J. Dent. 1991;
19: 263-271
263
Review
Can the oral flora adapt to sorbitol? S. D. Hogg and A. J. Rugg-Gunn Department
of Oral Biology, Dental School, Newcastle
upon Tyne, UK
ABSTRACT The number of non-sugar sweeteners that are approved for use in foods and drinks is increasing and manufacturers are using these as alternatives to cariogenic sugar. These non-sugar sweeteners are generally classed as non-cariogenic. The most frequently used non-sugar sweetener is sorbitol, and concern has been expressed that the oral flora may adapt to sorbitol so that it looses its ‘safe for teeth’ property. The purpose of this review is to describe the mechanisms whereby oral microorganisms, and mutans streptococci in particular, might metabolize sorbitol and to summarize published research into changes in plaque acid production and changes in plaque flora after exposure to sorbitol. Finally, the possibility that some groups of people may be especially ‘at-risk’ from adaptation of oral microorganisms to sorbitol is considered. It is concluded that frequent or long-term use of sorbitol is unlikely to present any increased risk of dental caries in normal people, but that frequent use of sorbitol may present a small cariogenic risk in people with low salivary flow. KEY WORDS: Oral flora, Non-sugar J. Dent. 1991;
19: 263-271
sweeteners,
Review
(Received 21 March
1991;
reviewed
10 May 1991;
accepted 23 May 1991)
Correspondence should be addressed to: Dr S. D. Hogg, Department of Oral Biology, University Tyne Dental School, Framlington Place, Newcastle upon Tyne NE2 4BW. UK.
INTRODUCTION Identification of dietary sugars as a key element in the aetiology of caries stimulated the development of a range of alternative, non-sugar sweeteners which are either not fermented by oral microorganisms or which are fermented only slowly. There is now a considerable body of opinion supporting the strategic use of these alternative sweeteners in a range of foods, drinks and medicines as a cariespreventive measure. Alternative sweeteners fall into two broad categories: the ‘intense sweeteners’, such as saccharin and aspartame, and ‘bulk sweeteners’, mainly a variety of polyols. Intense sweeteners are used in low concentrations and are well suited to particular applications such as soft drinks, while bulk sweeteners are more suitable for foods, such as confectionery, and medicines. Sorbitol and xylitol, which are examples of bulk sweeteners, are both sugar alcohols. Sorbitol can be fermented by a few of the many bacterial species which comprise the normal flora of the human mouth, including those which have been particularly implicated in the aetiology of caries. However, the amount and rate of acid production by oral microbes from sorbitol is significantly lower than that from common dietary sugars such as glucose, fructose and particularly sucrose. For this reason, @ 1991 Butterworth-Heinemann 0300-5712/91/050263-09
Ltd.
of Newcastle
upon
sorbitol has been referred to as hypoacidogenic. This distinguishes it from xylitol which, generally, is not fermented by oral microbes and, as such, has been called non-acidogenic. In addition, further studies have shown that xylitol can interfere with the fermentation of other sugars thereby reducing the amount of acid produced. For this reason, xylitol has also been described as cariostatic. The potential cariogenicity of sorbitol has been reviewed by Birkhed et al. (1984) and Birkhed and B2r (1991), who concluded that it was virtually non-cariogenic. Any cariogenic potential of sorbitol in man must be inferred from a number of published animal experiments and from data obtained fromin vitro fermentation studies because no long-term human clinical study of total substitution of dietary sugars by sorbitol has been attempted. The data, so far, indicate that, under certain circumstances, sorbitol may cause caries in rats, but that it is significantly less cariogenic than dietary sugars such as glucose and sucrose. The cariogenic effect of sorbitol in rats may be due either to the small, but nevertheless measurable, amount of acid formed by microorganisms when it is fermented, or by changes in the balance of the microbial ecology of the mouth. In the latter case, the growth of microorganisms which can utilize sorbitol will be favoured. This will
264
J. Dent. 1991;
19: No. 5
include Streptococcus mutans which is considered to be most closely associated with dental caries. There is some evidence from both animal and human studies that microbial adaptation to sorbitol can occur, but the clinical significance of this is not yet clear. The possibility that the oral flora can adapt to the presence of sorbitol has led to some doubts about its suitability as an alternative sweetener. The purpose of this report is to review the research which has suggested that oral microbial adaptation to sorbitol can occur and draw conclusions concerning the clinical significance of the information published so far. The subject is of considerable interest at the present time and many of the publications have appeared very recently. This review is structured so as to give: an overview of the role of microorganisms in the aetiology of dental caries, a description of the bacterial metabolism of sugars and some polyols, and the possible methods of adaptation of oral microorganisms to polyols and, finally, a review of the studies which have investigated this possible problem.
MICROORGANISMS
AND DENTAL CARIES
Some investigators argue that caries is caused by specific microorganisms within dental plaque, while others maintain that no one microorganism is responsible. The putative pathogens in the so-called ‘specific-plaque’ hypothesis are mutans streptococci which in gnotobiotic animal experiments invariably induce caries (Hamada and Slade, 1980). Cross-sectional epidemiological studies of human populations have implicated both mutans streptococci and lactobacilli in caries (Boyar and Bowden, 1985). However, only a longitudinal study can establish a causal relationship and these have consistently failed to show a unique association between caries and any particular microorganism. In some longitudinal studies, the finding that mutans streptococci can be isolated in high numbers from sites that do not become carious, together with an inability to isolate them from other carious sites, is evidence supporting the ‘non-specific plaque’ hypothesis (Hardie et al., 1977). Other supporting evidence comes from animal studies which have shown that a number of oral microbes can initiate caries to a greater or lesser extent (Gibbons and van Houte, 1975). Several experimental problems associated with clinical caries studies have been suggested to account for the lack of a clear association between caries and mutans streptococci. The most important of these has been the difficulties encountered by clinicians in identifying early lesions. This problem has been addressed by sampling plaque from teeth removed for orthodontic reasons and diagnosing early caries using histological techniques (Marsh et al., 1989). A unique association between mutans streptococci and early caries was not found, and it seems possible that different microorganisms may be involved at different stages in lesion development (Marsh et al., 1989). At the
present time, it is not possible to attribute human caries uniquely to mutans streptococci. However, this group of microorganisms is characterized by a number of potential virulence factors which, taken together, may account for their close involvement in the pathogenesis of caries compared with other oral bacteria. These include synthesis of insoluble extracellular polysaccharides which are thought to be important in maintaining the structural integrity of plaque, lactic acid production, overall acid production (acidogenicity), acid tolerance (aciduricity), and intracellular polysaccharide synthesis. Consequently, the mutans streptococci have been studied extensively and, in view of the central role of acid production in the aetiology of caries, much of the research effort has been directed at understanding their carbohydrate metabolism.
BACTERIAL CARBOHYDRATE METABOLISM The adaptation of the oral flora to sorbitol centres on the ability of a microorganism or group of microorganisms to use sorbitol as a substrate for energy production. In order to understand how oral microbial adaptation might occur and any significance this may have regarding tooth demineralization, it is necessary to describe some of the essential features of carbohydrate uptake and metabolism. Only a very few oral microorganisms have the ability to ferment sorbitol. Prominent among these are the mutans streptococci and Lactobacillus casei. Although other oral microorganisms are not noted for this ability, a low frequency of sorbitol fermentation capability should be expected, particularly among the more commonly encountered species, e.g. Actinomyces spp., Strep. sang& and Strep. mitis, but details are scarce (Kalfas et al., 1990a). The consensus view that the mutans streptococci are more closely involved in dental caries compared with other oral species has prompted a greater interest in the carbohydrate metabolism of this group and, as a consequence, very much more is known. Much of the detail of glycolysis and sugar transport described below relates, therefore, to the mutans streptococci.
Glycolysis Oral bacteria, in common with many other species, obtain their energy by a process known as glycolysis. The main sequence of reactions is called the Embden-Meyerhof pathway in which glucose is converted to pyruvic acid with the release of energy. This energy is used to convert adenosine diphosphate (ADP) to adenosine triphosphate (ATP). Thus, ATP can be thought of as an ‘energy-rich’ compound which can be used as a source of energy in other metabolic processes. The Embden-Meyerhofpathway includes one oxidation reaction in which nicotinamide adenine dinucleotide (NAD+) acts as an electron acceptor and in so doing is
Hogg and Rugg-Gunn:
reduced to NADH,. This conversion alters the oxidationreduction balance of the cell and, if allowed to proceed without redress, will ultimately cause cell metabolism to cease. The microorganism must therefore regenerate NAD’ by oxidizing NADH, in order to survive. It accomplishes this by reducing pyruvic acid. The endproduct of this reduction reaction is either lactic acid or a mixture of ethanol, formic acid and acetic acid, depending on the environmental conditions. These metabolic endproducts are expressed from the cell and it is the various acids which may ultimately attack the tooth mineral. Carbohydrate
uptake
Streptococcus mutans has two uptake systems for glucose. At high external glucose concentrations, glucose enters the cell via a carrier molecule called a permease which sits across the cell membrane. The uptake of a glucose molecule is coupled to the uptake of a proton and so the system is referred to as a ‘proton-linked permease’. At a low external glucose concentration, the protonlinked permease is inoperative. In this case the cell takes up glucose via a mechanism called the ‘phosphotransferase system’ (PTS). High external concentrations of glucose repress the glucose-PTS. Two similar systems have also been described in Strep. mutans for the uptake of sucrose. However, the sucrosePTS as well as being inhibited by high concentrations of sucrose, is also inhibited by glucose. This means that Strep. mutans will preferentially assimilate glucose rather than sucrose in the presence of both. Uptake
and metabolism
of sorbitol
Streptococcus mutans transports sorbitol across the cell membrane by a specific sorbitol-PTS. No proton-linked permease for sorbitol uptake has been described. However, the enzymes required for sorbitol-PTS are not routinely synthesized by the cell. They have to be induced and this requires the presence of sorbitol. The presence of glucose, or other sugar such as sucrose, will inhibit this induction. This means that if sorbitol and either glucose or sucrose are present together, the sorbitol will not be taken up until the other sugars have been metabolized. Once inside the cell, sorbitol, which has been converted to sorbitold-phosphate by the sorbitol-PTS, enters glycolysis as fructosed-phosphate and will ultimately contribute to acid formation. The conversion of sorbitol6-phosphate to fructose-dphosphate is catalysed by sorbitol-6-phosphate dehydrogenase. This enzyme is not constitutive and, like the sorbitol uptake system, must be induced by the presence of its substrate. Induction of any enzyme or protein takes about one generation to accomplish; thus production is subject to a lag period related to the growth rate of the cell. Control
of glycolysis
Oral microorganisms
are intermittently
exposed to very
Possible intra-oral
metabolism
of sorbitole
265
large increases in sugar. In the absence of effective control mechanisms this could cause substrate-accelerated death or ‘sugar killing’, which is brought about by the accumulation within the cell of toxic intermediates of glycolysis. To prevent this, microorganisms, such as oral streptococci, regulate intracellular sugar metabolism. This is achieved, first, by converting some glucose to intracellular polysaccharides (IPS) which are similar to animal glycogen, second, by regulating the rate of glycolysis through glucose-6-phosphate activation of pyruvate kinase which converts phosphoenolpyruvate to pyruvic acid and, finally, by switching pyruvic acid conversion from formic acid, ethanol and acetic acid production to lactic acid production. Pyruvic acid is converted to lactic acid by lactate dehydrogenase. This is a constitutive enzyme which is activated by the glycolytic intermediate fructose-l, 6diphosphate. Under anaerobic conditions and in the phosphate, so these end-products (formic acid, ethanol and acetic acid) will only be formed when environmental sugar is low. Furthermore, PFL is very oxygen sensitive and only operatives under anaerobic conditions, often found in dental plaque. Pyruvic acid is converted to a mixture of formic acid, ethanol and acetic acid by pyruvate-formate lyase (PFL). This enzyme is inhibited by the glycolytic intermediates glyceraldehyde-3-phosphate and dihydroxyacetonephosphate, so these end-products (formic acid, ethanol and acetic acid) will only be formed when environmental sugar is low. Furthermore, PFL is very oxygen sensitive and only operates under anaerobic conditions, often found in dental plaque. glycolytic intermediates to suppress PFL, activate lactate dehydrogenase and switch end-product formation to lactic acid. This increases the rate at which glycolysis proceeds and protects the cell from toxic intermediates. Metabolic end-products fermentation
of sorbitol
When pyruvic acid is reduced to lactic acid, sufficient NAD’ is regenerated to maintain the oxidation-reduction balance of the cell. The reduction of pyruvic acid to ethanol generates exactly twice as much NAD+. This means that when glucose is low, one-half of the available pyruvic acid can be converted to acetic acid. This reaction is favourable at low substrate concentrations because it yields a further ATP. Formic acid is produced as a byproduct of both reactions. Sorbitol is transported intostrep. mutans by an induced sorbitol-PTS. Inside the cell sorbitold-phosphate is dehydrogenated to fructosed-phosphate before it enters the Embden-Meyerhof pathway. This conversion generates NADH2. Thus, for each sorbitol molecule metabolized, an extra NADH, is produced compared with glucose. This means that, regardless of the substrate concentration, when sorbitol is being metabolized, the only end-products of anaerobic glycolysis are formic acid,
266
J. Dent. 1991;
19: No. 5
ethanol and acetic acid. If lactic acid were to be produced, insufficient NAD+ would be regenerated and the oxidationreduction balance of the cell would be compromised. The PFL-catalysed reduction of pyruvic acid is slower than that oflactate dehydrogenase, which explains why the rate of acid production from sorbitol is lower than that from glucose or sucrose. When Strep. mutans is metabolizing sorbitol it can convert the pyruvic acid to lactic acid but only in aerobic conditions. This is because it has NADH oxidase. However, such aerobic conditions are unlikely to be present in dental plaque. This raises an important pointin vitro comparisons of acid production by organisms such as Strep. mutans from sorbitol should be performed under anaerobic conditions in order that both the rate and type of acid produced reflect more closely the situation found within plaque. Furthermore, it may be very misleading to extrapolate from an in vitro pure culture situation to that of a mixed microbial community in vivo.
MICROBIAL
ADAPTATION
TO SORBITOL
Microbial populations, especially mixed populations, are well known for their ability to respond to environmental changes. First, a microorganism can acquire genetic information either spontaneously by mutation or by transfer of genetic material from other, competent organisms. Secondly, microorganisms can change their phenotype by a process known as enzymic induction. Finally, the whole microbial population, whether it is a mixed population or composed of a single species, may alter to meet the challenge. This whole population response may occur as a result of genetic or induced changes in individual members, or it may simply reflect the diversity of phenotypes already present in the environment. The oral microbial population is a particularly good example of this process, and fluctuations in the proportions of Strep. mutans and lactobacilli in response to changes in intake of dietary carbohydrates are well documented (Newbrun, 1989). The well-established ability of some oral microorganisms, especially mutans streptococci and lactobacilli, to ferment sorbitol, has prompted suggestions that frequent or long-term use of sorbitol-containing products will result in adaptive changes in the oral flora (Birkhedet al., 1984, 1987). Concern has been expressed that these changes will result in a flora with an increased sorbitolfermenting capability. Such changes might increase the low cariogenicity of sorbitol to unacceptable levels. There is no evidence in the scientific literature that genetic changes in the oral flora have occurred in response to either long-term or frequent use of sorbitol. Those changes which have been reported have been due to enzyme induction and subsequent shifts in the balance of the microbial population. The problem has been investigated by measuring changes in acid production in
dental plaque and changes in the microbial population of plaque. These changes have been measured following both short- and long-term sorbitol adaptation periods, mostly in normal healthy human subjects. A limited amount of information is available on the adaptation of oral flora in certain ‘at-risk’ groups, such as individuals suffering from hyposalivation, and diabetics with reduced dietary sugar. There is very little information available concerning a potential increase in caries risk, probably because of the numerous scientific and technical problems involved and the cost.
Review of studies in acid production
investigating changes in dental plaque
Birkhed et al. (1978) measured acid production by plaque in vivo andin vitro in 18 subjects who had been exposed to
10 ml of 10 per cent sorbitol solution six times each day for 6 weeks. Exposure of the plaque to 10 per cent sorbitol in viva caused lower pH values after sorbitol adaptation, which was statistically significant 5, 10, 20 and 30 min after rinsing. Although the pH values were statistically significantly lower, at no time did they approach the critical pH for demineralization. The greatest difference found was only 0.33 pH units which occurred at 20 min, and the lowest pH achieved by sorbitol-adapted plaque was 6.67. The in vitro acidogenicity was measured by removing plaque from several sites and homogenizing it in phosphate buffer to which was added either glucose or sorbitol. The rate of acid production was measured by titrating the incubation mixture with alkali. The amount of acid produced from sorbitol after sorbitol adaptation was significantly greater (P < 0.001) but was still only approximately 30 per cent of the rate of acid production from glucose under the same conditions. The in vivo effect the sorbitol and xylitol on homogenized plaque collected from long-term users of products containing both xylitol and sorbitol was investigated by Makinen and Virtanen (1978). The periods of use exceeded 4 years. The pH of the homogenized plaque when it was incubated with sorbitol was lower than when it was incubated with xylitol. This effect only achieved statistical significance after 4 h incubation which reflects the low rate of acid production from sorbitol. It is apparent, therefore, that frequent use of sorbitolcontaining products over a 4.5-year period did not result in greater plaque acid production from sorbitol in vitro compared with xylitol. However, the subjects’ plaque in this study was also conditioned to xylitol so there is an assumption that no adaptation to xylitol had occurred. Significant amounts of acid were produced from sorbitol in the incubation mixtures after 22 h but this is of little relevance to the in vivo situation. Birkhed et al. (1979) looked at the effects of frequent sorbitol consumption throughout a 3-month period on both in vivo andin vitro acid production by plaque. Thein
Hogg and Rugg-Gunn:
viva acid production from sorbitol after the adaptation period was significantly greater (P = 0.05) but the lowest pH value reached was about 6.75, substantially higher than the critical pH for demineralization. The rate of acid production by homogenized plaque in vitro was not significantly different from that recorded before the adaptation period. The pre- and post-sorbitol adaptation acid production rates from sorbitol in this study were, in fact, remarkably similar and were only 15.4 and 18 per cent, respectively, of the production rate when glucose was fermented. Topitsoglou et al. (1983) looked at the effect of sorbitol adaptation on the pH changes within dental plaque and on the amount of titratable acid produced when plaque was exposed to sorbitol in vitro. This was a double blind cross-over trial in which the subjects refrained from oral hygiene for 4 days during which they chewed either sorbitol gum, xylitol gum or xylitol/sorbitol gum 10 times each day. Overall, the in vivo pH change of sorbitoladapted plaque, when exposed to sorbitol, was greater than xylitol-adapted plaque exposed to sorbitol. The increase in acid reached statistically significant amounts after lo,20 and 30 min when the subjects chewed sorbitol gum, and at 5 and 10 min but not 20 and 30 min when the mouth was rinsed with a 25 per cent sorbitol solution. However, these increases were only very small. The difference in pH between sorbitol-fermenting sorbitoladapted plaque and xylitol-adapted plaque was never greater than 0.34 pH unit. Sorbitol-conditioned plaque exposed to sorbitol in vivo was always above pH 6.5. The rate of acid production by sorbitol-adapted plaque was compared to that of xylitol-adapted plaque, but there was no comparison with pre-adapted plaque. Using sorbitol as the substrate, sorbitol-adapted plaque produced acid at a significantly greater rate (P < 0.01) than xylitol-adapted plaque. However, similar increases occurred when glucose was used as the substrate so at least some of the difference in acid production by sorbitol-adapted plaque, compared to xylitol-adapted plaque, was due to a lower metabolic activity of the xylitol-adapted plaque. It is, therefore, not possible to attribute all the change in rate of acid production in this study to sorbitol adaptation. Sdderling et al. (1989) measured interdental plaque pH response to sorbitol at four different sites using a palladium touch electrode. Volunteers adapted their oral flora to either sorbitol or xylitol or a mixture of both polyols by chewing two sticks of appropriate gum five times each day for 2 weeks. The acidogenic response of plaque to sorbitol was small in all three groups, and the changes induced by the 2-week adaptation period were also small and not statistically significant. Birkhed et al. (1990) studied in vivo plaque pH changes to a sorbitol challenge and acid production activity in plaque from sorbitol, in selected subjects who had been consuming sorbitol for at least 2 years at a frequency of intake of at least four times each day. The in vivo fall in plaque pH after rinsing with 10 per cent sorbitol solution was minor and not statistically significant. The lowest pH
Possible intra-oral
metabolism
of sorbitole
267
values were between 6.4 and 7.3, which were similar to those seen in individuals who had rinsed their mouths with sorbitol solutions six times each day for 4 weeks (Birkhed et al., 1979). Thus no differences in acid production in plaque were apparent when short- and long-term sorbitol consumers were compared. by homogenized The in vitro rate of acid production plaque from sorbitol was 30.7 per cent of that from glucose. Short-term sorbitol adaptation studies gave a similar rate (Birkhed et al., 1978) suggesting that there is no progressive increase in the rate of acid production attributable to long-term sorbitol consumption.
Discussion production
of plaque pH and acid activity studies
Research has shown that frequent use of sorbitol can lead to a greater in vivo pH response when the plaque is subsequently challenged with sorbitol. The increase in plaque acid production was statistically significant in some cases (Birkhed er al., 1978, 1979; Topitsoglou et al., 1983) but not in others (Soderling et al., 1989; Birkhed et al., 1990). However, even when differences were statistically significant, the increase in acid production was very small and the plaque pH did not approach the critical pH for demineralization. The studies which measured the rate of acid production plaque in vitro (Birkhed et al., 1978, 1979; Topitsoglou ef al., 1983; Birkhed et al., 1990) have to be interpreted with some caution because the conditions of measurement were not similar to those in in viva plaque studies in two main respects. First, the presence of oxygen in the in vitro experiments should, theoretically, cause an increase in the amount of lactic acid produced from sorbitol but not from glucose which was used as a reference. This could mean that the acid produced by plaque microorganisms from sorbitol was being overestimated. However, it should be noted that Kalfas and Birkhed (1986) have reported that the rate of acid production by pure cultures of Strep. mutans from sorbitol under aerobic conditions is very much lower than that under anaerobic conditions, which suggests that acid production from sorbitol could actually be underestimated in the above studies. Plaque microbial communities are, however, extremely complex and, in the same study, Kalfas and Birkhed (1986) showed that there was, in fact, little difference in acid production when mixed suspensions of plaque microorganisms were examined. Nevertheless, there have been few studies in this area and prudence dictates that the results obtained in the presence of oxygen should be treated with some caution. Second, the incubating mixture in these studies was kept at a constant pH 6.8 throughout. This would have the effect of maximizing the rate of acid production from sorbitol compared to that from glucose, because sorbitol fermentation from mixed oral bacteria is more sensitive to lower pH (Kalfas et al., 1990a).
268
J. Dent 1991; 19: No. 5
Review of studies in plaque flora
investigating
changes
Harjola and Liesmaa (1978) measured the change in an index of lactobacilli concentration in 5.5normal children divided into a group consuming xylitol and sorbitol, a group consuming sugar and a non-sugar group. The experimental period was 2 weeks and the concentration of oral lactobacilli was measured using the Dentocult (L) system. The results were compared with baseline values measured before the start of the experimental period. High variability in the numbers of lactobacilli cultured from the saliva of children in all three groups made statistical analysis of the observed changes difficult. Nevertheless, there was a marked reduction in the numbers of lactobacilli in the group using the polyols, which suggests that sorbitol-fermenting species were not favoured. However, since both xylitol and sorbitol were used together it is not possible to exclude an effect of xylitol in suppressing lactobacilli growth. Birkhed et al. (1979) looked at the effect of 3 months’ frequent consumption of Lycasin, maltitol, sorbitol and xylitol on the numbers of total streptococci, Strep. mutans and lactobacilli in dental plaque. Few changes were found in samples taken before and after the test period. In the sorbitol group, a significant decrease in the number of lactobacilli was found (P < 0.05). In this study, as in others involving measuring relative numbers of oral bacteria, there were large variations in numbers between samples. It is possible that such variations concealed an effect of sorbitol consumption. The authors also point out that an increase in the frequency and duration of consumption of a sorbitol-containing product may produce measurable changes in the oral flora. The study by Rateitschak-Pl-iiss and Guggenheim (1982) differs from other investigations in that the subjects’ carbohydrate intake was limited to the substances under test. There were four experimental groups, with six subjects in each group. The groups consumed candies containing either sorbitol, xylitol or sucrose. The fourth group consumed no candies. Each subject participated in each group for 4 days, allowing 9 days’ recovery between each period. Counts were made of the number of bacteria which fermented sorbitol and xylitol, and this was expressed as a percentage of the total number of viable bacteria. A significantly greater percentage of sorbitol-fermenting bacteria were isolated from the sorbitol group compared with the sucrose group (P < 0.001). However, significant increases in the proportion of sorbitol-fermenters were also found in the xylitol group (P < 0.05) and in the sucrose group (P < 0.05). The study also found a significant increase in the proportion of xylitol-fermenting bacteria in the xylitol group when compared with the sucrose group (P < 0.05). Although significant differences were found, the authors pointed to the fact that only a small fragment of the flora was able to ferment sorbitol and an even smaller proportion could ferment xylitol.
Loesche et al. (1984) compared the effect of chewing gums containing xylitol, sorbitol/mannitol and fructose on oral microbiology in a parallel group study. Counts of Strep. mutans were made from samples of saliva and occlusal and approximal plaque before the chewing period, and after 2 and 4 weeks. At 4 weeks gum chewing ceased and the levels of Strep. mutans were monitored 2 and 4 weeks later. Sorbitol had no effect on the salivary levels of Strep. mutans. In occlusal plaque, the total count of Strep. mutans was unaffected, but the relative proportion increased although this was not statistically significant. In approximal plaque, the proportion of Strep. mutans increased significantly after 4 weeks (P < 0.05) and then decreased significantly (P < 0.05) after chewing of sorbitol gum ceased. No changes were found in salivary levels of lactobacilli, or in approximal or occlusal plaque lactobacilli levels, or proportions of Strep. sanguis or lactobacilli. Wennerholm and Emilson (1989) measured salivary levels of Strep. mutans, Strep. sanguis, Strep. salivatius and lactobacilli in patients attending a smoking cessation clinic in Gothenburg. Twenty-seven patients chewed a nicotine-containing sorbitol-sweetened (19 per cent sorbitol) chewing gum when they felt a desire for nicotine. The subjects used about five sticks of chewing gum each day. Salivary bacterial levels were measured at baseline, after 1.5 months and finally after 3 months of treatment. A small decrease in the salivary levels of all the bacterial species was found, but this was not statistically significant when compared to the baseline values. Sijderlinget al. (1989) compared the effect of 2 weeks use of sorbitol, xylitol and sorbitol/xylitol chewing gums on salivary and plaque levels of Strep. mutans. They stated that both plaque and salivary levels of Strep. mutans generally increased with sorbitol use, but detailed data were not presented and were not accompanied by statistical analyses. Birkhed et al. (1990) reported that in subjects who had consumed sorbitol-containing products at least four times each day for 2 years, there was a positive correlation (P < 0.05) between the number of mutans streptococci in saliva and the frequency of sorbitol intake. The sorbitolfermenting microorganisms isolated were mutans streptococci, sanguis-mitior-like organisms and Strep. salivarius.
Linke and Castle (1990) isolated sorbitol-fermenting bacteria from the dental plaque of people using sorbitol products. Most of these bacteria produced significant amounts of acid, which was defined as a decrease in the pH of the culture to approximately pH 4.5-4.8, but this was over an incubation period lasting 5 days. Sorbitolfermenting strains isolated were identified as Strep. acidominimus, Strep. sanguis, Strep. mutans and Micrococcus luteus. The authors reported that a large number of sorbitol-metabolizing bacteria were present in the plaque of patients preferentially using sorbitol-containing sweets, but do not relate the numbers to the total number of
Hogg and Rugg-Gunn: Possible intra-oral metabolism of sorbitole
bacteria cultured. They go on to say that ‘The bacteriological results suggest that frequent consumption of sorbitol-containing products, with moderate as well as heavy use, will lead to a shift in oral ecology, numerically favouring sorbitol-degrading bacteria’. No statistical treatment of the data was presented in support of this statement.
Discussion
of bacteriological
studies
There is very little evidence of statistically significant changes in the oral flora due to sorbitol consumption either in the short- or long-term. This is to be expected because both sorbitol uptake, and metabolism in oral streptococci, have to be induced. These occur in the presence of the substrate but are repressed by the presence of glucose (Slee and Tanzer, 1983), which is transported into the cell and metabolized by constitutive systems. In practical terms, this means that even small amounts of glucose in the diet will effectively prevent sorbitol utilization regardless of its concentration. In view of this, the paper by Rateitschak-Pliiss and Guggenheim (1982) is important because, in this study, the dietary sources of carbohydrate of the subjects were limited to the test sweeteners, and significant increases in sorbitol- and xylitol-fermenting strains were found after sorbitol- and xylitol-conditioning, respectively. Even the results of this study were equivocal because sorbitolfermenting strains also increased significantly after xylitol and sucrose conditioning, even in the absence of other dietary carbohydrates. Difficulties in demonstrating increases in sorbitol-fermenting strains, even in carefully controlled experiments such as this, may be due partly to endogenous salivary glucose levels (Jenkins, 1978) which are higher than the levels required for repression of sorbitol fermentation (Slee and Tanzer, 1983). Review of studies investigating sorbitol metabolism in the plaque of special ‘at-risk’ groups There have been suggestions that sorbitol fermentation may present a problem for particular groups such as xerostomic patients, who frequently consume sorbitolcontaining products as saliva stimulants and are known to harbour elevated numbers of mutans streptococci. Furthermore, they will lack much of the microbial control mechanisms present in people with normal saliva flow, and especially much of the capability for buffering acids in saliva. Two recent studies have examined the effects of sorbitol-conditioning on the oral environment. Kalfas et al. (1990b) selected 12 people with low and 11 people with normal salivary flow rates. Both groups rinsed frequently with 10 per cent sorbitol solution for 4 weeks. Sorbitolfermenting bacteria increased significantly, as did the proportion of mutans streptococci, in plaque samples taken from both groups after conditioning. Plaque acid
269
production from sorbitol increased in both groups, but more so in the group with low salivary flow rates. Values in three out of 12 subjects were less than pH 5.7, and in five out of 12 subjects, values were less than pH 6.0. Bacteriological studies of the group with low saliva flow revealed that the sorbitol-fermenting flora was almost exclusively composed of the genera Streptococcus, Lactobacillus and Actinomyces (Kalfas and Edwardsson, 1990). Another at-risk group could be patients on long-term low-sugar diets, such as some diabetics. Giilzow et al. (1990) examined this problem in 25 subjects with juvenile diabetes mellitus. Saliva samples from this group and from the same number of healthy individuals, were incubated under aerobic and anaerobic conditions, and the respiration rate of the bacteria measured using a Warburg respirometer. Sorbitol was metabolized faster and to a greater extent by salivary microorganisms in the diabetic group. Although this is evidence that some adaptation may have occurred, it is difficult to relate these findings, which are presented in terms of oxygen consumption and carbon dioxide evolution, to in viva plaque
PH. Review
of relevant
animal studies
Only one animal study appears to have been performed with the object of investigating sorbitol adaptation, but this was a well-designed and controlled experiment carried out over 2 years using macaque monkeys (Cornick and Bowen, 1972).In viva acid production by plaque from sorbitol was small at baseline and no increase was found after 8 weeks or 2 years when large amounts of sorbitol were consumed daily. Similarly, there was no increase over an g-week period in the number of sorbitolfermenting microorganisms. Information on the microbial composition of the plaque ceased at 8 weeks.
CONCLUSIONS The possibility of dental plaque adaptation to sorbitol has been examined in healthy, human subjects in a variety of studies by measuring changes in plaque acidogenicity in viva, plaque acid production activity in vitro, and population changes in the microbial composition of plaque. In addition, there has been one study which has looked at sorbitol adaptation in people with low salivary flow rate, one study which has looked at adaptation in diabetics on low-sugar diets, and one animal study. 1. Studies of plaque acid production in vivo have not provided consistent evidence of plaque adaptation to sorbitol There have been five in viva human plaque pH studies. In three of these studies, although there was a statistically significant increase in acid production by sorbitolconditioned plaque, the amount of acid produced was
270
J. Dent.
1991; 19: No. 5
unlikely to be sufficient to affect teeth clinically. The remaining two studies did not show any significant increase in plaque acid production. In one animal experiment, there was no increase in plaque acid over 2 years of frequent sorbitol exposure. 2. There is no firm evidence that long-term use of sorbitol results in increased acid production activity by plaque in vitro There have been five studies of in vitro acid production activity by sorbitol-conditioned plaque. One study showed an increase in the rate of acid production by sorbitol-adapted plaque, one showed an increase which could not be wholly attributable to sorbitol, two did not show any increase, and one showed that there was no progressive increase in acid production rate in the long term. 3. There is very little evidence in the current literature of an alteration in the microbial ecology of dental plaque which could be attributed to sorbitol consumption in the short or long term There have been eight studies of the effect of sorbitol on the microbial ecology of plaque and, although some changes have been reported, they were either not statistically significant, or could not be attributed to sorbitol. No changes found
in the
microbial
in a long-term
animal
composition
of plaque
were
experiment.
4. There is some evidence that frequent use of sorbitol may present a small problem in people with low salivary flow One study has shown that plaque acid production from sorbitol increased after short-term (Cweek) use of sorbitol, and that pH values reached levels close to the critical pH. This may be partly due to a decrease in the natural salivary buffering capacity inherent in this condition. 5. In the light of current evidence, frequent or long-term use of sorbitol is unlikely to present any increased risk of dental caries in normal people References Birkhed D. and Bar A (1991) Sorbitol and dental caries. World Rev. Nutr. Dietet 65, l-37. Birkhed D., Edwardsson S., Svensson B. et al. (1978) Acid production from sorbitol in human dental plaque. Arch. Oral BioJ. 23, 971-975. Birkhed D., Edwardsson S., Ahldtn M.-L. et al. (1979) Effects of 3 months frequent consumption of hydrogenated starch hydrolysate (Lycasin), maltitol, sorbitol and xylitol on human dental plaque. Acta Odontol. Stand. 37, 103-l 15.
Birkhed D., Edwardsson S.. Kalfas S. et al. (1984) Cariogenicity of sorbitol. Swed. Dent. .I 8, 147-154. Birkhed D., Svenstiter G., Kalfas S. et al. (1987) The risk of adaptation of the oral microflora to sorbitol. Dtsch. Zahnarztl. Zeit. 42, S141-S144. Birkhed D., Svensater G. and Edwardsson G. (1990) Cariological studies of individuals with long-term sorbitol consumption. Caries Res. 24, 220-223. Boyar R. M. and Bowden G. H. (1985) The microflora associated with the progression of incipient caries lesions in teeth of children living in a water-fluoridated area. Caries Res. 19, 298-306. Comick D. E. A. and Bowen W. H. (1972) The effect of sorbitol on the microbiology of the dental plaque in monkeys (Macaca irus). Arch. Oral Biol. 17, 1637-1648. Gibbons R. J. and van Houte J. (1975) Dental caries. Ann. Rev. Med. 26, 121-136. Gillzow H.-J., Kary H. and Schiffner U. (1990) Zum adapataionsverhalten von mikroorganismen der menschlichen mundhole gegenuber sorbit. Dtsch. Zahnarztl. Zeit. 45, 90-92. Hamada S. and Slade H. D. (1980) Biology, immunobiology, and cariology of Streptococcus mutans. Microbial. Rev. 44, 331-384. Hardie J. M., Thompson P. L., South R. J. et al. (1977) A longitudinal epidemiological study on dental plaque and the development of dental caries-interim results after two years. J. Dent Res. 56, (Special Issue C), C90-C98. Harjola U. and Liesmaa H. (1978) Effects of polyol and sucrose candies on plaque, gingivitis and lactobacillus scores. Observations on Helsinki school children. Acta Odontol. Stand. 36, 237-242. Jenkins G. N. (1978)The Physiology and Biochemistry of the Mouth, 4th edn. Oxford, Blackwell Scientific. Kalfas S. and Birkhed D. (1986) Effect of aerobic and anaerobic atmosphere on acid production from sorbitol and suspensions of dental plaque and oral streptococci. Caries Res. 20, 237-243. Kalfas F. and Edwardsson S. (1990) Sorbitol-fermenting predominant cultivable flora of human dental plaque in relation to sorbitol adaptation and salivary secretion rate. Oral Microbial. Immunol. 5, 33-38. Kalfas S., Maki Y.. Birkhed D. et al. (1990a) Effect of pH on acid production from sorbitol in washed cell suspensions of oral bacteria. Caries Res. 24, 107-l 12. Kalfas F., Svensater G., Birkhed D. et al. (1990b) Sorbitol adaptation of dental plaque in people with low and normal salivary-secretion rates. J. Dent. Res. 69, 442-446. Linke H. A. B. and Castle M. (1990) Isolation of acid producing sorbitol adapted bacteria from dental plaque using selective agar media. Microbios 61, 39-48. Loesche W. J., Earnest R., Grossman N. S. et al. (1984) The effect of chewing xylitol gum on the plaque and saliva levels of Streptococcus mutans. J. Am. Dent. Assoc. 108, 587-592. Makinen K. K. and Virtanen K. K. (1978) Effect of 4.5-year use of xylitol and sorbitol on plaque. J. De&. Res. 57, 441-446. Marsh P. D., Featherstone A., McKee A. S. et al. (1989) A microbiological study of early caries of approximal surfaces in schoolchildren. J. Dent Res. 68, 1151-1154. Newbrun E. (1989) Substrate: diet and caries. In: Cariology, 3rd edn. Quintessence, Chicago, pp. 99-l 34. Rateitschak-Pliiss E. M. and Guggenheim B. (1982) Effects of carbohydrate-free diet and sugar substitutes on dental plaque accumulation. J. Clin. Periodontal. 9, 239-251.
Zissis et al.: Accuracy
Slee A. M. and Tanzer J. M. (1983) The repressible metabolism of sorbitol (D-glucitol) by intact cells of the oral plaque-forming bacterium Streptococcus mutans. Arch. Oral Biol. 28, 839-845. Siiderling E., MBkinen K. K., Chen C.-Y. et al. (1989) Effect of sorbitol, xylitol and xylitol/sorbitol chewing gums on dental plaque. Caries Res. 23, 378-384.
Forthcoming Original
Research
and stability
Reports
The radiopacity of composites compared with human enamel and dentine G. Willems, N. J. Noack, S. Znokoshi, P. Lambrech ts, B. Van Meerbeek, M. Braem, J. G. Roulet and G. Vanherle
and endodontics
A. D. Walmsley, W. R. E. Laird and P, J. Lumley
Microleakage in porcelain laminate veneers A. Zaimoglu and L. KaraaEaqlioElu In vitro evaluation of two microleakage D. G. Charlton and B. K. Moore
detection tests
Marginal adaptation of four touth-coloured inlay systems in vivo B. Van Meerbeek, S. Znokoshi, G. Willems, M. J. Noack, M. Braem, Lambrechts, J-F. Roulet and G. Vanherle Reinforcement of poly(methylmethacrylate) polyethylene fibres
P.
with ultra-high-modulus
D. L. Gutteridge
Clinical bone densitometric study of mandibular atrophy using dental panoramic tomography K. Horner and H. Devlin
The effects of digital grey-scale approximal carious lesions
modification
base materials
271
Topitsoglou V., Birkhed D., Larsson L.-A. et al. (1983) Effect of chewing gums containing xylitol, sorbitol or a mixture of xylitol and sorbitol on plaque formation, pH changes and acid production in human dental plaque. Caries Res. 17,369-378. Wennerholm K. and Emilson C.-G. (1989) Effect of sorbitoland xylitol-containing chewing gum on salivary microflora, saliva and oral sugar clearance. Stand. J. Dent. Res. 97, 257-262.
Articles
Ultrasound in dentistry. Part 1. Periodontology
of denture
on the diagnosis
of small
E. H. Verdonschot, J. M. C. Kuijpers. B. J. Polder, M. H. De Leng-Worm and E. M. Bronkhorts
19: 263-271
263
Review
Can the oral flora adapt to sorbitol? S. D. Hogg and A. J. Rugg-Gunn Department
of Oral Biology, Dental School, Newcastle
upon Tyne, UK
ABSTRACT The number of non-sugar sweeteners that are approved for use in foods and drinks is increasing and manufacturers are using these as alternatives to cariogenic sugar. These non-sugar sweeteners are generally classed as non-cariogenic. The most frequently used non-sugar sweetener is sorbitol, and concern has been expressed that the oral flora may adapt to sorbitol so that it looses its ‘safe for teeth’ property. The purpose of this review is to describe the mechanisms whereby oral microorganisms, and mutans streptococci in particular, might metabolize sorbitol and to summarize published research into changes in plaque acid production and changes in plaque flora after exposure to sorbitol. Finally, the possibility that some groups of people may be especially ‘at-risk’ from adaptation of oral microorganisms to sorbitol is considered. It is concluded that frequent or long-term use of sorbitol is unlikely to present any increased risk of dental caries in normal people, but that frequent use of sorbitol may present a small cariogenic risk in people with low salivary flow. KEY WORDS: Oral flora, Non-sugar J. Dent. 1991;
19: 263-271
sweeteners,
Review
(Received 21 March
1991;
reviewed
10 May 1991;
accepted 23 May 1991)
Correspondence should be addressed to: Dr S. D. Hogg, Department of Oral Biology, University Tyne Dental School, Framlington Place, Newcastle upon Tyne NE2 4BW. UK.
INTRODUCTION Identification of dietary sugars as a key element in the aetiology of caries stimulated the development of a range of alternative, non-sugar sweeteners which are either not fermented by oral microorganisms or which are fermented only slowly. There is now a considerable body of opinion supporting the strategic use of these alternative sweeteners in a range of foods, drinks and medicines as a cariespreventive measure. Alternative sweeteners fall into two broad categories: the ‘intense sweeteners’, such as saccharin and aspartame, and ‘bulk sweeteners’, mainly a variety of polyols. Intense sweeteners are used in low concentrations and are well suited to particular applications such as soft drinks, while bulk sweeteners are more suitable for foods, such as confectionery, and medicines. Sorbitol and xylitol, which are examples of bulk sweeteners, are both sugar alcohols. Sorbitol can be fermented by a few of the many bacterial species which comprise the normal flora of the human mouth, including those which have been particularly implicated in the aetiology of caries. However, the amount and rate of acid production by oral microbes from sorbitol is significantly lower than that from common dietary sugars such as glucose, fructose and particularly sucrose. For this reason, @ 1991 Butterworth-Heinemann 0300-5712/91/050263-09
Ltd.
of Newcastle
upon
sorbitol has been referred to as hypoacidogenic. This distinguishes it from xylitol which, generally, is not fermented by oral microbes and, as such, has been called non-acidogenic. In addition, further studies have shown that xylitol can interfere with the fermentation of other sugars thereby reducing the amount of acid produced. For this reason, xylitol has also been described as cariostatic. The potential cariogenicity of sorbitol has been reviewed by Birkhed et al. (1984) and Birkhed and B2r (1991), who concluded that it was virtually non-cariogenic. Any cariogenic potential of sorbitol in man must be inferred from a number of published animal experiments and from data obtained fromin vitro fermentation studies because no long-term human clinical study of total substitution of dietary sugars by sorbitol has been attempted. The data, so far, indicate that, under certain circumstances, sorbitol may cause caries in rats, but that it is significantly less cariogenic than dietary sugars such as glucose and sucrose. The cariogenic effect of sorbitol in rats may be due either to the small, but nevertheless measurable, amount of acid formed by microorganisms when it is fermented, or by changes in the balance of the microbial ecology of the mouth. In the latter case, the growth of microorganisms which can utilize sorbitol will be favoured. This will
264
J. Dent. 1991;
19: No. 5
include Streptococcus mutans which is considered to be most closely associated with dental caries. There is some evidence from both animal and human studies that microbial adaptation to sorbitol can occur, but the clinical significance of this is not yet clear. The possibility that the oral flora can adapt to the presence of sorbitol has led to some doubts about its suitability as an alternative sweetener. The purpose of this report is to review the research which has suggested that oral microbial adaptation to sorbitol can occur and draw conclusions concerning the clinical significance of the information published so far. The subject is of considerable interest at the present time and many of the publications have appeared very recently. This review is structured so as to give: an overview of the role of microorganisms in the aetiology of dental caries, a description of the bacterial metabolism of sugars and some polyols, and the possible methods of adaptation of oral microorganisms to polyols and, finally, a review of the studies which have investigated this possible problem.
MICROORGANISMS
AND DENTAL CARIES
Some investigators argue that caries is caused by specific microorganisms within dental plaque, while others maintain that no one microorganism is responsible. The putative pathogens in the so-called ‘specific-plaque’ hypothesis are mutans streptococci which in gnotobiotic animal experiments invariably induce caries (Hamada and Slade, 1980). Cross-sectional epidemiological studies of human populations have implicated both mutans streptococci and lactobacilli in caries (Boyar and Bowden, 1985). However, only a longitudinal study can establish a causal relationship and these have consistently failed to show a unique association between caries and any particular microorganism. In some longitudinal studies, the finding that mutans streptococci can be isolated in high numbers from sites that do not become carious, together with an inability to isolate them from other carious sites, is evidence supporting the ‘non-specific plaque’ hypothesis (Hardie et al., 1977). Other supporting evidence comes from animal studies which have shown that a number of oral microbes can initiate caries to a greater or lesser extent (Gibbons and van Houte, 1975). Several experimental problems associated with clinical caries studies have been suggested to account for the lack of a clear association between caries and mutans streptococci. The most important of these has been the difficulties encountered by clinicians in identifying early lesions. This problem has been addressed by sampling plaque from teeth removed for orthodontic reasons and diagnosing early caries using histological techniques (Marsh et al., 1989). A unique association between mutans streptococci and early caries was not found, and it seems possible that different microorganisms may be involved at different stages in lesion development (Marsh et al., 1989). At the
present time, it is not possible to attribute human caries uniquely to mutans streptococci. However, this group of microorganisms is characterized by a number of potential virulence factors which, taken together, may account for their close involvement in the pathogenesis of caries compared with other oral bacteria. These include synthesis of insoluble extracellular polysaccharides which are thought to be important in maintaining the structural integrity of plaque, lactic acid production, overall acid production (acidogenicity), acid tolerance (aciduricity), and intracellular polysaccharide synthesis. Consequently, the mutans streptococci have been studied extensively and, in view of the central role of acid production in the aetiology of caries, much of the research effort has been directed at understanding their carbohydrate metabolism.
BACTERIAL CARBOHYDRATE METABOLISM The adaptation of the oral flora to sorbitol centres on the ability of a microorganism or group of microorganisms to use sorbitol as a substrate for energy production. In order to understand how oral microbial adaptation might occur and any significance this may have regarding tooth demineralization, it is necessary to describe some of the essential features of carbohydrate uptake and metabolism. Only a very few oral microorganisms have the ability to ferment sorbitol. Prominent among these are the mutans streptococci and Lactobacillus casei. Although other oral microorganisms are not noted for this ability, a low frequency of sorbitol fermentation capability should be expected, particularly among the more commonly encountered species, e.g. Actinomyces spp., Strep. sang& and Strep. mitis, but details are scarce (Kalfas et al., 1990a). The consensus view that the mutans streptococci are more closely involved in dental caries compared with other oral species has prompted a greater interest in the carbohydrate metabolism of this group and, as a consequence, very much more is known. Much of the detail of glycolysis and sugar transport described below relates, therefore, to the mutans streptococci.
Glycolysis Oral bacteria, in common with many other species, obtain their energy by a process known as glycolysis. The main sequence of reactions is called the Embden-Meyerhof pathway in which glucose is converted to pyruvic acid with the release of energy. This energy is used to convert adenosine diphosphate (ADP) to adenosine triphosphate (ATP). Thus, ATP can be thought of as an ‘energy-rich’ compound which can be used as a source of energy in other metabolic processes. The Embden-Meyerhofpathway includes one oxidation reaction in which nicotinamide adenine dinucleotide (NAD+) acts as an electron acceptor and in so doing is
Hogg and Rugg-Gunn:
reduced to NADH,. This conversion alters the oxidationreduction balance of the cell and, if allowed to proceed without redress, will ultimately cause cell metabolism to cease. The microorganism must therefore regenerate NAD’ by oxidizing NADH, in order to survive. It accomplishes this by reducing pyruvic acid. The endproduct of this reduction reaction is either lactic acid or a mixture of ethanol, formic acid and acetic acid, depending on the environmental conditions. These metabolic endproducts are expressed from the cell and it is the various acids which may ultimately attack the tooth mineral. Carbohydrate
uptake
Streptococcus mutans has two uptake systems for glucose. At high external glucose concentrations, glucose enters the cell via a carrier molecule called a permease which sits across the cell membrane. The uptake of a glucose molecule is coupled to the uptake of a proton and so the system is referred to as a ‘proton-linked permease’. At a low external glucose concentration, the protonlinked permease is inoperative. In this case the cell takes up glucose via a mechanism called the ‘phosphotransferase system’ (PTS). High external concentrations of glucose repress the glucose-PTS. Two similar systems have also been described in Strep. mutans for the uptake of sucrose. However, the sucrosePTS as well as being inhibited by high concentrations of sucrose, is also inhibited by glucose. This means that Strep. mutans will preferentially assimilate glucose rather than sucrose in the presence of both. Uptake
and metabolism
of sorbitol
Streptococcus mutans transports sorbitol across the cell membrane by a specific sorbitol-PTS. No proton-linked permease for sorbitol uptake has been described. However, the enzymes required for sorbitol-PTS are not routinely synthesized by the cell. They have to be induced and this requires the presence of sorbitol. The presence of glucose, or other sugar such as sucrose, will inhibit this induction. This means that if sorbitol and either glucose or sucrose are present together, the sorbitol will not be taken up until the other sugars have been metabolized. Once inside the cell, sorbitol, which has been converted to sorbitold-phosphate by the sorbitol-PTS, enters glycolysis as fructosed-phosphate and will ultimately contribute to acid formation. The conversion of sorbitol6-phosphate to fructose-dphosphate is catalysed by sorbitol-6-phosphate dehydrogenase. This enzyme is not constitutive and, like the sorbitol uptake system, must be induced by the presence of its substrate. Induction of any enzyme or protein takes about one generation to accomplish; thus production is subject to a lag period related to the growth rate of the cell. Control
of glycolysis
Oral microorganisms
are intermittently
exposed to very
Possible intra-oral
metabolism
of sorbitole
265
large increases in sugar. In the absence of effective control mechanisms this could cause substrate-accelerated death or ‘sugar killing’, which is brought about by the accumulation within the cell of toxic intermediates of glycolysis. To prevent this, microorganisms, such as oral streptococci, regulate intracellular sugar metabolism. This is achieved, first, by converting some glucose to intracellular polysaccharides (IPS) which are similar to animal glycogen, second, by regulating the rate of glycolysis through glucose-6-phosphate activation of pyruvate kinase which converts phosphoenolpyruvate to pyruvic acid and, finally, by switching pyruvic acid conversion from formic acid, ethanol and acetic acid production to lactic acid production. Pyruvic acid is converted to lactic acid by lactate dehydrogenase. This is a constitutive enzyme which is activated by the glycolytic intermediate fructose-l, 6diphosphate. Under anaerobic conditions and in the phosphate, so these end-products (formic acid, ethanol and acetic acid) will only be formed when environmental sugar is low. Furthermore, PFL is very oxygen sensitive and only operatives under anaerobic conditions, often found in dental plaque. Pyruvic acid is converted to a mixture of formic acid, ethanol and acetic acid by pyruvate-formate lyase (PFL). This enzyme is inhibited by the glycolytic intermediates glyceraldehyde-3-phosphate and dihydroxyacetonephosphate, so these end-products (formic acid, ethanol and acetic acid) will only be formed when environmental sugar is low. Furthermore, PFL is very oxygen sensitive and only operates under anaerobic conditions, often found in dental plaque. glycolytic intermediates to suppress PFL, activate lactate dehydrogenase and switch end-product formation to lactic acid. This increases the rate at which glycolysis proceeds and protects the cell from toxic intermediates. Metabolic end-products fermentation
of sorbitol
When pyruvic acid is reduced to lactic acid, sufficient NAD’ is regenerated to maintain the oxidation-reduction balance of the cell. The reduction of pyruvic acid to ethanol generates exactly twice as much NAD+. This means that when glucose is low, one-half of the available pyruvic acid can be converted to acetic acid. This reaction is favourable at low substrate concentrations because it yields a further ATP. Formic acid is produced as a byproduct of both reactions. Sorbitol is transported intostrep. mutans by an induced sorbitol-PTS. Inside the cell sorbitold-phosphate is dehydrogenated to fructosed-phosphate before it enters the Embden-Meyerhof pathway. This conversion generates NADH2. Thus, for each sorbitol molecule metabolized, an extra NADH, is produced compared with glucose. This means that, regardless of the substrate concentration, when sorbitol is being metabolized, the only end-products of anaerobic glycolysis are formic acid,
266
J. Dent. 1991;
19: No. 5
ethanol and acetic acid. If lactic acid were to be produced, insufficient NAD+ would be regenerated and the oxidationreduction balance of the cell would be compromised. The PFL-catalysed reduction of pyruvic acid is slower than that oflactate dehydrogenase, which explains why the rate of acid production from sorbitol is lower than that from glucose or sucrose. When Strep. mutans is metabolizing sorbitol it can convert the pyruvic acid to lactic acid but only in aerobic conditions. This is because it has NADH oxidase. However, such aerobic conditions are unlikely to be present in dental plaque. This raises an important pointin vitro comparisons of acid production by organisms such as Strep. mutans from sorbitol should be performed under anaerobic conditions in order that both the rate and type of acid produced reflect more closely the situation found within plaque. Furthermore, it may be very misleading to extrapolate from an in vitro pure culture situation to that of a mixed microbial community in vivo.
MICROBIAL
ADAPTATION
TO SORBITOL
Microbial populations, especially mixed populations, are well known for their ability to respond to environmental changes. First, a microorganism can acquire genetic information either spontaneously by mutation or by transfer of genetic material from other, competent organisms. Secondly, microorganisms can change their phenotype by a process known as enzymic induction. Finally, the whole microbial population, whether it is a mixed population or composed of a single species, may alter to meet the challenge. This whole population response may occur as a result of genetic or induced changes in individual members, or it may simply reflect the diversity of phenotypes already present in the environment. The oral microbial population is a particularly good example of this process, and fluctuations in the proportions of Strep. mutans and lactobacilli in response to changes in intake of dietary carbohydrates are well documented (Newbrun, 1989). The well-established ability of some oral microorganisms, especially mutans streptococci and lactobacilli, to ferment sorbitol, has prompted suggestions that frequent or long-term use of sorbitol-containing products will result in adaptive changes in the oral flora (Birkhedet al., 1984, 1987). Concern has been expressed that these changes will result in a flora with an increased sorbitolfermenting capability. Such changes might increase the low cariogenicity of sorbitol to unacceptable levels. There is no evidence in the scientific literature that genetic changes in the oral flora have occurred in response to either long-term or frequent use of sorbitol. Those changes which have been reported have been due to enzyme induction and subsequent shifts in the balance of the microbial population. The problem has been investigated by measuring changes in acid production in
dental plaque and changes in the microbial population of plaque. These changes have been measured following both short- and long-term sorbitol adaptation periods, mostly in normal healthy human subjects. A limited amount of information is available on the adaptation of oral flora in certain ‘at-risk’ groups, such as individuals suffering from hyposalivation, and diabetics with reduced dietary sugar. There is very little information available concerning a potential increase in caries risk, probably because of the numerous scientific and technical problems involved and the cost.
Review of studies in acid production
investigating changes in dental plaque
Birkhed et al. (1978) measured acid production by plaque in vivo andin vitro in 18 subjects who had been exposed to
10 ml of 10 per cent sorbitol solution six times each day for 6 weeks. Exposure of the plaque to 10 per cent sorbitol in viva caused lower pH values after sorbitol adaptation, which was statistically significant 5, 10, 20 and 30 min after rinsing. Although the pH values were statistically significantly lower, at no time did they approach the critical pH for demineralization. The greatest difference found was only 0.33 pH units which occurred at 20 min, and the lowest pH achieved by sorbitol-adapted plaque was 6.67. The in vitro acidogenicity was measured by removing plaque from several sites and homogenizing it in phosphate buffer to which was added either glucose or sorbitol. The rate of acid production was measured by titrating the incubation mixture with alkali. The amount of acid produced from sorbitol after sorbitol adaptation was significantly greater (P < 0.001) but was still only approximately 30 per cent of the rate of acid production from glucose under the same conditions. The in vivo effect the sorbitol and xylitol on homogenized plaque collected from long-term users of products containing both xylitol and sorbitol was investigated by Makinen and Virtanen (1978). The periods of use exceeded 4 years. The pH of the homogenized plaque when it was incubated with sorbitol was lower than when it was incubated with xylitol. This effect only achieved statistical significance after 4 h incubation which reflects the low rate of acid production from sorbitol. It is apparent, therefore, that frequent use of sorbitolcontaining products over a 4.5-year period did not result in greater plaque acid production from sorbitol in vitro compared with xylitol. However, the subjects’ plaque in this study was also conditioned to xylitol so there is an assumption that no adaptation to xylitol had occurred. Significant amounts of acid were produced from sorbitol in the incubation mixtures after 22 h but this is of little relevance to the in vivo situation. Birkhed et al. (1979) looked at the effects of frequent sorbitol consumption throughout a 3-month period on both in vivo andin vitro acid production by plaque. Thein
Hogg and Rugg-Gunn:
viva acid production from sorbitol after the adaptation period was significantly greater (P = 0.05) but the lowest pH value reached was about 6.75, substantially higher than the critical pH for demineralization. The rate of acid production by homogenized plaque in vitro was not significantly different from that recorded before the adaptation period. The pre- and post-sorbitol adaptation acid production rates from sorbitol in this study were, in fact, remarkably similar and were only 15.4 and 18 per cent, respectively, of the production rate when glucose was fermented. Topitsoglou et al. (1983) looked at the effect of sorbitol adaptation on the pH changes within dental plaque and on the amount of titratable acid produced when plaque was exposed to sorbitol in vitro. This was a double blind cross-over trial in which the subjects refrained from oral hygiene for 4 days during which they chewed either sorbitol gum, xylitol gum or xylitol/sorbitol gum 10 times each day. Overall, the in vivo pH change of sorbitoladapted plaque, when exposed to sorbitol, was greater than xylitol-adapted plaque exposed to sorbitol. The increase in acid reached statistically significant amounts after lo,20 and 30 min when the subjects chewed sorbitol gum, and at 5 and 10 min but not 20 and 30 min when the mouth was rinsed with a 25 per cent sorbitol solution. However, these increases were only very small. The difference in pH between sorbitol-fermenting sorbitoladapted plaque and xylitol-adapted plaque was never greater than 0.34 pH unit. Sorbitol-conditioned plaque exposed to sorbitol in vivo was always above pH 6.5. The rate of acid production by sorbitol-adapted plaque was compared to that of xylitol-adapted plaque, but there was no comparison with pre-adapted plaque. Using sorbitol as the substrate, sorbitol-adapted plaque produced acid at a significantly greater rate (P < 0.01) than xylitol-adapted plaque. However, similar increases occurred when glucose was used as the substrate so at least some of the difference in acid production by sorbitol-adapted plaque, compared to xylitol-adapted plaque, was due to a lower metabolic activity of the xylitol-adapted plaque. It is, therefore, not possible to attribute all the change in rate of acid production in this study to sorbitol adaptation. Sdderling et al. (1989) measured interdental plaque pH response to sorbitol at four different sites using a palladium touch electrode. Volunteers adapted their oral flora to either sorbitol or xylitol or a mixture of both polyols by chewing two sticks of appropriate gum five times each day for 2 weeks. The acidogenic response of plaque to sorbitol was small in all three groups, and the changes induced by the 2-week adaptation period were also small and not statistically significant. Birkhed et al. (1990) studied in vivo plaque pH changes to a sorbitol challenge and acid production activity in plaque from sorbitol, in selected subjects who had been consuming sorbitol for at least 2 years at a frequency of intake of at least four times each day. The in vivo fall in plaque pH after rinsing with 10 per cent sorbitol solution was minor and not statistically significant. The lowest pH
Possible intra-oral
metabolism
of sorbitole
267
values were between 6.4 and 7.3, which were similar to those seen in individuals who had rinsed their mouths with sorbitol solutions six times each day for 4 weeks (Birkhed et al., 1979). Thus no differences in acid production in plaque were apparent when short- and long-term sorbitol consumers were compared. by homogenized The in vitro rate of acid production plaque from sorbitol was 30.7 per cent of that from glucose. Short-term sorbitol adaptation studies gave a similar rate (Birkhed et al., 1978) suggesting that there is no progressive increase in the rate of acid production attributable to long-term sorbitol consumption.
Discussion production
of plaque pH and acid activity studies
Research has shown that frequent use of sorbitol can lead to a greater in vivo pH response when the plaque is subsequently challenged with sorbitol. The increase in plaque acid production was statistically significant in some cases (Birkhed er al., 1978, 1979; Topitsoglou et al., 1983) but not in others (Soderling et al., 1989; Birkhed et al., 1990). However, even when differences were statistically significant, the increase in acid production was very small and the plaque pH did not approach the critical pH for demineralization. The studies which measured the rate of acid production plaque in vitro (Birkhed et al., 1978, 1979; Topitsoglou ef al., 1983; Birkhed et al., 1990) have to be interpreted with some caution because the conditions of measurement were not similar to those in in viva plaque studies in two main respects. First, the presence of oxygen in the in vitro experiments should, theoretically, cause an increase in the amount of lactic acid produced from sorbitol but not from glucose which was used as a reference. This could mean that the acid produced by plaque microorganisms from sorbitol was being overestimated. However, it should be noted that Kalfas and Birkhed (1986) have reported that the rate of acid production by pure cultures of Strep. mutans from sorbitol under aerobic conditions is very much lower than that under anaerobic conditions, which suggests that acid production from sorbitol could actually be underestimated in the above studies. Plaque microbial communities are, however, extremely complex and, in the same study, Kalfas and Birkhed (1986) showed that there was, in fact, little difference in acid production when mixed suspensions of plaque microorganisms were examined. Nevertheless, there have been few studies in this area and prudence dictates that the results obtained in the presence of oxygen should be treated with some caution. Second, the incubating mixture in these studies was kept at a constant pH 6.8 throughout. This would have the effect of maximizing the rate of acid production from sorbitol compared to that from glucose, because sorbitol fermentation from mixed oral bacteria is more sensitive to lower pH (Kalfas et al., 1990a).
268
J. Dent 1991; 19: No. 5
Review of studies in plaque flora
investigating
changes
Harjola and Liesmaa (1978) measured the change in an index of lactobacilli concentration in 5.5normal children divided into a group consuming xylitol and sorbitol, a group consuming sugar and a non-sugar group. The experimental period was 2 weeks and the concentration of oral lactobacilli was measured using the Dentocult (L) system. The results were compared with baseline values measured before the start of the experimental period. High variability in the numbers of lactobacilli cultured from the saliva of children in all three groups made statistical analysis of the observed changes difficult. Nevertheless, there was a marked reduction in the numbers of lactobacilli in the group using the polyols, which suggests that sorbitol-fermenting species were not favoured. However, since both xylitol and sorbitol were used together it is not possible to exclude an effect of xylitol in suppressing lactobacilli growth. Birkhed et al. (1979) looked at the effect of 3 months’ frequent consumption of Lycasin, maltitol, sorbitol and xylitol on the numbers of total streptococci, Strep. mutans and lactobacilli in dental plaque. Few changes were found in samples taken before and after the test period. In the sorbitol group, a significant decrease in the number of lactobacilli was found (P < 0.05). In this study, as in others involving measuring relative numbers of oral bacteria, there were large variations in numbers between samples. It is possible that such variations concealed an effect of sorbitol consumption. The authors also point out that an increase in the frequency and duration of consumption of a sorbitol-containing product may produce measurable changes in the oral flora. The study by Rateitschak-Pl-iiss and Guggenheim (1982) differs from other investigations in that the subjects’ carbohydrate intake was limited to the substances under test. There were four experimental groups, with six subjects in each group. The groups consumed candies containing either sorbitol, xylitol or sucrose. The fourth group consumed no candies. Each subject participated in each group for 4 days, allowing 9 days’ recovery between each period. Counts were made of the number of bacteria which fermented sorbitol and xylitol, and this was expressed as a percentage of the total number of viable bacteria. A significantly greater percentage of sorbitol-fermenting bacteria were isolated from the sorbitol group compared with the sucrose group (P < 0.001). However, significant increases in the proportion of sorbitol-fermenters were also found in the xylitol group (P < 0.05) and in the sucrose group (P < 0.05). The study also found a significant increase in the proportion of xylitol-fermenting bacteria in the xylitol group when compared with the sucrose group (P < 0.05). Although significant differences were found, the authors pointed to the fact that only a small fragment of the flora was able to ferment sorbitol and an even smaller proportion could ferment xylitol.
Loesche et al. (1984) compared the effect of chewing gums containing xylitol, sorbitol/mannitol and fructose on oral microbiology in a parallel group study. Counts of Strep. mutans were made from samples of saliva and occlusal and approximal plaque before the chewing period, and after 2 and 4 weeks. At 4 weeks gum chewing ceased and the levels of Strep. mutans were monitored 2 and 4 weeks later. Sorbitol had no effect on the salivary levels of Strep. mutans. In occlusal plaque, the total count of Strep. mutans was unaffected, but the relative proportion increased although this was not statistically significant. In approximal plaque, the proportion of Strep. mutans increased significantly after 4 weeks (P < 0.05) and then decreased significantly (P < 0.05) after chewing of sorbitol gum ceased. No changes were found in salivary levels of lactobacilli, or in approximal or occlusal plaque lactobacilli levels, or proportions of Strep. sanguis or lactobacilli. Wennerholm and Emilson (1989) measured salivary levels of Strep. mutans, Strep. sanguis, Strep. salivatius and lactobacilli in patients attending a smoking cessation clinic in Gothenburg. Twenty-seven patients chewed a nicotine-containing sorbitol-sweetened (19 per cent sorbitol) chewing gum when they felt a desire for nicotine. The subjects used about five sticks of chewing gum each day. Salivary bacterial levels were measured at baseline, after 1.5 months and finally after 3 months of treatment. A small decrease in the salivary levels of all the bacterial species was found, but this was not statistically significant when compared to the baseline values. Sijderlinget al. (1989) compared the effect of 2 weeks use of sorbitol, xylitol and sorbitol/xylitol chewing gums on salivary and plaque levels of Strep. mutans. They stated that both plaque and salivary levels of Strep. mutans generally increased with sorbitol use, but detailed data were not presented and were not accompanied by statistical analyses. Birkhed et al. (1990) reported that in subjects who had consumed sorbitol-containing products at least four times each day for 2 years, there was a positive correlation (P < 0.05) between the number of mutans streptococci in saliva and the frequency of sorbitol intake. The sorbitolfermenting microorganisms isolated were mutans streptococci, sanguis-mitior-like organisms and Strep. salivarius.
Linke and Castle (1990) isolated sorbitol-fermenting bacteria from the dental plaque of people using sorbitol products. Most of these bacteria produced significant amounts of acid, which was defined as a decrease in the pH of the culture to approximately pH 4.5-4.8, but this was over an incubation period lasting 5 days. Sorbitolfermenting strains isolated were identified as Strep. acidominimus, Strep. sanguis, Strep. mutans and Micrococcus luteus. The authors reported that a large number of sorbitol-metabolizing bacteria were present in the plaque of patients preferentially using sorbitol-containing sweets, but do not relate the numbers to the total number of
Hogg and Rugg-Gunn: Possible intra-oral metabolism of sorbitole
bacteria cultured. They go on to say that ‘The bacteriological results suggest that frequent consumption of sorbitol-containing products, with moderate as well as heavy use, will lead to a shift in oral ecology, numerically favouring sorbitol-degrading bacteria’. No statistical treatment of the data was presented in support of this statement.
Discussion
of bacteriological
studies
There is very little evidence of statistically significant changes in the oral flora due to sorbitol consumption either in the short- or long-term. This is to be expected because both sorbitol uptake, and metabolism in oral streptococci, have to be induced. These occur in the presence of the substrate but are repressed by the presence of glucose (Slee and Tanzer, 1983), which is transported into the cell and metabolized by constitutive systems. In practical terms, this means that even small amounts of glucose in the diet will effectively prevent sorbitol utilization regardless of its concentration. In view of this, the paper by Rateitschak-Pliiss and Guggenheim (1982) is important because, in this study, the dietary sources of carbohydrate of the subjects were limited to the test sweeteners, and significant increases in sorbitol- and xylitol-fermenting strains were found after sorbitol- and xylitol-conditioning, respectively. Even the results of this study were equivocal because sorbitolfermenting strains also increased significantly after xylitol and sucrose conditioning, even in the absence of other dietary carbohydrates. Difficulties in demonstrating increases in sorbitol-fermenting strains, even in carefully controlled experiments such as this, may be due partly to endogenous salivary glucose levels (Jenkins, 1978) which are higher than the levels required for repression of sorbitol fermentation (Slee and Tanzer, 1983). Review of studies investigating sorbitol metabolism in the plaque of special ‘at-risk’ groups There have been suggestions that sorbitol fermentation may present a problem for particular groups such as xerostomic patients, who frequently consume sorbitolcontaining products as saliva stimulants and are known to harbour elevated numbers of mutans streptococci. Furthermore, they will lack much of the microbial control mechanisms present in people with normal saliva flow, and especially much of the capability for buffering acids in saliva. Two recent studies have examined the effects of sorbitol-conditioning on the oral environment. Kalfas et al. (1990b) selected 12 people with low and 11 people with normal salivary flow rates. Both groups rinsed frequently with 10 per cent sorbitol solution for 4 weeks. Sorbitolfermenting bacteria increased significantly, as did the proportion of mutans streptococci, in plaque samples taken from both groups after conditioning. Plaque acid
269
production from sorbitol increased in both groups, but more so in the group with low salivary flow rates. Values in three out of 12 subjects were less than pH 5.7, and in five out of 12 subjects, values were less than pH 6.0. Bacteriological studies of the group with low saliva flow revealed that the sorbitol-fermenting flora was almost exclusively composed of the genera Streptococcus, Lactobacillus and Actinomyces (Kalfas and Edwardsson, 1990). Another at-risk group could be patients on long-term low-sugar diets, such as some diabetics. Giilzow et al. (1990) examined this problem in 25 subjects with juvenile diabetes mellitus. Saliva samples from this group and from the same number of healthy individuals, were incubated under aerobic and anaerobic conditions, and the respiration rate of the bacteria measured using a Warburg respirometer. Sorbitol was metabolized faster and to a greater extent by salivary microorganisms in the diabetic group. Although this is evidence that some adaptation may have occurred, it is difficult to relate these findings, which are presented in terms of oxygen consumption and carbon dioxide evolution, to in viva plaque
PH. Review
of relevant
animal studies
Only one animal study appears to have been performed with the object of investigating sorbitol adaptation, but this was a well-designed and controlled experiment carried out over 2 years using macaque monkeys (Cornick and Bowen, 1972).In viva acid production by plaque from sorbitol was small at baseline and no increase was found after 8 weeks or 2 years when large amounts of sorbitol were consumed daily. Similarly, there was no increase over an g-week period in the number of sorbitolfermenting microorganisms. Information on the microbial composition of the plaque ceased at 8 weeks.
CONCLUSIONS The possibility of dental plaque adaptation to sorbitol has been examined in healthy, human subjects in a variety of studies by measuring changes in plaque acidogenicity in viva, plaque acid production activity in vitro, and population changes in the microbial composition of plaque. In addition, there has been one study which has looked at sorbitol adaptation in people with low salivary flow rate, one study which has looked at adaptation in diabetics on low-sugar diets, and one animal study. 1. Studies of plaque acid production in vivo have not provided consistent evidence of plaque adaptation to sorbitol There have been five in viva human plaque pH studies. In three of these studies, although there was a statistically significant increase in acid production by sorbitolconditioned plaque, the amount of acid produced was
270
J. Dent.
1991; 19: No. 5
unlikely to be sufficient to affect teeth clinically. The remaining two studies did not show any significant increase in plaque acid production. In one animal experiment, there was no increase in plaque acid over 2 years of frequent sorbitol exposure. 2. There is no firm evidence that long-term use of sorbitol results in increased acid production activity by plaque in vitro There have been five studies of in vitro acid production activity by sorbitol-conditioned plaque. One study showed an increase in the rate of acid production by sorbitol-adapted plaque, one showed an increase which could not be wholly attributable to sorbitol, two did not show any increase, and one showed that there was no progressive increase in acid production rate in the long term. 3. There is very little evidence in the current literature of an alteration in the microbial ecology of dental plaque which could be attributed to sorbitol consumption in the short or long term There have been eight studies of the effect of sorbitol on the microbial ecology of plaque and, although some changes have been reported, they were either not statistically significant, or could not be attributed to sorbitol. No changes found
in the
microbial
in a long-term
animal
composition
of plaque
were
experiment.
4. There is some evidence that frequent use of sorbitol may present a small problem in people with low salivary flow One study has shown that plaque acid production from sorbitol increased after short-term (Cweek) use of sorbitol, and that pH values reached levels close to the critical pH. This may be partly due to a decrease in the natural salivary buffering capacity inherent in this condition. 5. In the light of current evidence, frequent or long-term use of sorbitol is unlikely to present any increased risk of dental caries in normal people References Birkhed D. and Bar A (1991) Sorbitol and dental caries. World Rev. Nutr. Dietet 65, l-37. Birkhed D., Edwardsson S., Svensson B. et al. (1978) Acid production from sorbitol in human dental plaque. Arch. Oral BioJ. 23, 971-975. Birkhed D., Edwardsson S., Ahldtn M.-L. et al. (1979) Effects of 3 months frequent consumption of hydrogenated starch hydrolysate (Lycasin), maltitol, sorbitol and xylitol on human dental plaque. Acta Odontol. Stand. 37, 103-l 15.
Birkhed D., Edwardsson S.. Kalfas S. et al. (1984) Cariogenicity of sorbitol. Swed. Dent. .I 8, 147-154. Birkhed D., Svenstiter G., Kalfas S. et al. (1987) The risk of adaptation of the oral microflora to sorbitol. Dtsch. Zahnarztl. Zeit. 42, S141-S144. Birkhed D., Svensater G. and Edwardsson G. (1990) Cariological studies of individuals with long-term sorbitol consumption. Caries Res. 24, 220-223. Boyar R. M. and Bowden G. H. (1985) The microflora associated with the progression of incipient caries lesions in teeth of children living in a water-fluoridated area. Caries Res. 19, 298-306. Comick D. E. A. and Bowen W. H. (1972) The effect of sorbitol on the microbiology of the dental plaque in monkeys (Macaca irus). Arch. Oral Biol. 17, 1637-1648. Gibbons R. J. and van Houte J. (1975) Dental caries. Ann. Rev. Med. 26, 121-136. Gillzow H.-J., Kary H. and Schiffner U. (1990) Zum adapataionsverhalten von mikroorganismen der menschlichen mundhole gegenuber sorbit. Dtsch. Zahnarztl. Zeit. 45, 90-92. Hamada S. and Slade H. D. (1980) Biology, immunobiology, and cariology of Streptococcus mutans. Microbial. Rev. 44, 331-384. Hardie J. M., Thompson P. L., South R. J. et al. (1977) A longitudinal epidemiological study on dental plaque and the development of dental caries-interim results after two years. J. Dent Res. 56, (Special Issue C), C90-C98. Harjola U. and Liesmaa H. (1978) Effects of polyol and sucrose candies on plaque, gingivitis and lactobacillus scores. Observations on Helsinki school children. Acta Odontol. Stand. 36, 237-242. Jenkins G. N. (1978)The Physiology and Biochemistry of the Mouth, 4th edn. Oxford, Blackwell Scientific. Kalfas S. and Birkhed D. (1986) Effect of aerobic and anaerobic atmosphere on acid production from sorbitol and suspensions of dental plaque and oral streptococci. Caries Res. 20, 237-243. Kalfas F. and Edwardsson S. (1990) Sorbitol-fermenting predominant cultivable flora of human dental plaque in relation to sorbitol adaptation and salivary secretion rate. Oral Microbial. Immunol. 5, 33-38. Kalfas S., Maki Y.. Birkhed D. et al. (1990a) Effect of pH on acid production from sorbitol in washed cell suspensions of oral bacteria. Caries Res. 24, 107-l 12. Kalfas F., Svensater G., Birkhed D. et al. (1990b) Sorbitol adaptation of dental plaque in people with low and normal salivary-secretion rates. J. Dent. Res. 69, 442-446. Linke H. A. B. and Castle M. (1990) Isolation of acid producing sorbitol adapted bacteria from dental plaque using selective agar media. Microbios 61, 39-48. Loesche W. J., Earnest R., Grossman N. S. et al. (1984) The effect of chewing xylitol gum on the plaque and saliva levels of Streptococcus mutans. J. Am. Dent. Assoc. 108, 587-592. Makinen K. K. and Virtanen K. K. (1978) Effect of 4.5-year use of xylitol and sorbitol on plaque. J. De&. Res. 57, 441-446. Marsh P. D., Featherstone A., McKee A. S. et al. (1989) A microbiological study of early caries of approximal surfaces in schoolchildren. J. Dent Res. 68, 1151-1154. Newbrun E. (1989) Substrate: diet and caries. In: Cariology, 3rd edn. Quintessence, Chicago, pp. 99-l 34. Rateitschak-Pliiss E. M. and Guggenheim B. (1982) Effects of carbohydrate-free diet and sugar substitutes on dental plaque accumulation. J. Clin. Periodontal. 9, 239-251.
Zissis et al.: Accuracy
Slee A. M. and Tanzer J. M. (1983) The repressible metabolism of sorbitol (D-glucitol) by intact cells of the oral plaque-forming bacterium Streptococcus mutans. Arch. Oral Biol. 28, 839-845. Siiderling E., MBkinen K. K., Chen C.-Y. et al. (1989) Effect of sorbitol, xylitol and xylitol/sorbitol chewing gums on dental plaque. Caries Res. 23, 378-384.
Forthcoming Original
Research
and stability
Reports
The radiopacity of composites compared with human enamel and dentine G. Willems, N. J. Noack, S. Znokoshi, P. Lambrech ts, B. Van Meerbeek, M. Braem, J. G. Roulet and G. Vanherle
and endodontics
A. D. Walmsley, W. R. E. Laird and P, J. Lumley
Microleakage in porcelain laminate veneers A. Zaimoglu and L. KaraaEaqlioElu In vitro evaluation of two microleakage D. G. Charlton and B. K. Moore
detection tests
Marginal adaptation of four touth-coloured inlay systems in vivo B. Van Meerbeek, S. Znokoshi, G. Willems, M. J. Noack, M. Braem, Lambrechts, J-F. Roulet and G. Vanherle Reinforcement of poly(methylmethacrylate) polyethylene fibres
P.
with ultra-high-modulus
D. L. Gutteridge
Clinical bone densitometric study of mandibular atrophy using dental panoramic tomography K. Horner and H. Devlin
The effects of digital grey-scale approximal carious lesions
modification
base materials
271
Topitsoglou V., Birkhed D., Larsson L.-A. et al. (1983) Effect of chewing gums containing xylitol, sorbitol or a mixture of xylitol and sorbitol on plaque formation, pH changes and acid production in human dental plaque. Caries Res. 17,369-378. Wennerholm K. and Emilson C.-G. (1989) Effect of sorbitoland xylitol-containing chewing gum on salivary microflora, saliva and oral sugar clearance. Stand. J. Dent. Res. 97, 257-262.
Articles
Ultrasound in dentistry. Part 1. Periodontology
of denture
on the diagnosis
of small
E. H. Verdonschot, J. M. C. Kuijpers. B. J. Polder, M. H. De Leng-Worm and E. M. Bronkhorts
