Integrity of Proteins in Human Saliva after Sterilization ... · Department of Operative Dentistry and Periodontology, Dental School,2 Institute of Medical Microbiology and Hygiene,3

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2011, p. 749–755 Vol. 77, No. 30099-2240/11/$12.00 doi:10.1128/AEM.01374-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Integrity of Proteins in Human Saliva after Sterilizationby Gamma Irradiation�

Stefan Ruhl,1,2* Pereshia Berlenbach,2 Sabine Langenfelder,2 Dagmar Horl,2 Norbert Lehn,3Karl-Anton Hiller,2 Gottfried Schmalz,2 and Helmut Durchschlag4

Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York,1 andDepartment of Operative Dentistry and Periodontology, Dental School,2 Institute of Medical Microbiology and Hygiene,3

and Institute of Biophysics and Physical Biochemistry,4 University of Regensburg, Regensburg, Germany

Received 9 June 2010/Accepted 1 December 2010

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., whenutilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gammairradiation for its suitability to sterilize saliva and to investigate the treatment’s influence on the compositionand integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth bygamma irradiation, a sterility assurance level of 10�6 was determined to be reached at a dose of 3.5 kGy. Atthis dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoreticbanding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged.Filtration reduced the total protein concentration to about half of its original value and decreased lysozymeactivity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its nativeform.

Saliva, a complex biological fluid, is essential for the pro-cessing of food and maintenance of health in the oral cavityand the upper digestive tract (20). Many of these beneficialfunctions are mediated by the numerous proteins present insaliva (12, 41) that also modulate the microbial colonization ofthe mouth (32, 35). However, in vitro functional studies involv-ing human whole mixed saliva, the fluid that actually is presentin the mouth in vivo, are often hampered by the presence ofbacteria shed from resident oral microbial biofilms (1).

There have been longstanding and continuous efforts topurge saliva of viable microorganisms for various experimentalpurposes, such as its use as a substrate for bacterial or fungalgrowth (5, 6, 18, 22, 39), studies of microbial adhesion to toothor biomaterial surfaces (3, 23–25, 28, 30), bacterial coaggrega-tion (17), or biofilm formation (9, 27), measurement of anti-microbial activities (14), transformability of oral bacteria withDNA (21), evaluation of tissue regeneration or host responsein the presence of saliva (11, 13, 45), oral hygiene producttesting (19), and even for preparing autologous saliva as asupplement during radiotherapy for head and neck cancer(40).

Methods used to sterilize saliva include filtration, pasteur-ization, gamma irradiation, and UV irradiation, as well ashydrogen peroxide, ethylene oxide, or chlorhexidine treatment.The relative efficacies of most of these methods have beenevaluated by Williams and Kraus (46). Filtration has beendeemed to be preferable (16) and is now the most commonlyused method (9), even though it has been reported that the

amount of total salivary protein as well as enzyme activities canbe decreased by filtration (16, 40, 46).

In order to achieve sterilization of saliva with minimum lossof components or alteration of their functional activities,gamma irradiation, which has been increasingly employed forpreservation of foodstuffs, medical products, pharmaceuticals,cosmetics, and sterile packaging materials (47), deserves to beevaluated as a means of sterilizing saliva. Cobalt-60 is typicallyused as a source of gamma rays to ionize chemical bonds.There are two possible mechanisms of action in the destructionof microorganisms by gamma irradiation. One is the irrevers-ible damage to critical biomolecules of the bacterium, mostspecifically, the DNA. The other way, presumably more effec-tive, is the ionization of water molecules present in and aroundthe bacteria, which produces free radicals that attack macro-molecules (47). We hypothesized that irradiation of salivawould be a valid alternative to filter sterilization, provided thatthe radiation doses could be delineated that kill bacteria butleave salivary proteins unaffected. Thus, in the present inves-tigation, the influence of gamma irradiation sterilization onprotein integrity was studied and compared to filter steriliza-tion.

MATERIALS AND METHODS

Saliva collection. Subjects’ rights were protected by review through the EthicsCommittee of the Medical Faculty of the University of Regensburg, and in-formed consent was granted. Whole saliva was obtained separately from threedifferent individuals by expectoration into sterile 50-ml polypropylene vials (BlueMax, Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) with continuousstimulation of salivary flow through chewing of paraffin-like material (parafilm;Pechiney Plastic Packaging Company, Chicago, IL). Prior to collection of saliva,donors rinsed their mouth with water and refrained from eating or drinking forat least 2 h. For each irradiation and filtration experiment, 20 to 35 ml of salivawas freshly collected from each donor. All saliva samples were kept on ice duringcollection and throughout all further experimental procedures. To allow forprecise pipetting, the viscosity of saliva was broken by repeatedly drawing the

* Corresponding author. Mailing address: Department of Oral Bi-ology, School of Dental Medicine, State University of New York atBuffalo, Buffalo, NY 14214. Phone: (716) 829-6073. Fax: (716) 829-3942. E-mail: [email protected].

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samples through a sterile blunt needle (0.9 by 22 mm; Miraject, Duisburg,Germany) fit to a sterile 5-ml syringe (Luer-Lok; Becton Dickinson, Droghede,Ireland). After completion of the experimental treatments, aliquots of saliva formicrobial counting were plated onto culture plates, and aliquots for gel electro-phoresis were denatured in SDS sample buffer. Samples for analysis of proteinintegrity were frozen and stored at �80°C. For later use in the enzymatic activitytests, samples were slowly thawed at 4°C overnight. During the assays, sampleswere kept on ice at all times to preserve the activities of the enzymes. Totalprotein concentrations were determined with the use of the bicinchoninic acidprotein assay reagent (Pierce, Rockford, IL).

Irradiation and filtration. The salivary samples from three separate individ-uals were irradiated by the company Isotron (Allershausen, Germany). Aliquots(1 ml) of each donor’s saliva were kept on ice in sterile 2-ml polypropylenescrew-cap microcups (Sarstedt & Co., Numbrecht, Germany) and exposed todifferent doses of gamma irradiation by use of an industrial-sized cobalt-60source (Pallet Irradiator type JS 9000; Nordion International Inc., Kanata, On-tario, Canada) with an energy output of 1.17 MeV (99.9%) and 1.33 MeV(100%). Samples were placed at certain distances from the gamma radiationsource and were exposed for certain time intervals that had been previouslydetermined as optimal to obtain the calculated doses. Time intervals variedbetween 5 min (1 kGy) and 1 h (12 kGy). Two film dosimeters were mounted toeach set of samples and evaluated later to precisely record the doses actuallyobtained. Nonirradiated control samples were kept under the same conditionsexcept that they were not exposed to the radiation source. For the evaluation ofirradiation to larger volumes of saliva, 20-ml aliquots from each donor wereplaced in sterile syringes, capped, sealed, and exposed to the radiation source atdoses between 3 and 5 kGy as described above.

Filtration was performed under sterile conditions in a laminar flow hood.Saliva from two separate individuals was passed consecutively through sterilesyringe tip membrane filters of decreasing pore size (Minisart CE single-usesyringe filters, hydrophilic; 5.0, 1.2, 0.8, 0.45, and 0.2 �m; Sartorius AG, Gottin-gen, Germany) fit to a sterile 5-ml Luer-Lok syringe (Becton-Dickinson).

Bacterial strains, culture, and colony counting. All microbial cultures grownon plates were incubated for 2 days at 37°C. Deinococcus radiodurans ATCC13939 was cultured on low-salt Luria-Bertani medium containing 5 g/liter glucose(LSLB-Glc) followed by overnight liquid culture in LSLB-Glc medium at 37°Cunder constant motion. Escherichia coli ATCC 25922 was grown on MacConkeyagar (Merck, Darmstadt, Germany), Geobacillus stearothermophilus ATCC 7953was grown on CASO agar (casein-peptone soymeal-peptone agar; Merck, Darm-stadt, Germany), and Candida albicans ATCC 10231 was grown on Albicans ID2agar (bioMerieux Deutschland GmbH, Nurtingen, Germany).

Salivary samples were serially diluted in saline solution, and 50-�l aliquots ofeach sample were plated in triplicate on CASO agar. For spiking experiments,900 �l of saliva was mixed with 100-�l aliquots of suspensions of D. radioduransin saline solution to obtain an estimated final concentration of 106 deinococci perml. For determination of bacterial viability following experimental treatments,samples containing the pure bacterial strains or samples spiked with D. radio-durans were further cultured in their appropriate growth medium as describedabove. Bacterial colonies were counted manually from plates that containedbetween 50 and 100 colonies. For sterility tests, 500-�l aliquots of saliva sampleswere added to sterile blood culture flasks (20 ml; BacT/Alert PF; bioMerieuxDeutschland GmbH, Nurtingen, Germany). From the 20-ml volumes of irradi-ated saliva, 1-ml aliquots were added to triplicate blood culture flasks. Afterincubation for 48 h at 37°C, blood culture flasks were visually evaluated for colorchange, following the instructions of the manufacturer.

Gel electrophoresis. Salivary proteins were denatured under reducing condi-tions, separated by SDS-PAGE (0.75 �g per lane) on 4-to-20% gradient gels(Invitrogen, Karlsruhe, Germany) run under a constant voltage of 125 V for 90min in a vertical gel apparatus (X Cell II minicell; Novex Electrophoresis GmbH,Frankfurt, Germany), and visualized by means of an ammoniacal silver stain kit(SilverXpress; Invitrogen, Carlsbad, CA) or transferred (10 �g per lane) tonitrocellulose with a 0.4-�m pore size (Invitrogen) in a semidry electroblottingapparatus (X Cell II blot module; Novex) as previously described (31). Molecularweight standards, unstained for silver stains and prestained for blotting (BroadRange and Precision Plus; Bio-Rad Laboratories, Munchen, Germany), wereincluded on all gels. Glycoproteins were labeled by a modification of the hydra-zide method as previously described (33) using 10 mM sodium meta-periodate(Fisher, Fairlawn, NJ) for oxidation of sugars and biotin-LC-hydrazide (Pierce,Rockford, IL) together with avidin D-horseradish peroxidase (Vector, Burlin-game, CA) and 4-chloro-1-naphthol (Bio-Rad Laboratories) for detection andstaining, respectively. Gels and blots were scanned with a high-resolution scanner(Sharp color image scanner, model JX-330, fit with a Sharp film scanning unit,model JX-3F6) in the transparent and reflective modes, respectively.

Fluorescence spectroscopy and measurement of circular dichroism. Thirtymicroliters of saliva was mixed thoroughly with 970 �l of 17.5 mM sodiumpotassium phosphate buffer (pH 7.0), and fluorescence excitation and emissionof 1:33.3-diluted saliva samples were recorded with a spectrofluorometer (JobinYvon Spex FluoroMax-2; HORIBA Jobin Yvon GmbH, Unterhaching, Ger-many). A wavelength of 280 nm was chosen for excitation of the intrinsic flu-orophores tryptophan and tyrosine, and 295 nm was used for selective excitationof tryptophan. For evaluation of the emission data, a wavelength of 340 nm wasselected. Circular dichroism spectra of 1:5-diluted saliva samples were recordedin a spectropolarimeter (Aviv 62A-DS circular dichroism spectrometer; AVIVBiomedical, Lakewood, NJ). Spectroscopic data were evaluated and interpretedas described previously (7, 8).

Measurement of enzyme activities. Amylase activity was measured accordingto a modified version of the Wohlgemuth procedure (34). A range of 1:5 to 1:50dilutions of saliva with 17.5 mM sodium potassium phosphate buffer (pH 7.0) wasprepared. Seventy-five microliters was mixed with 300 �l 1% (wt/vol) starch(Merck, Darmstadt, Germany) and 375 �l buffer and kept at a constant temper-ature of 25°C by using a Dri-Block (DB-3; Techne Inc., Princeton, NJ). Thehydrolysis of starch was stopped at 5-min intervals over a period of 25 min byreaction with an iodine-potassium iodide test solution. Absorbance measure-ments at a wavelength of 578 nm were taken at 25°C by using a spectrophotom-eter (UV/VIS spectrophotometer V-530; JASCO GmbH Deutschland, Groß-Umstadt, Germany). Standard curves were obtained by using serial dilutions ofporcine pancreas �-amylase (Sigma-Aldrich, Munchen, Germany). Enzymaticactivity of lysozyme was measured using the method described by Shugar (36).Thirty microliters of saliva either undiluted or diluted 1:3 in 66 mM sodiumphosphate buffer containing 17 mM NaCl (phosphate-buffered saline [PBS], pH7.0) was added to 2,970 �l of a cell suspension of Micrococcus lysodeikticus(Sigma-Aldrich, Munchen, Germany) in PBS (0.2 mg/ml). The decrease in ex-tinction (turbidity) was measured in the spectrophotometer at a wavelength of450 nm for a continuous time period of 3 min at 25°C. Serial dilutions of chickenegg white lysozyme (Serva, Heidelberg, Germany) were used to obtain standardcurves.

Statistical evaluation. Statistical calculations were performed using the soft-ware SPSS PC� version 5.01 (SPSS Inc., Chicago, IL). Mean CFU were calcu-lated from triplicate culture plates. Medians together with the 25% and 75%quantiles for CFU, protein concentrations, and enzyme activities before andafter filtration or irradiation were calculated with the help of the SigmaPlotsoftware (version 8.0; SPSS Inc.) for two donors in a total of five independentexperiments and for three donors in a total of three independent experiments,respectively. The radiation dose that caused a 10-fold (90%) reduction in via-bility (D10) and the sterility assurance level (SAL) in saliva, as defined by areduction to a theoretical value of 10�6 CFU per ml, were calculated by linearregression of values, not including values below the theoretical detection limit.

RESULTS

Determination of irradiation or filtration parameters re-quired for sterilization of saliva. To determine the function-ality and effectiveness of the irradiation procedure, the well-characterized radio-resistant strain D. radiodurans ATCC13939 was used as a control and compared to suspensions of E.coli strain ATTC 25922 and G. stearothermophilus strain ATCC7953 (Fig. 1A). Viabilities of both E. coli and G. stearother-mophilus cells were reduced to below detection limits by irra-diation with doses of 1 kGy and above. Analogous results werefound for C. albicans (data not shown). In contrast, viability ofD. radiodurans was reduced by only approximately 10- to 100-fold, even at doses as high as 10 kGy. When D. radiodurans wassuspended in saliva instead of saline before irradiation, nosignificant differences in viability could be observed, thus ex-cluding the possibilities of a radio-protective or radio-sensitiz-ing effect of saliva suspension on bacterial viability. The declineof D. radiodurans viability was found to be almost linearlyrelated to the increase in irradiation dose (r2 � 0.73 and 0.84in saline and saliva, respectively), showing the reliability of theirradiation method over this wide dose range.

When whole saliva was irradiated, bacterial viability for all

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samples fell below detection limits at doses of more than 2.5kGy (Fig. 1B). Blood cultures from irradiated saliva sampleswere found to be negative at doses of 3.0 kGy and beyond.The cumulative value for the SAL in saliva was calculated to bereached at a dose of 3.54 kGy (Fig. 1B, insert). The D10 valuewas calculated to be an interval of 0.25 kGy in radiation dose.When larger volumes of saliva (20 ml instead of 1 ml) wereirradiated, 5.0 kGy was found to be sufficient for inhibitingbacterial growth, as evidenced by negative blood cultures (datanot shown).

When saliva was filtered successively through membranefilters of decreasing pore size, a 10- to 100-fold decrease inCFU was observed after filtration with pore diameters rangingfrom 5.0 to 0.80 �m (Fig. 1C). A significant drop in CFU tobelow detection limits was found only after use of a filter witha pore size of 0.45 �m. This was confirmed by the use of bloodcultures, which did not detect any residual bacterial growth insamples filtered with pore sizes of 0.45 or 0.20 �m (data notshown).

Effects of irradiation and filtration on integrity and activityof proteins. Salivary proteins were subjected to separation bySDS-PAGE and stained with silver to evaluate a possible effectof irradiation and filtration on protein denaturation and loss ofprotein components, respectively (Fig. 2). At doses up to 5kGy, no significant change of the protein subunits was ob-served. When salivary samples were irradiated with doseshigher than 5 kGy, however, protein bands appeared morediffuse, which is indicative of denaturation of proteins (Fig.2A). This effect became most pronounced at the highest dosesapplied (�10 kGy). Analogous results were obtained whennitrocellulose transfers were stained for glycoproteins by thehydrazide method (data not shown). Similarly, filtrationchanged the pattern of protein bands only slightly. However, atall pore sizes substantial losses of three protein components(approximately 11, 27, and 48 kDa) were observed. The mostpronounced effect was visible after initial filtration with a poresize of 5.0 �m, which resulted in the almost complete loss of alow-molecular-mass component of about 11 kDa (Fig. 2B).The loss of this band was consistently observed with saliva fromdifferent donors and at different sampling times. This effectcannot be accounted for by simple size exclusion of such asmall molecule, because of the large pore size of this filtermembrane. Although the reported molecular mass of humanlysozyme is 14.7 kDa, it is still possible that the protein band atthe molecular range of 11 kDa represents a salivary form oflysozyme. Notably, few alterations in the relative proportion oflarge-molecular-size glycoprotein bands were observed on hy-drazide-stained transfers (data not shown). Furthermore, mea-surement of far UV circular dichroism spectra of the salivaryprobes revealed no changes of the secondary structure (helixcontent) of the involved proteins, after either irradiation or asa consequence of the filtration process (data not shown).

The concentration of total proteins was not influenced byirradiation (Fig. 3A), but filtration through the 5.0-�m-poremembrane resulted in a substantial drop in total proteinconcentration, while subsequent steps with smaller-pore di-ameters had no further effect on total protein concentration(Fig. 3B), in agreement with the results from the SDS-PAGE. Increasing doses of radiation caused a decrease inintrinsic fluorescence by about 30% at 10 kGy (Fig. 3C),

FIG. 1. Effects of irradiation or filtration on bacterial viability.(A) Suspensions of Escherichia coli, Geobacillus stearothermophilus,and Deinococcus radiodurans were irradiated with doses up to 10 kGy.Irradiation was performed in three independent experiments on purebacterial suspensions in saline solution (saline data for D. radiodurans)or on salivary samples from three individuals spiked with D. radio-durans (saliva data). (B) Samples of whole saliva were irradiated withdoses up to 5.1 kGy. Irradiation was performed in three independentexperiments on samples from three individuals (data points are shownas F, Œ, and f). Each data point represents the result from one donorin one experiment. (C) Samples of whole saliva were filtrated in suc-cessive steps with membrane filters of decreasing pore size. Filtrationwas performed in two independent experiments on samples from twoindividuals (F and Œ). Each data point represents the result from onedonor in one experiment. Aliquots of the samples were plated on agar,and bacterial colonies grown after 48 h were counted and calculated asCFU per ml of saliva. The detection limit was determined to be 20CFU per ml of suspension and is indicated as a dashed horizontal line.Data below the limit of sensitivity were not considered for calculationof the D10 value or the SAL (insert in panel B).

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which indicates a decrease in the integrity of the fluoro-phores tryptophan and tyrosine and, thus, suggests thatthese amino acids become degraded at higher doses of ir-radiation. Initial filtration through the membrane with a5.0-�m pore size resulted in about a 50% loss of intrinsicfluorescence but remained unchanged after filtration withsmaller pore sizes (Fig. 3D). This observed decrease mostlikely results from the loss of total protein concentrationrather than from degradation of intrinsic fluorophores be-cause it parallels the drop in protein concentration seen inFig. 3B. The enzymatic activity of amylase was decreased byirradiation to less than 50% of its original level at 10 kGy(Fig. 3E), whereas filtration had no apparent effect on amy-lase activity (Fig. 3F). Interestingly, the opposite was ob-served for lysozyme, in that irradiation had only marginaleffects on enzyme activity whereas filtration reduced ly-sozyme activity to 10% or less of its original level alreadyafter the initial filtration step through the 5.0-�m pore size.

DISCUSSION

For investigating functional effects of saliva, filtration hasbeen widely, and without much critical evaluation, adopted asa standard method whenever sterile saliva is needed. With thecurrent advances in protein sterilization by gamma irradiation,developed largely in the food and medical industries, this tech-nique deserves to be reevaluated for the sterilization of wholehuman saliva. The results of the present study show that radi-ation doses can be delimited that will sufficiently inactivatebacterial viability in saliva but will not considerably influencesalivary protein composition, integrity, or function. In compar-ison, filtration caused not only a substantial decrease in overallprotein concentration but also a selective loss of certain pro-teins. Thus, depending on the intended experimental purpose,gamma irradiation sterilization of saliva could in certain in-stances be preferable to filter sterilization.

Irradiation doses of 3.5 kGy, which were found to be suffi-cient for suppressing bacterial growth in salivary samples,caused virtually no protein denaturation, as seen by the elec-trophoretic banding pattern, and only a modest decrease inenzymatic activities. However, not all proteins appeared to beequally sensitive to the effects of radiation, as evidenced by themuch higher radio-tolerance of lysozyme compared to amy-lase. On one hand, this may be due to intrinsic structuraldifferences that endow a given protein with less sensitivity toradiation. On the other hand, it could be that more-dilutesalivary proteins, exemplified by lysozyme (80 �g/ml in wholesaliva [15]), tend to be less affected by radiation damage thanthose which occur at a relatively higher concentration, i.e.,salivary amylase (650 to 800 �g/ml in parotid saliva [2]). Thewindow of radiation dosage sufficient for achieving sterility ofsaliva without causing significant damage to salivary proteinscan be estimated to range from 3 to �5 kGy and is similar towhat was found in a previous study that used electron beamradiation derived from a linear accelerator. The disadvantageof the latter method is the long time required to accumulatethe desired doses necessary for sterilization (10 h for 2.5 kGy)(40), whereas less than 20 min of exposure to the cobalt-60source used in the present study was required for sterilizing thesalivary samples. Such short irradiation periods also allow forbetter control of collateral heat generation throughout theirradiation process, because the samples are kept in an ice bathor exposed in a frozen state to the radiation source (47).

Filtration reduced the total protein concentration by aboutone-half. This confirmed the findings in a previous report inwhich a similar decrease in protein concentration was found(40). In that previous study, only a pore size of 0.45 �m wasused, whereas in the present investigation, the most significantdecrease in protein concentration was observed already by useof a much greater filter pore size of 5.0 �m. It is unlikely thatthe effect is solely due to protein being trapped by bacteria, ashad been suggested earlier (38), because for microbial reduc-tion the most significant drop was observed only after filtrationwith a 10-fold-smaller pore size of 0.45 �m (Fig. 1C). Since apore diameter of 5.0 �m is above the average size of a singlesalivary protein by several orders of magnitude, this suggeststhat a large part of protein in whole saliva is organized in verylarge multicomponent protein aggregates. Indeed, formationof supramolecular complexes involving salivary mucins has

FIG. 2. Effects of irradiation or filtration of whole saliva on theprofile and integrity of protein bands after SDS-PAGE. Salivary sam-ples were irradiated with doses up to 11.5 kGy (A) or filtrated insuccessive steps with membrane filters of decreasing pore size (B).Gels (two different donors) were stained with silver. Molecularmasses of protein subunits were estimated by comparison to stan-dard proteins (S).

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FIG. 3. Effects of irradiation or filtration of whole saliva on the concentration of proteins (A and B) and integrity of fluorophores (C and D), as wellas enzymatic activities of amylase (E and F) or lysozyme (G and H). Salivary samples were irradiated (A, C, E, and G) with doses up to 10 kGy or filtrated(B, D, F, and H) with membrane filters of decreasing pore size. Irradiation was performed in three independent experiments on samples from threeindividuals (F, Œ, and f). Each data point represents the result from one donor in one experiment. Filtration was performed in five (B) or two (D, F,and H) independent experiments on samples from two individuals (F and Œ). The dashed horizontal line shows the 50% margin.

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been reported (29, 37, 44). How those supramolecular struc-tures are related to salivary micelle-like globules (48) or therecently discovered exosomes in saliva (10, 26), and also whatrole bacterial aggregates play, deserves further investigation.The selective loss of lysozyme after filtration with the 5.0-�mpore size is perplexing as well and raises the question as to howsuch a small molecule could be filtered out by a comparativelygigantic pore size. Selective binding of lysozyme, which has apositive net charge, to the negatively charged cellulose acetatemembrane of the filter may occur but is unlikely to account forthe drastic drop to only 10% of its original activity, since themembrane’s binding capacity will be saturated while saliva stillcontinues to percolate. It is also possible that lysozyme isbound to larger bacterial aggregates retained in the 5-�m filter,but an alternative explanation could be that it is bound toprotein aggregates too large to pass the 5-�m pore size of thefilter. In this regard, it was suggested previously that thesupramolecular salivary mucin matrix is decorated with pro-tective factors which also include lysozyme (15, 43, 44). Indeed,in human bronchial secretions, lysozyme shows a strong ionicinteraction with mucins (4, 42).

If a sterile saliva is wanted that most closely resembles thetrue in vivo oral fluid, sterilization of saliva by gamma irradi-ation is preferable over filtration because it alters the originalcomposition and biological activity the least. This type of fluidwill still contain particulate matter and bacterial remnants asthey were present under in vivo conditions. The question ulti-mately will be whether this is appropriate for the intendedexperimental purpose. It will also be necessary to decide foreach experimental setting, depending on the bioburden of thesaliva and duration of the experiment planned, how high asterility assurance level will be needed versus how much of theoriginal biological or enzymatic activity needs to remain. Thiswill eventually determine the dose of radiation to which aparticular sample has to be exposed. If filtration is chosen tosterilize saliva, one has to be aware that the resulting fluid willbe significantly altered in its composition, particularly if sali-vary proteins are of importance. Fortunately, difficulties en-countered in the past when sterilizing saliva by gamma irradi-ation, including the scarcity of conveniently available andpowerful Co60 sources, can now be overcome by using speciallydesigned industrial facilities for irradiation that achieve safetystandards and system requirements set out by the NuclearRegulatory Commission.

ACKNOWLEDGMENTS

We are grateful to Verena Wittmann and Barbara Kellerer forexcellent technical help and to the employees of Gammamaster Deut-schland GmbH in Allershausen, Germany, particularly Reiner Eiden-berger, Albert Low, and Michael Spies for their friendly assistance inperforming the irradiation experiments. We further thank Frank Scan-napieco and Molakala Reddy for their critical reviews of the manu-script.

This work was supported by Deutsche Forschungsgemeinschaftgrants Ru409/4-1 and SFB 585, B5 (S.R.).

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