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Levan synthesis and accumulation by human dental plaque
Archs oral Bid. Vol. 15, pp. 563-567, 1970. Pergamon Press. Printed in Great Britain.
LEVAN SYNTHESIS AND ACCUMULATION BY HUMAN DENTAL PLAQUE M. HIGUCHI, Y. IWAMI,T. YAMADAand S. ARAYA Laboratory
of Oral Biochemistry, Dental Faculty, Tohoku University 162 Kita-6bancho, Sendai, Japan
IT IS WELL known that, on diets containing sucrose, a gelatinous and voluminous plaque develops on the tooth surface (CARLSSONand EGELBERG,1965). It has also been shown that extracellular polysaccharides, which consist of dextran and levan, are produced from sucrose by certain strains of cariogenic streptococci, whereas noncariogenic strains formed only trace amounts (ZINNERet a/., 1965; KRASSE,1965 ; GIBBONSet al., 1966). GIBBONSand BANGHART(1967) reported on the synthesis of extracellular dextran by cariogenic streptoccci and on its presence in human dental plaque. This paper deals with the formation of levan in dental plaque suspension by using uniformly labelled 14C-sucrose (“Sucrose-U-14C”) from the Daiichi Pure Chemicals Co., Japan and fructose labelled 14C-sucrose (“Sucrose-F-14C”) as substrates and with the levels of levan and dextran in human dental plaque. The sucrose-F-14C was prepared enzymatically from uniformly labelled 14Cfructose (0 * I mc, sp. act. 10 mc/mmole) according to the method of WOLOCHOW et al. (1949) and purified by paper chromatography, purity being confirmed by radioautographic localization. Dental plaque was obtained from four individuals who had not brushed their teeth for 24 hr and had not eaten for at least 12 hr. It was collected from all surfaces of the teeth and suspended in 0.1 M phosphate buffer (pH 7 -2). The formation of extracellular polysaccharide was studied by adding to 1 a0 ml of a suspension of plaque, 0 -35 ml of phosphate buffer (pH 7 *2,0 - 1 M) and 0 +15 ml of 0 -2 M solution of sucrose-U-‘4C or sucrose-F-14C, specific activity 0.025 mc/mmole. Extracellular polysaccharide synthesis took place at 35°C in an atmosphere of nitrogen and was terminated by heat treatment (1OO’C for 3 min). Samples at zero time were heated before the addition of substrate. The reaction mixtures were centrifuged for 15 min (13,000 rev/min) at 0°C. The precipitated cells were treated with 2 ml of 0.1 M acetic acid to dissolve extracellular polysaccharides (GIBBONS et al., 1966; HOBSONand MACPHERSON,1954): the best condition for polysaccharide extraction was not, however, elucidated. After deproteinization with trichloroacetic acid (to a final concentration of 5 per cent), the extracts were mixed with the supernatant, which was cell-free. The mixtures were then dialyzed against tap water for 18 hr in order to remove soluble carbohydrates. The non-dialyzable fraction was assayed for carbohydrates, using the anthrone method (HODGE and HOFREITER,1962) and its 563
564
M.
HIGUCHI,
Y.
JWAMI,
T. YAMADA and S. ARAY~
Amounts of polysaccharides
1000
I-
Colrnts in polysoccharides
Min a2?z?q%;40~
b=666-60-
0~204
FIG. 1. Formation of extracellular polysaccharides from sucrose-U-W and sucrose-F14Cby dental plaque bacteria. Upper(a) : Amounts of extracellular polysaccharides formed during the incubation. Lower(b) : Counts in the polysaccharides. The lowermost thin line shows the level of radioactivity derived from “%-sucrose remaining after dialysis for 18 hr. It therefore represents a blank value.
radioactivity determined by a Geiger-MiiiIer counter. The radioactivities of the non-dialysable fractions in the samples at zero time were ca. 60 c/min for both substrates, which represent the blank counts derived from sucrose-‘4C. Figure la shows the rate of formation of extracellular polysaccbaride (as nondialysabie anthrone-positive material) and Fig. lb the rate of incorporation of 14C. As the specific activities of both sucrose-UT-14C and sucrose-F-‘4C! added as substrate were the same, then the specific activity of the fructose moiety of the sucrose-F14C could be twice that of the fructose moiety of the uniformly-labelled sucrose. If levan is formed excIusiveIy from the fructose moiety of sucrose, then any levan formed from sucrose-F-14C would have a specific activity double that of levan formed from
LEVAN
SYNTHESIS AND AC CXMULATIONBYHUMANDENTALPLAQUE
Standard
Glucose
565
-
0
Dextran hydrolysate under D condition
Levan hydrolysate under L condition
l
L
Fructose
(I)
;:
D condition
.::: ..
53 cpm
u- “+c (
L condition
.:: .
i:. ..:
... ..i Xf
a: ..
202 cpm .. ..;:
D condition F- 14C I
L condition
.@: ::;.;j .f
.. :.
.N
::
485 cpm Sample
FIG. 2. Radioautogram
of hydrolysates of extracellular polysaccharidesderived from sucrose-I_J-‘%J and sucroseF-“‘C.
sucrose-U-14C. Therefore, as the rates of extracellular polysaccharide formation were similar for both substrates, the net radioactivity (2~) incorporated from sucrose-F-14C may be halved to give a value (a) for the formation of levan which can be compared with the radioactivity incorporated from sucrose-U- 14C, representing total (dextran + levan) polysaccharide synthesis. By subtraction of the value (a) for levan, a measure of the amount of dextran synthesized (b) is calculated : Values of a:b = 1evan:dextran = 2:l. Furthermore, samples obtained in the above experiments were divided into equal volumes. One portion was treated with 0.1 M H,SO,, the other with 2 N H,S04, before hydrolysis at 100°C for 3 hr. The weaker acid concentration was shown to be favourable to levan hydrolysis (L condition), preventing degradation of levan to hydroxymethylfurfural and levulinic acid and the stronger acid concentration was used for dextran hydrolysis (D condition) as described by GIBBONSet al. (1967) and WHELAN(1962). After hydrolysis the acid was neutralized with barium carbonate. The supernatant was separated by ascending paper chromatography with a solvent system: phenol : n-butanol : acetic acid : water. (20:20:8:40). Sugars formed during the hydrolysis were detected by radioautography and by calorimetric comparison with authentic samples. The upper figure of Fig. 2 shows the chromatograms of
566
M. HIGUCHI,Y. IWAMI,T. YAMADA and S. ARAYA
samples of standard and of hydrolysates and the lower figure the radioautogram of experimental samples and counts of extracts following localization by radioautography. In the upper figure, hydrolysate of levan standard under the L condition produced a major spot identified as fructose and only a faint spot of hydroxymethylfurfural derived from fructose. In the sample from sucrose-U-‘4C, the hydrolysis under the D condition produced 2 spots, fructose (major) and glucose (minor), but under the L condition there were, in addition to a very marked fructose spot, only faint spots corresponding to products of the partial hydrolysis of dextran and levan. The glucose spot under the D condition showed a strength of 53 c/min, and the fructose spot under the L condition a strength of 202 c/min. This result also supports the finding that levan synthesis activity is higher than that of dextran. In the sample from sucrose-F- 14C, the hydrolysate under the D condition shows a faint fructose spot and no glucose spot, while the hydrolysate under the L condition gave only a very marked fructose spot (485 c/min), in addition to two faint spots, corresponding to a partial hydrolysate of levan and hydroxymethylfurfural. These results suggest that the fructose component of sucrose is not transformed into glucose during extracellular polysaccharide synthesis. Polysaccharides in dental plaque were assayed from about 20 individual mixed plaques. An extract containing extracellular polysaccharide from this sample was divided into equal volumes and hydrolyzed under the D and L conditions, followed by analysis by paper chromatography. The chromatograms showed fructose and glucose, which were determined quantitatively. Dental plaque was thus shown to contain both dextran and levan, which together accounted for an average of 4.2 per cent by weight of dry plaque, levan being about one-eighth of the total polysaccharides. These results indicate that the activity of levan synthesis is higher than that of dextran in plaque, whereas accumulation is lower, as discussed by WOOD (1964; 1967), WOOD and CRITCHLEY (1967) and MCDOUGALL (1964). These authors reported that the amount of Ievan in dental plaque was about 5 per cent of total polysaccharide. However, the present investigation in which weak acid hydrolysis was used suggests that the accumulation of levan in dental plaque may be somewhat greater than this. Acknowledgements-The authors are indebted to Dr. K. MATSUDA and Dr. T. WATANABE of the Laboratory of Applied Biochemistry, Department of Agricultural Chemistry, Tohoku University, for supplying the pure levan and dextran, and for their advice. REFERENCES CARLSSON, J. and EGELBERG, J. 1965. Effect of diet on early plaque formation. Odont. Revy 16, 112125. GIBBONS,R. J. and BANGHART, S. B. 1967. Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Archs oral Biol. 12, 11-24. GIBBONS.R. J. BERMAN. K. S.. KNOETTNER. P. and KAPSIMALIS.B. 1966. Dental caries and alveolar bone loss in gnotodiotic r&s infected with capsule forming’streptococci of human origin. Archs oral Biol. 11,549-560. HOBSON,P. N. and MACPHERSON,M. J. 1954. Some serological and chemical studies on materials extracted from an amylolytic Streptococcus from the rumen of the sheep. Biochem. J. 57,145-151.
LEVANSYNTHESIS AND ACCUMULATION BY
HUMAN DENTALPLAQUe
567
HODGE, J. E. and HOFREITER,B. T. 1962. Determination of reducing sugars and carbohydrates. Methoris in Carbohydrate Chemistry (edited by WHISTLER,R. L. and WOLFROM,M. L.) Vol. 1, p. 380. Academic Press, New York. KRASSE, B. 1965. The effect of caries-inducing streptococci in hamsters fed diets with sucrose or glucose. Archs oral Biol. 10,223-226. MCDOUGALL,W. A. 1964. Studies on the dental plaque IV. Ievans and the dental plaque. Aust. dent. J. 9, l-5. WHELAN,W. J. 1962. Methods in Carbohydrate Chemistry (edited by WHISTLER,R. L. and WOLFROM M. L.) Vol. 1, p. 321. Academic Press, New York. WOLOCHOW,H., PIJTMAN,E. W., DOUDOROFF,M., HASSID,W. Z. and BARKER,H. A. 1949. Preparation of sucrose labelled with 14C in the glucose or fructose component. J. biol. Chem. 180, 1237-1242. WOOD, J. M. 1964. Polysaccharides synthesis and utilization of dental plaque. J. dent. Res. 43, 955. WOOD, J. M. 1967. The amount, distribution and metabolism of soluble polysaccharides in human dental plaque. Archs oral Biol. l&849-858. WOOD, J. M. and CRITCHLEY,P. 1967. The soluble carbohydrates of the plaque matrix. J. dent. Res. 46, supplemented, 129-130. ZINNER,D. D., JABLON,J. M., ARAN, A. P. and SASLAW,M. S. 1965. Experimental caries induced in animals by streptococci of human origin. Proc. Sot. exp. Biol. Med. 118, 766-770.
LEVAN SYNTHESIS AND ACCUMULATION BY HUMAN DENTAL PLAQUE M. HIGUCHI, Y. IWAMI,T. YAMADAand S. ARAYA Laboratory
of Oral Biochemistry, Dental Faculty, Tohoku University 162 Kita-6bancho, Sendai, Japan
IT IS WELL known that, on diets containing sucrose, a gelatinous and voluminous plaque develops on the tooth surface (CARLSSONand EGELBERG,1965). It has also been shown that extracellular polysaccharides, which consist of dextran and levan, are produced from sucrose by certain strains of cariogenic streptococci, whereas noncariogenic strains formed only trace amounts (ZINNERet a/., 1965; KRASSE,1965 ; GIBBONSet al., 1966). GIBBONSand BANGHART(1967) reported on the synthesis of extracellular dextran by cariogenic streptoccci and on its presence in human dental plaque. This paper deals with the formation of levan in dental plaque suspension by using uniformly labelled 14C-sucrose (“Sucrose-U-14C”) from the Daiichi Pure Chemicals Co., Japan and fructose labelled 14C-sucrose (“Sucrose-F-14C”) as substrates and with the levels of levan and dextran in human dental plaque. The sucrose-F-14C was prepared enzymatically from uniformly labelled 14Cfructose (0 * I mc, sp. act. 10 mc/mmole) according to the method of WOLOCHOW et al. (1949) and purified by paper chromatography, purity being confirmed by radioautographic localization. Dental plaque was obtained from four individuals who had not brushed their teeth for 24 hr and had not eaten for at least 12 hr. It was collected from all surfaces of the teeth and suspended in 0.1 M phosphate buffer (pH 7 -2). The formation of extracellular polysaccharide was studied by adding to 1 a0 ml of a suspension of plaque, 0 -35 ml of phosphate buffer (pH 7 *2,0 - 1 M) and 0 +15 ml of 0 -2 M solution of sucrose-U-‘4C or sucrose-F-14C, specific activity 0.025 mc/mmole. Extracellular polysaccharide synthesis took place at 35°C in an atmosphere of nitrogen and was terminated by heat treatment (1OO’C for 3 min). Samples at zero time were heated before the addition of substrate. The reaction mixtures were centrifuged for 15 min (13,000 rev/min) at 0°C. The precipitated cells were treated with 2 ml of 0.1 M acetic acid to dissolve extracellular polysaccharides (GIBBONS et al., 1966; HOBSONand MACPHERSON,1954): the best condition for polysaccharide extraction was not, however, elucidated. After deproteinization with trichloroacetic acid (to a final concentration of 5 per cent), the extracts were mixed with the supernatant, which was cell-free. The mixtures were then dialyzed against tap water for 18 hr in order to remove soluble carbohydrates. The non-dialyzable fraction was assayed for carbohydrates, using the anthrone method (HODGE and HOFREITER,1962) and its 563
564
M.
HIGUCHI,
Y.
JWAMI,
T. YAMADA and S. ARAY~
Amounts of polysaccharides
1000
I-
Colrnts in polysoccharides
Min a2?z?q%;40~
b=666-60-
0~204
FIG. 1. Formation of extracellular polysaccharides from sucrose-U-W and sucrose-F14Cby dental plaque bacteria. Upper(a) : Amounts of extracellular polysaccharides formed during the incubation. Lower(b) : Counts in the polysaccharides. The lowermost thin line shows the level of radioactivity derived from “%-sucrose remaining after dialysis for 18 hr. It therefore represents a blank value.
radioactivity determined by a Geiger-MiiiIer counter. The radioactivities of the non-dialysable fractions in the samples at zero time were ca. 60 c/min for both substrates, which represent the blank counts derived from sucrose-‘4C. Figure la shows the rate of formation of extracellular polysaccbaride (as nondialysabie anthrone-positive material) and Fig. lb the rate of incorporation of 14C. As the specific activities of both sucrose-UT-14C and sucrose-F-‘4C! added as substrate were the same, then the specific activity of the fructose moiety of the sucrose-F14C could be twice that of the fructose moiety of the uniformly-labelled sucrose. If levan is formed excIusiveIy from the fructose moiety of sucrose, then any levan formed from sucrose-F-14C would have a specific activity double that of levan formed from
LEVAN
SYNTHESIS AND AC CXMULATIONBYHUMANDENTALPLAQUE
Standard
Glucose
565
-
0
Dextran hydrolysate under D condition
Levan hydrolysate under L condition
l
L
Fructose
(I)
;:
D condition
.::: ..
53 cpm
u- “+c (
L condition
.:: .
i:. ..:
... ..i Xf
a: ..
202 cpm .. ..;:
D condition F- 14C I
L condition
.@: ::;.;j .f
.. :.
.N
::
485 cpm Sample
FIG. 2. Radioautogram
of hydrolysates of extracellular polysaccharidesderived from sucrose-I_J-‘%J and sucroseF-“‘C.
sucrose-U-14C. Therefore, as the rates of extracellular polysaccharide formation were similar for both substrates, the net radioactivity (2~) incorporated from sucrose-F-14C may be halved to give a value (a) for the formation of levan which can be compared with the radioactivity incorporated from sucrose-U- 14C, representing total (dextran + levan) polysaccharide synthesis. By subtraction of the value (a) for levan, a measure of the amount of dextran synthesized (b) is calculated : Values of a:b = 1evan:dextran = 2:l. Furthermore, samples obtained in the above experiments were divided into equal volumes. One portion was treated with 0.1 M H,SO,, the other with 2 N H,S04, before hydrolysis at 100°C for 3 hr. The weaker acid concentration was shown to be favourable to levan hydrolysis (L condition), preventing degradation of levan to hydroxymethylfurfural and levulinic acid and the stronger acid concentration was used for dextran hydrolysis (D condition) as described by GIBBONSet al. (1967) and WHELAN(1962). After hydrolysis the acid was neutralized with barium carbonate. The supernatant was separated by ascending paper chromatography with a solvent system: phenol : n-butanol : acetic acid : water. (20:20:8:40). Sugars formed during the hydrolysis were detected by radioautography and by calorimetric comparison with authentic samples. The upper figure of Fig. 2 shows the chromatograms of
566
M. HIGUCHI,Y. IWAMI,T. YAMADA and S. ARAYA
samples of standard and of hydrolysates and the lower figure the radioautogram of experimental samples and counts of extracts following localization by radioautography. In the upper figure, hydrolysate of levan standard under the L condition produced a major spot identified as fructose and only a faint spot of hydroxymethylfurfural derived from fructose. In the sample from sucrose-U-‘4C, the hydrolysis under the D condition produced 2 spots, fructose (major) and glucose (minor), but under the L condition there were, in addition to a very marked fructose spot, only faint spots corresponding to products of the partial hydrolysis of dextran and levan. The glucose spot under the D condition showed a strength of 53 c/min, and the fructose spot under the L condition a strength of 202 c/min. This result also supports the finding that levan synthesis activity is higher than that of dextran. In the sample from sucrose-F- 14C, the hydrolysate under the D condition shows a faint fructose spot and no glucose spot, while the hydrolysate under the L condition gave only a very marked fructose spot (485 c/min), in addition to two faint spots, corresponding to a partial hydrolysate of levan and hydroxymethylfurfural. These results suggest that the fructose component of sucrose is not transformed into glucose during extracellular polysaccharide synthesis. Polysaccharides in dental plaque were assayed from about 20 individual mixed plaques. An extract containing extracellular polysaccharide from this sample was divided into equal volumes and hydrolyzed under the D and L conditions, followed by analysis by paper chromatography. The chromatograms showed fructose and glucose, which were determined quantitatively. Dental plaque was thus shown to contain both dextran and levan, which together accounted for an average of 4.2 per cent by weight of dry plaque, levan being about one-eighth of the total polysaccharides. These results indicate that the activity of levan synthesis is higher than that of dextran in plaque, whereas accumulation is lower, as discussed by WOOD (1964; 1967), WOOD and CRITCHLEY (1967) and MCDOUGALL (1964). These authors reported that the amount of Ievan in dental plaque was about 5 per cent of total polysaccharide. However, the present investigation in which weak acid hydrolysis was used suggests that the accumulation of levan in dental plaque may be somewhat greater than this. Acknowledgements-The authors are indebted to Dr. K. MATSUDA and Dr. T. WATANABE of the Laboratory of Applied Biochemistry, Department of Agricultural Chemistry, Tohoku University, for supplying the pure levan and dextran, and for their advice. REFERENCES CARLSSON, J. and EGELBERG, J. 1965. Effect of diet on early plaque formation. Odont. Revy 16, 112125. GIBBONS,R. J. and BANGHART, S. B. 1967. Synthesis of extracellular dextran by cariogenic bacteria and its presence in human dental plaque. Archs oral Biol. 12, 11-24. GIBBONS.R. J. BERMAN. K. S.. KNOETTNER. P. and KAPSIMALIS.B. 1966. Dental caries and alveolar bone loss in gnotodiotic r&s infected with capsule forming’streptococci of human origin. Archs oral Biol. 11,549-560. HOBSON,P. N. and MACPHERSON,M. J. 1954. Some serological and chemical studies on materials extracted from an amylolytic Streptococcus from the rumen of the sheep. Biochem. J. 57,145-151.
LEVANSYNTHESIS AND ACCUMULATION BY
HUMAN DENTALPLAQUe
567
HODGE, J. E. and HOFREITER,B. T. 1962. Determination of reducing sugars and carbohydrates. Methoris in Carbohydrate Chemistry (edited by WHISTLER,R. L. and WOLFROM,M. L.) Vol. 1, p. 380. Academic Press, New York. KRASSE, B. 1965. The effect of caries-inducing streptococci in hamsters fed diets with sucrose or glucose. Archs oral Biol. 10,223-226. MCDOUGALL,W. A. 1964. Studies on the dental plaque IV. Ievans and the dental plaque. Aust. dent. J. 9, l-5. WHELAN,W. J. 1962. Methods in Carbohydrate Chemistry (edited by WHISTLER,R. L. and WOLFROM M. L.) Vol. 1, p. 321. Academic Press, New York. WOLOCHOW,H., PIJTMAN,E. W., DOUDOROFF,M., HASSID,W. Z. and BARKER,H. A. 1949. Preparation of sucrose labelled with 14C in the glucose or fructose component. J. biol. Chem. 180, 1237-1242. WOOD, J. M. 1964. Polysaccharides synthesis and utilization of dental plaque. J. dent. Res. 43, 955. WOOD, J. M. 1967. The amount, distribution and metabolism of soluble polysaccharides in human dental plaque. Archs oral Biol. l&849-858. WOOD, J. M. and CRITCHLEY,P. 1967. The soluble carbohydrates of the plaque matrix. J. dent. Res. 46, supplemented, 129-130. ZINNER,D. D., JABLON,J. M., ARAN, A. P. and SASLAW,M. S. 1965. Experimental caries induced in animals by streptococci of human origin. Proc. Sot. exp. Biol. Med. 118, 766-770.