Abstract: Thiols such as N-acetylcystein (NAC) are used to replenish glutathione (GSH) level, with regard to their function in the maintenance of cellular reduction-oxidation balance and control of oxidative stress. Thiols play a role in the reductive metabolism of nitrates to NO, an important signaling molecule in the cardiovascular system as well as other systems throughout the body. This study aimed to evaluate the influence of NAC on decomposition of different organic nitrate esters according to its potential i.e., pentaerythrityl tetranitrate (PETN) and isosorbide dinitrate (ISDN). The results showed that NAC gives a rapid and significant decrease of PETN and ISDN during the incubation period. During the experiment, about 85% of PETN were decomposed, while the decomposition of ISDN was about 20%. Detection of nitrite and elucidation of disulphide bond of NAC gives evidence that confirms the presence of reactions.
INTRODUCTION
N-acetylcystein (NAC) is a thiol compound which is an acetyl derivatives of the amino acid cystein. Since most tissue posses the ability to remove the acetyl group, NAC metabolizes rapidly into cystein which is a direct precursor in glutathione (GSH) synthesis. For cystein to become dietary supplement is problematic because its free sulfhydryl group is capable of spontaneous oxidation resulting in product instability (Dilger and Baker, 2007; Sun, 2010). Moreover, compared to cystein, NAC is less toxic, less susceptible to oxidation (and dimerization) and more water soluble. Therefore, protection of cystein by an acetyl group makes NAC more suitable for use as therapeutic agent (Atkuri et al., 2007). GSH, in the reductive state, plays essential roles as a protecting agent against xenobiotic compounds and as principle regulator of oxidative stress caused by the imbalance of antioxidant defense and oxidant production. NAC is beneficial for replenishing the intracellular GSH level that is lost while maintaining the cell oxidative state (Bindoli et al., 2008).
Organic nitrate ester is widely known as a prodrug to treat a variety of vascular ailments. Its active form is Nitric Oxide (NO), previously known as Endothelial-derived Relaxing Factor (EDRF) which is produced by the reductive metabolism of nitrate to nitrite and the further release of NO with the presence of GSH, involving GSH transferase. Thiol compound (e.g., GSH) enhances the reduction of organic nitrate ester (Wong and Fukuto, 1999). Endothelial NO, acting as a small chemical messenger with a short lifetime, produced at endothelial cells, could quickly penetrate smooth muscle and dilate blood vessel. Research findings show the importance of NO in pathophysiology as well as disease development, e.g., osteoarthritis (Feelisch, 2008), cancer (Coulter et al., 2008), osteoporosis (Wimalawansa, 2008), migraine (Olesen, 2008), diabetes and obesity (Bakker et al., 2009), human tumor (Fitzpatrick et al., 2008), brain inflammation (Mander and Brown, 2004), penile erection, bladder control, lung vasodilatation and peristalsis (Tuteja et al., 2004).
Organic nitrate esters were distinguished from each other not only because of different pharmacokinetics, pharmacodynamics, dosing regimen and routes of application but also because of its bioactivation mechanism. Its metabolism was linked to its bioactivation so-called high potency pathway and low-potency pathway. It is suggested that mitochondrial aldehyde dehydrogenase (ALDH-2) bioactivates high potency organic nitrates e.g., nitroglycerin (GTN), pentaerythrityl tetranitrate (PETN) and pentaerythrityl trinitrate (PETriN). Cytochrome P450s is responsible for NO formation in vascular tissue for low potency organic nitrates e.g., isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), pentaerythrityl dinitrate (PEDN) and pentaerythrityl mononitrate (PEMN) (Muenzel et al., 2005).
It has been reported that reaction mechanism, in general, indicates the role of thiol in NO released from organic nitrate. However, interaction of certain organic nitrate with specific thiol is not widely publicized while the metabolism of nitrates is still not completely resolved. Therefore, through this work we aim to describe the influence of NAC (in several concentrations) on organic nitrates, in vitro. Moreover, we will differentiate nitrates usages according to its potency. The decomposition profiles of nitrates (PETN and ISDN) resulted from the involvement of NAC are expected to be able to explain the potential of the nitrates along with the mechanisms of the reaction pathways.
MATERIALS AND METHODS
Solvents and solutions: All chemicals used in this experiment are analytical grade. PETN was a product from the synthesis of pentaerythritol according to Lange et al. (2009). All solvents used for reaction incubation are HPLC grade.
PETN solution was prepared to 0.6 mM concentration (189.68 mg L-1), by dissolving it with 500 ml methanol where liquid was then added with water to obtain the 1000 mL final volume. Solution of ISDN (354.21 mg L-1) was prepared in a similar way to the preparation of the PETN solution procedure. Since the nitrates used were not dissolved well in water, the use of organic solvent is necessary.
NAC solution was prepared to 6 mM liquid concentration (979.14 mg L-1) by using phosphate-buffered saline of pH 7.4 The solution was then diluted to 0.6, 1.2, 3 and 6 mM liquid concentrations of NAC which is equimolar, 2, 5 and 10 times of nitrate concentrations, respectively. Phosphate-buffered saline was prepared by dissolving 0.6 g sodium dihydrogenphosphate-dodecahydrate, 1.76 g disodium hydrogen-phosphate-dihydrate and 2.92 g sodium chloride in 1000 mL of double distilled water. The pH was adjusted to 7.4 with either 10% solution of sodium hydroxide or 10% phosphoric acid.
Samples: NAC is a donor of thiol (-SH) which is acetyl (-C[O]-C) derivative of cystein. PETN and ISDN are donor of nitrate (-ONO2), where PETN has 4 nitrate group and ISDN has only 2. Structure of investigated compound is presented in Fig. 1.
A solution of either PETN or ISDN was added to equal volume of each concentration ratios of NAC in a well-stoppered glass apparatus. The mixture was then incubated in thermostated water bath at 37°C. A solution of 0.3 mM nitrates in 25% methanol in phosphate-buffered saline was also incubated and treated as control. The aliquot was sampled (n = 3) at certain intervals of up to 240 min of incubation time and analyzed by means of High Performance Liquid Chromatography (HPLC) method.
Chromatographic condition: The samples were analyzed by means of HPLC method to determine the amount of nitrate as tracing substances in the solutions. Hitachi D-7000 HPLC system was used which was equipped with L-7400 detector, HPLC system manager V4.0 and reverse phase C18 column (phenomenex bond clone, 300x3.9 mm, 10 μm). Volume of HPLC sample loop was 20 μL. We used wavelength detection on 215 nm for HPLC quantitative analysis, obtained from spectrophotometer scanning. The validation of HPLC method was accomplished by calibrating it against the analytes concentration. As mobile phase, acetonitrile: water (50:50) ratio was applied at the flow rate of 1 mL min-1.
Preparation of griess reagent: Detection of nitrite as possible product of the reactions between nitrates and thiols was carried out by using modified Griess method according to Caringal (1980) and Miranda et al. (2001). Griess reagent was prepared by dissolving 1,5 g of sulfanilic acid in 450 mL of 10% of acetic acid and then the solution was added to the solution of 0.6 g alpha naphthylamine in 60 mL of boiling distilled water and finally filtered through Whatman No. 1 filter paper. This colorless solution would turn red when its activity was tested by adding a few drops of 10% sodium nitrite solution.
Mass spectroscopy measurement: Mass Spectroscopy (MS) analysis were conducted to verify that the reaction between nitrates (PETN and ISDN) and thiol (NAC) took place in the right path as assumed by means formation of disulphide bond with molecular weight identification of NAC in the form of disulfide bond.
| Fig. 1: | Interesting compounds of nitrates and NAC used in the experiment |
It was one of the expected results where an explanation of nitrate-thiol reaction mechanism might be established. A Waters Mass Spectroscopy, equipped with electrospray ionization (ESI) and Time of Flight (TOF) analysis, was utilized to collect Mass Spectra. The spectrum was processed by MassLynx V4.1 to have appropriate appearance.
RESULTS AND DISCUSSION
Calibration: Peak on chromatograms obtained for each injection on HPLC of PETN, ISDN and NAC were completely separated and persistent at retention time 12.7, 6.4 and 2.6 min, respectively (Fig. 2). Therefore, a simultaneous determination of nitrates and thiol would be possible. However, since there were reaction in between, we indicate newly peak around NAC retention time as product of reaction and it is deleterious one another, hence there would not possible to quantify neither NAC nor product of reaction by this HPLC condition.
A validation of the HPLC method was accomplished and an accurate and valid calibration was obtained. Linearity achieved from the graph by plotting concentration of PETN and ISDN versus value of area under the curves (AUC) obtained from HPLC chromatograms (Fig. 3a, b). It gives acceptance concentration range of 33.57 to 600 μM for PETN and 18.85 to 600 μM for ISDN from the working solution concentration. Calibration line equations were Y = 11011.67 X+100801 with r = 0.999 and Y = 12980 X-12062 with r = 0.999 for PETN and ISDN, respectively.
Peaks on chromatograms that were obtained for each of the various injections on HPLC of PETN, ISDN and NAC were completely separate and constant at retention time 12.7, 6.4 and 2.6 min, respectively. However, since there were reaction in between, new peaks were around the NAC retention time as product of the reaction and it is deleterious to one another. Therefore, it would not possible to quantify either the NAC or the product of the reaction in this HPLC condition.
Decomposition profiles of PETN and ISDN: Decomposition profiles of nitrates (e.g., PETN and ISDN) were derived as result of data collected from HPLC chromatogram on samples with several ratio of concentration of NAC and presented in Fig. 4a,b.
In the condition where no NAC is present, PETN and ISDN are shown to be stable, albeit its concentration is slightly decreased but without significant decomposition over time. In contrast, adding NAC results in a rapid and significant decrease of the concentration of nitrates during incubation period and from the profiles we conclude that there are reactions occurring between the nitrates used (PETN and ISDN) and the NAC. Indeed, interaction of nitrates with thiol is confirmed by these experiments.
| Fig. 2: | Overlaid of single chromatogram of PETN, ISDN and NAC |
| Fig. 3(a-b): | Calibration of reference of (a) PETN and (b) ISDN |
Thiol may act as cofactors in various enzymatic and non-enzymatic pathways of metabolizing of nitrates. Reduced sulfhydryl group of NAC seems to be responsible for the reaction that occurs while oxidized thiol is formed as a product of reaction.
Both profiles of nitrates (PETN and ISDN) are the same, except for the difference in the amount of nitrates converted. PETN was about 85% decomposed from the starting concentration (Fig. 5a), while ISDN was about 20% (Fig. 5b). This phenomenon might be correlated with both the number of nitrate (-ONO2) in the compound structure and its potential activity as described previously which is dependent on its metabolism pathways and is still unresolved due to the limited supporting data and method (Wenzel et al., 2007).
| Fig. 4(a-b): | Decomposition profile of (a) PETN and (b) ISDN |
PETN decomposed much more than ISDN and this is due to the fact that PETN acts as high potent organic nitrates.
After 1 h incubation time, we observed no more decomposition until the end of incubation period. One possible reason for this occurrence is the use of Phosphate-buffered Saline (PBS) or the strength of its buffer, since according to proposed reaction of nitrates and thiol, acid (H+) condition is likely to play a role for the reaction to carried out. And also, formation of disulphide form of NAC will probably change reduction-oxidation (redox) state of the solution and might influence the equilibrium of the reaction.
The use of different concentration of NAC on nitrates apparently shows similar profile although small differences were noted and for this reason, it is neglected.
Reaction confirmation: Inadequacies in the technical aspect for the detection as well as for the quantification of NO as the primary product from the reaction between nitrate and thiol, will result in the research being focused on its donors or compounds that have capacity to release NO. With this limitation, the research was done with some confirmation analysis on the nitrite (NO2¯) and the disulphide bond of the NAC as an intermediate product of the reaction. Nitrite and nitrate have been widely used as an index of indirect measurement of NO levels.
Scheme of the reaction of PETN and ISDN in the presence of NAC, as shown in Fig. 5, indicates formation of alcoholic nitrate derivatives (R-OH), nitrite (NO2¯)and disulphide form of NAC (S-S bond). Formation of nitrite was simply verified by qualitative Griess method and occurs on reaction of NAC with both PETN and ISDN The mechanism of this reaction involves formation of diazonium derivative by interaction of acidified nitrite and sulfanilamide. Addition of naphthylamine yields a chromophoric azo product that absorbs energy strongly at 540 nm (Bryan and Grisham, 2007).
| Fig. 5(a-b): | Proposed reaction of (a) PETN and (b) ISDN under the influence of NAC. The reaction of nitrate with NAC gives a disulphide form of NAC (MW = 324.04 amu) and some nitrite (NO2¯), besides the derivatives of nitrates |
| Fig. 6(a-b): | Mass spectra of reaction of (a) PETN-NAC and (b) ISDN-NAC. Structure of oxidize form of NAC with disulphide bond (MW = 323.04 amu) obtained from MS Spectra |
Nitrite is present in the entire body of mammals with various low concentrations ranging from 100 nM to 500 μM. At higher concentrations, nitrite has been considered a toxic compound (the oral median lethal dose, LD50, in rats is 180 mg kg-1) (Tsuchiya et al., 2005).
Detection of disulphide bond on samples was accomplished by Mass Spectroscopy (MS) analysis. Elucidation of the spectrum gives prediction of the presence of disulfide form of NAC at the peak signal of 323 m/z as shown in the Fig. 6, which is equivalent with molecular weight of that structure. Both reactions, PETN-NAC and ISDN-NAC, are indicating the occurrence of disulphide bond. However, PETN-NAC spectrum shows stronger response than ISDN-NAC and this is analogous to the previous results where the decomposition of PETN is much more than ISDN under NAC influences.
CONCLUSIONS
The identification and quantification of PETN and ISDN were performed using validated HPLC method. HPLC chromatograms show completely separated peaks of PETN, ISDN and NAC, although simultaneous determination would not be possible due to the presence of products of reaction between nitrates (PETN and ISDN) and NAC which are deleterious to NAC peak.
PETN and ISDN were rapidly decomposed under the influence of NAC. During the incubation period, the amount of decomposed PETN was about 85%, while ISDN was about 20%. Where there was no presence of NAC, PETN and ISDN were decreased in negligible amount. Confirmation of reaction of PETN-NAC and ISDN-NAC shows the existence of nitrite (NO2¯) and disulphide form of NAC (S-S bond) which corroborates the occurrence of the reaction of nitrates and thiols.
ACKNOWLEDGMENT
The authors would like to appreciate the contribution of Prof. Dr. Jochen Lehmann and Dr. Andreas Seeling from Friedrich-Schiller University of Jena, Germany, for their support and assistance. Also, funding support from DP2M DIKTI through Hibah S3 grant (1109/D3/PL/2010) is highly acknowledged. The authors have declared no conflict of interest.