Minoxidil: mechanisms of action on hair growth.

We have known for over 30 years that minoxidil stimulates hair growth, yet our understanding of its mechanism of action on the hair follicle is very limited. In animal studies, topical minoxidil shortens telogen, causing premature entry of resting hair follicles into anagen, and it probably has a similar action in humans. Minoxidil may also cause prolongation of anagen and increases hair follicle size. Orally administered minoxidil lowers blood pressure by relaxing vascular smooth muscle through the action of its sulphated metabolite, minoxidil sulphate, as an opener of sarcolemmal KATP channels. There is some evidence that the stimulatory effect of minoxidil on hair growth is also due to the opening of potassium channels by minoxidil sulphate, but this idea has been difficult to prove and to date there has been no clear demonstration that KATP channels are expressed in the hair follicle. A number of in vitro effects of minoxidil have been described in monocultures of various skin and hair follicle cell types including stimulation of cell proliferation, inhibition of collagen synthesis, and stimulation of vascular endothelial growth factor and prostaglandin synthesis. Some or all of these effects may be relevant to hair growth, but the application of results obtained in cell culture studies to the complex biology of the hair follicle is uncertain. In this article we review the current state of knowledge on the mode of action of minoxidil on hair growth and indicate lines of future research.

Hypertrichosis in females applying minoxidil topical solution and in normal controls.

BACKGROUND: Hypertrichosis has been reported more frequently in females than in males who use minoxidil topical solution (MTS) for the treatment of androgenetic alopecia (AGA). This article examines the occurrence of MTS-induced hypertrichosis in females. METHODS: Data from placebo-controlled clinical trials in females (up to 5% MTS) were analysed based on spontaneous reports of hypertrichosis/facial hair and investigators' inquiries (solicited) about the presence of any new hair growth on body parts other than the scalp. A postmarketing drug surveillance database for MTS was also examined for reports of hypertrichosis/facial hair. RESULTS: In the clinical trials involving a total of 1333 females, spontaneous reports of hypertrichosis/facial hair were noted for 50 (4%) females in a dose-related pattern of response (5% MTS > 2% MTS > placebo). Nine females (seven and two in the 5% MTS and 2% MTS groups, respectively) discontinued treatment because of hypertrichosis/facial hair. Solicited reports of excessive hair growth (primarily facial) also showed a dose-related pattern of response. Post-marketing data showed a lower occurrence (0.5%) of hypertrichosis/facial hair than in the clinical trials. Of interest, in one clinical trial, 27% of the females enrolled (MTS and placebo treated) had facial hair growth reported at baseline. CONCLUSIONS: Females with some hirsutism are particularly prone to seek treatment for AGA, and this may explain the high occurrence of hypertrichosis/facial hair found in the MTS clinical trials. Furthermore, some demographic groups of females are prone to develop facial hair and the problem of unwanted facial hair growth seems to be underestimated. Some females may have hair follicles that are very sensitive to MTS and should use the lowest strength of MTS (2%) to help avoid unwanted hair growth. The hypertrichotic effect of MTS on other sites than the scalp, including the face, is reversible and does not always require discontinuation of therapy.

Characterization of the binding of delmopinol to salivary precipitates.

The aim of this study was to quantify the amount of delmopinol recovered in different molecular weight ranges of salivary proteins in the supernatant and pellet of delmopinol-saliva mixes. Unstimulated whole saliva was collected from 5 subjects and mixed with radiolabeled delmopinol to obtain a final drug concentration of 9.7 mM. The salivary-delmopinol solutions were incubated and then centrifuged. The resulting pellets and supernatants were studied in an electrophoresis assay. Each individual sample was run in three different electrophoretic gel lanes. The first lane was stained with silver dyes; the second lane was used for electrophoretic blotting and autoradiography; the third lane was cut in standard slices that were dissolved and analyzed with scintillation counting. The scintillation results demonstrated that higher radioactivity levels were detected at high molecular weight ranges (700-600 kDa). Furthermore, pellet samples were found to contain the highest amounts of delmopinol. The autoradiography results confirmed that delmopinol was bound to proteins of high molecular weight (700-600 kDa).

The content of delmopinol and characterization of the molecular weight pattern of salivary protein precipitates.

The aims of this investigation were: a) To quantify the amount of radiolabelled delmopinol in precipitates formed in a mixture of saliva and delmopinol at two concentrations, b) To analyze the precipitates by polyacrylamide gel electrophoresis. Unstimulated whole saliva was collected from 5 individuals and the samples were clarified by centrifugation. In a first series of experiments radiolabelled delmopinol hydrochloride at the final concentrations of 1.6 mM and 6.4 mM was incubated with the saliva samples and centrifuged. The supernatants and the pellets were immediately transferred to scintillation vials and radiolabelled delmopinol was detected by using scintillation counting. In a second series of experiments, similar saliva-delmopinol mixtures were centrifuged and the supernatants and pellets were prepared for electrophoresis. Videodensitometry was used to quantify the relative density of stained protein in the pellet samples within four ranges of molecular weight (10-21.5, 21.5-26, 26-45, 45-300 kDa). The results of this study showed that the amounts of labeled delmopinol found in the precipitates were higher at 6.4 mM delmopinol than at 1.6 mM. The amount of salivary protein precipitated by delmopinol was found to depend on delmopinol concentration as well as salivary composition. Delmopinol interactions to salivary proteins could influence protein availability on oral cavity surfaces.

6-month use of 0.2% delmopinol hydrochloride in comparison with 0.2% chlorhexidine digluconate and placebo (II). Effect on plaque and salivary microflora.

This double-blind, randomised, 6-month clinical trial with parallel group design in 68 subjects with gingivitis was conducted to study the effects on the oral flora of delmopinol hydrochloride 2 mg/ml (0.2% w/v, Decapinol Mouthwash), when used for partly supervised mouthrinsing in comparison with placebo and chlorhexidine digluconate 2 mg/ml (0.2% w/v, Hibitane Dental, ICI Pharmaceuticals, UK). Apart from estimating the total cultivable microbial dental plaque flora and salivary flora, analyses were focused on bacterial groups associated with gingivitis/periodontitis and dental caries. Furthermore, the presence of staphylococci, gram-negative enteric bacteria and yeasts in saliva were evaluated. The minimal inhibitory concentration (MIC) was determined for isolates belonging to the predominating micro-organisms in samples of both dental plaque and saliva. In relation to the findings in the placebo group, the use of delmopinol during the rinsing period did not produce an undesirable shift in the bacterial populations considered to be related to dental caries or periodontal diseases. These groups remained virtually unchanged during the study. In relation to the observations in the placebo group, slight reductions in the total cultivable plaque and salivary flora were observed during the study and no change was found in the ratio total anaerobically/aerobically cultivable microbial flora. Furthermore, no increased growth in staphylococci, enteric bacteria or yeasts was observed in the saliva samples. The pattern of changes taking place in the composition of the plaque and salivary microbial flora in samples from the participants rinsing with chlorhexidine were in most aspects similar to that observed in the delmopinol group. In the delmopinol group, no microbiologically significant changes were observed over time in the MIC-values for the isolates, neither in the plaque nor in the saliva samples, which indicates that no adaptation to delmopinol had taken place during the rinsing period. Similar observations were made for the plaque isolates in samples from the participants in the chlorhexidine group. On the other hand, when gram-positive and catalase-negative cocci from the saliva samples of the latter group were tested against chlorhexidine, 4-6 times higher MIC-values were obtained at 3 and 6 months both when compared to baseline and in comparison with the other two rinsing groups (p<0.01 or p<0.05). Neither delmopinol nor chlorhexidine showed any residual effect on the studied microbial groups in the plaque and the saliva samples 3 months after the end of treatment. In conclusion, delmopinol was accompanied by a composition of the plaque and salivary flora associated with healthy conditions in the oral cavity.

Delmopinol hydrochloride- and chlorhexidine digluconate-induced precipitation of salivary proteins of different molecular weights.

Gel electrophoresis was used to analyze precipitates formed of delmopinol hydrochloride or chlorhexidine digluconate mixed with unstimulated whole saliva samples from five test subjects. Final concentrations of delmopinol (6.4 mM) or chlorhexidine (6.4 mM, 2.2 mM) mixed with whole saliva were incubated for 10 min at 37 degrees C. The precipitates were pelleted by centrifugation and resuspended to a similar protein density. The protein patterns in the pellets were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using 12.3% gels. The amount of pellet protein was determined by densitometry in four molecular weight ranges (10-21.5, 21.5-26, 26-45, and 45-300). The results indicated that high molecular weight (45-300) proteins dominated in the precipitate and that 2.2 mM chlorlhexidine precipitated more salivary protein than 6.4 mM. At equimolar concentration (6.4 mM) delmopinol precipitated more high molecular weight salivary proteins than chlorhexidine.

Effect of delmopinol hydrochloride mouthrinse on plaque formation and gingivitis in "rapid" and "slow" plaque formers.

The aim of the present study was to investigate differences in the plaque and gingivitis inhibiting effect of delmopinol rinsing between "rapid" and "slow" plaque formers. 23 subjects (12 "rapid" and 11 "slow" plaque formers) were selected from 71 healthy young adults. The selection was based on the plaque index on the buccal surfaces of all premolars and 1st molars after 3-days without plaque control. The 23 subjects were randomly assigned into 3 groups with different mouthrinses, i.e., 0.1% delmopinol, 0.2% delmopinol, and placebo. The study was double-blind with parallel design between the "rapid" and "slow" plaque formers and cross-over design between 2 active periods and a placebo period. Each rinsing period lasted for 5 days. During the 3 test periods, the subjects refrained from all oral hygiene and rinsed 2x daily with either one of the 3 solutions. Gingival crevicular fluid (GCF) was collected from buccal surfaces of upper canines and premolars and bleeding on probing (BOP) recorded at 6 sites around each tooth before and after each test period. Plaque assessment, including plaque index (PI) and standardized color slides for planimetric analyses obtained from the canines and premolars, were only recorded after each test period. Results showed that the mean PI and planimetry values for both the "rapid" and "slow" plaque formers were lower than the placebo, for either the 0.1% or the 0.2% delmopinol mouthrinse. The differences between the" rapid" and "slow" plaque formers were not statistically significant. There was a small reduction in BOP in both groups for the delmopinol periods, as against a slight increase in the placebo period; the difference between the placebo group and the 2 groups of plaque formers was not statistically significant (p>0.6 for both 0.1% and 0.2% delmopinol). Results suggested that both 0.1% and 0.2% delmopinol reduce plaque formation and gingivitis to a similar extent in subjects with extreme rates of plaque formation.

Delmopinol interactions with cell walls of gram-negative and gram-positive oral bacteria.

The main purpose of the present study was to investigate the influence of delmopinol hydrochloride on the cell surface morphology of gram-negative and gram-positive bacterial cells by using transmission electron microscopy. A second purpose was to evaluate the extraction of cell wall material caused by delmopinol and the binding of radiolabelled delmopinol to the various strains. Fresh isolates and type strains of gram-negative rods associated with periodontal disease, Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and strains of the gram-positive streptococci Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius, were exposed to 3.2 mM (0.1%) or 6.4 mM (0.2%) delmopinol hydrochloride from 1 to 90 min. For electron microscopy the cells were fixed and negatively contrast-stained. Treatment with 6.4 mM delmopinol for 1 min resulted in marked ultrastructural changes of cell wall components and the outer cell membrane of the 3 gram-negative species compared with control cells, whereas the gram-positive streptococci treated with delmopinol showed little or no morphologic alteration as compared with untreated cells. The result from the electron microscopy was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins released from bacterial cells treated as for electron microscopy. More material was extracted from the gram-negative rods than from the gram-positive cocci. Significantly more delmopinol bound to the gram-negative rods than to the streptococci. It appears that the amphiphilic properties of delmopinol make gram-negative rods more vulnerable to delmopinol than gram-positive streptococci.

The binding of delmopinol and chlorhexidine to Streptococcus mutans and Actinobacillus actinomycetemcomitans strains with varying degrees of surface hydrophobicity.

This study evaluated the binding of chlorhexidine and the new surface-active anti-plaque agent delmopinol hydrochloride to Streptococcus mutans and Actinobacillus actinomycetemcomitans cells with various cell surface hydrophobicities. The influence of saliva concentration on the binding of these compounds was also investigated. The radiolabeled compounds were incubated with bacteria and the cells were recovered using a centrifugal filtering technique. Delmopinol had higher binding to the hydrophilic variant strains than to the hydrophobic parent strains; chlorhexidine had higher binding to hydrophobic than to hydrophilic A. actinomycetemcomitans strains and higher binding to hydrophilic than to hydrophobic S. mutans strains. The presence of salivary films decreased the binding of both compounds. Both delmopinol and chlorhexidine had stronger affinity to A. actinomycetemcomitans cells than to S. mutans cells. At equimolar concentrations, delmopinol had a lower binding to all strains tested than chlorhexidine. The high reversibility of the delmopinol binding might be related to a higher diffusion rate and solubility compared with that of chlorhexidine. The amphiphilicity of both molecules is an important feature in their retention to S. mutans and A. actinomycetemcomitans strains of varying hydrophobicities and could play an important role in the substantivity of delmopinol or chlorhexidine in the oral cavity.

Agglutination of Streptococcus mutans serotype C cells but inhibition of Porphyromonas gingivalis autoaggregation by human lactoferrin.

The ability of various forms of human lactoferrin (LF) to agglutinate oral Streptococcus mutans, Strep. sobrinus, Strep. rattus, Strep. sanguis, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans cells was studied spectrophotometrically. Fe3+ saturated LF was unable to agglutinate these bacteria, whereas iron-free LF (apo LF) effectively agglutinated Strep. mutans cells but not the other bacteria. The efficiency and rate of agglutination of Strep. mutans were somewhat lower with apo LF than with human whole saliva. However, secretory IgA, phosphate and whole saliva almost totally abolished the apo LF-mediated agglutination of Strep. mutans, suggesting binding to the same target sites on bacterial cell surfaces, or to each other. The presence of exogenous iron (Fe2+, Fe3+), lactoperoxidase or serum albumin did not affect the agglutination by apo LF. Low Ca2+ (50-100 microns) slightly enhanced the agglutination by apo LF but higher concentrations (0.5-1.0 mM) totally blocked the apo LF-mediated agglutination of Strep. mutans. Both saliva and apo LF significantly delayed the rapid autoaggregation of P. gingivalis cells. Aggregation of P. gingivalis is considered a potential virulence factor and a protective mechanism against the host's cellular defences in the gingival crevice. These findings show a novel, strain-specific antibacterial mechanism for LF against Strep. mutans and P. gingivalis and adds a new compound to the group of agglutinating proteins in human saliva.

In vitro interactions of delmopinol hydrochloride with salivary films adsorbed at solid/liquid interfaces.

Delmopinol hydrochloride is a highly surface active substance which has been shown to reduce the amount of plaque in vitro and in vivo and the level of gingivitis in vivo. Ellipsometry was used to evaluate the effect of delmopinol on films formed from whole, parotid and sublingual/submandibular saliva on solid surfaces. Hydrophilic plasma cleaned silica and methylated hydrophobic substrates were used. The adsorption processes of the salivary proteins and their interactions with delmopinol were monitored in situ with a Rudolph thin-film ellipsometer. The adsorption of delmopinol on bare substrates with out previous salivary adsorption was also investigated and the results indicate that delmopinol molecules were adsorbed on both hydrophilic and hydrophobic surfaces. When delmopinol interacted with the films formed from the different types of saliva an initial increase in the adsorbed amounts was observed in the majority of the cases indicating binding to the salivary pellicles and/or substrate surface. The noted increases were largest for the films adsorbed from whole and sublingual/submandibular saliva. After rinsing in the system, partial desorption of the salivary films took place. It is thus indicated that delmopinol binds to salivary proteins within the pellicles and alters the cohesive and adhesive properties of these films.

Effect of delmopinol on the cohesion of glucan-containing plaque formed by Streptococcus mutans in a flow cell system.

Glucan-containing plaque was formed by Streptococcus mutans adhering to saliva-coated glass slides in flow cells thermostated at 37 degrees C. The substrate was Brain Heart Infusion broth containing 1% sucrose and 10% sterile saliva. During the build-up of the plaque, which lasted for 29 h, the plaque was subjected to three two-minute exposures to either 0.1 mol/L sodium acetate buffer, pH 6.0, or the same buffer containing 6.4 mmol/L (0.2%) of the surface-active anti-plaque substance delmopinol hydro-chloride. The glass slides carrying the plaque were weighed, and plaques subjected to delmopinol treatment weighed only seven percent of the control plaques. The glass slides were then mounted in a beaker containing buffer, subjected to ultrasonication, and re-weighed. The delmopinol-treated plaques lost 59% of their wet weight upon sonication, while the controls lost only 19%. Control plaques having the same weight as delmopinol-treated plaques were not different from the control plaques grown for 29 h with regard to reduction of plaque weight after sonication. Transmission electron micrographs (TEM) showed a plaque dominated by globular or fibrillar matrix components in controls, while the delmopinol-treated plaque showed empty or unordered matrix areas between more densely packed cells. The TEM results were confirmed by scanning electron micrographs, which showed amorphous material associated with the bacterial cells in the control but not in the delmopinol-treated plaque. In conclusion, delmopinol reduced surface-associated glucan synthesis and lowered the cohesion of the plaque, indicating that glucan-containing plaque formed during repeated rinsings with delmopinol may be easier to remove by mechanical means than a non-treated plaque of this type.

In vitro interactions of anionic and cationic surfactants with salivary fractions on well-defined solid surfaces.

Ellipsometry was used to study the interaction of one anionic (SDS) and one cationic (CTAB) surfactant with films adsorbed from six different salivary fractions obtained after fractionation of whole unstimulated saliva on a Superdex 200 Hiload gel filtration column. Experiments were performed on both hydrophilic silica and hydrophobic methylated silica surfaces. The results of this study indicate that the adhesive and cohesive properties of the films adsorbed from the individual fractions were strongly dependent on the surface characteristics of the substrates and that the outcome of protein/surfactant interactions was dependent on factors such as protein composition, surfactant charge, and substrate characteristics. These interactions probably involve replacement of the adsorbed proteins by surfactants or protein/surfactant complex formation. The anionic surfactant seemed to be more efficient in removing adsorbed salivary proteins than the cationic one.

Effect of 4 days of mouth rinsing with delmopinol or chlorhexidine on the vitality of plaque bacteria.

Delmopinol is a new surface active anti-plaque agent that has demonstrated a low antimicrobial effect in vitro. By use of a vitality staining technique, the antimicrobial effect on bacteria in plaque samples was tested after rinsing with delmopinol or chlorhexidine. 6 healthy male subjects volunteered to rinse for 4 days using a double-blind cross-over study design with a wash-out period between the rinsing regimens. No oral hygiene measures were allowed during the test periods and each test period started with a professional tooth cleaning procedure 2 days before the start of rinsing to allow for plaque formation. Rinsing was performed with 0.2% delmopinol hydrochloride or 0.2% chlorhexidine digluconate 2 x a day. Small samples of plaque were collected from the buccal surfaces of premolars and 1st molars before the first rinse on day 1 and then before and 1, 2, 4, 7, and 24 h after the last rinse on the 4th day. The plaque samples were immediately stained with propidium iodide and fluorescein diacetate to visualize dead and vital microorganisms respectively. The vitality of the microflora was evaluated using a fluorescence microscope. The baseline vitality values were 91% for chlorhexidine and 86% for delmopinol. At day 4, the plaque vitality for chlorhexidine was approximately 40% up to 4 h and 50% at 7 h and 60% at 24 h after the last rinse. Corresponding values for plaque vitality after delmopinol rinsing were between 70 and 80% on all sampling occasions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of delmopinol on the viscosity of extracellular glucans produced by Streptococcus mutans.

The surfactant delmopinol, which is a new antiplaque agent with a low anti-microbial profile, was tested for its effects on the viscosity of bacterial extracellular glucans. Glucans were isolated from Streptococcus mutans broth supernatants incubated with 0.15 M sucrose in 50 mM sodium phosphate buffer at pH 6. The viscosity was measured in a shear rate range from 15 to 230 reciprocal seconds. The viscosity of the water-soluble glucan was found to be independent of shear rate whereas the water-insoluble glucan showed a strong shear thinning. The addition of delmopinol to preformed glucans did not affect the viscosity nor the shear rate dependence of the glucans. However, when present during synthesis of the polysaccharides, delmopinol was found to reduce the viscosity of both water-soluble and water-insoluble glucans by approximately 50% at the shear rates investigated. The reduction in viscosity for the water-soluble glucans was obtained at a delmopinol concentration of 0.32 mM (0.01%) and for the water-insoluble glucans at 3.2 mM delmopinol. The observed reduction of viscosity of glucans indicates that the in vivo stability of plaque matrix after delmopinol treatment would be lowered, which may lead to a reduction of plaque cohesion and thus facilitate mechanical plaque removal.

Adsorption from salivary fractions at solid/liquid and air/liquid interfaces.

Ellipsometry and the drop-volume technique were used to study the interfacial behaviour of fractions obtained from unstimulated whole saliva. Fractionation was by gel filtration on a Superdex 200 Hiload column equilibrated with 10 mM potassium phosphate buffer, pH 6.8, containing 0.15 M NaCl. The fractions were reconstituted to have the same absorbance at 215 nm (estimated molecular-weight range, F1 greater than 760-460 K, F2 205-39 K, F3 14-4.5 K, F4 4.5-2.5 K, F5 1.5-0.85 K, F6 0.85 less than or equal to 0.5 K). The fractions were analysed for amino acid composition and studied by hydrophobic interaction chromatography on a Phenyl-Superose column. Fraction 3 contained the largest amounts of proline, followed by fractions 4 and 2. Fraction 3 showed the highest relative hydrophobicity. Ellipsometric measurements on negatively charged silica surfaces and methylated hydrophobic surfaces revealed that larger amounts of material adsorbed on hydrophobic than on hydrophilic surfaces. On hydrophilic surfaces the largest amounts were adsorbed from the high molecular-weight fraction 1. Fractions 4 and 6 did not give any adsorption at all on these surfaces. Fraction 3 gave the largest amounts adsorbed on the hydrophobic surfaces. Drop-volume measurements showed distinct differences in the ability of the salivary fractions to lower the surface tension. Fractions 2 and 3 showed the greatest reduction in surface tension. It was concluded that the adsorption behaviour of salivary proteins showed a wide variation among the different fractions and that it is influenced by the physicochemical characteristics of the interfaces present in the mouth.

Effect of whole saliva on the rheologic behavior of extracellular water-soluble glucan produced by Streptococcus mutans.

The viscosity of mixtures of Streptococcus mutans water-soluble glucan and stimulated whole saliva or buffer was measured at pH 5, 6, 7, and 8. The viscosity was measured as a function of shear rate in the range 15 s-1-230 s-1. Though the centrifuged saliva had a viscosity close to that of water it increased the viscosity of the glucan up to 65% at pH 6 and 55% at pH 7 and at a shear rate of 20 s-1, indicating an interaction between saliva components and glucan that could be an important part of the cohesive forces of plaque matrix. The interaction between saliva and glucan was less pronounced at pH 5 and 8, which indicates a charge-dependent interaction. The viscosity increase at pH 6 and 7 was higher at low than at high shear rates, suggesting a higher contribution to plaque stability when weak as opposed to high mechanical forces are exerted on the plaque.

Effect of delmopinol on in vitro dental plaque formation, bacterial acid production and the number of microorganisms in human saliva.

This study investigated the effect of a surface active compound, delmopinol, on plaque formation and established plaque in vitro, on the bacterial acid production from glucose and on the total viable bacterial counts in saliva. The antimicrobial effect was compared with that of chlorhexidine. The plaque-inhibiting effect was evaluated in an artificial mouth system, and the effect on bacterial acid production was registered as a decrease of pH in bacterial suspensions with various concentrations of delmopinol. It was shown that delmopinol is able to prevent plaque formation, to dissolve established plaque in vitro, and that it has 5-125 times higher minimum inhibitory concentrations than chlorhexidine. Saliva samples collected 1 min after rinsing with delmopinol showed on differences in the total number of bacteria in saliva as compared with controls. It was also shown that the bacterial acid production from glucose was reduced successively with increasing concentrations of delmopinol. The results indicate that delmopinol might be as effective as chlorhexidine against plaque formation and that delmopinol is capable of penetrating established plaque, thus promoting a more effective mechanical cleansing.

Biological qualities of saliva sterilized by filtration or ethylene oxide treatment.

Stimulated whole saliva was centrifuged and sterilized either by filtration in the presence or absence of dithiothreitol (DTT) or by ethylene oxide treatment. Filtration was accomplished by 2 different techniques: conventional positive pressure filtration through prefilters and membranes with decreasing pore sizes and tangential flow microporous filtration. The sterile saliva samples were analyzed for enzyme activities of phosphatases, esterases, glucuronidase, and lysozyme. The samples were also tested by polyacrylamide electrophoresis, isoelectric focusing and for IgA concentration. Moreover, the samples were tested for support of bacterial growth of strains from the genera Actinomyces, Lactobacillus, Neisseria, Rothia and Streptococcus after the addition of glucose at a final concentration of 0.5%. The samples treated with ethylene oxide appeared to be more affected by this treatment than samples treated otherwise, as demonstrated by the electrophoretic analyses and analyses of enzyme activities. Ethylene oxide-treated saliva also was the least favorable saliva sample to support growth of bacteria. The positive pressure technique gave more rapid filtration than the tangential flow technique. DTT treatment of saliva facilitated filtration and, in contrast to ethylene oxide treatment, it affected the qualities of saliva only to a minor extent compared with the original saliva sample.