For investigating functional effects of saliva, filtration has been widely, and without much critical evaluation, adopted as a standard method whenever sterile saliva is needed. With the current advances in protein sterilization by gamma irradiation, developed largely in the food and medical industries, this technique deserves to be reevaluated for the sterilization of whole human saliva. The results of the present study show that radiation doses can be delimited that will sufficiently inactivate bacterial viability in saliva but will not considerably influence salivary protein composition, integrity, or function. In comparison, filtration caused not only a substantial decrease in overall protein concentration but also a selective loss of certain proteins. Thus, depending on the intended experimental purpose, gamma irradiation sterilization of saliva could in certain instances be preferable to filter sterilization.
Irradiation doses of 3.5 kGy, which were found to be sufficient for suppressing bacterial growth in salivary samples, caused virtually no protein denaturation, as seen by the electrophoretic banding pattern, and only a modest decrease in enzymatic activities. However, not all proteins appeared to be equally sensitive to the effects of radiation, as evidenced by the much higher radio-tolerance of lysozyme compared to amylase. On one hand, this may be due to intrinsic structural differences that endow a given protein with less sensitivity to radiation. On the other hand, it could be that more-dilute salivary proteins, exemplified by lysozyme (80 μg/ml in whole saliva [15
]), tend to be less affected by radiation damage than those which occur at a relatively higher concentration, i.e., salivary amylase (650 to 800 μg/ml in parotid saliva [2
]). The window of radiation dosage sufficient for achieving sterility of saliva without causing significant damage to salivary proteins can be estimated to range from 3 to <5 kGy and is similar to what was found in a previous study that used electron beam radiation derived from a linear accelerator. The disadvantage of the latter method is the long time required to accumulate the desired doses necessary for sterilization (10 h for 2.5 kGy) (40
), whereas less than 20 min of exposure to the cobalt-60 source used in the present study was required for sterilizing the salivary samples. Such short irradiation periods also allow for better control of collateral heat generation throughout the irradiation process, because the samples are kept in an ice bath or exposed in a frozen state to the radiation source (47
Filtration reduced the total protein concentration by about one-half. This confirmed the findings in a previous report in which a similar decrease in protein concentration was found (40
). In that previous study, only a pore size of 0.45 μm was used, whereas in the present investigation, the most significant decrease in protein concentration was observed already by use of a much greater filter pore size of 5.0 μm. It is unlikely that the effect is solely due to protein being trapped by bacteria, as had been suggested earlier (38
), because for microbial reduction the most significant drop was observed only after filtration with a 10-fold-smaller pore size of 0.45 μm (Fig. ). Since a pore diameter of 5.0 μm is above the average size of a single salivary protein by several orders of magnitude, this suggests that a large part of protein in whole saliva is organized in very large multicomponent protein aggregates. Indeed, formation of supramolecular complexes involving salivary mucins has been reported (29
). How those supramolecular structures are related to salivary micelle-like globules (48
) or the recently discovered exosomes in saliva (10
), and also what role bacterial aggregates play, deserves further investigation. The selective loss of lysozyme after filtration with the 5.0-μm pore size is perplexing as well and raises the question as to how such a small molecule could be filtered out by a comparatively gigantic pore size. Selective binding of lysozyme, which has a positive net charge, to the negatively charged cellulose acetate membrane of the filter may occur but is unlikely to account for the drastic drop to only 10% of its original activity, since the membrane's binding capacity will be saturated while saliva still continues to percolate. It is also possible that lysozyme is bound to larger bacterial aggregates retained in the 5-μm filter, but an alternative explanation could be that it is bound to protein aggregates too large to pass the 5-μm pore size of the filter. In this regard, it was suggested previously that the supramolecular salivary mucin matrix is decorated with protective factors which also include lysozyme (15
). Indeed, in human bronchial secretions, lysozyme shows a strong ionic interaction with mucins (4
If a sterile saliva is wanted that most closely resembles the true in vivo oral fluid, sterilization of saliva by gamma irradiation is preferable over filtration because it alters the original composition and biological activity the least. This type of fluid will still contain particulate matter and bacterial remnants as they were present under in vivo conditions. The question ultimately will be whether this is appropriate for the intended experimental purpose. It will also be necessary to decide for each experimental setting, depending on the bioburden of the saliva and duration of the experiment planned, how high a sterility assurance level will be needed versus how much of the original biological or enzymatic activity needs to remain. This will eventually determine the dose of radiation to which a particular sample has to be exposed. If filtration is chosen to sterilize saliva, one has to be aware that the resulting fluid will be significantly altered in its composition, particularly if salivary proteins are of importance. Fortunately, difficulties encountered in the past when sterilizing saliva by gamma irradiation, including the scarcity of conveniently available and powerful Co60 sources, can now be overcome by using specially designed industrial facilities for irradiation that achieve safety standards and system requirements set out by the Nuclear Regulatory Commission.