The importance of the composition of saliva as a factor influencing dental caries has repeatedly attracted the attention of investigators studying this disease." 2 Since the demonstration that dental caries is unquestionably of an infectious nature, interest in salivary components that serve as essential metabolites and growth factors for members of the oral flora is especially apropos. Such contributing compounds as amino acids and vitamins have been identified in saliva. 4-10 However, to date, the metabolism of inorganic nitrogen in saliva (by the oral flora) has received little attention. The fact that both nitrate" and ammonia2 -nitrogen in its most reduced state and assumed to be the final product in the reduction of nitrate 3-have been reported to occur in saliva suggests that the reduction of salivary nitrate might play a role in bacterial growth in the oral cavity. It is generally believed that inorganic nitrogen, only in the form of ammonia, can be synthesized into organic nitrogen compounds; 14 hence the reduction of nitrate by saliva may possibly lead to the assimilation of nitrogen. At the same time it is possible that alternately, or in addition, nitrate and/or one of its reduction compounds may serve as hydrogen acceptors in reactions concerned with the energy metabolism'5 of certain oral bacteria. On the other hand, salivary nitrate may act as a hydrogen acceptor in place of a usual substance and thereby interfere with a normal energy yielding reaction"' of the oral flora. This paper describes some preliminary observations of the reduction of endogenous and added nitrate by whole saliva, as well as the presumptive reduction of the resulting nitrite. However, the reduction product(s) of nitrite have not, at this time, been identified in saliva. EXPERIMENTAL METHODSThe saliva examined in this study was obtained by paraffin stimulation and was collected in test tubes immersed in crushed ice. The saliva was maintained in the ice bath until it was used, this period usually being less than 1 hour.The nitrate in saliva was determined by the method described by Whelan'7 and was deproteinized for this analysis according to this author's recommendation for plasma, serum, and ascitic fluid. The method is based on the oxidation of colorless diphenylbenzidine by nitric or nitrous acid to its intensely blue quinoidimonium salt. Although nitrite, as well as nitrate, gives the characteristic blue color of oxidized diphenylbenzidine, the method is quantitative for both nitrate and nitrite. Parallel nitrite analyses were made with each nitrate determination whenever absolute values for nitrate were desired.
SYNOPSIS IN INTERLINGUA EFFECTO ANTICARIOGENE DE UN SOLUTION DE MONOFLUOROPHOSPHATO DE NATRIUM IN JUVENILES POST CURSOS DE APPLICATION DE VINTI-UN MENSES.—Un gruppo de 263 juveniles de etates de inter 6 e 14 annos e habitante un area a aqua fluorurate esseva dividite in duo gruppos de 129 e 134 individuos. Le juveniles in gruppo I continuava lor usual methodo de brossar lor dentes supplementate per le uso, un vice per die, de un solution de 6 pro cento de fluoruro in un preparation a aroma e a coloration rosa. Le subjectos in gruppo II recipeva le mesme instructiones, sed le solution in lor caso esseva chloruro de natrium. Le conclusiones del studio es basate super 77 juveniles usante fluoruro e 75 usante chloruro. Es presentate plur indices reflectente le plus favorabile resultatos obtenite in gruppo I. In gruppo II le reversiones esseva 66 pro cento plus frequente que in gruppo I.
The fundamental objective of many experimental investigations is to characterize and measure the relationships between independent and dependent variables.* Usually, in the systems studied, a large number of factors (independent variables) influence the dependent variable. The customary approach to this type of problem has been to determine the effect of each independent variable one at a time while holding the other independent variables constant or to perform a set of experiments composed of specific combinations of the independent variables. These two techniques are known as the "univariate"' and "factorial" methods,2 3 respectively. However, the results so obtained are, in most cases, dependent on the conditions at which the other independent variables are held constant. One should be cognizant of these interactions when evaluating the results of such an experiment in which the univariate method is utilized. A more accurate appraisal of the interrelationships can be obtained if the effects of several independent variables are investigated simultaneously and in such a way that it is possible to estimate the specific effect of each factor, at different levels of each of the other factors considered in the investigation. Although techniques such as the univariate method and the factorial methods are capable of measuring these effects and interactions, they necessitate an experiment of considerable magnitude. In the last few years, however, a methodology that permits the experimental determination of the effects of several variables simultaneously and subsequent approximation of their response surface has been developed.t This technique has been described as the method of steepest ascent and was conceived and developed by G.E.P. In general, the procedure is as follows: first, the method of steepest ascent is used to determine the conditions for a maximal (or minimal) response of the dependent
Microbiological assay techniques' 2 have proved valuable in the examination of saliva for vitamins and amino acids.3 4 5 Inasmuch as the interest in saliva is in its content of free stimulatory compound the application of these analytical methods did not appear to present any particular problems in regard to special preparatory treatment of the samples.5 It has therefore been customary to use the more or less standard and proved assay procedures.3 8 However, those who have used microbiological assay methods readily appreciate that small variations in procedure may sometimes exert profound effects upon the response of the test micro-organism.With these considerations in mind some of the initial technical steps of the microbiological assay were examined in an effort to improve the application of this method of analysis to saliva. By the very nature of the test, any modification that does not violate the principles of the microbiological assay but which will result in a greater growth response of the test micro-organism lends improvement to the procedure. These considerations relate to the recognition and elimination of artifacts which are likely to develop as the result of manipulating any biological material and which must be appreciated when evaluating a study of such material.The results given in this report are not presented in terms of a microbiological assay but rather in terms of growth response. It should be noted, however, that growth curves may be used as typical assay results by comparing growth responses at any given interval. To evaluate the assay procedure the effect of autoclaving and dilution on the growth response to saliva and its crude fractions has been examined. EXPERIMENTAL METHODSThe test micro-organism used was Lactobacillus case, strain L652, originally obtained from the Zoller Dental Clinic, University of Chicago. Stock cultures were maintained on slants of tomato juice-yeast extract medium.The double-strength assay medium employed was of the following composition: glucose, 20 gm.; casamino acids,t 10 gm.; L-cystine, 20 mg.; L-tryptophane, 10 mg.; L-asparagine, 50 mg.; adenine, 20 mg.; guanine, 20 mg.; xanthine, 20 mg.; uracil, 20
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