J. Tonzetich

Direct gas chromatographic analysis of sulphur compounds in mouth air in man

Abstract A gas Chromatograph equipped with a sensitive photometric detection system was successfully applied to the direct determination of sulphur compounds in mouth air. A measurable amount of sulphur-containing compounds, that is detectable by organoleptic perception, was found in air samples of all 15 subjects studied. Large differences in concentration were observed among subjects and in the individual subjects during different periods of day. The maximum levels correspond to the reported peaks of objectionability of mouth odour. The gas Chromatographic analysis of mouth air revealed the presence of three sulphur-containing compounds. Two of these, which accounted for approximately 90 per cent of the sulphur content, were positively identified as hydrogen sulphide and methyl mercaptan. Both compounds emanate an objectionable putrid odour. The third, a minor odoriferous component, was tentatively identified on the basis of retention time on a Teflon column as dimethyl sulphide. The saliva tests indicate that the same three components are produced during the initial 60—90 min of putrefaction. Since the oral hygiene measures markedly reduced the sulphur content of mouth air, it is concluded that the determined sulphur compounds are produced by a putrefactive process occurring within the oral cavity.

Characterization of volatile sulphur production by pathogenic and non-pathogenic strains of oral Bacteroides.

Marked differences were observed in intermediate sulphur metabolism between non-pathogenic strains of Bacteroides melaninogenicus var melaninogenicus (CP-) and pathogenic Bacteroides melaninogenicus asaccharolyticus (CP+). The CP+ strains, which produced collagenase and protease and caused formation of abscesses when injected subcutaneously into groins of guinea pigs, produced copious amounts of volatile sulphur compounds (VSC) which consisted predominantly of CH3SH and (CH3S)2. Hydrogen sulphide occurred in considerably lesser amounts. CP+ cultures yielded 8-fold more total volatile S, 15-fold more CH3SH and 260-fold more (CH3S)2 during 24 h of incubation in trypticase-yeast extract medium. Whereas H2S accounted for 60 per cent of the total volatile S content of the head-space of CP- cultures, it represented only 8 per cent of the volatile S in CP + systems. Although the CP-organisms did not grow as well as CP +, the differences in concentration of VSC may be only partly related to the disparity in growth rates. When the VSC concentrations were calculated on the basis of equivalent optical density of 1.0, the CP + strains still produced over 3-fold more total volatile S, 6-fold more CH3SH and 100-fold more (CH3S)2. A similar allowance for growth rate suggests that CP-strains may possess a greater potential to produce H2S. Both groups metabolized S-containing amino acids and serine, resulting in appreciable increases in H2S production by CP-. However, the two groups appeared to metabolize the carbon moiety of cystine an cysteine by different pathways. The addition of glucose to the medium depressed total volatile S production by both CP+ and CP-strains, attributable mostly to lower H2S levels. Whereas the omission of yeast extract and charcoal treatment of trypticase did not adversely effect the activity of CP+, it further markedly reduced the capacity of CP-cultures to produce VSC. These results suggest that VSC analysis offers a convenient means of assessing strain differences and pathogenic potential of B. melaninogenicus.

Production of volatile sulphur compounds from cysteine, cystine and methionine by human dental plaque

Abstract A gas Chromatographic technique was employed for the evaluation of volatile sulphur compounds produced by dental plaque deposits from cysteine, cystine and methionine. The results indicated that the major components from the tested substrates firmly established their predominance within the initial 24 hr of incubation. While similar chromatographic profiles were recorded on the head-space samples of cysteine and cystine systems, their major products of putrefaction differed markedly from those derived from methionine. Of the seven components derived from cysteine and cystine, methyl mercaptan and hydrogen sulphide, the latter predominating, accounted for more than 95 per cent of the volatile sulphur content. The sulphur levels of eight components formed from methionine were related principally to the presence of methyl mercaptan and dimethyl disulphide and to considerably lower amounts of hydrogen sulphide and an unidentified compound. Since whole saliva exhibited prominent peaks corresponding to all of the mentioned major components, the study suggests that cysteine, cystine and methionine probably serve as contributing sources of volatile sulphur compounds in putrefying saliva.

Changes in Concentration of Volatile Sulphur Compounds of Mouth Air During the Menstrual Cycle

Five female subjects were studied to determine the applicability of volatile sulphur analysis of mouth air to monitor chemical, cytological and physiological changes observed during the menstrual cycle. Volatile sulphur results were compiled over twelve ovulatory cycles derived from two or three consecutive cycles from each subject. The results of mouth air evaluations were compared with concurrently determined levels of hormones in blood serum and organic metabolites in vaginal secretions. Distinct cyclic variations were observed in concentrations of all three volatile sulphur components (hydrogen sulphide, methyl mercaptan and dimethyl sulphide) of mouth air. There was a definite overall trend for the compounds to increase two- to four-fold immediately around mid-cycle and menstruation as well as during mid-proliferative and mid-luteal phases of each menstrual cycle. In those cycles in which hormonal profiles were obtained, the increase in volatile sulphur content closely coincided with the mid-cycle surge in luteinizing hormone, while the peak during the mid-luteal phase corresponded to a period of maximum level of progesterone and elevated oestrogens. The concentrations of lactic acid and urea in vaginal secretions also underwent cyclic changes analogous to those described for volatile sulphur components of mouth air. The occurrence of malodourous concentrations of hydrogen sulphide and methyl mercaptan immediately around menses in most of the cycles studied satisfactorily accounts for the reported incidence of breath malodour observed during this time.

Stimulation of enzyme and cytokine production by methyl mercaptan in human gingival fibroblast and monocyte cell cultures

The volatile sulphur compound methyl mercaptan (CH3SH) is a by-product of protein metabolism and a principal component of oral malodour. This investigation examines the effect of CH3SH on the enzymatic activities of cathepsins B and G and elastase, and on the production by human gingival fibroblasts of two key factors, prostaglandin E (PGE) and cAMP, of the PGE2-cAMP-dependent pathway, which may contribute to the increased production of collagenase and tissue destruction in human periodontal disease. The results demonstrate that CH3SH alone, or in combination with interleukin-1 (IL-1) or lipopolysaccharide, can significantly enhance the secretion of PGE2, cAMP and procollagenase by human gingival fibroblasts. CH3SH also stimulated mononuclear cells to produce IL-1, which can increase cAMP production, and act in synergism with the direct effect of CH3SH on cAMP. CH3SH also significantly enhanced the activity of cathepsin B, moderately suppressed that of cathepsin G, but did not significantly affect elastase. These results provide evidence that CH3SH could be a contributing factor in the enzymatic and immunological cascade of events leading to tissue degradation in periodontal diseases.

Chemical analysis of thiol, disulphide and total sulphur content of human saliva

Abstract Salivary fractions were prepared from untreated, dialyzed and sonicated saliva and were assayed for thiol, disulphide, total sulphur and inorganic sulphate. Individual fractions of untreated and dialyzed preparations were analyzed for their capacity to serve as substrate sources for the production of volatile sulphur compounds, namely, H 2 S and CH 3 SH. The relative order of volatile sulphur production was: whole saliva > sediment > supernate > filtrate. The order correlates with the determined thiol and disulphide content of the tested fractions. Dialysis did not significantly affect the thiol and disulphide content or the capacity of the whole saliva, sediment and supernatant fractions to yield volatile sulphur compounds. As filtration through 0.2 μm membrane removed approximately 90 per cent of the thiol and disulphide content of saliva and yielded a filtrate with an extremely low capacity to serve as substrate for the production of H 2 S and CH 3 SH, it appears that the substrate is associated almost entirely with the cellular and particulate matter of leucocytes and epithelial cells. Sonication and filtration studies suggest that most of the substrate is in the disulphide state and derived primarily from the epithelial cell membranes in whole saliva and the sediment.

Volatiles of exogenous origin from the human oral cavity.

The volatiles found in the headspace above male and female saliva were examined by combined gas chromatography--mass spectrometry. This had led to the identification of a number of constituents of exogenous origin. The most likely source of these products are atmospheric and water pollutants as well as food stuffs and cosmetic products. Volatiles from saliva represent a potential medium for the detection of reproductive states as well as local and systemic diseases. Consequently, knowledge of compounds not arising from the bodys metabolic process if important to prevent their identification as anomalous metabolites.

Reaction of hydrogen sulphide with proteins associated with the human mouth

Abstract Radioactive, electrophoretic and chromatographic techniques were used to characterize the reaction of H 2 S with saliva, collagen and gelatin. H 2 S readily reacted with salivary cellular elements and proteins, most of the activity being associated with the cellular fraction. Electrophoresis suggested that H 2 S-binding by salivary proteins is a selective phenomenon as the major concentration of 35 S-activity was localized in 3 of the 18 separated anionic proteins. Incubation of saliva produced a marked reduction in H 2 S binding and the disappearance of protein bands detectable by Coomassie Blue staining. The remaining protein fragments consisted of small peptides soluble in TCA, but sufficiently large to be retained during dialysis. Considerable H 2 35 S-binding occurred in insoluble tendon collagen and gelatin preparations. The findings support the involvement of sulphur compounds in the aetiology of periodontal disease.

Metabolism of [35S]-thiosulphate and [35S]-thiocyanate by human saliva and dental plaque

Abstract Chromatographic and radioisotope techniques were employed to characterize the mechanisms involved in the metabolism of thiosulphate and thiocyanate by human saliva and plaque. The criteria for determining the utilization of substrates were based on measurements of 35S-binding by salivary proteins, formation and identification of volatile and non-volatile 35S compounds, and production of inorganic [35S]-sulphate. Through selective 35S-labelling at the beta (35S.SO32−) and alpha (S.35SO32−) positions, it was demonstrated that the two S portions of thiosulphate are metabolized through different pathways. Whereas the alpha S yielded only traces of intermediate volatile S products and was completely recovered as sulphate, the beta S was first converted to the volatile fraction which was subsequently reabsorbed and oxidized by the incubated reaction mixtures to several non-volatile products. Gas chromatographic (GC) analyses of the head-space of the 35S.SO32− systems detected a single prominent peak which corresponded to the retention time for H2S. Paper chromatographic analysis of the non-volatile components showed that the salivary metabolism of 35S.SO32− gave rise to 6 principal zones of radioactivity which were identified on the basis of RF values and staining reactions as: protein, inorganic sulphate, trithionate, tetrathionate, pentathionate and hexathionate. In addition to these components, a very prominent 35S peak was also obtained for thiosulphate when H235S was employed as the sole source of 35S. It appears that H2S and S2O32− are intermediate products of oral organic sulphur metabolism leading to the formation of inorganic sulphate and polythionates. The GC and 35S analyses showed that 35SCN− was not a precursor of volatile S compounds and yielded only traces of [35S]-sulphate in putrescent systems. Similarly, the paper chromatographic studies showed that, except for the uptake of approximately 1 per cent of 35SCN− radioactivity by the salivary proteins, the remainder of 35SCN− appeared inert. The addition of SCN− had no discernible effect on the normal putrefactive activity or oxidation of l -methionine, l -cysteine and l -cystine.

Pathways of cystine metabolism in human saliva.

Abstract Salivary fractions were analyzed by paper Chromatographie, radioisotope, and staining techniques to determine the intermediates produced by dethiolation of cystine by putrescent saliva. Filtrate and whole-saliva fractions yielded identical results except for significantly greater incorporation of 35 S and 14 C activity into the cellular debris-protein fraction of whole saliva. Most of this 14 C-uptake was attributed to metabolites and not to intact cystine. The evidence indicates that metabolism of the de-thiolated portion of the cystine molecule proceeds via the serine pathway. Serine, the main intermediate 14 C-containing product from de-thiolated cystine, was broken down into 5 non-volatile products that accounted for 80 per cent of the total 14 C-activity of the added substrate. The remainder was attributed to volatile components. The two predominant non-volatile metabolites, represented by the protein-cellular binding peak at the origin ( R f = 0) and an unknown peak with R f = 0.32, were ninhydrin-positive. Peaks corresponding to thiosulphate, inorganic sulphate and polythionates were consistently obtained as major products derived from the 35 S-portion of the cystine molecule.