Nobuhiko Takagi

Leaching and cytotoxicity of formaldehyde and methyl methacrylate from acrylic resin denture base materials

Acrylic resin dentures have the potential to elicit irritation, inflammation, and an allergic response of the oral mucosa. Studies of substances leachable from acrylic resins, their cytotoxicity to cultured cells, and means of reducing their leaching were systematically conducted. Under in vivo and in vitro conditions, formaldehyde and methyl methacrylate were significantly leached into human saliva and saliva-substitute buffer, especially from autopolymerized resins. Both leachable substances showed cytotoxic potentials in the range of their leaching concentrations. Formaldehyde was cytotoxic at lower concentrations than methyl methacrylate. Preleaching in water reduced subsequent leaching of both formaldehyde and methyl methacrylate, and the amount of reduction depended on an increase in the preleaching temperatures. Immersion of acrylic resin dentures in hot water (50 degrees C) before insertion is recommended, especially for autopolymerized resins used either for rebasing or as denture base materials, to minimize the risk of adverse reactions in patients who wear acrylic resin dentures.

High-performance liquid chromatography of carboxylic acids using 4-bromomethyl-7-acetoxycoumarin as fluorescence reagent

Abstract A sytem for the high-performance liquid chromatography of carboxylic acids using 4-bromomethyl-7-acetoxycoumarin (Br-Mac) as the fluorescence reagents is described. Br-Mac reacts with carboxylic acids to give the ester derivatives, which are separated using reversed-phase liquid chromatography. The eluate from the column is mixed with an alkaline solution. The labelled carboxylic acids are hydrolysed to the fluorescent coumarin derivatives, which are introduced into a flow-through fluorimeter. In this system, the fluorescent hydrolysate, equimolar to a carboxylic acid, is detected and this fluorophor is common to every carboxylic acid. Only a slight variation is found in the peak areas for various carboxylic acids. A gradient elution technique is effectively used in this system because the fluorescence quantum yield of the fluorescent hydrolysate is not affected by the constitution of the mobile phase. Low femtomole levels of carboxylic acids can be detected.

Leachability of denture-base acrylic resins in artificial saliva☆

We studied the influence of salivary acidity on leachability of denture-base acrylic resins with etiological interest in denture stomatitis because denture surfaces are frequently exposed to acidic conditions in the oral cavities. Auto-, heat-, and microwave-polymerized resins were immersed in artificial saliva with pH ranging from 4.0 to 6.8 at 37 degrees C, and leachables were pursued quantitatively with time. Methyl methacrylate, methacrylic acid, and benzoic acid leached from all resins. Their concentrations in the saliva were markedly high for auto-polymerized resins, while leachability of heat- and microwave-polymerized resins was so low that quantitative analysis of leachables was impossible. Lower pH showed higher concentrations of methyl methacrylate, although no apparent association was confirmed between salivary acidity and its own leachability. The concentrations of methacrylic acid increased remarkably with an increase in pH, which was probably due to hydrolysis of methyl methacrylate. These results suggest that chemotoxic actions of auto-polymerized resins are potentially ascribable to methyl methacrylate under more acidic conditions and to methacrylic acid under less acidic conditions.

Pharmacological Studies on the Anti-inflammatory Action of Phenolic Compounds:

The mechanism of the anti-inflammatory action of phenolic compounds was examined using neutrophil chemotaxis. Chemotactic activity of guinea pig peritoneal neutrophils to N-formylmethionyl-leucylphenylalanine (FMLP) was suppressed in a concentration-dependent manner. The order of drug potency in inhibiting the neutrophil chemotaxis was eugenol » thymol > guaiacol » phenol. The concentrations of phenolic compounds used in these experiments did not induce lactate dehydrogenase (LDH) release and did not affect neutrophil viability. There was a consistent positive relation between the ID50 of superoxide anion generation in neutrophils and the inhibitory dose for neutrophil chemotaxis by phenolic compounds. A free phenolic hydroxyl group is essential for scavenging oxygen free-radicals and is also essential for inhibiting leukocyte chemotaxis, as was demonstrated in these experiments. These findings suggest that inhibition of leukocyte chemotaxis may be involved in the anti-inflammatory action of phenolic compounds, and that one of the anti-inflammatory actions of phenolic compounds is the prevention of the production of oxygen free-radicals by leukocytes.

In Vitro Comparison of Cone Beam Computed Tomography with Digital Periapical Radiography for Detection of Vertical Root Fracture in Posterior Teeth.

Statement of the Problem: The diagnosis of vertical root fracture (VRF) is a challenging task. Purpose: This in vitro study compared cone beam computed tomography (CBCT) imaging with digital periapical radiography (DPR) made by three different horizontal angels (20°mesial, 0° and 20° distal) for accurate diagnosis of VRF. Materials and Method: Among 120 posterior teeth included in this study, 60 were vertically fractured. Fractured and non-fractured teeth were randomly distributed into three groups defined as group 1 with no filling in the root canal, group 2 with gutta-percha in the canal, and group 3 with the intracanal post. All samples were placed in a dry mandible and imaged with CBCT and DPR techniques. Two blind observers investigated the images. Results: CBCT had higher sensitivity but lower specificity compared with DPR, except for the intracanal post group in which the sensitivity of DPR was higher; though the chi-square test showed the differences to be statistically insignificant. The sensitivity, specificity, and accuracy of CBCT and DPR were reduced in the cases that gutta-percha or post were present in the canal. Inter-observer agreement was higher for CBCT. A set of three DPRs with different horizontal angels were significantly more sensitive for VRF recognition than a single orthogonal DPR. Conclusion: Based on our results, there was no significant difference between CBCT and a set of three DPRs with different angulations for VRF detection in posterior teeth. Therefore, it is suggested to consider DPRs with three different horizontal angels (20°mesial, 0° and 20° distal) for radiographic evaluation before CBCT examination.

Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography

Fatty acids are separated by reversed-phase high-performance liquid chromatography after derivatization with a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin. Each derivative eluted from a column is successively hydrolysed by mixing it with an alkaline solution, and the produced fluorescence is detected. The derivatives of series of both saturated and unsaturated fatty acids (C6:0--C20:4) are simultaneously separated by a continuous gradient elution method using a methanol-based solvent containing acetonitrile. The quantitative detection of fatty acids is over a range of 5-1000 pmol per derivatization mixture. This method is applicable to the quantitative analysis of free fatty acids in normal human blood samples and blood samples from diabetic patients. Ten microliters of blood plasma are sufficient to carry out the determination. The analytical results show good recovery and good reproducibility. This sensitive method is very useful for the analysis of fatty acids in very low concentrations.

Reversed-phase ion-pair chromatographic analysis of tetracycline antibiotics. Application to discolored teeth

A high-performance liquid chromatographic method with diode array detection was developed to simultaneously separate tetracycline antibiotics and applied to the analysis of discolored teeth. By a reversed-phase ion-pair chromatographic system using pentanesulfonate as a counter ion, minocycline, oxytetracycline, tetracycline and demeclocycline were eluted in this order, and they showed base-line separation within 9 min. When using oxytetracycline as an internal standard, the quantitative ranges were between 2.5 ng/ml and 7.5 microg/ml. Powdered dentine (10 mg) and enamel (40 mg) prepared from discolored primary teeth were sonicated in 0.25 ml of 10 mM HCl containing oxytetracycline (0.75 microg/ml) and 50 mM EDTA-2Na, thereafter the supernatants were chromatographed. Eluates from both discolored tooth samples were identified as minocycline based on diode array spectra of their peaks, while minocycline was not detected in any samples from nondiscolored normal teeth, indicating that discoloration of the tested teeth was due to minocycline incorporated into dentine and enamel. Replicate quantitative analyses of the identical tooth substances showed that intra- and inter-assay C.V.s were 2.63 and 4.95% for dentine, and 5.42 and 10.88% for enamel. Application of the developed method to nine discolored teeth revealed that the incorporated minocycline ranged from 20.13 to 84.62 ng/mg of dentine and 0.89 to 7.87 ng/mg of enamel.

Sensitive high-performance liquid chromatographic method for prostaglandins using a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin

High-performance liquid chromatography of prostaglandins is developed in which a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin is used to perform the high-sensitivity detection. The reagent reacts with prostaglandins and related compounds to form the ester derivatives, which are separated using a reversed-phase system. Each labeled compound eluted from the column is successively hydrolyzed to the fluorescent coumarin derivative, and this fluorophore is introduced into a flow-through fluorometer. Prostaglandins can be determined in the range of at least 1 nmol to 5 pmol, and the detection limit is about 10 fmol. This system is applied to the analysis of prostaglandins in human seminal fluid.

Determination of formaldehyde in reagents and beverages using flow injection

A flow injection procedure has been developed for the sensitive and selective determination of formaldehyde in reagents and beverages. The method involves on-line condensation of formaldehyde with 4-amino-3-penten-2-one, followed by fluorescence detection of the product 3,5-diacetyl-2,6-dihydrolutidine. Under optimized conditions, the relative standard deviation in peak height variation is 0.26% for formaldehyde (40.0 nmol ml–1), and the calibration graph is linear in the range 0.5–100.0 nmol ml–1. The detection is so specific for formaldehyde that the other compounds, including acetaldehyde, give no fluorescence response. Results obtained with the proposed method are in good agreement with those obtained by a standard spectrophotometric method. The flow injection procedure enables 12 samples to be analysed per hour on direct injection of the sample solutions, and it does not suffer from the high blank responses or interferences by contamination from the other aldehydes, which are found for previous methods. Its application to the analysis of reagents and beverages has revealed the widespread occurrence of formaldehyde as an impurity or a constituent.

Flow injection analysis of formaldehyde leached from denture-base acrylic resins

Formaldehyde is responsible for allergic inflammation in acrylic denture wearers and the quantitation of formaldehyde is necessary to study its leaching from denture-base materials. Flow injection analysis was developed to quantify the formaldehyde leached from acrylic resins. Different resins were immersed in aqueous solvents at 37 degrees C and the immersion solutions were directly injected into the flow system, in which formaldehyde was converted on-line to a fluorescent derivative and its fluorescence was detected. Under the optimized conditions, the leached formaldehyde could be quantified in a short time (within 4 min) with high sensitivity (pmol levels per injection) and high specificity (no fluorescent response to the other leachables). In leaching experiments, significant amounts of formaldehyde were leached from autopolymerized resins, but not from heat- and microwave-polymerized resins.