Polyamines of Dental Plaque in Caries-resistant vs.

Caries-susceptible Adults
S. M. VRATSANOS and I.D. MANDEL
Division

of Preventive Dentistry, School of Dental and Oral Surgery, Columbia University, 630 West 168 Street, New York, New York 10032 indicated consumption of protons, the conversion of an Utilizing a sensitive liquid chromatographic system, polyamines were quantitated in three-day plaque from 13 caries-resistant (CR) amino acid a-amino group to an aliphatic amine group that and 35 caries-susceptible (CSJ adults after they had fasted for is a stronger base, and the role of CO2. At acidic, neutral, 12 hours. The values for putrescine and cadaverine were significantand mildly alkaline pH, an increase in the amino group's ly higher in the CR group. This may be attributed to a greater basicity is of no importance, because, under these pH consubstrate precursors of polyamines and/or availability of salivary this group is practically completely protonated. ditions, higher levels of biosynthetic decarboxylase activities in the CR CO2 evolution in acid media plays no role; in neutral or subjects, or both. mildly alkaline media, where CO2 is retained to yield carbonic acid, this acid can only partially compensate for the J Dent Res 64(3):422-424, March, 1985

Introduction.
In comparative studies of plaque acidogenesis in cariesresistant (CR) vs. caries-susceptible (CS) adults, we found (Vratsanos and Mandel, 1982) that the amount and rate of production of lactic acid were lower, and the levels of acetic acid higher, in the CR than in the CS group. High pK acids, such as acetic, propionic, and butyric, provide a buffering system (e.g., acetate-acetic acid) capable of absorbing the hydrogen ions generated by the low pK acids (lactic, formic, and pyruvic) (Vratsanos, 1981; Vratsanos and Mandel, 1982; Margolis and Moreno, 1983). Such a buffering system could act to reduce the continued plaque pH fall (below pH 5.5) that can occur with unlimited carbohydrate substrates. Other mechanisms, however, must function in the pH region above 5.5. These would include buffer systems operating at higher pH, such as: imidazole moieties, phosphate and bicarbonate groups; and generation of ammonia and decarboxylation of amino acids to form mono-amines and polyamines. The loss of carboxylate and the transformation to amines that are inherent in the process of amino acid decarboxylation are important means of pH regulation in bacterial metabolism (Gale, 1946; Morris and Fillinghame, 1974; Recsei and Snell, 1972; Hayes and Hyatt, 1974). In the presence of substrate and low pH, biodegradative decarboxylases can be induced to "consume protons and neutralize acid products of carbohydrate fermentation", according to the scheme,

consumption of protons concomitant with decarboxylation. Accordingly, the consumption of protons and its pHrise effect remain the dominant features. In this study, we examined the effect of decarboxylase activity in plaque by quantitating the levels of polyamines in three-day plaque of a group of CR and CS subjects after a 12-hour fast.

Materials and methods. The study was conducted with 13 caries-resistant and 35 caries-susceptible subjects. Caries-resistant subjects had a DMFS of zero as determined by clinical and radiographic examination, had not been exposed to systemic fluoride during tooth development, and were between 24 and 34 years of age. The caries-susceptible subjects were of similar age and had a minimum DMFS index of 20 (with a range of 20-35), with at least one active lesion currently present, or a history of lesions restored within the past year. All subjects were given a thorough dental prophylaxis one to two weeks prior to plaque collection in order to remove all deposits, to allow for resolution of gingival inflammation, and to minimize the influence of gingival crevicular fluid on plaque composition. The subjects were instructed to refrain from all oral hygiene for three days prior to a test run, and to report for the experiment at least 12 hours after their last food intake. This provided a threeday plaque accumulation in a fasting state, at minimal metabolic activity, for study purposes. Samples were removed with a curette from all of the labial and buccal sur-

R
+

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COO-+ H R- CH2 +CO

NH3

NH3

At neutral or slightly alkaline pH, biosynthetic decarboxylases have been shown to function in a similar manner, utilizing ornithine, arginine, and lysine as substrates (Gale, 1946), with mediation of S-adenosylmethionine to yield higher polyamines, e.g., spermidine and spermine. Under the pH conditions of normal plaque, amino acid decarboxylations have a pH-rise effect involving three factors: the
Received for publication August 1, 1984 Accepted for publication January 28, 1985 This investigation was supported in part by USPHS Research Grant DE-01554-22 from the National Institute of Dental Research, NIH, Bethesda, MD 20205, and in part by the Basic Research Support Grant, S07RR05331, to the School of Dental and Oral Surgery, Columbia University, New York, NY.

faces, were immediately weighed in tared plastic capsules, and were stored at -5 C (no more than one week until analyzed for polyamines). The polyamines were analyzed by liquid column chromatography using a system developed in this laboratory (Fig. 1, analytical system). The system employs pellicularly sulfonated, divinylbenzene-cross-linked polystyrene beads*, step-gradient ionic strength and pH elution, and fluorometric detection based on the reaction of primary amines with o-phthalicdicarboxaldehyde. The system has a limiting sensitivity to measure 3 picomoles of a polyamine, thus permitting the analysis for putrescine and cadaverine in as little as one microgram of wet plaque (Vratsanos, unpublished). As in all polyamine analyzers using sulfonated polystyrene resin, in the polyamine profile obtained, monoamines and ammonia are immediately eluted as a single peak at the beginning, as expected on the basis of charge.
*Dionex Corp.,

Sunnyvale, CA

422

Vol. 64 No. 3

POL YAMINES IN PLA QUE

423

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Diagrammatic representation used for analyzing polyamines.

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single positive charge. One to three mg wet weight of three-day fasting plaque from the CR and CS subjects was dispersed in one ml of 0.02 N HC1 by fast-stirring for 30 min at 250C. The dispersion was then filtered through a 0.2-ptm Millex GS millipore filter, and an aliquot of the filtrate, usually corresponding to 0.1 mg of wet plaque, was analyzed. Results. A typical polyamine profile from the analysis of plaque (1.2 mg wet weight) from a CS subject is shown in Fig. 2. Peaks with the retention times of putrescine, cadaverine, and spermidine were always present. Spermine was frequently observed. Putrescine was always the dominant amine. Unidentified minor peaks in the spermidine region were noted frequently. Qualitatively, the polyamine profile of CR subjects was similar to that of CS, but differed quantitatively. The mean values ( S.D.) of polyamines in threeday plaque samples of 13 CR and 35 CS subjects are shown in the Table. Mean values for putrescine and cadaverine were significantly higher (Student's t test) in CR than in CS subjects. These elevated values resulted in a significantly higher content of total polyamines and total polyamine base per mg wet plaque in the CR group. As "polyamine base", we define the amount of basicity calculated not as moles of polyamine(s) per se, but rather as the total basic groups occurring in a number of polyamine moles.

However, tryptamine is the only mono-amine appearing within the polyamine spectrum (before spermidine), on account of its large hydrophobic character, and despite its

Fig. 2 - Typical chromatogram of a polyamine analysis of threeday fasting plaque.

TABLE POLYAMINE CONTENT OF THREE-DAY PLAQUE FROM CARIES-RESISTANT (CR) AND CARIES-SUSCEPTIBLE (CS) SUBJECTS WHO HAVE FASTED (CR n = 13; CS n = 35)

Nanomol/mg Wet Plaque

Mean
Putrescine Cadaverine

S.D. 1.35 1.01 0.27 0.18 1.46

Mean

S.D. 0.93 0.44 0.18 0.29 1.29

Significance*
P < 0.05 P < 0.05 NS NS P < 0.01

2.36 1.02
0.50 0.14

Spermidine Spermine
Total

4.02

1.49 0.40 0.40 0.16 2.45

Polyamine Base (CR n = 13;CS n = 35)

Total Nanoequivalents of Base/mg Wet Plaque Mean S.D. Mean S.D. Significance*

8.82
*Statistical

3.10

5.57

2.68

P < 0.01

significance according to Student's t test.

Discussion.
The most likely source of polyamines in plaque from fasting subjects (with a pH usually from 6.7 to 7.0) is from

424

VRA TSANOS & MANDEL

J Den t R es March 1 985

biosynthetic decarboxylation of arginine and lysine precursors derived from salivary peptides. The significantly higher levels of putrescine and cadaverine in the CR group would suggest that appropriate salivary precursors are more readily available in CR than in CS subjects, or the decarboxylases are more abundant in the CR (reflecting differences in the microflora), or both. The process of formation of the polyamines (amino acid to amine transformation by decarboxylation) could readily affect plaque pH and contribute to the higher resting pH often seen in CR subjects. It should be noted that, contrary to the conventional wisdom, the polyamines - and amines in general formed by decarboxylation - are not of value in counter-acting acidity, since at normal plaque pH these amines are already fully protonated, and are incapable of acting as scavengers of additional protons. Thus, polyamines found in fasting plaque are not the neutralizing agents, but only the residual evidence of an earlier defense against acidity. Base formation, e.g., the generation of ammonia, may accompany polyamine formation, as in the conversion of arginine to putrescine, but the formation of polyamines per se is important in terms of pH, because amino acid decarboxylation requires consumption of protons. In comparing our putrescine and cadaverine data for CS plaque with the values reported by Hyatt and Hayes (1975), our putrescine value is about 50% higher, and our cadaverine value about double. No spermidine and spermine values were given by Hyatt and Hayes in their report on amino acids and amines of the plaque, although these polyamines were identified as dansylated derivatives using thin layer chromatography. The quantitative differences from our results may be attributed to the facts that these authors worked with 24-hour plaque, differing in micro-organisms from the three-day plaque, and also to the difference in procedure and in sensitivity of the analytical system of thin

layer chromatography followed by elution, vs. high-performance liquid column chromatography.

The absence of histamine and tryptamine in our chromatographic patterns is a reflection of the near-neutral pH of plaque samples from fasting subjects. As the pH falls, biodegradative decarboxylases can utilize histidine and tryptophan (Gale, 1946) and generate amines. These conversions should appear in plaque with declining pH and need to be considered in the examination of pH regulation in plaque.
REFERENCES

Adv Enzymol 6:1-32. HAYES, M.L. and HYATT, A.T. (1974): Free Amino Acids and Amines in Human Dental Plaque, Arch Oral Biol 19:361-369. HYATT, A.T. and HAYES, M.L. (1975): Free Amino Acids and Amines in Human Dental Plaque, Arch Oral Biol 20:203-209. MANDEL, LD. and ZENGO, A.N. (1973): Genetic and Chemical Aspects of Caries Resistance. In: Comparative Immunology of the Oral Cavity, H. Scherp and S. Mergenhagen, Eds., Washington, DC: U.S. Government Printing Office, pp. 118-137. MARGOLIS, H.C. and MORENO, E.C. (1983): The Effect of High pKa Acids on Cariogenic Potential of Plaque Fluid, IADR Progr 62:No. 214. MORRIS, D.R. and FILLINGHAME, R.H. (1974): Regulation of Amino Acid Decarboxylation, Ann Rev Biochem 43:303-325. RECSEI, P.A. and SNELL, E.E. (1972): Histidine Decarboxylaseless Mutants of Lactobacillus 30a: Isolation and Growth Properties, JBacteriol1 12:624-626. VRATSANOS, S.M. (1981): Chromatographic Microanalysis of Organic Acids in Plaque Related to Food Cariogenicity. In: Foods, Nutrition, and Dental Health, Vol. 3, Park Forest South, IL: Pathotox, pp. 88-91. VRATSANOS, S.M. and MANDEL, I.D. (1982): Comparative Plaque Acidogenesis of Caries-resistant vs. Caries-susceptible Adults, JDent Res 61:465-468.

GALE, E.F. (1946):

The Bacterial Amino Acid

Decarboxylases,