Glutaraldehyde purity and stability: implications for preparation, storage, and use as a pulpotomy agent.

Glutaraldehyde can be prepared and stored for use as a pulpotomy agent in a variety of ways. This study was designed to determine the most effective and practical procedures in the clinical setting. The purity and efficacy of several glutaraldehyde solutions were analyzed before and after six months of storage. Commercial glutaraldehyde (25%) contained considerable organic impurities, but solutions prepared from it proved more effective at fixing protein than the solutions prepared from pure glutaraldehyde. Comparison of refrigerated and room temperature storage of dilute buffered and unbuffered solutions demonstrated that the buffered, unrefrigerated preparations developed organic impurities, some of which were different chromatographically from those seen in stock solution. In addition, fixation of pulp protein by the aged buffered preparations was diminished, as determined by measurements of residual enzyme activity. The results suggest that while some of the impurities in stock glutaraldehyde solutions are polymers which enhance fixation, some that appear in buffered solutions stored at room temperature are converted species which have lost their cross-linking properties. The search for a substitute for formocresol (FC) had been prompted by a series of negative reports questioning both its local tissue effects TM and its extrapulpal toxicity. 5,6 Recently, glutaraldehyde (GA), standard fixative for electron microscopy,7 has been recommended 8,9 and tested ..... as an alternative pulpotomy medicament for deciduous teeth. Its efficacy as a protein cross-linking agent has been demonstrated with bovine pulp,12-14 and several clinical studies are ongoing’~,~6 or completed. 11 Therefore, it is likely that the use of GA as a pulpotomy agent will become more widespread. Since users of GA for histologic fixation have noted that purified solutions of GA are unstable, resulting in a variable composition of stock solutions, 1 .... this study was initiated to determine if these concerns might have clinical implications. Specifically, this report will deal with the effects of preparation and storage on the accumulation of chemical impurities in GA solutions and their impact on the fixation of bovine pulp. Methods and Materials Preparation and Storage of Solutions An aliquot of a 25% stock solution of commercial glutaraldehyde (pH 2.9) a was vacuum distilled at 80°C to obtain the pure dialdehyde. The distillate was diluted immediately with either distilled water to make 2 and 5% unbuffered solutions or with 0.1 M phosphate buffer (pH 7.2) to make 2 and 5% buffered solutions. The initial pH of the freshly made unbuffered preparations was 5.8. An aliquot of each was refrigerated at 4°C; another aliquot of each was stored at room temperature (25°C). Analysis of Impurities Immediately after preparation and monthly for six months the aliquots were analyzed spectrophotometrically for the presence of impurities, determined by an increase in the 235/280 nm absorbance ratio (impurity index). This index was chosen because has been reported that the dialdehyde is responsible for absorption at 280 nm and extraneous material for the absorption at 235 nm.’9,~° Thus, in this study an increase of the ratio 235/280 nm denotes a conversion of glutaraldehyde into contaminating molecules or polymeric forms of GA. Cross-Linking Assay The ability of various preprations of GA to fix pulps was determined by comparing the degree of cross linking following exposure of pulp tissue to each of the solutions. The procedure for the cross linking followed the protocol of Hassel and Hand.2l After thawing and mincing frozen samples of calf molar pulp, weighed aliquots (-0.5 g) were placed in vials containing 10 ml of 0.1 M phosphate buffer or one of two preparations of 2% buffered GA, prepared either from purified distillate or a stock solution with demonstrated impurities. The samples were agitated on a Aldrich Chemical Co., Milwaukee, WI. PEDIATRIC DENTISTRY: lune 1984/Vol. 6 No. 2 83 reciprocal shaker for four hours at room temperature, after which any extTacted protein was separated by filtration. Protein in the filtrate (extractable fraction) was precipitated with 10% trichloroacetic acid (TCA) and washed several times with 5% TCA. The final precipitates were solubilized in 1 NaOH at 60°C overnight and assayed for total protein. The filtered residue from the original treated samples was rinsed and homogenized in distilled water using a hand homogenizer and then centrifuged at 750 x g at 4C for 20 minutes. The supernatant (soluble fraction) and the remaining pellet (insoluble fraction) were treated described above. The amount of protein in the fractions was determined by the Bradford assay = using bovine serum albumin (BSA) as a standard, and the mean _+S.D. was calculated for five samples at each condition. Enzyme Assay Lactic dehydrogenase activity was determined as a measure of tissue viability following fixation. The procedure for the assay of lactate dehydrogenase activity was modified from a previous study. ’2 In initial experiments comparing enzyme inactivation by stock and pure preparations, pulp samples were incubated for one hour in 0.2% buffered GA solutions diluted from either stock solution or fresh distillate. Phosphate buffer (0.1 M, pH 7.2) served as the control solution. The residual fixative property of stored GA was determined by treating pulp samples with the 2% preparations that had been stored for six months. In this latter experiment, buffer, 2% buffered and unbuffered glutaraldehyde (stock), and 2% buffered and unbuffered glutaraldehyde (distillate) served as control solutions. After treatment, each sample was washed and homogenizedb for 1 minute in 10 ml of 0.2M Tris HC1, pH 7.3. Following centrifugation ~ for 10 minutes at 300 rpm the supernatant was decanted. Fifty microliters of the supernate was added to a cuvette containing 2.8 ml of Tris HC1, 1.0 ml of 6.6 mM NaOH, and 0.1 ml of 30 mM sodium pyruvate, then vortexed for 10 seconds. Change in absorbance per minute was measured with a spectrophotometer at 345 nm at room temperature. The activities were calculated as units/mg of wet weight, and a mean _ S.D. was calculated for five samples in each group. Chromatographic Analysis The presence of chemical impurities was determined by comparison of thin layer chromatograms ot each of the test solutions with freshly distilled GA. Thin layer chromatograms were prepared by application of distilled GA, stock GA, or stored GA solub Tekmar Tissumizer, Tekmar Co., Cincinnati, OH. c IEC clinical centrifuge, International Equipment Co., Needham, MA. tions to chromatography plates, d The mobile phase consisted of benzene: methanol 4:1, v/v. Spots wer~ visualized following oxidation with 50% sulphuric acid and heat.