Decomposition of l-Valine under Nonthermal ... - ACS Publications

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Decomposition of L‑Valine under Nonthermal Dielectric Barrier Discharge Plasma Yingying Li, Arben Kojtari, Gary Friedman, Ari D. Brooks,† Alex Fridman, and Hai-Feng Ji* Department of Chemistry, Drexel University, Philadelphia, Pennsylvania 19104, United States S Supporting Information *

ABSTRACT: L-Valine solutions in water and phosphate buffer were treated with nonthermal plasma generated by using a dielectric barrier discharge (DBD) device and the products generated after plasma treatments were characterized by 1H NMR and GC-MS. Our results demonstrate that L-valine is decomposed to acetone, formic acid, acetic acid, threomethylaspartic acid, erythro-methlyaspartic acid, and pyruvic acid after direct exposure to DBD plasma. The concentrations of these compounds are time-dependent with plasma treatment. The mechanisms of L-valine under the DBD plasma are also proposed in this study. Acetone, pyruvic acid, and organic radicals • CHO, CH 3 COCH 2 OO • (acetonylperoxy), and CH3COC(OH)2OO• (1,1-dihydroxypropan-2-one peroxy) may be the determining chemicals in DNA damage.

1. INTRODUCTION

thermal plasma is required to promote the development of clinical applications of nonthermal plasma. As the first attempt of a series of study of these reactions, we demonstrate in this work the decomposition products and decomposition mechanism of L-valine under nonthermal DBD plasma. L-Valine was selected in the study because of a direct correlation between the peroxidation efficiency13,14 of amino acids and the level of DNA damage. DNA damage was directly proportional to the peroxidation efficiency of the amino acids, with L-valine producing the most significant level of damage and serine and methionine producing no detectable DNA damage. L-Valine is one of the branched-chain amino acids (BCAA), with a methyl-branch in the side chain. Its peroxidation efficiency of 49 is the highest among the 20 common amino acids.

Dielectric barrier discharge (DBD) plasma is generated when high voltage of sinusoidal waveform or short duration pulses are applied between two electrodes, with at least one electrode being insulated.1,2 The plasma can be formed in air or other gases at atmospheric pressure and room temperature. The insulator prevents build-up of current between the electrodes, creating electrically safe plasma without substantial gas heating. Since nonthermal plasma produces no heat, its effects can be selective. When nonthermal atmospheric pressure plasma is applied directly to living cells and tissues, the species created in plasma such as ions, neutral species, reactive oxygen and nitrogen species can penetrate and dissolve into the liquid surface, resulting in bacteria inactivation and blood coagulation without significant heating.3,4 Nonthermal plasma treatment has also been demonstrated to promote cell proliferation,5 enhance cell transfection,6,7 sterilize root canals,8−10 wound healing,11 skin sterilization,3,12 and so on. Although clinical applications of plasma are becoming clear, understanding the chemicals species generated from DBD and their interaction with living cells and tissues is required to fully develop the clinical application DBD. Recently, it was observed that DBD nonthermal plasma had chemical-dependent effects on the damage of DNA in cell culture.13 It is believed that DNA damage induced by DBD nonthermal plasma is initiated by production of active neutral species that induces formation of organic peroxides in cell medium. Cell culture medium is composed of amino acids, glucose, vitamins, growth factors, and inorganic salts, as well as serum. Understanding of the reaction intermediates and products of these chemicals under non© 2014 American Chemical Society

2. EXPERIMENTAL SECTION 2.1. Chemicals. L-Valine was purchased from Alfa Aesar and was recrystallized from ethanol−water solution before use. N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% of trimethylchlorosilane, phthaldialdehyde (OPA), Boc-L-cysteine, and deteurium oxide (D2O) were purchased from SigmaAldrich. Formic acid, acetic acid, pyruvic acid, acetone, boric acid, sodium hydroxide, and phosphate buffered saline (PBS, 10×, powder) were purchased from Fisher Scientific. PBS buffer solution was prepared immediately before plasma treatment. Dichloromethane (DCM) of GC quality was from Received: November 20, 2013 Revised: January 21, 2014 Published: January 22, 2014 1612

dx.doi.org/10.1021/jp411440k | J. Phys. Chem. B 2014, 118, 1612−1620

The Journal of Physical Chemistry B

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2.3. Sample Preparation. For 1H NMR tests, the plasmatreated L-valine D2O solutions were tested directly without dilution. Following plasma treatment, methanol was added to the solutions (1 mM) as an internal standard to quantify the chemical concentrations in the solutions. For GC-MS, 10 mL of each plasma-treated D2O solution was extracted with CH2Cl2 (3×) and then evaporated at room temperature to reduce to ∼0.5 mL volume. A total of 100 μL of the CH2Cl2 solution was mixed with an equal volume of BSTFA and then derivatized at 50 °C for 1 h. The solution was diluted to 0.5 mL using CH2Cl2 for GC-MS. Several standard solutions of formic acid, acetic acid, pyruvic acid, and acetone were also derivatized by using BSTFA with the same procedure for comparison. To prepare samples for mass spectrometry experiments, the derivatizing reagent was prepared freshly by dissolving 10 mg of OPA and 10 mg of Boc-L-Cys in 1 mL of methanol. A borate buffer was made using 0.4 M boric acid adjusted to pH 9.0 with sodium hydroxide. A 100-μL of Boc-L-Cys-OPA reagent and 300 μL of the borate buffer were added to a vial containing 100 μL of the plasma treated D2O solution. The derivitization reaction was performed for 2 min under ambient conditions, after which the products were used for analyses directly. 2.4. Instrumentation. 1H NMR spectra were recorded on a Varian Gemini 500 MHz spectrometer. GC-MS analyses were performed using a GC-FID/MS (PerkinElmer Clarus 500 GCMS) equipped with an autosampler and a split/splitless injector. Separations were accomplished using a 30 m long Elite-5MS capillary column, 0.25 mm internal diameter (I.D.), and 0.25 μm film thickness (PerkinElmer, U.S.A.) at a constant helium flow rate of 1.2 mL/min. Samples were injected in 1.0 μL volumes with a split ratio of 50:1 at 250 °C The column temperature was kept at 35 °C for the 12 min acquisition. The MS data of the Boc-L-Cys-OPA derivatized sample were acquired by using a Waters high-resolution mass spectrometer

Pharmco-AAPER. Nitrogen and oxygen gas were obtained from Airgas. 2.2. Plasma Setup and Treatment. DBD plasma was produced by using an experimental setup as shown in Figure 1.

Figure 1. Scheme of the DBD plasma setup.

Plasma was generated by applying alternating polarity pulsed (500 Hz to 1.5 kHz) voltage of 20 kV magnitude (peak to peak), 10 ns pulse width, and a rise time of 5 V/ns between the high voltage electrodes using a variable voltage and variable frequency power supply (FID Technology). A total of 1 mm thick quartz glass was used as an insulating dielectric barrier covering the 1 inch diameter copper electrode. The discharge gap between the bottom of the quartz and the treated sample surface was fixed at 1 mm. A quartz plate (87 × 52 mm) with a 1 mm deep groove (57 × 32 mm) was used as a sample holder. The input energy was 10 mJ/pulse, and it is approximately the same for all the gases used here. No gas flow in the system. The parameters of the plasma setup were set to be 11.2 kV (R = 75 Ohm) and 690 fHz for all the experiments. The L-valine D2O solutions (20 mM) were treated with air-, N2- and O2-plasma for different times.

Figure 2. 1H NMR spectra of the untreated and air-plasma-treated L-valine solutions in D2O. The initial concentration of L-valine was 20 mM. 1613



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Figure 3. 1H NMR spectra of a 1 mL D2O solution of L-valine after treated with air-plasma for 10 min.

Figure 4. GC chromatograms of the BSTFA-derivatized solutions: (a) the L-valine solution after a 10 min treatment with air-plasma; (b) the mixture of standard solutions including acetone (1.68 min), formic acid (2.10 min), acetic acid (2.86 min), and pyruvic acid (9.77 min).

plasma. In these spectra, the peak at 4.79 ppm is the solvent residual of water in D2O. In the 1H NMR spectrum of the untreated L-valine (1), the three peaks, a doublet at 3.60 ppm, a multiplet at 2.27 ppm, and a doublet−doublet at 1.03 and 0.98 ppm, are attributed to protons bonded to the α, β, and γ carbons of L-valine, respectively. After the plasma treatment, the three peaks shifted to lower field at different degree due to the pH decrease of the solution (see the pH changes in the Supporting Information). A significant chemical shift was observed for the α-proton. 1H NMR spectra of the plasmatreated solutions in Figure 2 show that the three peaks of Lvaline disappeared after a 20 min air-plasma treatment, suggesting the total decomposition of L-valine in the solution.

(Micromass AutoSpec Ultima-Q) with EI, CI, ESI, and FAB ion sources available. In this work, FAB ion source (fast atom bombardment) was utilized.

3. RESULTS AND DISCUSSION 3.1. Characterization of Compounds in the AirPlasma-Treated L-Valine Solutions. After treatment with DBD nonthermal plasma in the air, compounds in the L-valine D2O solutions were analyzed by using 1H NMR spectroscopy. This method assured that all of the compounds remained in the solutions after the treatment so that the NMR spectra provide the full information of all the chemical species in the treated solutions. Figure 2 shows the 1H NMR spectra of L-valine D2O solutions before and after various treatment times with air1614



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Several new peaks appeared in the 1H NMR spectra of the airplasma treated solutions. Figures 3 and 4 show the enlarged 1H NMR spectra and GC chromatograms of the plasma-treated L-valine solution, respectively. Combining the NMR and GC-MS spectra, we conclude that six compounds were produced in the L-valine solution after a 10 min air-plasma treatment. They are acetone (2), acetic acid (3), pyruvic acid (4), threo-methylaspartic acid (5), erythro-methlyaspartic acid (6), and formic acid (7). Detailed analyses are summarized as follows. 1 H NMR Results. In 1H NMR spectra (Figure 3), four singlet peaks at 8.20, 2.26, 2.19, and 2.06 ppm are assigned to formic acid, pyruvic acid, acetone, and acetic acid, respectively. The three peaks in Figure 3, a doublet at 4.36 ppm, a multiplet at 3.30 ppm, and a doublet at 1.32 ppm belong to threomethylaspartic acid. The three peaks, a doublet at 4.41 ppm, a multiplet at 3.38 ppm, and a doublet at 1.34 ppm, are from erythro-methlyaspartic acid. The assignments of these peaks are based on the reported chemical shifts, splitting patterns, and coupling constants of these chemicals.15−17 1H NMR spectra show that compounds 2−7 were produced in the solution after treated with air-plasma for 1, 5, 10, and 20 min. The concentrations of the compounds are dependent on the airplasma treatment time: the concentrations of acetic acid and pyruvic acid increased from 0 to 20 min in the solution; the concentrations of acetone, threo-methylaspartic acid, erythromethlyaspartic acid, and formic acid increased initially and then subsequently decreased with longer plasma treatments. The details will be discussed in the kinetics study section. These results suggest that the intermediates acetone, threo-methylaspartic acid, erythro-methlyaspartic acid, and formic acid are further converted to other compounds, mostly acetic acid, pyruvic acid, and CO2 in 20 min. It is possible that acetic acid and pyruvic acid will subsequently decompose with longer treatment times; however, we did not observe their decomposition within 20 min plasma treatment time. We also compared the 1H NMR spectra 5 min and 3 h after the plasma treatment. The two spectra are identical, suggesting that the progress of the oxidation reactions either stopped or significantly slowed down once the plasma source was removed. GC-MS Results. The GC chromatograms of the BSTFAderivatized solution and the control sample are shown in Figure 4. The retention time (Rt) at 1.68 min was due to the carrier solvent CH2Cl2, and the Rt at 1.78, 1.98, 2.43, and 3.83 min were attributed to the byproducts of the trimethylsilyl esterification reaction and the derivatives of the residual water in the CH2Cl2 solution.18 The broad peak between 7 and 8 min was from BSTFA and the peak at 1.72 min was from trimethylchlorosilane (TMCS) added in the commercial BSTFA. These peaks were observed both in the CH2Cl2 solution of air-plasma-treated sample and in the mixture solution of control samples. They were also shown in the GC chromatogram of pure BSTFA. By comparing the retention times and mass spectrometry characters of the plasma-treated sample (Figure 4a) and the control sample (Figure 4b), we concluded that the retention times at 1.61 (m/z 43, 58), 2.11 (m/z 45, 75, 103), 2.88 (m/z 45, 75, 117), and 9.58 min (m/z 43, 45, 73, 75, 145) in the chromatogram of the derivatized plasma-treated sample (Figure 4a) are attributed to acetone, the trimethylsilyl ester derivatives of formic acid, acetic acid, and pyruvic acid, respectively. These are largely in agreement with the NMR results on the formation of acetone, acetic acid, pyruvic acid, and formic acid in the plasma-treated solution.

threo-Methylaspartic acid and erythro-methlyaspartic acid were not observed in the GC chromatogram of the BSTFAderivatized plasma-treated sample, which was due to the extraction process during the sample preparation. Since BSTFA, the derivatization reagent, is sensitive to water, the acids cannot directly be derivatized in D2O. Instead, dichloromethane was used to extract organic species in the D2O solution, and then the organic phase was derivatized with BSTFA. Because of the highly water-soluble character of threomethylaspartic acid and erythro-methlyaspartic acid, they remained in D2O during the dichloromethane extraction. In order to confirm the production of threo-methylaspartic acid and erythro-methlyaspartic acid, we used Boc-L-Cys-OPA (OPA and Boc-L-cysteine) to derivatize threo-methylaspartic acid and erythro-methlyaspartic acid via amino terminal reaction19,20 directly in the plasma-treated aqueous solutions. The Boc-L-Cys-OPA derivatized plasma-treated solution was analyzed by means of mass spectrometry and a major ion (m/z 490.1) was found in the mass spectra (see the Supporting Information), which was attributed to M + Na+ ion of the BocL-Cys-OPA derivatives of threo-methylaspartic acid and erythromethlyaspartic acid (C21H26N2O8S, M + Na+, m/z 490.1). 3.2. Kinetics Study of L-Valine Decomposition in AirPlasma. The kinetics of L-valine decomposition was obtained from 15 equal aliquots of L-valine in D2O (20 mM) that were air-plasma treated for different times (0, 10, 20, and 40 s, and 1, 2, 4, 5, 7, 9, 10, 13, 15, 18, and 20 min). Each treated solution was immediately analyzed by using 1H NMR to quantify the concentrations of compounds 1−7. It is noteworthy that, although GC-MS is the more accepted technique to determine the quantity of chemicals, it is not appropriate to quantitatively measure the concentrations of chemicals in this work because of the loss of water-soluble species during the extraction and derivatization process. As shown in Figure 5, the relationship

Figure 5. First-order degradation kinetics curves of L-valine under airplasma treatment.

between the concentration of L-valine and the air-plasma treatment time is well-fitted to the first-order degradation kinetics equation ln(ct/c0) = −kt, that is, the decomposition of L-valine in air plasma follows the first-order kinetic law. From the slope of the plot the rate constant (k) of L-valine decomposition under air-plasma treatment is calculated to be 4.77 × 10−3 s−1. The decomposition of a 20 mM L-valine solution in air plasma is thus elucidated with the equation ln(ct/ c0) = −4.77 × 10−3t or rateL‑valine = 4.77 × 10−3[L-valine]. The concentration changes of intermediates and products 2− 7 versus the air-plasma treatment time are also shown in Figure 6. The concentration of acetone increased from 0 to 5 min during the air-plasma treatment (Figure 6, left). The concentration of acetone was directly proportional to time 1615



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Figure 6. Concentration changes of compounds 2−4 (left) and 5−7 (right) with the air-plasma treatment time.

Figure 7. Proposed mechanisms of L-valine oxidized by air-plasma to form acetone and the oxidation of acetone to form acetic acid and pyruvic acid.

and 2.25 × 10−3 mM·s−1 for acetic acid and pyruvic acid, respectively. The formation rate of pyruvic acid was significantly faster than that of acetic acid within 1 min airplasma treatment. After 1 min, the formation rates of the two acids become comparable and their concentrations were closer with longer air-plasma treatment time. It is noteworthy that although the concentrations of acetic acid and pyruvic acid kept increasing during the 20 min air-plasma exposure time, we cannot exclude possible further decomposition of these species. In fact, slow decomposition of these acids on exposure to H2O2 and •OH has been reported.21,22 The concentration changes of threo-methylaspartic acid, erythro-methlyaspartic acid, and formic acid with the air-plasma treatment time are presented in Figure 6, right. The concentrations of these three compounds increased from 0 to 13 min and then decreased from 13 to 20 min during the airplasma treatment. The phenomena will be discussed in the following mechanisms study section. The data of the range of concentration increase for threo-methylaspartic acid, erythromethlyaspartic acid, and formic acid are well-fitted by using the

within 2 min air-plasma treatment, the increase of acetone during this period can be depicted by using an equation cacetone = kacetonet, in which k is calculated from the slope of the plot equals 3.86 × 10−3 mM·s−1. After 5 min, the concentration of acetone decreased slowly with the air-plasma treatment time, suggesting that acetone was initially generated from the decomposition of L-valine under air-plasma treatment and subsequently consumed in reactions with the active species from air-plasma. The concentrations of acetic acid and pyruvic acid increased during the 20 min plasma treatment time in our experiments (Figure 6, left). The changes in concentration of the two acids with the plasma treatment time exhibit two regions, 0−1 min and 2−20 min. In the two regions, both the concentrations of acetic acid and pyruvic acid can be illustrated by the equation c = c0 + kt. In the 0−1 min region, the values of c0 and k are 0 mM and 1.35 × 10−3 mM·s−1, 0 mM and 7.02 × 10−3 mM·s−1 for acetic acid and pyruvic acid, respectively. In the range of 2− 20 min air-plasma treatment, the values of c0 and k are estimated to be 0.15 mM and 2.44 × 10−3 mM·s−1, 0.48 mM 1616



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Figure 8. Proposed mechanism of L-valine oxidized by air-plasma to form formic acid.

Figure 9. Proposed peroxidation mechanism of L-valine and decomposition mechanism of compounds threo-methylaspartic acid and erythromethlyaspartic acid under air-plasma treatment.

equation c = kt, and the rate constant k is estimated to be 3.53 × 10−3, 3.68 × 10−3, and 7.45 × 10−3 mM·s−1 for threomethylaspartic acid, erythro-methlyaspartic acid, and formic acid, respectively. Similarly, the concentration decrease of compounds threo-methylaspartic acid, erythro-methlyaspartic acid, and formic acid can be described by the equation c = c0 − kt, where c0 and k are equal to 3.54 mM and 1.97 × 10−3 mM·s−1, 3.83 mM and 2.16 × 10−3 mM·s−1, and 6.25 mM and 1.83 × 10−3 mM·s−1 for threo-methylaspartic acid, erythromethlyaspartic acid, and formic acid, respectively. 3.3. Decomposition Mechanisms of L-Valine under Air-Plasma Treatment. It is known that plasma is an ionized gas composed of UV photons, electrons, ions, electronically

excited atoms and molecules, and reactive oxygen and nitrogen species (ROS and RNS), including ozone (O3), •NO, •OH, singlet oxygen (1O2), and so on.23,24 When the L-valine solution is exposed to air-plasma, hydrogen peroxide (H2O2), ozone (O3), the charged species and radicals such as hydroperoxyl radicals (HO2•), peroxynitrite (ONOO−), superoxide (O2−•), nitric oxide (•NO), and hydroxyl radicals (•OH) are generated in the solution. Therefore, possible reactions of L-valine with most if not all of these species have been examined and the reaction mechanisms are proposed as follows. Formation of Acetone, Acetic Acid, and Pyruvic Acid. The possible decomposition mechanism of L-valine to acetone on exposure to air-plasma is shown in Figure 7. In the presence of 1617



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erythro-methylaspartic acid increased within the first 13 min on exposure to air-plasma and then decreased with longer plasma treatment time, suggesting that threo-methylaspartic acid and erythro-methlyaspartic acid slowly decomposed or consumed under air-plasma treatment. We hypothesize that, on reacting with oxidative species, threo-methylaspartic acid and erythromethlyaspartic acid would undergo β-scission of the aspartate backbone to form pyruvic acid, as demonstrated in Figure 9. This hypothesis is supported by the oxidation mechanism of aspartate,25,26 which has a very similar molecular structure to methylaspartic acids such as threo-methylaspartic acid and erythro-methylaspartic acid. 3.4. Chemicals Contributing to Enhanced DNA Damage. Understanding the quantity of the initial products, such as in several seconds to tens of seconds, generated in a plasma treated solution is critical for the biological and medical applications of plasma because the biological samples are typically exposed to plasma for a brief period of time. A previous study showed that DNA damage is most significant in the presence of valine than other amino acids and initial DNA damage was detected after a 15 s plasma treatment. To correlate the biological effect and reaction products of the plasma-treated solution, we further investigated the initial products in the solution produced from the decomposition of L-valine within 1 min air-plasma treatment and their quantities are summarized in Table 1.

these oxidative species, L-valine decomposes to acetone via βscission of alkoxyl radicals formed at the β carbon position of Lvaline.25−27 From the kinetics study, it is known that the amount of acetone increases in the first 5 min and decreases with longer air-plasma treatment time, which indicates acetone can progress through further oxidative reactions via a variety of oxidants, including •OH, O2−•, ONOO−, H2O2, and O3, in the air-plasma treated solution. Acetic acid and pyruvic acid are the expected oxidation products of acetone.21,28 Figure 7 also shows the oxidation mechanisms of acetone to form acetic acid and pyruvic acid. Acetone was oxidized in the presence of •OH and O2, and a tetraoxide radical, •5, was formed. The tetraoxide degraded to form methylglyoxal (CH3COCHO) through the elimination of hydrogen peroxide or the release of oxygen. Then methylglyoxal, the central intermediate, further degraded to acetic acid and pyruvic acid. The production of acetic acid may be explained with two possible reaction mechanisms. One possible pathway is the reaction of methylglyoxal and H2O2 to form a peroxide, followed by the elimination of a HCOOH molecule. Another pathway is the attack of •OH on the keto carbon of methylglyoxal to form an alkoxyl radical, •6, followed by the elimination of a •CHO radical. The formation of pyruvic acid may be due to the elimination of a HO2• from methylglyoxal after the hydrogen abstraction of the aldehydic hydrogen. Formation of pyruvic acid may also arise from the decomposition of threo-Methylaspartic acid and erythromethlyaspartic acid, which will further be discussed in the Formation of threo-Methylaspartic Acid and erythro-Methlyaspartic Acid” section. Formation of Formic Acid. In the presence of strong oxidants such as hydroxyl radical (•OH) and peroxynitrite (ONOO−), L-valine can also decompose to release methyl radical (•CH3) via β-scission of alkoxyl radicals formed at the β carbon position.25−27 The generated •CH3 can be oxidized to methanol and formaldehyde, and eventually formic acid, as shown in Figure 8. Formic acid may slowly decompose to CO2 and H2O in the presence of the active species (•OH, H2O2) created from air-plasma. Formation of threo-Methylaspartic Acid and erythroMethlyaspartic Acid. Besides the β-scission reaction, the peroxidation of valine occurs readily in the presence of strong oxidants,29−31 so peroxidation would be one of the main reaction pathways of L-valine decomposition on exposure to airplasma. Compounds 5 and 6 are expected to be the peroxidation products of L-valine, as shown in Figure 9. Isomers of L-valine hydroperoxides, •OOCH2CH(CH3)CH(NH2)COOH, were formed through the hydrogen abstraction of the methyl H-atom by •OH and the following addition of oxygen. Decomposition of these hydroperoxides on the terminal methyl groups of L-valine yielded primary alkoxyl radicals, •OCH2CH(CH3)CH(NH2)COOH. These alkoxyl radical species underwent a solvent-assisted, rapid 1,2-hydrogen atom shift reaction to give α-hydroxyalkyl radicals, HOCH•CH(CH3)CH(NH2)COOH. Addition reaction of αhydroxyalkyl radicals and oxygen, and the subsequent elimination of a HO2· gave threo-methylaspartic acid and erythro-methylaspartic acid. This hypothesis is supported by the observation that the concentrations of threo-methylaspartic acid and erythro-methylaspartic acid are nearly the same during the air-plasma treatment (Figure 6) since they are a pair of stereoisomers.2930 Kinetics study of L-valine decomposition (Figure 6 right) shows that the concentrations of threo-methylaspartic acid and

Table 1. Quantities of the Initial Chemicals in the Solution Produced from the Decomposition of L-Valine (20 mM) within 1 min Air-Plasma Treatment concentration of products 2−7 (mM) time (s)

acetone

acetic acid

pyruvic acid

threo-methyl aspartic acid

erythro-methyl aspartic acid

formic acid

10 20 40 60

0.055 0.1 0.195 0.26

0.01 0.02 0.05 0.08

0.06 0.14 0.32 0.40

0.00 0.00 0.06 0.12

0.00 0.00 0.06 0.12

0.00 0.06 0.18 0.33

After 10 s air-plasma treatment of L-valine, only acetone, acetic acid, and pyruvic acid were detected in the solution, with acetone and pyruvic acid the most abundant chemical species. In the 20 s air-plasma-treated solution, formation of formic acid was observed as well as acetone and pyruvic acid. threoMethylaspartic acid and erythro-methlyaspartic acid appeared in the solution until after 40 s air-plasma treatment. In air-plasma treated samples with 40 and 60 s exposure, acetone, pyruvic acid, and formic acid generated from the decomposition of Lvaline were still the major analytes detectable via NMR. These results suggested that acetone, pyruvic acid, and organic radicals • 1−•8 in Figure 7 may play major roles in peroxidative damage of DNA. We further hypothesized that •CHO, peroxide radicals • 4 and •8, and also possibly tetraoxide •5 and radical •6, may be the determining chemicals in DNA damage because smaller, neutral particles diffuse faster than larger, amphiphilic radicals. Organic radicals •9−•15 in Figures 8 and 9 may not contribute significantly to the early state DNA damage since their concentrations were significantly lower in the first 20 s, assuming their oxidation power is not significantly stronger than •CHO, •4, and •8. However, one would predict that •CH3 and CH3OO• formed in the subsequent reactions may contribute more to the DNA damage should the biological samples be further treated with plasma. 1618



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3.5. Decomposition of L-Valine in PBS-D2O under AirPlasma Treatment. The 1H NMR spectra of the products formed in the PBS-D2O solution of L-valine after air-plasma treatment are the same as those of the products formed in the D2O solution of L-valine, which implies that the decomposition mechanisms of L-valine under air-plasma treatment were not affected by the buffer and pH of the solution. However, the decomposition rates of L-valine in these two solutions under air-plasma treatment show a significant difference. The decomposition rate constant of L-valine in PBS-D2O under air-plasma treatment is 1.29 × 10−3 s−1, estimated from the slope of the plot ln(ct/c0) versus t (Figure 10), which is nearly

Table 2. Decomposition Efficiencies and Rate Constants of L-Valine under Air-, N2-, and O2-Plasma Treatment plasma treatment time O2-plasma air-plasma N2-plasma

1 min

5 min

10 min

k (s−1)

0.28 0.21 0.13

0.89 0.69 0.50

1.00 0.94 0.76

7.77 × 10−3 4.77 × 10−3 2.59 × 10−3

× 10−3 s−1) > air-plasma (4.77 × 10−3 s−1) > N2-plasma (2.59 × 10−3 s−1). The decomposition rate of L-valine in N2-plasma is much slower than that of L-valine in O2-plasma, indicating that the reactive oxygen species play a more significant role in the decomposition of L-valine under plasma treatment than the reactive nitrogen species.

4. CONCLUSION Our work provides insight on the products formed from nonthermal plasma treatment of L-valine, including acetone, formic acid, acetic acid, threo-methylaspartic acid, erythromethlyaspartic acid, and pyruvic acid. The mechanisms of Lvaline under the DBD plasma were proposed. The results will aid in understanding the mechanisms and overall effects of the plasma acting on biological species and to expand the applications of nonthermal plasma in medicine.

Figure 10. First-order degradation kinetics curves of L-valine under air-, N2-, and O2-plasma treatment.

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four times slower than that of L-valine in D2O (k = 4.77 × 10−3 s−1). Therefore, the decomposition rate of L-valine in air-plasma displays a pH-dependence when other experimental conditions and parameters are the same, and the decomposition rate of Lvaline in air-plasma is slower in neutral buffered pH solution. 3.6. Decomposition of L-Valine under N2-Plasma and O 2 -Plasma Treatment. To determine the effects of atmospheric composition on plasma treatment of D2O solutions of L-valine, samples were also treated with N2-plasma and O2-plasma for different times and characterized using NMR spectra after plasma treatment. The 1H NMR spectra (in Supporting Information) revealed that compounds 2−7 were also generated when L-valine solutions were treated with N2plasma and O2-plasma. However, more peaks were displayed in the 1H NMR spectra of L-valine solutions treated with N2plasma and O2-plasma, suggesting the decomposition processes of L-valine under N2-plasma and O2-plasma treatment are more complicated than those of L-valine under air-plasma treatment. We also found that organic species disappeared in the valine solution when the solution was treated with O2-plasma for 20 min, as shown in the 1H NMR spectra in the Supporting Information, which implies that all of L-valine had been converted to CO2 and H2O under the strong oxidative environment of O2-plasma. Studies on the decomposition mechanisms of L-valine in N2-plasma and O2-plasma are ongoing in our lab. The concentration changes of L-valine with time under N2-plasma and O2-plasma treatment also obey the first-order degradation kinetics law, as illustrated in Figure 10. The decomposition rate constants of L-valine in N2-plasma and O2-plasma are calculated and shown in Table 2. After 10 min plasma treatment, L-valine had mostly or completely decomposed in O2-plasma (∼100%) and air-plasma (94%), while decomposition in N2-plasma under identical time conditions was slower (76% decomposed). The decomposition rate constant of L-valine decreases in the order, O2-plasma (7.77

ASSOCIATED CONTENT

S Supporting Information *

Additional analytical details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 01-215-895-2562. Fax: 01215-895-1265. Present Address †

(A.D.B.) Department of Surgery, University of Pennsylvania, Philadelphia, PA 19104. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Keck Foundation for support of the project. REFERENCES

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