Free amino acids and amines in human dental plaque

FRjEE AMINO

ACIDS AND AMINES DENTAL PLAQUE

IN HUMAN

A. T. HYATT and M. L. HAYES Department of Biochemistry (Oral Biology). University of Bristol, Bristol, England

Summary-Human, 24 hr dental plaque was extracted with either trichloroacetic acid or saline and the free amino acids and amines determined after partial purification by ion-exchange chromatography. Total amino acids, as represented by acid extractions. were measured by automated amino-acid analysis and gave a mean value (n = 6) of 12 pmoles/g wet wt of plaque, whereas the saline extracts contained a mean (n = 4) of 9 pmoles/g wet wt. The most prominent amino acids in the total extract were glutamate, 28 per cent, aspartate, 13 per cent, proline and glycine, each 12 per cent, and alanine 9 per cent. The main amino acids extracted with saline from the extracellular fluid were glutamate, 29 per cent, glycine, 18 per cent, alanine, 13 per cent, proline, 10 per cent and aspartate. 8 per cent. Acid-extractable amines were assayed fluorimetrically as their dansyl derivatives. They represented a mean (n varying between 7 and 28) of 2 pmoles/g wet wt comprising putrcscine 71 per cent, cadaverine I I per cent. and y-aminobutyric acid I3 per cent. Histamine was also detected. 5 per cent, when assayed with o-phthalaldehyde. Agmatine and p-alanine were not detected in these extracts. The average concentrations of the amino acids and amines in the extracellular and intracellular pools were also estimated and the significance of these results has been discussed in terms of plaque amino-acid metabolism.

IYTRODUCTION Gale and Epps (1942)and Gale (1946) were the first to show that certain bacteria, when cultured in media containing amino acids, were able to induce enzymes which catalysed the removal of the a-carboxyl group from an L-amino acid to produce carbon dioxide and a basic amine. These amino acid decarboxylases (EC 4.1.1. 1I-28) are typically produced by species of Clostridium, Streptococcus. Lactohacillus, Proteus and coliforms (Barman, 1969) and the initial evidence that, under certain circumsiances, the oral bacteria can produce these enzymes was provided by Gochman et al. (1959). Bibb and Straughn (1961) and Blomquist and WLinge (1963). Since then, it has been suggested that these reactions may make a significant contribution to the acid-base balance in the dental plaque (Kleinberg, 1970a, 1970b; Kleinberg, Craw and Komiyama, 1973) and to the putrefactive processes that occur in the mouth (Frostell and !;oder, 1970). The latter authors argued that, since the initiation of inflammatory reactions at the gingival margin was paralleled by an increased formation of proteolytic enzymes, the direct and indirect products of proteolysis. which include peptides, amino acids. .imines and ammonia. will probably contribute to halitosis and to the processes involved in the initiation of periodontal disease. In addition, free amino acids Ire found in the gingival crevice fluid (Brill, 1962) and. perhaps not surprisingly, the crevice flora includes species which can ferment amino acids and have a nutritional requirement for certain decarboxylation products such as putrescine and the spermidine related polyamines. spermine and (Loesche, 1968). Some dental plaque organisms, when cultured in media containing an amino acid and pyridoxal phosphate, induce amino acid decarboxylases (Hayes and Hyatt. 1974). These er,zymes were found to be active

over the pH range 4.5-6.0 and produced amines from glutamate, aspartate, ornithine, lysine, arginine and histidine. These results support the suggestion that. if these reactions occur in Guo, the fall in pH after consumption of dietary carbohydrates would promote decarboxylase activity and the subsequent accumulation of basic amines would help to restore the pH to neutrality. The effectiveness of this base-producing system will depend, however, on the availability of the amino acids to act as inducers and as substrates for these enzymes, and this paper describes the isolation and identification of free amino acids and amines in plaque extracts.

MATERIALS AND METHODS Human dental plaque This was allowed to accumulate on the teeth of two subjects for 24 hr, scraped off all available smooth surfaces at least 2 hr after exposure to food, weighed and used as soon as possible. Chemicals Chromatographically homogeneous amino acids were obtained from B.D.H. Chemicals Ltd.; spermidine, 1,5-pentanedidmine (cadaverine) dihydrochloride. tetramethylenediamine (putrescine). /j-alanine and y-aminobutyric acid were from Koch-Light Ltd.; spermine tetrahydrochloride and l-amino 4-guanidobutane (agmatine) sulphate from Sigma Chemical Company Ltd. ; histamine dihydrochloride, O-phthalaldchyde (OPT) and I-dimethylamino-naphthalene-5sulphonyl (dansyl) chloride were obtained from B.D.H. Chemicals Ltd. Solutions of OPT (1 per cent w/v in methanol) were freshly prepared before use. Laboratory reagents (A.R. grade) were obtained from B.D.H. Chemicals Ltd.

A. T. Hyatt and M. L. Hayes

204

[U-‘sC]-glutamic acid and -1ysine (25 mCi/m-atom of labelled carbon) were obtained from the Radiochemica1 Centre. Amersham. [U-‘4C]-Y-aminobutyric acid (GABA) and [U-‘“Cl-cadaverine were prepared from these amino acids by incubation with lysine and glutamate decarboxylases as described by Hayes and Hyatt (1974). After the reactions were complete, the solutions were adjusted to 5 per cent (w/v) with trichloroacetic acid (TCA), allowed to stand overnight at 4-C. centrifuged (15,000g. at 4 ‘C for 15 min) and the protein-free supernatant liquids used as solutions of the [U-‘4C]-amines. E.stractior~

ofpluyue

I. With TCA.

Plaque (2&50 mg wet wt) was homogenized for 5 min at 4°C in 2.0 ml of 5 per cent (w/v) TCA and left. with occasional mixing, for 2.5 hr. The suspension was centrifuged (5000g. 4’C, IOmin), the precipitate re-extracted with TCA, re-centrifuged and the protein-free supernatant solutions combined. 2. With suline. Plaque (20-50 mg wet wt) was homogenized in 1.0 ml of 0.16 M sodium chloride solution for 5 min at 4’C. The suspension was centrifuged as above, re-extracted with saline. and the supernatant solutions combined and then deproteinized with 5 per cent (w/v) TCA.

The protein-free extracts (pH 1.2) were applied to 100 mm columns of Dowex 50 H+ form X 8 resin (2@ 50 mesh. Bio-Rad Laboratories) to retain the amines and amino acids and the eluates were discarded. The columns were washed with 20ml of twice-distilled water and the bound cations were subsequently eluted with IO ml of concentrated hydrochloric acid followed by 5 ml of water. The eluates were collected, combined and evaporated to dryness at 60°C in vucuo. The dried residues were taken up in 200 ~1 of 0.1 M hydrochloric acid and their amine and amino acid contents determined. A modification of this procedure was used to obtain a fraction suitable for histamine determinations. Plaque was homogenized with 0.75 ml of 5 per cent (w,/ v) TCA and the supernatant solutions obtained as above. These were adjusted to 2.0 ml and pH 4.5 with NaOH. Samples (I.0 ml) were applied to 100 mm AGlX 8 Dowex carbonate columns (200-400 mesh, BioRad Laboratories) to retain histidine as described by Weissbach. Lovenberg and Udenfriend (1961). Any histamine left on the column was washed off with 1.75 ml of water, the eluates combined and made up to 3.0 ml with distilled water. These preparations are referred to below as the plaque fractions. Prepuratiorl.s

ofdansylated

derivatives

of‘amirws

Procedures for the identification and the fluorimetric assay of nanomole amounts of amines by the production of their dansylated derivatives have been reviewed by Seiler and Wiechmann (1970). Samples (100~1)of the plaque fractions and standard solutions (1 mM solutions of either ornithine, lysine, putrescine, cadaverine, spermine, spermidine, agmatine, histamine or GABA) were dansylated as recommended by Smith (1973). except that sodium carbonate was substituted for sodium bicarbonate when determining agmatine.

Standard amino acid solutions and the plaque fractions were treated with dansyl chloride by the method of Zanetta et al. (1970).

of dunsyluted

Chrorllatoyraphy

amiws

rrml tr~rli~~oacids

The dansylated amines and the dansylated plaque fractions were separated by thin-layer chromatography on plastic sheets (200 x 200 mm) precoated with 0.25 mm layers of silica gel G (Camlab Ltd.). The plates were activated at IOVC for 2 hr immediately before use. Samples (10 {tl) of the dansylated amines were applied to the plates and R, values were dctermined after one and two dimensional separations in the following solvents: (A) chloroform: triethylaminc. 5: I (v/v);(B) cyclohexane: ethyl acetate. 3:2 (v/v): two consecutive runs. Two series of plates were prepared from these fractions: (1) the original extracts in toluene (SX ~tl samples); and (2) an eight-fold concentration of the original extract (l-20 ~1). Dansylated amino acids were separated and identified separately by the method of Zanetta c’t al. internal

standards

Presence of the amines was confirmed by the inclusion of an internal standard of each dansylated amine prior to chromatography of the dansylated plaque: fractions. Comparison with plates not containing internal standards showed intensified fluorescence from spots corresponding to positively identified components. [U-‘4C]-cadaverine and [U-“Cl-GABA samples were also dansylated and included with the dansylated plaque extracts. Locution

cfdansylated

derivutires

Non-radioactive spots were located by their tluorescence under U.V. light after spraying each plate with 25 ml of a mixture of triethanolamine and isopropanol, I :4 (v/v), immediately after removal from the chromatography tank. The isopropanol was allowed to evaporate before the plates were examined. Radioactive spots were located by radioautography against Kodirex Estar X-ray films (I 65 x 216 mm) for I week.

Spots corresponding to the ddnsylated amines were located by their fluorescence under U.V. light on unsprayed plates, cut out and shaken with 5 ml of ethylacetate for 15 min. The resulting suspension was centrifuged and the supernatant liquid decanted. The silica gel was rc-extracted, the tube rccentrifuged. and the supernatant fractions combined. The ethyl acetate was evaporated in vacua at 60°C. and the dansylatcd amines retained as dried residues. Measurement

of:fiuorescence

The dried, dansylated amine residues were dissolved in 3,Oml ofa mixture of toluene and triethylamine. 19: I (v/v). The fluorescence of the solutions was measured in a Baird Atomic Fluorimet fluorimeter using a Chance FMllO-OX1 filter (peak transmission 360 nm) for incident light, and a Wratten 58 filter (peak transmission 510 nm) supplied by Kodak Ltd.. for emitted light. The latter was replaced by a Wratten 98

Amino acids and amines in dental plaque filter (peak transmission 450 nm) for measurement the histamine-OPT complex.

of

Assay qf’putrescine and cadaverine Samples of the dansylated plaque fractions, equivalent to 50 per cent (wrt wt of plaque/vol) in toluene, and a range of dansylate’d putrescine and cadaverine standard solutions in toluene were separated by twodimensional chromatography. The spots corresponding to dansyl putrescine and dansyl cadaverine were eluted and their flue:-escence determined.

citrate buffer (200,~1,0.1 M, pH 3.25), and aliquots (1Ck 30 ~1) were analysed for amino acids using a Technicon rSM2 amino-acid analyser. A known mixture of amino acids acted as an external standard for each analysis and these results were used to determine the amino acid concentrations in the fractions.

RESULTS

Chromatography of‘ the dansylated arnines

The dansylated plaque fractions in toluene were concentrated by evaporation to dryness at 60°C in ~acuo. and resuspended in 504 of toluene. The total extracts. equivalent to 4(f120 per cent (wet wt of plaque/vol) in toluene, and a range of dansylated GABA standard solutions were partially purified by one-dimensional chromatography in chloroform-cyclohexane (solvent Cl, 19: I (v/v). The dansyl GABA spots were located, eluted, and rechromatographed in solvent B. The dansyl GABA fractions were then recovered and found to be homogeneous in solvent A and in benzeneemethanol (solvent D). 9: 1 (v/v).

The plaque extracts prepared for histamine determinations, as well as standard solutions of histamine, were treated with the OPT reagent and the fluorescence of the acid-stable, histamine-OPT fluorophore determined as described by Shore (1971). Recovery of histamine after ion-exchange chromatography was proportional to the concentration, up to 5OOnmoles/ ml. and was independent of the presence of histidine, up to 3 ~moles/ml. T’stal recovery was 85 per cent of the histamine applied to the columns and the plaque results were corrected accordingly.

Saline and TCA elctracts of the plaque were prepared and fractionated as described. The plaque fractions were reduced to 0.5 ml by evaporation at 60°C in vacua. and then dried ‘over phosphorous pentoxide and potassium hydroxide The residue was dissolved in Table I. Separations

20.5

The relative mobilities of the dansylated derivatives of the amines are shown in Table 1. The R, values for all the first-dimensional separations are in close agreement with those quoted by Seiler and Wiechmann (1970). A comparison with the R, values observed after chromatography of the dansylated plaque fractions suggested the presence of the dansyl derivatives of putrescine, cadaverine, ornithine, lysine, spermine. spermidine, GABA and histamine in the latter and this was confirmed by the inclusion of the appropriate internal standard. Ammonia and ethanolamine were also observed but dansylated agmatine was not detected under these conditions. Chromatography

of dansylated amino acids

Separations of the standard solutions of the dansylated amino acids gave R, values in good agreement with those reported by Zanetta et ul. However standard solutions of r-alanine, fl-alanine and GABA could not be separated from each other. Chromatography of plaque fractions obtained after TCA extraction and dansylated as described gave positive results for glutamate. asparate, alanine, glycine, threonine. serine, lycine and ornithine. Fbrirnetric

ussays

The fluorescence of standard solutions of putrescine, cadaverine, GABA and histamine was directly proportional to their concentrations over the range OIO nmol. Each result represented three or more determinations and was expressed as the mean i: 1 S.D. The mean values for these amines in a number (n varying between 7 and 28) of plaque fractions obtained after TCA extraction are shown in Table 2.

of dansyl amine derivatives by thin-layer chromatography solvent systems (AWD) are described in the text

on 0.25 mm silica gel G. The

Rf values First dimension Derivative Ornithine Lysine Putrescine Cddaverine GABA Ammonia Histamine Spermine Spermidine Ethanolamine Agmatine GABA

of

Second dimension

(A)

(B)

(A)

(B)

0.76 0.84 052 0.63 0.92 0.46 0.75 0.85 0.73 @29 (C) 0.00 027

044

0.63 0.77 0.40 0.56 0.59 0.26 0.64 0.83 0.70 0.16

0.40 0.55 0.18 0.20 0.46 0.26 0.13 0.10 0.17

0.56 0.27 0.28 0.26 0.37 0.09 0.11 0.17 0.09 (D) 0.19 0.65

A. T. Hyatt and M. L. Hayes

2M

An analysis of the mean results (II = 6) obtained with standard amino acid solutions gave coefficients of variation ranging from 3.3 to 19.5 per cent. but proline was exceptionally high at 30.7 per cent. Figure I illustrates the estimated, average concentrations of free amino acids in the extra- and intracellular plaque pools and was based on the following assumptions. Firstly. that the TCA extraction yields total, free amino acids from both the intracellular and extracellular spaces and that acid hydrolysis of protein is negligible. Secondly. that the extraction with physiological saline removes free amino acids only from the extracellular space, and, finally. that the total available water in the plaque represents 820&g wet wt. of which 490 pi/g wet wt is intracellular and 330 &g wet wt is extracellular (Edgar and Tatevossian, 197 I).

Table 2. Amine content of plaque extracted with 5 per cent (w/v) trichloroacetic acid. The number of determinations is given in parentheses Plaque content (pmoles/g wet wt) Mean SD.

Amine Putrescine Cadaverine GABA Histamine Agmatine $Alanine Total

I.1 0.2 0.2 0.1

0.5 0.1 (7) (8) @2 0.1 (28) not detectable not detectable (10)

I.6

DISCL SSION

Table 3 shows the mean results of the amino acid analyses of the plaque fractions obtained after separate TCA (n = 6) and saline (n = 4) extractions of the same pooled plaque samples. This technique confirmed the results obtained by chromatography and was able to distinguish between GABA, a-alanine and ,&alanine, however the latter was not detected in these fractions. Table 3. Amino acid content

Amino acid Aspartate Thrconine Serine Glutamate Proline Glycine Alanine Valine Histidine Lysine Total

Amho

ucids iu pluyw

Blackwell, Fosdick and Namajuska. in 1954, used chromatography to demonstrate that a variety of amino acids existed in a free form in ethanol-water extracts of plaque. Critchley c’f rrl. (1967) and Silverman and Kleinberg (1967) showed that extracted

of plaque extracted with either 5 per cent (w/v) trichloroacetic (w/v) sodium chloride solution

Mean

(n

Extract in TCA (pmoles/g wet wt) = 6) S.D. 0.5 0.2 0.3 I.1 0.7 0.4 04 0.1 0.1 0.2

1.6 0.6 0.6 3.4 1.5 1.4

I .o 0.2 0.2 0.6 1 I.6

Extrocellular

acid or 0.9 per cent

Extract in NaCl @moles/g wet wt) Mean (n = 4) S.D. 0.8 0.4 0.7 2.7 0.9 I.6 1.2 0.2 0.2 0.3 9.2

0.4 0.2 @4 I.1 0.6 0.6 0.6 0.1 0.1 0.2

I~t~~C~llUl9~

Fig. I. Estimated free amino acid concentrations in the intra- and extracellular compartments of plaque. Calculations are based on the space determinations of Edgar and Tatevossian (1971) and the results for y-aminobutyric acid (GABA) are also included. fi-Alanine was not detectable.

Amino acids and amines in dental plaque plaque contained a mixture of saccharides. proteins, peptides and amino acids and that a large proportion of the extract could be removed by dialysis and was, therefore, of small molecular weight. This information led Critchley (1969) to identify the amino acids present in aqueous extracts of unhydrolysed plaque whereas Singer and Kleinberg (1973) performed a similar analysis on hydrochloric acid extracts. Their estimations were carried out by automated amino acid analysis and this method wzs used in the present study. The extraction procedures described in this paper were, in essence, very similar to those developed by the above authors but it was thought that extraction into sodium chloride solutions would reduce the extent of any osmotic shock to the intact bacteria and that trichloroacetic acid could be used to extract the total amino acid pool while precipitating soluble protein. The acid and saline extractions, since they were performed on separate fractions of the same plaque sample, not only showed the differences between the two techniques but made it possible to estimate the average concentrations of the amino acids in the extracellular and intracellular fluid compartments. The assumptions on which these estimates were made have been described above and are recognized to be acceptable procedures in this type of distribution space determination (Morgan et a/., 196 1). The results shown in Table 3 agree in substance with those obtained by Critchley (1969); Singer and Kleinberg (1973) and by L.ongton, Carroll and Cole (1974). There were high levels of the amino acids which are involved in intermediary metabolism, notably glutamic acid, but with prominent amounts of aspartic acid, alanine and glycine. Glutamic acid represented 28 per cent of the total amino acid pool in these plaque samples taken after food and was, therefore, lower than the figure of 50 per cent quoted by Singer and Kleinberg for fasting plaque. This discrepancy may be accounted for by their observation that prior exposure to glucose caused a sharp fall in the levels of glutamic and aspartic acids and a rise in alanine. Intracellular concentrations The estimated valu8:s shown in Fig. 1 made it apparent that there were considerable differences between the extracellular and intracellular concentrations of amino acids, although it must be noted that the intracellular measurements depended on the differences between the acid and saline values and, as such, could have exaggerated any error inherent in these results. In spite of this, the total concentration (approximately 7 mM) and the spectrum of amino acids were both typical of bacterial intracellular amino acid pools. These are chan.cterized by a relatively high level of glutamate, whereas the other common amino acids are either present in smaller amounts or are often undetectable (Holden, 1!)62; Tempest, Meers and Brown, 1970). Furthermore, the composition of this pool is not constant but is known to change in response to differences in substrate availability, growth rate and ionic strength and these results can only represent the conditions found in non-fasting plaque with a poorly defined dietary history. The low values for total amino acids, the high glutamate to alanine ratio and the relative predominance of basic amino acids are all considered to be typical of bacteria when sampled either under conditions of a low gr,Dwth rate or at the end of batch

207

culture (Dawson, 1965; Tempest et al., 1970). It is probable that these features represent reduced protein synthesis and are consistent with reports that the plaque bacteria in uivo have a very slow turn-over time (Gibbons, 1964; Tanzer, Wood and Krichevsky, 1969; Van Houte and Saxton, 197 1). Extracellular

concentrations

The total extracellular amino acid concentration was some 4 times greater than the intracellular concentration and indicated a gradient between the two pools, particularly for glutamate, glycine, alanine, proline, aspartate and serine. Not all common amino acids were represented and there was a notable absence of the sulphur-containing and aromatic amino acids, supporting the suggestion (Critchley, 1969, 1970) that bacterial protein synthesis was limited by deficiencies in these amino acids. These concentrations are also very much higher than the equivalent concentrations in the saliva (Battistone and Burnett, 1961; Eastoe, 1961X in the plasma (Krebs, 1950) and, if the latter can be taken to reflect the composition of the gingival sulcus fluid (Brill, 1962) in this also. These differences indicate that constraints on free diffusion probably exist between the plaque fluid and the rest of the mouth and may be either because the plaque matrix has properties which can attract and retain charged species or because the amino acids become available within the plaque at a rate greater than that at which they are washed away by the saliva. Edgar and Tatevossian (1971) and Tatevossian (1974) have reported that a number of other small molecules can occur at high concentrations in the plaque fluid, probably trapped within a sponge-like mesh of protein and polysaccharide. The source of these amino acids is not clear but it is known that a number of organisms can synthesize large quantities of extracellular glutamate from carbohydrate (Holden, 1962) and alanine is often formed in excess of the cell needs and appears in the medium. The accumulation of metabolic amino acids is probably a consequence of the extent to which pyruvate is accumulating as a catabolic intermediate and as a function of the supply of ammonia or amino nitrogen, provided reduced pyridine nucleotides are available for amination reactions (Umbarger, 1969). In addition, an important supplementary source of amino acids must be the extensive proteolytic activity of the plaque bacteria (Mlkinen, 1966; Mgkinen and Paunio, 1966; Soder et al. 1966; Soder, 1967) and the range of free amino acids produced by hydrolysis will reflect the amino acid composition of the protein components of the plaque matrix. It may be noted that the relative concentrations of extracellular amino acids were similar to those found in hydrolysates of salivery proteins, of the acquired pellicle and of plaque extracts (Leach et al., 1967; Armstrong, 1970; Oppenheim, Hay and Franzblau, 197 1). Amines in plaque A number of these amino acids act as substrates for plaque decarboxylation reactions in vitro and it seems reasonable to assume that the corresponding amines will be present in the plaque extracts, although this argument does not preclude the possibility that they could have arisen by other means. The amines were identified and measured by chromatographic and

20x

A. T. Hyatt

and M. L. Hayes

fluorimetric techniques in association with the appropriate internal standard and the results shown in Table 2 suggest that putrcscine could have been derived from ornithine. cadaverine from lysine. GABA from glutamate. and histamine from histidine. Preliminary distribution space determinations indicated that putrescine was more or less evenly distributed between the intracellular and extracellular compartments while the bulk ofthe cddaverine and GABA was found to be extracellular. The presence of the latter in the plaque fluid helps to explain the observation of Rose and Kerr (1957) that GABA. although not present in sterile whole saliva. could be identified in mixed saliva and supports their suggestion that its appearance was due to bacterial contamination. The absence of the amino acid. argininc. and the amine, agmatine, indicates that the decarboxylation of this amino acid is unlikely irl l?uo even though previous work (Hayes and Hyatt, 1974) suggests that arginine decarboxylase can be induced under suitable irk citvo conditions. These measurements of the amines may not indicate the full extent of plaque decarboxylations. however, since it cannot be assumed that. once produced. they will remain unaltered in the plaque. It may be. as already described, that they are produced under acidic conditions when the plaque is metabolically inactive but are subsequently metabolized when neutral conditions are restored. Consequently. the values in Table 2 only represent the average plaque concentrations at the time of collection and these may vary from site to site in the mouth and from time to time in the same individual. It is known. for example. that microorganisms can metabolize GABA to succinic semialdehyde and succinate (Jakoby and Scott, 1959) and the failure to identify /I-alanine and agmatine may reflect active pathways for the degradation of these amines (Meister. 1965; Morris and Pardee. 1966; Hayaishi et LII., 1961). The polyamines putrescine. spermine and spermidine are nutritionally important to a wide varicty of microorganisms. including Luctohacillus. !Vci.ssrj’itr and Veillor~rlln species. They facilitate the emergence of bacteria from a lag phase, increase the rate 01 cell growth and are thought to act by playing an essential role in stabilizing cell structures (Cohen, 1971; Smith, 1972).

Attention should be drawn to speculations (Frostell and Soder. 1970) that these amines could contribute to the initiation of periodontal disease or. at least. modify a pre-existing injury at the gingival margin. Many amines arc toxic when applied elsewhere in the body and exert considerable pharmacological effects. Plaque histamine could have a superficial effect on the gingiva and its microcirculation but would need to exert its effect through the crevicular epithelium. Enzymes exist that can convert certain of the amines, particularly putrescine, spermine and spermidine, into a variety of oxidation products. for example dialdehydes which are highly toxic to bacterial and mammalian cells (Cohen, 1971). However. it is not known if these degradation products are produced in the plaque and their pathogenicity can only be a subject for speculation. It must also bc noted that the observation that a high plaque pH is generally associated with gingival pathology (Kleinberg and Jenkins, 1964) contrasts with the acid conditions which favour induced amine production.

although it could be argued that their basic nature will ultimately raise the pH. Ack,lo\~/rngc,r?~r,~t.s-Thanks are specially due to Mr. D. W. Robinson for his outstanding technical assistance during the course of this work. Financial support from the Medical Research Council is also gratefully acknowledged. REFERENCES

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