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Kinetic Analysis on Decomposition of Pentaerythrityl Tetranitrate under Influence of Thiols

Proceedings International Scholars Conference, Volume 7 Issue 1, October 2019, pp. 1901-1907 1902 In the formation of nitric oxide (NO) as the endothelium-derived relaxing factor (EDRF), high potency of organic nitrates e.g. nitroglycerin (NTG), pentaerythrityl tetranitrate (PETN) and pentaerythrityl trinitrate (PETriN), were suggested to be bioactivated by mitrochondrial aldehyde dehydrogenase (ALDH-2), where cytochrome P450s is responsible for low potency of organic nitrate e.g. isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), pentaerythrityl dinitrate (PEDN), and pentaerythrityl mononitrate (PEMN) (Münzel et al., 2011). Due to lack of technical aspect to detect direct NO, as well as for quantification of NO as primary product in the reaction of nitrate and thiol, thus, analysis is being focuses on its donor as indirect measurement of NO levels (Suwitono et al., 2011). This study aims to study decomposition reaction kinetic of PETN under influence of thiol, e.g. N-acetyl cysteine (NAC) and thiosalicylic acid (TSA), in the physiological condition. High performance liquid chromatography (HPLC) method was used to quantify the amount of PETN change due to its reaction with thiols.


INTRODUCTION
Pentaerythrityl tetranitrate (PETN) is an long acting organic nitrate used in the therapy as anti-anginal medication caused by endothelial dysfunction in patients with coronary artery disease (Rutherford and Struthers, 2013). In animal models of diabetes and hypertension, PETN showed to improved vascular function. PETN has fewer side effects on the vascular, particularly in endothelial cell, than other nitrates like NTG (nitroglycerin), isosorbide-5mononitrate (ISMN), or isosorbide dinitrate (ISDN) (Steven et al., 2017).
Reducing compounds especially thiols, e.g. N-acetyl cysteine (NAC), play a role in the metabolism in activating organic nitrates to nitrite then liberate NO. Thiols acts as cofactors in the enzymatic bioactivation, and as reducing agents in the non-enzymatic via formation of nitro-and nitroso-thioles as precursors (Kartasasmita, 2002;Seeling and Lehmann, 2006).
In the formation of nitric oxide (NO) as the endothelium-derived relaxing factor (EDRF), high potency of organic nitrates e.g. nitroglycerin (NTG), pentaerythrityl tetranitrate (PETN) and pentaerythrityl trinitrate (PETriN), were suggested to be bioactivated by mitrochondrial aldehyde dehydrogenase (ALDH-2), where cytochrome P450s is responsible for low potency of organic nitrate e.g. isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN), pentaerythrityl dinitrate (PEDN), and pentaerythrityl mononitrate (PEMN) (Münzel et al., 2011). Due to lack of technical aspect to detect direct NO, as well as for quantification of NO as primary product in the reaction of nitrate and thiol, thus, analysis is being focuses on its donor as indirect measurement of NO levels (Suwitono et al., 2011).
This study aims to study decomposition reaction kinetic of PETN under influence of thiol, e.g. N-acetyl cysteine (NAC) and thiosalicylic acid (TSA), in the physiological condition.
High performance liquid chromatography (HPLC) method was used to quantify the amount of PETN change due to its reaction with thiols.

Solution
Solution of PETN 0.6 mM were prepared by dissolving PETN with methanol, and added with water to have 50% methanol solution of PETN. Then, the solution was filtered through 0.45 µm membrane filter. Solution of 6 mM of TSA or NAC were prepared by dissolving TSA or NAC with small amount of methanol, and PBS solution was added to have concentrations ratios, which is equimolar, two, five and ten times of nitrates concentration.
Phosphate buffered saline (PBS) solution (pH 7.4, 10 mmol) were prepared by mixing NaH2PO4 with Na2HPO4 and NaCl in aqua bidestillata, to have phosphate strength of 10 mmol in physiological solution, and adjusted with either 10% NaOH or 10% phosphoric acid solution to have pH 7.4.

Incubation
Decomposition of PETN was analyzed by adding PETN solution with equal volume to each concentration ratios of TSA and NAC in a well-stoppered glass apparatus. The mixture was then incubated in thermostated water bath at 37 °C. Solution of PETN was added equivolume with PBS solution, and treated as control. The aliquot was sampled (n=3) at certain intervals of up to 240 minutes of incubation time, samples then freezed below -20 °C until ready to be analyzed by HPLC, but not more than 1 week.

HPLC Condition
HPLC condition is optimized and validated to analyzed PETN. The HPLC system was Hitachi D-7000 with L-7400 UV/Vis detector, using RP C-18 column (phenomenex bond clone, 300 x 3.9 mm, 10 µm). Volume of HPLC sample loop was 20 µl. PETN was detected at 215 nm wavelength. Mobile phase was isocratic acetonitrile:water with ratio 1:1, 1 ml/min

Validation of HPLC Condition
Validation of HPLC method was performed, and some parameter of HPLC condition to analyze PETN were obtained ( PETN showed significant linear regression along its concentration from 12.5 uM up to 300 uM, (Figure 1) thus, this calibration equation could be used in conversion of concentration while detecting PETN. Table 1). PETN was eluted at 12.7 min, where TSA and NAC were around 2.6 min.
Examination of either TSA and NAC could not implement since newly product peak emerged at around TSA and NAC retention time, and piled up each other, therefore PETN used as tracing compound. PETN showed significant linear regression along its concentration from 12.5 uM up to 300 uM, (Figure 1   Reaction order kinetic were calculated based on equilibrium condition, which is showing a nearly stable condition (no more significant reduction of PETN). Calculation of reaction rate is attempted with several kinetic models with curve fitting method, using several reaction order equations, which is narrowed to 1 st , 2 nd and composite 1 st order, showed in Table 2.
Result of kinetic calculation from ordinate equation were shown in Table 3, showing linearity significant value r, which is indicating acceptance of reaction order, with graphical plot of its mathematical derivation were shown in

Conclusion
Analysis of PETN in this reaction scheme were performed using validated HPLC method.
Chromatogram peak of PETN were completely separated from TSA and NAC, although simultaneous determination would not be possible due to the presence of products of reaction which are deleterious to NAC peak.
Result of this research confirmed that nitrate tolerance is occur when organic nitrate reacted with thiols, i.e. thiosalicylic acid (TSA) and n-acetyl-cystein (NAC). Nevertheless, the result showed that thiols plays important role in bioactivation of nitrates, as key on NO pathway.