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Basic Research Technology

Chemical Interaction of Alexidine and Sodium Hypochlorite

Hyeon-Sik Kim, DDS, PhD, * Qiang Zhu, DDS, PhD, Seung-Ho Baek, DDS, PhD, * Il-Young Jung, DDS, PhD, Won-Jun Son, DDS, PhD, * Seok-Woo Chang, DDS, PhD, Woocheol Lee, DDS, PhD, * Yu Gu, MD, MSD,* Yoon Lee, DDS, MSD, Sung-Tae Hong, DDS, PhD, k Kwang-Shik Bae, DDS, PhD, * Ji-Woong Kim, BDS, MSD,* Kun Cho, BDS, MS, ** and Kee-Yeon Kum, DDS, PhD *

§

Abstract

Introduction: Recent studies have reported the color change and formation of precipitates containing para -chloroaniline (PCA) after a reaction of sodium hypochlorite (NaOCl) and chlorhexidine (CHX). Alexi- dine (ALX), a biguanide disinfectant similar to CHX, has greater affinity for bacterial virulence factors than CHX. This study determined by electrospray ionization mass spectrometry (ESI-MS) and scanning electron microscopy (SEM) whether the chemical interaction between ALX and NaOCl results in PCA or precipitates. Methods: ESI-MS was performed on 4 different concen- trations of ALX (1%, 0.5%, 0.25%, and 0.125%) with 4% NaOCl to detect the presence of PCA. As control groups, 1% ALX, 0.5% PCA, and a mixture of 2% CHX and 4% NaOCl were analyzed. The formation of precip- itates on the dentinal surfaces of premolar root canals treated with the solutions of ALX and NaOCl (AN) or CHX and NaOCl (CN) was observed by SEM and the color change in the reaction solutions was also analyzed. Results: ESI-MS showed that the peak (mass/charge ratio = 128.026) in the PCA spectrum was not detected in any of the 4 AN solutions, whereas the peak was found in the CN solution. SEM revealed precipitates covering dentinal surfaces in the CN solu- tion. The AN solutions produced no precipitate. The AN solutions changed in color from light yellow to trans- parent with decreasing ALX concentration, whereas peach-brown discoloration was observed in the CN solu- tion. Conclusions: The interaction of ALX and NaOCl did not produce PCA or precipitates, and the color of the re- acted solution changed transparent with decreasing ALX concentration. (J Endod 2012;38:112–116)

Key Words

Alexidine, color change, electrospray mass spectrometry, interaction, precipitate

T he major aim of endodontic treatment is to remove microorganisms and their by-products from the root canal system through the combined action of endodontic

instruments and auxiliary chemicals (1–3). Therefore, the use of various antimicrobial irrigants, sequentially or in combination, is needed to enhance their antimicrobial effect

during mechanical instrumentation (4) . Sodium hypochlorite (NaOCl) is the most commonly used irrigating solution because of its tissue-dissolving capability as well as its broad antimicrobial action and ability to neutralize toxic products (4–8). On the other hand, NaOCl does not impart antimicrobial substantivity (9). Chlorhexidine gluconate (CHX) is a cationic bisbiguanide with antimicrobial efficacy against certain NaOCl-resistant bacteria and against its virulence factor (10–13). Furthermore, CHX has antimicrobial substantivity (9, 14), which is beneficial for more effective root canal disinfection. Therefore, CHX can be considered an advantageous conjunctive root canal irrigant, even though it lacks tissue-dissolving properties (15). For this reason, the effect of a root canal disinfection regimen with a combination of CHX and NaOCl was investigated (16–18). However, this association results in the formation of a dense-brown precip- itate, para-chloroaniline (PCA) (4, 17). The precipitate formed is of clinical relevance with regard to staining the dentin, hampering the seal of obturation, and potentially leaching PCA into the periapex (18, 19) . Similar to CHX, alexidine (ALX) is a bisbiguanide disinfectant that helps to inhibit the immune response of the major virulence factors (lipopolysaccharide and lipoteichoic acid) of bacteria (20) and contains 2 hydrophobic ethylhexyl groups in its structure (21). ALX has been previously used as a mouthwash solution (22) and contact lens solution (23). To date, however, no reports have been published on the antimicrobial effects of ALX or its chemical interaction with NaOCl as an endodontic irrigant. Therefore, the aim of this study was to determine by electrospray ionization mass spectrometry (ESI-MS) and scanning electron micros- copy (SEM) whether the chemical interaction between ALX and NaOCl results in PCA or precipitates.

From the *Department of Conservative Dentistry, Dental Research Institute, School of Dentistry, Seoul National University Dental Hospital, Seoul National University, Seoul, South Korea; Department of Endodontology, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut; Department of Conservative Dentistry, Oral Science Institute, College of Dentistry, Yonsei University, Seoul; § Department of Conservative Dentistry, Institute of Oral Health Science, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul; Department of Conservative Dentistry, Wonju College of Medicine, Yonsei University, Wonju; k Department of Dentistry, School of Medicine, Ajou University, Suwon; and **Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang, South Korea. Supported by grants from the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology (MEST) (no. 2009- 0086835), Republic of Korea. Address requests for reprints to Dr Kee-Yeon Kum, Associate Professor, 25-9 Jongro-Gu Yungun-Dong, Department of Conservative Dentistry, Dental Research Institute and BK 21 Program, School of Dentistry, Seoul National University Dental Hospital, Seoul National University, Seoul, South Korea. E-mail address:

0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi: 10.1016/j.joen.2011.10.003

112 Kim et al.

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Materials and Methods

Assessment of Color Changes or Precipitates

Solutions of 4% NaOCl (Sigma-Aldrich, St Louis, MO), 2% CHX (Sung-Kwang Pharm, Bucheon, Korea), and 1% ALX (alexidine dihy- drochloride; Molekula, Gillingham, Dorset, UK) were prepared with distilled water. NaOCl was kept refrigerated to maintain the chloride ion activity, which was confirmed immediately before the experiment by using chlorine test paper (Advantec, Tokyo, Japan). Six Falcon tubes (Falcon 2052 polystyrene round-bottom tube, 12 75 mm; Becton Dickinson, Franklin Lakes, NJ) were prepared. The first 2 tubes were filled with 2 mL of 2% CHX (tube 1) and 1 mL of 2% CHX with 1 mL of 4% NaOCl (tube 2) and stirred. For the last 4 tubes (tubes 3–6), 1 mL of 4% NaOCl was added to 1 mL of 1%, 0.5%, 0.25%, and 0.125% ALX and stirred. All 6 solutions were kept at 36.5 C with 95% humidity for 2 weeks.

One individual observed and recorded the formation of precipi- tates or color changes in the reaction solutions every 15 minutes for the first 2 hours and until 2 weeks. This process was repeated 5 times. To check for any precipitate, all 6 tested solutions were centrifuged at 1000 rpm for 10 minutes. For additional confirmation of any precipi- tates in the reacted solutions, 6 extracted lower premolar teeth were selected. The working length was determined by using a #10 K-file, and the samples were prepared to 40/.06 size by using K3 nickel- titanium rotary instruments (SybronEndo, Orange, CA) with a crown- down technique. Irrigation was performed by using 5 mL of 4% NaOCl solution (Sigma-Aldrich) and 25-mm 30-gauge NaviTip (Ultradent Products Inc, South Jordan, UT) between each instrument. The root canal surfaces were conditioned for smear layer removal by using a 17% ethylenediaminetetraacetic acid solution (Pulpdent, Watertown, MA) for 2 minutes, followed by a final flush with distilled water to

minutes, followed by a final flush with distilled water to Figure 1. Analysis of the color

Figure 1. Analysis of the color changes and precipitates in 6 tested solutions (A, tubes 1–6). Note the peach-brown precipitate in the mixed solution of NaOCl and CHX (tube 2). The color of the mixed solutions of ALX and NaOCl became transparent as the concentration of ALX decreased (from tube 3 to tube 6). SEM images show the precipitates covering on dentinal surfaces in the CHX/NaOCl mixture group ( B and C ; original magnification, 500), whereas no precipitates were formed in the ALX/NaOCl mixture group ( D and E ; original magnification, 500).

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remove any trace of the demineralizing solution. All teeth were dried and irrigated alternatively with the following solutions for 3 minutes:

10 mL of 4% NaOCl and 10 mL of 2% CHX, or 10 mL of 4% NaOCl and 10 mL of 1% ALX. The root surfaces were grooved with a disk, split by using a chisel, and observed for the presence of any precipitates on the dentinal surfaces by SEM (Hitachi S-4700, Tokyo, Japan). The central beam of the scanning electron microscope was directed to the surface area of each tooth under 500 magnification.

ESI-MS Analysis

For an analysis of the molecular peaks after the chemical reaction, 1 mL of 1% ALX and 1 mL of 4% NaOCl were mixed and analyzed by ESI-MS. All mass spectra were acquired by using a hybrid ion-trap time-of-flight mass spectrometer (Shimadzu LCMS-IT-TOF, Kyoto, Japan) equipped with an ESI source (ESI-IT-TOFMS) in negative ion mode at a mass resolution of 10,000 full-width at half maximum. Accu- rate masses were corrected by calibration by using sodium trifluoroa- cetate clusters as internal references. The sample solutions were prepared by dissolving each sample in a solution of methyl alcohol to a final concentration of 50 mg/ m L. All ions produced were finally intro- duced to the TOF instrument for accurate mass determinations. Data acquisition and analysis were performed by using LC Solution 3.0 soft- ware (Shimadzu, Kyoto, Japan). The Shimadzu Composition Formula Predictor (Shimadzu) was also used to verify the identifications. As control groups, 2 mL of 1% ALX, 2 mL of 0.5% (wt/wt) PCA, and a mixed solution of 1 mL of 2% CHX and 1 mL of 4% NaOCl were also analyzed. The mass spectra were interpreted by nano high-performance liquid chromatography separation, ionized by an ion spray or nanospray method, and detected according to the mass/charge ratio (m/z) (24).

Results

Assessment of the Color Changes and Reaction Precipitates

Figure 1A shows the color changes and formation of precipitates in the 6 tested solutions. The 2% CHX solution was pink, but there was no precipitate found (tube 1). A peach-brown discoloration was observed in the mixed solution of NaOCl and CHX (tube 2). The color change was noted immediately and did not change with time. The color of the 4 mixed solutions of ALX and NaOCl ranged from light yellow to trans- parent as the ALX concentration decreased (tubes 3–6). Regarding the reaction precipitates, a precipitate was only observed in the mixed solution of CHX and NaOCl after centrifuging. SEM views also showed dense precipitates covering the dentinal surface and occluding the dentinal tubules in this association (Fig. 1B and C). However, there was no precipitate covering the dentinal tubules under the SEM views (Fig. 1 D and E ) in the mixture of ALX and NaOCl.

ESI-MS Analysis

The mass spectra showed a clear peak for PCA (m/z = 128.026; Fig. 2 A), which was identified as C 6 H 6 NCl by using a Formula Predictor. The mass spectrum of a mixture of CHX/NaOCl also displayed the same peak (C 6 H 6 NCl, m/z = 128.026; Fig. 2 B ) shown in the PCA spectrum. The molecular formula of ALX dihydrochloride is C 26 H 56 N 10 (HCl) 2 (molecular weight, 581.70). The mass spectrum of ALX showed char- acteristic peaks at 255.248, 509.473, and 545.447 m/z (Fig. 2C ). The 509.473 peak represented C 26 H 56 N 10 , and the 545.447 peak repre- sented C 26 H 56 N 10 (HCl), as confirmed by using a Formula Predictor. The 255.248 peak was the same peak from the 509.473 peak because the former peak was the double charge form of [M+H] +2 . Interestingly, there was no 128.026 peak observed in the spectrum of the ALX/NaOCl mixture (Fig. 2 D).

114 Kim et al.

of the ALX/NaOCl mixture ( Fig. 2 D ). 114 Kim et al. Figure 2. The
of the ALX/NaOCl mixture ( Fig. 2 D ). 114 Kim et al. Figure 2. The
of the ALX/NaOCl mixture ( Fig. 2 D ). 114 Kim et al. Figure 2. The

Figure 2. The mass spectrum results showing a typical peak (m/z = 128.026, black arrow) in the PCA solution ( A ) and in the mixed solution of NaOCl and ALX ( B ). The mass spectrum of ALX ( C ) shows peaks at 255.248, 509.473, and 545.447. The 128.026 peak was not found in the NaOCl and ALX ( D).

Discussion

This study examined the reaction product of the ALX/NaOCl mixture by using ESI-MS and SEM to determine whether PCA or precipitate was formed. Basrani et al (17, 25) demonstrated that PCA (4-chloroaniline) was the reaction product of NaOCl and CHX by using the time-of-flight secondary ion MS (TOF-SIMS) and x-ray photon spectroscopy. Krishnamurthy and Sudhakaran (19) also demonstrated that PCA was produced after an interaction between NaOCl and CHX on the basis of Beilstein and HCl solubility test along with nuclear magnetic resonance (NMR) spectroscopy. On the other hand, Thomas and Sem (26) and Nowicki and Sem (27) reported that this association did not produce PCA by using NMR spectros-

copy. The present results demonstrated that the mass spectrum of the NaOCl/CHX mixture displayed the same peak (m/z =128.026) in the PCA spectrum. This is in agreement with the results of previous studies (17, 18) . On the other hand, the mass spectra of an ALX/NaOCl mixture did not display the typical peak (m/z = 128.026) in the PCA spectrum, whereas numerous peaks ranging from 201–705 m/z were shown. The observed non-PCA peaks might have resulted from fragmentation

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caused by bond breaking between the carbon and nitrogen of ALX because of the low-dissociation energy between the 2 atoms and bonding of ions dissociated from NaOCl. Regarding the formation of reaction precipitates, the association of ALX/NaOCl showed no precipitates covering the dentinal surface under SEM observations and also in the reaction solution after centrifuging. In the CHX/NaOCl mixture, however, the reaction precipitate tended to occlude the dentinal tubules under SEM, and a peach-brown colored material precipitated in the reaction solution after centrifuging. The

formation of precipitate in the mixed solution of CHX/NaOCl could be explained by an acid-base reaction (17). In particular, CHX, a dicationic acid, has the ability to donate protons, whereas NaOCl is alkaline and can accept protons from the dicationic acid. This proton exchange results in the formation of a neutral and insoluble substance, which is referred to as the precipitate (17, 28). This precipitate acts as

a chemical smear layer and can compromise the dentin permeability,

the diffusion of intracanal medication, and sealing after obturation (18, 19, 29) . ALX has a slightly different structure, containing 2 hydrophobic ethylhexyl groups, whereas CHX contains

p -chlorophenyl end groups (Fig. 3). Consequently, ALX cannot produce

a PCA precipitate when mixed with NaOCl (28). Thomas and Sem (26) argued that TOF-SIMS might not be

a conclusive method for determining the presence of degradation prod-

ucts because it relies on gas phase ionization, which can fragment mole- cules. In contrast, NMR spectroscopy could analyze reaction of the precipitate in a noninvasive and nondestructive manner and determine the chemical composition of the breakdown products (27). ESI-MS used in this study is less invasive and has a higher resolution than TOF-SIMS. This technique is extremely useful for analyzing large, non-volatile, chargeable molecules. Another reason for the extraordi- nary high-mass capability of ESI can be found in the characteristic formation of multiply charged ions in case of high-mass analytes. Multiple charging folds up the m/z scale by the number of charges and compress the ions into the m/z range of standard mass analyzers

(30). MS itself has limitation in that molecular weight can be identified, but the molecular structure of the material cannot be identified through the spectrum (30). Therefore, further studies will be needed to deter- mine the structures of the non-PCA peaks observed in the ALX/NaOCl spectra by using NMR spectroscopy as well as their possible effects on the dental and periapical tissues. It has been reported that CHX itself slowly hydrolyzes and forms PCA when placed in an aqueous solution (31). Tanaka et al (32, 33) reported that the degradation of CHX occurs through the cleavage of molecules along the hexane diamine linker between the aromatic rings at either end of the molecule. Such cleavage results in smaller degradation products, each of which contains an aromatic ring, and can cause PCA formation (26). Nevertheless, this study did not observe any PCA precipitation in the CHX solution. Recently there has been concern regarding the clinical relevance of the color changes associated with tooth staining. In the present study, peach-brown discoloration occurred in the CHX/NaOCl mixture. Vivacqua-Gomes et al (34) reported that the viscous dark-brown precipitate formed in the mixture of NaOCl/CHX stained dentin and could not be removed completely from the root canals, probably acting as a residual film. Souza et al (35) suggested that a mixture of CHX/NaOCl should be avoided because of the possibility of color changes in the dental structures. In the ALX/NaOCl mixed solutions, the color changed from light yellow to transparent with decreasing ALX concentration. In addition, even the discolored solution (yellow color) did not stain dentin and was easy to remove from the root canals by irrigation. This suggests that ALX is a promising irrigant that can be used with NaOCl during irrigation. ALX and CHX are cationic molecules that exert their antibacterial effects by disrupting the integrity of the bacterial cytoplasmic membrane, causing the leakage of the intracellular contents (20). Indeed, gram-positive bacteria are more sensitive to cations because they are more negatively charged (22). ALX has greater affinity for the major virulence factors of bacteria than CHX. The difference in

virulence factors of bacteria than CHX. The difference in Figure 3. Molecular formula of CHX (

Figure 3. Molecular formula of CHX ( A ), PCA ( B ), and ALX ( C). CHX contains phenol rings, ammonia, and chlorine ( A ), which are components of PCA, whereas ALX does not contain phenol rings ( C).

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the hydrophobic moieties between these 2 compounds is believed to be responsible for the more rapid bactericidal action of ALX (20). ALX had been tested in periodontologic and ophthalmologic fields. When used as mouth rinse, it reduced salivary bacterial counts and had residual antibacterial activity (22). ALX has disinfection efficacy against all bacteria and fungi tested by contact lens disinfection standard (23). A recent study has suggested the potential use of ALX as a novel anti- cancer compound (36). Although ALX has less effect on plaque accu- mulation and shorter residual antibacterial activity than CHX (37), it might be valuable for endodontic irrigation purposes. Within the limitation of this in vitro study, the association of ALX/ NaOCl did not produce PCA or any precipitate. If the antimicrobial activity or possible effects on the dental and periapical tissues are further confirmed to be effective and safe, ALX might be used as an effec- tive canal irrigant sequentially or in combination with NaOCl.

Acknowledgments

The authors deny any conflicts of interest related to this study.

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