The production of l (+) and d (−) lactic acid and volatile acids by human dental plaque and the effect of plaque buffering and acidic strength on pH

Archs oral Bid. Vol. 17,pp. 537-545, 1972. Pergamon Press.Printedin GreatBritain.

THE PRODUCTION OF L(+) AND D(-) LACTIC ACID AND VOLATILE ACIDS BY HUMAN DENTAL PLAQUE AND THE EFFECT OF PLAQUE BUFFERING AND ACIDIC STRENGTH ON pH DOROTHY A. M. GEDDES

Department

of Oral Physiology, University of Newcastle Newcastle upon Tyne, NE2 4A.I

upon

Tyne,

Summary-Both L(+) and D(-) lactate was identified from all plaques after in vitro incubation with sucrose. When three subjects were repeatedly sampled, the L(+): D(-) lactate ratios varied between 1.1 and 3 ‘9, the inter-subject variation being greater than the intra-subject variation. Acetic, propionic and n-butyric acids were also identified from all samples. The total 1actate:volatile acid ratios varied between 1.1 and 1.9. The poor correlation between final pH and total identified acid may be due to variations in plaque-buffering from sample to sample, the presence of unidentified acids, possibly formic, and the effect of the acidic strengths of the acids formed. Addition of equivalent amounts of the individual organic acids to replicate samples of plaque produced consistently different pH decreases within the physiological range, lactate producing a significantly lower pH than the volatile acids. INTRODUCTION DENTAL

plaque is of complex composition, considering the bacterial component alone, although the cultivation of 27 different types of organisms by HEMMENS and her coworkers (1946) emphasises the large number of species involved; even that extensive analysis was by no means complete. One feature of microbial ecosystems is that an exogenous nutrient may be utilized by some types of bacterium with production of waste products which, in turn, serve as nutrients for other species (HUNGATE, 1962). For example, lactic acid released as an end-product of sugar fermentation by lactobacilli and certain streptococci can be utilized by Veillonella and Peptostreptococcus species and converted to acetic and propionic acids (HOBSON, 1969). Microorganisms can produce lactic acid in the L( +) and D(-) forms : some species will form only the L(+) and others only the D(-) isomer while some produce the racemic mixture. The optical activity of the lactate formed varies with the organism, depending upon the stereospecificity of the lactate dehydrogenase and upon whether the organism possesses lactate racemase, an enzyme which reversibly converts D(-) lactate to the L(+) form (ROSE, 1968). A shift in the proportion of L(+) or D(-) lactate-forming organisms without a concomitant shift in the proportion of L(+) or @-)-utilizing organisms could lead to an accumulation of lactate and a lowering of pH (HOBSON, 1969). If this occurred in the human mouth, it could influence the cariogenicity of plaque. Previous studies on acid production from plaque have not distinguished between the lactate isomers (MUNTZ, 1943; MOORE et al., 1956; RANKE, BRAMSTEDT and NAUJOKS, 1964; GILMOUR and POOLE, 1967; GEDDES and GILMOUR, 537 A.O.B. 17/3-L

538

D. A. M.

GEDDES

1969). Volatile acids are also produced by plaque organisms and, as lactic, acetic and propionic acids have different pK values, the type of acid present may affect the final pH of plaque. MATERIALS

AND

METHODS

Plaque fermentations

Plaque samples were taken with a nickel microspatula from all the enamel surfaces of young adults who had not brushed their teeth for 24 hr. They were weighed and immediately incubated aerobically in 5 per cent sucrose in 7.0 mM phosphate buffer at pH 7.0 at a concentration of 1 mg wet wt. per 20 ~1 of medium. After 15 min at 37”C, the samples were centrifuged (3000 g for 15 min) the supernatant retained, the pH measured potentiometrically using a glass combination microelectrode (Beckman 39030) and the individual acids analysed. Acid analysis

The lactate isomers were determined enzymically using a modification of a method for L(+) lactate (HOHORST,1963). Duplicate 25 ~1 samples, standards and reagent blanks were added to 3 ml of reaction mixture of the following composition: two volumes of buffer (0.5 M glycine, 0.4 M hydrazine at pH 9.0) to one volume aqueous NAD + solution (2 mg/ml); to every 24 ml of buffer-NAD+ solution was added 100 ~1 (500 rg) of enzyme preparation (Boehringer, Mannheim L(+) or D(-) lactate dehydrogenase). Because the D(-) dehydrogenase preparation had a lower specific activity (300 IU at 25°C) and optimum pH than the L(+) dehydrogenase (360 IU at 25”C), the optimum incubation time was longer for the D(-) assay. After incubation at 25°C (1 hr for L(+) and 3 hr for D(-) LDH), the absorbancy at 366 nm was measured using a glass cuvette (1.0 cm light path) employing a Unicam SP 500 ultraviolet spectrophotometer, the absorbancy of the reagent blank was subtracted from each standard and sample. Standard curves for L(+) and D(-) lactate were determined for each assay. From the same supernatant, the volatile acids, except formic, were determined as free acids, by gas-liquid chromatography (G.L.C.) employing a Pye 104 chromatograph using a 2 per cent phosphoric acid on a Phase Pak Q column with formic acid vapour in the Nz carrier gas to prevent solute-ghosting (GEDDESand GILMOUR,1970). The analyses were isothermal at 180°C using a flame ionization detector; standard solutions of mixtures of authentic acids were chromatographed to give absolute calibration for qualitative and quantitative analysis. identification of formate was performed by a modified spot test (FEIGL. 1947). Plaque samples were vacuum-distilled (TYLER, 19?‘0) and to tde distillates 5d ~1 2 N HCl and 10 mg o? powdered magnesium were added followed by 1.0 ml 72 per cent H,SO, and 5-O mg of chromotropic acid. The mixture was heated in a water bath at 70°C for 10 min; violet-pink colour indicated the presence of formic acid. The limit of detection was 3.0 pg. Acetic, propionic, n-butyric and lactic acids did not interfere. Plaque was collected as described above. Samples (15.0 mg wet wt.) were placed either (Series 1) into microfermentation tubes or (Series 2) into the well of a one-drop electrode (Beckman 40316). To the plaque, 10 pl of a standard acid solution was added and the pH decrease measured. In order that the pH decrease would be within the physiological range, in Series 1, an inorganic acid was used in the same weak phosphate buffer as was employed for fermentation and in Series 2 an organic acid without buffer was employed. Acidic strength

Plaque was sampled from 20 subjects, pooled into a small-volume, plastic, humid container and kept on ice for not more than 30 min. Replicate plaque samples (15.0 mg wet wt.) were placed in the one-drop electrode and standard 0.3 M solutions of acetic, propionic, lactic and equimolar mixture were added and the pH decrease measured. Each acid was tested 5 times. RESULTS

In preliminary fermentation studies using samples of pooled plaque from 20 subjects and samples of individual plaques from four subjects, both L(+) and D(-)

mean

mean

mean

I.5 7.6 I.2 7.4

4.9 4.9 5.0

~0.5

5.8 5.1 5.6 5.7

7.9 7.8

7.7 I.9

6.5 5.5 5.9 6.0

3.6 3.8 3.9 3.8

3.4 2.2 2.0 2.5

4.0 5.6 3.8 4.5

4.4 4.4 5.1 4.6

3.8 4.6 4.6 4.3

4.8 5.0 2.9 4.2

3.8 3.9 3.4 3.7

2.8 4.2 3.3 3.4

Propionate D( -) Lactate Acetate (x 1O-5 m-mol acid/mg wet wt. plaque)

x low5 mmol/mg wet wt.

L( +) Lactate

5.0 5.3 4.9

5.3 4.6 4.6

Final pH

* Present in all samples but in concentration

D. C.

B. W.

A. T.

Subject

* * *

* * *

* * *

n-Butyrate

1.1 1.4 1.2 1.2

1.6 1.5 1.4 1.5

2.2 3.6 3.9 3.2

Lactate

1.1 1.1 1.1 1.1

1.6 1.2 1.9 1.6

1.7 1.1 1.2 1.3

Lactate: volatile

Molar ratios L(+):D(-)

TABLE1. ACID PRODUCTION FROM9 PLAQUESINCUBATED WITH 5 PER CENT SUCROSE

540

D.A.

M.

GEDDES

isomers of lactic acid were consistently identified. Samples from three of the subjects were then studied individually and repeatedly on different days. Plaques were routinely collected 2-24 hr after breakfast, weighed and incubated immediately. Greater amounts of L(+) than D(-) lactic acid were found in each sample (Table 1). For all three subjects, the L(+) isomer gave less day-to-day variation. Subjects B. W. and D. C. had similar L(+):D(-) ratios with a small individual day-to-day variation, whereas subject A. T. consistently gave a higher ratio and greater variation. From Lfolatile acid analysis, greater amounts of acetic than propionic acid were found from each sample, only traces of n-butyric acid being detected. In each case, the total lactate exceeded total volatiles; from subject D. C. the ratio was the same on all 3 days. The total acid identified was compared with the corresponding final pH values (Table 2). The data from the 9 plaques have been ranked in decreasing order of pH. There is poor correlation. For subject A. T., the extreme case, equivalent amounts of total acid were obtained from plaques with final pH values differing by 0.7 units. TABLE2. pH AND TOTAL IDENTIFIED ACID FROM9 PLAQUESAFTERINCUBATIONWITH 5 PER CENTSUCROSE

Subject

PH

Acid*

A. B. B. D. B. D. D. A. A.

5.3 5.3 5.0 5.0 4.9 4.9 4.9 4.6 4.6 4.9 0.25

17.7 17.8 17.6 19.8 18.0 23.7 22.8 18.9 17.8

T. W. W. C. W. C. C. T. T. Mean S.D.

* Total acid (mmol x 10-5/mg wet wt.) identified asacetic,propionicandlactic.

There are several possible explanations for this apparent anomaly. Firstly, other acids may have been produced but not identified. Secondly, the buffering capacity of the plaque samples may have varied between subjects and from day to day. Thirdly, the different strength of the organic acids produced might affect the final pH obtained. Considering the first hypothesis, the analytical techniques employed would have detected iso-butyric, n- and iso-valeric but not formic acid. The latter has a pK of 3.75 similar to that of lactic acid, pK 3 -86 at 25°C and is a recognized end-product of bacterial fermentations. Repeated attempts to identify formate from similar samples of both individual and pooled plaque by the spot-test method have proved inconclusive. In order to compare plaque buffering capacity, acid solutions were added to fresh samples of plaque. The decrease in pH produced upon the addition of the standard a_cid solutions, in amounts calculated to simulate that from plaque fermentation, was

L(+),D(-)

LACTIC AND VOLATILEACIDSAND

PLAQUE

BUFFERING

541

used as a measure of plaque buffering. The reproducibility of the method was determined using duplicate samples from the same plaque collection from the two subjects who produced a sufficient quantity to permit such duplication; the error was within the reproducibility of pH measurement, i.e. fO-03 units (Table 3). The procedure TABLE ~.PLAQUE ~HBEFORE ANDAFTERADDITION

OFSTANDARD ACID SOLUTION* TO DUPLICATEALlQUOTSOFPLAQUES

Subject

Initial pH

Final pH

* PH

A. T.

5.85 5.90

4.50 4.49

1.35 l-41

D. C.

5.95 5.95

4.62 4.63

1.33 1.32

* 10 ~1 O-3 M acid/l5

mg wet wt. plaque.

was repeated on plaques from each of the subjects sampled on different days. Within each series, the equivalents of acid added was constant, permitting variation in the buffering capacity of the plaques to be measured. The pH range from the two series covers that obtained from the plaque fermentations (Table 2). The plaque buffering capacity was similar in both series and varied between subjects by O-6 units and within subjects by 0 - 5 units (Table 4). TABLE 4. PLAQUE pH PRODUCED BY ADDITION OF lOpI STANDARD ACID SOLUTIONS TO 15 MC WET WT. OF PLAQUE

Series 1’ Subject D. C. B. W. D. C. A. T. B. w. B. W. D. C. A. T. A. T. Mean S.D.

Series 2t PH 5.60 5.40 5.30 5.30 5.20 5.10 5.10 5.10 5.00 5.20 0.15

Subject D.C. B. W. D. C. A. T. B. W. A. T. B. W. A. T. D. C. Mean SD.

PH 4.62 4-52 4.52 4.52 4.50 4.50 4.40 4.40 4.25 4.47 0.11

* 0.2 M HCI in 7-O mM phosphate buffer, pH 4.0. t O-3 M acetic acid in water, pH 3-O.

A third possible explanation for the lack of correlation in the fermentation experiments, the effect on plaque of the different strength of the organic acid produced, was investigated by pooling plaque from 20 subjects, taking replicate samples and adding a standard acid solution, assuming that only one type of acid had been produced. In

D.A.M.

542

GEDDES

this series, the acid molarity was kept constant and plaque buffering was assumed to be constant because aliquots of pooled plaque were used; the acidic strength was varied by using acids with different pK values. Lactic acid, as would be expected from its dissociation constant, produced the greatest decrease in pH giving a final pH 0.5 of a unit lower than the weakest acid, namely propionic (Table 5). TABLE 5. PLAQUE pH PRODUCED BY ADDITION OF VARIOUS STANDARD ACID SOLUTIONS* TOPOOLEDPLAQUE(MEANAND S.D. OF 5 SAMPLES)

Acids

pK at 25°C

Acetic

PH

APH

4.76

4.59 0.09

1.24 0.15

Propionic

4.87

4.65 0.08

1.13 0.09

D-L Lactic

3.86

4.15 0.05

1.66 0.18

Equimolar mixture

-

4.47 0.05

1.31 o-02

* 10 ~1 O-3 M acid/l5

mg wet wt. plaque.

The difference between the decrease in pH with lactic and with any of the other acids was statistically significant (P < 0.01). The difference between the pH decrease produced with the mixture and propionic was also significant (P < 0.01). Therefore, when the individual acids were present in concentrations equivalent to that of total acid from plaque (20 x 10e5 mmol acid/mg wet wt.), the type of acid produced could affect the final pH. DISCUSSION

In order to obtain sufficient plaque material to permit enzymic analyses of both lactate isomers and G.L.C. analysis of the volatile acids, the sampling technique employed inevitably resulted in disruption of the plaque structure and loss of intermicrobial relationship established during plaque development (GILMOUR and POOLE, 1967). Consequently, the fermentation patterns observed can only approximate the situation in vivo at the plaque enamel interface. The production of both isomers of lactic acid is in accordance with the fact that the majority of the most frequently isolated oral species of lactobacilli (ROGOSA et al., 1953) produce either L(+) lactic acid or the racemic mixture (ROGOSA and SHARPE, 1959). However, the proportion of lactobacilli in plaque is low (HEMMENS et al., 1946) and other lactic acid bacteria from plaque can also produce both the L(+) and the D(-) isomer. For instance, the cariogenic Streptococcus rnutans strain PKI has been shown to produce L( +) : D( -) lactate ratios of 2.3 and 1 - 3 from glucose

L(+), D(-)

LACTIC AND VOLATILE

ACIDSANDPLAQUE BUFFERING

543

under anaerobic and aerobic conditions respectively (YAMADAet al., 1970). The L( +): D(-) lactate ratios reported here indicate that either the L(+) isomer was predominantly produced or the D(-) isomer preferentially utilized. The subject-to-subject variation in total lactate production and L( +):D(-) lactate ratio may be due to subject variation in plaque microbial composition. The proportion of lactobacilli is generally higher in saliva than in plaque, however. SANDHAM and KLEINBERG (1970) concluded that only L( +) lactic acid was produced in their salivary sediment system. The conclusion was based on their finding that analyses of a number of samples both by an enzymic method IL(+) only] and by the less sensitive Barker and Summerson method [L(+) and D(-)] gave almost the same results. Absence of the D(-) isomer from salivary sediment would be evidence of the metabolic differences between the two biological materials. Formic acid produced from plaque was tentatively identified by MUNTZ (1943) and RANKE et al. (1964) and positively identified by TYLER (1971). Failure to demonstrate formate in this and in earlier studies (GEDDES and GILMOUR, 1969) can be attributed to inadequate methods of detection rather than absence of formate. Inter-subject variation in plaque buffering has also been reported by STRALFORS (1948) and can in part explain the lack of correlation between final pH and total identified acid (Table 2) found in the in vitro fermentations. The relative importance in vivo of these variations will, of course, be modified by the additional buffering action of the individual subject’s saliva. The addition to plaque of equimolar solutions of the different organic acids in concentrations equal to those found from plaque fermentation influenced the final pH. The effect of pH in controlling the type of microorganism predominating in mixed cultures is well known and moreover control by organic acids themselves has also been reported (see HOBSON, 1969). To what extent pH and the organic acid anions themselves regulate the proportion of bacterial types in plaque is not known. In relation to enamel dissolution even small differences in plaque pH such as have been demonstrated with lactic and the volatile acids could be important in relation to caries at the “critical pH” value (JENKINS, 1971). Additionally, in vitro studies indicate that dissolution of hydroxyapatite is also affected by the acid anion (BUONOCORE, 1961; GRAY, 1966). Ackrlowledgements-This work was supported by a grant from the Medical Research Council. I wish to thank Professor G. N. JENKINS for his encouragement and advice, Mrs. A. THOMPSON for her skilled technical assistance and the subjects for their cooperation. RBsum6-Les lactates L (+) et D (-) sont identifies dans tomes les plaques, apres incubation in vitro avec le saccharose. Le prtlevement rep&? chez trois sujets montfe des rapports de lactases L (+) : D (-) qui varient de 1, 1 a 3, 9; les variations inter-sujets sont plus &levees que les variations intra-sujets. Les acides acetiques, propioniques et n-butyriques sont egalement recherches dans tous les 6chantilIons. Les rapports totaux lactate acide volatil varient entre 1,l et 1,9. La correlation faible entre le pH final et les acides identifies peut etre like a des variations du pouvoir tampon des plaques, d’un echantillon a I’autre, a la presence d’acides non identifies, peut-&tre formique, et a l’effet

544

D. A. M. GEDDES des forces acidiques des acides form&s. L’adjonction de quantites Cquivalentes d’acides organiques, pour reproduire des tchantillons de plaques, provoque de facon constante des chutes variables de pH dans des proportions physiologiques: le lactate produit unc chute du pH plus significative que les acides volatils. in vitro-Inkubation von Plaques mit Rohrzucker wurde in aIlen Proben L (+) und D (-) Laktat gefunden. Wenn solche Proben bei 3 Personen wiederholt gesammelt und untersucht wurden, variierte dasverhal tnis zwischen L (;-) und D (-) Laktat von 1,l bis 3,9; dabei waren die Schwankungen zwischen verschiedenen Personen griil3er als dieVariabilit& der Ergebnisse beieinem Probanden. Weiterhin wurden in allen Proben Essigsaure, Propionslure und n-Butters&ire identifiziert. Die Verhlltniszahlen zwischen Laktat und fliichtigen Sauren schwankten zwischen I,1 und 1,9. Die schwache Korrelation zwischen dem pH am Ende der Inkubation und der gesamten Sauremenge diirfteauf die von Probezu Probe unterschiedliche Plaquepufferung, auf die Gegenwart nichtidentitizierter Sauren (wie z.B. moglicherweise Ameisensaure) und auf die Saurestarke der gebildten Sauren zurtickzufiihren sein. Der Zusatz aequivalenter Mengen der einzelnen organischen Sluren zur Regeneration der Plaqueproben verursachte sehr unterschiedliche pH-Abfalle innerhalb des physiologischen Bereiches, wobei Laktat ein signifikant niedrigeres pH herbeiftihrte als die fltichtigen Sauren. Zusammenfassung-Nach

REFERENCES BUONOCORE,M. G. 1961. Dissolution rates of enamel and dentine in acid buffers. J. dent. Res. 40, 561-570. FEIGL, F. 1947. Qualitative Analysis by Spot Tests, Third Ed, Chap. 6, pp. 397. Elsevier, New York. GEDDES,D. A. M. and GILMOUR,M. N. 1969. The reproducibility of acid production by human, unhomogenized, dental plaques. J. dent. Res. 48, 1106. GEDDES,D. A. M. and GILMOUR,M. N. 1970. The control of ghosting, a major source of error in gas liquid chromatographic determinations of C,-C5 acids. J. chromutogr. Sci. 8, 394-397. GILMOUR,M. N. and POOLE,A. E. 1967. The fermentation capabilities of dental plaque. Curies. Res. 1,247-260.

GRAY, J. A. 1966. Kinetics of enamel dissolution

during formation

of incipient caries-like lesions.

Archs oral Biol. 11. 397421.

HEMMENS,E. S., BLA~N&, J. R., BRADEL,S. F. and HARRISON,R. W. 1946. The microbic flora of the dental plaque in relation to the beginning of caries. J. dent. Res. 25, 195-205. HOBSON,P. N. 1969. Growth of mixed cultures and their biological control. Symp. Sot. Gerr. Microbial. No. XIX Microbial Growth, pp. 43-64. Cambridge Univ. Press. HOHORST,H. J. 1963. Methods of Enzymic Analysis p. 268 (edited by BERGMEYER, H. U.). Academic Press, New York. HUNGATE, R. E. 1962. The Bacteria, Vol. 4, chap. 3 pp. 97-98 (edited by GUNSALUS, I. C. and STANIER,R. Y.). Academic Press, New York. JENKINS,G. N. 1971. The present status of the fluoridation question. The Sri. Basis of Med. Ann. Rev. Chap. XXI, p. 398 (edited by GILLILAND,I. and FRANCIS,J.), Athlone Press, London. MOORE,B. W., CARTER,W. J., DUNN, 3. K. and FOSDICK,L. S. 1956. The formation of lactic acid in dental plaques. 1. Caries-active individuals. J. dent. Res. 35, 778-785. MUNTZ, J. A. 1943. Production of acids from glucose by dental plaque material. J. biol. Chem. 148, 225-236.

RANKE, B., BRAMSTEDT,F. and NAUJOKS, R. 1964. Untersuchungen mit markierten Verbindungen iiber den Kohlenhydratabbau in den Plaques. Advances in Fluorine Research and Dental Caries Prevention, Vol. 2, pp. 189-193 (edited by HARDWICK, J. L., DUSTIN, J.-P. and HELD, H. S.) Pergamon Press, Oxford. ROGOSA,M. and SHARPE,M. E. 1959. An approach to the classification of lactobacilli. J. appl. Bact. 22,329-340.

ROGOSA,M., WISEMAN,R. F., MITCHELL,J. A., DISRAELY,M. N. and BEARMAN,A. J. 1953. Species differentiation of oral lactobacilli from man including descriptions of Lactobacillas sulivarius nov spec and Lactobacillus cellobiosus nov spec. J. Bact. 65,681~699. ROSE, A. H. 1968. Chemical Microbiology, 2nd Ed, Chap. 6, p. 138. Butterworths, London. SANDHAM,H. J. and KLEINBERG,I. 1970. Contribution of lactic and other acids to the pH of a human salivary sediment system during glucose catabolism. Archs oral Biol. 15, 1263-1283.

L(+), D(-)LACTIC

AND

VOLATILE ACIDS

AND PLAQUEBUFFERING

545

STR~LFORS,A. 1948. Studies of the microbiology of caries. III. The buffer capacity of the dental plaques. J. dent. Res. 27, 587-592. TYLER, J. E. 1970. Quantitative estimation of volatile fatty acids in carious enamel by gas liquid chromatography. Internat. Ass. for Dent. Res., Brit. Div. Preprinted Abstracts 18th Meeting, Abstract 125. TYLER,J. E. 1971. Quantitative estimation of volatile fatty acids in carious enamel by gas chromatography of their methyl esters. J. dent. Res. 50, 694-695. Abstract. YAMADA,T.,HOJO, S.,KOBAYASHI,K., ASANO,~. and ARAYA,S. 1970. Studies on the carbohydrate metabolism of cariogenetic Streptococcus mutam strain PK-1. Arch oral Biol. 15, 1205-l 217.