Plasma and Developing Enamel Fluoride Concentrations During Chronic Acid-base Disturbances

GARY M. WHITFORD and KEITH E. REYNOLDS

Department of Oral Biology-Physiology, School of Dentistry, Medical College of Georgia, Augusta, Georgia 30912

Mild acid-base disturbances were induced in rats for 30 days. These disturbances did not affect % ash of maxillary incisors or % P of the developing enamel from mandibular incisors. Total fluoride intake (food and water) among groups drinking fluoride-free water was constant. Nevertheless, average plasma and developing enamel fluoride concentrations were highest in the acidotic group and lowest in the alkalotic group. Among groups drinking water containing 50 ppm fluoride, total fluoride intake was highest by the alkalotic group and lowest by the acidotic group. Plasma and enamel fluoride concentrations, however, were highest in the acidotic group. It is concluded that plasma and developing enamel fluoride levels can be independent of, or inversely related to, fluoride intake. J Dent Res 58(11):2058-2065, November 1979

Introduction.
The plasma ,2 ,3 , 5 and calcified tissue6 7,8 ionic fluoride concentrations in experimental animals and human subjects have generally been found to reflect fluoride intake. These concentrations, however, are not related to intake in a simple linear fashion and, further, most reports indicate considerable individual variation in these concentrations.3,'56'9 These findings suggest that fluoride balance or retention is variable, not only among individuals or groups but also within individuals, over time. Fluoride retention can be increased by a decreased efficiency with which the kidneys excrete fluoride (renal clearance rate) or by an increase in the rate of calcified tissue uptake and vice versa. Several factors which
Received for publication December 7, 1978. Accepted for publication May 10, 1979. This work was supported by NIDR Grants DE 04332 and DE 04832. 2058

influence the latter process have been identified. Principal among these are the rate of growth101 1 and previous exposure to fluoride.12 Until recently, the renal clearance of fluoride was thought to be mainly determined by, and a direct function of, urine flow rate1 3,14 or the renal clearance rate of chloride. 15 Additional studies,16'17'18 however, have shown that fluoride renal clearance can be easily dissociated from both urine flow rate and chloride clearance. These studies identified urine pH as a major determinant of the rate of fluoride renal clearance in the rat and dog and of fluoride excretion rate by humans. In a chronic acid-base disturbance study using rats, Whitford and Pashley18 found that plasma fluoride levels, and those of eight soft tissues, were highest in the acidotic group and lowest in the alkalotic group. Urinary fluoride excretion by alkalotic rats, however, was twice that of acidotic rats while the control group was intermediate. It was calculated that fluoride was removed from the body four times more rapidly in alkalosis than in acidosis. This greater efficiency in fluoride excretion was consistent with the lower plasma and soft tissue fluoride levels of the alkalotic group, but the role of the calcified tissue compartment was not defined. Chronic metabolic acidosis has been reported to increase the rate of bone resorptionl9'20 while alkalosis may stabilize bone mineral.20 Thus, the high plasma fluoride levels in acidosis, as well as the lower values in alkalosis,18 may have been partially due to the effects of the acid-base disturbances on calcified tissue fluoride handling. The current study was designed to provide information relevant to fluoride

Vol. S8 No. 11

FLUORIDE LEVELS DURING ACID-BASE DISTURBANCES

2059

uptake by calcified tissue during chronic acid-base disturbances using the unerupted incisor enamel of the rat as the model.21 This tissue provides the opportunity to examine fluoride concentrations in developing partially and fully mineralized, calcified tissue.

(Orion, Model 94-09A) following hexamethyldisiloxane (HMDS) diffusion.22 The

details of the method were as follows: Two ml of doubly de-ionized, distilled water were placed in the bottom of a non-wettable petri dish (Falcon, #1007). Known volumes of plasma or a fluoride standard solution, or known weights of enamel, were added to the water. The base trap, 50 pl of 0.05 N NaOH, was placed in five separate drops on the inside of the top half of the dish. The inside periphery of the lid was ringed with Vaseline and sealed to the bottom. Two ml of 3.0 N H2 SO4, saturated with HMDS, were then added to the bottom by injection through a small hole previously burned in the top half of the diffusion dish. The hole was immediately sealed with Vaseline. The diffusion and trapping of fluoride continued for at least 18 hours. The tops were then removed, and 25 pl of 0.20 N acetic acid were added to the NaOH trap which adjusted the pH to 4.8. The buffered solution was drawn into a calibrated polyethylene tube, and water was added to a final volume of 75 p1. This is an important step, since volume loss of the trap during prolonged diffusion can be large and variable. (Alternatively, the fluoride-containing trap can be dried and known volume of acetic acid added.) The solution was then returned to the lid and the fluoride and calomel reference electrodes were immersed for analysis. The acid solutions were recovered from the dish bottoms containing the dissolved enamel samples for phosphorus analysis, according to the method of Chen et al.23 Data are expressed as mean SEM; significance testing was done usiiig Student's two-tailed t-test or analysis of variance followed by Duncan's Multiple Range Test, and a significance level of p < .05 was selected.
The average percent increase in body weight for groups 1-5 during the 30-day study was approximately 20%, while the increase for group 6 was 7.4%, as shown in Table 1. The cause of the lower weight gain of group 6 was not apparent. Water consumption was highest by the alkalotic groups (3 and 6) and, between these, it was higher by group 3 whose drinking water

Materials and methods.

Eighteen female, random-bred Wistar rats (187 4 gm) were randomly assigned to six groups of three rats each. Each group was housed in a single cage. The rats were allowed free access to distilled water and food (36 ppm fluoride) during two weeks of acclimatization to their new environment. For the next 30 days, groups 1, 2 and 3 continued on fluoride-free water, while groups 4, 5 and 6 received water containing 50 ppm fluoride (as NaF) ad libitum. Groups 1 and 4 were made acidotic by adding NH4Cl (0.25 M) to their drinking water, while groups 3 and 6 were made alkalotic by adding NaHCO3 (0.15 M) to their drinking water.18 No additional salts were added to the water of groups 2 and 5. Body weights and food consumption were monitored weekly. Water consumption was measured three times each week. On the final day of the experiment, food and water were denied each group, starting at 7 A.M. Four hours later, each animal was decapitated, blood was collected from the torso in polypropylene test tubes, and the maxillary and mandibular incisors were extracted. The maxillary incisors were dried to constant weight at 1000 C and ashed in a muffle furnace at 6000 C overnight. Only the ash content was determined on these incisors. The developing enamel of the unerupted portions of the mandibular incisors was dissected from the teeth with a scalpel blade and divided into three sections. Anatomically, these sections were as follows: El, root apex to 1 mm of the transitional zone or white opaque boundary; E2, the transitional zone representing the next 2 mm; and E3, the next 4-5 mm of maturing enamel. The corresponding enamel sections from the incisors of each animal were pooled in a single microbeaker and dried to constant weight (Cahn Model 23) at 100 C. Fluoride in plasma and enamel was determined using the ion-specific electrode

Results.

2060

WHITFORD AND REYNOLDS

J Dent Res November 19 79

was fluoride-free. The presence of 50 ppm fluoride in the drinking water of groups 4 and 5, however, had no influence on water intake, as seen by comparison with groups 1 and 2 (Table 1). There were no statistically significant differences among the groups for food intake.

same relationship to acid-base balance, i.e., group 1 > 2 > 3, although these differences did not achieve statistical significance. The percent ash content of the dried whole maxillary incisors revealed no statistically significant differences between any of

TABLE 1 SUMMARY OF 30-DAY BODY WEIGHT GAIN AND DAILY WATER AND FOOD INTAKE

Groupa
1 2 3

Body Weight, % Gain

Water Intake, ml/group/day

Food Intake, gm/group/day 53 +2

54

21.6

61

+2.4
19.6

+1
71

+2.7
20.4

+3
151 +7

+2
57

+1.8
4
5

+3
53 +3 53

18.7

+4.2
20.0

57 +1

+1.2
6

67 2

+2
48

7.4b
+3.8

83b
+4

+3

a- Three rats per group. Groups 1-3 received fluoride-free water, and groups 4-6 received water containing 50 ppm fluoride during the 30-day study. In addition, the drinking water of groups 1 and 4 contained 0.25 M NH4C1, and that of groups 3 and 6 contained 0.15 M NaHCO3.

p < .05. Significance testing (t-test) compares group 1 with 4, group 2 with 5, and group 3 with 6.

Table 2 shows the average daily fluoride intake with food, water, total fluoride intake, and the terminal plasma fluoride concentrations of each group. Due to greater water intake, group 5 received 10% more fluoride than group 4, and group 6 received 23% more than group 4. In spite of its lower intake of fluoride, the plasma fluoride of group 4 (acidotic) was significantly higher than that of both groups 5 (normal acid-base balance) and 6 (alkalotic). Further, the plasma level of group 5 was 20% higher than that of group 6 (not significant), while its fluoride intake was 1 1 % lower. The terminal plasma fluoride concentrations of the groups drinking fluoride-free water showed the

six groups. However, the combined data of the groups drinking 50 ppm fluoride water had slightly lower values. Comparing the data of groups 1-3 and groups 4-6, the latter group had a significantly lower ash content (69.8 + 0.4% vs. 68.6 0.4%, respectively). This finding was consistent with the presence of striations in the erupted enamel (fluorosis) in each of the mandibular incisors, and about 50% of the maxillary incisors of the rats in group 4-6. While no attempt was made to quantitate the degree of enamel fluorosis, examination with a dissecting scope (x3) revealed that the striations were most apparent in group 4.

Vol.58 No.11I

FL UORIDE LEVELS DURING ACID-BASE DISTURBANCES

2061

The figure presents the enamel fluoride Among the groups drinking water fluoriand phosphorus data for each of the three dated at 50 ppm, group 4 had the highest developing enamel sections. The values fluoride levels in each enamel section as for phosphorus and the generally higher well as the highest plasma level. The diffluoride concentrations in the enamel ferences in the enamel fluoride levels were of the E2 section (transitional zone) are significant in several cases (E2: 4 vs. 5, in close agreement with the findings of 4 vs. 6; E3: 4 vs. 5, 4 vs. 6). These conWeatherell et al.21 There were no statis- centration differences occurred even tically significant differences for percent though the animals of group 4 ingested phosphorus among the six groups. Com- less fluoride than those of groups 5 or 6 bining the percent phosphorus data of (Table 2). The average fluoride concentrations in the El and E3 sections of groups groups 1-3 and of groups 4-6, however, it 5 and 6 were nearly identical. The relatively was found that the average value of the latter animals was significantly lower in each high fluoride level of the E2 section, found of the three enamel sections. in every other group, was absent in group 6, The fluoride data in the figure suggest where it averaged 41% and 22% lower than that the effects of the acid-base distur- the levels of groups 4 and 5, respectively. bances on enamel fluoride concentrations Overall, the enamel fluoride concentrations paralleled those observed in plasma (Table of groups 4, 5, and 6 were inversely re2). The amounts of fluoride consumed lated to fluoride intake and directly by the groups drinking fluoride-free water related to the plasma fluoride levels (Table 2). were not significantly different (Table 2). Nevertheless, the average plasma and enamel fluoride concentrations were highest Discussion. in the acidotic group and lowest in the We have reported that urinary pH is alkalotic group. The differences in the an important determinant of fluoride renal enamel fluoride levels reached statistical clearance in the ratl6'18 and the dog,17'18 significance in several instances (E 1: 1 vs. and of urinary fluoride excretion by 3; E2: 1 vs. 2, 1 vs. 3, 2 vs. 3; E3: 1 vs. 2, humans.18 The rate of fluoride absorption 1 vs. 3, 2 vs. 3). from the urinary bladder of the rat was TABLE 2 SUMMARY OF DAILY FLUORIDE INTAKES AND TERMINAL PLASMA FLUORIDE
CONCENTRATIONS

Fluoride Intake,

mg/group/day
Group
1

Plasma Fluoride Concentration, AM

Food

1.92 +.07 1.93

Water 0

Total 1.92 .07

3.2 .8 2.6 .1 2.4 .4

.4

+.07
3
4
5

1.93 .07 2.05 .09 4.75 .11 5.24a .13

5.86a .23

2.05

+.09
1.90 + .09
1.89 + .08

2.85 .07

8.4

3.35a
+ .10

5.0a .1 .3

6
a
p <

1.71 .12

4.15a .20

4.0a

.05 compared to group 4 as determined by analysis of variance and Duncan's Multiple Range Test. There were no statistically significant differences among groups 1, 2 and 3.

2062

WHITFORD AND REYNOLDS

J Dent Res November 19 79

found to be inversely related to pH24 and, using Streptococcus mutans, fluoride uptake was found to be a function of the magnitude of the transcellular pH gradient.25 A pre-existing metabolic acidosis was found to sensitize rats to acute fluoride toxicity, while a pre-existing metabolic alkalosis afforded significant protection.26 Furthermore, there is theoretical and experimental evidence that the steady-state distribution of fluoride between the extra- and intracellular compartments of several mammalian soft tissues is determined by the magnitude of transmembrane pH gradients.18'26'27 The influence of mild systemic acid-base disturbances on fluoride metabolism in hard tissues has received little attention, however, and the present work was done to partially fill this hiatus. The current results indicate that chronic, mild acid-base disturbances do not markedly alter hard tissue fluoride metabolism and that, under these conditions, enamel fluoride concentrations simply reflect those of plasma. Insofar as fluoride uptake by enamel is representative of uptake elsewhere in the calcified tissue compartment, the urinary pH-dependence of the renal handling of fluoride16-18'26 becomes the most important determinant of both soft and hard tissue fluoride concentrations when intakes are not markedly different. Additional studies on bone fluoride metabolism are indicated to further clarify these relationships. The present results also indicate that plasma and enamel fluoride concentrations and severity of enamel fluorosis are not always directly related to fluoride intake. Intake by groups 1, 2 and 3 (fluoride-free drinking water) was similar throughout the study (Table 2). In spite of this similarity, plasma and enamel fluoride concentrations were highest in the acidotic group and lowest in the alkalotic group (Table 2 and Figure). Among the groups drinking water fluoridated at 50 ppm, fluoride intake was highest by the alkalotic group and lowest by the acidotic group. Based on these intake patterns, one would have predicted that plasma and enamel concentrations would have been highest in the alkalotic group. However, exactly the reverse was found (Table 2 and Figure). From these data it is concluded that, when fluoride intake is not greatly different

(compare groups 1, 2 and 3 or groups 4, 5 and 6), plasma and enamel fluoride concentrations can be independent of, or inversely related to, fluoride intake. It should be noted that urinary fluoride excretion rates and blood and urine pH values were not monitored in the current study. However, in the chronic study described in the Introduction,18 in which the NH4Cl and NaHCO3 drinking water concentrations were the same as used in the present study, the average blood pH values of the acidotic, control, and alkalotic groups were 7.41, 7.46 and 7.53, respectively. The blood pH of the control group was in close agreement with the findings of other investigators (7.41-7.45).28,29 The average urine pH values were 5.38, 5.80, and 6.51, respectively. These data are representative of mild acid-base disturbances. It is not unreasonable to assume that those group pH differences and fluoride excretion rate differences were closely approximated in the current study. Fecal excretion of fluoride (or of other ions of potential importance, e.g., calcium and phosphate) was not evaluated in the present study. If there were differences among the groups, which would indicate absorption differences, they could have contributed to the results obtained. However, in the previous study,18 all groups received identical fluoride doses subcutaneously two hours prior to sacrifice. This fluoride was never in contact with the lumen of the gastrointestinal tract. In agreement with the present results, the plasma fluoride concentration of the acidotic group was twice that of the alkalotic group. It is, therefore, unlikely that group differences in the absorption of fluoride contributed significantly to the current results. Finally, the lower values of maxillary incisor percent ash and mandibular incisor enamel percent phosphorus values of groups 4, 5 and 6, compared to those of groups 1, 2 and 3, were consistent with the observation of enamel fluorosis in the former groups. The fact that there were no differences among groups 4, 5 and 6 for these variables (percent ash and phosphorus) perhaps was due to the small sizes of the groups. An elevated protein content and decreased mineral content is characteristic of fluorosed enamel30'31 and, as described in the Results, the fluorotic striations

Vol. S8 No. II

FLUORIDE LEVELS DURING A CID-BASE DISTURBANCES

2063

400r
340

140r
E 100 0.
CL

E
U.

a-

0.

[
I~~~~~~~~~~~~~~~~

2800

60F

220

20 0
11
X 13
r

GROUPS 1-3

GROUPS 4-6

CL

113

9
E, E E I '2

aI

0'
'3

E1

E2

E3

Fig. 1 -Average values of fluoride concentrations (bars, SEM) and percent phosphorus in the three sections of the developing enamel of rat mandibular incisors. Groups 1, 2 and 3 received fluoride-free drinking water and groups 4, 5 and 6 received water containing 50 ppm. Groups 1 and 4 (0-*) were acidotic, groups 2 and 5 (o-o) were in normal acid-base balance, and groups 3 and 6 (o-o) were alkalotic.

2064

WHITFORD AND REYNOLDS

J Dent Res November 1 9 79

ride in the Drinking Water and the Plasma Fluoride Concentration in Man, Caries Res 12:123-127, 1978. ZIPKIN, I; MCCLURE, F. J.; LEONE, N. C.; and LEE, W. A.: Fluoride Disposition in Human Bones After Prolonged Ingestion of Fluoride in Drinking Water, Pub Hlth Rept US 73:732-740,1958. SMITH, F. A.: Metabolism of Inorganic Fluoride. In: Handbook of Experimental Pharmacology, Pharmacology of Fluorides (ed. F. A. Smith), vol XX, Part I, New York: Springer-Verlag, 1966, pp. 107-117. JACKSON, D. and WEIDMANN, S. M.: The Relationship Between Age and the Fluorine Content of Human Dentine and Enamel: A Regional Survey, Brit Dent J 107:303-306, 1959. WEATHERELL, J. A.; DEUTSCH, D.; ROBINSON, C.; and HALLSWORTH, A. S.: Assimilation of Fluoride by Enamel Throughout the Life of the Tooth, Caries Res 11 (Suppl 1): 85-115, 1977. GEDALIA, I.; BRZEZINSKI, N.; PORTUGUESE, N.; and BERCOVICI, B.: The Fluoride Content of Teeth and Bone of Human Foetuses, Arch Oral Biol 9:331340, 1964. ZIPKIN, I. and MCCLURE, F. J.: Deposition of Fluorine in the Teeth and Bones of the Growing Rat, J Nutr 47:6 11-6 29, 1 95 2. SUTTIE, J. W. and PHILLIPS, P. H.: The Effect of Age on the Rate of Fluoride Deposition in the Femur of the Rat, Arch Biochem 83:355-359, 1959. CARLSON, C. H.; ARMSTRONG, W. D.; SINGER, L.; and HINSHAW, L. B.: Renal Excretion of Radiofluoride in the Dog, Am JPhysiol 198:829-832, 1960. CHEN, P. S., JR.; SMITH, F. A.; GARDNER, D. E.; O'BRIEN, J. A.; and HODGE, H. C.: Renal Clearance of Fluoride, Proc Soc Exper Biol Med 92:879-883, 1956. WALSER, M.; and RAHILL, W. J.: Renal Tubular Transport of Fluoride Compared with Chloride, Am J Physiol 210:'12901292, 1966. WHITFORD, G. M.; PASHLEY, D. H.; and STRINGER, G. I.: Fluoride Renal Clearance:a pH-dependent Event, Am J Physiol 230:527-5 32, 1976. HARMON, C.; WHITFORD, G. M.; PASHLEY, D. H.; REYNOLDS, K. E.; and STOCK, H.: pH-dependence of Fluoride Renal Clearance in Dogs, Soc Exper Biol Med (Southeastern Section): Abstract 13, 1976. WHITFORD, G. M. and PASHLEY, D. H.: The Effect of Body Fluid pH on Fluoride Distribution, Toxicity and Renal Clearance. In: Continuing Evaluation of the Use of Fluorides, Am Assoc Advan Sci Monograph, Selected Symposium 11, 187221, 1979.

were most pronounced in the incisors of the acidotic group. This observation was associated with the higher plasma and enamel fluoride concentrations of this group. Based on these findings, studies are in progress to determine a fluoride intake level which will produce enamel fluorosis in mildly acidotic animals, but no fluorosis in normal or alkalotic animals. The results of such studies might have direct bearing on the endemic fluorosis reported among humans residing in areas where fluoride intake is apparently not excessive.32-34

6.

7.

8.

Conclusions.
The results of this study permit the

following conclusions:
1. Chronic metabolic acidosis is associated with elevated plasma and developing enamel fluoride concentrations, compared to the values associated with normal acid-base balance or chronic metabolic alkalosis. 2. These relationships obtain even when fluoride intake is greater by the normal or alkalotic animals. 3. Enamel fluoride concentrations reflect those of plasma and these, in turn, appear to be mainly determined by the efficiency with which the kidneys excrete fluoride.

9.

10.

11.

12.

Acknowledgment.
Mr. R. Harp assisted with the statistical analyses.
13.

1.

2.

3.

4.

5.

REFERENCES SMITH, F. A.; GARDNER, D. E.; and HODGE, H. C.: Investigations on the Metabolism of Fluoride: II. Fluoride Content of Blood and Urine as a Function of the Fluorine in Drinking Water, J Dent Res 29:596600, 1950. SHEARER, T. R. and SUTTIE, J. W.: Effect of Fluoride Administration on Plasma Fluoride and Food Intake in the Rat, Am J Physiol 212:1165-1168, 1967. GUY, W. S.; TAVES, D. R.; and BREY, W. S.: Organic Fluorocompounds in Human Plasma: Prevalence and Characterization. In: Biochemistry Involving Carbon- Fluorine Bonds (ed. Robert Fuller), ACS Symposium Series, No. 28, pp 117-134, 1965. OPHAUG, R. H. and SINGER, L.: Influence of Variations in Fluoride Intake on the Ionic and Bound Fractions of Plasma and Muscle Fluoride, Proc Soc Exper Biol Med 155: 23-26, 1977. EKSTRAND, J.: Relationship between Fluo-

14.

15.

16.

17.

18.

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19. ALBRIGHT, F. and REIFENSTEIN, F. G., JR.: In: The Parathyroid Glands and Metabolic Bone Disease, Baltimore. Williams and Wilkins Co., 194 8, pp. 24 1-247. 20. BARZEL, U.S. and JOWSEY, J.: The Effects of Chronic Acid and Alkali Administration on Bone Turnover in Adult Rats, Gin Sci 36:517-524, 1969. 21. WEATHERELL, J. A.; DEUTSCH, D.; ROBINSON, C.; and HALLSWORTH, A. S.: Fluoride Concentrations in Developing Enamel, Nature, Lond. 256:230-232, 1975. 22. TAVES, D. R.: Determination of Submicromolar Concentrations of Fluoride in Biological Samples, Talanta 15:1015-1023, 1968. 23. CHEN, P. S., JR.; TORIBARA, T. Y.; and WARNER, H.: Microdetermination of Phosphorus, Analyt Chem 28:1756-1758, 1956. 24. WHITFORD, G. M.; PASHLEY, D. H.; and REYNOLDS, K. E.: Fluoride Absorption from the Rat Urinary Bladder: a pHdependent Event, Am J Physiol 232:F10F15, 1977. 25. WHITFORD, G. M.; SCHUSTER, G. S.; PASHLEY, D. H.; and VENKATESWARLU, P.: Fluoride Uptake by Streptococcus mutans 6715, Infect Immun 18:680.687, 1977. 26. REYNOLDS, K. E.; WHITFORD, G. M.; and PASHLEY, D. H.: Acute Fluoride Toxicity: The Influence of Acid-Base Status, Toxicol Appl Pharmacol 45:415427, 1978. 27. WHITFORD, G. M.; PASHLEY, D. H.; and REYNOLDS, K. E.: Fluoride Tissue Dis-

28.

29.

30.

31.

32.

33.

34.

tribution: Short Term Kinetics, Am JPhysiol 236:F141-F149, 1979. IRVINE, R. 0. H.; DOW, J.; and FONG, J.: Effect of Anesthesia on the Collection of Blood for Acid-Base Studies in the Rat, Med PharmacolExp 14:55 7-562, 1966. ROLLINS, D. E.; WITHROW, C. D.; and WOODBURY, D. M.: Tissue Acid-Base Balance in Acetazolamide-Treated Rats, JPharmacol Exptl Ther 174:535-540, 1970. SHEARER, T. R.; KOLSTAD, D. L.; and SUTTIE, J. W.: Bovine Dental Fluorosis: Histologic and Physical Characteristics, Am J Vet Res 39:597b602, 1978. SHINODA, H.: Effect of Long-Term Administration of Fluoride on Physico-Chemical Properties of Rat Incisor Enamel, Calcif Tiss Res 18:91-100, 1975. LEATHERWOOD, E. C.; BURNETT, G. W.; CHANDRAVEJJSMARN, R.;and SIRIKAYA, P.: Dental Caries and Dental Fluorosis in Thailand, Am J Pub Itith 55:1792-1799, 1965. NANDA, R. S.; ZIPKIN, I.; DOYLE, J.; and HOROWITZ, H. S.: Factors Affecting the Prevalence of Dental Fluorosis in Lucknow, India, Arch Oral Biol 19:781792, 1974. GRAHNEN, H.; LYSELL, L.; MYRBERG, N.; and OLLINEN, P.: Fluoride, Mineralization Defects of the Enamel, and Tooth Width, Acta Pediat Scand 63:188-192, 1974.

ANNOUNCEMENT
Combined First Annual Scientific Session Society For Clinical Trials AND Seventh Annual Symposium For Coordinating Clinical Trials May 6-8, 1980

Philadelphia, Pennsylvania
The Sessions will focus on the design, organization, management, and analyses of clinical trials. Abstracts must be received by January 21, 1980. For more information write to: Christian R. Klimt, M.D., Secretary Society for Clinical Trials, Inc. 600 Wyndhurst Avenue Baltimore, MD 21210