High-performance liquid chromatography analysis of salivary bisphenol A levels from light-cured and chemically cured orthodontic adhesives

Introduction

Our objective was to assess the levels of bisphenol A (BPA) released from light-cured and chemically cured resins used for orthodontic bracket bonding in 1 month.

Methods

Saliva specimens were obtained at 5 time periods from 40 healthy patients treated with orthodontic mechanotherapeutics. The periods of collections were just before bonding orthodontic brackets, followed by 30 minutes, 1 day, 1 week, and 1 month after orthodontic bonding. The specimens were analyzed with the high-performance liquid chromatography/mass spectrometry method for quantitative evaluation of BPA levels.

Results

We observed a large increase in BPA levels 30 minutes after orthodontic bonding in the 2 groups. Thereafter, there was sudden decline in BPA levels as time passed, and the levels reached a statistically significant level at 1 month after orthodontic bonding. Interestingly, the amount of BPA released from chemically cured resin was much higher; this was also significant statistically compared with light-cured resins.

Conclusions

The results of this in-vivo approach with high-performance liquid chromatography on salivary specimens confirmed continued release of BPA after bonding brackets for 1 month, although in smaller quantities. The release during the initial 30 minutes is high, making it essential to introduce measures to dilute it for better patient safety.

Highlights

  • Bisphenol A is found in composites used for bonding.

  • Level of BPA in saliva is significantly increased immediately after bonding.

  • There is a drastic fall from peak to base level within a month.

  • Base level is still higher than as shown in many studies.

  • Chemically cured composites show higher levels of leaching.

Bisphenol A (BPA), an important component of many manufactured products, including polycarbonate plastics, the inner coating of food cans, and cosmetics, is a basic material in dental resins as a precursor for the generation of BPA glycidyldimethacrylate (Bis GMA) and BPA dimethacrylate. Researchers have removed the smoke screen regarding the deleterious effects of BPA on almost all organs and systems in the human body, and concerned authorities have issued appropriate statements at national and international levels. There is evidence that BPA mimics 17β-estradiol and aims at all estrogen target organs in the body. BPA also mimics synthetic estrogens such as diethylstilbestrol, and experiments have shown that its dimethacrylate derivative Bis-DMA, BADGE, and related diphenylalkanes are estrogenic in different bioassays and systems. Recently, reports demonstrating BPA’s effect on thyroid hormones as well as reduction in testosterone levels in boys have also been published. All hormone-mimicking effects can lead to obesity, widespread fertility problems, feminization of boys, accelerated maturational changes in girls, and increased diabetes risks and breast cancer incidences.

Orthodontic resin-based adhesives consist of 2 main components: an organic monomer matrix based on functional dimethacrylate such as Bis GMA, urethane dimethacrylates, or triethyleneglycol-dimethacrylate; and inorganic filler components such as silica, glass, and ceramic. These are used for bonding various attachments because of their better mechanical and esthetic properties as well as lower failure rates. The risk associated from the systemic intake of BPA is due to the orthodontic brackets bonded to the dentition that stay for approximately 2 years as well as bonded lingual retainers that stay longer than that. The literature abounds with documented evidence of leaching BPA components from orthodontic bonding agents used for bracket bonding and placement of permanent lingual retainers.

The aim of this study was to assess quantitatively the release of BPA from Bis GMA-based chemically cured and visible light-cured composite resins from the same manufacturer after obtaining saliva of patients undergoing orthodontic treatment at different time periods.

Material and methods

A total of 40 healthy volunteers—21 male subjects (mean age, 19.9 years; range, 13-30 years) and 19 female subjects (mean age, 23.1 years; range, 13-30 years)—were included in the study. They were diagnosed with Angle skeletal Class I malocclusion with moderate to severe crowding requiring maxillary and mandibular first premolar extractions as part of orthodontic treatment. All subjects in the study agreed to receive fixed appliance orthodontic treatment with metal brackets (Victory series, 0.022 × 0.028-in brackets; 3M Unitek, Monrovia, Calif). Written informed consents were obtained from all subjects with confidentiality of personal information strictly maintained. Excluded were users of tobacco in any form (smoking or chewing), and those with hepatic or renal disease or acrylic-related work or resin restorations on any surface of the teeth, and those taking any medications. The study protocol was approved by the ethical committee (PMS/IEC/2012/14). The power of the sample was calculated using the formula as described previously.

The study subjects were randomly grouped by lot into 2 groups: group 1, 20 subjects had brackets bonded with Unite (3M Unitek) no-mix adhesive; and group 2, 20 subjects bonded brackets with Transbond XT (3M Unitek) light-cured adhesive. One operator (S.B.) performed all bonding procedures in the maxillary and mandibular dentitions according to the manufacturer’s recommendations.

With calibrated pipettes, unstimulated saliva was collected from the buccal vestibule and the sublingual area and stored in glass vials to prevent any background contamination. The samples were collected immediately before bonding and used as the controls. A second saliva sample was collected 30 minutes after bracket placement, followed by samples collected 1 day, 1 week, and 1 month after bonding to measure the kinetics of BPA release. A total of 5 samples of unstimulated saliva were collected from each participant. To this 10 mL of unstimulated saliva, 6 mL of methanol was added to extract the BPA. Standard solutions were prepared by diluting known amounts of the BPA-methanol solution in the saliva at various concentrations. The vials were kept refrigerated until analysis. The saliva samples were brought to room temperature before analysis and then vortexed for 1 minute, and 0.1 N of hydrogen chloride and 2 mL of methyl t-butyl ether were added to 500-mL aliquots. The mixture was centrifuged at 3000 rpm for 5 minutes, and 1 mL of the supernatant was collected. The supernatant was evaporated to dryness in a nitrogen gas flow at 40°C. The dried residues were dissolved in 50 mL of methanol, and 10 mL was injected into the high-performance liquid chromatography column.

A high-performance liquid chromatography/mass spectrometry system (model 1100; Shimadzu, Kyoto, Japan) was used to determine the levels of BPA. The resultant data were processed with ChemStation software (Shimadzu, Kyoto, Japan). The mobile phase was a 60:40 (volume/volume) mixture of 0.1% acetic acid and acetonitrile. The flow rate was 0.7 mL per millimeter. The peak was detected in the selected ion monitoring mode, and the electrospray positive mode was used for ionization. Nitrogen gas was used as the nebulizing gas at a nebulizing pressure of 40 psi with a gas flow rate of 12 L per minute, gas temperature of 300°C, and capillary voltage of 3500 V. The BPA signal was set at mass to charge ratio of 227 with a 140-V fragment voltage. The limit of detection in liquid chromatography/mass spectrometry was 0.5 ng per cubic milliliter of volume from the subjects.

Quantitative data obtained from the liquid chromatography analyses of samples from both the groups at different times were tabulated and analyzed using 1-way analysis of variance (ANOVA) followed by Tukey HSD post-hoc analysis to determine whether variations in BPA leached out at different times have significance statistically. Intergroup comparisons at different times were assessed using unpaired t tests.

Results

The overall observation showed that the group 1 specimens leached out more BPA than did the group 2 specimens over the 1-month evaluation period. Specifically, the group 1 specimens showed the maximum leach out at 30 minutes (mean ± SD, 19.6 ± 8 μg/mL) ( Table 1 ). Minimum leach out of BPA at 30 minutes was 8 ng per milliliter, and maximum leach out was 36 μg per milliliter in group 1. We observed sudden declines in BPA levels after 1 day and 1 week with mean values of 5.0 ± 1.3 and 4.0 ± 1.3 μg per milliliter, respectively. But the values never returned to the control levels even after 1 month of bracket bonding, with a value of 1.2 ± 0.8 μg per milliliter in the group 1 subjects. Group 2 specimens also showed the same pattern as group 1, with maximum leach out observed at 30 minutes, but the values were lower than those of group 1 (11.2 ± 4.2 μg/mL). Unpaired t tests showed a statistically significant difference between groups 1 and 2 of BPA leach out values at 30 minutes after bonding ( Table II ). After 1 day, we observed a significant reduction in BPA release in the group 2 specimens (3.1 ± 1.0 μg/mL), which showed a statistically significant difference compared with the amount released from the group 1 specimens. By 1 week, we observed a further reduction in the salivary BPA levels (2 ± 1 μg/mL), which was also a significant difference statistically compared with group 1. At 1 month, the values in group 2 dropped to very low levels compared with group 1 (0.6 ± 0.32 μg/mL) and still showed significant differences statistically compared with the group 1 specimens ( Table II ).

Table I
BPA levels in groups 1 and 2 specimens
Leach out Group 1 Group 2 Group 1 Group 2
Mean SD Mean SD Minimum Maximum Minimum Maximum
A Just before bonding 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
B After 30 minutes 19.6 8.0 11.2 4.2 8.0 36.2 6.0 19.0
C After 1 day 5.0 1.3 3.1 1.0 3.0 7.4 1.4 5.0
D After 1 week 4.0 1.3 2.0 1.0 1.9 6.2 0.6 4.1
E After 1 month 1.2 0.8 0. 6 0.32 0.45 2.3 0.12 0.98

Table II
Results of unpaired t tests from groups 1 and 2 specimens
Group 1
Mean ± SD
Group 2
Mean ± SD
P value
A Before bonding 0.0 0.0 NS
B 30 minutes after bonding 19.6 ± 8 11.2 ± 4.2 <0.0002
C 1 day after bonding 5 ± 1.3 3.1 ± 1 <0.0001
D 1 week after bonding 4 ± 1.3 2 ± 1 <0.0001
E 1 month after bonding 1.2 ± 0.8 0.6 ± 0.32 0.0021
NS , Not significant.

Significant at 0.05 level.

Comparison of leach out at different time periods were evaluated in both groups using 1-way ANOVA. The results from the group 1 specimens at different time periods showed significant results statistically ( Table III ). Evaluation with Tukey HSD post hoc analysis showed significant differences with a P value less than 0.005 compared with values from the control group at all other time periods: 30 minutes, 1 day, 1 week, and 1 month. The same results were observed when values were compared between 30 minutes and the other time periods such as 1 week and 1 month. The values between 1 week and 1 month did not show a statistically significant difference. Group 2 specimens had a different scenario in the ANOVA analysis followed by the Tukey HSD post hoc analysis. We observed a statistically significant difference between the control group and values obtained at evaluations after 30 minutes and 1 day, but no significant differences between control values and those at 1 week and 1 month. The values at 30 minutes were statistically significant throughout their comparisons with the other 3 time periods (1 day, 1 week, and 1 month). No difference was observed statistically when the time periods of 1 day, 1 week, and 1 month from group 2 were compared ( Table IV ).

Dec 8, 2018 | Posted by in Orthodontics | Comments Off on High-performance liquid chromatography analysis of salivary bisphenol A levels from light-cured and chemically cured orthodontic adhesives
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