Avhs oral Bid. Vol. i’wgamon ~‘ress WI.

24, pp. 15 to 20. ,979. Printed in Great Br~tam.

ADENOSINE TRIPHOSPHATE CARIOUS HUMAN

IN NORMAL DENTINE

AND

1. LXIKKB and M. LARMAS Institute of Dentistry, University of Turku, Turku, and Institute of Dentistry,

University of Oulu, Oulu, Finland Summary-Adenosine triphosphate (ATP) levels were quantitatively measured in various layers of freeze-dried human dentine. Developing, intact, fully-formed, carious and gangrenous teeth were studied. ATP was extracted from the dentine powder with 0.1 M perchloric acid solution. Using a iiquid scintillation counter, the amount of ATP (pmole/mg dentine) was determined from lo-mg samples of dentine by means of the luciferase reaction. The highest ATP level in hard dentine was observed in coronal and apical peripulpal areas of developing teeth (2.0pmole/mg dentine). In coronal hard dentine, ATP concentration decreased towards the dentineenamel junction, where practically no ATP could be observed. In intact teeth, the corresponding peripulpal values were statistically lower (0.4 pmole/mg dentine). ATP levels of 1.7 pmole/mg dentine were observed in coronal peripulpal dentine under an incipient caries lesion. When the hard dentine became soft and carious, 5-8 times higher ATP values were measurable (8.7-14.5 pmole/mg dentine).

INTRODUCTION

tine under conditions.

The significance of adenosine 5’-triphosphoric acid (ATP) in the mineralization of biological tissues was first observed in connection with mineralizing cartilage (Albaum, Hirshfield and Sobel, 1952; Cartier and Picard, 1955a; Whitehead and Weidman, 1957). Cartier and Picard (1955b) assumed that high concentrations of inorganic pyrophosphate in mineralizing tissues are derived from ATP and may provide phosphate to the mineralizing organic matrix (Polonovski and Cartier, 1951; Glimcher and Krane, 1962; Krane and Glimcher, 1962). However, inhibition of mineraliza.tion by inorganic pyrophosphate and ATP was also shown by Fleisch and Neuman (1961) Fleisch (1964), Fleisch et al. (1966) Russel et al. (1971) and Posner, Betts and Blumenthal (1977). Membrane-bound bodies (matrix vesicles) have been described in cartilage (Anderson, 1969; Bonucci, 1970; Ali, 1976) bone (Bernard and Pease, 1969) predentine (Bernard, 1972; Eisenmann and Glick, 1972; Larsson and Bloom, 1973; Katchburian, 1973a, b) and the cytoplasm of odontoblasts (Reith, 1976). They are thought to be related to the initiation of mineralization because of the occurrence of crystal-like inclusions, presumably calcium phosphates, within the vesicles. These calcium and phosphate ions are possibly transported via matrix vesicles (Sayegh, Davis and Solomon, 1974) with ATP and mitochondria probably playing some part (Lehninger, 1970; Matthews and Martin, 1971). ATP may also be important in hydroxyapatite production as Blumenthal, Belts and Posner (1975) showed that synthetic amorphous calcium phosphate can be converted in water to hydroxyapatite by removal of hydroxyl ions from the solution. Posner, Betts and Blumenthal (1977) postulated that, by regulating the ATP level, the cells can prevent this hydroxyapatite formation. Our aim was to throw light on this problem by determining the ATP concentrations in human den-

various

physiological

and pathological

MATERIAL AND METHODS

Healthy, carious and amalgam-filled permanent human teeth (about 30 for preliminary studies and 99 for quantitative analysis) were extracted under local anaesthesia (Citanest-Octapressin@ or XylocainAdrenalin, Astra Ab, Sodertllje, Sweden) at the University Students’ Health Centre and at the Dental Health Centre in Turku. Immediately after extraction, the teeth were rinsed with cold tap water to remove the blood and immersed in liquid nitrogen. The teeth were then lyophilized without thawing (Hetofrig, Heto, Birkerod, Denmark) and stored dry at +2”C for not more than two days before use. Clamjkation

of the teeth

Using criteria previously described (Le Bell and Larmas, 1976; Llikkii and Larmas, 1978a), the teeth were classified both clinically and under a dissecting microscope as follows : (1) intact developing teeth with less than half of the root formed; (2) intact developing teeth with about 3/4 of the root formed: (3) intact, non-carious fully-formed teeth; (4) teeth with caries affecting enamel only; (5) teeth with caries extending to dentine; (6) teeth with caries extending to the pulp; (7) teeth with gangrenous pulps; (8) teeth with gangrenous roots; (9) amalgam-restored teeth. The teeth were split mechanically with forceps along their long axes; their classification was then reconsidered. The pulp and periodontal tissues were carefully removed with excavators and curettes. The carious lesions were cleaned by scraping the surface with an excavator to avoid contamination with loose dental plaque. The fillings were removed from amalgam-restored teeth without the use of dental burs; if caries was found under the amalgam, the tooth was discarded. 15

16

I. LSiikkSand M. Larmas

Preparation of the dentine samples

lO.O-mg dentine samples were obtained from 3 to 6 areas of each tooth (Figs. 2a-c) and served as material for a series of analyses, The samples were drilled with round No. 2 and 3 steel burs (0.86 and 1.03 mm) at room temperature at speeds not exceeding 4000 r.p.m. A fresh bur (Hager & Meisinger, Diisseldorf, FRG) was used for each sample. When drilling the peripulpal zone, the thinnest possible layer of dentine was left against the pulp chamber. Extraction of ATP from the dentine powder

ATP was extracted from the dentine powder using (a) water; (b) isotonic 0.9 per cent NaCl; (c) 0.01-5 per cent Triton@ X-100; (d) 75 mM tris-HCl buffer, pH 7.3 (I 0 mM MgCl,, 5 mM EDTA); (e) 50 mM glycyl-glycine buffer, pH 7.5 (10 mM MgCl,, 5 mM EDTA); (f) 0.01-1.0 M HClO, solutions. Extraction mixtures with solutions (a-e) were transferred to a boiling-water bath ( + 96°C) for 3 min immediately after the adding of the solution to avoid possible enzymic hydrolysis of ATP. For the quantitative analysis, the extraction method was as follows: lO.Omg of dry dentine powder was dissolved in 1.1 ml of cold (+ 2°C) 0.1 M HC104 solution and homogenized for 15 s with a Teflon-glass homogenizer (Karppinen, Turku, Finland). After an extraction time of 3 h (15min-24 h were tried) at +2”C, the dentine particles were removed by centrifugation in a Sorvall Superspeed Refrigerated Centrifuge (Ivar Sorvall inc. Norwalk, Conn.) 23,500 g for 15 min at + 1-3°C. 1.0 ml of the supernatant solution was neutralized according to Bagnara and Finch (1972) with 0.4 ml of 0.7 M KOH, containing 0.16 M KHCO,. The samples were stirred, kept at +2”C for 15 min and centrifuged for 10 min at 4000g. Determination of ATP

The ATP concentration in the supernatant solution was determined by means of the luciferase reaction (Strehler, 1968) by the modified method of Chapman, Fall and Atkinson (1971). 200 ~1 of the supernatant solution and 50 ~1 of 75 mM tris-HCl buffer, pH 7.3, containing 15 mM MgC12, was incubated for 15 min at +3O”C. After cooling in an ice bath (+2”(Z), lOO$ of this mixture (and another 100~1 for the duplicate determination) and 1.85 ml of 0.05 M glycyl-glycine buffer (+2O”C), pH 7.5, were pipetted into scintillation bottles. 50~1 of luciferase enzyme reagent (6mg of the firefly lantern extract/l ml of water) which had been left to stabilize for 24 h at +2”C (Stanley and Williams, 1969) was then added and the solutions were mixed carefully as suggested by Lin and Cohen (1968). Samples were counted in a Liquid Scintillation Counter (1210 Ultrobeta, LKB-Wallac, Turku, Finland) for 20 s starting exactly 15 s after the addition of the luciferase reagent. Standard curves (0-50pmole ATP) were prepared separately for each determination and were handled similarly. In addition, calcium phosphate was added to the standard solutions, 3-12mg in preliminary studies and 8 mg in the quantitative analysis series. Other methods

The calcium (Ca”) content of the dentine samples was measured using atomic absorption spectrophoto-

metry (Perk&Elmer, Model 303, Norwalk, Conn.) and inorganic phosphate by a modified method of Lowry and Lopez (1946). The results were calculated as the means and SE of the means, and Student’s t-test was used to determine the statistical significance of any differences between the means. Chemicals

TrisHCl buffer and firefly lantern extract (FLEJO) were purchased from Sigma Chemical Co. (St. Louis. MO.). Glycyl-glycine buffer was obtained from Fluka AG (Buchs AG, Switzerland) and adenosine-5’-triphosphate (disodiumsalt) was a product of Boehringer (Mannhein Gmbh, FRG). Calcium phosphate, Ca,,(OH)#O,),, was purchased from J. T. Baker Chemical Co. (Phillipsburg, N.J.). All other reagents were obtained from E. Merck AG (Darmstadt, FRG). RESULTS

Solubilization and homogenization The maximum amount of ATP was extracted with a 0.1 M HCIO, solution both from hard and soft dentine. Mechanical treatment with a glass homogenizer before the extraction with perchloric acid increased the level of ATP by 10-15 per cent. After the second mechanical treatment, the amount of ATP did not increase. The extraction time of 3 h released the most ATP from the dentine powder. Water, isotonic 0.9 per cent NaCl and Triton X-100 (0.01-5 per cent) solutions extracted 65 per cent ATP from hard dentine and 5-10 per cent from soft carious dentine when compared with the perchloric acid extraction. However, 75mM trisHC1 buffer, pH 7.3 (10 mM mg Cl*, 5 mM EDTA) and 50 mM glycy-glytine buffer, pH 7.5 (1OmM MgC12, 5 mM EDTA) extracted 40-50 per cent of the quantity of ATP extracted by HC104. The efict curve

of hydroxyapatite on the standard ATP

Eight milligrams of hydroxyapatite caused about the same percentage reduction on the standard ATP curve as did 1Omg of hard dentine powder from the enamel-dentine layer (Fig. 1). l.Oml of 0.1 M HClO* extract of dentine powder contained 1.85 mg calcium and 1.02 mg inorganic phosphate. The quantitative analysis

In developing teeth, the highest concentrations of ATP were observed in the coronal and apical peripulpal dentine (2.0pmole/mg dentine; Fig. 2a). The values in the peripulpal zone between the coronal and apical regions were significantly lower (p c 0.05). A highly significant decrease (p < 0.001) was observed in the peripulpal ATP level when the tooth became fully-formed and remained intact. ATP values throughout the peripulpal area of developing teeth were statistically significantly higher than those in intact teeth (Fig. 2a). In coronal dentine layers of developing and intact teeth, ATP was less towards the dentineenamel junction, where practically no measurable ATP could be observed. In teeth with caries of enamel only, the ATP level began to be greater in all parts of the tooth when

17

ATP in human dentine

0

5

10

30

20 pmoles

40

50

ATP

Fig. 1. The effect of hard dentine powder and hydroxyapatite on the standard ATP curve: (0) control; (A) 6 mg of hydroxyapatite; (0) 8 mg of hydroxyapatite; (0) 10 mg of dentine (from the dentin-name1 border) was added.

compared to intact teeth (Fig. 2b). This rise was statistically significant in the crown peripulpal dentine. In amalgam-restored teeth, the ATP level was similar to that in intact teeth (Fig. 2b). In teeth with caries extending to involve the pulp, the ATP level of hard dentine showed lower values when compared to teeth with incipient dentine caries (Figs. 2b and 2~). In the hard dentine of gangrenous teeth, the ATP level was near zero. In soft carious dentine, a highly significant greater ATP content was present (p < 0.001) when compared to the same area of intact teeth. In teeth with caries extending to the pulp, soft carious dentine revealed the highest values (14.5 pmole/mg dentine; Fig. 2~). However, when the caries cavity was more open (gangrenous pulps and gangrenous roots), the ATP in soft dentine was significantly less (Fig. 2~).

DISCUSSION

Of the numerous methods reported for the analyses of ATP in various tissues, the modification of perchloric acid hydrolysis (Bagnara and Finch, 1972) was thr: most suitable for our study. However, the relatively long extraction procedure suggests that the ATP is tightly bound to the dentine structures. Our findings concerning the distribution of high ATP concentration in the peripulpal zone of mineralizing dentine, both in the coronal area and in the mineralizing apex (Fig. 2a), suggest that ATP has a role in the mineralizing process. The substantial increase in the coronal section of the peripulpal zone under the lesion gives indirect support to the views O.B.24/l--

13

of Fish (1930) about the formation of hypermineralized sclerotic dentine under caries. It is surprising, however, that the highly significant higher values in the peripulpal dentine were measurable even in teeth with incipient caries of the enamel only with no signs of demineralization in the dentine under a dissecting microscope. This may indicate that a still undiscovered information chain starts the production of ATP molecules in the very early stages of carious attack in the dentine. ATP is then demonstrable even at the dentine-enamel junction, possibly reflecting passing of the molecules via the dentinal fluid into the whole thickness of the dentine. It seems probable that in mineralizing tissues generally initial mineral deposition begins inside membrane-bound bodies. Phosphohydrolytic enzyme activity towards simple phosphate esters and ATP (Ali, Sajdera and Anderson, 1970; Majeska and Wuthier, 1975) enriched calcium and inorganic phosphate levels (Ali, 1976) ATPase activity (Matsuzawa and Anderson (1971) and alkaline phosphatase activity (Larsson, 1973; Yanagisawa, 1975) during early stages of mineralization have been reported in these bodies. There also appears to be a relationship between ATP and initial calcium uptake (Anderson and Reynolds, 1973). LHikkG and Larmas (1978a) observed significantly greater phosphatase activity and free inorganic phosphate content (Llikkii and Larmas, 1979) in the mineralizing peripulpal dentine of human teeth. Our present study revealed high ATP concentrations in corresponding areas. Thus, our biochemical observations support the concept of mineralization based on electronmicroscopical findings.

18

I. LkiikkS and M. Larmas DEVELOPING

E NAMEL

ib)

CAR’ ES

4 PULP-

I NVDLVED

DEVE LOPIN

DENTINE

CARIES

-

I N TACT

AMALGAM - RESTORED

GANGR ENUUS

Fig. 2~ Areas from which the dentine samples were drilld. The values express the amount of ATP liberated from the dentine samples (pmoles!mg f SE) The level nf prubability is expressed by means of asterisk indicating the ranges of probability (p) BS bllows: “0.01 c p < 0.05: “0.001 < p < 0.01;

l**p -z cml.

ATP in human

The great increase of ATP in soft carious dentine is probably due to the microbial production of ATP molecules. However, the ATP level decreased to zero in the hard dentine of necrotic and gangrenous teeth as a whole. Furthermore, we have observed a significantly lower ATP level in the necrotic pulpal tissues of gangrenous teeth. These observations support the total loss of possible energy sources in the pulp and dentine of gangrenous state of the teeth. Achnowledgement-The Pyi-klri is gratefully

technical acknowledged.

assistance

of Miss

K.

REFERENCES

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