.3 Experimental (■) and fitted (--) kinetic curves on reaction of EP with dissolved O₂ in alkaline solution. Upper panel, 30ºC 0.5 mol L^-1 NaOH; middle panel, 40ºC 1 mol L^-1 NaOH; lower panel, 40ºC 0.1 mol L^-1 NaOH. Other conditions as described in experimental section.

2.2 Reactions with O₂ in alkaline solution
EP and NE were found to react with dissolved oxygen in alkaline solution to give a highly fluorescent species in the presence of a proper reducer, such as ascorbic acid etc.. The maximum wavelength of excitation (l_ex) was found to be 410 nm, and that of emission (l_em) was 510 nm. The fluorescent species was known as trihydroxyindole species and the structure was also known (4'). When no reductant was present, the trihydroxyindole species was unstable and went on further oxidation to products with no fluorescence, so the fluorescence intensity of the solution decreased. The structure of the final product is unknown, but may be as oxoaminochrome (6). The overall process can be followed by monitoring the variation of fluorescence intensity with time using FISF technique. A number of kinetic curves were obtained for EP by this method at different conditions and the kinetic data were fitted to the kinetic equation of B, good results were obtained and reported in a previous paper^[11].Some of the unreported results are given in Fig.3. The rise and fall of the fluorescence intensity confirms the successive reaction of O₂ with EP, which can be used in kinetic analysis and to study oxidation pathway.

Fig.4 Experimental data (■) and fitted kinetic curve (--) on the reaction of EP (upper panel) and NE (lower panel) with Fe(CN)₆^3-. Experimental conditions for EP: [EP] = 2 × 10^-5 mol L^-1, [Fe(CN)₆^3-] = 4 × 10^-3 mol L^-1, pH = 3; for NE: [NE] = 1.563 × 10^-4 mol L^-1, [Fe(CN)₆^3-] = 8 × 10^-2 mol L^-1, pH = 4.5. Temperature = 40ºC.

2.3 Reactions with Fe(CN)₆^3- in weakly acid media
EP and NE were found to react with Fe(CN)₆^3- in weakly acid media to form red products which exhibit an absorption peak at 490 nm. The products have long been known as aminochromees(4). The reactions can be followed by monitoring the variation of absorbance with time using FISF technique. A number of kinetic curves were recorded for EP and NE at different pHs and found that at lower pHs, an obvious induction period was observed at the start phase of the oxidation reaction; but at higher pHs, the induction period was diminished and the kinetic curve can be explained by simple first order kinetics. After the reaction has been reached equilibrium, no absorption decrease at 490 nm was observed, indicating that the reaction did not pass the aminochrome stage. This is different from the fact that in alkaline solution, without the protection of a reductant, the reaction of EP and NE with Fe(CN)₆^3- pass the fluorescent trihydroxyindole stage, which is the rearrangement form of aminochrome, to form a further oxidation product with no fluorescence ^[6]. The kinetic curves with an obvious induction period can be explained by the successive reaction kinetics with detection of final products. Fig.4 was the experimental kinetic data and fitted kinetic curve using kinetic equation of C. As can be seen, the kinetic data was fitted well, the rate constants for EP at pH 3 were: k₁ = 0.111 s^-1, k₂ = 0.0254 s^-1 and for NE at pH 4 are: k₁ = 2.697 × 10^-2 s^-1, k₂ = 9.726 × 10^-3 s^-1. This was exploited in a two-rate method for the simultaneous determination of EP and NE ^[12]. The presence of successive reactions indicated that an intermediate product was formed during the course of developing the final product of aminochrome. According to the oxidation pathway, the intermediate must be 2. This is supported by the observation that a sharp increase in initial rate is present for EP at pH about 4, and for NE at pH about 5 (Fig.5). The fact that higher pH is needed for the rapid oxidation of 2 in the case of NE agree with the structure difference between EP and NE, as explained in previous section.

Fig.5 Effect of pH on the initial rate of EP and NE.[NE] = [EP] = 2 × 10^-5 mol L^-1, [Fe(CN)₆^3-] = 8 × 10^-2 mol L^-1, Temperature = 40ºC.

Fig.6 Experimntal kinetic data (■) and fitted kinetic curve (--) on the reaction of NE with HRP/H₂O₂ system. Conditions as desribed in experimental section.

2.4 Reactions with HRP/H₂O₂ in weakly alkaline medium
The HRP catalyzed reaction of H₂O₂ with EP and NE was studied in the phosphate buffer of pH 7.4. red products were formed with one absorption peak at 490 nm and charaterized as aminochrome (4). The reaction was followed by the FISF technique and found that an induction period was observed for NE, but not for EP. When the reaction has reached equilibrium, no dcrease of absorption at 490 nm was observed for both EP and NE within 10 min.. This indicated that the reaction did not pass the aminochrome stage. The kinetic data of NE can be fitted to kinetic equation of C very well (Fig.6), indicating that an intermediate product is present during the course of devoloping the red products. Althrough no further efforts was done to elucidate the intermediate, it must probably be 2 in oxidation pathway proposed in previous section. The rate constants for NE is: k₁ = 1.660 × 10^-2 s^-1, k₂ = 7.059 × 10^-3 s^-1.

3 CONCLUSION
The successive reactions of EP and NE with different oxidizers were confirmed in this paper by fitting the kinetic data to the coresponding successive kinetic equations. The presence of intermediate product was naturally indicated by the presence of a successive reaction. Additional information about the oxidation state of the intermediate and final products could be obtained by studies on the kinetic of detecting the reagent in successive reactions, as described in the case studies of the reaction of EP and NE with Fe(phen). This make us realized that the complex oxidation pathway could be investigated by using the kinetics of successive reactions. It must be emphasized that the oxidation pathway and structures proposed for EP and NE were merely deduced based on results from kinetic studies, it may be the correct one, but the validity must be confirmed by further studies.

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